Hv_Apo ) , whereas the GO term ‘metabolic process’ was enriched among down-regulated contigs ( Hv_Sym < Hv_Apo ) ( Figure 1D ) .","More specifically , the up-regulated contigs included many genes related to ‘transmembrane transporter activity’ , ‘transmembrane transport’ , ‘transposition’ , ‘cilium’ and ‘protein binding , bridging’ ( Figure 1E ) .","In the down-regulated contig set , the GO classes ‘cellular amino acid metabolic process’ , ‘cell wall organization or biogenesis’ and ‘peptidase activity’ were overrepresented ( Figure 1E ) .","These results suggest that the Chlorella symbiont affects core metabolic processes and pathways in Hydra .","Particularly , carrier proteins and active membrane transport appear to play a prominent role in the symbiosis .","As next step , we used GO terms , domain search and similarity search to further analyze the differentially expressed contigs between Hv_Sym and Hv_Apo ( Supplementary file 1 ) .","As the genes with GO terms related to localization and transport , we identified 27 up-regulated contigs in Hv_Sym ( Table 1 ) .","Interestingly , this gene set included a contig showing sequence similarity to the glucose transporter GLUT8 gene , which was previously reported to be up-regulated in the symbiotic state of the sea anemone Aiptasia ( Lehnert et al . , 2014; Sproles et al . , 2018 ) .","Thus , a conserved mechanism may be responsible for photosynthate transport from the symbiont into the host cytoplasm across the symbiosome membrane .","Further , a contig encoding a carbonic anhydrase ( CA ) enzyme was up-regulated in Hv_Sym ( Table 1 ) .","CA catalyzes the interconversion of HCO3 and CO2 .","Similar to the GLUT8 gene , carbonic anhydrase also appears to be up-regulated in symbiotic corals and anemones ( Weis et al . , 1989; Grasso et al . , 2008; Ganot et al . , 2011; Lehnert et al . , 2014 ) .","It appears plausible that for efficient photosynthesis in symbiotic algae , the host may need to convert CO2 to the less freely diffusing inorganic carbon ( HCO3 ) to maintain it in the symbiosome ( Lucas and Berry , 1985; Weis et al . , 1989; Barott et al . , 2015 ) .","We also observed up-regulation of contigs encoding proteins involved in vesicular and endosomal trafficking , such as spe-39 protein , otoferlin , protein fam194b and V-type proton ATPase 21 kda proteolipid , which may have a function in nutrition exchange between host and symbiont and maintenance of proper condition in the symbiosome .","Upregulated genes also include genes encoding rhamnospondin and fibrillin , known to be involved in cell adhesion and extracellular matrix , and retention of the symbiont at the proper site in the Hydra cells .","Photosynthesis by symbiotic algae imposes Reactive Oxygen Species ( ROS ) that can damage lipids , proteins and DNA in the host cells ( Lesser , 2006 ) .","Therefore , in symbiosis with photosynthetic organisms an appropriate oxidative stress response of the host is required for tolerance of the symbiont .","Indeed , an increase of antioxidant activities in symbiotic states of cnidarians has been reported previously ( Richier et al . , 2005 ) and it has been suggested that ROS produced by stress could be the major trigger of symbiosis breakdown during coral bleaching ( Lesser , 2006; Weis , 2008 ) .","To understand the oxidative stress response in green hydra , we searched the differentially expressed genes with the GO terms ‘response to oxidative stress’ , ‘oxidation-reduction process’ and ‘oxidoreductase activity’ .","In Hv_Sym , contigs for peroxidase , methionine-r-sulfoxide reductase/selenoprotein and glutaredoxin , which are known to be related to oxidative stress response were up-regulated ( Table 1 ) .","On the other hand , some contigs encoding glutathione S-transferase and ascorbate peroxidase were down-regulated in Hv_Sym .","In addition , two contigs encoding polycystin were down-regulated in Hv_Sym .","Polycystin is an intracellular calcium release channel that is inhibited by ROS ( Montalbetti et al . , 2008 ) and is also down-regulated in a different strain of symbiotic green hydra ( Ishikawa et al . , 2016 ) .","In addition , six contigs encoding metalloproteinases showed differential expression between Hv_Sym and Hv_Apo .","Although metalloproteinases have many functions such as cleavage of cell surface proteins and remodeling of the extracellular matrix , in a previous study they also were found to play a role in the oxidative stress response ( Császár et al . , 2009 ) .","A key antioxidant in the oxidative stress response in symbiotic cnidarians turns out to be glutathione ( Sunagawa et al . , 2009; Meyer and Weis , 2012 ) .","The gene encoding glutathione S-transferase was previously observed to be downregulated in corals , sea anemones , different strains of green hydra and Paramecium ( Kodama et al . , 2014; Lehnert et al . , 2014; Ishikawa et al . , 2016; Mohamed et al . , 2016 ) .","Our study supports this view ( Table","1 ) and may point to a conserved feature of oxidative stress response in algal-animal symbiosis .","Previous studies have suggested that during establishment of coral–algal symbiosis the host immune response may be partially suppressed ( Weis et al . , 2008; Mohamed et al . , 2016 ) .","Our observations in Hydra together with previous findings in corals indicate that regulation of symbiosis by innate immunity pathways indeed may be a general feature of cnidarian symbiosis .","Among the differentially expressed contigs we identified a number of genes involved in innate immunity and apoptosis .","Pattern recognition receptors ( PRRs ) and the downstream innate immunity and apoptosis pathways are thought to play important roles in various symbiotic interactions including cnidarian-dinoflagellate symbiosis ( Davy et al . , 2012 ) .","In Hv_Sym we found two up-regulated contigs that contain a Toll/interleukin-1 receptor ( TIR ) domain ( Table 1 ) .","TIR is a known PRR that is inserted in the host cell membrane and plays an important role in the innate immune system by specifically recognizing microbial-associated molecular patterns , such as flagellin , lipopolysaccharide ( LPS ) and peptidoglycan ( Hoving et al . , 2014 ) .","Furthermore , we found one up-regulated contig with similarity to a mannose receptor gene with C-type lectin domain ( Table 1 ) .","This is worth noting since C-type lectin receptors bind carbohydrates and some of them are known to function as PRRs .","Host lectin-algal glycan interactions have been proposed to be involved in infection and recognition of symbionts in some cnidarians including green hydra , sea anemones and corals ( Meints and Pardy , 1980; Lin et al . , 2000; Wood-Charlson et al . , 2006 ) .","Interestingly , up-regulation of C-type lectin genes was also observed during onset of cnidarian–dinoflagellate symbiosis ( Grasso et al . , 2008; Schwarz et al . , 2008; Sunagawa et al . , 2009; Mohamed et al . , 2016 ) .","Furthermore , contigs encoding chitinase enzymes also were differentially expressed between Hv_Sym and Hv_Apo ( Table 1 ) .","Chitinases are involved in degradation of chitin , which is a component of the exoskeleton of arthropods and the cell wall of fungi , bacteria and some Chlorella algae ( Kapaun and Reisser , 1995 ) , and also might play a role in host-defense systems for pathogens which have chitinous cell wall .","Chitinases are classified into two glycoside hydrolase families , GH18 and GH19 , with different structures and catalytic mechanisms .","In Hv_Sym two contigs encoding GH18 chitinases were up-regulated , while one contig encoding a GH19 chitinase was down-regulated , suggesting that the enzymes involved in chitin degradation are sensitive to the presence or absence of symbiotic Chlorella .","To narrow down the number of genes specifically affected by the presence of the native symbiont Chlorella A99 , we identified 12 contigs that are differentially expressed in symbiosis with Chlorella A99 , but not in presence of foreign Chlorella NC64A ( Figure 1C A99-specific ) .","Independent qPCR confirmed the differential expression pattern for 10 of these genes ( Table 2 ) .","The genes up-regulated by the presence of the symbiont encode a Spot_14 protein , a glutamine synthetase ( GS ) and a sodium-dependent phosphate ( Na/Pi ) transport protein in addition to a H . viridissima specific gene ( rc_12891: Sym-1 ) and a Hydra genus specific gene ( rc_13570: Sym-2 ) ( Table 2 ) .","Hydra genes down-regulated by the presence of Chlorella A99 were two H . viridissima-specific genes and three metabolic genes encoding histidine ammonia-lyase , acetoacetyl-CoA synthetase and 2-isopropylmalate synthase ( Table 2 ) .","Of the up-regulated genes , Spot_14 is described as thyroid hormone-responsive spot 14 protein reported to be induced by dietary carbohydrates and glucose in mammals ( Tao and Towle , 1986; Brown et al . , 1997 ) .","Na/Pi transport protein is a membrane transporter actively transporting phosphate into cells ( Murer and Biber , 1996 ) .","GS plays an essential role in the metabolism of nitrogen by catalyzing the reaction between glutamate and ammonia to form glutamine ( Liaw et al . , 1995 ) .","Interestingly , out of the three GS genes H . viridissima contains only GS-1 was found to be up-regulated by the presence of the symbiont ( Figure 1—figure supplement 3 ) .","The discovery of these transcriptional responses points to an intimate metabolic exchange between the partners in a species-specific manner .","To better understand the specificity of Hydra´s response to the presence of the foreign symbiont , we also identified the genes differentially expressed in Hydra polyps hosting a non-native Chlorella NC64A ( Hv_NC64A ) compared to both polyps hosting the obligate symbiont Chlorella A99 ( Hv_A99 ) and aposymbiotic Hydra ( Hv_Apo ) .","We found 19 contigs that were up-regulated and 45 contigs that were down-regulated in presence of NC64A , which strikingly did not include any genes related to immunity or oxidative stress response ( Supplementary file 1 ) .","Instead , the differentially expressed contigs showed similarity to methylase genes involved in ubiquinone menaquinone biosynthesis and secondary metabolite synthesis such as n- ( 5-amino-5-carboxypentanoyl ) -l-cysteinyl-d-valine synthase and non-ribosomal peptide synthase .","Four differentially expressed contigs specifically up-regulated in Hv_NC64A encoded ubiquitin carboxyl-terminal hydrolases , ( Table 3 ) .","To test whether photosynthetic activity of the symbiont is required for up-regulation of gene expression , Hv_Sym was either cultured under a standard 12 hr light/dark alternating regime or continuously in the dark for 1 to 4 days prior to RNA extraction ( Figure 2A ) .","Interestingly , four ( GS1 , Spot14 , Na/Pi and Sym-1 ) of five genes specifically activated by the presence of Chlorella A99 showed significant up-regulation when exposed to light ( Figure 2B ) , indicating the relevance of photosynthetic activity of Chlorella .","This up-regulation was strictly dependent on presence of the algae , as in aposymbiotic Hv_Apo the response was absent ( Figure 2B ) .","On the other hand , symbiosis-regulated Hydra genes not specific for Chlorella A99 ( Figure 1C Symbiosis-regulated , Table 4 ) appear to be not up-regulated in a light-dependent manner ( Figure 2—figure supplement 1 ) .","These genes are involved in Hydra´s innate immune system ( e . g . proteins containing Toll/interleukin-1 receptor domain or Death domain ) or in signal transduction ( C-type mannose receptor , ephrin receptor , proline-rich transmembrane protein 1 , ‘protein-kinase , interferon-inducible double stranded RNA dependent inhibitor , repressor of ( p58 repressor ) ’ ) .","That particular transcriptional changes observed in Hydra rely solely on the photosynthetic activity of Chlorella A99 was confirmed by substituting the dark incubation with selective chemical photosynthesis inhibitor DCMU ( Dichorophenyl-dimethylurea ) ( Vandermeulen et al . , 1972 ) , which resulted in a similar effect ( Figure 2C , D ) .","To further characterize the symbiont induced Hydra genes , we performed whole mount in situ hybridization ( Figure 3A–F ) and quantified transcripts by qPCR using templates from isolated endoderm and ectoderm ( Figure 3—figure supplement 1 ) , again comparing symbiotic and aposymbiotic polyps ( Figure 3G–I ) .","The GS-1 gene and the Spot14 gene are expressed both in ectoderm and in endoderm ( Figure 3A , B ) and both genes are strongly up-regulated in the presence of the symbiont ( Figure 3G , H ) .","In contrast , the Na/Pi gene was expressed only in the endoderm ( Figure 3C ) and there it was strongly up-regulated by the symbiont ( Figure 3I ) .","Since Chlorella sp .","A99 colonizes endodermal epithelial cells only , the impact of algae on symbiosis-dependent genes in both the ectodermal and the endodermal layer indicates that photosynthetic products can be transported across these two tissue layers or some signals can be transduced by cell-cell communication .","To more closely dissect the nature of the functional interaction between Hydra and Chlorella and to explore the possibility that maltose released from the algae is involved in A99-specific gene regulation , we cultured aposymbiotic polyps ( Hv_Apo ) for 2 days in medium containing various concentrations of maltose ( Figure 3J ) .","Of the five A99 specific genes , GS-1 and the Spot14 gene were up-regulated by maltose in a dose-dependent manner; the Na/Pi gene was only up-regulated in 100 mM maltose and the Hydra specific genes Sym-1 and Sym-2 did not show significant changes in expression by exposure to maltose ( Figure 3J ) .","This provides strong support for previous views that maltose excretion by symbiotic algae contributes to the stabilization of this symbiotic association ( Cernichiari et al . , 1969 ) .","When polyps were exposed to glucose instead of maltose , the genes of interest were also transcriptionally activated in a dose-dependent manner , while sucrose had no effect ( Figure 3—figure supplement 2A–D ) .","Exposure to low concentrations of galactose increased transcriptional activity but at high concentration it did not , indicating a substrate inhibitor effect for this sugar .","That the response to glucose is similar or even higher compared to maltose after 6 hr of treatment ( Figure 3—figure supplement 2E ) , suggests that Hydra cells transform maltose to glucose as a source of energy .","In animals including cnidarians , several glucose transporters have been identified ( Sproles et al . , 2018 ) , but yet no maltose transporters .","This is consistent with the view that maltose produced by the symbiont is digested to glucose in the symbiosome and translocated to the host cytoplasm through glucose transporters .","To better understand the symbiosis between H . viridissima and Chlorella and to refine our knowledge of the functions that are required in this symbiosis , we sequenced the genome of Chlorella sp .","strain A99 and compared it to the genomes of other green algae .","The genome of Chlorella sp .","A99 was sequenced to approximately 211-fold coverage , enabling the generation of an assembly comprising a total of 40 . 9 Mbp ( 82 scaffolds , N50 = 1 . 7 Mbp ) ( Table 5 ) .","Chlorella sp .","A99 belongs to the family Chlorellaceae ( Figure 4A ) and of the green algae whose genomes have been sequenced it is most closely related to Chlorella variabilis NC64A ( NC64A ) ( Merchant et al . , 2007; Palenik et al . , 2007; Worden et al . , 2009; Blanc et al . , 2010; Prochnik et al . , 2010; Blanc et al . , 2012; Gao et al . , 2014; Pombert et al . , 2014 ) .","The genome size of the total assembly in strain A99 was similar to that of strain NC64A ( 46 . 2 Mb ) ( Figure 4B ) .","By k-mer analysis ( k-mer = 19 ) , the genome size of A99 was estimated to be 61 Mbp ( Marçais and Kingsford , 2011 ) .","Its GC content of 68% , is the highest among the green algae species recorded ( Figure 4B ) .","In the A99 genome , 8298 gene models were predicted .","As shown in Figure 4C , about 80% of these predicted genes have extensive sequence similarity to plant genes , while 13% so far have no similarity to genes of any other organisms ( Figure 4C ) .","It is also noteworthy that 7% of the A99 genes are similar to genes of other kingdoms but not to Hydra , indicating the absence of gene transfer from Hydra to the symbiont genome ( Figure 4C ) .","Several independent lines of evidence demonstrate that nitrogen limitation and amino-acid metabolism have a key role in the Chlorella–Hydra symbiosis and that symbiotic Chlorella A99 depends on glutamine provided by its host ( Rees , 1986; McAuley , 1987a; 1987b; McAuley , 1991; Rees , 1991;1989 ) .","To identify Chlorella candidate factors for the development and maintenance of the symbiotic life style , we therefore used the available genome information to assess genes potentially involved in amino acid transport and the nitrogen metabolic pathway .","When performing a search for the Pfam domain ‘Aa_trans’ or ‘AA_permease’ to find amino acid transporter genes in the A99 genome , we discovered numerous genes containing the Aa_trans domain ( Table 6A ) .","In particular , A99 contains many orthologous genes of amino acid permease 2 and of transmembrane amino acid transporter family protein ( solute carrier family 38 , sodium-coupled neutral amino acid transporter ) , as well as NC64A ( Table 6B , Supplementary file 2 ) .","Both of these gene products are known to transport neutral amino acids including glutamine .","This observation is supporting the view that import of amino acids is an essential feature for the symbiotic way of life of Chlorella .","In symbiotic organisms , loss of genes often occurs due to the strictly interdependent relationship ( Ochman and Moran , 2001; Wernegreen , 2012 ) , raising the possibility that Chlorella A99 might have lost some essential genes .","To test this hypothesis , we searched the Chlorella A99 genome for genes conserved across free-living green algae Coccomyxa subellipsoidea C169 ( C169 ) , Chlamydomonas reinhardtii ( Cr ) and Volvox carteri ( Vc ) .","In a total of 9851 C169 genes , we found 5701 genes to be conserved in Cr and Vc ( Supplementary file 3 ) .","Of these , 238 genes did not match to any gene models and genomic regions in Chlorella A99 and thus were considered as gene loss candidates .","Interestingly , within these 238 candidates , genes with the GO terms ‘transport’ in biological process and ‘transporter activity’ in molecular function were overrepresented ( Figure 5 ) .","In particular , the 28 genes annotated to these GO terms encoded nitrate transporter , urea transporter and molybdate transporter , which are known to be involved in nitrogen metabolism ( Table 7 ) .","Beside ammonium , nitrate and urea are major nitrogen sources for plants , whereas molybdate is a co-factor of the nitrate reductase , an important enzyme in the nitrate assimilation pathway .","These transporter genes are conserved across green algae including Chlorella NC64A ( Sanz-Luque et al . , 2015; Gao et al . , 2014 ) and appear to be lost in the Chlorella A99 genome .","In nitrogen assimilation processes , plants usually take up nitrogen in the form of nitrate ( NO3- ) via nitrate transporters ( NRTs ) or as ammonium ( NH4+ ) via ammonium transporters ( AMT ) ( Figure 6A ) .","In higher plants , two types of nitrate transporters , NRT1 and NRT2 , have been identified ( Krapp et al . , 2014 ) .","Some NRT2 require nitrate assimilation-related component 2 ( NAR2 ) to be functional ( Quesada et al . , 1994 ) .","NO3- is reduced to nitrite by nitrate reductase ( NR ) , NO2- is transported to the chloroplast by nitrate assimilation-related component1 ( NAR1 ) , and NO2- is reduced to NH4+ by nitrite reductase ( NiR ) .","NH4+ is incorporated into glutamine ( Gln ) by glutamine synthetase ( GS ) , and Gln is incorporated into glutamate ( Glu ) by NADH-dependent glutamine amide-2-oxoglutarate aminotransferase ( GOGAT ) , also known as glutamate synthase .","This pathway is highly conserved among plants and all of its major components , including NRT1 and NRT2 , NAR1 and NAR2 , NR , NiR , AMT , GOGAT and GS , are present in the 10 green algae species that have been genome-sequenced so far ( with the exception of NRT1 , which is absent in Micromonas pusilla ) ( Sanz-Luque et al . , 2015 ) .","In Symbiodinium , the photosynthetic symbiont of marine invertebrates , all these components of the nitrogen assimilation pathway were also observed ( Supplementary file 4 ) ( Shoguchi et al . , 2013; Lin et al . , 2015; Aranda et al . , 2016; Sproles et al . , 2018 ) .","Based on the annotation by Sanz-Luque et al . ( Sanz-Luque et al . , 2015 ) , we searched these nitrogen assimilation genes in the Chlorella A99 genome , using ortholog grouping and a reciprocal BLAST search using the protein sequences from other green algae ( Figure 6B , Supplementary file 5 ) .","As expected , the Chlorella A99 genome contains many homologues of the genes involved in nitrogen assimilation in plants including genes encoding NRT1 , NAR1 , NR , AMT , GS and GOGAT ( Figure 6B ) .","Intriguingly , our systematic searches failed to identify representative genes for NRT2 , NAR2 and NiR in the Chlorella A99 genome ( Figure 6B ) .","We confirmed the absence of the NRT2 and NiR genes by PCR using primers designed for the conserved regions of these genes and which failed to produce a product with genomic DNA as a template ( Figure 6—figure supplement 1 ) .","Due to the weak sequence conservation of the NAR2 gene in the three algae genomes , PCR of that gene was not performed .","Taken together , our observations indicate that Chlorella A99 algae appears to lack NRT2 , NAR2 and NiR .","Since in many fungi , cyanobacteria and algae species , nitrate assimilation genes are known to act in concert and a gene cluster of NR and NiR genes is conserved between different green algae ( Sanz-Luque et al . , 2015 ) , we next investigated the level of genomic clustering of the nitrate assimilation pathway genes in the Chlorella genome .","Comparing the genomes of NC64A and C169 revealed the presence of a cluster of NR and NiR genes ( Figure 6C ) .","In NC64A , two NRT2 genes , together with genes for NAR2 , NR and NiR are clustered on scaffold 21 .","In C169 , one of the NR genes and NiR are clustered together , whereas the second NR gene is separate .","Interestingly , analysis of the sequences around the NR gene in the Chlorella A99 genome provided no evidence for the presence of a co-localized NiR gene or any other nitrate assimilation genes , nor any conserved gene synteny to NC64A and C169 ( Figure 6C ) .","Therefore , our comparative genomic analyses points to an incomplete and scattered nitrogen metabolic pathway in symbiotic Chlorella A99 , which lacks essential transporters and enzymes for nitrate assimilation as well as the clustered structure of nitrate assimilation genes .","The absence of genes essential for nitrate assimilation in the Chlorella A99 genome ( Figure 6 ) is consistent with its inability to grow outside the Hydra host cell ( Habetha and Bosch , 2005 ) and indicates that Chlorella symbionts are dependent on metabolites provided by their host .","We hypothesized that Chlorella is unable to use nitrite and ammonium as a nitrogen source , and that it relies on Hydra assimilating ammonium to glutamine to serve as the nitrogen source .","To test this hypothesis and to examine utilization of nitrogen compounds of A99 , we isolated Chlorella A99 from Hv_Sym and cultivated it in vitro using modified bold basal medium ( BBM ) ( Nichols and Bold , 1965 ) containing the same amount of nitrogen in the form of NO3- , NH4+ , Gln or casamino acids ( Figure 7 ) .","As controls , Chlorella variabilis NC64A ( NC64A ) isolated from Hv_NC64A and free-living C169 were used .","To confirm that the cultured A99 is not contamination , we amplified and sequenced the genomic region of the 18S rRNA gene by PCR ( Figure 7—figure supplement","1 ) and checked this against the genomic sequence of A99 .","Kamako et al . reported that free-living alga Chlorella vulgaris Beijerinck var .","vulgaris grow in media containing only inorganic nitrogen compounds as well as in media containing casamino acids as a nitrogen source , while NC64A required amino acids for growth ( Kamako et al . , 2005 ) .","Consistent with these observations , C169 grew in all tested media and NC64A grew in media containing casamino acids and Gln , although its growth rate was quite low in presence of NH4+ and NO3- ( Figure 7 ) .","Remarkably , Chlorella A99 increased in cell number for up to 8 days in media containing casamino acids and Gln ( Figure 7 ) .","Similar to NC64A , A99 did not grow in presence of NH4+ and NO3- .","The growth rates of both A99 and NC64A were higher in medium containing a mixture of amino acids ( casamino acids ) than the single amino acid Gln .","In contrast to NC64A , A99 could not be cultivated permanently in casamino acids or glutamine supplemented medium , indicating that additional growth factors are necessary to maintain in vitro growth of this obligate symbiont .","Thus , although in vitro growth of A99 can be promoted by adding Glu and amino acids to the medium , A99 cannot be cultured permanently in this enriched medium , indicating that other host derived factors remain to be uncovered ."],["Sequencing of the Chlorella A99 genome in combination with the transcriptome analyses of symbiotic , aposymbiotic and NC64A-infected H . viridissima polyps has enabled the identification of genes with specific functions in this symbiotic partnership .","The Hydra-Chlorella symbiosis links carbohydrate supply from the photosynthetic symbiont to glutamine synthesis by the host .","Characteristics of the symbiont genome obviously reflect its adaptation to this way of life , including an increase in amino acid transporters and degeneration of the nitrate assimilation pathway .","This conclusion is based on six observations:","( i ) Expression of some genes including GS-1 , Spot 14 and NaPi is specifically up-regulated in the presence of Chlorella A99 ( Figure 1C , Table 2 ) , and","( ii ) they are induced by both , photosynthetic activity of Chlorella and by supplying exogenous maltose or glucose ( Figures 2 and 3J , Figure 3—figure supplement 2 ) .","Maltose produced by the symbiont is likely to be digested to glucose in symbiosome and translocated to the host cytoplasm through glucose transporters ( Figure 8A ) .","Upregulation of a GLUT8 gene in the symbiotic state of green hydra may reflect activation of sugar transport ( Table 1 ) .","These results indicate that maltose release by photosynthesis of the symbiont enhances nutrition supply including glutamine by the host ( Figure 8A ) .","( iii ) Symbiotic Chlorella A99 cannot be cultivated in vitro in medium containing a single inorganic nitrogen source ( Figure 7 ) .","Since medium containing glutamine supports in vitro growth of A99 , this organism appears to depend on glutamine provided by the Hydra host .","( iv ) The genome of Chlorella A99 contains multiple amino acid transporter genes ( Table 6 ) , but lacks genes involved in nitrate assimilation ( Figure 6 ) , pointing to amino acids as main source of nitrogen and a degenerated nitrate assimilation pathway .","As for ammonium , which is one of the main nitrogen sources in plants , previous studies have reported the inability of symbiotic algae to take up ammonium because of the low peri-algal pH ( pH 4–5 ) that stimulates maltose release ( Douglas and Smith , 1984; Rees , 1989; McAuley , 1991; Dorling et al . , 1997 ) .","Since Chlorella apparently cannot use nitrite and ammonium as a nitrogen source , it seems that Hydra has to assimilate ammonium to glutamine and provides it to Chlorella A99 ( Figure 8A ) .","( v ) While polyps with native symbiont Chlorella A99 grew faster than aposymbiotic ones , symbiosis with foreign algae NC64A had no effect on the growth of polyps at all ( Figure 1B ) .","( vi ) Hydra endodermal epithelial cells host significantly fewer NC64A algae than A99 ( Figure 1—figure supplement 1 ) providing additional support for the view of a tightly regulated codependent partnership in which exchange of nutrients appears to be the primary driving force .","Previous studies have reported that symbiotic Chlorella in green hydra releases significantly larger amounts of maltose than NC64A ( Mews and Smith , 1982; Rees , 1989 ) .","In addition , Rees reported that Hydra polyps containing high maltose releasing algae had a high GS activity , whereas aposymbiotic Hydra or Hydra with a low maltose releasing algae had lower GS activity ( Rees , 1986 ) .","Although the underlying mechanism of how maltose secretion and transportation from Chlorella is regulated is still unclear , the amount of maltose released by the symbiont could be an important symbiont-derived driver or stabilizer of the Hydra–Chlorella symbiosis .","Transcriptome comparison between symbiotic and aposymbiotic H . viridissima demonstrated that symbiosis-regulated genes are involved in oxidative stress response and innate immunity .","The fact that PRRs and apoptosis-related genes , are also differentially expressed in a number of other symbiotic cnidarians ( Table 1 ) , suggests innate immunity as conserved mechanism involved in controlling the development and maintenance of stable symbiotic interactions .","Furthermore , the exchange of nitrogenous compounds and photosynthetic products between host and symbiont observed here in the Hydra-Chlorella symbiosis is also observed in marine invertebrates such as corals , sea anemones and giant clams associated with Symbiodinium algae ( Figure 8B , C ) .","Despite these similarities , however , there are also conspicuous differences among symbiotic cnidarians in particular with respect to the nutrients provided by the symbiont to the host .","For example , symbiotic Chlorella algae in green hydra , Paramecium and fresh water sponges provide their photosynthetic products in form of maltose and glucose ( Figure 8B ) ( Brown and Nielsen , 1974; Wilkinson , 1980; Kamako and Imamura , 2006 ) .","In contrast , Symbiodinium provides glucose , glycerol , organic acids , amino acids as well as lipids to its marine hosts ( Figure 8C ) ( Muscatine and Cernichiari , 1969; Lewis and Smith , 1971; Trench , 1971; Kellogg and Patton , 1983 ) .","A former transcriptome analysis of amino acid biosynthetic pathways suggested that Symbiodinium can synthesize almost all amino acids ( Shinzato et al . , 2014 ) .","Gene loss in cysteine synthesis pathway in the coral host Acropora digitifera seems to reflect the dependency on the amino acids provided by the Symbiodinium symbiont ( Shinzato et al . , 2011 ) .","In contrast to Symbiodinium which can assimilate inorganic nitrogen such as nitrate and ammonium ( Lipschultz and Cook , 2002; Grover et al . , 2003; Tanaka et al . , 2006; Yellowlees et al . , 2008 ) , the symbiotic Chlorella algae in Hydra and Paramecium can only use amino acids as a nitrogen source ( Figure","6 ) ( Kamako et al . , 2005 ) .","In efforts to explain the metabolic efficiency of nitrogen use in symbiotic organisms , two models have been proposed: the ‘nitrogen conservation’ and the ‘nitrogen recycling’ hypothesis .","The nitrogen conservation hypothesis suggests that photosynthetic carbon compounds from the symbiont are used preferentially by the host respiration , which reduces catabolism of nitrogenous compounds ( Rees and Ellard , 1989; Szmant et al . , 1990; Wang and Douglas , 1998 ) .","The ‘nitrogen recycling’ hypothesis suggests that symbionts assimilate nitrogenous waste ( ammonium ) of the host into valuable , organic compounds , which then are translocated back to the host ( Figure 8C Symbiont nitrogen assimilation ) ( Lewis and Smith , 1971; Muscatine and Porter , 1977; Falkowski et al . , 1993; Wang and Douglas , 1998 ) .","Our observation that in symbiotic green hydra many genes involved in amino acid metabolism are down-regulated ( Figure 1E ) is consistent with the assumption of reduction of amino acid consumption by respiration .","In addition to the nitrogen recycling hypothesis , it has been proposed that also corals , sea anemones , Paramecium and green hydra hosts can assimilate ammonium into amino acids ( Figure 8B , C Host nitrogen assimilation ) ( Miller and Yellowlees , 1989; Rees , 1989; Szmant et al . , 1990; Rees , 1991; Wang and Douglas , 1998; Lipschultz and Cook , 2002 ) .","Ammonia assimilation by the host implies that the host controls the nitrogen status to regulate metabolism of the symbionts , which may be involved in controlling the number of symbionts within the host cell .","For organisms such as corals living in oligotrophic sea , inorganic nitrogen assimilation and recycling may be necessary to manage the nitrogen sources efficiently .","In contrast , for Hydra and Paramecium living in a relatively nutrient-rich environment may be advantageous in terms of metabolic efficiency that the symbiont abandons its ability to assimilate inorganic nitrogen and specializes in the supply of photosynthetic carbohydrate to the host .","Metabolic dependence of symbionts on host supply occasionally results in genome reduction and gene loss .","For example , symbiotic Buchnera bacteria in insects are missing particular genes in essential amino acid pathways ( Shigenobu et al . , 2000; Hansen and Moran , 2011 ) .","The fact that the corresponding genes of the host are up-regulated in the bacteriocyte , indicates complementarity and syntrophy between host and symbiont .","Similarly , in Chlorella A99 the nitrogen assimilation system could have been lost as a result of continuous supply of nitrogenous amino acids provided by Hydra .","Compared to Chlorella NC64A , the closest relative to Chlorella A99 among the genome-sequenced algae , genome size and total number of genes in Chlorella A99 were found to be smaller ( Figure 4B ) .","Although both A99 and NC64A cannot be cultivated using inorganic nitrogen sources ( Figure","7 ) ( Kamako et al . , 2005 ) , NC64A , unlike A99 , obtains all major nitrogen assimilation genes and their cluster structure on the chromosome ( Figure","6 ) ( Sanz-Luque et al . , 2015 ) .","NR and NiR activities were found to be induced by nitrate in free-living Chlorella , but not in Chlorella NC64A , indicating mutations in the regulatory region ( Kamako et al . , 2005 ) .","Considering the phylogenetic position of NC64A and the symbiotic Chlorella of green hydra ( Kawaida et al . , 2013 ) , the disability of nitrate assimilation in A99 and NC64A seems to have evolved independently , suggesting convergent evolution in a similar symbiotic environment .","Although our findings indicate that genome reduction in Chlorella A99 is more advanced than in Chlorella NC64A , genome size and total number of genes do not differ much between the Trebouxiophyceae ( A99 , NC64A and C169 ) ( Figure 4B ) .","By contrast , the parasitic algae Helicosporidium and Auxochlorella have significantly smaller genome sizes and number of genes indicating extensive genome reduction ( Gao et al . , 2014; Pombert et al . , 2014 ) .","The apparently unchanged complexity of the Chlorella A99 genome suggests a relatively early stage of this symbiotic partnership .","Thus , gene loss in metabolic pathways could occur as a first step of genome reduction in symbionts caused by the adaptation to continuous nutrient supply from the host .","Taken together , our study suggests metabolic-codependency as the primary driving force in the evolution of symbiosis between Hydra and Chlorella ."],["Experiments were carried out with the Australian Hydra viridissima strain A99 , which was obtained from Dr . Richard Campbell , Irvine .","Polyps were maintained at 18°C on a 12 hr light/dark cycle and fed with Artemia two or three times a week .","Aposymbiotic ( algae free ) polyps were obtained by photobleaching using 5 μM DCMU ( 3- ( 3 , 4-dichlorophenyl ) −1 , 1-dimethylurea ) as described before ( Pardy , 1976; Habetha et al . , 2003 ) .","Experiments were carried out with polyps starved for 3–6 days .","Isolation of endodermal layer and ectodermal layer was performed as described by Kishimoto et al . ( Kishimoto et al . , 1996 ) .","Symbiotic Chlorella were isolated as described before by Muscatine and McAuley ( Muscatine , 1983; McAuley , 1986 ) .","Chlorella variabilis NC64A ( NIES-2541 ) , Coccomyxa subellipsoidea C-169 ( NIES-2166 ) and Chlamydomonas reinhardtii ( NIES-2235 ) were obtained from the Microbial Culture Collection at the National Institute for Environmental Studies ( Tsukuba , Japan ) .","Total RNA of Hydra was extracted by use of the Trizol reagent and PureLink RNA Mini Kit ( Thermo Fisher Scientific ) after lysis and removal of algae by centrifugation .","The genomic DNA of green algae was extracted using ISOPLANT II ( Nippon Gene , Tokyo , Japan ) following DNase I treatment to degrade contaminant DNA .","Quantity and quality of DNA and RNA were checked by NanoDrop ( Thermo Scientific Inc . , Madison , USA ) and BioAnalyzer ( Agilent Technologies , Santa Clara , USA ) .","Total RNA for synthesis of cRNA targets was extracted from about 100 green hydra for each experimental group .","Experiments were carried out using three biological replicates .","cRNA labeled with cyanine-3 were synthesized from 400 ng total Hydra RNA using a Low Input Quick Amp Labeling Kit for one color detection ( Agilent Technologies ) .","A set of fluorescently labeled cRNA targets was employed in a hybridization reaction with 4 × 44K Custom-Made Hydra viridissima Microarray ( Agilent Technologies ) contributing a total of 43 , 222 transcripts that was built by mRNA-seq data ( NCBI GEO Platform ID: GPL23280 ) ( Bosch et al . , 2009 ) .","Hybridization and washing were performed using the GE Hybridization Kit and GE Wash Pack ( Agilent Technologies ) after which the arrays were scanned on an Agilent Technologies G2565BA microarray scanner system with SureScan technology following protocols according to the manufacturer's instructions .","The intensity of probes was extracted from scanned microarray images using Feature Extraction 10 . 7 software ( Agilent Technologies ) .","All algorithms and parameters used in this analysis were used with default conditions .","Background-subtracted signal-intensity values ( gProcessedSignal ) generated by the Feature Extraction software were normalized using the 75th percentile signal intensity among the microarray .","Those genes differentially expressed between two samples were determined by average of fold change ( cut of >2 . 0 ) and Student's t-test ( p<0 . 1 ) .","The data series are accessible at NCBI GEO under accession number GSE97633 .","Total RNA was extracted from 50 green hydra polyps for each biological replicate independently .","For reverse transcription of total RNA First Strand cDNA Synthesis Kit ( Fermentas , Ontario , Canada ) was used .","Real-time PCR was performed using GoTaq qPCR Master Mix ( Promega , Madison , USA ) and ABI Prism 7300 ( Applied Biosystems , Foster City , USA ) .","All qPCR experiments were performed in duplicate with three biological replicates each .","Values were normalized using the expression of the tubulin alpha gene .","Primers used for these experiments are listed in Supplementary file 6A .","Expression patterns of specific Hydra genes were detected by whole mount in situ hybridization with digoxigenin ( DIG ) -labelled RNA probes .","Specimens were fixed in 4% paraformaldehyde .","Hybridization signal was visualized using anti-DIG antibodies conjugated to alkaline phosphatase and NBT/BCIP staining solution ( Roche ) .","DIG-labeled sense probes ( targeting the same sequences as the antisense probes ) were used as a control .","Primers used for these experiments are listed in Supplementary file 6B .","For genome sequencing of Chlorella sp .","A99 , Chlorella sp .","A99 was isolated from H . viridissima A99 and genomic DNA was extracted .","Paired-end library ( insert size: 740 bp ) and mate-pair libraries ( insert size: 2 . 2 and 15 . 2 kb ) were made using Illumina TruSeq DNA LT Sample Prep Kit and Nextera Mate Pair Sample Preparation Kit respectively ( Illumina Inc . , San Diego , USA ) , following the manufacturer's protocols .","Genome sequencing was performed using Illumina Miseq and Hiseq 2000 platforms .","Sequence reads were assembled using Newbler Assembler version 2 . 8 ( Roche , Penzberg , Germany ) and subsequent scaffolding was performed by SSPACE ( Boetzer et al . , 2011 ) .","Gaps inside the scaffolds were closed with the paired-end and mate-pair data using GapCloser of Short Oligonucleotide Analysis Package ( Luo et al . , 2012 ) .","To overcome potential assembly errors arising from tandem repeats , sequences that aligned to another sequence by more than 50% of the length using blastn ( 1e-50 ) were removed from the assembly .","The completeness of the genome was evaluated using CEGMA v2 . 4 ( Core Eukaryotic Genes Mapping Approach ) based on mapping of the 248 most highly conserved core eukaryotic genes ( CEGs ) on the assembled genome ( Parra et al . , 2007 ) .","The completeness of complete and partial CEGs in the A99 scaffolds was 80 and 88% , respectively .","The fraction of repetitive sequences was 12% .","Gene model was predicted by AUGUSTUS 3 . 0 . 1 using model parameters for NC64A ( Stanke et al . , 2006 ) .","This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession PCFQ00000000 ( BioProject ID: PRJNA412448 ) .","Genome sequences and gene models are also accessible at the website of OIST Marine Genomics Unit Genome Project ( http://marinegenomics . oist . jp/chlorellaA99/viewer/info ? project_id=65 ) .","Annotation of transcriptome contigs and prediction of gene models was performed by use of BLAST , Gene Ontology ( Ashburner et al . , 2000 ) and blast2go ( Conesa et al . , 2005 ) .","To examine the conservation of H . viridissima contigs among metazoans , homology searches by blastx ( evalue 1E-5 ) were performed using protein databases obtained from NCBI for Drosophila melanogaster and Homo sapiens , from the JGI genome portal ( http://genome . jgi . doe . gov/ ) for Branchiostoma floridae , Nematostella vectensis , from Echinobase ( http://www . echinobase . org/EchinoBase/ ) for Strongylocentrotus pupuratus , from Compagen for Hydra magnipapillata , and from the OIST marine genomics Genome browser ver . 1 . 1 ( http://marinegenomics . oist . jp/coral/viewer/info ? project_id=3 ) for Acropora digitifera .","For comparative analysis of gene models of Chlorella sp .","A99 and other algae , domain searches against the Pfam database ( Pfam-A . hmm ) were performed using HMMER ( Eddy , 1998; Finn et al . , 2016 ) , and ortholog gene grouping was done using OrthoFinder ( Emms and Kelly , 2015 ) .","The sequences of the reference genes and genomes were obtained from the database of the JGI genome portal for Chlorella variabilis NC64A , Coccomyxa subellipsoidea C-169 , Volvox carteri , Micromonas pusilla , and Ostreococcus tauri , from NCBI for Auxenochlorella protothecoides 0710 , from Phytozome ( http://phytozome . jgi . doe . gov/pz/portal . html ) for Chlamydomonas reinhardtii , from OIST Marine Genomics ( http://marinegenomics . oist . jp/symb/viewer/info ? project_id=21 ) for Symbiodinium minutum , Symbiodinium kawagutti genome , from Dinoflagellate Resources ( http://web . malab . cn/symka_new/ ) for Symbiodinium kawagutti and Reefgenomics ( http://reefgenomics . org/ ) for Symbiodinium microadriaticum ) ( Merchant et al . , 2007; Palenik et al . , 2007; Worden et al . , 2009; Blanc et al . , 2010; Prochnik et al . , 2010; Blanc et al . , 2012 ) Nitrogen assimilation genes in Chlorella A99 were identified by orthologous gene groups and reciprocal blast searches .","The number of genes for nitrate assimilation genes , glutamine synthetase and glutamate synthetase , and clustering of such genes were systematically reported by ( Sanz-Luque et al . , 2015 ) .","We used these data as reference for searches of nitrogen assimilation genes , and further nitrogen assimilation genes were searched by Kyoto Encyclopedia of Genes and Genomes ( KEGG ) pathway ( Kanehisa and Goto , 2000 ) .","JGI genome browsers of Chlorella variabilis NC64A and Coccomyxa subellipsoidea C-169 were also used for retrieving genes and checking gene order on the scaffolds .","For a phylogenetic tree of chlorophyte green algae , the sequences of 18S rRNA gene , ITS1 , 5 . 8S rRNA gene , ITS2 and 28S rRNA gene were obtained from scaffold20 of Chlorella A99 genome sequence , and from NCBI nucleotide database entries for Chlorella variabilis NC64A ( FM205849 . 1 ) , Auxenochlorella protothecoides 0710 ( NW_011934479 . 1 ) , Coccomyxa subellipsoidea C169 ( AGSI01000011 . 1 ) , Volvox carteri f .","nagariensis ( NW_003307662 . 1 ) , Chlamydomonas reinhardtii ( FR865576 . 1 ) , Ostreococcus tauri ( GQ426340 . 1 ) and Micromonas pusilla ( FN562452 . 1 ) .","Multiple alignments were produced with CLUSTALX ( 2 . 1 ) with gap trimming ( Larkin et al . , 2007 ) .","Sequences of poor quality that did not well align were deleted using BioEdit ( Hall , 1999 ) .","Phylogenetic analyses were performed using the Neighbor-Joining method by CLUSTALX with the default parameters ( 1000 bootstrap tests and 111 seeds ) .","Representative phylogenetic trees were drawn by using NJ plot ( Perrière and Gouy , 1996 ) .","Primers were designed based on the conserved region of the NRT2 gene , NiR and NR genes ( positive control ) identified by comparison of genes from Chlorella variabilis NC64A ( NC64A ) , Coccomyxa subellipsoidea C169 ( C169 ) , and Chlamydomonas reinhardtii ( Cr ) which belongs to Chlorophyceae class of green algae .","Primers for NAR2 could not be designed because of insufficient conservation .","As positive controls , amplicons were produced for NR of all the green algae examined and of NRT2 and NiR from NC64A , C169 and Cr , after which their sequences were checked .","KOD FX Neo ( TOYOBO , Tokyo , Japan ) was used under the following conditions: an initial denaturation phase ( 94°C for 120 s ) followed by 36 cycles of ( 98°C for 30 s , 69°C for 100 s ) for NiR , ( 98°C for 30 s , 58°C for 30 s and 68°C for 210 s ) for NRT2 and ( 98°C for 30 s , 59°C for 30 s and 68°C for 60 s ) for NR .","In each case , 10 ng gDNA was used as a template .","The primers used are described in Supplementary file 6C .","PCR products were sequenced to confirm amplification of the target genes using ABI PRISM 3100 Genetic Analyzer ( Thermo Fisher Scientific Inc . , Madison , USA ) using BigDye Terminator v3 . 1 Cycle Sequencing Kit ( Thermo Fisher Scientific ) .","To isolate symbiotic algae , polyps were quickly homogenized in 0 . 25% sodium dodecyl sulfate ( SDS ) solution and centrifuged at 3000 g for 1 min .","The pellet was resuspended in 0 . 05% SDS and centrifuged at 500 g for 5 min .","Isolated A99 , NC64A and C169 were washed by sterilized Bold Basal Medium ( Bischoff and Bold , 1963 ) modified by the addition of 0 . 5% glucose , 1 . 2 mg/L vitamine B1 ( Thiaminhydrochloride ) , 0 . 01 mg/L vitamine B12 ( Cyanocobalamin ) ( Supplementary file 7 ) and incubated for two days in modified Bold Basal Medium with 50 mg/l ampicillin and streptomycin .","The algae were cultivated in 5 ml of modified Bold Basal Medium ( BBM ) with the same amount of nitrogen ( 2 . 9 mM NaNO3 , NH4Cl , glutamine or 426 mg/l casamino acids ) and 5 mg/l Carbendazim ( anti-fungal ) with fluorescent illumination ( 12 hr light , 12 hr dark ) at 20˚C .","Mean numbers of algae per ml were calculated from three tubes enumerated at 4 , 8 , and 12 days after inoculation with 106 cell/sml using a hemocytometer .","After cultivation , gDNA was isolated from the A99 cultured in Gln-containing BBM and casamino acid-containing BBM and A99 was isolated from green hydra directly .","A partial genomic region of the 18S rRNA gene was amplified by PCR and sequenced to confirm absence of contamination by other algae .","PCR was performed using AmpliTaq Gold ( Thermo Fisher Scientific ) .","Sequencing was performed as described above .","The primers used are described in Supplementary file 6D ."]],"string":"[\n [\n \"Symbiosis has been a prevailing force throughout the evolution of life , driving the diversification of organisms and facilitating rapid adaptation of species to divergent new niches ( Moran , 2007; Joy , 2013; McFall-Ngai et al . , 2013 ) .\",\n \"In particular , symbiosis with photosynthetic symbionts is observed in many species of cnidarians such as corals , jellyfish , sea anemones and hydra , contributing to the ecological success of these sessile or planktonic animals ( Douglas , 1994; Davy et al . , 2012 ) .\",\n \"Among the many animals dependent on algal symbionts , inter-species interactions between green hydra Hydra viridissima and endosymbiotic unicellular green algae of the genus Chlorella have been a subject of interest for decades ( Muscatine and Lenhoff , 1963; Roffman and Lenhoff , 1969 ) .\",\n \"Such studies not only provide insights into the basic ‘tool kit’ necessary to establish symbiotic interactions , but are also of relevance in understanding the resulting evolutionary selective processes ( Muscatine and Lenhoff , 1965a; 1965b; Thorington and Margulis , 1981 ) .\",\n \"The symbionts are enclosed in the host endodermal epithelial cells within perialgal vacuoles called ‘symbiosomes’ .\",\n \"The interactions at play here are clearly metabolic: the algae depend on nutrients that are derived from the host or from the environment surrounding the host , while in return the host receives a significant amount of photosynthetically fixed carbon from the algae .\",\n \"Previous studies have provided evidence that the photosynthetic symbionts provide their host with maltose , enabling H . viridissima to survive periods of starvation ( Muscatine and Lenhoff , 1963; Muscatine , 1965; Roffman and Lenhoff , 1969; Cook and Kelty , 1982; Huss et al . , 1994 ) .\",\n \"Chlorella-to-Hydra translocation of photosynthates is critical for polyps to grow ( Muscatine and Lenhoff , 1965b; Mews , 1980; Douglas and Smith , 1983; 1984 ) .\",\n \"Presence of symbiotic algae also has a profound impact on hydra´s fitness by promoting oogenesis ( Habetha et al . , 2003; Habetha and Bosch , 2005 ) .\",\n \"Pioneering studies performed in the 1980 s ( McAuley and Smith , 1982; Rahat and Reich , 1984 ) showed that there is a great deal of adaptation and specificity in this symbiotic relationship .\",\n \"All endosymbiotic algae found in a single host polyp are clonal and proliferation of symbiont and host is tightly correlated ( Bossert and Dunn , 1986; McAuley , 1986a ) .\",\n \"Although it is not yet known how Hydra controls cell division in symbiotic Chlorella , Chlorella strain A99 is unable to grow outside its polyp host and is transmitted vertically to the next generation of Hydra , indicating loss of autonomy during establishment of its symbiotic relationship with this host ( Muscatine and McAuley , 1982; Campbell , 1990; Habetha et al . , 2003 ) .\",\n \"Molecular phylogenetic analyses suggest that H . viridissima is the most basal species in the genus Hydra and that symbiosis with Chlorella was established in the ancestral viridissima group after their divergence from non-symbiotic Hydra groups ( Martínez et al . , 2010; Schwentner and Bosch , 2015 ) .\",\n \"A recent phylogenetic analysis of different strains of green hydra resulted in a phylogenetic tree that is topologically equivalent to that of their symbiotic algae ( Kawaida et al . , 2013 ) , suggesting these species co-evolved as a result of their symbiotic relationship .\",\n \"Although our understanding of the factors that promote symbiotic relationships in cnidarians has increased ( Shinzato et al . , 2011; Davy et al . , 2012; Lehnert et al . , 2014; Baumgarten et al . , 2015; Ishikawa et al . , 2016 ) , very little is known about the molecular mechanisms allowing this partnership to persist over millions of years .\",\n \"Recent advances in transcriptome and genome analysis allowed us to identify the metabolic interactions and genomic evolution involved in achieving the Hydra-Chlorella symbiotic relationship .\",\n \"We present here the first characterization , to our knowledge , of genetic complementarity between green Hydra and Chlorella algae that explains the emergence and/or maintenance of a stable symbiosis .\",\n \"We also provide here the first report of the complete genome sequence from an obligate intracellular Chlorella symbiont .\",\n \"Together , our results show that exchange of nutrients is the primary driving force for the symbiosis between Chlorella and Hydra .\",\n \"Subsequently , reduction of metabolic pathways may have further strengthened their codependency .\",\n \"Our findings provide a framework for understanding the evolution of a highly codependent symbiotic partnership in an early emerging metazoan .\"\n ],\n [\n \"As tool for our study we used the green hydra H . viridissima ( Figure 1A ) colonized with symbiotic Chlorella sp .\",\n \"strain A99 ( abbreviated here as Hv_Sym ) , aposymbiotic H . viridissima from which the symbiotic Chlorella were removed ( Hv_Apo ) , as well as aposymbiotic H . viridissima , which have been artificially infected with Chlorella variabilis NC64A ( Hv_NC64A ) .\",\n \"The latter is symbiotic to the single-cellular protist Paramecium ( Karakashian and Karakashian , 1965 ) .\",\n \"Although an association between H . viridissima and Chlorella NC64A can be maintained for some time , both their growth rate ( Figure 1B ) and the number of NC64A algae per Hydra cell ( Figure 1—figure supplement\",\n \"1 ) are significantly reduced compared to the symbiosis with native symbiotic Chlorella A99 .\",\n \"H . H . viridissima genes involved in the symbiosis with Chlorella algae were identified by microarray based on the contigs of H . viridissima A99 transcriptome ( NCBI GEO Platform ID: GPL23280 ) .\",\n \"For the microarray analysis , total RNA was extracted from the polyps after light exposure for six hours .\",\n \"By comparing the transcriptomes of Hv_Sym and Hv_Apo , we identified 423 contigs that are up-regulated and 256 contigs that are down-regulated in presence of Chlorella A99 ( Figure 1C ) .\",\n \"To exclude genes involved in oogenesis and embryogenesis , only contigs differently expressed with similar patterns in both sexual and asexual Hv_Sym were recorded .\",\n \"Interestingly , contigs whose predicted products had no discernible homologs in other organisms including other Hydra species were overrepresented in these differentially expressed contigs ( Chi-squared test p<0 . 001 ) ( Figure 1—figure supplement 2 ) .\",\n \"Such taxonomically restricted genes ( TRGs ) are thought to play important roles in the development of evolutionary novelties and morphological diversity within a given taxonomic group ( Khalturin et al . , 2009; Tautz and Domazet-Lošo , 2011 ) .\",\n \"We further characterized functions of the differentially expressed Hydra genes by Gene Ontology ( GO ) terms ( Ashburner et al . , 2000 ) and found the GO term ‘localization’ overrepresented among up-regulated contigs ( Hv_Sym > Hv_Apo ) , whereas the GO term ‘metabolic process’ was enriched among down-regulated contigs ( Hv_Sym < Hv_Apo ) ( Figure 1D ) .\",\n \"More specifically , the up-regulated contigs included many genes related to ‘transmembrane transporter activity’ , ‘transmembrane transport’ , ‘transposition’ , ‘cilium’ and ‘protein binding , bridging’ ( Figure 1E ) .\",\n \"In the down-regulated contig set , the GO classes ‘cellular amino acid metabolic process’ , ‘cell wall organization or biogenesis’ and ‘peptidase activity’ were overrepresented ( Figure 1E ) .\",\n \"These results suggest that the Chlorella symbiont affects core metabolic processes and pathways in Hydra .\",\n \"Particularly , carrier proteins and active membrane transport appear to play a prominent role in the symbiosis .\",\n \"As next step , we used GO terms , domain search and similarity search to further analyze the differentially expressed contigs between Hv_Sym and Hv_Apo ( Supplementary file 1 ) .\",\n \"As the genes with GO terms related to localization and transport , we identified 27 up-regulated contigs in Hv_Sym ( Table 1 ) .\",\n \"Interestingly , this gene set included a contig showing sequence similarity to the glucose transporter GLUT8 gene , which was previously reported to be up-regulated in the symbiotic state of the sea anemone Aiptasia ( Lehnert et al . , 2014; Sproles et al . , 2018 ) .\",\n \"Thus , a conserved mechanism may be responsible for photosynthate transport from the symbiont into the host cytoplasm across the symbiosome membrane .\",\n \"Further , a contig encoding a carbonic anhydrase ( CA ) enzyme was up-regulated in Hv_Sym ( Table 1 ) .\",\n \"CA catalyzes the interconversion of HCO3 and CO2 .\",\n \"Similar to the GLUT8 gene , carbonic anhydrase also appears to be up-regulated in symbiotic corals and anemones ( Weis et al . , 1989; Grasso et al . , 2008; Ganot et al . , 2011; Lehnert et al . , 2014 ) .\",\n \"It appears plausible that for efficient photosynthesis in symbiotic algae , the host may need to convert CO2 to the less freely diffusing inorganic carbon ( HCO3 ) to maintain it in the symbiosome ( Lucas and Berry , 1985; Weis et al . , 1989; Barott et al . , 2015 ) .\",\n \"We also observed up-regulation of contigs encoding proteins involved in vesicular and endosomal trafficking , such as spe-39 protein , otoferlin , protein fam194b and V-type proton ATPase 21 kda proteolipid , which may have a function in nutrition exchange between host and symbiont and maintenance of proper condition in the symbiosome .\",\n \"Upregulated genes also include genes encoding rhamnospondin and fibrillin , known to be involved in cell adhesion and extracellular matrix , and retention of the symbiont at the proper site in the Hydra cells .\",\n \"Photosynthesis by symbiotic algae imposes Reactive Oxygen Species ( ROS ) that can damage lipids , proteins and DNA in the host cells ( Lesser , 2006 ) .\",\n \"Therefore , in symbiosis with photosynthetic organisms an appropriate oxidative stress response of the host is required for tolerance of the symbiont .\",\n \"Indeed , an increase of antioxidant activities in symbiotic states of cnidarians has been reported previously ( Richier et al . , 2005 ) and it has been suggested that ROS produced by stress could be the major trigger of symbiosis breakdown during coral bleaching ( Lesser , 2006; Weis , 2008 ) .\",\n \"To understand the oxidative stress response in green hydra , we searched the differentially expressed genes with the GO terms ‘response to oxidative stress’ , ‘oxidation-reduction process’ and ‘oxidoreductase activity’ .\",\n \"In Hv_Sym , contigs for peroxidase , methionine-r-sulfoxide reductase/selenoprotein and glutaredoxin , which are known to be related to oxidative stress response were up-regulated ( Table 1 ) .\",\n \"On the other hand , some contigs encoding glutathione S-transferase and ascorbate peroxidase were down-regulated in Hv_Sym .\",\n \"In addition , two contigs encoding polycystin were down-regulated in Hv_Sym .\",\n \"Polycystin is an intracellular calcium release channel that is inhibited by ROS ( Montalbetti et al . , 2008 ) and is also down-regulated in a different strain of symbiotic green hydra ( Ishikawa et al . , 2016 ) .\",\n \"In addition , six contigs encoding metalloproteinases showed differential expression between Hv_Sym and Hv_Apo .\",\n \"Although metalloproteinases have many functions such as cleavage of cell surface proteins and remodeling of the extracellular matrix , in a previous study they also were found to play a role in the oxidative stress response ( Császár et al . , 2009 ) .\",\n \"A key antioxidant in the oxidative stress response in symbiotic cnidarians turns out to be glutathione ( Sunagawa et al . , 2009; Meyer and Weis , 2012 ) .\",\n \"The gene encoding glutathione S-transferase was previously observed to be downregulated in corals , sea anemones , different strains of green hydra and Paramecium ( Kodama et al . , 2014; Lehnert et al . , 2014; Ishikawa et al . , 2016; Mohamed et al . , 2016 ) .\",\n \"Our study supports this view ( Table\",\n \"1 ) and may point to a conserved feature of oxidative stress response in algal-animal symbiosis .\",\n \"Previous studies have suggested that during establishment of coral–algal symbiosis the host immune response may be partially suppressed ( Weis et al . , 2008; Mohamed et al . , 2016 ) .\",\n \"Our observations in Hydra together with previous findings in corals indicate that regulation of symbiosis by innate immunity pathways indeed may be a general feature of cnidarian symbiosis .\",\n \"Among the differentially expressed contigs we identified a number of genes involved in innate immunity and apoptosis .\",\n \"Pattern recognition receptors ( PRRs ) and the downstream innate immunity and apoptosis pathways are thought to play important roles in various symbiotic interactions including cnidarian-dinoflagellate symbiosis ( Davy et al . , 2012 ) .\",\n \"In Hv_Sym we found two up-regulated contigs that contain a Toll/interleukin-1 receptor ( TIR ) domain ( Table 1 ) .\",\n \"TIR is a known PRR that is inserted in the host cell membrane and plays an important role in the innate immune system by specifically recognizing microbial-associated molecular patterns , such as flagellin , lipopolysaccharide ( LPS ) and peptidoglycan ( Hoving et al . , 2014 ) .\",\n \"Furthermore , we found one up-regulated contig with similarity to a mannose receptor gene with C-type lectin domain ( Table 1 ) .\",\n \"This is worth noting since C-type lectin receptors bind carbohydrates and some of them are known to function as PRRs .\",\n \"Host lectin-algal glycan interactions have been proposed to be involved in infection and recognition of symbionts in some cnidarians including green hydra , sea anemones and corals ( Meints and Pardy , 1980; Lin et al . , 2000; Wood-Charlson et al . , 2006 ) .\",\n \"Interestingly , up-regulation of C-type lectin genes was also observed during onset of cnidarian–dinoflagellate symbiosis ( Grasso et al . , 2008; Schwarz et al . , 2008; Sunagawa et al . , 2009; Mohamed et al . , 2016 ) .\",\n \"Furthermore , contigs encoding chitinase enzymes also were differentially expressed between Hv_Sym and Hv_Apo ( Table 1 ) .\",\n \"Chitinases are involved in degradation of chitin , which is a component of the exoskeleton of arthropods and the cell wall of fungi , bacteria and some Chlorella algae ( Kapaun and Reisser , 1995 ) , and also might play a role in host-defense systems for pathogens which have chitinous cell wall .\",\n \"Chitinases are classified into two glycoside hydrolase families , GH18 and GH19 , with different structures and catalytic mechanisms .\",\n \"In Hv_Sym two contigs encoding GH18 chitinases were up-regulated , while one contig encoding a GH19 chitinase was down-regulated , suggesting that the enzymes involved in chitin degradation are sensitive to the presence or absence of symbiotic Chlorella .\",\n \"To narrow down the number of genes specifically affected by the presence of the native symbiont Chlorella A99 , we identified 12 contigs that are differentially expressed in symbiosis with Chlorella A99 , but not in presence of foreign Chlorella NC64A ( Figure 1C A99-specific ) .\",\n \"Independent qPCR confirmed the differential expression pattern for 10 of these genes ( Table 2 ) .\",\n \"The genes up-regulated by the presence of the symbiont encode a Spot_14 protein , a glutamine synthetase ( GS ) and a sodium-dependent phosphate ( Na/Pi ) transport protein in addition to a H . viridissima specific gene ( rc_12891: Sym-1 ) and a Hydra genus specific gene ( rc_13570: Sym-2 ) ( Table 2 ) .\",\n \"Hydra genes down-regulated by the presence of Chlorella A99 were two H . viridissima-specific genes and three metabolic genes encoding histidine ammonia-lyase , acetoacetyl-CoA synthetase and 2-isopropylmalate synthase ( Table 2 ) .\",\n \"Of the up-regulated genes , Spot_14 is described as thyroid hormone-responsive spot 14 protein reported to be induced by dietary carbohydrates and glucose in mammals ( Tao and Towle , 1986; Brown et al . , 1997 ) .\",\n \"Na/Pi transport protein is a membrane transporter actively transporting phosphate into cells ( Murer and Biber , 1996 ) .\",\n \"GS plays an essential role in the metabolism of nitrogen by catalyzing the reaction between glutamate and ammonia to form glutamine ( Liaw et al . , 1995 ) .\",\n \"Interestingly , out of the three GS genes H . viridissima contains only GS-1 was found to be up-regulated by the presence of the symbiont ( Figure 1—figure supplement 3 ) .\",\n \"The discovery of these transcriptional responses points to an intimate metabolic exchange between the partners in a species-specific manner .\",\n \"To better understand the specificity of Hydra´s response to the presence of the foreign symbiont , we also identified the genes differentially expressed in Hydra polyps hosting a non-native Chlorella NC64A ( Hv_NC64A ) compared to both polyps hosting the obligate symbiont Chlorella A99 ( Hv_A99 ) and aposymbiotic Hydra ( Hv_Apo ) .\",\n \"We found 19 contigs that were up-regulated and 45 contigs that were down-regulated in presence of NC64A , which strikingly did not include any genes related to immunity or oxidative stress response ( Supplementary file 1 ) .\",\n \"Instead , the differentially expressed contigs showed similarity to methylase genes involved in ubiquinone menaquinone biosynthesis and secondary metabolite synthesis such as n- ( 5-amino-5-carboxypentanoyl ) -l-cysteinyl-d-valine synthase and non-ribosomal peptide synthase .\",\n \"Four differentially expressed contigs specifically up-regulated in Hv_NC64A encoded ubiquitin carboxyl-terminal hydrolases , ( Table 3 ) .\",\n \"To test whether photosynthetic activity of the symbiont is required for up-regulation of gene expression , Hv_Sym was either cultured under a standard 12 hr light/dark alternating regime or continuously in the dark for 1 to 4 days prior to RNA extraction ( Figure 2A ) .\",\n \"Interestingly , four ( GS1 , Spot14 , Na/Pi and Sym-1 ) of five genes specifically activated by the presence of Chlorella A99 showed significant up-regulation when exposed to light ( Figure 2B ) , indicating the relevance of photosynthetic activity of Chlorella .\",\n \"This up-regulation was strictly dependent on presence of the algae , as in aposymbiotic Hv_Apo the response was absent ( Figure 2B ) .\",\n \"On the other hand , symbiosis-regulated Hydra genes not specific for Chlorella A99 ( Figure 1C Symbiosis-regulated , Table 4 ) appear to be not up-regulated in a light-dependent manner ( Figure 2—figure supplement 1 ) .\",\n \"These genes are involved in Hydra´s innate immune system ( e . g . proteins containing Toll/interleukin-1 receptor domain or Death domain ) or in signal transduction ( C-type mannose receptor , ephrin receptor , proline-rich transmembrane protein 1 , ‘protein-kinase , interferon-inducible double stranded RNA dependent inhibitor , repressor of ( p58 repressor ) ’ ) .\",\n \"That particular transcriptional changes observed in Hydra rely solely on the photosynthetic activity of Chlorella A99 was confirmed by substituting the dark incubation with selective chemical photosynthesis inhibitor DCMU ( Dichorophenyl-dimethylurea ) ( Vandermeulen et al . , 1972 ) , which resulted in a similar effect ( Figure 2C , D ) .\",\n \"To further characterize the symbiont induced Hydra genes , we performed whole mount in situ hybridization ( Figure 3A–F ) and quantified transcripts by qPCR using templates from isolated endoderm and ectoderm ( Figure 3—figure supplement 1 ) , again comparing symbiotic and aposymbiotic polyps ( Figure 3G–I ) .\",\n \"The GS-1 gene and the Spot14 gene are expressed both in ectoderm and in endoderm ( Figure 3A , B ) and both genes are strongly up-regulated in the presence of the symbiont ( Figure 3G , H ) .\",\n \"In contrast , the Na/Pi gene was expressed only in the endoderm ( Figure 3C ) and there it was strongly up-regulated by the symbiont ( Figure 3I ) .\",\n \"Since Chlorella sp .\",\n \"A99 colonizes endodermal epithelial cells only , the impact of algae on symbiosis-dependent genes in both the ectodermal and the endodermal layer indicates that photosynthetic products can be transported across these two tissue layers or some signals can be transduced by cell-cell communication .\",\n \"To more closely dissect the nature of the functional interaction between Hydra and Chlorella and to explore the possibility that maltose released from the algae is involved in A99-specific gene regulation , we cultured aposymbiotic polyps ( Hv_Apo ) for 2 days in medium containing various concentrations of maltose ( Figure 3J ) .\",\n \"Of the five A99 specific genes , GS-1 and the Spot14 gene were up-regulated by maltose in a dose-dependent manner; the Na/Pi gene was only up-regulated in 100 mM maltose and the Hydra specific genes Sym-1 and Sym-2 did not show significant changes in expression by exposure to maltose ( Figure 3J ) .\",\n \"This provides strong support for previous views that maltose excretion by symbiotic algae contributes to the stabilization of this symbiotic association ( Cernichiari et al . , 1969 ) .\",\n \"When polyps were exposed to glucose instead of maltose , the genes of interest were also transcriptionally activated in a dose-dependent manner , while sucrose had no effect ( Figure 3—figure supplement 2A–D ) .\",\n \"Exposure to low concentrations of galactose increased transcriptional activity but at high concentration it did not , indicating a substrate inhibitor effect for this sugar .\",\n \"That the response to glucose is similar or even higher compared to maltose after 6 hr of treatment ( Figure 3—figure supplement 2E ) , suggests that Hydra cells transform maltose to glucose as a source of energy .\",\n \"In animals including cnidarians , several glucose transporters have been identified ( Sproles et al . , 2018 ) , but yet no maltose transporters .\",\n \"This is consistent with the view that maltose produced by the symbiont is digested to glucose in the symbiosome and translocated to the host cytoplasm through glucose transporters .\",\n \"To better understand the symbiosis between H . viridissima and Chlorella and to refine our knowledge of the functions that are required in this symbiosis , we sequenced the genome of Chlorella sp .\",\n \"strain A99 and compared it to the genomes of other green algae .\",\n \"The genome of Chlorella sp .\",\n \"A99 was sequenced to approximately 211-fold coverage , enabling the generation of an assembly comprising a total of 40 . 9 Mbp ( 82 scaffolds , N50 = 1 . 7 Mbp ) ( Table 5 ) .\",\n \"Chlorella sp .\",\n \"A99 belongs to the family Chlorellaceae ( Figure 4A ) and of the green algae whose genomes have been sequenced it is most closely related to Chlorella variabilis NC64A ( NC64A ) ( Merchant et al . , 2007; Palenik et al . , 2007; Worden et al . , 2009; Blanc et al . , 2010; Prochnik et al . , 2010; Blanc et al . , 2012; Gao et al . , 2014; Pombert et al . , 2014 ) .\",\n \"The genome size of the total assembly in strain A99 was similar to that of strain NC64A ( 46 . 2 Mb ) ( Figure 4B ) .\",\n \"By k-mer analysis ( k-mer = 19 ) , the genome size of A99 was estimated to be 61 Mbp ( Marçais and Kingsford , 2011 ) .\",\n \"Its GC content of 68% , is the highest among the green algae species recorded ( Figure 4B ) .\",\n \"In the A99 genome , 8298 gene models were predicted .\",\n \"As shown in Figure 4C , about 80% of these predicted genes have extensive sequence similarity to plant genes , while 13% so far have no similarity to genes of any other organisms ( Figure 4C ) .\",\n \"It is also noteworthy that 7% of the A99 genes are similar to genes of other kingdoms but not to Hydra , indicating the absence of gene transfer from Hydra to the symbiont genome ( Figure 4C ) .\",\n \"Several independent lines of evidence demonstrate that nitrogen limitation and amino-acid metabolism have a key role in the Chlorella–Hydra symbiosis and that symbiotic Chlorella A99 depends on glutamine provided by its host ( Rees , 1986; McAuley , 1987a; 1987b; McAuley , 1991; Rees , 1991;1989 ) .\",\n \"To identify Chlorella candidate factors for the development and maintenance of the symbiotic life style , we therefore used the available genome information to assess genes potentially involved in amino acid transport and the nitrogen metabolic pathway .\",\n \"When performing a search for the Pfam domain ‘Aa_trans’ or ‘AA_permease’ to find amino acid transporter genes in the A99 genome , we discovered numerous genes containing the Aa_trans domain ( Table 6A ) .\",\n \"In particular , A99 contains many orthologous genes of amino acid permease 2 and of transmembrane amino acid transporter family protein ( solute carrier family 38 , sodium-coupled neutral amino acid transporter ) , as well as NC64A ( Table 6B , Supplementary file 2 ) .\",\n \"Both of these gene products are known to transport neutral amino acids including glutamine .\",\n \"This observation is supporting the view that import of amino acids is an essential feature for the symbiotic way of life of Chlorella .\",\n \"In symbiotic organisms , loss of genes often occurs due to the strictly interdependent relationship ( Ochman and Moran , 2001; Wernegreen , 2012 ) , raising the possibility that Chlorella A99 might have lost some essential genes .\",\n \"To test this hypothesis , we searched the Chlorella A99 genome for genes conserved across free-living green algae Coccomyxa subellipsoidea C169 ( C169 ) , Chlamydomonas reinhardtii ( Cr ) and Volvox carteri ( Vc ) .\",\n \"In a total of 9851 C169 genes , we found 5701 genes to be conserved in Cr and Vc ( Supplementary file 3 ) .\",\n \"Of these , 238 genes did not match to any gene models and genomic regions in Chlorella A99 and thus were considered as gene loss candidates .\",\n \"Interestingly , within these 238 candidates , genes with the GO terms ‘transport’ in biological process and ‘transporter activity’ in molecular function were overrepresented ( Figure 5 ) .\",\n \"In particular , the 28 genes annotated to these GO terms encoded nitrate transporter , urea transporter and molybdate transporter , which are known to be involved in nitrogen metabolism ( Table 7 ) .\",\n \"Beside ammonium , nitrate and urea are major nitrogen sources for plants , whereas molybdate is a co-factor of the nitrate reductase , an important enzyme in the nitrate assimilation pathway .\",\n \"These transporter genes are conserved across green algae including Chlorella NC64A ( Sanz-Luque et al . , 2015; Gao et al . , 2014 ) and appear to be lost in the Chlorella A99 genome .\",\n \"In nitrogen assimilation processes , plants usually take up nitrogen in the form of nitrate ( NO3- ) via nitrate transporters ( NRTs ) or as ammonium ( NH4+ ) via ammonium transporters ( AMT ) ( Figure 6A ) .\",\n \"In higher plants , two types of nitrate transporters , NRT1 and NRT2 , have been identified ( Krapp et al . , 2014 ) .\",\n \"Some NRT2 require nitrate assimilation-related component 2 ( NAR2 ) to be functional ( Quesada et al . , 1994 ) .\",\n \"NO3- is reduced to nitrite by nitrate reductase ( NR ) , NO2- is transported to the chloroplast by nitrate assimilation-related component1 ( NAR1 ) , and NO2- is reduced to NH4+ by nitrite reductase ( NiR ) .\",\n \"NH4+ is incorporated into glutamine ( Gln ) by glutamine synthetase ( GS ) , and Gln is incorporated into glutamate ( Glu ) by NADH-dependent glutamine amide-2-oxoglutarate aminotransferase ( GOGAT ) , also known as glutamate synthase .\",\n \"This pathway is highly conserved among plants and all of its major components , including NRT1 and NRT2 , NAR1 and NAR2 , NR , NiR , AMT , GOGAT and GS , are present in the 10 green algae species that have been genome-sequenced so far ( with the exception of NRT1 , which is absent in Micromonas pusilla ) ( Sanz-Luque et al . , 2015 ) .\",\n \"In Symbiodinium , the photosynthetic symbiont of marine invertebrates , all these components of the nitrogen assimilation pathway were also observed ( Supplementary file 4 ) ( Shoguchi et al . , 2013; Lin et al . , 2015; Aranda et al . , 2016; Sproles et al . , 2018 ) .\",\n \"Based on the annotation by Sanz-Luque et al . ( Sanz-Luque et al . , 2015 ) , we searched these nitrogen assimilation genes in the Chlorella A99 genome , using ortholog grouping and a reciprocal BLAST search using the protein sequences from other green algae ( Figure 6B , Supplementary file 5 ) .\",\n \"As expected , the Chlorella A99 genome contains many homologues of the genes involved in nitrogen assimilation in plants including genes encoding NRT1 , NAR1 , NR , AMT , GS and GOGAT ( Figure 6B ) .\",\n \"Intriguingly , our systematic searches failed to identify representative genes for NRT2 , NAR2 and NiR in the Chlorella A99 genome ( Figure 6B ) .\",\n \"We confirmed the absence of the NRT2 and NiR genes by PCR using primers designed for the conserved regions of these genes and which failed to produce a product with genomic DNA as a template ( Figure 6—figure supplement 1 ) .\",\n \"Due to the weak sequence conservation of the NAR2 gene in the three algae genomes , PCR of that gene was not performed .\",\n \"Taken together , our observations indicate that Chlorella A99 algae appears to lack NRT2 , NAR2 and NiR .\",\n \"Since in many fungi , cyanobacteria and algae species , nitrate assimilation genes are known to act in concert and a gene cluster of NR and NiR genes is conserved between different green algae ( Sanz-Luque et al . , 2015 ) , we next investigated the level of genomic clustering of the nitrate assimilation pathway genes in the Chlorella genome .\",\n \"Comparing the genomes of NC64A and C169 revealed the presence of a cluster of NR and NiR genes ( Figure 6C ) .\",\n \"In NC64A , two NRT2 genes , together with genes for NAR2 , NR and NiR are clustered on scaffold 21 .\",\n \"In C169 , one of the NR genes and NiR are clustered together , whereas the second NR gene is separate .\",\n \"Interestingly , analysis of the sequences around the NR gene in the Chlorella A99 genome provided no evidence for the presence of a co-localized NiR gene or any other nitrate assimilation genes , nor any conserved gene synteny to NC64A and C169 ( Figure 6C ) .\",\n \"Therefore , our comparative genomic analyses points to an incomplete and scattered nitrogen metabolic pathway in symbiotic Chlorella A99 , which lacks essential transporters and enzymes for nitrate assimilation as well as the clustered structure of nitrate assimilation genes .\",\n \"The absence of genes essential for nitrate assimilation in the Chlorella A99 genome ( Figure 6 ) is consistent with its inability to grow outside the Hydra host cell ( Habetha and Bosch , 2005 ) and indicates that Chlorella symbionts are dependent on metabolites provided by their host .\",\n \"We hypothesized that Chlorella is unable to use nitrite and ammonium as a nitrogen source , and that it relies on Hydra assimilating ammonium to glutamine to serve as the nitrogen source .\",\n \"To test this hypothesis and to examine utilization of nitrogen compounds of A99 , we isolated Chlorella A99 from Hv_Sym and cultivated it in vitro using modified bold basal medium ( BBM ) ( Nichols and Bold , 1965 ) containing the same amount of nitrogen in the form of NO3- , NH4+ , Gln or casamino acids ( Figure 7 ) .\",\n \"As controls , Chlorella variabilis NC64A ( NC64A ) isolated from Hv_NC64A and free-living C169 were used .\",\n \"To confirm that the cultured A99 is not contamination , we amplified and sequenced the genomic region of the 18S rRNA gene by PCR ( Figure 7—figure supplement\",\n \"1 ) and checked this against the genomic sequence of A99 .\",\n \"Kamako et al . reported that free-living alga Chlorella vulgaris Beijerinck var .\",\n \"vulgaris grow in media containing only inorganic nitrogen compounds as well as in media containing casamino acids as a nitrogen source , while NC64A required amino acids for growth ( Kamako et al . , 2005 ) .\",\n \"Consistent with these observations , C169 grew in all tested media and NC64A grew in media containing casamino acids and Gln , although its growth rate was quite low in presence of NH4+ and NO3- ( Figure 7 ) .\",\n \"Remarkably , Chlorella A99 increased in cell number for up to 8 days in media containing casamino acids and Gln ( Figure 7 ) .\",\n \"Similar to NC64A , A99 did not grow in presence of NH4+ and NO3- .\",\n \"The growth rates of both A99 and NC64A were higher in medium containing a mixture of amino acids ( casamino acids ) than the single amino acid Gln .\",\n \"In contrast to NC64A , A99 could not be cultivated permanently in casamino acids or glutamine supplemented medium , indicating that additional growth factors are necessary to maintain in vitro growth of this obligate symbiont .\",\n \"Thus , although in vitro growth of A99 can be promoted by adding Glu and amino acids to the medium , A99 cannot be cultured permanently in this enriched medium , indicating that other host derived factors remain to be uncovered .\"\n ],\n [\n \"Sequencing of the Chlorella A99 genome in combination with the transcriptome analyses of symbiotic , aposymbiotic and NC64A-infected H . viridissima polyps has enabled the identification of genes with specific functions in this symbiotic partnership .\",\n \"The Hydra-Chlorella symbiosis links carbohydrate supply from the photosynthetic symbiont to glutamine synthesis by the host .\",\n \"Characteristics of the symbiont genome obviously reflect its adaptation to this way of life , including an increase in amino acid transporters and degeneration of the nitrate assimilation pathway .\",\n \"This conclusion is based on six observations:\",\n \"( i ) Expression of some genes including GS-1 , Spot 14 and NaPi is specifically up-regulated in the presence of Chlorella A99 ( Figure 1C , Table 2 ) , and\",\n \"( ii ) they are induced by both , photosynthetic activity of Chlorella and by supplying exogenous maltose or glucose ( Figures 2 and 3J , Figure 3—figure supplement 2 ) .\",\n \"Maltose produced by the symbiont is likely to be digested to glucose in symbiosome and translocated to the host cytoplasm through glucose transporters ( Figure 8A ) .\",\n \"Upregulation of a GLUT8 gene in the symbiotic state of green hydra may reflect activation of sugar transport ( Table 1 ) .\",\n \"These results indicate that maltose release by photosynthesis of the symbiont enhances nutrition supply including glutamine by the host ( Figure 8A ) .\",\n \"( iii ) Symbiotic Chlorella A99 cannot be cultivated in vitro in medium containing a single inorganic nitrogen source ( Figure 7 ) .\",\n \"Since medium containing glutamine supports in vitro growth of A99 , this organism appears to depend on glutamine provided by the Hydra host .\",\n \"( iv ) The genome of Chlorella A99 contains multiple amino acid transporter genes ( Table 6 ) , but lacks genes involved in nitrate assimilation ( Figure 6 ) , pointing to amino acids as main source of nitrogen and a degenerated nitrate assimilation pathway .\",\n \"As for ammonium , which is one of the main nitrogen sources in plants , previous studies have reported the inability of symbiotic algae to take up ammonium because of the low peri-algal pH ( pH 4–5 ) that stimulates maltose release ( Douglas and Smith , 1984; Rees , 1989; McAuley , 1991; Dorling et al . , 1997 ) .\",\n \"Since Chlorella apparently cannot use nitrite and ammonium as a nitrogen source , it seems that Hydra has to assimilate ammonium to glutamine and provides it to Chlorella A99 ( Figure 8A ) .\",\n \"( v ) While polyps with native symbiont Chlorella A99 grew faster than aposymbiotic ones , symbiosis with foreign algae NC64A had no effect on the growth of polyps at all ( Figure 1B ) .\",\n \"( vi ) Hydra endodermal epithelial cells host significantly fewer NC64A algae than A99 ( Figure 1—figure supplement 1 ) providing additional support for the view of a tightly regulated codependent partnership in which exchange of nutrients appears to be the primary driving force .\",\n \"Previous studies have reported that symbiotic Chlorella in green hydra releases significantly larger amounts of maltose than NC64A ( Mews and Smith , 1982; Rees , 1989 ) .\",\n \"In addition , Rees reported that Hydra polyps containing high maltose releasing algae had a high GS activity , whereas aposymbiotic Hydra or Hydra with a low maltose releasing algae had lower GS activity ( Rees , 1986 ) .\",\n \"Although the underlying mechanism of how maltose secretion and transportation from Chlorella is regulated is still unclear , the amount of maltose released by the symbiont could be an important symbiont-derived driver or stabilizer of the Hydra–Chlorella symbiosis .\",\n \"Transcriptome comparison between symbiotic and aposymbiotic H . viridissima demonstrated that symbiosis-regulated genes are involved in oxidative stress response and innate immunity .\",\n \"The fact that PRRs and apoptosis-related genes , are also differentially expressed in a number of other symbiotic cnidarians ( Table 1 ) , suggests innate immunity as conserved mechanism involved in controlling the development and maintenance of stable symbiotic interactions .\",\n \"Furthermore , the exchange of nitrogenous compounds and photosynthetic products between host and symbiont observed here in the Hydra-Chlorella symbiosis is also observed in marine invertebrates such as corals , sea anemones and giant clams associated with Symbiodinium algae ( Figure 8B , C ) .\",\n \"Despite these similarities , however , there are also conspicuous differences among symbiotic cnidarians in particular with respect to the nutrients provided by the symbiont to the host .\",\n \"For example , symbiotic Chlorella algae in green hydra , Paramecium and fresh water sponges provide their photosynthetic products in form of maltose and glucose ( Figure 8B ) ( Brown and Nielsen , 1974; Wilkinson , 1980; Kamako and Imamura , 2006 ) .\",\n \"In contrast , Symbiodinium provides glucose , glycerol , organic acids , amino acids as well as lipids to its marine hosts ( Figure 8C ) ( Muscatine and Cernichiari , 1969; Lewis and Smith , 1971; Trench , 1971; Kellogg and Patton , 1983 ) .\",\n \"A former transcriptome analysis of amino acid biosynthetic pathways suggested that Symbiodinium can synthesize almost all amino acids ( Shinzato et al . , 2014 ) .\",\n \"Gene loss in cysteine synthesis pathway in the coral host Acropora digitifera seems to reflect the dependency on the amino acids provided by the Symbiodinium symbiont ( Shinzato et al . , 2011 ) .\",\n \"In contrast to Symbiodinium which can assimilate inorganic nitrogen such as nitrate and ammonium ( Lipschultz and Cook , 2002; Grover et al . , 2003; Tanaka et al . , 2006; Yellowlees et al . , 2008 ) , the symbiotic Chlorella algae in Hydra and Paramecium can only use amino acids as a nitrogen source ( Figure\",\n \"6 ) ( Kamako et al . , 2005 ) .\",\n \"In efforts to explain the metabolic efficiency of nitrogen use in symbiotic organisms , two models have been proposed: the ‘nitrogen conservation’ and the ‘nitrogen recycling’ hypothesis .\",\n \"The nitrogen conservation hypothesis suggests that photosynthetic carbon compounds from the symbiont are used preferentially by the host respiration , which reduces catabolism of nitrogenous compounds ( Rees and Ellard , 1989; Szmant et al . , 1990; Wang and Douglas , 1998 ) .\",\n \"The ‘nitrogen recycling’ hypothesis suggests that symbionts assimilate nitrogenous waste ( ammonium ) of the host into valuable , organic compounds , which then are translocated back to the host ( Figure 8C Symbiont nitrogen assimilation ) ( Lewis and Smith , 1971; Muscatine and Porter , 1977; Falkowski et al . , 1993; Wang and Douglas , 1998 ) .\",\n \"Our observation that in symbiotic green hydra many genes involved in amino acid metabolism are down-regulated ( Figure 1E ) is consistent with the assumption of reduction of amino acid consumption by respiration .\",\n \"In addition to the nitrogen recycling hypothesis , it has been proposed that also corals , sea anemones , Paramecium and green hydra hosts can assimilate ammonium into amino acids ( Figure 8B , C Host nitrogen assimilation ) ( Miller and Yellowlees , 1989; Rees , 1989; Szmant et al . , 1990; Rees , 1991; Wang and Douglas , 1998; Lipschultz and Cook , 2002 ) .\",\n \"Ammonia assimilation by the host implies that the host controls the nitrogen status to regulate metabolism of the symbionts , which may be involved in controlling the number of symbionts within the host cell .\",\n \"For organisms such as corals living in oligotrophic sea , inorganic nitrogen assimilation and recycling may be necessary to manage the nitrogen sources efficiently .\",\n \"In contrast , for Hydra and Paramecium living in a relatively nutrient-rich environment may be advantageous in terms of metabolic efficiency that the symbiont abandons its ability to assimilate inorganic nitrogen and specializes in the supply of photosynthetic carbohydrate to the host .\",\n \"Metabolic dependence of symbionts on host supply occasionally results in genome reduction and gene loss .\",\n \"For example , symbiotic Buchnera bacteria in insects are missing particular genes in essential amino acid pathways ( Shigenobu et al . , 2000; Hansen and Moran , 2011 ) .\",\n \"The fact that the corresponding genes of the host are up-regulated in the bacteriocyte , indicates complementarity and syntrophy between host and symbiont .\",\n \"Similarly , in Chlorella A99 the nitrogen assimilation system could have been lost as a result of continuous supply of nitrogenous amino acids provided by Hydra .\",\n \"Compared to Chlorella NC64A , the closest relative to Chlorella A99 among the genome-sequenced algae , genome size and total number of genes in Chlorella A99 were found to be smaller ( Figure 4B ) .\",\n \"Although both A99 and NC64A cannot be cultivated using inorganic nitrogen sources ( Figure\",\n \"7 ) ( Kamako et al . , 2005 ) , NC64A , unlike A99 , obtains all major nitrogen assimilation genes and their cluster structure on the chromosome ( Figure\",\n \"6 ) ( Sanz-Luque et al . , 2015 ) .\",\n \"NR and NiR activities were found to be induced by nitrate in free-living Chlorella , but not in Chlorella NC64A , indicating mutations in the regulatory region ( Kamako et al . , 2005 ) .\",\n \"Considering the phylogenetic position of NC64A and the symbiotic Chlorella of green hydra ( Kawaida et al . , 2013 ) , the disability of nitrate assimilation in A99 and NC64A seems to have evolved independently , suggesting convergent evolution in a similar symbiotic environment .\",\n \"Although our findings indicate that genome reduction in Chlorella A99 is more advanced than in Chlorella NC64A , genome size and total number of genes do not differ much between the Trebouxiophyceae ( A99 , NC64A and C169 ) ( Figure 4B ) .\",\n \"By contrast , the parasitic algae Helicosporidium and Auxochlorella have significantly smaller genome sizes and number of genes indicating extensive genome reduction ( Gao et al . , 2014; Pombert et al . , 2014 ) .\",\n \"The apparently unchanged complexity of the Chlorella A99 genome suggests a relatively early stage of this symbiotic partnership .\",\n \"Thus , gene loss in metabolic pathways could occur as a first step of genome reduction in symbionts caused by the adaptation to continuous nutrient supply from the host .\",\n \"Taken together , our study suggests metabolic-codependency as the primary driving force in the evolution of symbiosis between Hydra and Chlorella .\"\n ],\n [\n \"Experiments were carried out with the Australian Hydra viridissima strain A99 , which was obtained from Dr . Richard Campbell , Irvine .\",\n \"Polyps were maintained at 18°C on a 12 hr light/dark cycle and fed with Artemia two or three times a week .\",\n \"Aposymbiotic ( algae free ) polyps were obtained by photobleaching using 5 μM DCMU ( 3- ( 3 , 4-dichlorophenyl ) −1 , 1-dimethylurea ) as described before ( Pardy , 1976; Habetha et al . , 2003 ) .\",\n \"Experiments were carried out with polyps starved for 3–6 days .\",\n \"Isolation of endodermal layer and ectodermal layer was performed as described by Kishimoto et al . ( Kishimoto et al . , 1996 ) .\",\n \"Symbiotic Chlorella were isolated as described before by Muscatine and McAuley ( Muscatine , 1983; McAuley , 1986 ) .\",\n \"Chlorella variabilis NC64A ( NIES-2541 ) , Coccomyxa subellipsoidea C-169 ( NIES-2166 ) and Chlamydomonas reinhardtii ( NIES-2235 ) were obtained from the Microbial Culture Collection at the National Institute for Environmental Studies ( Tsukuba , Japan ) .\",\n \"Total RNA of Hydra was extracted by use of the Trizol reagent and PureLink RNA Mini Kit ( Thermo Fisher Scientific ) after lysis and removal of algae by centrifugation .\",\n \"The genomic DNA of green algae was extracted using ISOPLANT II ( Nippon Gene , Tokyo , Japan ) following DNase I treatment to degrade contaminant DNA .\",\n \"Quantity and quality of DNA and RNA were checked by NanoDrop ( Thermo Scientific Inc . , Madison , USA ) and BioAnalyzer ( Agilent Technologies , Santa Clara , USA ) .\",\n \"Total RNA for synthesis of cRNA targets was extracted from about 100 green hydra for each experimental group .\",\n \"Experiments were carried out using three biological replicates .\",\n \"cRNA labeled with cyanine-3 were synthesized from 400 ng total Hydra RNA using a Low Input Quick Amp Labeling Kit for one color detection ( Agilent Technologies ) .\",\n \"A set of fluorescently labeled cRNA targets was employed in a hybridization reaction with 4 × 44K Custom-Made Hydra viridissima Microarray ( Agilent Technologies ) contributing a total of 43 , 222 transcripts that was built by mRNA-seq data ( NCBI GEO Platform ID: GPL23280 ) ( Bosch et al . , 2009 ) .\",\n \"Hybridization and washing were performed using the GE Hybridization Kit and GE Wash Pack ( Agilent Technologies ) after which the arrays were scanned on an Agilent Technologies G2565BA microarray scanner system with SureScan technology following protocols according to the manufacturer's instructions .\",\n \"The intensity of probes was extracted from scanned microarray images using Feature Extraction 10 . 7 software ( Agilent Technologies ) .\",\n \"All algorithms and parameters used in this analysis were used with default conditions .\",\n \"Background-subtracted signal-intensity values ( gProcessedSignal ) generated by the Feature Extraction software were normalized using the 75th percentile signal intensity among the microarray .\",\n \"Those genes differentially expressed between two samples were determined by average of fold change ( cut of >2 . 0 ) and Student's t-test ( p<0 . 1 ) .\",\n \"The data series are accessible at NCBI GEO under accession number GSE97633 .\",\n \"Total RNA was extracted from 50 green hydra polyps for each biological replicate independently .\",\n \"For reverse transcription of total RNA First Strand cDNA Synthesis Kit ( Fermentas , Ontario , Canada ) was used .\",\n \"Real-time PCR was performed using GoTaq qPCR Master Mix ( Promega , Madison , USA ) and ABI Prism 7300 ( Applied Biosystems , Foster City , USA ) .\",\n \"All qPCR experiments were performed in duplicate with three biological replicates each .\",\n \"Values were normalized using the expression of the tubulin alpha gene .\",\n \"Primers used for these experiments are listed in Supplementary file 6A .\",\n \"Expression patterns of specific Hydra genes were detected by whole mount in situ hybridization with digoxigenin ( DIG ) -labelled RNA probes .\",\n \"Specimens were fixed in 4% paraformaldehyde .\",\n \"Hybridization signal was visualized using anti-DIG antibodies conjugated to alkaline phosphatase and NBT/BCIP staining solution ( Roche ) .\",\n \"DIG-labeled sense probes ( targeting the same sequences as the antisense probes ) were used as a control .\",\n \"Primers used for these experiments are listed in Supplementary file 6B .\",\n \"For genome sequencing of Chlorella sp .\",\n \"A99 , Chlorella sp .\",\n \"A99 was isolated from H . viridissima A99 and genomic DNA was extracted .\",\n \"Paired-end library ( insert size: 740 bp ) and mate-pair libraries ( insert size: 2 . 2 and 15 . 2 kb ) were made using Illumina TruSeq DNA LT Sample Prep Kit and Nextera Mate Pair Sample Preparation Kit respectively ( Illumina Inc . , San Diego , USA ) , following the manufacturer's protocols .\",\n \"Genome sequencing was performed using Illumina Miseq and Hiseq 2000 platforms .\",\n \"Sequence reads were assembled using Newbler Assembler version 2 . 8 ( Roche , Penzberg , Germany ) and subsequent scaffolding was performed by SSPACE ( Boetzer et al . , 2011 ) .\",\n \"Gaps inside the scaffolds were closed with the paired-end and mate-pair data using GapCloser of Short Oligonucleotide Analysis Package ( Luo et al . , 2012 ) .\",\n \"To overcome potential assembly errors arising from tandem repeats , sequences that aligned to another sequence by more than 50% of the length using blastn ( 1e-50 ) were removed from the assembly .\",\n \"The completeness of the genome was evaluated using CEGMA v2 . 4 ( Core Eukaryotic Genes Mapping Approach ) based on mapping of the 248 most highly conserved core eukaryotic genes ( CEGs ) on the assembled genome ( Parra et al . , 2007 ) .\",\n \"The completeness of complete and partial CEGs in the A99 scaffolds was 80 and 88% , respectively .\",\n \"The fraction of repetitive sequences was 12% .\",\n \"Gene model was predicted by AUGUSTUS 3 . 0 . 1 using model parameters for NC64A ( Stanke et al . , 2006 ) .\",\n \"This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession PCFQ00000000 ( BioProject ID: PRJNA412448 ) .\",\n \"Genome sequences and gene models are also accessible at the website of OIST Marine Genomics Unit Genome Project ( http://marinegenomics . oist . jp/chlorellaA99/viewer/info ? project_id=65 ) .\",\n \"Annotation of transcriptome contigs and prediction of gene models was performed by use of BLAST , Gene Ontology ( Ashburner et al . , 2000 ) and blast2go ( Conesa et al . , 2005 ) .\",\n \"To examine the conservation of H . viridissima contigs among metazoans , homology searches by blastx ( evalue 1E-5 ) were performed using protein databases obtained from NCBI for Drosophila melanogaster and Homo sapiens , from the JGI genome portal ( http://genome . jgi . doe . gov/ ) for Branchiostoma floridae , Nematostella vectensis , from Echinobase ( http://www . echinobase . org/EchinoBase/ ) for Strongylocentrotus pupuratus , from Compagen for Hydra magnipapillata , and from the OIST marine genomics Genome browser ver . 1 . 1 ( http://marinegenomics . oist . jp/coral/viewer/info ? project_id=3 ) for Acropora digitifera .\",\n \"For comparative analysis of gene models of Chlorella sp .\",\n \"A99 and other algae , domain searches against the Pfam database ( Pfam-A . hmm ) were performed using HMMER ( Eddy , 1998; Finn et al . , 2016 ) , and ortholog gene grouping was done using OrthoFinder ( Emms and Kelly , 2015 ) .\",\n \"The sequences of the reference genes and genomes were obtained from the database of the JGI genome portal for Chlorella variabilis NC64A , Coccomyxa subellipsoidea C-169 , Volvox carteri , Micromonas pusilla , and Ostreococcus tauri , from NCBI for Auxenochlorella protothecoides 0710 , from Phytozome ( http://phytozome . jgi . doe . gov/pz/portal . html ) for Chlamydomonas reinhardtii , from OIST Marine Genomics ( http://marinegenomics . oist . jp/symb/viewer/info ? project_id=21 ) for Symbiodinium minutum , Symbiodinium kawagutti genome , from Dinoflagellate Resources ( http://web . malab . cn/symka_new/ ) for Symbiodinium kawagutti and Reefgenomics ( http://reefgenomics . org/ ) for Symbiodinium microadriaticum ) ( Merchant et al . , 2007; Palenik et al . , 2007; Worden et al . , 2009; Blanc et al . , 2010; Prochnik et al . , 2010; Blanc et al . , 2012 ) Nitrogen assimilation genes in Chlorella A99 were identified by orthologous gene groups and reciprocal blast searches .\",\n \"The number of genes for nitrate assimilation genes , glutamine synthetase and glutamate synthetase , and clustering of such genes were systematically reported by ( Sanz-Luque et al . , 2015 ) .\",\n \"We used these data as reference for searches of nitrogen assimilation genes , and further nitrogen assimilation genes were searched by Kyoto Encyclopedia of Genes and Genomes ( KEGG ) pathway ( Kanehisa and Goto , 2000 ) .\",\n \"JGI genome browsers of Chlorella variabilis NC64A and Coccomyxa subellipsoidea C-169 were also used for retrieving genes and checking gene order on the scaffolds .\",\n \"For a phylogenetic tree of chlorophyte green algae , the sequences of 18S rRNA gene , ITS1 , 5 . 8S rRNA gene , ITS2 and 28S rRNA gene were obtained from scaffold20 of Chlorella A99 genome sequence , and from NCBI nucleotide database entries for Chlorella variabilis NC64A ( FM205849 . 1 ) , Auxenochlorella protothecoides 0710 ( NW_011934479 . 1 ) , Coccomyxa subellipsoidea C169 ( AGSI01000011 . 1 ) , Volvox carteri f .\",\n \"nagariensis ( NW_003307662 . 1 ) , Chlamydomonas reinhardtii ( FR865576 . 1 ) , Ostreococcus tauri ( GQ426340 . 1 ) and Micromonas pusilla ( FN562452 . 1 ) .\",\n \"Multiple alignments were produced with CLUSTALX ( 2 . 1 ) with gap trimming ( Larkin et al . , 2007 ) .\",\n \"Sequences of poor quality that did not well align were deleted using BioEdit ( Hall , 1999 ) .\",\n \"Phylogenetic analyses were performed using the Neighbor-Joining method by CLUSTALX with the default parameters ( 1000 bootstrap tests and 111 seeds ) .\",\n \"Representative phylogenetic trees were drawn by using NJ plot ( Perrière and Gouy , 1996 ) .\",\n \"Primers were designed based on the conserved region of the NRT2 gene , NiR and NR genes ( positive control ) identified by comparison of genes from Chlorella variabilis NC64A ( NC64A ) , Coccomyxa subellipsoidea C169 ( C169 ) , and Chlamydomonas reinhardtii ( Cr ) which belongs to Chlorophyceae class of green algae .\",\n \"Primers for NAR2 could not be designed because of insufficient conservation .\",\n \"As positive controls , amplicons were produced for NR of all the green algae examined and of NRT2 and NiR from NC64A , C169 and Cr , after which their sequences were checked .\",\n \"KOD FX Neo ( TOYOBO , Tokyo , Japan ) was used under the following conditions: an initial denaturation phase ( 94°C for 120 s ) followed by 36 cycles of ( 98°C for 30 s , 69°C for 100 s ) for NiR , ( 98°C for 30 s , 58°C for 30 s and 68°C for 210 s ) for NRT2 and ( 98°C for 30 s , 59°C for 30 s and 68°C for 60 s ) for NR .\",\n \"In each case , 10 ng gDNA was used as a template .\",\n \"The primers used are described in Supplementary file 6C .\",\n \"PCR products were sequenced to confirm amplification of the target genes using ABI PRISM 3100 Genetic Analyzer ( Thermo Fisher Scientific Inc . , Madison , USA ) using BigDye Terminator v3 . 1 Cycle Sequencing Kit ( Thermo Fisher Scientific ) .\",\n \"To isolate symbiotic algae , polyps were quickly homogenized in 0 . 25% sodium dodecyl sulfate ( SDS ) solution and centrifuged at 3000 g for 1 min .\",\n \"The pellet was resuspended in 0 . 05% SDS and centrifuged at 500 g for 5 min .\",\n \"Isolated A99 , NC64A and C169 were washed by sterilized Bold Basal Medium ( Bischoff and Bold , 1963 ) modified by the addition of 0 . 5% glucose , 1 . 2 mg/L vitamine B1 ( Thiaminhydrochloride ) , 0 . 01 mg/L vitamine B12 ( Cyanocobalamin ) ( Supplementary file 7 ) and incubated for two days in modified Bold Basal Medium with 50 mg/l ampicillin and streptomycin .\",\n \"The algae were cultivated in 5 ml of modified Bold Basal Medium ( BBM ) with the same amount of nitrogen ( 2 . 9 mM NaNO3 , NH4Cl , glutamine or 426 mg/l casamino acids ) and 5 mg/l Carbendazim ( anti-fungal ) with fluorescent illumination ( 12 hr light , 12 hr dark ) at 20˚C .\",\n \"Mean numbers of algae per ml were calculated from three tubes enumerated at 4 , 8 , and 12 days after inoculation with 106 cell/sml using a hemocytometer .\",\n \"After cultivation , gDNA was isolated from the A99 cultured in Gln-containing BBM and casamino acid-containing BBM and A99 was isolated from green hydra directly .\",\n \"A partial genomic region of the 18S rRNA gene was amplified by PCR and sequenced to confirm absence of contamination by other algae .\",\n \"PCR was performed using AmpliTaq Gold ( Thermo Fisher Scientific ) .\",\n \"Sequencing was performed as described above .\",\n \"The primers used are described in Supplementary file 6D .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Many multicellular organisms rely on symbiotic associations for support of metabolic activity , protection , or energy .","Understanding the mechanisms involved in controlling such interactions remains a major challenge .","In an unbiased approach we identified key players that control the symbiosis between Hydra viridissima and its photosynthetic symbiont Chlorella sp .","A99 .","We discovered significant up-regulation of Hydra genes encoding a phosphate transporter and glutamine synthetase suggesting regulated nutrition supply between host and symbionts .","Interestingly , supplementing the medium with glutamine temporarily supports in vitro growth of the otherwise obligate symbiotic Chlorella , indicating loss of autonomy and dependence on the host .","Genome sequencing of Chlorella sp .","A99 revealed a large number of amino acid transporters and a degenerated nitrate assimilation pathway , presumably as consequence of the adaptation to the host environment .","Our observations portray ancient symbiotic interactions as a codependent partnership in which exchange of nutrients appears to be the primary driving force ."],"string":"[\n \"Many multicellular organisms rely on symbiotic associations for support of metabolic activity , protection , or energy .\",\n \"Understanding the mechanisms involved in controlling such interactions remains a major challenge .\",\n \"In an unbiased approach we identified key players that control the symbiosis between Hydra viridissima and its photosynthetic symbiont Chlorella sp .\",\n \"A99 .\",\n \"We discovered significant up-regulation of Hydra genes encoding a phosphate transporter and glutamine synthetase suggesting regulated nutrition supply between host and symbionts .\",\n \"Interestingly , supplementing the medium with glutamine temporarily supports in vitro growth of the otherwise obligate symbiotic Chlorella , indicating loss of autonomy and dependence on the host .\",\n \"Genome sequencing of Chlorella sp .\",\n \"A99 revealed a large number of amino acid transporters and a degenerated nitrate assimilation pathway , presumably as consequence of the adaptation to the host environment .\",\n \"Our observations portray ancient symbiotic interactions as a codependent partnership in which exchange of nutrients appears to be the primary driving force .\"\n]"},"summary":{"kind":"list like","value":["All animals host microorganisms; some of which form ‘symbiotic’ relationships with their host that are mutually beneficial .","For instance , the human gut shelters tens of thousands of species of bacteria that break down our food for us , and corals , jellyfish or sea anemones can extract energy directly from sunlight thanks to the algae that live inside their cells .","Hydra , a small freshwater animal , lives in a symbiotic relationship with algae called Chlorella that it carries inside its cells .","Once an independent organism , Chlorella has evolved in such a way that , in nature , it cannot exist without Hydra anymore .","In turn , the algae produce sugars to fuel the animal when it cannot get food from the environment .","Yet , despite over 30 years of research , it still remains unclear how exactly the relationship between Hydra and Chlorella works , and how it came to be .","Understanding how these two organisms live together could help researchers to figure out the general principles that guide symbiotic interactions .","Nitrogen is an element that is essential for life , and organisms can extract it from various sources , such as nitrates or the amino acid glutamine .","Here , Hamada , Schröder et al . sequenced the entire genome of Chlorella .","This revealed that Chlorella has lost someof the genes required to obtain nitrates , and to process them into nitrogen .","However , the genetic analysis showed that the algae express genes that allow them to import amino acids .","In turn , analysis of the genes expressed by Hydra when it lives in symbiosis with Chlorella showed that the animal turns on genetic information needed to make glutamine .","It thus seems that Hydra creates glutamine which Chlorella can import; the algae then process this amino acid to obtain the nitrogen they need .","Hamada , Schröder et al . also discovered that if the environment was artificially enriched in glutamine , Chlorella could live on their own outside of Hydra for a while .","The results suggest that symbiotic relationships , such as the one between Hydra and Chlorella , were established because the organisms became dependent on each other for essential nutrients .","This co-dependency is strengthened if the organisms lose the ability to produce the nutrients on their own .","However , this partnership may be altered when the environment changes too much , especially if the balance of nutrients available gets tipped .","For example , if seas that are normally poor in nutrients become suddenly rich in these elements , this may disrupt the existence of symbiotic organisms such as corals ."],"string":"[\n \"All animals host microorganisms; some of which form ‘symbiotic’ relationships with their host that are mutually beneficial .\",\n \"For instance , the human gut shelters tens of thousands of species of bacteria that break down our food for us , and corals , jellyfish or sea anemones can extract energy directly from sunlight thanks to the algae that live inside their cells .\",\n \"Hydra , a small freshwater animal , lives in a symbiotic relationship with algae called Chlorella that it carries inside its cells .\",\n \"Once an independent organism , Chlorella has evolved in such a way that , in nature , it cannot exist without Hydra anymore .\",\n \"In turn , the algae produce sugars to fuel the animal when it cannot get food from the environment .\",\n \"Yet , despite over 30 years of research , it still remains unclear how exactly the relationship between Hydra and Chlorella works , and how it came to be .\",\n \"Understanding how these two organisms live together could help researchers to figure out the general principles that guide symbiotic interactions .\",\n \"Nitrogen is an element that is essential for life , and organisms can extract it from various sources , such as nitrates or the amino acid glutamine .\",\n \"Here , Hamada , Schröder et al . sequenced the entire genome of Chlorella .\",\n \"This revealed that Chlorella has lost someof the genes required to obtain nitrates , and to process them into nitrogen .\",\n \"However , the genetic analysis showed that the algae express genes that allow them to import amino acids .\",\n \"In turn , analysis of the genes expressed by Hydra when it lives in symbiosis with Chlorella showed that the animal turns on genetic information needed to make glutamine .\",\n \"It thus seems that Hydra creates glutamine which Chlorella can import; the algae then process this amino acid to obtain the nitrogen they need .\",\n \"Hamada , Schröder et al . also discovered that if the environment was artificially enriched in glutamine , Chlorella could live on their own outside of Hydra for a while .\",\n \"The results suggest that symbiotic relationships , such as the one between Hydra and Chlorella , were established because the organisms became dependent on each other for essential nutrients .\",\n \"This co-dependency is strengthened if the organisms lose the ability to produce the nutrients on their own .\",\n \"However , this partnership may be altered when the environment changes too much , especially if the balance of nutrients available gets tipped .\",\n \"For example , if seas that are normally poor in nutrients become suddenly rich in these elements , this may disrupt the existence of symbiotic organisms such as corals .\"\n]"},"year":{"kind":"string","value":"2018"}}},{"rowIdx":4286,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["cell biology"],"string":"[\n \"cell biology\"\n]"},"title":{"kind":"string","value":"Optogenetic control of PRC1 reveals its role in chromosome alignment on the spindle by overlap length-dependent forces"},"id":{"kind":"string","value":"elife-61170-v2"},"sections":{"kind":"list like","value":[["Pre-anaphase chromosome movements culminate with chromosome alignment at the spindle equator , a distinctive feature of mitosis important for the synchronous anaphase poleward movement of chromatids and proper telophase nuclear reformation ( Fonseca et al . , 2019; Maiato et al . , 2017 ) .","Chromosome congression to the metaphase plate , a process of directed chromosome movement from the polar regions toward the spindle equator , has been explored extensively ( Barisic et al . , 2014; Cai et al . , 2009; Kapoor et al . , 2006; Maiato et al . , 2017 ) .","Yet , the maintenance of chromosome alignment at the spindle equator is less understood .","This is a dynamic process given that the chromosomes constantly make small oscillatory excursions across the equator ( Skibbens et al . , 1993 ) .","Two mechanisms have been proposed to underlie the maintenance of chromosome alignment at the equator , namely length-dependent dynamics of kinetochore fiber ( k-fiber ) microtubules and polar ejection forces .","Pulling forces exerted by k-fiber tips on kinetochores are thought to be regulated by kinesin-8 motor proteins , which are needed for proper kinetochore alignment in various organisms from yeast to humans ( Garcia et al . , 2002; Stumpff et al . , 2008; Wargacki et al . , 2010 ) .","These motors can ‘measure’ microtubule length because they bind to microtubules along their length and move toward the plus end , leading to more kinesin-8 accumulated at the plus end of longer microtubules , where they promote microtubule catastrophe , that is , a switch from growth to shrinkage ( Tischer et al . , 2009; Varga et al . , 2006 ) .","During kinetochore movements across the spindle equator , the leading k-fiber shrinks , while the trailing one grows and accumulates kinesin-8 , resulting in microtubule catastrophe , followed by the movement of the trailing kinetochore back toward the equator .","Although this mechanism can explain kinetochore alignment at the spindle equator ( Klemm et al . , 2018 ) , the switching dynamics characteristic for this model , where the trailing kinetochore initiates the change of direction of motion , differs from the observations in mammalian cells where the leading kinetochore typically changes the direction before the trailing one ( Armond et al . , 2015; Civelekoglu-Scholey et al . , 2013; Wan et al . , 2012 ) .","In addition to the forces produced by k-fibers , polar ejection forces push chromosome arms away from the pole , powered by arm-bound chromokinesins that walk toward the plus end of microtubules ( Bajer et al . , 1982; Brouhard and Hunt , 2005 ) .","Because microtubule density increases toward the pole , these forces help the chromosomes to stay away from the poles , but most likely have little effect on kinetochore movements close to the spindle equator ( Cane et al . , 2013; Ke et al . , 2009 ) .","Thus , the current models do not provide a complete picture of kinetochore alignment at the spindle center .","K-fibers are surrounded by a dense network of spindle microtubules with which they have multiple interactions ( McDonald et al . , 1992; O'Toole et al . , 2020 ) , resulting in forces acting on k-fibers and thus also on kinetochores .","In particular , each pair of sister k-fibers is tightly linked by the bridging fiber , a bundle of antiparallel microtubules that balances the tension on sister kinetochores ( Kajtez et al . , 2016; Polak et al . , 2017; Vukušić et al . , 2017 ) .","However , the role of forces exerted by bridging fiber in chromosome alignment at the metaphase plate is unknown .","In metaphase , overlap regions within bridging fibers are crosslinked by protein regulator of cytokinesis 1 ( PRC1 ) ( Kajtez et al . , 2016; Polak et al . , 2017; Tolić , 2018 ) .","PRC1 , like other non-motor microtubule-associated proteins from Ase1/PRC1/MAP65 family , selectively bundles antiparallel microtubules and provides stable overlaps in vitro ( Bieling et al . , 2010b; Janson et al . , 2007; Mollinari et al . , 2002; Subramanian et al . , 2010 ) .","Cellular studies of its function show that PRC1 is associated with the spindle midzone in anaphase , where its activity is essential for stable microtubule organization , localization of numerous microtubule-associated proteins within this structure , and successful completion of cytokinesis , while its microtubule-binding and -bundling affinities are regulated by phosphorylation and dephosphorylation events ( Gruneberg et al . , 2006; Jiang et al . , 1998; Kurasawa et al . , 2004; Liu et al . , 2009; Mollinari et al . , 2002; Mollinari et al . , 2005; Neef et al . , 2007; Subramanian et al . , 2013; Subramanian et al . , 2010; Zhu and Jiang , 2005; Zhu et al . , 2006 ) .","In this work , we developed an optogenetic approach for acute and reversible removal of PRC1 from the spindle to the cell membrane , building upon ideas of dimerization or dissociation induced chemically ( Cheeseman et al . , 2013; Haruki et al . , 2008; Robinson et al . , 2010; Wordeman et al . , 2016 ) or by light ( Fielmich et al . , 2018; Guntas et al . , 2015; Okumura et al . , 2018; van Haren et al . , 2018; Yang et al . , 2013; Zhang et al . , 2017 ) to rapidly redistribute proteins .","By using our assay on metaphase spindles , we found that bridging fibers promote kinetochore alignment .","PRC1 removal resulted in partial disassembly of bridging fibers and elongation of their overlap zones .","Moreover , the metaphase plate widened , inter-kinetochore distance decreased , and lagging chromosomes appeared more frequently , showing that PRC1 indirectly regulates forces acting on kinetochores .","Kif4A/kinesin-4 and Kif18A/kinesin-8 were found to localize in the bridging fiber during metaphase and were largely lost upon PRC1 removal , with Kif4A showing a greater reduction .","These results , together with the finding that Kif4A or Kif18A depletion by siRNA led to elongated overlaps , suggest that these proteins regulate the overlap length of bridging microtubules .","PRC1 removal did not affect the localization of Kif4A on the chromosomes and Kif18A , CLASP1 , and CENP-E/kinesin-7 on the plus ends of k-fibers , arguing against perturbed polar ejection forces or molecular events at the kinetochore microtubule plus ends as origins of the observed kinetochore misalignment .","In conclusion , our optogenetic experiments show that bridging microtubules buffer chromosome movements , thus promoting their alignment .","We propose that this occurs via length-dependent forces , which depend on the antiparallel overlap length within the bridging fiber ."],["To study the role of PRC1 and the forces arising from coupling of bridging and k-fibers in chromosome alignment , we developed an optogenetic tool for fast and reversible removal of PRC1 from the spindle to the cell membrane , based on the previously designed improved light inducible dimer ( iLID ) system ( Guntas et al . , 2015 ) .","We attached PRC1 to the red fluorescent protein tgRFPt and the bacterial protein SspB , while the iLID , which contains the bacterial peptide SsrA and the light-oxygen-voltage ( LOV2 ) domain , is bound to the cell membrane by a short peptide , CAAX .","In this system , LOV2 adopts a conformation that allows dimerization of SsrA and SspB upon exposure to the blue light ( Figure 1A ) .","After cessation of exposure to the blue light , LOV2 adopts its initial conformation leading to decreased affinity of SsrA to SspB .","Therefore , exposure to the blue light should induce translocation of PRC1 from the central region of the metaphase spindle , which we will refer to as the spindle midzone , to the cell membrane , whereas cessation of exposure to blue light should restore PRC1 localization on the spindle ( Figure 1A ) .","To test our optogenetic approach , we used U2OS cells with stable expression of CENP-A-GFP , transient expression of PRC1-tgRFPt-SspB ( henceforth opto-PRC1 ) and iLID-CAAX ( henceforth opto cells; Figure 1B; Figure 1—video 1 ) .","Endogenous PRC1 was depleted 90 ± 2% ( all results are mean ± s . e . m . ) by siRNA before addition of opto-PRC1 ( Figure 1—figure supplement 1A ) .","Before exposure to the blue light , opto-PRC1 had normal localization on the microtubule bundles in the spindle midzone ( Figure 1B; 0:00 min ) , with the length of PRC1 streaks of 3 . 77 ± 0 . 08 µm ( n = 193 bundles , N = 30 cells ) , consistent with that of endogenous and fluorescently labeled PRC1 in metaphase ( Kajtez et al . , 2016; Polak et al . , 2017 ) , although the total signal intensity of opto-PRC1 on the spindle was higher compared to endogenous PRC1 ( Figure 1—figure supplement 1B ) .","Addition of opto-PRC1 did not change the duration of metaphase , as inferred from the fraction of cells that entered anaphase during image acquisition , which was similar in cells with endogenous PRC1 and cells treated with PRC1 siRNA and containing opto-PRC1 ( 79 ± 6% , N = 37 , and 71 ± 5% , N = 72 , respectively; p = 0 . 4 , Pearson’s Chi-squared test; Figure 1—figure supplement 1C ) .","After exposure to the blue light , opto cells were able to progress to cytokinesis ( Figure 1—figure supplement 1D ) .","Taken together , these data suggest that opto-PRC1 replaces the depleted endogenous PRC1 .","Upon exposure to the blue light , opto-PRC1 signal on the spindle decreased and its signal on the membrane increased ( Figure 1B; 0:20-20:00 min ) .","After the blue light was switched off , opto-PRC1 returned to the spindle midzone ( Figure 1B; 20:40-30:10 min ) .","To validate our system , we performed two sets of test experiments .","The first one was performed on the same cell line containing opto-PRC1 , but without iLID , imaged with the same imaging protocol as the opto cells , which we refer to as control throughout the paper .","The second test experiment was on the opto cells but without the blue light ( Figure 1—figure supplement 1E ) .","In both cases , opto-PRC1 remained on the spindle ( Figure 1—figure supplement 1E ) .","Thus , our optogenetic approach allows for acute and reversible control of PRC1 localization in metaphase .","To quantify the dynamics and spatial pattern of opto-PRC1 removal and return , we measured the intensity of opto-PRC1 on the metaphase spindle ( Figure 1—figure supplement 1F , G ) .","We found that 88 ± 3% of opto-PRC1 was removed after 20 min of exposure to the blue light with a half-time of 4 . 2 ± 0 . 1 min ( Figure 1C ) .","During the opto-PRC1 removal , there was simultaneous decrease in both signal intensity and length of the overlap region ( Figure 1D , left; Figure 1—figure supplement 1G , top ) , which may be due to fewer antiparallel regions being positioned laterally than in the central part of the bundle ( Mastronarde et al . , 1993 ) .","The signal of the outermost midzone bundles typically lasted longer than of the inner ones ( Figure 1B; 3:40-6:30 ) .","After the blue light was switched off , opto-PRC1 signal restored to 65 ± 1% of the initial intensity within 10 min , with return half-time being 0 . 71 ± 0 . 04 min ( Figure 1C ) .","The faster PRC1 return to the spindle in comparison with its removal may be due to the higher affinity difference between PRC1 binding to the spindle and to the membrane in the dark than under light .","During the opto-PRC1 return , it initially localized throughout the spindle , with gradual increase in intensity in the spindle midzone ( Figure 1D , right; Figure 1—figure supplement 1G , bottom ) , suggesting that PRC1 has higher unbinding rate outside than within the overlap bundles in the spindle .","This result is consistent with PRC1 having a life-time of several seconds on single microtubules and a 10-fold preference for overlap regions in vitro ( Subramanian et al . , 2010 ) .","To test whether bridging fiber has a role in the maintenance of chromosome alignment , acute optogenetic removal of PRC1 , a depletion of which is known to perturb bridging fibers ( Polak et al . , 2017 ) should affect chromosome positioning at the spindle equator ( Figure 2A ) .","Surprisingly , we observed that the acute removal of opto-PRC1 resulted in movement of sister kinetochore pairs away from the metaphase plate ( Figure 2B; Figure 2—video 1 ) , which is not found after long-term PRC1 depletion by siRNA ( Polak et al . , 2017 ) .","Upon opto-PRC1 removal , the distances of sister kinetochore midpoints from the equatorial plane increased ( dEQ , Figure 2B , C , D; Figure 2—figure supplement 1A , B ) .","While >95% of kinetochore pairs were found within the region of PRC1 streaks , that is , less than 2 µm away from the equatorial plane before removal of opto-PRC1 , 9 . 3 ± 2 . 7% of kinetochore pairs made excursions far outside this region after opto-PRC1 removal ( Figure 2—figure supplement 1B ) .","The displaced kinetochores were found more often in the inner part of the spindle , that is , close to the long spindle axis , than in the outer regions ( Figure 2E ) .","Kinetochores fluctuated to a similar extent in the presence and absence of opto-PRC1 , but in its absence the displaced kinetochores fluctuated within a region that was offset from the equatorial plane ( Figure 2—figure supplement 1C ) .","These displaced kinetochores upon opto-PRC1 removal had lower inter-kinetochore distance in comparison to non-displaced ones , suggesting that kinetochore displacement was related to a more severe reduction of tension ( Figure 2F ) .","On average , kinetochores remained displaced even after opto-PRC1 return ( Figure 2D ) .","We conclude that PRC1 has a role in keeping kinetochores in tight alignment on the metaphase plate .","The mean inter-kinetochore distance ( dKC , Figure 2C ) was reduced when opto-PRC1 was removed ( Figure 2G; Figure 2—figure supplement 1D ) , and the distance after 20 min of exposure to the blue light ( 0 . 79 ± 0 . 01 µm ) was closer to metaphase ( 0 . 87 ± 0 . 01 µm ) than prometaphase ( 0 . 66 ± 0 . 01 µm ) values ( see Materials and methods ) , suggesting that tension was not completely lost and that these changes were not due to kinetochore detachment from k-fibers ( Figure 2—figure supplement 1D ) .","In agreement with this , the fraction of cells that entered anaphase during image acquisition was similar in control and opto cells ( 71 ± 5% , N = 72 , and 60 ± 5% , N = 93 , respectively; p = 0 . 1 , Pearson’s Chi-squared test; Figure 1—figure supplement 1C ) , indicating that PRC1 removal did not prevent spindle assembly checkpoint satisfaction .","After opto-PRC1 return to the spindle , inter-kinetochore distance increased , implying restoration of tension , although not to the original value ( Figure 2G ) .","To investigate the influence of acute PRC1 removal on the orientation of sister kinetochores , we measured the angle between sister kinetochore axis and long spindle axis ( αKC , Figure 2C ) .","We observed that removal of opto-PRC1 caused misorientation of sister kinetochores , that is , increased αKC ( Figure 2B , H; Figure 2—figure supplement 1A , E ) .","Misoriented kinetochores were found at a larger distance from the equatorial plane and closer to the long spindle axis ( Figure 2—figure supplement 1F ) .","Interestingly , sister kinetochore pairs remained misoriented even after opto-PRC1 return ( Figure 2H ) .","Similarly , PRC1 bundles were misoriented upon PRC1 return ( see Materials and methods , Figure 2—figure supplement 1G ) .","These results suggest that when PRC1 returns to the overlaps whose geometry was perturbed by PRC1 removal , it likely confines the chromosomes in new positions and orientations .","The observed effects of PRC1 removal on inter-kinetochore distance , kinetochore alignment and orientation did not change when SiR-tubulin was added ( dKC p = 0 . 82 , dEQ p = 0 . 27 , αKC p = 0 . 61 , respectively; t-test ) .","The effects of PRC1 removal were found neither in control experiments in cells without iLID , which were stained with SiR-tubulin , nor in a different set of control experiments where cells expressing only CENP-A-GFP without SiR-tubulin were imaged with the same illumination protocol ( Figure 2D , G , H; Figure 2—figure supplement 1A , B , D , E ) .","Therefore , the observed effects in opto cells were not a consequence of SiR-tubulin or laser photodamage ( Douthwright and Sluder , 2017 ) .","As previous reports have shown that acute rapamycin-dependent protein translocation and long-term depletion by siRNA can yield different and even opposite phenotypes ( Cheeseman et al . , 2013; Wordeman et al . , 2016 ) , we compared kinetochore parameters after acute removal of PRC1 with those obtained from cells after long-term depletion of PRC1 by siRNA .","Strikingly , in contrast to acute removal , the long-term depletion did not cause kinetochore misalignment or misorientation ( Figure 2D , H; Figure 2—figure supplement 1H , I; Table 1 ) .","The two methods decreased the inter-kinetochore distance to a similar extent ( Figure 2G; Figure 2—figure supplement 1J; Table 1 ) , even though unlike acute removal , long-term removal reduced the fraction of cells that entered anaphase ( 35 ± 8% , N = 37 , and 79 ± 6% , N = 37 , for PRC1 siRNA treated and untreated , respectively; p = 0 . 046 , Pearson’s Chi-squared test; Figure 1—figure supplement 1C ) .","Thus , acute removal of PRC1 results in different effects in comparison with a long-term depletion .","To test to what extent the acute removal of PRC1 during metaphase affects chromosome segregation , we measured the frequency of lagging kinetochores .","We found that lagging kinetochores occurred more frequently when opto-PRC1 was being removed than in control cells ( Figure 2I , J; Figure 2—video 2 ) .","Similarly , long-term depletion of PRC1 by siRNA also increased the frequency of lagging kinetochores during early anaphase ( Figure 2—figure supplement 2A; Table 1 ) .","Opto cells that showed lagging kinetochores in anaphase had a slightly smaller inter-kinetochore distance before anaphase than opto cells without lagging kinetochores ( Figure 2—figure supplement 2B ) , suggesting that a decrease in tension may be involved in the imperfect kinetochore segregation .","The cells with lagging kinetochores did not have a larger average kinetochore misalignment in metaphase ( Figure 2—figure supplement 2C ) , which indicates that misalignment and lagging kinetochores are not linked on the cell level , although they may be linked locally on individual kinetochores .","As perturbation of the PRC1-CLASP1 interaction and the consequent absence of CLASP1 from the spindle midzone results in lagging chromosomes ( Liu et al . , 2009 ) , we inspected the localization of CLASP1 and found that it did not accumulate between segregating chromosomes in opto HeLa cells stably expressing EGFP-CLASP1 ( Figure 2—figure supplement 2D ) .","Thus , the observed higher occurrence of lagging kinetochores could be attributed to changes in tension during metaphase , perturbed recruitment of CLASP1 to the spindle midzone by PRC1 during early anaphase , or a combination of both effects .","Factors that could contribute to the altered chromosome alignment and occurrence of lagging kinetochores upon opto-PRC1 removal are changes related to ( 1 ) microtubules in the bridging fibers , ( 2 ) polar ejection forces , and/or ( 3 ) proteins that modulate the dynamics of k-fiber plus-ends ( Figure 2K ) .","Because PRC1 crosslinks microtubules within bridging fibers ( Kajtez et al . , 2016; Polak et al . , 2017 ) , we first tested the effects of PRC1 removal on those microtubules .","An important aspect of the bridging fiber that may affect chromosome alignment is the dynamics of microtubules that make up these fibers .","To explore their dynamics , we developed an assay to track the growing plus ends of individual microtubules in the bridging fiber by using cells expressing the plus end marker EB3 ( Stepanova et al . , 2003 ) tagged with GFP ( Figure 3 ) .","We followed single EB3 spots in the spindle and identified the ones belonging to a bridging fiber as the spots that move toward a kinetochore , cross the region between this kinetochore and its sister , and move beyond it toward the other spindle pole ( Figure 3A–C; Figure 3—video 1 ) .","We found 1 . 8 ± 0 . 2 EB3 spots per minute per bridging fiber in control cells , showing that bridging fibers are dynamically remodeled during metaphase ( Figure 3D ) .","This number was similar after opto-PRC1 removal , 2 . 0 ± 0 . 3 spots/min ( p = 0 . 59; t-test , Figure 3D ) , suggesting that the number of dynamic microtubules in the bridge is largely unaffected by acute PRC1 removal .","However , in PRC1 siRNA-treated cells fewer EB3 spots were observed to move to opposite spindle half , 1 . 5 ± 0 . 1 EB3 spots/min , compared to untreated cells , where 1 . 91 ± 0 . 09 spots/min were observed ( p = 0 . 01; t-test , Figure 3—figure supplement 1A ) , indicating that long-term PRC1 depletion slightly decreases the number of dynamic microtubules in the bridge .","To assess the changes in the dynamics of bridging microtubules , we followed EB3 spots in the bridge from the time when they can be distinguished from neighboring spots near the pole until they disappear in the opposite spindle half , which we interpret as the moment when the microtubule stops growing ( Maurer et al . , 2012 ) .","Interestingly , EB3 tracks were longer after opto-PRC1 removal than in control cells ( Figure 3B , C; Figure 3—video 1 ) , but the velocities of the EB3 spots were not affected by opto-PRC1 removal or long-term depletion when compared to untreated cells ( Figure 3E; Figure 3—figure supplement 1B ) .","In agreement with these results , kymographs of the central region of the spindle show that EB3 spots reach deeper into the opposite half of the spindle after opto-PRC1 removal ( Figure 3F; Figure 3—figure supplement 1C ) .","Thus , acute removal of PRC1 results in longer bridging microtubules , which is not a consequence of altered microtubule growth rate , but most likely due to a reduced microtubule catastrophe rate .","EB3 tracks allowed us to estimate the length of the overlap zone of antiparallel microtubules in the bridging fiber , which currently cannot be measured after PRC1 removal because PRC1 itself is the only available marker of antiparallel overlaps in the spindle .","We define the overlap half-length as the distance beyond the equatorial plane that an EB3 spot covers while moving within the bridging fiber from one spindle half into the other ( Figure 3G , left ) , where this microtubule can form antiparallel overlaps with the oppositely oriented microtubules extending from the other pole .","In control cell , the overlap half-length was 1 . 8 ± 0 . 2 µm ( n = 17 spots in N = 9 cells; Figure 3G , right ) , which corresponds well to the overlap half-length measured as the half-length of PRC1 streaks in this cell line , 0 . 5x ( 3 . 8 ± 0 . 4 ) µm ( N = 9 cells; Figure 3H ) , validating our method for overlap length measurement based on EB3 tracks .","After opto-PRC1 removal , the overlap half-length increased to 2 . 6 ± 0 . 2 µm ( n = 23 spots in N = 9 cells; Figure 3G ) .","In contrast to opto cells , treatment with PRC1 siRNA did not result in a change of overlap length with respect to untreated cells ( Figure 3—figure supplement 1D–F ) .","Thus , the antiparallel overlaps became on average 40% longer after acute , but unchanged after long-time PRC1 removal .","Interestingly , the overlaps were especially long in the inner part of the spindle , whereas those on the spindle periphery were similar to overlaps in control cells ( Figure 3I ) .","This spatial difference is correlated with our findings that PRC1 is removed faster from the inner bundles ( see Figure 1B ) and that displaced and misoriented kinetochores are found more often in the inner than in the outer spindle region ( see Figure 2D , E , H ) , suggesting a mechanistic link between the overlap length and kinetochore positioning .","To test to what extent PRC1 removal in metaphase affects the number of microtubules in the bridging fibers ( Figure 4A ) , we visualized microtubules by using SiR-tubulin , a far-red tubulin dye excited by red light ( Lukinavičius et al . , 2014 ) , which allowed us to observe microtubules both when the blue light is turned on and switched off .","Intensity profiles across the spindle midzone revealed that SiR-tubulin intensity maxima were lower upon PRC1 removal and increased after its return ( Figure 4—figure supplement 1A; Figure 4—video 1 ) .","Measurements of SiR-tubulin signal intensity between and lateral from sister kinetochores showed that upon PRC1 removal the tubulin signal was reduced specifically in the bridging fibers ( Figure 4A–C; Figure 4—video 1 ) .","As an alternative to SiR-tubulin , which is a taxol-based dye that may affect microtubule dynamics ( Lukinavičius et al . , 2014 ) , we tested YFP-tubulin , but the excitation laser for YFP also activated the optogenetic system ( Wang and Hahn , 2016; Figure 4—figure supplement 1B ) .","Finally , we used tubulin-GFP to determine tubulin signal intensities of the bridging fibers and k-fibers upon acute removal of PRC1 ( Figure 4A , D–H; Figure 4—figure supplement 1C–F; see Materials and methods ) .","Upon exposure to the blue light , tubulin signal intensity in the bridging fibers decreased ~2 . 5-fold , which corresponds to 5 . 6 ± 0 . 9 microtubules given that the average number of microtubules in the bridging fiber is 14 ( Kajtez et al . , 2016 ) .","Together with the finding that the number of growing microtubules in the bridging fiber is similar with and without PRC1 ( Figure 3D ) , this result implies that in the presence of PRC1 the bridge contains more microtubules with a smaller fraction of them being dynamic than without PRC1 , and that PRC1 removal leads mainly to disassembly of non-dynamic microtubules .","Upon PRC1 return the intensity and thus the number of microtubules remained low ( Figure 4D–G; Figure 4—figure supplement 1D ) , possibly as a consequence of the perturbed spindle architecture in the absence of PRC1 , which may be able to recover after a longer time period .","Importantly , the intensity of k-fiber was unaltered ( Figure 4H , see Materials and methods ) , suggesting that the k-fibers were not affected by PRC1 removal .","Thus , acute removal of PRC1 and its return change the number of microtubules specifically in the bridging fibers .","Long-term depletion of PRC1 by siRNA in HeLa cells with stable expression of tubulin-GFP and transient expression of mRFP-CENP-B resulted in a similar reduction in bridging fiber tubulin intensity as after acute PRC1 removal ( Figure 4—figure supplement 1G , H ) .","Long-term depletion resulted in a decreased number of microtubules in the bridge , 7 . 5 ± 1 . 1 , which was similar to the microtubule number after acute removal ( p = 0 . 21 , t-test; Figure 4—figure supplement 1I , Table 1 ) .","As in acute removal , the intensity of k-fibers did not change after long-term depletion ( p = 0 . 49 , t-test ) .","Given that the bridging fiber is under compression ( Kajtez et al . , 2016 ) , reduction of number of microtubules in the bridging fiber is expected to reduce this compression and thus to straighten the k-fibers ( Figure 4—figure supplement 1J ) .","Tracking of the pole-to-pole contour of the outermost k-fibers revealed that PRC1 removal indeed straightened the k-fibers and thus made the spindle diamond-shaped ( Figure 4—figure supplement 1K–M ) .","Similar to acute removal , long-term depletion of PRC1 caused straightening of outermost k-fibers , although to a smaller extent , whereas spindle length and width were unchanged after both treatments ( Figure 4—figure supplement 1L , N , O; Table 1 ) .","This result supports that compression in the bridging fibers enables the spindle to obtain a curved shape .","Since bridging fibers are , nevertheless , present upon PRC1 removal , we asked how the residual microtubules are bundled together .","Eg5/kinesin-5 , which localizes in the bridging fibers ( Kajtez et al . , 2016; Mann and Wadsworth , 2018 ) , was still detectable in these fibers after PRC1 siRNA ( Figure 4—figure supplement 1P ) .","Thus , we propose that microtubule crosslinkers such as Eg5 crosslink the remaining microtubules in the bridge after acute PRC1 removal .","To investigate the mechanism of bridging microtubule regulation via PRC1 , we analyzed the localization of major proteins that regulate spindle microtubule dynamics and/or are binding partners of PRC1: Kif4A , Kif18A , and MKLP1 ( Bieling et al . , 2010b; Bringmann et al . , 2004; Gruneberg et al . , 2006; Kurasawa et al . , 2004; Mayr et al . , 2007; Stumpff et al . , 2008; Stumpff et al . , 2012 ) before and after PRC1 removal in metaphase ( Figure 5; Figure 5—figure supplement 1; Figure 5—figure supplement 2 and Table 1 ) .","We first looked at the localization of these proteins in the bridging fiber .","Surprisingly , we found that Kif4A and Kif18A localize in the bridge during metaphase , visible as thin lines across or next to the location of sister kinetochores where PRC1-labeled bundles are found ( Polak et al . , 2017; Figure 5A; Figure 5—figure supplement 1A ) .","Similar localization was observed for MKLP1 , spanning the region between two sister k-fibers ( Figure 5A ) .","In vertically oriented spindles , whose long axis was roughly perpendicular to the imaging plane , Kif4A and Kif18A colocalized with opto-PRC1 , now visible as spots in the cross-section of the spindle ( Figure 5—figure supplement 1B ) .","To explore whether PRC1 removal affects the localization of Kif4A , Kif18A , and MKLP1 , we analyzed their intensities within bridging fibers .","Measurements of Kif4A intensity upon acute PRC1 removal and long term-depletion , in regions lateral from chromosomes which correspond to peripheral parts of bridging fibers , showed that its intensity in the bridging fibers decreased ( Figure 5A–C; Figure 5—figure supplement 1A ) .","Additional analysis of Kif4A intensity in HeLa cells after long-term PRC1 removal corroborated this result ( Figure 5—figure supplement 1C ) .","Interestingly , there was a larger reduction in Kif4A intensity after acute PRC1 removal , where it decreased by 76 ± 5% , in comparison to long-term depletion where it decreased by 52 ± 3% ( p = 1×10−4; Figure 5C; Table 1 ) .","Kif18A intensity in the bridging fibers also decreased upon acute PRC1 removal and long-term depletion ( Figure 5A–C; Figure 5—figure supplement 1A , C ) .","Yet , in contrast to Kif4A , Kif18A intensities in the bridging fiber decreased to a similar extent , that is by 43 ± 11% and 38 ± 6% , after acute and long-term PRC1 depletion , respectively ( p = 0 . 68; Figure 5C; Table 1 ) .","MKLP1 intensities were also similarly reduced after both approaches , 87 ± 5% after acute PRC1 removal and 83 ± 11% after long-term depletion ( p = 0 . 68; Figure 5D; Figure 5—figure supplement 1C ) .","Among the tested proteins , only MKLP1 was exclusively localized in the bridging fibers , co-localizing with PRC1 , both before PRC1 removal and after its return ( Figure 5A , D; Figure 5—figure supplement 1C–G ) .","Even though MKLP1 was removed to a large extent from the spindle by acute PRC1 removal , it was not detected at the cell membrane together with PRC1 .","It may be that MKLP1 binds rather weakly to PRC1 in metaphase and/or that the absence of PRC1 decreases its affinity for microtubules .","In addition , the ability of MKLP1 to bind along scaffold of antiparallel overlaps could depend on the role of PRC1 in dictating 35-nm-inter-microtubule spacing , proposed to be important to enable localization of specific proteins within these structures ( Kellogg et al . , 2016; Subramanian et al . , 2010 ) .","As Kif4A and Kif18A are known to regulate microtubule length ( Mayr et al . , 2007; Varga et al . , 2006; Wandke et al . , 2012 ) , we set out to explore their roles in overlap length regulation .","We hypothesized that these kinesins may regulate the length of bridging microtubules and hence their antiparallel overlaps during metaphase .","Indeed , removal of Kif4A or Kif18A by siRNA resulted in longer PRC1-labeled overlaps and longer spindles ( Figure 5E ) , with a small but not significant increase in the ratio of overlap length to spindle length ( Figure 5F ) .","However , the increase in the overlap and spindle length was significantly different after the two treatments ( Figure 5G ) .","Kif4A siRNA treatment increased overlap length for 0 . 9 ± 0 . 2 µm compared with control , which was similar to spindle length increase of 1 . 2 ± 0 . 3 µm ( p = 0 . 48 , t-test; Figure 5G ) .","In contrast , Kif18A siRNA treatment increased overlap length for 1 . 8 ± 0 . 3 µm , whereas the spindle length increased to a larger extent , for 3 . 7 ± 0 . 6 µm ( p = 0 . 004 , t-test; Figure 5G ) .","These results suggest that both Kif4A and Kif18A regulate the length of bridging microtubules and their overlap , but Kif18A has a greater effect on overall spindle microtubules and spindle length than Kif4A .","As an alternative to the changes in the bridging fiber , disruption of polar ejection forces may be the cause of kinetochore misalignment upon PRC1 removal ( Figure 2K ) , in particular because these forces are modulated by Kif4A ( Stumpff et al . , 2012 ) .","The most prominent Kif4A localization in metaphase is on chromosomes ( Gruneberg et al . , 2006; Kurasawa et al . , 2004; Zhu and Jiang , 2005; Figure 5A , B; Figure 5—figure supplement 1A–C; Figure 5—figure supplement 2A–C ) .","Neither acute nor long-term PRC1 removal had an extensive effect on the Kif4A signal on chromosome arms , although there was a slight decrease in Kif4A signal after PRC1 siRNA treatment ( Figure 5B; Figure 5—figure supplement 1A , C; Figure 5—figure supplement 2C , D; Table 1 ) .","In early anaphase , the amount of Kif4A on segregated chromosomes was similar in opto and control cells ( Figure 5—figure supplement 2E ) , indicating that increased occurrence of lagging kinetochores was neither due to perturbed polar ejection forces in that phase nor defects in chromosome architecture/condensation .","Finally , kinetochore misalignment and lagging kinetochores upon PRC1 removal may be due to disrupted localization of proteins that regulate microtubule dynamics at the plus-end of the k-fiber ( Figure 2K ) .","To investigate this possibility , we analyzed the k-fiber localization of the key proteins with this function , Kif18A , CLASP1 , and CENP-E ( Al-Bassam et al . , 2010; Maffini et al . , 2009; Maiato et al . , 2003; Mayr et al . , 2007; Stumpff et al . , 2008; Stumpff et al . , 2012; Yu et al . , 2016 ) .","These proteins were localized at the plus-ends of k-fibers before and after acute and long-term removal of PRC1 ( Figure 5A , B; Figure 5—figure supplement 1A–C; Figure 5—figure supplement 2F–I ) .","This indicates that kinetochore misalignment upon PRC1 removal is not caused by simultaneous removal of proteins that regulate microtubule dynamics at the k-fiber plus ends ."],["We developed an optogenetic approach that offers acute light-controlled removal of proteins from a normally formed spindle at a precise phase of mitosis .","The main advantages of this approach over chemically-induced protein translocation ( Cheeseman et al . , 2013; Haruki et al . , 2008; Robinson et al . , 2010; Wordeman et al . , 2016 ) are its reversibility , allowing the protein to return to its initial location within about a minute , and applicability to individual cells .","Unlike previous optogenetic approaches ( Fielmich et al . , 2018; Okumura et al . , 2018; van Haren et al . , 2018; Yang et al . , 2013; Zhang et al . , 2017 ) , this method allows for global loss-of-function of full-length spindle proteins , relying on simple protein tagging rather than domain splitting , with no need of chromophore addition .","Moreover , this method may be implemented with other optical perturbations ( Milas et al . , 2018 ) and used as ‘in vivo pull-down’ for probing protein-protein interactions in different phases of the cell cycle .","However , this approach depends on high turnover of the protein in comparison with the time scale of interest .","Acute PRC1 removal from the spindle by optogenetics and long-term PRC1 depletion by siRNA led to partially different phenotypes .","These differences are hard to explain by different levels of PRC1 on the spindle as both methods decreased PRC1 by ~90% .","It is also unlikely that the differences are caused by the interaction of the membrane-translocated PRC1 with astral microtubules because of the uniform PRC1 signal on the membrane , fast return to the spindle , and no change in spindle positioning .","Therefore , the generally weaker effects of siRNA in comparison with the acute optogenetic removal are most likely due to compensatory mechanisms acting during long-term depletion .","By overcoming temporal limitations of siRNA , our work reveals an unexpected role of PRC1 and bridging fibers in the regulation of chromosome alignment on the metaphase spindle via overlap length-dependent forces ( Figure 6A ) .","We propose that the interactions between k-fibers and bridging fibers regulate the movement of bi-oriented chromosomes along the pole-to-pole axis .","If a kinetochore pair is displaced toward one spindle pole , more motors and/or crosslinkers are expected to accumulate in the overlap facing the opposite pole because this overlap is longer , and pull the kinetochores back to the center ( Figure 6B ) .","As the efficiency of this type of centering depends on the relative asymmetry in the overlap length on either side , shorter overlap length leads to more precise centering of the kinetochores ( Figure 6A , B ) .","Our finding that acute PRC1 removal leads to chromosome misalignment and elongation of overlap zones supports this model .","Moreover , elongation of overlaps correlates with chromosome misalignment within spindles , as elongated overlaps and misaligned chromosomes are mostly found in the central part of the spindle rather than on the periphery .","In contrast to the acute PRC1 removal , long-term PRC1 depletion by siRNA leads neither to overlap elongation nor chromosome misalignment , providing further support to our model .","These differences between the acute and long-term depletion indicate the existence of compensatory mechanisms that regulate overlap length and hence maintain chromosome alignment during long-term PRC1 depletion .","How is the length of the bridging microtubules and their overlaps controlled ?","Interestingly , we found that Kif4A and Kif18A localize in the bridging fibers in metaphase and their intensities decreased following partial disassembly of the bridging fiber by optogenetic or siRNA-mediated PRC1 removal .","Both Kif4A and Kif18A suppress microtubule dynamics ( Bieling et al . , 2010b; Bringmann et al . , 2004; Stumpff et al . , 2008; Stumpff et al . , 2012 ) , and our experiments showed that depletion of either of them by siRNA leads to longer PRC1-labeled overlaps .","These elongated PRC1-labeled overlaps provide another line of evidence in support of our model for chromosome alignment by overlap length-dependent forces , as Kif4A and Kif18A siRNA-treated cells exhibit chromosome misalignment ( Stumpff et al . , 2008; Wandke et al . , 2012 ) .","Even though it was reported that Kif4A does not directly interact with PRC1 before late mitosis ( Gruneberg et al . , 2006; Kurasawa et al . , 2004; Zhu and Jiang , 2005 ) , our experiments suggest that there is a small pool of Kif4A that does so and binds to the antiparallel overlaps of bridging microtubules in metaphase .","Acute PRC1 removal likely results in the removal of this pool of Kif4A from the bridge .","Our result that Kif4A was reduced by ~76% , which is similar to the reduction of PRC1 by ~85% within the same time interval ( 12 min ) of acute removal supports this picture .","This reduction of Kif4A in the bridge may lead to excessive microtubule polymerization and thus longer overlap zones in metaphase , similar to the long overlaps in anaphase after Kif4A depletion ( Hu et al . , 2011; Kurasawa et al . , 2004; Zhu and Jiang , 2005 ) .","As the spindle length remained constant after acute PRC1 removal , we suggest that the overlap elongation is due to Kif4A acting specifically on bridging microtubules rather than overall spindle microtubules .","In contrast to Kif4A , Kif18A in the bridging fiber was reduced less than PRC1 within 12 min of acute PRC1 removal , ~43% compared with ~85% , respectively .","This difference in the behavior of Kif18A and PRC1 is consistent with the fact that Kif18A is not known to be a binding partner of PRC1 .","Thus , we infer that the reduction of Kif18A in the bridge was a consequence of the fewer microtubules in the bridging fiber , and that the amount of Kif18A per microtubule remained largely unaffected by PRC1 removal .","Finally , MKLP1 , which is a binding partner of PRC1 ( Gruneberg et al . , 2006 ) , was reduced to a similar extent as PRC1 , ~87% and ~88% within 20 min of acute PRC1 removal , respectively .","Based on these results , we conclude that the localization of MKLP1 and a small pool of Kif4A in the bridging fiber are directly PRC1-dependent , whereas the localization of Kif18A is not .","We propose that both Kif4A and Kif18A regulate the length of bridging microtubules and their overlaps under normal conditions ( Figure 6C ) , as their depletion results in longer overlaps .","Intriguingly , we found that Kif4A signal in the bridging fibers decreased to a larger extent after acute than after long-term PRC1 depletion , whereas Kif18A signal decreased to a similar extent by the two methods of PRC1 depletion .","Moreover , Kif4A depletion by siRNA resulted in an increase of ~1 µm in overlap length , which was similar to the increase observed after acute PRC1 removal , whereas Kif18A depletion by siRNA led to a larger overlap increase .","Thus , we suggest that the observed overlap elongation after acute PRC1 removal is a consequence of the strong reduction of Kif4A in the bridging fibers .","In contrast , after long-term PRC1 depletion , we speculate that a larger fraction of Kif4A is present on the microtubules during overlap formation , contributing to the compensatory mechanism maintaining normal overlap length .","The proteins that we found in the bridging fiber , Kif4A , Kif18A , and MKLP1 , may also slide apart and/or stabilize the bridging microtubules ( Figure 6C ) .","In addition to these kinesins , we observed Eg5 in the bridging fiber ( Kajtez et al . , 2016; Mann and Wadsworth , 2018 ) .","During early anaphase , Kif4A and Eg5 drive the sliding of antiparallel microtubules that elongates the spindle ( Vukušić et al . , 2019 ) .","Thus , these kinesins may have a similar role during metaphase .","This possibility is in agreement with previous work showing that Kif4A depletion reduces microtubule poleward flux in metaphase ( Wandke et al . , 2012 ) .","Similarly , Kif18A in the bridging fiber may have microtubule sliding and crosslinking activities equivalent to those of the yeast kinesin-8 ( Su et al . , 2013 ) .","Furthermore , MKLP1 contributes to the stabilization of bridging microtubules in early anaphase ( Vukušić et al . , 2017 ) , and we suggest that it performs a similar function in metaphase together with other motors and crosslinkers including Eg5 and PRC1 .","The roles of these and other proteins within bridging fibers in the regulation of microtubule dynamics and sliding will be an intriguing topic for future studies .","The localization of Kif4A on chromosome arms , where it is involved in polar ejection forces ( Bieling et al . , 2010a; Brouhard and Hunt , 2005 ) was preserved after acute PRC1 removal .","Similarly , Kif18A , CLASP1 , and CENP-E remained on plus-ends of k-fibers .","However , as we cannot exclude potential subtle changes in the localization of these proteins or mislocalization of other proteins , the observed chromosome misalignment and overlap elongation after acute PRC1 removal could also be promoted by the changes of the dynamics of other microtubules within the spindle consequently affecting forces produced by k-fibers and polar ejection forces .","The changes upon acute PRC1 removal were not spatially uniform across the spindle .","The most affected part was the inner part of the spindle , where the PRC1 signal disappeared faster and the bridging microtubules became longer than on the periphery of the spindle .","The inner bridging fibers were more severely affected by PRC1 removal possibly because they are made up of fewer microtubules than the outer bridges .","Severely misaligned kinetochores that moved more than 2 µm away from the equatorial plane and lagging kinetochores occurred also more often in the inner part of the spindle .","This local effect is in line with weak mechanical coupling between neighboring k-fibers yet strong coupling between sister k-fibers ( Elting et al . , 2017; Suresh et al . , 2020; Vladimirou et al . , 2013 ) , and indicative of a mechanistic link between the bridging fiber geometry and kinetochore alignment .","In conclusion , we propose that overlap length-dependent forces help to position the chromosomes at the equatorial plane of the spindle .","The overlap length is regulated by the PRC1-dependent Kif4A , and by Kif18A within the bridging fiber .","Kif4A , Eg5 , and possibly other kinesins may slide bridging microtubules apart , whereas PRC1 together with the kinesins stabilizes the overlaps .","Thus , in addition to the forces generated at k-fiber tips and polar ejection forces , proper chromosome alignment requires forces generated within the bridging fiber , which are transferred to the k-fiber and rely on precise regulation of the overlap region ."],["Experiments were performed using: unlabeled human osteosarcoma U2OS cell line , U2OS cells expressing CENP-A-GFP , mCherry-α-tubulin and PA-GFP-tubulin and U2OS cell line stably expressing CENP-A-GFP , used in our previous work ( Vukušić et al . , 2017 ) , which were a gift from Marin Barišić and Helder Maiato ( Institute for Molecular Cell Biology , University of Porto , Portugal ) ; U2OS cell line stably expressing 2xGFP-EB3 and mCherry-CENP-A , a gift from Julie Welburn ( University of Edinburgh , United Kingdom ) ( Kajtez et al . , 2016 ) ; HeLa-Kyoto BAC lines stably expressing MKLP1-GFP , Kif4A-GFP , Kif18A-GFP , CENP-E-GFP and PRC1-GFP were a courtesy of Ina Poser and Tony Hyman ( MPI-CBG , Dresden , Germany ) ; unlabeled HeLa-TDS cells from the High-Throughput Technology Development Studio ( MPI-CBG , Dresden ) ; HeLa-TDS cells , permanently transfected with pEGFP-α-tubulin , used in our previous work ( Kajtez et al . , 2016 ) ; HeLa cells stably expressing YFP-tubulin , a courtesy of Lars Jansen ( University of Oxford , United Kingdom ) ; HeLa cells permanently transfected with EGFP-CLASP1 , which was a gift from Helder Maiato ( Institute for Molecular Cell Biology , University of Porto , Portugal ) .","Cells were grown in flasks in Dulbecco’s Modified Eagle’s Medium ( DMEM; Lonza , Basel , Switzerland ) with 1 g/L D-glucose , L-glutamine , and pyruvate , supplemented with 10% of heat-inactivated Fetal Bovine Serum ( FBS; Sigma Aldrich , St . Louis , MO , USA ) , 100 IU/mL penicillin and 100 mg/mL streptomycin solution ( Lonza ) .","For cell lines with stable expression of fluorescently labeled proteins , 50 µg/mL geneticin ( Life Technologies , Waltham , MA , USA ) was added .","Cells were kept at 37°C and 5% CO2 in a Galaxy 170 R humidified incubator ( Eppendorf , Hamburg , Germany ) .","All used cell lines were confirmed to be mycoplasma free by using MycoAlert Mycoplasma Detection Kit ( Lonza ) .","To make PRC1-tgRFPt-SspB WT ( opto-PRC1 ) plasmid , PRC1 fragment was amplified from His6-PRC1 plasmid ( RRID:Addgene_69111 ) ( Nixon et al . , 2015 ) using the primers GCTAGAATTGACCGGATGAGGAGAAGTGAGGTGCTG ( FWD ) and CATGGTGGCGACCGGTAAATTCGAAGCTTGAGCTCGAGATCTGAGGGACTGGATGTTGGTTGAATTGAGG ( REV ) and inserted into plasmid tgRFPt-SspB WT ( RRID:Addgene_60415 ) ( Guntas et al . , 2015 ) using AgeI restriction site .","This step was performed using commercially available In-Fusion HD Cloning Kit ( Clontech , Mountain View , CA , USA ) .","The produced plasmid expresses PRC1 tagged with tgRFPt and SspB at the C-terminus .","Plasmid iLID-CAAX was purchased ( RRID:Addgene_85680 ) ( O'Neill et al . , 2016 ) .","Plasmids pEGFP-C1Kif4a-sires and EGFP-Kif18A were a gift from Jason Stumpff ( University of Vermont , Burlington , VT , USA ) ( Stumpff et al . , 2008; Stumpff et al . , 2012 ) .","Plasmid GFP-CENP-E was a gift from Marin Barišić ( Danish Cancer Society Research Center , Copenhagen , Denmark ) .","Plasmid mRFP-CENP-B ( pMX234; RRID:Addgene_23006 ) was provided by Linda Wordeman ( University of Washington ) .","For depletion of endogenous PRC1 before opto experiments , cells were transfected 72 hr ( U2OS cells ) or 24 hr ( HeLa cells ) prior to imaging with 25 µL of 20 µM Accell PRC1 siRNA ( A-019491-15-0020 , Dharmacon , Lafayette , CO , USA ) targeting 3’ UTR of PRC1 mRNA .","A day prior to imaging , siRNA-treated cells were transfected with corresponding plasmids in following amounts: 0 . 3 μg of iLID-CAAX , 5 . 5 μg PRC1-tgRFPt-SspB-WT ( resistant to the used RNAi ) , 0 . 5 µg pEGFP-C1Kif4a-sires , 1 µg EGFP-Kif18A , 1 µg GFP-CENP-E , and 2 . 5 µg mRFP-CENP-B .","In HeLa BAC lines , 24 hr prior to imaging , mock experiment cells were transfected with 100 nM Accell Non-targeting Pool ( D-001910-10-05; Dharmacon ) , PRC1 siRNA treated with 100 nM Accell PRC1 siRNA ( Dharmacon ) , Kif4A siRNA treated with 100 nM Kif4A siRNA ( sc-60888; Santa Cruz Biotechnologies , Dallas , TX , USA ) , whereas Kif18A siRNA treated with 100 nM Silencer Select Validated Kif18A siRNA ( s37882; Ambion , Austin , TX , USA ) .","All transfections were performed using Nucleofector Kit R with the Nucleofector 2b Device ( Lonza ) using X-001 program for U2OS and O-005 ( high viability ) program for HeLa cells .","Following transfection , the cells were seeded on 35 mm glass coverslip uncoated dishes with 0 . 17 mm ( 1 . 5 coverglass ) glass thickness ( MatTek Corporation , Ashland , MA , USA ) in 1 . 5 mL DMEM medium with appropriate supplements .","To visualize microtubules , cells were stained with silicon rhodamine ( SiR ) -tubulin ( Lukinavičius et al . , 2014; Spirochrome AG , Stein am Rhein , Switzerland ) , a far-red tubulin dye , at a concentration of 50 nM 12–16 hr prior to imaging .","To prevent dye efflux , verapamil , a broad-spectrum efflux-pump inhibitor ( Spirochrome Ltd . ) , was added in U2OS cells at a concentration of 1 μM .","To visualize chromosomes and determine the phase of the mitosis , 20 min prior to imaging SiR-DNA ( Lukinavičius et al . , 2015; Spirochrome AG , Stein am Rhein , Switzerland ) was added to a final concentration of 150 nM .","For experiments on U2OS cells expressing 2xGFP-EB3 and mCherry-CENP-A , the cells were synchronized by adding 20 µM of the proteasome inhibitor MG-132 ( Sigma-Aldrich ) to arrest the cells in metaphase .","Imaging was started 15 min after adding MG-132 .","Cells were fixed with ice-cold methanol for 3 min , washed three times with phosphate buffer saline ( PBS ) , followed by 15 min permeabilization with 0 . 5% Triton in PBS .","Cells were washed three times with PBS and blocked in 1% Normal Goat Serum ( NGS ) except in experiment for intensity of opto-PRC1 where cells were blocked in BSA in PBS for 1 hr at 4°C .","Cells were washed three times with PBS and then incubated in primary antibody solution in blocking buffer over night at 4°C .","Following primary antibodies were used: mouse anti-PRC1 monoclonal antibody ( 1:100; C-1; sc-376983 , Santa Cruz Biotechnology ) , rabbit anti-α-tubulin polyclonal antibody ( 1:100; RRID:AB_10743646; SAB4500087; Sigma-Aldrich Corporation , St . Louis , MO , USA ) , mouse monoclonal anti-Kif4A antibody ( 1:100; RRID:AB_10707683; E-8; sc-365144; Santa Cruz Biotechnology ) , rabbit polyclonal anti-MKLP1 antibody ( 1:100; RRID:AB_631959; N-19; sc-867 , Santa Cruz Biotechnology ) , mouse monoclonal anti-Eg5 antibody ( 1:100; RRID:AB_10841907; A-1; sc-365681 , Santa Cruz Biotechnology ) , rabbit polyclonal anti-Kif18A antibody ( 1:100; RRID:AB_2296551; A301-080A; Bethyl ) , rabbit polyclonal anti-Kif4A antibody ( 1:200; RRID:AB_2280904; A301-074A; Bethyl ) .","After washing off primary antibodies with PBS , cells were incubated in a solution of secondary antibodies in 2% NGS or BSA in PBS for 1 hr at room temperature protected from light .","Following secondary antibodies were used: donkey anti-mouse IgG Alexa Fluor 488 ( 1:250; ab150109 , Abcam , Cambridge , UK ) , donkey anti-rabbit IgG Alexa Fluor 594 ( 1:250; ab150064 , Abcam ) , donkey anti-rabbit IgG Alexa Fluor 405 ( 1:250; RRID:AB_2715515; ab175649 , Abcam ) , and goat anti-mouse IgG Alexa Fluor 647 ( 1:250; RRID:AB_2811129; ab150119 , Abcam ) .","After washing off the secondary antibodies three times in PBS , cells were incubated with a solution of 4’ , 6-diamidino-2-phenylindole ( DAPI ) ( 1:1000 ) in PBS for 20 min and washed three times in PBS or SiR-DNA ( 150 nM ) in PBS for 15 min before imaging .","Note that we used immunocytochemistry for PRC1 rather than Western blot analysis because the efficiency of opto-PRC1 plasmid transfection is low , and as Western blot analysis provides information about the complete cell population , these results may not be relevant for the cells used in the optogenetic experiments .","In contrast , by using immunocytochemistry we analyzed only the cells with a similar opto-PRC1 level and in the same phase as those in our optogenetic experiments .","Immunocytochemistry imaging and live imaging of unlabeled U2OS , U2OS stably expressing CENPA-GFP , HeLa-TDS pEGFP-α-tubulin and HeLa BAC CENP-E-GFP cells was performed using Bruker Opterra Multipoint Scanning Confocal Microscope ( Bruker Nano Surfaces , Middleton , WI , USA ) , described previously ( Buđa et al . , 2017 ) .","In brief , the system was mounted on a Nikon Ti-E inverted microscope equipped with a Nikon CFI Plan Apo VC 100x/1 . 4 numerical aperture oil objective ( Nikon , Tokyo , Japan ) .","During imaging , live cells were maintained at 37°C using H301-K-frame heating chamber ( Okolab , Pozzuoli , NA , Italy ) .","In order to obtain the optimal balance between spatial resolution and signal-to-noise ratio , 60 µm pinhole aperture was used .","Opterra Dichroic and Barrier Filter Set 405/488/561/640 was used to separate the excitation light from the emitted fluorescence .","Following emission filters were used: BL HC 525/30 , BL HC 600/37 , and BL HC 673/11 ( all from Semrock , Rochester , NY , USA ) .","Images were captured with an Evolve 512 Delta Electron Multiplying Charge Coupled Device ( EMCCD ) Camera ( Photometrics , Tucson , AZ , USA ) using a 200 ms exposure time .","Electron multiplying gain was set on 400 .","Camera readout mode was 20 MHz .","No binning was performed .","The xy-pixel size in the image was 83 nm .","The system was controlled with the Prairie View Imaging Software ( Bruker ) .","For kinetics experiments on U2OS cells ( Figure 1 ) , 561 and 488 nm diode laser lines were used every 10 s with 200 ms exposure time .","In all other optogenetic experiments , stacks were acquired using sequentially the following diode laser lines: 561 nm ( to visualize opto-PRC1 ) , 488 nm ( to activate the optogenetic system and to visualize GFP ) , and 640 nm ( to visualize SiR-tubulin or SiR-DNA , when applicable ) , with time interval between z-stacks of 60 s and with 200 ms exposure time per laser line .","To prevent dissociation of PRC1 from the cell membrane between acquiring two consecutive z-stacks , only blue light was turned on for 200 ms every 10 s .","Cells were imaged this way for 20 min in order to achieve almost complete removal of PRC1 from the spindle , after which the blue light was turned off and imaging was continued for another 10 min at 60 s intervals .","The total imaging time of 30 min was chosen to be close to the typical metaphase duration of 29 . 7 ± 2 . 3 min , which was measured from the metaphase plate formation until anaphase onset in U2OS cells expressing CENP-A-GFP , mCherry-α-tubulin and PA-GFP-tubulin ( N = 187 ) imaged after nuclear envelope breakdown every minute by obtaining 15 z-slices with 0 . 5 µm spacing and 150 ms exposure time .","After 30 min of imaging , one z-stack in each of the three channels was taken in order to visualize the spindle and kinetochores after PRC1 return .","In all cells except HeLa cells expressing pEGFP-α-tubulin , three focal planes with spacing between adjacent planes of 1 µm were acquired .","Live imaging of HeLa cells stably expressing pEGFP-α-tubulin for measurements of tubulin intensities after acute PRC1 removal was performed in the same manner as described above for optogenetic experiments .","Additionally , before turning the blue light on every 10 s , one z-stack was acquired using 561 , 488 , and 640 nm diode laser lines with averaging 8 , and seven focal planes with spacing between adjacent planes of 0 . 5 µm .","Stack was taken in the same way after 20 min of exposure to the blue light and again 10 min after the blue light was switched off .","The same HeLa cells were used for measurements of tubulin intensities after long-term PRC1 removal and imaged by acquiring one z-stack using 561 , 488 , and 640 nm diode laser lines with averaging 8 , and seven focal planes with spacing between adjacent planes of 0 . 5 µm .","Imaging of HeLa BAC CENP-E-GFP mock and PRC1 siRNA-treated cells was performed by acquiring one z-stack of 3 focal planes with spacing between adjacent planes of 1 µm .","For imaging of immunostained cells , five focal planes with spacing between adjacent planes of 0 . 5 µm were acquired .","Live imaging of U2OS cells stably expressing 2x-GFP-EB3 and mCherry-CENP-A and U2OS cells with transient expression of opto-PRC1 , iLID-CAAX and GFP-Kif4A or EGFP-Kif18A was performed on a spinning disk confocal microscope system ( Dragonfly , Andor Technology , Belfast , UK ) using 63x/1 . 47 HC PL APO glycerol objective ( Leica ) and Zyla 4 . 2P scientific complementary metal oxide semiconductor ( sCMOS ) camera ( Andor Technologies ) .","During imaging cells were maintained at 37° and 5% CO2 within H301-T heating chamber ( Okolab , Pozzuoli , Italy ) .","Images were acquired using Fusion software ( v 2 . 2 . 0 . 38 ) .","For live imaging of U2OS cells expressing 2x-GFP-EB3 , mCherry-CENP-A and opto-PRC1 , 488 nm and 561 nm laser lines were used for excitation to visualize GFP , and mCherry and opto-PRC1 , respectively .","In order to achieve PRC1 removal from the spindle , 3 z-planes with a z-spacing of 1 µm were acquired sequentially with both laser lines , every 10 s with 200 ms exposure time for 10 min .","This imaging protocol was followed by five minutes of faster imaging , every 1 . 5 s with both laser lines on a central z-plane in order to visualize EB3 dynamics and to prevent the opto-PRC1 return .","Control , mock and PRC1 siRNA-treated cells were imaged with the same imaging protocol .","Live imaging of U2OS cells with transient expression of opto-PRC1 , iLID-CAAX and GFP-Kif4A or EGFP-Kif18A was performed in the similar manner as described above .","Additionally , to better visualize the localization of the Kif4A and Kif18A , before turning the blue light on every 10 s , one z-stack was acquired using 561 , 488 and 640 nm diode laser lines with frame averaging 4 , and three focal planes with spacing between adjacent planes of 1 µm .","Stack was taken in the same way after 12 min of exposure to the blue light and again 5 min after the blue light was switched off .","Note that the exposure to the blue light was shorter than in previous experiments regarding opto-PRC1 kinetics so that the majority of the cells remained in metaphase during the experiment .","Live imaging of unlabeled , BAC ( except CENP-E-GFP ) , YFP-tubulin , and EGFP-CLASP1 HeLa cell lines was performed on Leica TCS SP8 X laser scanning confocal microscope with a HC PL APO 63x/1 . 4 oil immersion objective ( Leica , Wetzlar , Germany ) heated with an objective integrated heater system ( Okolab , Burlingame , CA , USA ) .","During imaging , cells were maintained at 37°C in Okolab stage top heating chamber ( Okolab , Burlingame , CA , USA ) .","The system was controlled with the Leica Application Suite X software ( LASX , 1 . 8 . 1 . 13759 , Leica , Wetzlar , Germany ) .","For GFP- and YFP-labeled proteins , a 488 nm and 514 nm Argon laser was used , respectively , and for SiR-DNA or SiR-tubulin , a 652 nm white light laser was used .","For AF-647 , a 637 nm white light laser was used .","GFP , and SiR-DNA , SiR-tubulin or AF-647 emissions were detected with hybrid detector .","For mock , PRC1 siRNA , Kif4A siRNA , and Kif18A siRNA experiments , images were acquired at 1–3 focal planes with 1 μm spacing and 0 . 05 µm pixel size .","In optogenetic experiments 3 z-stacks with 1 μm spacing were acquired sequentially every 10 s in the same manner as in optogenetic experiments in U2OS cells .","One z-stack with line averaging of 6 or 16 was acquired before system activation , 20 min after exposure to blue light and 10 min after the light was switched off .","Since the cells were transiently transfected with opto-PRC1 , we observed variability in PRC1 expression levels and therefore we imaged and analyzed only those metaphase spindles with PRC1 localization consistent with endogenous and fluorescently labeled PRC1 ( Kajtez et al . , 2016; Polak et al . , 2017 ) .","Cells were not synchronized in order to avoid additional chemical treatment of cells , and metaphase was determined by alignment of kinetochores in the equatorial plane .","For determination of kinetics of PRC1 removal and return ( Figure 1C ) , intensity of opto-PRC1 was measured in each time frame on one focal plane .","We used Polygon selection tool in Fiji ( National Institutes of Health , Bethesda , MD , USA ) to encompass the area of the spindle , Aspindle , and measure mean spindle intensity , Mspindle .","Mean background intensity in the cytoplasm was measured using 2 . 5 × 2 . 5 µm rectangle , Mcyto .","Spindle intensity was background corrected by subtracting Mcyto from Mspindle to obtain Mspindle corr .","In order to calculate the sum of PRC1 intensity on the spindle , Mspindle corr was multiplied with Aspindle for each timeframe .","The background intensity outside of the cell was negligible , thus we did not take it into account .","Note that for the measurements of kinetic parameters in Figure 1C , four outliers were excluded ( see Figure 1—figure supplement 1F ) .","The percentage of PRC1 removal was calculated from the mean value of intensity of all cells at time 20 min , that is , the last time point of the continuous exposure to the blue light .","The percentage of return was calculated from the mean value of intensity of all cells in the interval 25–30 min , that is , during last 5 min of PRC1 return .","Intensity profiles of opto-PRC1 removal and return ( Figure 1D ) were obtained on sum intensity projections of all three z-planes by drawing a pole-to-pole line along the long axis of the spindle by using Line tool in Fiji .","The width of the line corresponded to the width of each individual spindle .","Intensities were normalized to position of the poles .","For quantification of PRC1 knock-down by siRNA and intensity level of opto-PRC1 ( in Figure 1—figure supplement 1A , B ) PRC1 intensity on fixed cells was measured on a sum-intensity projection of five focal planes by the procedure described above , in a way that mean spindle intensity was background corrected by subtracting mean intensity in the cytoplasm .","For measuring opto-PRC1 intensity on the spindle , cells where PRC1 was visible on astral microtubules were not analyzed , nor imaged in live experiments .","For quantification of PRC1 knock-down by siRNA in HeLa BAC MKLP1-GFP cell line , PRC1 intensity was quantified in the same manner as in U2OS cells .","Inter-kinetochore distance was measured using Line tool in Fiji on individual or maximum intensity z-projections of up to two z-planes as a distance between centers of sister kinetochore signals .","Peripheral kinetochores were defined as three outermost pairs on each side of the spindle with respect to spindle long axis , while the remaining were considered as central .","Measurement of inter-kinetochore distances in prometaphase was performed on U2OS cells expressing CENP-A-GFP , mCherry-α-tubulin and PA-GFP-tubulin , just after nuclear envelope breakdown in one imaging plane where both sister kinetochores could be clearly distinguished .","For measuring kinetochore alignment and orientation , as well as orientation and length of PRC1 bundles , Multipoint tool in Fiji was used .","A point was placed in the center of signal of each sister kinetochore or edges of PRC1 signal for each bundle .","Before measuring , images were rotated in order to achieve perpendicular direction of the equatorial plane with respect to x-axis .","The equatorial plane was defined with two points placed between outermost pairs of kinetochores on the opposite sides of the spindle .","For all measurements regarding kinetochores , those located at the spindle poles were not taken into account .","All measurements in opto and control cells were performed in three time-points: before the blue light was switched on , after 20 min of continuous exposure to the blue light , and 10 min after the blue light was turned off .","In untreated and PRC1 siRNA-treated cells measurements were performed at the beginning of imaging .","Kymographs of kinetochore oscillations were produced by Low Light Tracking Tool ( LLTT ) , an ImageJ plugin ( Krull et al . , 2014 ) .","Tracking of kinetochores in x , y plane was performed on maximum intensity of 2–3 z-planes .","Sigma value ( standard deviation of the Gaussian used to approximate the Point Spread Function ( PSF ) of the tracked objects ) was set to Automatic .","EB3 spots in U2OS cells stably expressing 2x-GFP-EB3 and mCherry-CENP-A were tracked by obtaining their xy coordinates using Multipoint tool in Fiji from the frame when a spot appeared until it disappeared or was no longer clearly distinguishable from its neighbors .","Only spots belonging to bridging fibers were traced , which were defined as those passing between sister kinetochores or moving along PRC1 streaks .","Half-overlap length was measured as the distance between the last location where a tracked EB3 spot was visible and the spindle equator .","The number of spots in time was obtained by visually inspecting time frames where individual kinetochore pair or PRC1 bundle was visible , and dividing the total number of the observed EB3 spots in the bridging fibers by the total time of observation of individual kinetochore pairs or PRC1 bundles .","Kymographs were generated using KymographBuilder plugin in Fiji , and half-overlap length was measured from kymographs as the distance between a spot's trajectory end point and the mid-line between the poles corresponding to the equatorial plane .","These measurements were performed in the first 2 min of a fast imaging sequence , which followed after the 10 min imaging protocol required for PRC1 removal .","The tubulin-GFP signal intensity of a cross-section of a bridging fiber was measured by drawing a 3-pixel-thick line between and perpendicular to the tubulin signal joining the tips of sister k-fibers .","The same method was used in cells containing a kinetochore marker , that is , the profile intensity of the bridging fiber was extracted by looking at the tubulin channel and drawing the line between tips of k-fibers .","For confirmation , we subsequently checked the kinetochore channel , and observed that this line was always placed between sister kinetochores .","This validates our measurements of bridging fibers in opto cells using tubulin-GFP only .","The tubulin intensity profile was corrected by subtracting mean background signal present in the cytoplasm ( see Figure 4A and Figure 4—figure supplement 1C , G ) .","The signal intensity of the bridging fiber was calculated as the area under the peak using SciDavis ( Free Software Foundation Inc , Boston , MA , USA ) ( Figure 4E ) .","The signal intensity in the region lateral from k-fiber tip was measured in a similar manner , 0 . 882 ± 0 . 04 µm away from the k-fiber tip ( Figure 4—figure supplement 1C , G ) .","This intensity corresponds to the sum of the bridging and k-fiber , given that PRC1 overlap half-length is longer and EB3 spots pass further than the position where the intensity profiles were measured .","Therefore , the intensity of the k-fiber was calculated by subtracting the bridging fiber intensity from the corresponding sum of bridging and k-fiber intensity .","The profile intensity of bridging fiber and at the position lateral from k-fiber tip in the cells stained with SiR-tubulin ( Figure 4B , C ) was performed in the same manner .","All the measurements were performed on a single z-plane .","Note that mostly outermost fibers were used for these measurements because of being most easily distinguished from neighboring fibers .","All measurements in opto and control cells were performed at three time-points: before the blue light was switched on , after 20 min of continuous exposure to the blue light , and 10 min after the blue light was turned off .","Shapes of the spindle were quantified by tracking outermost k-fiber contours in the central z-slice of the spindle , or maximum intensity z-projection of two central z-slices .","All spindles were rotated to have horizontal long axis .","Pole-to-k-fiber-tip tracking was done using Multipoint tool by placing five roughly equidistant points along contour length , first point being at the pole and the last point being at the k-fiber tip .","First point of each contour was translated to ( 0 , 0 ) .","This was done for maximum of 4 trackable outermost k-fibers in the spindle .","Curvature of the contour was calculated by fitting a circle to the contour points of individual k-fibers and retrieving reciprocal value of its radius .","To test the effect of tracking errors on curvature , we introduced 1-pixel noise to the x and y values of tracked points , which did not change the result .","Angle between outermost k-fibers ( θ ) was calculated as the angle between lines passing through last two points along the contour of sister k-fibers .","Spindle length and width were measured using Line tool in Fiji .","For length measurements , a line was drawn from pole to pole .","The position of the pole was defined as the location of the strongest tubulin signal .","Width was measured by drawing a line between outermost kinetochore pairs on the opposite sides of the spindle , perpendicular to the spindle long axis .","To avoid the signal of Kif4A at the chromosomes , Kif4A bridging fiber intensity was measured on individual z-planes with 1 × 0 . 5 µm rectangles at the positions lateral from sister kinetochores and chromosomes , where no DNA ( DAPI or SiR-DNA ) was found , yet lateral parts of PRC1 streaks are present .","This approach was applied in cases when PRC1 was not depleted or relocated to the cell membrane , that is , in opto cells before acute PRC1 removal and untreated cells .","When PRC1 was not present on the spindle , that is , in opto cells after acute PRC1 removal and in PRC1 siRNA-treated cells , we placed rectangles at positions lateral from kinetochores and chromosomes , where antiparallel zones are thought to be positioned , considering their location before removal .","The rectangles were placed parallel to the overlap .","Given that bridging fiber intensity was measured mostly in the inner parts of the spindle , mean values from the spindle poles were considered as the Kif4A background and subtracted from Kif4A intensity retrieved from the positions of bridging fibers .","For the measurements of Kif4A in the bridging fiber , we chose only cells in which Kif4A showed previously known localization on chromosomes and at comparable levels .","This was important as immunostaining protocol sometimes resulted in cells with no or low chromosome staining , yet high spindle staining , and HeLa BAC Kif4A-GFP cells vary in expression levels among cells .","The intensity of Kif4A on chromosome arms in metaphase was measured in Kif4A channel on sum-intensity projections of all z-planes ( nine planes in immunostained , and three in opto and BAC cells ) using Polygon selection tool in Fiji by encompassing chromosomes in DAPI or SiR-DNA channel .","In order to avoid the Kif4A signal on the spindle microtubules , the signal was measured only on the parts of the chromosome arms protruding into cytoplasm away from the spindle .","For each cell , mean value of Kif4A arm intensity was calculated and corrected for background cytoplasm intensity measured in 2 . 5 × 2 . 5 µm rectangle by subtracting it from mean intensity of Kif4A .","In immunostained cells only those with Kif4A localized on chromosomes were taken into account as in some sessions the Kif4A signal was very strong on the whole spindle and not present on the chromosomes both in untreated and PRC1 siRNA-treated cells .","The intensity of GFP-Kif4A on chromosomes in anaphase was measured in GFP-Kif4A channel on sum-intensity projections of all three z-planes using Polygon selection tool in Fiji by encompassing chromosomes in SiR-DNA channel .","Background cytoplasm intensities in GFP-Kif4A channel were measured in 2 . 5 × 2 . 5 µm rectangle and subtracted from measured mean intensities of GFP-Kif4A .","Corrected intensities were divided by the number of focal planes .","In anaphase , measurements were performed 4 min after anaphase onset .","Anaphase onset was defined as the timeframe when separation of majority of sister chromatids in SiR-DNA channel occurred .","The mean bridging fiber intensities of Kif18A in all treatments were obtained on a single z-plane using 0 . 4 × 0 . 4 µm rectangles in Fiji covering the Kif18A signal in the bridge .","The rectangles of the same dimension were used to obtain the Kif18A signal from the sister k-fiber tips and the mean value for the pair was calculated .","Since the measurements of Kif18A were mostly obtained on the outermost fibers , these values were corrected by subtracting mean background cytoplasm intensity measured in a 2 . 5 × 2 . 5 µm rectangle .","The intensity of MKLP1 in the bridging fibers was measured in GFP channel on a single z-plane using 0 . 4 × 0 . 4 µm rectangle in Fiji by placing it on each bridging fiber visible in the SiR-tubulin channel .","These intensities were corrected for background GFP intensity in cytoplasm measured in 2 . 5 × 2 . 5 µm rectangle .","For quantification of PRC1 knock-down by siRNA in HeLa BAC MKLP1-GFP cell line , PRC1 intensity was quantified in the same manner as in U2OS cells: on a sum-intensity projection of five focal planes in a way that mean spindle intensity was background corrected by subtracting mean intensity in the cytoplasm , .","The intensity of CENP-E and CLASP1 on plus ends-of k-fibers was measured using 0 . 4 × 0 . 4 µm rectangle encompassing plus-end of k-fibers on a single z-plane .","Intensities were corrected for the intensity in cytoplasm measured using 2 . 5 × 2 . 5 µm rectangle in the central z-plane as this intensity was similar between different z-planes .","For analysis of CLASP1 and CENP-E intensity on plus-ends of k-fibers only cells with similar intensities on the spindle within each treatment were taken into account as there was a variation in expression level among cells .","Measurements regarding Kif4A , Kif18A , MKLP1 , CENP-E , and CLASP1 were performed in two time-points in metaphase: before the blue light was switched on and at the end of continuous exposure to the blue light .","The intensities were normalized on the mean value of control at each treatment , that is , before the blue light was switched on for acute PRC1 removal and untreated for long-term depletion .","Quantification of the signal length of PRC1-GFP in mock , Kif4A , and Kif18A siRNA-treated HeLa cells was performed by drawing a 5-pixel-thick pole-to-pole line along individual bundles and calculated as the width of the peak of the PRC1-GFP signal intensity .","Statistical analysis was performed in MATLAB ( RRID:SCR_001622; MathWorks , Natick , MA , USA ) and RStudio ( RRID:SCR_000432; R Foundation for Statistical Computing , Vienna , Austria ) .","Normality of the data was inspected by Q-Q plots and Shapiro-Wilk test of normality .","Two groups with normally distributed data were tested with two-tailed t-test , while more than two groups were tested with one-way ANOVA followed by Tukey HSD post hoc test .","Two groups with non-normally distributed data were tested with Mann-Whitney test , while more than two groups were tested with Kruskal-Wallis rank sum test followed by pairwise Wilcoxon rank sum test .","Used statistical analysis is noted in figure captions .","Proportions were statistically compared with test for equality of proportions , two-proportions z-test .","For data with expected count smaller than 5 , Yates's correction for continuity was used .","Graphs were generated in MATLAB ( MathWorks ) and RStudio ( R Foundation for Statistical Computing ) .","ImageJ ( RRID:SCR_003070; National Institutes of Health , Bethesda , MD , USA ) was used to crop and rotate images , and to adjust brightness and contrast to the entire image , which was applied equally to all images in the same panel .","The images are rotated in order for the spindle to be horizontal in every time frame .","To remove high-frequency noise in displayed images a Gaussian blur filter with a 0 . 5-pixel sigma ( radius ) was applied where stated .","Figures were assembled in Adobe Illustrator CS5 ( RRID:SCR_010279; Adobe Systems , Mountain View , CA , USA ) ."]],"string":"[\n [\n \"Pre-anaphase chromosome movements culminate with chromosome alignment at the spindle equator , a distinctive feature of mitosis important for the synchronous anaphase poleward movement of chromatids and proper telophase nuclear reformation ( Fonseca et al . , 2019; Maiato et al . , 2017 ) .\",\n \"Chromosome congression to the metaphase plate , a process of directed chromosome movement from the polar regions toward the spindle equator , has been explored extensively ( Barisic et al . , 2014; Cai et al . , 2009; Kapoor et al . , 2006; Maiato et al . , 2017 ) .\",\n \"Yet , the maintenance of chromosome alignment at the spindle equator is less understood .\",\n \"This is a dynamic process given that the chromosomes constantly make small oscillatory excursions across the equator ( Skibbens et al . , 1993 ) .\",\n \"Two mechanisms have been proposed to underlie the maintenance of chromosome alignment at the equator , namely length-dependent dynamics of kinetochore fiber ( k-fiber ) microtubules and polar ejection forces .\",\n \"Pulling forces exerted by k-fiber tips on kinetochores are thought to be regulated by kinesin-8 motor proteins , which are needed for proper kinetochore alignment in various organisms from yeast to humans ( Garcia et al . , 2002; Stumpff et al . , 2008; Wargacki et al . , 2010 ) .\",\n \"These motors can ‘measure’ microtubule length because they bind to microtubules along their length and move toward the plus end , leading to more kinesin-8 accumulated at the plus end of longer microtubules , where they promote microtubule catastrophe , that is , a switch from growth to shrinkage ( Tischer et al . , 2009; Varga et al . , 2006 ) .\",\n \"During kinetochore movements across the spindle equator , the leading k-fiber shrinks , while the trailing one grows and accumulates kinesin-8 , resulting in microtubule catastrophe , followed by the movement of the trailing kinetochore back toward the equator .\",\n \"Although this mechanism can explain kinetochore alignment at the spindle equator ( Klemm et al . , 2018 ) , the switching dynamics characteristic for this model , where the trailing kinetochore initiates the change of direction of motion , differs from the observations in mammalian cells where the leading kinetochore typically changes the direction before the trailing one ( Armond et al . , 2015; Civelekoglu-Scholey et al . , 2013; Wan et al . , 2012 ) .\",\n \"In addition to the forces produced by k-fibers , polar ejection forces push chromosome arms away from the pole , powered by arm-bound chromokinesins that walk toward the plus end of microtubules ( Bajer et al . , 1982; Brouhard and Hunt , 2005 ) .\",\n \"Because microtubule density increases toward the pole , these forces help the chromosomes to stay away from the poles , but most likely have little effect on kinetochore movements close to the spindle equator ( Cane et al . , 2013; Ke et al . , 2009 ) .\",\n \"Thus , the current models do not provide a complete picture of kinetochore alignment at the spindle center .\",\n \"K-fibers are surrounded by a dense network of spindle microtubules with which they have multiple interactions ( McDonald et al . , 1992; O'Toole et al . , 2020 ) , resulting in forces acting on k-fibers and thus also on kinetochores .\",\n \"In particular , each pair of sister k-fibers is tightly linked by the bridging fiber , a bundle of antiparallel microtubules that balances the tension on sister kinetochores ( Kajtez et al . , 2016; Polak et al . , 2017; Vukušić et al . , 2017 ) .\",\n \"However , the role of forces exerted by bridging fiber in chromosome alignment at the metaphase plate is unknown .\",\n \"In metaphase , overlap regions within bridging fibers are crosslinked by protein regulator of cytokinesis 1 ( PRC1 ) ( Kajtez et al . , 2016; Polak et al . , 2017; Tolić , 2018 ) .\",\n \"PRC1 , like other non-motor microtubule-associated proteins from Ase1/PRC1/MAP65 family , selectively bundles antiparallel microtubules and provides stable overlaps in vitro ( Bieling et al . , 2010b; Janson et al . , 2007; Mollinari et al . , 2002; Subramanian et al . , 2010 ) .\",\n \"Cellular studies of its function show that PRC1 is associated with the spindle midzone in anaphase , where its activity is essential for stable microtubule organization , localization of numerous microtubule-associated proteins within this structure , and successful completion of cytokinesis , while its microtubule-binding and -bundling affinities are regulated by phosphorylation and dephosphorylation events ( Gruneberg et al . , 2006; Jiang et al . , 1998; Kurasawa et al . , 2004; Liu et al . , 2009; Mollinari et al . , 2002; Mollinari et al . , 2005; Neef et al . , 2007; Subramanian et al . , 2013; Subramanian et al . , 2010; Zhu and Jiang , 2005; Zhu et al . , 2006 ) .\",\n \"In this work , we developed an optogenetic approach for acute and reversible removal of PRC1 from the spindle to the cell membrane , building upon ideas of dimerization or dissociation induced chemically ( Cheeseman et al . , 2013; Haruki et al . , 2008; Robinson et al . , 2010; Wordeman et al . , 2016 ) or by light ( Fielmich et al . , 2018; Guntas et al . , 2015; Okumura et al . , 2018; van Haren et al . , 2018; Yang et al . , 2013; Zhang et al . , 2017 ) to rapidly redistribute proteins .\",\n \"By using our assay on metaphase spindles , we found that bridging fibers promote kinetochore alignment .\",\n \"PRC1 removal resulted in partial disassembly of bridging fibers and elongation of their overlap zones .\",\n \"Moreover , the metaphase plate widened , inter-kinetochore distance decreased , and lagging chromosomes appeared more frequently , showing that PRC1 indirectly regulates forces acting on kinetochores .\",\n \"Kif4A/kinesin-4 and Kif18A/kinesin-8 were found to localize in the bridging fiber during metaphase and were largely lost upon PRC1 removal , with Kif4A showing a greater reduction .\",\n \"These results , together with the finding that Kif4A or Kif18A depletion by siRNA led to elongated overlaps , suggest that these proteins regulate the overlap length of bridging microtubules .\",\n \"PRC1 removal did not affect the localization of Kif4A on the chromosomes and Kif18A , CLASP1 , and CENP-E/kinesin-7 on the plus ends of k-fibers , arguing against perturbed polar ejection forces or molecular events at the kinetochore microtubule plus ends as origins of the observed kinetochore misalignment .\",\n \"In conclusion , our optogenetic experiments show that bridging microtubules buffer chromosome movements , thus promoting their alignment .\",\n \"We propose that this occurs via length-dependent forces , which depend on the antiparallel overlap length within the bridging fiber .\"\n ],\n [\n \"To study the role of PRC1 and the forces arising from coupling of bridging and k-fibers in chromosome alignment , we developed an optogenetic tool for fast and reversible removal of PRC1 from the spindle to the cell membrane , based on the previously designed improved light inducible dimer ( iLID ) system ( Guntas et al . , 2015 ) .\",\n \"We attached PRC1 to the red fluorescent protein tgRFPt and the bacterial protein SspB , while the iLID , which contains the bacterial peptide SsrA and the light-oxygen-voltage ( LOV2 ) domain , is bound to the cell membrane by a short peptide , CAAX .\",\n \"In this system , LOV2 adopts a conformation that allows dimerization of SsrA and SspB upon exposure to the blue light ( Figure 1A ) .\",\n \"After cessation of exposure to the blue light , LOV2 adopts its initial conformation leading to decreased affinity of SsrA to SspB .\",\n \"Therefore , exposure to the blue light should induce translocation of PRC1 from the central region of the metaphase spindle , which we will refer to as the spindle midzone , to the cell membrane , whereas cessation of exposure to blue light should restore PRC1 localization on the spindle ( Figure 1A ) .\",\n \"To test our optogenetic approach , we used U2OS cells with stable expression of CENP-A-GFP , transient expression of PRC1-tgRFPt-SspB ( henceforth opto-PRC1 ) and iLID-CAAX ( henceforth opto cells; Figure 1B; Figure 1—video 1 ) .\",\n \"Endogenous PRC1 was depleted 90 ± 2% ( all results are mean ± s . e . m . ) by siRNA before addition of opto-PRC1 ( Figure 1—figure supplement 1A ) .\",\n \"Before exposure to the blue light , opto-PRC1 had normal localization on the microtubule bundles in the spindle midzone ( Figure 1B; 0:00 min ) , with the length of PRC1 streaks of 3 . 77 ± 0 . 08 µm ( n = 193 bundles , N = 30 cells ) , consistent with that of endogenous and fluorescently labeled PRC1 in metaphase ( Kajtez et al . , 2016; Polak et al . , 2017 ) , although the total signal intensity of opto-PRC1 on the spindle was higher compared to endogenous PRC1 ( Figure 1—figure supplement 1B ) .\",\n \"Addition of opto-PRC1 did not change the duration of metaphase , as inferred from the fraction of cells that entered anaphase during image acquisition , which was similar in cells with endogenous PRC1 and cells treated with PRC1 siRNA and containing opto-PRC1 ( 79 ± 6% , N = 37 , and 71 ± 5% , N = 72 , respectively; p = 0 . 4 , Pearson’s Chi-squared test; Figure 1—figure supplement 1C ) .\",\n \"After exposure to the blue light , opto cells were able to progress to cytokinesis ( Figure 1—figure supplement 1D ) .\",\n \"Taken together , these data suggest that opto-PRC1 replaces the depleted endogenous PRC1 .\",\n \"Upon exposure to the blue light , opto-PRC1 signal on the spindle decreased and its signal on the membrane increased ( Figure 1B; 0:20-20:00 min ) .\",\n \"After the blue light was switched off , opto-PRC1 returned to the spindle midzone ( Figure 1B; 20:40-30:10 min ) .\",\n \"To validate our system , we performed two sets of test experiments .\",\n \"The first one was performed on the same cell line containing opto-PRC1 , but without iLID , imaged with the same imaging protocol as the opto cells , which we refer to as control throughout the paper .\",\n \"The second test experiment was on the opto cells but without the blue light ( Figure 1—figure supplement 1E ) .\",\n \"In both cases , opto-PRC1 remained on the spindle ( Figure 1—figure supplement 1E ) .\",\n \"Thus , our optogenetic approach allows for acute and reversible control of PRC1 localization in metaphase .\",\n \"To quantify the dynamics and spatial pattern of opto-PRC1 removal and return , we measured the intensity of opto-PRC1 on the metaphase spindle ( Figure 1—figure supplement 1F , G ) .\",\n \"We found that 88 ± 3% of opto-PRC1 was removed after 20 min of exposure to the blue light with a half-time of 4 . 2 ± 0 . 1 min ( Figure 1C ) .\",\n \"During the opto-PRC1 removal , there was simultaneous decrease in both signal intensity and length of the overlap region ( Figure 1D , left; Figure 1—figure supplement 1G , top ) , which may be due to fewer antiparallel regions being positioned laterally than in the central part of the bundle ( Mastronarde et al . , 1993 ) .\",\n \"The signal of the outermost midzone bundles typically lasted longer than of the inner ones ( Figure 1B; 3:40-6:30 ) .\",\n \"After the blue light was switched off , opto-PRC1 signal restored to 65 ± 1% of the initial intensity within 10 min , with return half-time being 0 . 71 ± 0 . 04 min ( Figure 1C ) .\",\n \"The faster PRC1 return to the spindle in comparison with its removal may be due to the higher affinity difference between PRC1 binding to the spindle and to the membrane in the dark than under light .\",\n \"During the opto-PRC1 return , it initially localized throughout the spindle , with gradual increase in intensity in the spindle midzone ( Figure 1D , right; Figure 1—figure supplement 1G , bottom ) , suggesting that PRC1 has higher unbinding rate outside than within the overlap bundles in the spindle .\",\n \"This result is consistent with PRC1 having a life-time of several seconds on single microtubules and a 10-fold preference for overlap regions in vitro ( Subramanian et al . , 2010 ) .\",\n \"To test whether bridging fiber has a role in the maintenance of chromosome alignment , acute optogenetic removal of PRC1 , a depletion of which is known to perturb bridging fibers ( Polak et al . , 2017 ) should affect chromosome positioning at the spindle equator ( Figure 2A ) .\",\n \"Surprisingly , we observed that the acute removal of opto-PRC1 resulted in movement of sister kinetochore pairs away from the metaphase plate ( Figure 2B; Figure 2—video 1 ) , which is not found after long-term PRC1 depletion by siRNA ( Polak et al . , 2017 ) .\",\n \"Upon opto-PRC1 removal , the distances of sister kinetochore midpoints from the equatorial plane increased ( dEQ , Figure 2B , C , D; Figure 2—figure supplement 1A , B ) .\",\n \"While >95% of kinetochore pairs were found within the region of PRC1 streaks , that is , less than 2 µm away from the equatorial plane before removal of opto-PRC1 , 9 . 3 ± 2 . 7% of kinetochore pairs made excursions far outside this region after opto-PRC1 removal ( Figure 2—figure supplement 1B ) .\",\n \"The displaced kinetochores were found more often in the inner part of the spindle , that is , close to the long spindle axis , than in the outer regions ( Figure 2E ) .\",\n \"Kinetochores fluctuated to a similar extent in the presence and absence of opto-PRC1 , but in its absence the displaced kinetochores fluctuated within a region that was offset from the equatorial plane ( Figure 2—figure supplement 1C ) .\",\n \"These displaced kinetochores upon opto-PRC1 removal had lower inter-kinetochore distance in comparison to non-displaced ones , suggesting that kinetochore displacement was related to a more severe reduction of tension ( Figure 2F ) .\",\n \"On average , kinetochores remained displaced even after opto-PRC1 return ( Figure 2D ) .\",\n \"We conclude that PRC1 has a role in keeping kinetochores in tight alignment on the metaphase plate .\",\n \"The mean inter-kinetochore distance ( dKC , Figure 2C ) was reduced when opto-PRC1 was removed ( Figure 2G; Figure 2—figure supplement 1D ) , and the distance after 20 min of exposure to the blue light ( 0 . 79 ± 0 . 01 µm ) was closer to metaphase ( 0 . 87 ± 0 . 01 µm ) than prometaphase ( 0 . 66 ± 0 . 01 µm ) values ( see Materials and methods ) , suggesting that tension was not completely lost and that these changes were not due to kinetochore detachment from k-fibers ( Figure 2—figure supplement 1D ) .\",\n \"In agreement with this , the fraction of cells that entered anaphase during image acquisition was similar in control and opto cells ( 71 ± 5% , N = 72 , and 60 ± 5% , N = 93 , respectively; p = 0 . 1 , Pearson’s Chi-squared test; Figure 1—figure supplement 1C ) , indicating that PRC1 removal did not prevent spindle assembly checkpoint satisfaction .\",\n \"After opto-PRC1 return to the spindle , inter-kinetochore distance increased , implying restoration of tension , although not to the original value ( Figure 2G ) .\",\n \"To investigate the influence of acute PRC1 removal on the orientation of sister kinetochores , we measured the angle between sister kinetochore axis and long spindle axis ( αKC , Figure 2C ) .\",\n \"We observed that removal of opto-PRC1 caused misorientation of sister kinetochores , that is , increased αKC ( Figure 2B , H; Figure 2—figure supplement 1A , E ) .\",\n \"Misoriented kinetochores were found at a larger distance from the equatorial plane and closer to the long spindle axis ( Figure 2—figure supplement 1F ) .\",\n \"Interestingly , sister kinetochore pairs remained misoriented even after opto-PRC1 return ( Figure 2H ) .\",\n \"Similarly , PRC1 bundles were misoriented upon PRC1 return ( see Materials and methods , Figure 2—figure supplement 1G ) .\",\n \"These results suggest that when PRC1 returns to the overlaps whose geometry was perturbed by PRC1 removal , it likely confines the chromosomes in new positions and orientations .\",\n \"The observed effects of PRC1 removal on inter-kinetochore distance , kinetochore alignment and orientation did not change when SiR-tubulin was added ( dKC p = 0 . 82 , dEQ p = 0 . 27 , αKC p = 0 . 61 , respectively; t-test ) .\",\n \"The effects of PRC1 removal were found neither in control experiments in cells without iLID , which were stained with SiR-tubulin , nor in a different set of control experiments where cells expressing only CENP-A-GFP without SiR-tubulin were imaged with the same illumination protocol ( Figure 2D , G , H; Figure 2—figure supplement 1A , B , D , E ) .\",\n \"Therefore , the observed effects in opto cells were not a consequence of SiR-tubulin or laser photodamage ( Douthwright and Sluder , 2017 ) .\",\n \"As previous reports have shown that acute rapamycin-dependent protein translocation and long-term depletion by siRNA can yield different and even opposite phenotypes ( Cheeseman et al . , 2013; Wordeman et al . , 2016 ) , we compared kinetochore parameters after acute removal of PRC1 with those obtained from cells after long-term depletion of PRC1 by siRNA .\",\n \"Strikingly , in contrast to acute removal , the long-term depletion did not cause kinetochore misalignment or misorientation ( Figure 2D , H; Figure 2—figure supplement 1H , I; Table 1 ) .\",\n \"The two methods decreased the inter-kinetochore distance to a similar extent ( Figure 2G; Figure 2—figure supplement 1J; Table 1 ) , even though unlike acute removal , long-term removal reduced the fraction of cells that entered anaphase ( 35 ± 8% , N = 37 , and 79 ± 6% , N = 37 , for PRC1 siRNA treated and untreated , respectively; p = 0 . 046 , Pearson’s Chi-squared test; Figure 1—figure supplement 1C ) .\",\n \"Thus , acute removal of PRC1 results in different effects in comparison with a long-term depletion .\",\n \"To test to what extent the acute removal of PRC1 during metaphase affects chromosome segregation , we measured the frequency of lagging kinetochores .\",\n \"We found that lagging kinetochores occurred more frequently when opto-PRC1 was being removed than in control cells ( Figure 2I , J; Figure 2—video 2 ) .\",\n \"Similarly , long-term depletion of PRC1 by siRNA also increased the frequency of lagging kinetochores during early anaphase ( Figure 2—figure supplement 2A; Table 1 ) .\",\n \"Opto cells that showed lagging kinetochores in anaphase had a slightly smaller inter-kinetochore distance before anaphase than opto cells without lagging kinetochores ( Figure 2—figure supplement 2B ) , suggesting that a decrease in tension may be involved in the imperfect kinetochore segregation .\",\n \"The cells with lagging kinetochores did not have a larger average kinetochore misalignment in metaphase ( Figure 2—figure supplement 2C ) , which indicates that misalignment and lagging kinetochores are not linked on the cell level , although they may be linked locally on individual kinetochores .\",\n \"As perturbation of the PRC1-CLASP1 interaction and the consequent absence of CLASP1 from the spindle midzone results in lagging chromosomes ( Liu et al . , 2009 ) , we inspected the localization of CLASP1 and found that it did not accumulate between segregating chromosomes in opto HeLa cells stably expressing EGFP-CLASP1 ( Figure 2—figure supplement 2D ) .\",\n \"Thus , the observed higher occurrence of lagging kinetochores could be attributed to changes in tension during metaphase , perturbed recruitment of CLASP1 to the spindle midzone by PRC1 during early anaphase , or a combination of both effects .\",\n \"Factors that could contribute to the altered chromosome alignment and occurrence of lagging kinetochores upon opto-PRC1 removal are changes related to ( 1 ) microtubules in the bridging fibers , ( 2 ) polar ejection forces , and/or ( 3 ) proteins that modulate the dynamics of k-fiber plus-ends ( Figure 2K ) .\",\n \"Because PRC1 crosslinks microtubules within bridging fibers ( Kajtez et al . , 2016; Polak et al . , 2017 ) , we first tested the effects of PRC1 removal on those microtubules .\",\n \"An important aspect of the bridging fiber that may affect chromosome alignment is the dynamics of microtubules that make up these fibers .\",\n \"To explore their dynamics , we developed an assay to track the growing plus ends of individual microtubules in the bridging fiber by using cells expressing the plus end marker EB3 ( Stepanova et al . , 2003 ) tagged with GFP ( Figure 3 ) .\",\n \"We followed single EB3 spots in the spindle and identified the ones belonging to a bridging fiber as the spots that move toward a kinetochore , cross the region between this kinetochore and its sister , and move beyond it toward the other spindle pole ( Figure 3A–C; Figure 3—video 1 ) .\",\n \"We found 1 . 8 ± 0 . 2 EB3 spots per minute per bridging fiber in control cells , showing that bridging fibers are dynamically remodeled during metaphase ( Figure 3D ) .\",\n \"This number was similar after opto-PRC1 removal , 2 . 0 ± 0 . 3 spots/min ( p = 0 . 59; t-test , Figure 3D ) , suggesting that the number of dynamic microtubules in the bridge is largely unaffected by acute PRC1 removal .\",\n \"However , in PRC1 siRNA-treated cells fewer EB3 spots were observed to move to opposite spindle half , 1 . 5 ± 0 . 1 EB3 spots/min , compared to untreated cells , where 1 . 91 ± 0 . 09 spots/min were observed ( p = 0 . 01; t-test , Figure 3—figure supplement 1A ) , indicating that long-term PRC1 depletion slightly decreases the number of dynamic microtubules in the bridge .\",\n \"To assess the changes in the dynamics of bridging microtubules , we followed EB3 spots in the bridge from the time when they can be distinguished from neighboring spots near the pole until they disappear in the opposite spindle half , which we interpret as the moment when the microtubule stops growing ( Maurer et al . , 2012 ) .\",\n \"Interestingly , EB3 tracks were longer after opto-PRC1 removal than in control cells ( Figure 3B , C; Figure 3—video 1 ) , but the velocities of the EB3 spots were not affected by opto-PRC1 removal or long-term depletion when compared to untreated cells ( Figure 3E; Figure 3—figure supplement 1B ) .\",\n \"In agreement with these results , kymographs of the central region of the spindle show that EB3 spots reach deeper into the opposite half of the spindle after opto-PRC1 removal ( Figure 3F; Figure 3—figure supplement 1C ) .\",\n \"Thus , acute removal of PRC1 results in longer bridging microtubules , which is not a consequence of altered microtubule growth rate , but most likely due to a reduced microtubule catastrophe rate .\",\n \"EB3 tracks allowed us to estimate the length of the overlap zone of antiparallel microtubules in the bridging fiber , which currently cannot be measured after PRC1 removal because PRC1 itself is the only available marker of antiparallel overlaps in the spindle .\",\n \"We define the overlap half-length as the distance beyond the equatorial plane that an EB3 spot covers while moving within the bridging fiber from one spindle half into the other ( Figure 3G , left ) , where this microtubule can form antiparallel overlaps with the oppositely oriented microtubules extending from the other pole .\",\n \"In control cell , the overlap half-length was 1 . 8 ± 0 . 2 µm ( n = 17 spots in N = 9 cells; Figure 3G , right ) , which corresponds well to the overlap half-length measured as the half-length of PRC1 streaks in this cell line , 0 . 5x ( 3 . 8 ± 0 . 4 ) µm ( N = 9 cells; Figure 3H ) , validating our method for overlap length measurement based on EB3 tracks .\",\n \"After opto-PRC1 removal , the overlap half-length increased to 2 . 6 ± 0 . 2 µm ( n = 23 spots in N = 9 cells; Figure 3G ) .\",\n \"In contrast to opto cells , treatment with PRC1 siRNA did not result in a change of overlap length with respect to untreated cells ( Figure 3—figure supplement 1D–F ) .\",\n \"Thus , the antiparallel overlaps became on average 40% longer after acute , but unchanged after long-time PRC1 removal .\",\n \"Interestingly , the overlaps were especially long in the inner part of the spindle , whereas those on the spindle periphery were similar to overlaps in control cells ( Figure 3I ) .\",\n \"This spatial difference is correlated with our findings that PRC1 is removed faster from the inner bundles ( see Figure 1B ) and that displaced and misoriented kinetochores are found more often in the inner than in the outer spindle region ( see Figure 2D , E , H ) , suggesting a mechanistic link between the overlap length and kinetochore positioning .\",\n \"To test to what extent PRC1 removal in metaphase affects the number of microtubules in the bridging fibers ( Figure 4A ) , we visualized microtubules by using SiR-tubulin , a far-red tubulin dye excited by red light ( Lukinavičius et al . , 2014 ) , which allowed us to observe microtubules both when the blue light is turned on and switched off .\",\n \"Intensity profiles across the spindle midzone revealed that SiR-tubulin intensity maxima were lower upon PRC1 removal and increased after its return ( Figure 4—figure supplement 1A; Figure 4—video 1 ) .\",\n \"Measurements of SiR-tubulin signal intensity between and lateral from sister kinetochores showed that upon PRC1 removal the tubulin signal was reduced specifically in the bridging fibers ( Figure 4A–C; Figure 4—video 1 ) .\",\n \"As an alternative to SiR-tubulin , which is a taxol-based dye that may affect microtubule dynamics ( Lukinavičius et al . , 2014 ) , we tested YFP-tubulin , but the excitation laser for YFP also activated the optogenetic system ( Wang and Hahn , 2016; Figure 4—figure supplement 1B ) .\",\n \"Finally , we used tubulin-GFP to determine tubulin signal intensities of the bridging fibers and k-fibers upon acute removal of PRC1 ( Figure 4A , D–H; Figure 4—figure supplement 1C–F; see Materials and methods ) .\",\n \"Upon exposure to the blue light , tubulin signal intensity in the bridging fibers decreased ~2 . 5-fold , which corresponds to 5 . 6 ± 0 . 9 microtubules given that the average number of microtubules in the bridging fiber is 14 ( Kajtez et al . , 2016 ) .\",\n \"Together with the finding that the number of growing microtubules in the bridging fiber is similar with and without PRC1 ( Figure 3D ) , this result implies that in the presence of PRC1 the bridge contains more microtubules with a smaller fraction of them being dynamic than without PRC1 , and that PRC1 removal leads mainly to disassembly of non-dynamic microtubules .\",\n \"Upon PRC1 return the intensity and thus the number of microtubules remained low ( Figure 4D–G; Figure 4—figure supplement 1D ) , possibly as a consequence of the perturbed spindle architecture in the absence of PRC1 , which may be able to recover after a longer time period .\",\n \"Importantly , the intensity of k-fiber was unaltered ( Figure 4H , see Materials and methods ) , suggesting that the k-fibers were not affected by PRC1 removal .\",\n \"Thus , acute removal of PRC1 and its return change the number of microtubules specifically in the bridging fibers .\",\n \"Long-term depletion of PRC1 by siRNA in HeLa cells with stable expression of tubulin-GFP and transient expression of mRFP-CENP-B resulted in a similar reduction in bridging fiber tubulin intensity as after acute PRC1 removal ( Figure 4—figure supplement 1G , H ) .\",\n \"Long-term depletion resulted in a decreased number of microtubules in the bridge , 7 . 5 ± 1 . 1 , which was similar to the microtubule number after acute removal ( p = 0 . 21 , t-test; Figure 4—figure supplement 1I , Table 1 ) .\",\n \"As in acute removal , the intensity of k-fibers did not change after long-term depletion ( p = 0 . 49 , t-test ) .\",\n \"Given that the bridging fiber is under compression ( Kajtez et al . , 2016 ) , reduction of number of microtubules in the bridging fiber is expected to reduce this compression and thus to straighten the k-fibers ( Figure 4—figure supplement 1J ) .\",\n \"Tracking of the pole-to-pole contour of the outermost k-fibers revealed that PRC1 removal indeed straightened the k-fibers and thus made the spindle diamond-shaped ( Figure 4—figure supplement 1K–M ) .\",\n \"Similar to acute removal , long-term depletion of PRC1 caused straightening of outermost k-fibers , although to a smaller extent , whereas spindle length and width were unchanged after both treatments ( Figure 4—figure supplement 1L , N , O; Table 1 ) .\",\n \"This result supports that compression in the bridging fibers enables the spindle to obtain a curved shape .\",\n \"Since bridging fibers are , nevertheless , present upon PRC1 removal , we asked how the residual microtubules are bundled together .\",\n \"Eg5/kinesin-5 , which localizes in the bridging fibers ( Kajtez et al . , 2016; Mann and Wadsworth , 2018 ) , was still detectable in these fibers after PRC1 siRNA ( Figure 4—figure supplement 1P ) .\",\n \"Thus , we propose that microtubule crosslinkers such as Eg5 crosslink the remaining microtubules in the bridge after acute PRC1 removal .\",\n \"To investigate the mechanism of bridging microtubule regulation via PRC1 , we analyzed the localization of major proteins that regulate spindle microtubule dynamics and/or are binding partners of PRC1: Kif4A , Kif18A , and MKLP1 ( Bieling et al . , 2010b; Bringmann et al . , 2004; Gruneberg et al . , 2006; Kurasawa et al . , 2004; Mayr et al . , 2007; Stumpff et al . , 2008; Stumpff et al . , 2012 ) before and after PRC1 removal in metaphase ( Figure 5; Figure 5—figure supplement 1; Figure 5—figure supplement 2 and Table 1 ) .\",\n \"We first looked at the localization of these proteins in the bridging fiber .\",\n \"Surprisingly , we found that Kif4A and Kif18A localize in the bridge during metaphase , visible as thin lines across or next to the location of sister kinetochores where PRC1-labeled bundles are found ( Polak et al . , 2017; Figure 5A; Figure 5—figure supplement 1A ) .\",\n \"Similar localization was observed for MKLP1 , spanning the region between two sister k-fibers ( Figure 5A ) .\",\n \"In vertically oriented spindles , whose long axis was roughly perpendicular to the imaging plane , Kif4A and Kif18A colocalized with opto-PRC1 , now visible as spots in the cross-section of the spindle ( Figure 5—figure supplement 1B ) .\",\n \"To explore whether PRC1 removal affects the localization of Kif4A , Kif18A , and MKLP1 , we analyzed their intensities within bridging fibers .\",\n \"Measurements of Kif4A intensity upon acute PRC1 removal and long term-depletion , in regions lateral from chromosomes which correspond to peripheral parts of bridging fibers , showed that its intensity in the bridging fibers decreased ( Figure 5A–C; Figure 5—figure supplement 1A ) .\",\n \"Additional analysis of Kif4A intensity in HeLa cells after long-term PRC1 removal corroborated this result ( Figure 5—figure supplement 1C ) .\",\n \"Interestingly , there was a larger reduction in Kif4A intensity after acute PRC1 removal , where it decreased by 76 ± 5% , in comparison to long-term depletion where it decreased by 52 ± 3% ( p = 1×10−4; Figure 5C; Table 1 ) .\",\n \"Kif18A intensity in the bridging fibers also decreased upon acute PRC1 removal and long-term depletion ( Figure 5A–C; Figure 5—figure supplement 1A , C ) .\",\n \"Yet , in contrast to Kif4A , Kif18A intensities in the bridging fiber decreased to a similar extent , that is by 43 ± 11% and 38 ± 6% , after acute and long-term PRC1 depletion , respectively ( p = 0 . 68; Figure 5C; Table 1 ) .\",\n \"MKLP1 intensities were also similarly reduced after both approaches , 87 ± 5% after acute PRC1 removal and 83 ± 11% after long-term depletion ( p = 0 . 68; Figure 5D; Figure 5—figure supplement 1C ) .\",\n \"Among the tested proteins , only MKLP1 was exclusively localized in the bridging fibers , co-localizing with PRC1 , both before PRC1 removal and after its return ( Figure 5A , D; Figure 5—figure supplement 1C–G ) .\",\n \"Even though MKLP1 was removed to a large extent from the spindle by acute PRC1 removal , it was not detected at the cell membrane together with PRC1 .\",\n \"It may be that MKLP1 binds rather weakly to PRC1 in metaphase and/or that the absence of PRC1 decreases its affinity for microtubules .\",\n \"In addition , the ability of MKLP1 to bind along scaffold of antiparallel overlaps could depend on the role of PRC1 in dictating 35-nm-inter-microtubule spacing , proposed to be important to enable localization of specific proteins within these structures ( Kellogg et al . , 2016; Subramanian et al . , 2010 ) .\",\n \"As Kif4A and Kif18A are known to regulate microtubule length ( Mayr et al . , 2007; Varga et al . , 2006; Wandke et al . , 2012 ) , we set out to explore their roles in overlap length regulation .\",\n \"We hypothesized that these kinesins may regulate the length of bridging microtubules and hence their antiparallel overlaps during metaphase .\",\n \"Indeed , removal of Kif4A or Kif18A by siRNA resulted in longer PRC1-labeled overlaps and longer spindles ( Figure 5E ) , with a small but not significant increase in the ratio of overlap length to spindle length ( Figure 5F ) .\",\n \"However , the increase in the overlap and spindle length was significantly different after the two treatments ( Figure 5G ) .\",\n \"Kif4A siRNA treatment increased overlap length for 0 . 9 ± 0 . 2 µm compared with control , which was similar to spindle length increase of 1 . 2 ± 0 . 3 µm ( p = 0 . 48 , t-test; Figure 5G ) .\",\n \"In contrast , Kif18A siRNA treatment increased overlap length for 1 . 8 ± 0 . 3 µm , whereas the spindle length increased to a larger extent , for 3 . 7 ± 0 . 6 µm ( p = 0 . 004 , t-test; Figure 5G ) .\",\n \"These results suggest that both Kif4A and Kif18A regulate the length of bridging microtubules and their overlap , but Kif18A has a greater effect on overall spindle microtubules and spindle length than Kif4A .\",\n \"As an alternative to the changes in the bridging fiber , disruption of polar ejection forces may be the cause of kinetochore misalignment upon PRC1 removal ( Figure 2K ) , in particular because these forces are modulated by Kif4A ( Stumpff et al . , 2012 ) .\",\n \"The most prominent Kif4A localization in metaphase is on chromosomes ( Gruneberg et al . , 2006; Kurasawa et al . , 2004; Zhu and Jiang , 2005; Figure 5A , B; Figure 5—figure supplement 1A–C; Figure 5—figure supplement 2A–C ) .\",\n \"Neither acute nor long-term PRC1 removal had an extensive effect on the Kif4A signal on chromosome arms , although there was a slight decrease in Kif4A signal after PRC1 siRNA treatment ( Figure 5B; Figure 5—figure supplement 1A , C; Figure 5—figure supplement 2C , D; Table 1 ) .\",\n \"In early anaphase , the amount of Kif4A on segregated chromosomes was similar in opto and control cells ( Figure 5—figure supplement 2E ) , indicating that increased occurrence of lagging kinetochores was neither due to perturbed polar ejection forces in that phase nor defects in chromosome architecture/condensation .\",\n \"Finally , kinetochore misalignment and lagging kinetochores upon PRC1 removal may be due to disrupted localization of proteins that regulate microtubule dynamics at the plus-end of the k-fiber ( Figure 2K ) .\",\n \"To investigate this possibility , we analyzed the k-fiber localization of the key proteins with this function , Kif18A , CLASP1 , and CENP-E ( Al-Bassam et al . , 2010; Maffini et al . , 2009; Maiato et al . , 2003; Mayr et al . , 2007; Stumpff et al . , 2008; Stumpff et al . , 2012; Yu et al . , 2016 ) .\",\n \"These proteins were localized at the plus-ends of k-fibers before and after acute and long-term removal of PRC1 ( Figure 5A , B; Figure 5—figure supplement 1A–C; Figure 5—figure supplement 2F–I ) .\",\n \"This indicates that kinetochore misalignment upon PRC1 removal is not caused by simultaneous removal of proteins that regulate microtubule dynamics at the k-fiber plus ends .\"\n ],\n [\n \"We developed an optogenetic approach that offers acute light-controlled removal of proteins from a normally formed spindle at a precise phase of mitosis .\",\n \"The main advantages of this approach over chemically-induced protein translocation ( Cheeseman et al . , 2013; Haruki et al . , 2008; Robinson et al . , 2010; Wordeman et al . , 2016 ) are its reversibility , allowing the protein to return to its initial location within about a minute , and applicability to individual cells .\",\n \"Unlike previous optogenetic approaches ( Fielmich et al . , 2018; Okumura et al . , 2018; van Haren et al . , 2018; Yang et al . , 2013; Zhang et al . , 2017 ) , this method allows for global loss-of-function of full-length spindle proteins , relying on simple protein tagging rather than domain splitting , with no need of chromophore addition .\",\n \"Moreover , this method may be implemented with other optical perturbations ( Milas et al . , 2018 ) and used as ‘in vivo pull-down’ for probing protein-protein interactions in different phases of the cell cycle .\",\n \"However , this approach depends on high turnover of the protein in comparison with the time scale of interest .\",\n \"Acute PRC1 removal from the spindle by optogenetics and long-term PRC1 depletion by siRNA led to partially different phenotypes .\",\n \"These differences are hard to explain by different levels of PRC1 on the spindle as both methods decreased PRC1 by ~90% .\",\n \"It is also unlikely that the differences are caused by the interaction of the membrane-translocated PRC1 with astral microtubules because of the uniform PRC1 signal on the membrane , fast return to the spindle , and no change in spindle positioning .\",\n \"Therefore , the generally weaker effects of siRNA in comparison with the acute optogenetic removal are most likely due to compensatory mechanisms acting during long-term depletion .\",\n \"By overcoming temporal limitations of siRNA , our work reveals an unexpected role of PRC1 and bridging fibers in the regulation of chromosome alignment on the metaphase spindle via overlap length-dependent forces ( Figure 6A ) .\",\n \"We propose that the interactions between k-fibers and bridging fibers regulate the movement of bi-oriented chromosomes along the pole-to-pole axis .\",\n \"If a kinetochore pair is displaced toward one spindle pole , more motors and/or crosslinkers are expected to accumulate in the overlap facing the opposite pole because this overlap is longer , and pull the kinetochores back to the center ( Figure 6B ) .\",\n \"As the efficiency of this type of centering depends on the relative asymmetry in the overlap length on either side , shorter overlap length leads to more precise centering of the kinetochores ( Figure 6A , B ) .\",\n \"Our finding that acute PRC1 removal leads to chromosome misalignment and elongation of overlap zones supports this model .\",\n \"Moreover , elongation of overlaps correlates with chromosome misalignment within spindles , as elongated overlaps and misaligned chromosomes are mostly found in the central part of the spindle rather than on the periphery .\",\n \"In contrast to the acute PRC1 removal , long-term PRC1 depletion by siRNA leads neither to overlap elongation nor chromosome misalignment , providing further support to our model .\",\n \"These differences between the acute and long-term depletion indicate the existence of compensatory mechanisms that regulate overlap length and hence maintain chromosome alignment during long-term PRC1 depletion .\",\n \"How is the length of the bridging microtubules and their overlaps controlled ?\",\n \"Interestingly , we found that Kif4A and Kif18A localize in the bridging fibers in metaphase and their intensities decreased following partial disassembly of the bridging fiber by optogenetic or siRNA-mediated PRC1 removal .\",\n \"Both Kif4A and Kif18A suppress microtubule dynamics ( Bieling et al . , 2010b; Bringmann et al . , 2004; Stumpff et al . , 2008; Stumpff et al . , 2012 ) , and our experiments showed that depletion of either of them by siRNA leads to longer PRC1-labeled overlaps .\",\n \"These elongated PRC1-labeled overlaps provide another line of evidence in support of our model for chromosome alignment by overlap length-dependent forces , as Kif4A and Kif18A siRNA-treated cells exhibit chromosome misalignment ( Stumpff et al . , 2008; Wandke et al . , 2012 ) .\",\n \"Even though it was reported that Kif4A does not directly interact with PRC1 before late mitosis ( Gruneberg et al . , 2006; Kurasawa et al . , 2004; Zhu and Jiang , 2005 ) , our experiments suggest that there is a small pool of Kif4A that does so and binds to the antiparallel overlaps of bridging microtubules in metaphase .\",\n \"Acute PRC1 removal likely results in the removal of this pool of Kif4A from the bridge .\",\n \"Our result that Kif4A was reduced by ~76% , which is similar to the reduction of PRC1 by ~85% within the same time interval ( 12 min ) of acute removal supports this picture .\",\n \"This reduction of Kif4A in the bridge may lead to excessive microtubule polymerization and thus longer overlap zones in metaphase , similar to the long overlaps in anaphase after Kif4A depletion ( Hu et al . , 2011; Kurasawa et al . , 2004; Zhu and Jiang , 2005 ) .\",\n \"As the spindle length remained constant after acute PRC1 removal , we suggest that the overlap elongation is due to Kif4A acting specifically on bridging microtubules rather than overall spindle microtubules .\",\n \"In contrast to Kif4A , Kif18A in the bridging fiber was reduced less than PRC1 within 12 min of acute PRC1 removal , ~43% compared with ~85% , respectively .\",\n \"This difference in the behavior of Kif18A and PRC1 is consistent with the fact that Kif18A is not known to be a binding partner of PRC1 .\",\n \"Thus , we infer that the reduction of Kif18A in the bridge was a consequence of the fewer microtubules in the bridging fiber , and that the amount of Kif18A per microtubule remained largely unaffected by PRC1 removal .\",\n \"Finally , MKLP1 , which is a binding partner of PRC1 ( Gruneberg et al . , 2006 ) , was reduced to a similar extent as PRC1 , ~87% and ~88% within 20 min of acute PRC1 removal , respectively .\",\n \"Based on these results , we conclude that the localization of MKLP1 and a small pool of Kif4A in the bridging fiber are directly PRC1-dependent , whereas the localization of Kif18A is not .\",\n \"We propose that both Kif4A and Kif18A regulate the length of bridging microtubules and their overlaps under normal conditions ( Figure 6C ) , as their depletion results in longer overlaps .\",\n \"Intriguingly , we found that Kif4A signal in the bridging fibers decreased to a larger extent after acute than after long-term PRC1 depletion , whereas Kif18A signal decreased to a similar extent by the two methods of PRC1 depletion .\",\n \"Moreover , Kif4A depletion by siRNA resulted in an increase of ~1 µm in overlap length , which was similar to the increase observed after acute PRC1 removal , whereas Kif18A depletion by siRNA led to a larger overlap increase .\",\n \"Thus , we suggest that the observed overlap elongation after acute PRC1 removal is a consequence of the strong reduction of Kif4A in the bridging fibers .\",\n \"In contrast , after long-term PRC1 depletion , we speculate that a larger fraction of Kif4A is present on the microtubules during overlap formation , contributing to the compensatory mechanism maintaining normal overlap length .\",\n \"The proteins that we found in the bridging fiber , Kif4A , Kif18A , and MKLP1 , may also slide apart and/or stabilize the bridging microtubules ( Figure 6C ) .\",\n \"In addition to these kinesins , we observed Eg5 in the bridging fiber ( Kajtez et al . , 2016; Mann and Wadsworth , 2018 ) .\",\n \"During early anaphase , Kif4A and Eg5 drive the sliding of antiparallel microtubules that elongates the spindle ( Vukušić et al . , 2019 ) .\",\n \"Thus , these kinesins may have a similar role during metaphase .\",\n \"This possibility is in agreement with previous work showing that Kif4A depletion reduces microtubule poleward flux in metaphase ( Wandke et al . , 2012 ) .\",\n \"Similarly , Kif18A in the bridging fiber may have microtubule sliding and crosslinking activities equivalent to those of the yeast kinesin-8 ( Su et al . , 2013 ) .\",\n \"Furthermore , MKLP1 contributes to the stabilization of bridging microtubules in early anaphase ( Vukušić et al . , 2017 ) , and we suggest that it performs a similar function in metaphase together with other motors and crosslinkers including Eg5 and PRC1 .\",\n \"The roles of these and other proteins within bridging fibers in the regulation of microtubule dynamics and sliding will be an intriguing topic for future studies .\",\n \"The localization of Kif4A on chromosome arms , where it is involved in polar ejection forces ( Bieling et al . , 2010a; Brouhard and Hunt , 2005 ) was preserved after acute PRC1 removal .\",\n \"Similarly , Kif18A , CLASP1 , and CENP-E remained on plus-ends of k-fibers .\",\n \"However , as we cannot exclude potential subtle changes in the localization of these proteins or mislocalization of other proteins , the observed chromosome misalignment and overlap elongation after acute PRC1 removal could also be promoted by the changes of the dynamics of other microtubules within the spindle consequently affecting forces produced by k-fibers and polar ejection forces .\",\n \"The changes upon acute PRC1 removal were not spatially uniform across the spindle .\",\n \"The most affected part was the inner part of the spindle , where the PRC1 signal disappeared faster and the bridging microtubules became longer than on the periphery of the spindle .\",\n \"The inner bridging fibers were more severely affected by PRC1 removal possibly because they are made up of fewer microtubules than the outer bridges .\",\n \"Severely misaligned kinetochores that moved more than 2 µm away from the equatorial plane and lagging kinetochores occurred also more often in the inner part of the spindle .\",\n \"This local effect is in line with weak mechanical coupling between neighboring k-fibers yet strong coupling between sister k-fibers ( Elting et al . , 2017; Suresh et al . , 2020; Vladimirou et al . , 2013 ) , and indicative of a mechanistic link between the bridging fiber geometry and kinetochore alignment .\",\n \"In conclusion , we propose that overlap length-dependent forces help to position the chromosomes at the equatorial plane of the spindle .\",\n \"The overlap length is regulated by the PRC1-dependent Kif4A , and by Kif18A within the bridging fiber .\",\n \"Kif4A , Eg5 , and possibly other kinesins may slide bridging microtubules apart , whereas PRC1 together with the kinesins stabilizes the overlaps .\",\n \"Thus , in addition to the forces generated at k-fiber tips and polar ejection forces , proper chromosome alignment requires forces generated within the bridging fiber , which are transferred to the k-fiber and rely on precise regulation of the overlap region .\"\n ],\n [\n \"Experiments were performed using: unlabeled human osteosarcoma U2OS cell line , U2OS cells expressing CENP-A-GFP , mCherry-α-tubulin and PA-GFP-tubulin and U2OS cell line stably expressing CENP-A-GFP , used in our previous work ( Vukušić et al . , 2017 ) , which were a gift from Marin Barišić and Helder Maiato ( Institute for Molecular Cell Biology , University of Porto , Portugal ) ; U2OS cell line stably expressing 2xGFP-EB3 and mCherry-CENP-A , a gift from Julie Welburn ( University of Edinburgh , United Kingdom ) ( Kajtez et al . , 2016 ) ; HeLa-Kyoto BAC lines stably expressing MKLP1-GFP , Kif4A-GFP , Kif18A-GFP , CENP-E-GFP and PRC1-GFP were a courtesy of Ina Poser and Tony Hyman ( MPI-CBG , Dresden , Germany ) ; unlabeled HeLa-TDS cells from the High-Throughput Technology Development Studio ( MPI-CBG , Dresden ) ; HeLa-TDS cells , permanently transfected with pEGFP-α-tubulin , used in our previous work ( Kajtez et al . , 2016 ) ; HeLa cells stably expressing YFP-tubulin , a courtesy of Lars Jansen ( University of Oxford , United Kingdom ) ; HeLa cells permanently transfected with EGFP-CLASP1 , which was a gift from Helder Maiato ( Institute for Molecular Cell Biology , University of Porto , Portugal ) .\",\n \"Cells were grown in flasks in Dulbecco’s Modified Eagle’s Medium ( DMEM; Lonza , Basel , Switzerland ) with 1 g/L D-glucose , L-glutamine , and pyruvate , supplemented with 10% of heat-inactivated Fetal Bovine Serum ( FBS; Sigma Aldrich , St . Louis , MO , USA ) , 100 IU/mL penicillin and 100 mg/mL streptomycin solution ( Lonza ) .\",\n \"For cell lines with stable expression of fluorescently labeled proteins , 50 µg/mL geneticin ( Life Technologies , Waltham , MA , USA ) was added .\",\n \"Cells were kept at 37°C and 5% CO2 in a Galaxy 170 R humidified incubator ( Eppendorf , Hamburg , Germany ) .\",\n \"All used cell lines were confirmed to be mycoplasma free by using MycoAlert Mycoplasma Detection Kit ( Lonza ) .\",\n \"To make PRC1-tgRFPt-SspB WT ( opto-PRC1 ) plasmid , PRC1 fragment was amplified from His6-PRC1 plasmid ( RRID:Addgene_69111 ) ( Nixon et al . , 2015 ) using the primers GCTAGAATTGACCGGATGAGGAGAAGTGAGGTGCTG ( FWD ) and CATGGTGGCGACCGGTAAATTCGAAGCTTGAGCTCGAGATCTGAGGGACTGGATGTTGGTTGAATTGAGG ( REV ) and inserted into plasmid tgRFPt-SspB WT ( RRID:Addgene_60415 ) ( Guntas et al . , 2015 ) using AgeI restriction site .\",\n \"This step was performed using commercially available In-Fusion HD Cloning Kit ( Clontech , Mountain View , CA , USA ) .\",\n \"The produced plasmid expresses PRC1 tagged with tgRFPt and SspB at the C-terminus .\",\n \"Plasmid iLID-CAAX was purchased ( RRID:Addgene_85680 ) ( O'Neill et al . , 2016 ) .\",\n \"Plasmids pEGFP-C1Kif4a-sires and EGFP-Kif18A were a gift from Jason Stumpff ( University of Vermont , Burlington , VT , USA ) ( Stumpff et al . , 2008; Stumpff et al . , 2012 ) .\",\n \"Plasmid GFP-CENP-E was a gift from Marin Barišić ( Danish Cancer Society Research Center , Copenhagen , Denmark ) .\",\n \"Plasmid mRFP-CENP-B ( pMX234; RRID:Addgene_23006 ) was provided by Linda Wordeman ( University of Washington ) .\",\n \"For depletion of endogenous PRC1 before opto experiments , cells were transfected 72 hr ( U2OS cells ) or 24 hr ( HeLa cells ) prior to imaging with 25 µL of 20 µM Accell PRC1 siRNA ( A-019491-15-0020 , Dharmacon , Lafayette , CO , USA ) targeting 3’ UTR of PRC1 mRNA .\",\n \"A day prior to imaging , siRNA-treated cells were transfected with corresponding plasmids in following amounts: 0 . 3 μg of iLID-CAAX , 5 . 5 μg PRC1-tgRFPt-SspB-WT ( resistant to the used RNAi ) , 0 . 5 µg pEGFP-C1Kif4a-sires , 1 µg EGFP-Kif18A , 1 µg GFP-CENP-E , and 2 . 5 µg mRFP-CENP-B .\",\n \"In HeLa BAC lines , 24 hr prior to imaging , mock experiment cells were transfected with 100 nM Accell Non-targeting Pool ( D-001910-10-05; Dharmacon ) , PRC1 siRNA treated with 100 nM Accell PRC1 siRNA ( Dharmacon ) , Kif4A siRNA treated with 100 nM Kif4A siRNA ( sc-60888; Santa Cruz Biotechnologies , Dallas , TX , USA ) , whereas Kif18A siRNA treated with 100 nM Silencer Select Validated Kif18A siRNA ( s37882; Ambion , Austin , TX , USA ) .\",\n \"All transfections were performed using Nucleofector Kit R with the Nucleofector 2b Device ( Lonza ) using X-001 program for U2OS and O-005 ( high viability ) program for HeLa cells .\",\n \"Following transfection , the cells were seeded on 35 mm glass coverslip uncoated dishes with 0 . 17 mm ( 1 . 5 coverglass ) glass thickness ( MatTek Corporation , Ashland , MA , USA ) in 1 . 5 mL DMEM medium with appropriate supplements .\",\n \"To visualize microtubules , cells were stained with silicon rhodamine ( SiR ) -tubulin ( Lukinavičius et al . , 2014; Spirochrome AG , Stein am Rhein , Switzerland ) , a far-red tubulin dye , at a concentration of 50 nM 12–16 hr prior to imaging .\",\n \"To prevent dye efflux , verapamil , a broad-spectrum efflux-pump inhibitor ( Spirochrome Ltd . ) , was added in U2OS cells at a concentration of 1 μM .\",\n \"To visualize chromosomes and determine the phase of the mitosis , 20 min prior to imaging SiR-DNA ( Lukinavičius et al . , 2015; Spirochrome AG , Stein am Rhein , Switzerland ) was added to a final concentration of 150 nM .\",\n \"For experiments on U2OS cells expressing 2xGFP-EB3 and mCherry-CENP-A , the cells were synchronized by adding 20 µM of the proteasome inhibitor MG-132 ( Sigma-Aldrich ) to arrest the cells in metaphase .\",\n \"Imaging was started 15 min after adding MG-132 .\",\n \"Cells were fixed with ice-cold methanol for 3 min , washed three times with phosphate buffer saline ( PBS ) , followed by 15 min permeabilization with 0 . 5% Triton in PBS .\",\n \"Cells were washed three times with PBS and blocked in 1% Normal Goat Serum ( NGS ) except in experiment for intensity of opto-PRC1 where cells were blocked in BSA in PBS for 1 hr at 4°C .\",\n \"Cells were washed three times with PBS and then incubated in primary antibody solution in blocking buffer over night at 4°C .\",\n \"Following primary antibodies were used: mouse anti-PRC1 monoclonal antibody ( 1:100; C-1; sc-376983 , Santa Cruz Biotechnology ) , rabbit anti-α-tubulin polyclonal antibody ( 1:100; RRID:AB_10743646; SAB4500087; Sigma-Aldrich Corporation , St . Louis , MO , USA ) , mouse monoclonal anti-Kif4A antibody ( 1:100; RRID:AB_10707683; E-8; sc-365144; Santa Cruz Biotechnology ) , rabbit polyclonal anti-MKLP1 antibody ( 1:100; RRID:AB_631959; N-19; sc-867 , Santa Cruz Biotechnology ) , mouse monoclonal anti-Eg5 antibody ( 1:100; RRID:AB_10841907; A-1; sc-365681 , Santa Cruz Biotechnology ) , rabbit polyclonal anti-Kif18A antibody ( 1:100; RRID:AB_2296551; A301-080A; Bethyl ) , rabbit polyclonal anti-Kif4A antibody ( 1:200; RRID:AB_2280904; A301-074A; Bethyl ) .\",\n \"After washing off primary antibodies with PBS , cells were incubated in a solution of secondary antibodies in 2% NGS or BSA in PBS for 1 hr at room temperature protected from light .\",\n \"Following secondary antibodies were used: donkey anti-mouse IgG Alexa Fluor 488 ( 1:250; ab150109 , Abcam , Cambridge , UK ) , donkey anti-rabbit IgG Alexa Fluor 594 ( 1:250; ab150064 , Abcam ) , donkey anti-rabbit IgG Alexa Fluor 405 ( 1:250; RRID:AB_2715515; ab175649 , Abcam ) , and goat anti-mouse IgG Alexa Fluor 647 ( 1:250; RRID:AB_2811129; ab150119 , Abcam ) .\",\n \"After washing off the secondary antibodies three times in PBS , cells were incubated with a solution of 4’ , 6-diamidino-2-phenylindole ( DAPI ) ( 1:1000 ) in PBS for 20 min and washed three times in PBS or SiR-DNA ( 150 nM ) in PBS for 15 min before imaging .\",\n \"Note that we used immunocytochemistry for PRC1 rather than Western blot analysis because the efficiency of opto-PRC1 plasmid transfection is low , and as Western blot analysis provides information about the complete cell population , these results may not be relevant for the cells used in the optogenetic experiments .\",\n \"In contrast , by using immunocytochemistry we analyzed only the cells with a similar opto-PRC1 level and in the same phase as those in our optogenetic experiments .\",\n \"Immunocytochemistry imaging and live imaging of unlabeled U2OS , U2OS stably expressing CENPA-GFP , HeLa-TDS pEGFP-α-tubulin and HeLa BAC CENP-E-GFP cells was performed using Bruker Opterra Multipoint Scanning Confocal Microscope ( Bruker Nano Surfaces , Middleton , WI , USA ) , described previously ( Buđa et al . , 2017 ) .\",\n \"In brief , the system was mounted on a Nikon Ti-E inverted microscope equipped with a Nikon CFI Plan Apo VC 100x/1 . 4 numerical aperture oil objective ( Nikon , Tokyo , Japan ) .\",\n \"During imaging , live cells were maintained at 37°C using H301-K-frame heating chamber ( Okolab , Pozzuoli , NA , Italy ) .\",\n \"In order to obtain the optimal balance between spatial resolution and signal-to-noise ratio , 60 µm pinhole aperture was used .\",\n \"Opterra Dichroic and Barrier Filter Set 405/488/561/640 was used to separate the excitation light from the emitted fluorescence .\",\n \"Following emission filters were used: BL HC 525/30 , BL HC 600/37 , and BL HC 673/11 ( all from Semrock , Rochester , NY , USA ) .\",\n \"Images were captured with an Evolve 512 Delta Electron Multiplying Charge Coupled Device ( EMCCD ) Camera ( Photometrics , Tucson , AZ , USA ) using a 200 ms exposure time .\",\n \"Electron multiplying gain was set on 400 .\",\n \"Camera readout mode was 20 MHz .\",\n \"No binning was performed .\",\n \"The xy-pixel size in the image was 83 nm .\",\n \"The system was controlled with the Prairie View Imaging Software ( Bruker ) .\",\n \"For kinetics experiments on U2OS cells ( Figure 1 ) , 561 and 488 nm diode laser lines were used every 10 s with 200 ms exposure time .\",\n \"In all other optogenetic experiments , stacks were acquired using sequentially the following diode laser lines: 561 nm ( to visualize opto-PRC1 ) , 488 nm ( to activate the optogenetic system and to visualize GFP ) , and 640 nm ( to visualize SiR-tubulin or SiR-DNA , when applicable ) , with time interval between z-stacks of 60 s and with 200 ms exposure time per laser line .\",\n \"To prevent dissociation of PRC1 from the cell membrane between acquiring two consecutive z-stacks , only blue light was turned on for 200 ms every 10 s .\",\n \"Cells were imaged this way for 20 min in order to achieve almost complete removal of PRC1 from the spindle , after which the blue light was turned off and imaging was continued for another 10 min at 60 s intervals .\",\n \"The total imaging time of 30 min was chosen to be close to the typical metaphase duration of 29 . 7 ± 2 . 3 min , which was measured from the metaphase plate formation until anaphase onset in U2OS cells expressing CENP-A-GFP , mCherry-α-tubulin and PA-GFP-tubulin ( N = 187 ) imaged after nuclear envelope breakdown every minute by obtaining 15 z-slices with 0 . 5 µm spacing and 150 ms exposure time .\",\n \"After 30 min of imaging , one z-stack in each of the three channels was taken in order to visualize the spindle and kinetochores after PRC1 return .\",\n \"In all cells except HeLa cells expressing pEGFP-α-tubulin , three focal planes with spacing between adjacent planes of 1 µm were acquired .\",\n \"Live imaging of HeLa cells stably expressing pEGFP-α-tubulin for measurements of tubulin intensities after acute PRC1 removal was performed in the same manner as described above for optogenetic experiments .\",\n \"Additionally , before turning the blue light on every 10 s , one z-stack was acquired using 561 , 488 , and 640 nm diode laser lines with averaging 8 , and seven focal planes with spacing between adjacent planes of 0 . 5 µm .\",\n \"Stack was taken in the same way after 20 min of exposure to the blue light and again 10 min after the blue light was switched off .\",\n \"The same HeLa cells were used for measurements of tubulin intensities after long-term PRC1 removal and imaged by acquiring one z-stack using 561 , 488 , and 640 nm diode laser lines with averaging 8 , and seven focal planes with spacing between adjacent planes of 0 . 5 µm .\",\n \"Imaging of HeLa BAC CENP-E-GFP mock and PRC1 siRNA-treated cells was performed by acquiring one z-stack of 3 focal planes with spacing between adjacent planes of 1 µm .\",\n \"For imaging of immunostained cells , five focal planes with spacing between adjacent planes of 0 . 5 µm were acquired .\",\n \"Live imaging of U2OS cells stably expressing 2x-GFP-EB3 and mCherry-CENP-A and U2OS cells with transient expression of opto-PRC1 , iLID-CAAX and GFP-Kif4A or EGFP-Kif18A was performed on a spinning disk confocal microscope system ( Dragonfly , Andor Technology , Belfast , UK ) using 63x/1 . 47 HC PL APO glycerol objective ( Leica ) and Zyla 4 . 2P scientific complementary metal oxide semiconductor ( sCMOS ) camera ( Andor Technologies ) .\",\n \"During imaging cells were maintained at 37° and 5% CO2 within H301-T heating chamber ( Okolab , Pozzuoli , Italy ) .\",\n \"Images were acquired using Fusion software ( v 2 . 2 . 0 . 38 ) .\",\n \"For live imaging of U2OS cells expressing 2x-GFP-EB3 , mCherry-CENP-A and opto-PRC1 , 488 nm and 561 nm laser lines were used for excitation to visualize GFP , and mCherry and opto-PRC1 , respectively .\",\n \"In order to achieve PRC1 removal from the spindle , 3 z-planes with a z-spacing of 1 µm were acquired sequentially with both laser lines , every 10 s with 200 ms exposure time for 10 min .\",\n \"This imaging protocol was followed by five minutes of faster imaging , every 1 . 5 s with both laser lines on a central z-plane in order to visualize EB3 dynamics and to prevent the opto-PRC1 return .\",\n \"Control , mock and PRC1 siRNA-treated cells were imaged with the same imaging protocol .\",\n \"Live imaging of U2OS cells with transient expression of opto-PRC1 , iLID-CAAX and GFP-Kif4A or EGFP-Kif18A was performed in the similar manner as described above .\",\n \"Additionally , to better visualize the localization of the Kif4A and Kif18A , before turning the blue light on every 10 s , one z-stack was acquired using 561 , 488 and 640 nm diode laser lines with frame averaging 4 , and three focal planes with spacing between adjacent planes of 1 µm .\",\n \"Stack was taken in the same way after 12 min of exposure to the blue light and again 5 min after the blue light was switched off .\",\n \"Note that the exposure to the blue light was shorter than in previous experiments regarding opto-PRC1 kinetics so that the majority of the cells remained in metaphase during the experiment .\",\n \"Live imaging of unlabeled , BAC ( except CENP-E-GFP ) , YFP-tubulin , and EGFP-CLASP1 HeLa cell lines was performed on Leica TCS SP8 X laser scanning confocal microscope with a HC PL APO 63x/1 . 4 oil immersion objective ( Leica , Wetzlar , Germany ) heated with an objective integrated heater system ( Okolab , Burlingame , CA , USA ) .\",\n \"During imaging , cells were maintained at 37°C in Okolab stage top heating chamber ( Okolab , Burlingame , CA , USA ) .\",\n \"The system was controlled with the Leica Application Suite X software ( LASX , 1 . 8 . 1 . 13759 , Leica , Wetzlar , Germany ) .\",\n \"For GFP- and YFP-labeled proteins , a 488 nm and 514 nm Argon laser was used , respectively , and for SiR-DNA or SiR-tubulin , a 652 nm white light laser was used .\",\n \"For AF-647 , a 637 nm white light laser was used .\",\n \"GFP , and SiR-DNA , SiR-tubulin or AF-647 emissions were detected with hybrid detector .\",\n \"For mock , PRC1 siRNA , Kif4A siRNA , and Kif18A siRNA experiments , images were acquired at 1–3 focal planes with 1 μm spacing and 0 . 05 µm pixel size .\",\n \"In optogenetic experiments 3 z-stacks with 1 μm spacing were acquired sequentially every 10 s in the same manner as in optogenetic experiments in U2OS cells .\",\n \"One z-stack with line averaging of 6 or 16 was acquired before system activation , 20 min after exposure to blue light and 10 min after the light was switched off .\",\n \"Since the cells were transiently transfected with opto-PRC1 , we observed variability in PRC1 expression levels and therefore we imaged and analyzed only those metaphase spindles with PRC1 localization consistent with endogenous and fluorescently labeled PRC1 ( Kajtez et al . , 2016; Polak et al . , 2017 ) .\",\n \"Cells were not synchronized in order to avoid additional chemical treatment of cells , and metaphase was determined by alignment of kinetochores in the equatorial plane .\",\n \"For determination of kinetics of PRC1 removal and return ( Figure 1C ) , intensity of opto-PRC1 was measured in each time frame on one focal plane .\",\n \"We used Polygon selection tool in Fiji ( National Institutes of Health , Bethesda , MD , USA ) to encompass the area of the spindle , Aspindle , and measure mean spindle intensity , Mspindle .\",\n \"Mean background intensity in the cytoplasm was measured using 2 . 5 × 2 . 5 µm rectangle , Mcyto .\",\n \"Spindle intensity was background corrected by subtracting Mcyto from Mspindle to obtain Mspindle corr .\",\n \"In order to calculate the sum of PRC1 intensity on the spindle , Mspindle corr was multiplied with Aspindle for each timeframe .\",\n \"The background intensity outside of the cell was negligible , thus we did not take it into account .\",\n \"Note that for the measurements of kinetic parameters in Figure 1C , four outliers were excluded ( see Figure 1—figure supplement 1F ) .\",\n \"The percentage of PRC1 removal was calculated from the mean value of intensity of all cells at time 20 min , that is , the last time point of the continuous exposure to the blue light .\",\n \"The percentage of return was calculated from the mean value of intensity of all cells in the interval 25–30 min , that is , during last 5 min of PRC1 return .\",\n \"Intensity profiles of opto-PRC1 removal and return ( Figure 1D ) were obtained on sum intensity projections of all three z-planes by drawing a pole-to-pole line along the long axis of the spindle by using Line tool in Fiji .\",\n \"The width of the line corresponded to the width of each individual spindle .\",\n \"Intensities were normalized to position of the poles .\",\n \"For quantification of PRC1 knock-down by siRNA and intensity level of opto-PRC1 ( in Figure 1—figure supplement 1A , B ) PRC1 intensity on fixed cells was measured on a sum-intensity projection of five focal planes by the procedure described above , in a way that mean spindle intensity was background corrected by subtracting mean intensity in the cytoplasm .\",\n \"For measuring opto-PRC1 intensity on the spindle , cells where PRC1 was visible on astral microtubules were not analyzed , nor imaged in live experiments .\",\n \"For quantification of PRC1 knock-down by siRNA in HeLa BAC MKLP1-GFP cell line , PRC1 intensity was quantified in the same manner as in U2OS cells .\",\n \"Inter-kinetochore distance was measured using Line tool in Fiji on individual or maximum intensity z-projections of up to two z-planes as a distance between centers of sister kinetochore signals .\",\n \"Peripheral kinetochores were defined as three outermost pairs on each side of the spindle with respect to spindle long axis , while the remaining were considered as central .\",\n \"Measurement of inter-kinetochore distances in prometaphase was performed on U2OS cells expressing CENP-A-GFP , mCherry-α-tubulin and PA-GFP-tubulin , just after nuclear envelope breakdown in one imaging plane where both sister kinetochores could be clearly distinguished .\",\n \"For measuring kinetochore alignment and orientation , as well as orientation and length of PRC1 bundles , Multipoint tool in Fiji was used .\",\n \"A point was placed in the center of signal of each sister kinetochore or edges of PRC1 signal for each bundle .\",\n \"Before measuring , images were rotated in order to achieve perpendicular direction of the equatorial plane with respect to x-axis .\",\n \"The equatorial plane was defined with two points placed between outermost pairs of kinetochores on the opposite sides of the spindle .\",\n \"For all measurements regarding kinetochores , those located at the spindle poles were not taken into account .\",\n \"All measurements in opto and control cells were performed in three time-points: before the blue light was switched on , after 20 min of continuous exposure to the blue light , and 10 min after the blue light was turned off .\",\n \"In untreated and PRC1 siRNA-treated cells measurements were performed at the beginning of imaging .\",\n \"Kymographs of kinetochore oscillations were produced by Low Light Tracking Tool ( LLTT ) , an ImageJ plugin ( Krull et al . , 2014 ) .\",\n \"Tracking of kinetochores in x , y plane was performed on maximum intensity of 2–3 z-planes .\",\n \"Sigma value ( standard deviation of the Gaussian used to approximate the Point Spread Function ( PSF ) of the tracked objects ) was set to Automatic .\",\n \"EB3 spots in U2OS cells stably expressing 2x-GFP-EB3 and mCherry-CENP-A were tracked by obtaining their xy coordinates using Multipoint tool in Fiji from the frame when a spot appeared until it disappeared or was no longer clearly distinguishable from its neighbors .\",\n \"Only spots belonging to bridging fibers were traced , which were defined as those passing between sister kinetochores or moving along PRC1 streaks .\",\n \"Half-overlap length was measured as the distance between the last location where a tracked EB3 spot was visible and the spindle equator .\",\n \"The number of spots in time was obtained by visually inspecting time frames where individual kinetochore pair or PRC1 bundle was visible , and dividing the total number of the observed EB3 spots in the bridging fibers by the total time of observation of individual kinetochore pairs or PRC1 bundles .\",\n \"Kymographs were generated using KymographBuilder plugin in Fiji , and half-overlap length was measured from kymographs as the distance between a spot's trajectory end point and the mid-line between the poles corresponding to the equatorial plane .\",\n \"These measurements were performed in the first 2 min of a fast imaging sequence , which followed after the 10 min imaging protocol required for PRC1 removal .\",\n \"The tubulin-GFP signal intensity of a cross-section of a bridging fiber was measured by drawing a 3-pixel-thick line between and perpendicular to the tubulin signal joining the tips of sister k-fibers .\",\n \"The same method was used in cells containing a kinetochore marker , that is , the profile intensity of the bridging fiber was extracted by looking at the tubulin channel and drawing the line between tips of k-fibers .\",\n \"For confirmation , we subsequently checked the kinetochore channel , and observed that this line was always placed between sister kinetochores .\",\n \"This validates our measurements of bridging fibers in opto cells using tubulin-GFP only .\",\n \"The tubulin intensity profile was corrected by subtracting mean background signal present in the cytoplasm ( see Figure 4A and Figure 4—figure supplement 1C , G ) .\",\n \"The signal intensity of the bridging fiber was calculated as the area under the peak using SciDavis ( Free Software Foundation Inc , Boston , MA , USA ) ( Figure 4E ) .\",\n \"The signal intensity in the region lateral from k-fiber tip was measured in a similar manner , 0 . 882 ± 0 . 04 µm away from the k-fiber tip ( Figure 4—figure supplement 1C , G ) .\",\n \"This intensity corresponds to the sum of the bridging and k-fiber , given that PRC1 overlap half-length is longer and EB3 spots pass further than the position where the intensity profiles were measured .\",\n \"Therefore , the intensity of the k-fiber was calculated by subtracting the bridging fiber intensity from the corresponding sum of bridging and k-fiber intensity .\",\n \"The profile intensity of bridging fiber and at the position lateral from k-fiber tip in the cells stained with SiR-tubulin ( Figure 4B , C ) was performed in the same manner .\",\n \"All the measurements were performed on a single z-plane .\",\n \"Note that mostly outermost fibers were used for these measurements because of being most easily distinguished from neighboring fibers .\",\n \"All measurements in opto and control cells were performed at three time-points: before the blue light was switched on , after 20 min of continuous exposure to the blue light , and 10 min after the blue light was turned off .\",\n \"Shapes of the spindle were quantified by tracking outermost k-fiber contours in the central z-slice of the spindle , or maximum intensity z-projection of two central z-slices .\",\n \"All spindles were rotated to have horizontal long axis .\",\n \"Pole-to-k-fiber-tip tracking was done using Multipoint tool by placing five roughly equidistant points along contour length , first point being at the pole and the last point being at the k-fiber tip .\",\n \"First point of each contour was translated to ( 0 , 0 ) .\",\n \"This was done for maximum of 4 trackable outermost k-fibers in the spindle .\",\n \"Curvature of the contour was calculated by fitting a circle to the contour points of individual k-fibers and retrieving reciprocal value of its radius .\",\n \"To test the effect of tracking errors on curvature , we introduced 1-pixel noise to the x and y values of tracked points , which did not change the result .\",\n \"Angle between outermost k-fibers ( θ ) was calculated as the angle between lines passing through last two points along the contour of sister k-fibers .\",\n \"Spindle length and width were measured using Line tool in Fiji .\",\n \"For length measurements , a line was drawn from pole to pole .\",\n \"The position of the pole was defined as the location of the strongest tubulin signal .\",\n \"Width was measured by drawing a line between outermost kinetochore pairs on the opposite sides of the spindle , perpendicular to the spindle long axis .\",\n \"To avoid the signal of Kif4A at the chromosomes , Kif4A bridging fiber intensity was measured on individual z-planes with 1 × 0 . 5 µm rectangles at the positions lateral from sister kinetochores and chromosomes , where no DNA ( DAPI or SiR-DNA ) was found , yet lateral parts of PRC1 streaks are present .\",\n \"This approach was applied in cases when PRC1 was not depleted or relocated to the cell membrane , that is , in opto cells before acute PRC1 removal and untreated cells .\",\n \"When PRC1 was not present on the spindle , that is , in opto cells after acute PRC1 removal and in PRC1 siRNA-treated cells , we placed rectangles at positions lateral from kinetochores and chromosomes , where antiparallel zones are thought to be positioned , considering their location before removal .\",\n \"The rectangles were placed parallel to the overlap .\",\n \"Given that bridging fiber intensity was measured mostly in the inner parts of the spindle , mean values from the spindle poles were considered as the Kif4A background and subtracted from Kif4A intensity retrieved from the positions of bridging fibers .\",\n \"For the measurements of Kif4A in the bridging fiber , we chose only cells in which Kif4A showed previously known localization on chromosomes and at comparable levels .\",\n \"This was important as immunostaining protocol sometimes resulted in cells with no or low chromosome staining , yet high spindle staining , and HeLa BAC Kif4A-GFP cells vary in expression levels among cells .\",\n \"The intensity of Kif4A on chromosome arms in metaphase was measured in Kif4A channel on sum-intensity projections of all z-planes ( nine planes in immunostained , and three in opto and BAC cells ) using Polygon selection tool in Fiji by encompassing chromosomes in DAPI or SiR-DNA channel .\",\n \"In order to avoid the Kif4A signal on the spindle microtubules , the signal was measured only on the parts of the chromosome arms protruding into cytoplasm away from the spindle .\",\n \"For each cell , mean value of Kif4A arm intensity was calculated and corrected for background cytoplasm intensity measured in 2 . 5 × 2 . 5 µm rectangle by subtracting it from mean intensity of Kif4A .\",\n \"In immunostained cells only those with Kif4A localized on chromosomes were taken into account as in some sessions the Kif4A signal was very strong on the whole spindle and not present on the chromosomes both in untreated and PRC1 siRNA-treated cells .\",\n \"The intensity of GFP-Kif4A on chromosomes in anaphase was measured in GFP-Kif4A channel on sum-intensity projections of all three z-planes using Polygon selection tool in Fiji by encompassing chromosomes in SiR-DNA channel .\",\n \"Background cytoplasm intensities in GFP-Kif4A channel were measured in 2 . 5 × 2 . 5 µm rectangle and subtracted from measured mean intensities of GFP-Kif4A .\",\n \"Corrected intensities were divided by the number of focal planes .\",\n \"In anaphase , measurements were performed 4 min after anaphase onset .\",\n \"Anaphase onset was defined as the timeframe when separation of majority of sister chromatids in SiR-DNA channel occurred .\",\n \"The mean bridging fiber intensities of Kif18A in all treatments were obtained on a single z-plane using 0 . 4 × 0 . 4 µm rectangles in Fiji covering the Kif18A signal in the bridge .\",\n \"The rectangles of the same dimension were used to obtain the Kif18A signal from the sister k-fiber tips and the mean value for the pair was calculated .\",\n \"Since the measurements of Kif18A were mostly obtained on the outermost fibers , these values were corrected by subtracting mean background cytoplasm intensity measured in a 2 . 5 × 2 . 5 µm rectangle .\",\n \"The intensity of MKLP1 in the bridging fibers was measured in GFP channel on a single z-plane using 0 . 4 × 0 . 4 µm rectangle in Fiji by placing it on each bridging fiber visible in the SiR-tubulin channel .\",\n \"These intensities were corrected for background GFP intensity in cytoplasm measured in 2 . 5 × 2 . 5 µm rectangle .\",\n \"For quantification of PRC1 knock-down by siRNA in HeLa BAC MKLP1-GFP cell line , PRC1 intensity was quantified in the same manner as in U2OS cells: on a sum-intensity projection of five focal planes in a way that mean spindle intensity was background corrected by subtracting mean intensity in the cytoplasm , .\",\n \"The intensity of CENP-E and CLASP1 on plus ends-of k-fibers was measured using 0 . 4 × 0 . 4 µm rectangle encompassing plus-end of k-fibers on a single z-plane .\",\n \"Intensities were corrected for the intensity in cytoplasm measured using 2 . 5 × 2 . 5 µm rectangle in the central z-plane as this intensity was similar between different z-planes .\",\n \"For analysis of CLASP1 and CENP-E intensity on plus-ends of k-fibers only cells with similar intensities on the spindle within each treatment were taken into account as there was a variation in expression level among cells .\",\n \"Measurements regarding Kif4A , Kif18A , MKLP1 , CENP-E , and CLASP1 were performed in two time-points in metaphase: before the blue light was switched on and at the end of continuous exposure to the blue light .\",\n \"The intensities were normalized on the mean value of control at each treatment , that is , before the blue light was switched on for acute PRC1 removal and untreated for long-term depletion .\",\n \"Quantification of the signal length of PRC1-GFP in mock , Kif4A , and Kif18A siRNA-treated HeLa cells was performed by drawing a 5-pixel-thick pole-to-pole line along individual bundles and calculated as the width of the peak of the PRC1-GFP signal intensity .\",\n \"Statistical analysis was performed in MATLAB ( RRID:SCR_001622; MathWorks , Natick , MA , USA ) and RStudio ( RRID:SCR_000432; R Foundation for Statistical Computing , Vienna , Austria ) .\",\n \"Normality of the data was inspected by Q-Q plots and Shapiro-Wilk test of normality .\",\n \"Two groups with normally distributed data were tested with two-tailed t-test , while more than two groups were tested with one-way ANOVA followed by Tukey HSD post hoc test .\",\n \"Two groups with non-normally distributed data were tested with Mann-Whitney test , while more than two groups were tested with Kruskal-Wallis rank sum test followed by pairwise Wilcoxon rank sum test .\",\n \"Used statistical analysis is noted in figure captions .\",\n \"Proportions were statistically compared with test for equality of proportions , two-proportions z-test .\",\n \"For data with expected count smaller than 5 , Yates's correction for continuity was used .\",\n \"Graphs were generated in MATLAB ( MathWorks ) and RStudio ( R Foundation for Statistical Computing ) .\",\n \"ImageJ ( RRID:SCR_003070; National Institutes of Health , Bethesda , MD , USA ) was used to crop and rotate images , and to adjust brightness and contrast to the entire image , which was applied equally to all images in the same panel .\",\n \"The images are rotated in order for the spindle to be horizontal in every time frame .\",\n \"To remove high-frequency noise in displayed images a Gaussian blur filter with a 0 . 5-pixel sigma ( radius ) was applied where stated .\",\n \"Figures were assembled in Adobe Illustrator CS5 ( RRID:SCR_010279; Adobe Systems , Mountain View , CA , USA ) .\"\n ]\n]"},"abstract":{"kind":"list like","value":["During metaphase , chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces .","However , the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown .","Here , we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment .","Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal , which was accompanied by misaligned and lagging kinetochores .","Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal , suggesting that these proteins regulate the overlap length of bridging microtubules .","We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promote chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers ."],"string":"[\n \"During metaphase , chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces .\",\n \"However , the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown .\",\n \"Here , we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment .\",\n \"Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal , which was accompanied by misaligned and lagging kinetochores .\",\n \"Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal , suggesting that these proteins regulate the overlap length of bridging microtubules .\",\n \"We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promote chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers .\"\n]"},"summary":{"kind":"list like","value":["Before cells divide to create copies of themselves , they need to duplicate their genetic material .","To help split their DNA evenly , they build a machine called the mitotic spindle .","The mitotic spindle is made of fine , tube-like structures called microtubules , which catch the chromosomes containing the genetic information and line them up at the center of the spindle .","Microtubules push and pull the chromosomes by elongating or shortening their tips .","But it remains unclear how the microtubules know when the chromosomes have reached center point .","One way to find out is to remove proteins that accumulate in the middle of the spindle during division , such as the protein PRC1 , which helps to assemble a subset of microtubules called bridging fibers , and the proteins Kif4A and Kif18A , which work like molecular rulers , shortening long microtubules .","Usually , scientists would delete one of these proteins to see what impact this has .","However , these experiments take days , giving the cell enough time to adapt and thus making it difficult to study the role of each of the proteins .","Here , Jagrić , Risteski , Martinčić et al . used light to manipulate proteins at the exact moment of chromosome alignment and to move PRC1 from the spindle to the cell membrane .","Consequently , Kif4A and Kif18A were removed from the spindle center .","This caused the bridging fibers , which overlap with the microtubules that connect to the chromosomes , to become thinner .","Jagrić et al . discovered that without the molecular ruler proteins , the bridging fibers were also too long .","This increased the overlap between the microtubules in the center of the spindle , causing the chromosomes to migrate away from the center .","This suggests that the alignment of chromosomes in the middle of the spindle depends on the bridging microtubules , which need to be of a certain length to effectively move and keep the chromosomes at the center .","Thus , forces that move the chromosomes are generated both at the tips of the microtubules and along the wall of microtubules .","These results might inspire other researchers to reassess the role of bridging fibers in cell division .","The optogenetic technique described here could also help to determine the parts other proteins have to play .","Ultimately , this might allow researchers to identify all the proteins needed to align the chromosomes ."],"string":"[\n \"Before cells divide to create copies of themselves , they need to duplicate their genetic material .\",\n \"To help split their DNA evenly , they build a machine called the mitotic spindle .\",\n \"The mitotic spindle is made of fine , tube-like structures called microtubules , which catch the chromosomes containing the genetic information and line them up at the center of the spindle .\",\n \"Microtubules push and pull the chromosomes by elongating or shortening their tips .\",\n \"But it remains unclear how the microtubules know when the chromosomes have reached center point .\",\n \"One way to find out is to remove proteins that accumulate in the middle of the spindle during division , such as the protein PRC1 , which helps to assemble a subset of microtubules called bridging fibers , and the proteins Kif4A and Kif18A , which work like molecular rulers , shortening long microtubules .\",\n \"Usually , scientists would delete one of these proteins to see what impact this has .\",\n \"However , these experiments take days , giving the cell enough time to adapt and thus making it difficult to study the role of each of the proteins .\",\n \"Here , Jagrić , Risteski , Martinčić et al . used light to manipulate proteins at the exact moment of chromosome alignment and to move PRC1 from the spindle to the cell membrane .\",\n \"Consequently , Kif4A and Kif18A were removed from the spindle center .\",\n \"This caused the bridging fibers , which overlap with the microtubules that connect to the chromosomes , to become thinner .\",\n \"Jagrić et al . discovered that without the molecular ruler proteins , the bridging fibers were also too long .\",\n \"This increased the overlap between the microtubules in the center of the spindle , causing the chromosomes to migrate away from the center .\",\n \"This suggests that the alignment of chromosomes in the middle of the spindle depends on the bridging microtubules , which need to be of a certain length to effectively move and keep the chromosomes at the center .\",\n \"Thus , forces that move the chromosomes are generated both at the tips of the microtubules and along the wall of microtubules .\",\n \"These results might inspire other researchers to reassess the role of bridging fibers in cell division .\",\n \"The optogenetic technique described here could also help to determine the parts other proteins have to play .\",\n \"Ultimately , this might allow researchers to identify all the proteins needed to align the chromosomes .\"\n]"},"year":{"kind":"string","value":"2021"}}},{"rowIdx":4287,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["biochemistry and chemical biology","structural biology and molecular biophysics"],"string":"[\n \"biochemistry and chemical biology\",\n \"structural biology and molecular biophysics\"\n]"},"title":{"kind":"string","value":"Regulation of multispanning membrane protein topology via post-translational annealing"},"id":{"kind":"string","value":"elife-08697-v2"},"sections":{"kind":"list like","value":[["The cotranslational integration and topogenesis of EmrE and its mutants is simulated using a recently developed CG model ( Zhang and Miller , 2012a ) , which we employ essentially unchanged from its original introduction .","Figure 1 illustrates the CG representation of a nascent protein and the protocol for simulating membrane integration .","The ribosome , translocon , and nascent protein are all composed of CG beads .","Each bead has a diameter of σ = 0 . 8 nm to represent approximately three amino-acid residues .","This bead diameter is similar to the Kuhn length of polypeptides ( Staple et al . , 2008 ) so that the nascent protein can be treated as a freely jointed chain .","The surrounding solvent and lipid bilayer are included implicitly , a technique that is used in other CG models of the translocon ( Rychkova and Warshel , 2013 ) .","The time-evolution of nascent protein configurations is calculated using Brownian dynamics with a 100 ns timestep .","The kinetics of the LG are modeled as stochastic transitions between a closed conformation , which prevents the nascent protein from exiting from the channel interior to the membrane , and an open conformation , which removes the barrier to membrane entry .","All bead positions are projected onto the plane that passes along the translocon channel axis between the helices forming the LG .","This off-lattice 2D approximation reflects the cylindrical geometry of the channel and is inspired by previous models of biopolymer translocation through nanopores ( Huopaniemi et al . , 2006 ) .","Beads representing the ribosome enclosure and translocon are placed to approximate their structures ( Van den Berg et al . , 2004; Frauenfeld et al . , 2011 ) .","Two negative charges are placed on a bead at the cytosolic end of the translocon LG , whereas two positive charges are placed on a bead at the periplasmic end of the translocon LG .","This charge distribution reflects the position of conserved charged residues ( White and von Heijne , 2004 ) near the translocon LG that have been previously shown to affect single-spanning protein topogenesis ( Goder et al . , 2004 ) .","Full details of the model are provided in Appendix 1 . 10 . 7554/eLife . 08697 . 003Figure 1 . Schematic of Sec-mediated cotranslational integration of EmrE and corresponding simulation representation .","( A ) At top , an illustration of the structural motifs in EmrE , including indication of the charged residues in the soluble loops with black circles and the transmembrane domain ( TMD ) /loop numbering scheme that is employed in the text; below , the corresponding sequence of coarse-grained ( CG ) beads that represent the EmrE amino-acid sequence .","TMDs and loops are assigned based on the hydropathy plot and consensus topology prediction shown in Figure 1—figure supplement 1 .","( B ) At top , a schematic illustration of the sequential integration of TMDs to obtain a multispanning Nperi/Cperi topology , in which both the N- and C-terminal loops are positioned in the periplasm , according to the cotranslational model; below , representative simulation snapshots of EmrE as the nascent protein grows during translation , integrates into the membrane , and exits the channel in the Nperi/Cperi multispanning topology .","The nascent protein is colored according to the legend at top , the ribosome is brown , and the translocon is green with translocon charges labeled explicitly . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 00310 . 7554/eLife . 08697 . 004Figure 1—figure supplement 1 . Hydropathy plot for EmrE . The amino-acid sequence is shown on the right with positive charges highlighted in red .","The transfer free energy according to the Wimley-White scale ( Wimley et al . , 1996 ) is plotted as a black line with a 7-residue moving average overlaid as a red line .","Regions predicted by TOPCONS to correspond to loops ( L1-L5 ) or TMDs ( TMD1-TMD4 ) are shaded according to the legend at right . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 00410 . 7554/eLife . 08697 . 005Figure 1—figure supplement 2 . Simulation snapshot illustrating the initial configuration comprised of 9 CG beads . Positions of several beads in the ribosome ( brown ) and translocon ( green ) are labeled , with each labeled bead indicated by a black dot .","Positions are labeled in units of σ .","DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 005 The CG model is well-suited to simulating the kinetics of cotranslational IMP integration , a process that is challenging for atomistic models ( Zhang and Miller , 2010; Gumbart et al . , 2011; Zhang and Miller , 2012b; Rychkova and Warshel , 2013 ) due to the large system size ( >100 , 000 atoms ) and the long timescale ( minutes ) of translation .","We note that the model does not include nascent protein secondary/tertiary structure , charged lipids , protein chaperones , or an electrostatic potential across the membrane .","However , the model does include explicit LG/translation dynamics , electrostatic interactions with the translocon , water/bilayer transfer free energies , and a direct mapping between the nascent protein sequence and the CG representation .","The model thus captures the major physicochemical features of the translocon-membrane system ( White and von Heijne , 2004 ) .","Moreover , the model has been shown to accurately predict features of single-spanning IMP integration and topogenesis ( Zhang and Miller , 2012a ) , including the sigmoidal dependence of stop-transfer efficiency on TMD hydrophobicity ( Hessa et al . , 2005 ) , the inversion of signal-anchor orientation during translation ( Goder and Spiess , 2003 ) , and the effect of translation rates and sequence features on signal-anchor orientation ( Goder and Spiess , 2003 ) .","In particular , the model has been shown ( Zhang and Miller , 2012a ) to correctly describe integration processes that are governed either by thermodynamics ( Hessa et al . , 2005 ) or kinetics ( Goder and Spiess , 2003 ) , and it has provided a means of understanding the competition between such effects .","The model has also been shown to correctly predict the dominant topology for a three-TMD multispanning IMP with a strong positive-inside bias ( Zhang and Miller , 2012a ) .","The strong agreement between simulation and experimental results presented in this work further indicates that IMP topological determinants are captured at this CG resolution ."],["For all 16 of the EmrE and nEmrE mutants with single added charges studied by Seppälä et al . ( 2010 ) , Figure 2 compares the experimentally observed IMP topologies with the prediction from the CG model .","Specifically , the figure compares the fraction of fully integrated proteins that adopt the Ncyto/Ccyto topology , with the remainder in the Nperi/Cperi topology .","The top and bottom rows show variants of EmrE and nEmrE respectively .","Each mutant differs only in the number and location of charges in the hydrophilic loops .","A schematic of each mutant drawn with the dominant topology predicted from simulations is included; positive charges are indicated as filled-in circles with additional charges relative to EmrE ( top row ) or nEmrE ( bottom row ) highlighted in red .","The topologies determined experimentally in Seppälä et al . ( 2010 ) are expressed as the fraction of Ncyto/Ccyto topologies by dividing the cell activity of each protein coexpressed with the EmrE ( Nperi ) mutant by the total growth of the protein coexpressed with either the EmrE ( Nperi ) or EmrE ( Ncyto ) mutant ( Seppälä et al . , 2010 ) , as described in the Experimental interpretation of EmrE topology section of the ‘Materials and methods’ . 10 . 7554/eLife . 08697 . 006Figure 2 . Topologies determined from simulations ( blue ) and compared to the experiments of Seppälä et al . ( 2010 ) ( red ) , reporting the fraction of fully integrated integral membrane protein ( IMP ) configurations in the Ncyto/Ccyto topology . Error bars indicate the standard error measured from independent blocks of simulations or taken from Seppälä et al . ( 2010 ) .","The dominant topology for each mutant is indicated schematically with additional positive charges relative to EmrE ( top ) or nEmrE ( bottom ) drawn as red dots . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 00610 . 7554/eLife . 08697 . 007Figure 2—figure supplement 1 . Robustness of the distribution of topologies to the trajectory termination criteria . The distribution of topologies for the EmrE , K3 , nK3 , and T28R mutants are computed using alternative trajectory termination criteria and compared to the distribution of topologies using the original protocol ( shown in Figure 2 ) .","At left , the plot illustrates the effect of extending each original CG trajectory by 50 s .","At right , the plot illustrates the effect of terminating trajectories at a distance of 20σ , rather than 4 . 5σ , from the origin .","The dashed line indicates perfect correlation .","Additional details on these calculations are included in the Robustness checks for the trajectory termination criteria section of the ‘Materials and methods’ .","DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 00710 . 7554/eLife . 08697 . 008Figure 2—figure supplement 2 . Correlation between the topologies determined from simulations ( x-axis ) and compared to the experiments of Seppälä et al . ( 2010 ) ( y-axis ) , reporting the fraction of fully integrated IMP configurations in the Ncyto/Ccyto topology . Mutants lying in the shaded quadrants have the same dominant topology in both the experiments and simulations .","The experimental and simulation measurements are linearly correlated with a Pearson correlation coefficient of r = 0 . 92 . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 008 It is clear from Figure 2 that the simulations are in excellent qualitative agreement with the experiments by correctly predicting the near 1:1 stoichiometry of wild-type EmrE and identifying the dominant topology for nearly all of the proteins considered .","Figure 2—figure supplement 2 illustrates that the distribution of topologies determined experimentally and the distribution of topologies measured from the simulations are linearly correlated ( Pearson correlation coefficient , r = 0 . 92 ) ; points lying in the two shaded quadrants of the graph correspond to proteins for which the simulations and experiments predict consistent topologies .","All mutants , with the exception of A52K , have the same dominant topology in the simulations as in the experiments within the statistical error .","The agreement between simulations and experiments suggests that the CG model correctly reproduces the essential molecular features of topogenesis; in the following , we analyze the ensembles of CG trajectories that give rise to these computed IMP topologies .","To investigate the molecular processes that govern the establishment of EmrE topology , we first examine the kinetics by which fully integrated topologies are reached .","As a function of time , Figure 3A shows the fraction of CG trajectories in which the studied protein reaches a fully integrated topology for several EmrE mutants and the CB mutant .","0 s corresponds to the end of translation and negative values of time correspond to the period that precedes the end of ribosomal translation in which the nascent protein is still attached to the ribosome .","Over 90% of the CB mutant trajectories reach the Ncyto/Ccyto topology within 3 s following the completion of translation and thus rapidly integrate as expected for the cotranslational model ( Blobel , 1980; Wessels and Spiess , 1988; Sadlish et al . , 2005 ) ; mechanistic features of individual TMD integration steps are discussed in the Cotranslational integration pathways section of the ‘Materials and methods’ .","In contrast , all variants of EmrE reach a fully integrated topology much more slowly , requiring hundreds of seconds for some CG trajectories to fully integrate ( see also Figure 3—figure supplement 2 ) . 10 . 7554/eLife . 08697 . 009Figure 3 . Kinetics of EmrE topogenesis .","( A ) Fraction of CG trajectories in which all TMDs are fully integrated in a multispanning topology , plotted as a function of time for several mutants .","( B ) Fraction of CG trajectories in which each TMD is integrated , plotted as a function of time for the cotranslationally-biased ( CB ) mutant ( top ) and EmrE ( bottom ) .","The snapshots show an example of a simulation in which TMD4 of EmrE does not integrate during translation .","In both panels , 0 s corresponds to the end of translation and negative values of time correspond to the period that precedes the end of ribosomal translation . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 00910 . 7554/eLife . 08697 . 010Figure 3—figure supplement 1 . Pathways for the cotranslational integration of TMDs into the membrane . At left , three distinct integration pathways are illustrated; the definition of each pathway is described in the Cotranslational integration pathways section of the ‘Materials and methods’ .","In the ‘channel-sliding’ pathway , the TMD partially enters the channel , then crosses the lateral gate ( LG ) , then fully integrates into the membrane .","In the ‘interface-sliding’ pathway , the TMD enters the cytoplasm through the gap between the translocon and ribosome , prior to undergoing membrane integration .","In the ‘in-out’ pathway , the TMD fully spans the channel prior to membrane integration .","At right , the fraction of cotranslational TMD integration events that exhibit each of these three pathways is presented for all four TMDs and for both the EmrE and nEmrE mutants .","The dominant cotranslational integration pathway is the ‘channel-sliding’ pathway for all TMDs in both mutants . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 01010 . 7554/eLife . 08697 . 011Figure 3—figure supplement 2 . Simulation time necessary for 50% , 90% , and 95% of the CG trajectories to reach fully integrated topologies for each mutant . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 01110 . 7554/eLife . 08697 . 012Figure 3—figure supplement 3 . Effect of loop length on integration trajectories .","( A ) Schematic illustrations of a new EmrE mutant with elongated , 10-bead loops , and a new CB mutant with shortened , 5-bead loops .","( B ) Fraction of CG trajectories that reach fully integrated topologies as a function of time for the EmrE and CB mutants with both loop lengths .","The integration kinetics for the EmrE mutants clearly show that longer loops increase the timescale of post-translational annealing , due to the reduction of the loop-flipping frequency for the longer soluble loops .","The corresponding effect is less clear for the two CB mutants , since the timescale for the loop-flipping frequency is dominated by the large positive charges on these loops , masking the effect of changing the loop length .","( C ) Average loop positions in the end-of-translation ( EOT ) ensemble for both the EmrE and CB mutants , presented in terms of the fraction of configurations for which each loop occupies the cytoplasm .","For the EmrE mutants , changing the loop length leads to relatively small shifts in the EOT ensemble; however , for the CB mutants , the effect of loop length on the EOT ensemble is much larger , with the new short-loop CB mutant exhibiting a much weaker bias towards the Ncyto/Ccyto topology in the EOT ensemble . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 012 The slow post-translational integration of the dual-topology EmrE mutants is due to the fact that a significant fraction of trajectories exhibit configurations in which some TMDs are not fully integrated at the end of translation .","As a function of time , Figure 3B shows the fraction of CG trajectories in which each TMD is integrated for both the CB mutant ( top ) and EmrE ( bottom ) .","TMDs in the CB mutant integrate sequentially with near 100% efficiency during translation , which is consistent with the standard cotranslational model of topogenesis ( c . f . Figure 1 ) and explains the rapid timescale for fully integrating into a multispanning topology shown in Figure 3A .","In contrast , the TMDs of EmrE exhibit only partial integration , even at long times following the completion of translation .","Snapshots of a typical misintegrated TMD in EmrE are shown in Figure 3B .","Various experiments have indicated that such configurations with misintegrated TMDs arise due to frustration from charges placed in consecutive loops ( Gafvelin and von Heijne , 1994 ) , the strong orientational preference of a neighboring TMD ( Öjemalm et al . , 2012 ) , or the weak stop-transfer efficiency of marginally hydrophobic TMDs ( Moss et al . , 1998 ) .","Consistent with these experimental observations , the simulations in Figure 3B find that the weakly hydrophobic TMD1 of EmrE integrates the least efficiently , followed by TMD4 which is flanked by two charged loops ."],["In this work , we utilize a recently developed CG computational approach ( Zhang and Miller , 2012a ) that enables the direct simulation of Sec-facilitated membrane integration of proteins on biological timescales to investigate the topogenesis of the dual-topology EmrE protein and its mutants .","In addition to demonstrating excellent agreement with the experimentally observed topologies of EmrE and its mutants ( Seppälä et al . , 2010 ) , the simulations reveal a novel mechanism for the regulation of topogenesis in multi-spanning membrane proteins , in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach final , fully integrated topologies .","The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins .","The inclusion of charged residues on the soluble loops of the IMP can lead to significant changes in the distribution of fully integrated topologies by both altering the ensemble of protein configurations at the end of ribosomal translation , as well as by altering the available kinetic pathways that lead to fully integrated topologies .","This analysis leads to a number of experimentally testable predictions regarding IMP topogenesis .","In particular , the results of Figure 8 predict that the mutation of charged residues near the cytoplasmic or periplasmic openings of the translocon channel will lead to significant shifts in the observed topology of several EmrE mutants .","More generally , we note that any effect of channel mutations on the fully integrated IMP topology would indicate that kinetic effects during translation influence topogenesis , as suggested by the proposed mechanism .","Additionally , we predict that the introduction of IMP mutations that significantly alter the EOT ensemble with respect to the cytosolic localization of the slowest-flipping soluble loop , either by introducing charge mutations or by changing TMD hydrophobicity , will influence the multispanning IMP topology .","Although the current manuscript primarily focuses on the mechanism of topogenesis in the dual-topology EmrE protein , the mechanism and simulation analysis presented here has broader implications for topogenesis in other multispanning IMPs .","For EmrE and its mutants , we find that a significant fraction of the IMP configurations are misintegrated upon completion of ribosomal translation and undergo subsequent post-translational annealing to reach fully integrated topologies .","In contrast , a CB mutant exhibits an essentially fully integrated ensemble of configurations at the time that ribosomal translation completes .","For other IMPs , a combination of these behaviors may well be expected ( Lu et al . , 2000; Lambert and Prange , 2001; Kanki et al . , 2002; Skach , 2009; Öjemalm et al . , 2012; Bowie , 2013; Virkki et al . , 2014 ) , with some fraction of the nascent protein configurations reaching fully integrated topologies at the completion of ribosomal translation and some fraction reaching misintegrated configurations that subsequently undergo post-translational annealing .","Indeed , the importance of chaperone proteins such as YidC or Sec62 that post-translationally rescue misintegrated TMDs ( Sommer et al . , 2013; Zhu et al . , 2013; Aviram and Schuldiner , 2014; Jung et al . , 2014 ) may be connected to this necessary process of annealing initially misintegrated IMP configurations towards fully integrated topologies .","The emerging understanding of the role of the Sec translocon in regulating IMP topogenesis , as well as advances in the methodologies for probing and modifying interactions between the nascent protein and the translocon complex , hold intriguing possibilities for the prediction and control of protein folding in cellular environments ."],["Loop-flipping frequencies are calculated by monitoring loop positions with respect to the membrane in 1-ms time intervals .","A loop-flipping event is counted if a given loop switches from a cytoplasmic to periplasmic ( or vice versa ) position according to the definitions in the main text and if the y-position of the reference bead in the loop ( defined in the main text ) is at least 4 . 5σ after the flip ( to exclude counting the rapid flipping of loops within the translocon channel ) .","The loop-flipping frequencies presented in Figure 6 are obtained by averaging the frequency of loop-flipping events in each trajectory .","Sufficiently infrequent loop flips will not occur in every trajectory , but such events are observed in the combined ensemble of trajectories .","To examine the effect of neighboring TMDs on the loop-flipping frequency , we separately calculate the loop-flipping frequency of each loop for configurations in which zero , one , or two of the neighboring TMDs is misintegrated ( discussed in the Rate of loop-flipping depends on charge mutations section of the ‘Results’ ) .","In Seppälä et al . ( 2010 ) , the dominant topologies of EmrE mutants are determined by measuring the growth of E . coli cells in the presence of ethidium bromide ( EtBr ) .","EtBr is toxic to E . coli , but antiparallel EmrE dimers , in which the two monomers forming the dimer have opposite topologies , confer drug resistance .","EmrE dimerization can also be suppressed by including an E14D mutation .","The topology of an EmrE variant with the E14D mutation can thus be inferred by coexpressing the mutant with another EmrE variant of known topology , as any resulting drug resistance ( and cell growth ) can be attributed to the formation of antiparallel dimers .","To enable a direct comparison between the topologies measured from simulations and the experimental results , we convert the experimentally-measured cell activities from Seppälä et al . ( 2010 ) to the fraction of Ncyto/Ccyto topologies by assuming a linear relationship between cell growth and the population of antiparallel EmrE dimers .","The fraction of Ncyto/Ccyto topologies is calculated as ( 1 ) f ( Ncyto/Ccyto ) =A ( Nperi ) A ( Ncyto ) +A ( Nperi ) , where A ( Ncyto ) and A ( Nperi ) are the experimentally-measured cell activities for cells coexpressing the EmrE ( Ncyto ) and EmrE ( Nperi ) mutants , respectively .","Greater cell growth in the presence of the EmrE ( Nperi ) mutant , which exhibits a single dominant Nperi/Cperi topology , indicates that the mutant of interest exhibits a larger fraction of the opposite Ncyto/Ccyto topology , and vice versa for growth in the presence of the EmrE ( Ncyto ) mutant .","Experimental values for the activities of the EmrE and nEmrE mutants are taken from Figure 2 and Figure S1 of Seppälä et al . ( 2010 ) , respectively; these values are used to compute the fraction of Ncyto/Ccyto topologies reported in Figure 2 of the current paper .","Values for the activities of the ( Nout ( E14D ) + Nin ) and the Nout constructs from Figure 1 of Seppälä et al . ( 2010 ) are used to approximate the topology of the EmrE ( Nperi ) mutant , while the activities of the ( Nin ( E14D ) + Nout ) and the Nin constructs from the same figure are used to approximate the topology of the EmrE ( Ncyto ) mutant .","Error bars are approximated via standard error propagation techniques based on Equation","1 . In Figure 7A , the average position of the slowest-flipping loop relative to the membrane in the EOT ensemble is compared with the average position of that same loop in the ensemble of fully integrated configurations at the end of the CG trajectories .","Four mutants ( K3 , L85R , nEmrE , and nT28R1 ) , however , have two slow-flipping loops with similar loop-flipping frequencies ( Figure 6 ) , and two of these mutants ( K3 and L85R ) deviate most significantly in terms of the correlation in Figure 7A .","To better understand the effect of multiple slow-flipping loops on the correlation between the EOT ensemble and the final topology , the current section provides additional analysis in which a more sophisticated definition of the ‘slowest-flipping loop’ is employed for the four mutants that exhibit a pair of slow-flipping loops .","Below , we present this alternative definition , which leads to a slightly better correlation between the EOT ensemble and the ensemble of fully integrated configurations , as plotted in Figure 7—figure supplement","1 . The alternative definition of the slowest-flipping loop for mutants with two slow-flipping loops is given by ϕEOT and ϕFI , which report on the average position of the two slow-flipping loops in the EOT ensemble and in the ensemble of fully integrated configurations , respectively .","The quantity ϕFI reports on the average position of the two slow-flipping loops in the ensemble of fully integrated configurations at the end of the CG trajectories .","For the L85R and nEmrE mutants , the two slow-flipping loops ( L2/L4 and L1/L5 , respectively ) reach positions on the same side of the membrane in either fully integrated topology; for these mutants , ϕFI is defined as the fraction of fully integrated configurations for which both slow-flipping loops are positioned in the cytoplasm .","For the K3 and nT28R1 mutants , the two slow-flipping loops ( L1 and L2 ) reach positions on opposite sides of the membrane in either fully integrated topology; for these mutants , ϕFI is defined as the fraction of fully integrated configurations for which L1 is positioned in the cytoplasm and L2 is positioned in the periplasm .","For the nEmrE , K3 , and nT28R1 mutants , ϕFI is equivalent to the fraction of CG trajectories that reach the fully integrated Ncyto/Ccyto topology , whereas for the L85R mutant , ϕFI is equivalent to the fraction of CG trajectories that reach the fully integrated Nperi/Cperi topology .","The quantity ϕEOT reports on the average position of the two slow-flipping loops in the EOT ensemble .","For each mutant , ϕEOT is defined as ( 2 ) ϕEOTL85R=0 . 5[f ( cyto ) L2+f ( cyto ) L4]ϕEOTnEmrE=0 . 5[f ( cyto ) L1+f ( cyto ) L5]ϕEOTK3=0 . 5[1+f ( cyto ) L1−f ( cyto ) L2]ϕEOTnT28R1=0 . 5[1+f ( cyto ) L1−f ( cyto ) L2] , where f ( cyto ) Li is the fraction of configurations in the EOT ensemble for which loop Li is in the cytoplasm .","As for the previous definition of ϕFI , this definition accounts for the fact that the two slow-flipping loops of the L85R and nEmrE mutants reach the same side of the membrane in the fully integrated topologies , while the two slow-flipping loops of the K3 and nT28R1 mutants reach opposite sides of the membrane in the fully integrated topologies .","The definition in Equation 2 additionally assumes that the post-translational annealing of misintegrated configurations in the EOT ensemble is equally rate-limited by the two slow-flipping loops .","Using these alternative definitions for the position of the slowest-flipping loop ( i . e . , ϕFI and ϕEOT ) , Figure 7—figure supplement 1 compares the average position of the slowest-flipping loop in the EOT ensemble to the average position of that same loop in the ensemble of fully integrated configurations .","Having more carefully accounted for the effect of both slow-flipping loops in the K3 , L85R , nEmrE , and nT28R1 mutants , this figure reveals a slight improvement in the correlation ( R2 = 0 . 88 vs R2 = 0 . 85 ) in comparison to the results in Figure 7A .","Alternative trajectory termination criteria are tested to ensure the robustness of the simulated distribution of multispanning topologies presented in Figure","2 . As a first alternative , the original set of CG trajectories are extended by 50 s , and the distribution of topologies is determined from the position of the slowest-flipping loop at the end of the extended trajectories .","As a second alternative , the distribution of topologies is calculated from the subset of original CG trajectories that reach fully integrated topologies in which all beads in the TMDs are at least 20σ , rather than 4 . 5σ , from the origin .","These robustness checks are presented in Figure 2—figure supplement 1 and exhibit excellent correlation with the results obtained using the original protocol .","Additionally , Figure 7B shows results in which the CG trajectories are terminated at a range of fixed times following the end of ribosomal translation .","Again , the results using this alternative trajectory termination criterion are in good agreement with the results obtained using the original protocol ( indicated in dashed lines in Figure 7B ) .","From the ensemble of CG trajectories , it is possible to examine the pathways by which individual TMDs undergo Sec-facilitated cotranslational integration .","In particular , following the definitions of Cymer et al . ( 2014 ) , it is possible to characterize each cotranslational TMD integration event as corresponding to either the ‘channel-sliding’ , ‘interface-sliding’ , or ‘in-out’ pathways .","Simulation snapshots illustrating the three pathways are shown in Figure 3—figure supplement","1 . Each pathway is defined in terms of the series of intermediate states that are visited by the TMD prior to membrane integration .","To characterize these intermediate states , the following geometric regions are defined ( see Figure 1—figure supplement 2 ) .","The channel region is defined as that for which −2σ ≤ x ≤ 2σ and −2σ ≤ y ≤ 2σ , the membrane region is defined as that for which −2σ ≤ x ≤ 2σ and y > 2σ , the ribosome region is defined as that for which −11σ ≤ x < −2σ and −8 . 5σ ≤ y ≤ 4 . 5σ , and the cytoplasm region is defined as the region outside of the ribosome for which x < −2σ .","Finally , a bead is considered to overlap the LG if it is within a distance of σ to any lateral-gate bead .","We now define the four intermediate states .","Intermediate state 1 ( IS1 ) is that for which the TMD partially enters the channel; it is defined as the set of configurations for which at least two TMD beads are in the channel region and zero TMD beads are in the membrane region .","Intermediate state 2 ( IS2 ) is that for which the TMD fully spans the membrane while in the channel; it is defined as the set of configurations for which all four TMD beads are in the channel region and the two hydrophilic beads that flank the TMD occupy opposite sides of the membrane .","Intermediate state 3 ( IS3 ) is that for which the TMD accesses the membrane interior via the LG; it is defined as the set of configurations for which at least one TMD bead occupies the membrane region , the remaining three TMD beads occupy either the channel or membrane regions , and at least one TMD bead overlaps with the LG .","Intermediate state 4 ( IS4 ) is that for which the TMD accesses the cytoplasm region without accessing the channel region; it is defined as the set of configurations for which each of the four TMD beads occupies either the ribosome , membrane , or cytoplasm regions and for which at least one of the hydrophilic beads that flank the TMD occupies the cytoplasm region .","In this analysis , cotranslational TMD integration events are defined as those for which the TMD reaches a membrane integrated configuration ( for which all four beads of the TMD span the membrane region and the two hydrophilic flanking beads occupy opposite sides of the membrane ) before reaching a misintegrated configuration ( for which both hydrophilic flanking beads occupy the same side of the membrane and for which all TMD beads and both flanking beads lie outside of the channel and ribosome regions ) .","Using the definitions of intermediate states , the cotranslational integration pathways are defined as follows .","In the ‘channel-sliding’ pathway , the TMD partially enters the channel , then crosses the LG , then fully integrates into the membrane; a trajectory thus exhibits this pathway if a TMD visits IS1 , IS2 , and membrane integration in chronological order and without visiting any other intermediate states .","In the ‘interface-sliding’ pathway , the TMD enters the cytoplasm through the gap between the translocon and ribosome , prior to undergoing membrane integration; a trajectory thus exhibits this pathway if a TMD visits IS4 on the way to membrane integration .","In the ‘in-out’ pathway , the TMD fully spans the channel prior to membrane integration; a trajectory thus exhibits this pathway if a TMD visits IS3 on the way to membrane integration without visiting IS4 .","At right , Figure 3—figure supplement 1 shows the relative fraction of cotranslational TMD integration events that exhibit each of these three pathways .","It is clear that the dominant cotranslational integration pathway for all four TMDs in both the EmrE and nEmrE mutants is the ‘channel-sliding’ pathway .","This same pathway was also observed in the previous study of single-spanning proteins using the CG model ( Zhang and Miller , 2012a ) and similar configurations were observed in long-timescale atomistic molecular dynamics simulations of the early stages of cotranslational membrane integration ( Zhang and Miller , 2012b ) .","We find that only a small number of CG trajectories exhibit the ‘interface-sliding’ pathway .","Finally , we note that the dominant cotranslational integration pathway is likely to depend on the IMP sequence , and the ‘channel-sliding’ behavior may be less dominant in other IMPs with less hydrophobic TMDs ."]],"string":"[\n [\n \"The cotranslational integration and topogenesis of EmrE and its mutants is simulated using a recently developed CG model ( Zhang and Miller , 2012a ) , which we employ essentially unchanged from its original introduction .\",\n \"Figure 1 illustrates the CG representation of a nascent protein and the protocol for simulating membrane integration .\",\n \"The ribosome , translocon , and nascent protein are all composed of CG beads .\",\n \"Each bead has a diameter of σ = 0 . 8 nm to represent approximately three amino-acid residues .\",\n \"This bead diameter is similar to the Kuhn length of polypeptides ( Staple et al . , 2008 ) so that the nascent protein can be treated as a freely jointed chain .\",\n \"The surrounding solvent and lipid bilayer are included implicitly , a technique that is used in other CG models of the translocon ( Rychkova and Warshel , 2013 ) .\",\n \"The time-evolution of nascent protein configurations is calculated using Brownian dynamics with a 100 ns timestep .\",\n \"The kinetics of the LG are modeled as stochastic transitions between a closed conformation , which prevents the nascent protein from exiting from the channel interior to the membrane , and an open conformation , which removes the barrier to membrane entry .\",\n \"All bead positions are projected onto the plane that passes along the translocon channel axis between the helices forming the LG .\",\n \"This off-lattice 2D approximation reflects the cylindrical geometry of the channel and is inspired by previous models of biopolymer translocation through nanopores ( Huopaniemi et al . , 2006 ) .\",\n \"Beads representing the ribosome enclosure and translocon are placed to approximate their structures ( Van den Berg et al . , 2004; Frauenfeld et al . , 2011 ) .\",\n \"Two negative charges are placed on a bead at the cytosolic end of the translocon LG , whereas two positive charges are placed on a bead at the periplasmic end of the translocon LG .\",\n \"This charge distribution reflects the position of conserved charged residues ( White and von Heijne , 2004 ) near the translocon LG that have been previously shown to affect single-spanning protein topogenesis ( Goder et al . , 2004 ) .\",\n \"Full details of the model are provided in Appendix 1 . 10 . 7554/eLife . 08697 . 003Figure 1 . Schematic of Sec-mediated cotranslational integration of EmrE and corresponding simulation representation .\",\n \"( A ) At top , an illustration of the structural motifs in EmrE , including indication of the charged residues in the soluble loops with black circles and the transmembrane domain ( TMD ) /loop numbering scheme that is employed in the text; below , the corresponding sequence of coarse-grained ( CG ) beads that represent the EmrE amino-acid sequence .\",\n \"TMDs and loops are assigned based on the hydropathy plot and consensus topology prediction shown in Figure 1—figure supplement 1 .\",\n \"( B ) At top , a schematic illustration of the sequential integration of TMDs to obtain a multispanning Nperi/Cperi topology , in which both the N- and C-terminal loops are positioned in the periplasm , according to the cotranslational model; below , representative simulation snapshots of EmrE as the nascent protein grows during translation , integrates into the membrane , and exits the channel in the Nperi/Cperi multispanning topology .\",\n \"The nascent protein is colored according to the legend at top , the ribosome is brown , and the translocon is green with translocon charges labeled explicitly . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 00310 . 7554/eLife . 08697 . 004Figure 1—figure supplement 1 . Hydropathy plot for EmrE . The amino-acid sequence is shown on the right with positive charges highlighted in red .\",\n \"The transfer free energy according to the Wimley-White scale ( Wimley et al . , 1996 ) is plotted as a black line with a 7-residue moving average overlaid as a red line .\",\n \"Regions predicted by TOPCONS to correspond to loops ( L1-L5 ) or TMDs ( TMD1-TMD4 ) are shaded according to the legend at right . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 00410 . 7554/eLife . 08697 . 005Figure 1—figure supplement 2 . Simulation snapshot illustrating the initial configuration comprised of 9 CG beads . Positions of several beads in the ribosome ( brown ) and translocon ( green ) are labeled , with each labeled bead indicated by a black dot .\",\n \"Positions are labeled in units of σ .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 005 The CG model is well-suited to simulating the kinetics of cotranslational IMP integration , a process that is challenging for atomistic models ( Zhang and Miller , 2010; Gumbart et al . , 2011; Zhang and Miller , 2012b; Rychkova and Warshel , 2013 ) due to the large system size ( >100 , 000 atoms ) and the long timescale ( minutes ) of translation .\",\n \"We note that the model does not include nascent protein secondary/tertiary structure , charged lipids , protein chaperones , or an electrostatic potential across the membrane .\",\n \"However , the model does include explicit LG/translation dynamics , electrostatic interactions with the translocon , water/bilayer transfer free energies , and a direct mapping between the nascent protein sequence and the CG representation .\",\n \"The model thus captures the major physicochemical features of the translocon-membrane system ( White and von Heijne , 2004 ) .\",\n \"Moreover , the model has been shown to accurately predict features of single-spanning IMP integration and topogenesis ( Zhang and Miller , 2012a ) , including the sigmoidal dependence of stop-transfer efficiency on TMD hydrophobicity ( Hessa et al . , 2005 ) , the inversion of signal-anchor orientation during translation ( Goder and Spiess , 2003 ) , and the effect of translation rates and sequence features on signal-anchor orientation ( Goder and Spiess , 2003 ) .\",\n \"In particular , the model has been shown ( Zhang and Miller , 2012a ) to correctly describe integration processes that are governed either by thermodynamics ( Hessa et al . , 2005 ) or kinetics ( Goder and Spiess , 2003 ) , and it has provided a means of understanding the competition between such effects .\",\n \"The model has also been shown to correctly predict the dominant topology for a three-TMD multispanning IMP with a strong positive-inside bias ( Zhang and Miller , 2012a ) .\",\n \"The strong agreement between simulation and experimental results presented in this work further indicates that IMP topological determinants are captured at this CG resolution .\"\n ],\n [\n \"For all 16 of the EmrE and nEmrE mutants with single added charges studied by Seppälä et al . ( 2010 ) , Figure 2 compares the experimentally observed IMP topologies with the prediction from the CG model .\",\n \"Specifically , the figure compares the fraction of fully integrated proteins that adopt the Ncyto/Ccyto topology , with the remainder in the Nperi/Cperi topology .\",\n \"The top and bottom rows show variants of EmrE and nEmrE respectively .\",\n \"Each mutant differs only in the number and location of charges in the hydrophilic loops .\",\n \"A schematic of each mutant drawn with the dominant topology predicted from simulations is included; positive charges are indicated as filled-in circles with additional charges relative to EmrE ( top row ) or nEmrE ( bottom row ) highlighted in red .\",\n \"The topologies determined experimentally in Seppälä et al . ( 2010 ) are expressed as the fraction of Ncyto/Ccyto topologies by dividing the cell activity of each protein coexpressed with the EmrE ( Nperi ) mutant by the total growth of the protein coexpressed with either the EmrE ( Nperi ) or EmrE ( Ncyto ) mutant ( Seppälä et al . , 2010 ) , as described in the Experimental interpretation of EmrE topology section of the ‘Materials and methods’ . 10 . 7554/eLife . 08697 . 006Figure 2 . Topologies determined from simulations ( blue ) and compared to the experiments of Seppälä et al . ( 2010 ) ( red ) , reporting the fraction of fully integrated integral membrane protein ( IMP ) configurations in the Ncyto/Ccyto topology . Error bars indicate the standard error measured from independent blocks of simulations or taken from Seppälä et al . ( 2010 ) .\",\n \"The dominant topology for each mutant is indicated schematically with additional positive charges relative to EmrE ( top ) or nEmrE ( bottom ) drawn as red dots . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 00610 . 7554/eLife . 08697 . 007Figure 2—figure supplement 1 . Robustness of the distribution of topologies to the trajectory termination criteria . The distribution of topologies for the EmrE , K3 , nK3 , and T28R mutants are computed using alternative trajectory termination criteria and compared to the distribution of topologies using the original protocol ( shown in Figure 2 ) .\",\n \"At left , the plot illustrates the effect of extending each original CG trajectory by 50 s .\",\n \"At right , the plot illustrates the effect of terminating trajectories at a distance of 20σ , rather than 4 . 5σ , from the origin .\",\n \"The dashed line indicates perfect correlation .\",\n \"Additional details on these calculations are included in the Robustness checks for the trajectory termination criteria section of the ‘Materials and methods’ .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 00710 . 7554/eLife . 08697 . 008Figure 2—figure supplement 2 . Correlation between the topologies determined from simulations ( x-axis ) and compared to the experiments of Seppälä et al . ( 2010 ) ( y-axis ) , reporting the fraction of fully integrated IMP configurations in the Ncyto/Ccyto topology . Mutants lying in the shaded quadrants have the same dominant topology in both the experiments and simulations .\",\n \"The experimental and simulation measurements are linearly correlated with a Pearson correlation coefficient of r = 0 . 92 . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 008 It is clear from Figure 2 that the simulations are in excellent qualitative agreement with the experiments by correctly predicting the near 1:1 stoichiometry of wild-type EmrE and identifying the dominant topology for nearly all of the proteins considered .\",\n \"Figure 2—figure supplement 2 illustrates that the distribution of topologies determined experimentally and the distribution of topologies measured from the simulations are linearly correlated ( Pearson correlation coefficient , r = 0 . 92 ) ; points lying in the two shaded quadrants of the graph correspond to proteins for which the simulations and experiments predict consistent topologies .\",\n \"All mutants , with the exception of A52K , have the same dominant topology in the simulations as in the experiments within the statistical error .\",\n \"The agreement between simulations and experiments suggests that the CG model correctly reproduces the essential molecular features of topogenesis; in the following , we analyze the ensembles of CG trajectories that give rise to these computed IMP topologies .\",\n \"To investigate the molecular processes that govern the establishment of EmrE topology , we first examine the kinetics by which fully integrated topologies are reached .\",\n \"As a function of time , Figure 3A shows the fraction of CG trajectories in which the studied protein reaches a fully integrated topology for several EmrE mutants and the CB mutant .\",\n \"0 s corresponds to the end of translation and negative values of time correspond to the period that precedes the end of ribosomal translation in which the nascent protein is still attached to the ribosome .\",\n \"Over 90% of the CB mutant trajectories reach the Ncyto/Ccyto topology within 3 s following the completion of translation and thus rapidly integrate as expected for the cotranslational model ( Blobel , 1980; Wessels and Spiess , 1988; Sadlish et al . , 2005 ) ; mechanistic features of individual TMD integration steps are discussed in the Cotranslational integration pathways section of the ‘Materials and methods’ .\",\n \"In contrast , all variants of EmrE reach a fully integrated topology much more slowly , requiring hundreds of seconds for some CG trajectories to fully integrate ( see also Figure 3—figure supplement 2 ) . 10 . 7554/eLife . 08697 . 009Figure 3 . Kinetics of EmrE topogenesis .\",\n \"( A ) Fraction of CG trajectories in which all TMDs are fully integrated in a multispanning topology , plotted as a function of time for several mutants .\",\n \"( B ) Fraction of CG trajectories in which each TMD is integrated , plotted as a function of time for the cotranslationally-biased ( CB ) mutant ( top ) and EmrE ( bottom ) .\",\n \"The snapshots show an example of a simulation in which TMD4 of EmrE does not integrate during translation .\",\n \"In both panels , 0 s corresponds to the end of translation and negative values of time correspond to the period that precedes the end of ribosomal translation . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 00910 . 7554/eLife . 08697 . 010Figure 3—figure supplement 1 . Pathways for the cotranslational integration of TMDs into the membrane . At left , three distinct integration pathways are illustrated; the definition of each pathway is described in the Cotranslational integration pathways section of the ‘Materials and methods’ .\",\n \"In the ‘channel-sliding’ pathway , the TMD partially enters the channel , then crosses the lateral gate ( LG ) , then fully integrates into the membrane .\",\n \"In the ‘interface-sliding’ pathway , the TMD enters the cytoplasm through the gap between the translocon and ribosome , prior to undergoing membrane integration .\",\n \"In the ‘in-out’ pathway , the TMD fully spans the channel prior to membrane integration .\",\n \"At right , the fraction of cotranslational TMD integration events that exhibit each of these three pathways is presented for all four TMDs and for both the EmrE and nEmrE mutants .\",\n \"The dominant cotranslational integration pathway is the ‘channel-sliding’ pathway for all TMDs in both mutants . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 01010 . 7554/eLife . 08697 . 011Figure 3—figure supplement 2 . Simulation time necessary for 50% , 90% , and 95% of the CG trajectories to reach fully integrated topologies for each mutant . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 01110 . 7554/eLife . 08697 . 012Figure 3—figure supplement 3 . Effect of loop length on integration trajectories .\",\n \"( A ) Schematic illustrations of a new EmrE mutant with elongated , 10-bead loops , and a new CB mutant with shortened , 5-bead loops .\",\n \"( B ) Fraction of CG trajectories that reach fully integrated topologies as a function of time for the EmrE and CB mutants with both loop lengths .\",\n \"The integration kinetics for the EmrE mutants clearly show that longer loops increase the timescale of post-translational annealing , due to the reduction of the loop-flipping frequency for the longer soluble loops .\",\n \"The corresponding effect is less clear for the two CB mutants , since the timescale for the loop-flipping frequency is dominated by the large positive charges on these loops , masking the effect of changing the loop length .\",\n \"( C ) Average loop positions in the end-of-translation ( EOT ) ensemble for both the EmrE and CB mutants , presented in terms of the fraction of configurations for which each loop occupies the cytoplasm .\",\n \"For the EmrE mutants , changing the loop length leads to relatively small shifts in the EOT ensemble; however , for the CB mutants , the effect of loop length on the EOT ensemble is much larger , with the new short-loop CB mutant exhibiting a much weaker bias towards the Ncyto/Ccyto topology in the EOT ensemble . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 012 The slow post-translational integration of the dual-topology EmrE mutants is due to the fact that a significant fraction of trajectories exhibit configurations in which some TMDs are not fully integrated at the end of translation .\",\n \"As a function of time , Figure 3B shows the fraction of CG trajectories in which each TMD is integrated for both the CB mutant ( top ) and EmrE ( bottom ) .\",\n \"TMDs in the CB mutant integrate sequentially with near 100% efficiency during translation , which is consistent with the standard cotranslational model of topogenesis ( c . f . Figure 1 ) and explains the rapid timescale for fully integrating into a multispanning topology shown in Figure 3A .\",\n \"In contrast , the TMDs of EmrE exhibit only partial integration , even at long times following the completion of translation .\",\n \"Snapshots of a typical misintegrated TMD in EmrE are shown in Figure 3B .\",\n \"Various experiments have indicated that such configurations with misintegrated TMDs arise due to frustration from charges placed in consecutive loops ( Gafvelin and von Heijne , 1994 ) , the strong orientational preference of a neighboring TMD ( Öjemalm et al . , 2012 ) , or the weak stop-transfer efficiency of marginally hydrophobic TMDs ( Moss et al . , 1998 ) .\",\n \"Consistent with these experimental observations , the simulations in Figure 3B find that the weakly hydrophobic TMD1 of EmrE integrates the least efficiently , followed by TMD4 which is flanked by two charged loops .\"\n ],\n [\n \"In this work , we utilize a recently developed CG computational approach ( Zhang and Miller , 2012a ) that enables the direct simulation of Sec-facilitated membrane integration of proteins on biological timescales to investigate the topogenesis of the dual-topology EmrE protein and its mutants .\",\n \"In addition to demonstrating excellent agreement with the experimentally observed topologies of EmrE and its mutants ( Seppälä et al . , 2010 ) , the simulations reveal a novel mechanism for the regulation of topogenesis in multi-spanning membrane proteins , in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach final , fully integrated topologies .\",\n \"The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins .\",\n \"The inclusion of charged residues on the soluble loops of the IMP can lead to significant changes in the distribution of fully integrated topologies by both altering the ensemble of protein configurations at the end of ribosomal translation , as well as by altering the available kinetic pathways that lead to fully integrated topologies .\",\n \"This analysis leads to a number of experimentally testable predictions regarding IMP topogenesis .\",\n \"In particular , the results of Figure 8 predict that the mutation of charged residues near the cytoplasmic or periplasmic openings of the translocon channel will lead to significant shifts in the observed topology of several EmrE mutants .\",\n \"More generally , we note that any effect of channel mutations on the fully integrated IMP topology would indicate that kinetic effects during translation influence topogenesis , as suggested by the proposed mechanism .\",\n \"Additionally , we predict that the introduction of IMP mutations that significantly alter the EOT ensemble with respect to the cytosolic localization of the slowest-flipping soluble loop , either by introducing charge mutations or by changing TMD hydrophobicity , will influence the multispanning IMP topology .\",\n \"Although the current manuscript primarily focuses on the mechanism of topogenesis in the dual-topology EmrE protein , the mechanism and simulation analysis presented here has broader implications for topogenesis in other multispanning IMPs .\",\n \"For EmrE and its mutants , we find that a significant fraction of the IMP configurations are misintegrated upon completion of ribosomal translation and undergo subsequent post-translational annealing to reach fully integrated topologies .\",\n \"In contrast , a CB mutant exhibits an essentially fully integrated ensemble of configurations at the time that ribosomal translation completes .\",\n \"For other IMPs , a combination of these behaviors may well be expected ( Lu et al . , 2000; Lambert and Prange , 2001; Kanki et al . , 2002; Skach , 2009; Öjemalm et al . , 2012; Bowie , 2013; Virkki et al . , 2014 ) , with some fraction of the nascent protein configurations reaching fully integrated topologies at the completion of ribosomal translation and some fraction reaching misintegrated configurations that subsequently undergo post-translational annealing .\",\n \"Indeed , the importance of chaperone proteins such as YidC or Sec62 that post-translationally rescue misintegrated TMDs ( Sommer et al . , 2013; Zhu et al . , 2013; Aviram and Schuldiner , 2014; Jung et al . , 2014 ) may be connected to this necessary process of annealing initially misintegrated IMP configurations towards fully integrated topologies .\",\n \"The emerging understanding of the role of the Sec translocon in regulating IMP topogenesis , as well as advances in the methodologies for probing and modifying interactions between the nascent protein and the translocon complex , hold intriguing possibilities for the prediction and control of protein folding in cellular environments .\"\n ],\n [\n \"Loop-flipping frequencies are calculated by monitoring loop positions with respect to the membrane in 1-ms time intervals .\",\n \"A loop-flipping event is counted if a given loop switches from a cytoplasmic to periplasmic ( or vice versa ) position according to the definitions in the main text and if the y-position of the reference bead in the loop ( defined in the main text ) is at least 4 . 5σ after the flip ( to exclude counting the rapid flipping of loops within the translocon channel ) .\",\n \"The loop-flipping frequencies presented in Figure 6 are obtained by averaging the frequency of loop-flipping events in each trajectory .\",\n \"Sufficiently infrequent loop flips will not occur in every trajectory , but such events are observed in the combined ensemble of trajectories .\",\n \"To examine the effect of neighboring TMDs on the loop-flipping frequency , we separately calculate the loop-flipping frequency of each loop for configurations in which zero , one , or two of the neighboring TMDs is misintegrated ( discussed in the Rate of loop-flipping depends on charge mutations section of the ‘Results’ ) .\",\n \"In Seppälä et al . ( 2010 ) , the dominant topologies of EmrE mutants are determined by measuring the growth of E . coli cells in the presence of ethidium bromide ( EtBr ) .\",\n \"EtBr is toxic to E . coli , but antiparallel EmrE dimers , in which the two monomers forming the dimer have opposite topologies , confer drug resistance .\",\n \"EmrE dimerization can also be suppressed by including an E14D mutation .\",\n \"The topology of an EmrE variant with the E14D mutation can thus be inferred by coexpressing the mutant with another EmrE variant of known topology , as any resulting drug resistance ( and cell growth ) can be attributed to the formation of antiparallel dimers .\",\n \"To enable a direct comparison between the topologies measured from simulations and the experimental results , we convert the experimentally-measured cell activities from Seppälä et al . ( 2010 ) to the fraction of Ncyto/Ccyto topologies by assuming a linear relationship between cell growth and the population of antiparallel EmrE dimers .\",\n \"The fraction of Ncyto/Ccyto topologies is calculated as ( 1 ) f ( Ncyto/Ccyto ) =A ( Nperi ) A ( Ncyto ) +A ( Nperi ) , where A ( Ncyto ) and A ( Nperi ) are the experimentally-measured cell activities for cells coexpressing the EmrE ( Ncyto ) and EmrE ( Nperi ) mutants , respectively .\",\n \"Greater cell growth in the presence of the EmrE ( Nperi ) mutant , which exhibits a single dominant Nperi/Cperi topology , indicates that the mutant of interest exhibits a larger fraction of the opposite Ncyto/Ccyto topology , and vice versa for growth in the presence of the EmrE ( Ncyto ) mutant .\",\n \"Experimental values for the activities of the EmrE and nEmrE mutants are taken from Figure 2 and Figure S1 of Seppälä et al . ( 2010 ) , respectively; these values are used to compute the fraction of Ncyto/Ccyto topologies reported in Figure 2 of the current paper .\",\n \"Values for the activities of the ( Nout ( E14D ) + Nin ) and the Nout constructs from Figure 1 of Seppälä et al . ( 2010 ) are used to approximate the topology of the EmrE ( Nperi ) mutant , while the activities of the ( Nin ( E14D ) + Nout ) and the Nin constructs from the same figure are used to approximate the topology of the EmrE ( Ncyto ) mutant .\",\n \"Error bars are approximated via standard error propagation techniques based on Equation\",\n \"1 . In Figure 7A , the average position of the slowest-flipping loop relative to the membrane in the EOT ensemble is compared with the average position of that same loop in the ensemble of fully integrated configurations at the end of the CG trajectories .\",\n \"Four mutants ( K3 , L85R , nEmrE , and nT28R1 ) , however , have two slow-flipping loops with similar loop-flipping frequencies ( Figure 6 ) , and two of these mutants ( K3 and L85R ) deviate most significantly in terms of the correlation in Figure 7A .\",\n \"To better understand the effect of multiple slow-flipping loops on the correlation between the EOT ensemble and the final topology , the current section provides additional analysis in which a more sophisticated definition of the ‘slowest-flipping loop’ is employed for the four mutants that exhibit a pair of slow-flipping loops .\",\n \"Below , we present this alternative definition , which leads to a slightly better correlation between the EOT ensemble and the ensemble of fully integrated configurations , as plotted in Figure 7—figure supplement\",\n \"1 . The alternative definition of the slowest-flipping loop for mutants with two slow-flipping loops is given by ϕEOT and ϕFI , which report on the average position of the two slow-flipping loops in the EOT ensemble and in the ensemble of fully integrated configurations , respectively .\",\n \"The quantity ϕFI reports on the average position of the two slow-flipping loops in the ensemble of fully integrated configurations at the end of the CG trajectories .\",\n \"For the L85R and nEmrE mutants , the two slow-flipping loops ( L2/L4 and L1/L5 , respectively ) reach positions on the same side of the membrane in either fully integrated topology; for these mutants , ϕFI is defined as the fraction of fully integrated configurations for which both slow-flipping loops are positioned in the cytoplasm .\",\n \"For the K3 and nT28R1 mutants , the two slow-flipping loops ( L1 and L2 ) reach positions on opposite sides of the membrane in either fully integrated topology; for these mutants , ϕFI is defined as the fraction of fully integrated configurations for which L1 is positioned in the cytoplasm and L2 is positioned in the periplasm .\",\n \"For the nEmrE , K3 , and nT28R1 mutants , ϕFI is equivalent to the fraction of CG trajectories that reach the fully integrated Ncyto/Ccyto topology , whereas for the L85R mutant , ϕFI is equivalent to the fraction of CG trajectories that reach the fully integrated Nperi/Cperi topology .\",\n \"The quantity ϕEOT reports on the average position of the two slow-flipping loops in the EOT ensemble .\",\n \"For each mutant , ϕEOT is defined as ( 2 ) ϕEOTL85R=0 . 5[f ( cyto ) L2+f ( cyto ) L4]ϕEOTnEmrE=0 . 5[f ( cyto ) L1+f ( cyto ) L5]ϕEOTK3=0 . 5[1+f ( cyto ) L1−f ( cyto ) L2]ϕEOTnT28R1=0 . 5[1+f ( cyto ) L1−f ( cyto ) L2] , where f ( cyto ) Li is the fraction of configurations in the EOT ensemble for which loop Li is in the cytoplasm .\",\n \"As for the previous definition of ϕFI , this definition accounts for the fact that the two slow-flipping loops of the L85R and nEmrE mutants reach the same side of the membrane in the fully integrated topologies , while the two slow-flipping loops of the K3 and nT28R1 mutants reach opposite sides of the membrane in the fully integrated topologies .\",\n \"The definition in Equation 2 additionally assumes that the post-translational annealing of misintegrated configurations in the EOT ensemble is equally rate-limited by the two slow-flipping loops .\",\n \"Using these alternative definitions for the position of the slowest-flipping loop ( i . e . , ϕFI and ϕEOT ) , Figure 7—figure supplement 1 compares the average position of the slowest-flipping loop in the EOT ensemble to the average position of that same loop in the ensemble of fully integrated configurations .\",\n \"Having more carefully accounted for the effect of both slow-flipping loops in the K3 , L85R , nEmrE , and nT28R1 mutants , this figure reveals a slight improvement in the correlation ( R2 = 0 . 88 vs R2 = 0 . 85 ) in comparison to the results in Figure 7A .\",\n \"Alternative trajectory termination criteria are tested to ensure the robustness of the simulated distribution of multispanning topologies presented in Figure\",\n \"2 . As a first alternative , the original set of CG trajectories are extended by 50 s , and the distribution of topologies is determined from the position of the slowest-flipping loop at the end of the extended trajectories .\",\n \"As a second alternative , the distribution of topologies is calculated from the subset of original CG trajectories that reach fully integrated topologies in which all beads in the TMDs are at least 20σ , rather than 4 . 5σ , from the origin .\",\n \"These robustness checks are presented in Figure 2—figure supplement 1 and exhibit excellent correlation with the results obtained using the original protocol .\",\n \"Additionally , Figure 7B shows results in which the CG trajectories are terminated at a range of fixed times following the end of ribosomal translation .\",\n \"Again , the results using this alternative trajectory termination criterion are in good agreement with the results obtained using the original protocol ( indicated in dashed lines in Figure 7B ) .\",\n \"From the ensemble of CG trajectories , it is possible to examine the pathways by which individual TMDs undergo Sec-facilitated cotranslational integration .\",\n \"In particular , following the definitions of Cymer et al . ( 2014 ) , it is possible to characterize each cotranslational TMD integration event as corresponding to either the ‘channel-sliding’ , ‘interface-sliding’ , or ‘in-out’ pathways .\",\n \"Simulation snapshots illustrating the three pathways are shown in Figure 3—figure supplement\",\n \"1 . Each pathway is defined in terms of the series of intermediate states that are visited by the TMD prior to membrane integration .\",\n \"To characterize these intermediate states , the following geometric regions are defined ( see Figure 1—figure supplement 2 ) .\",\n \"The channel region is defined as that for which −2σ ≤ x ≤ 2σ and −2σ ≤ y ≤ 2σ , the membrane region is defined as that for which −2σ ≤ x ≤ 2σ and y > 2σ , the ribosome region is defined as that for which −11σ ≤ x < −2σ and −8 . 5σ ≤ y ≤ 4 . 5σ , and the cytoplasm region is defined as the region outside of the ribosome for which x < −2σ .\",\n \"Finally , a bead is considered to overlap the LG if it is within a distance of σ to any lateral-gate bead .\",\n \"We now define the four intermediate states .\",\n \"Intermediate state 1 ( IS1 ) is that for which the TMD partially enters the channel; it is defined as the set of configurations for which at least two TMD beads are in the channel region and zero TMD beads are in the membrane region .\",\n \"Intermediate state 2 ( IS2 ) is that for which the TMD fully spans the membrane while in the channel; it is defined as the set of configurations for which all four TMD beads are in the channel region and the two hydrophilic beads that flank the TMD occupy opposite sides of the membrane .\",\n \"Intermediate state 3 ( IS3 ) is that for which the TMD accesses the membrane interior via the LG; it is defined as the set of configurations for which at least one TMD bead occupies the membrane region , the remaining three TMD beads occupy either the channel or membrane regions , and at least one TMD bead overlaps with the LG .\",\n \"Intermediate state 4 ( IS4 ) is that for which the TMD accesses the cytoplasm region without accessing the channel region; it is defined as the set of configurations for which each of the four TMD beads occupies either the ribosome , membrane , or cytoplasm regions and for which at least one of the hydrophilic beads that flank the TMD occupies the cytoplasm region .\",\n \"In this analysis , cotranslational TMD integration events are defined as those for which the TMD reaches a membrane integrated configuration ( for which all four beads of the TMD span the membrane region and the two hydrophilic flanking beads occupy opposite sides of the membrane ) before reaching a misintegrated configuration ( for which both hydrophilic flanking beads occupy the same side of the membrane and for which all TMD beads and both flanking beads lie outside of the channel and ribosome regions ) .\",\n \"Using the definitions of intermediate states , the cotranslational integration pathways are defined as follows .\",\n \"In the ‘channel-sliding’ pathway , the TMD partially enters the channel , then crosses the LG , then fully integrates into the membrane; a trajectory thus exhibits this pathway if a TMD visits IS1 , IS2 , and membrane integration in chronological order and without visiting any other intermediate states .\",\n \"In the ‘interface-sliding’ pathway , the TMD enters the cytoplasm through the gap between the translocon and ribosome , prior to undergoing membrane integration; a trajectory thus exhibits this pathway if a TMD visits IS4 on the way to membrane integration .\",\n \"In the ‘in-out’ pathway , the TMD fully spans the channel prior to membrane integration; a trajectory thus exhibits this pathway if a TMD visits IS3 on the way to membrane integration without visiting IS4 .\",\n \"At right , Figure 3—figure supplement 1 shows the relative fraction of cotranslational TMD integration events that exhibit each of these three pathways .\",\n \"It is clear that the dominant cotranslational integration pathway for all four TMDs in both the EmrE and nEmrE mutants is the ‘channel-sliding’ pathway .\",\n \"This same pathway was also observed in the previous study of single-spanning proteins using the CG model ( Zhang and Miller , 2012a ) and similar configurations were observed in long-timescale atomistic molecular dynamics simulations of the early stages of cotranslational membrane integration ( Zhang and Miller , 2012b ) .\",\n \"We find that only a small number of CG trajectories exhibit the ‘interface-sliding’ pathway .\",\n \"Finally , we note that the dominant cotranslational integration pathway is likely to depend on the IMP sequence , and the ‘channel-sliding’ behavior may be less dominant in other IMPs with less hydrophobic TMDs .\"\n ]\n]"},"abstract":{"kind":"list like","value":["The canonical mechanism for multispanning membrane protein topogenesis suggests that protein topology is established during cotranslational membrane integration .","However , this mechanism is inconsistent with the behavior of EmrE , a dual-topology protein for which the mutation of positively charged loop residues , even close to the C-terminus , leads to dramatic shifts in its topology .","We use coarse-grained simulations to investigate the Sec-facilitated membrane integration of EmrE and its mutants on realistic biological timescales .","This work reveals a mechanism for regulating membrane-protein topogenesis , in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies .","The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins .","The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis ."],"string":"[\n \"The canonical mechanism for multispanning membrane protein topogenesis suggests that protein topology is established during cotranslational membrane integration .\",\n \"However , this mechanism is inconsistent with the behavior of EmrE , a dual-topology protein for which the mutation of positively charged loop residues , even close to the C-terminus , leads to dramatic shifts in its topology .\",\n \"We use coarse-grained simulations to investigate the Sec-facilitated membrane integration of EmrE and its mutants on realistic biological timescales .\",\n \"This work reveals a mechanism for regulating membrane-protein topogenesis , in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies .\",\n \"The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins .\",\n \"The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis .\"\n]"},"summary":{"kind":"list like","value":["Proteins are long chains of smaller molecules called amino acids , and are built inside cells by a molecular machine called the ribosome .","Many important proteins must be inserted into the membrane that surrounds each cell in order to carry out their role .","As these proteins are being built by the ribosome , they thread their way into a membrane-spanning channel ( called the translocon ) from the inner side of the membrane .","Short segments of these integral membrane proteins ( called transmembrane domains ) then become embedded in the membrane , while other parts of the protein remain on either side of the membrane .","For a membrane protein to work properly , the end of each of its transmembrane domains must be on the correct side of the membrane ( i . e . , the protein must obtain the correct ‘topology’ ) .","The conventional model for this process suggests that topology is fixed when the first transmembrane domain of a protein is initially integrated into the membrane , while the ribosome is still building the protein .","This model can explain most integral membrane proteins , which only have a single topology .","However , it cannot explain the family of membrane proteins that have an almost equal chance of adopting one of two different topologies ( so-called ‘dual-topology proteins’ ) .","Van Lehn et al . have now used computer modeling to simulate how a bacterial protein called EmrE ( which is a dual-topology protein ) integrates into the membrane via the translocon .","The results reveal that a few transmembrane domains in EmrE do not fully integrate into the membrane while the ribosome is building the protein .","Instead , these transmembrane domains slowly integrate after the ribosome has finished its job .","These findings contradict the conventional model and suggest that some membrane proteins only become fully integrated after the protein-building process is complete .","The next step in this work is to experimentally test predictions from the computer simulations ."],"string":"[\n \"Proteins are long chains of smaller molecules called amino acids , and are built inside cells by a molecular machine called the ribosome .\",\n \"Many important proteins must be inserted into the membrane that surrounds each cell in order to carry out their role .\",\n \"As these proteins are being built by the ribosome , they thread their way into a membrane-spanning channel ( called the translocon ) from the inner side of the membrane .\",\n \"Short segments of these integral membrane proteins ( called transmembrane domains ) then become embedded in the membrane , while other parts of the protein remain on either side of the membrane .\",\n \"For a membrane protein to work properly , the end of each of its transmembrane domains must be on the correct side of the membrane ( i . e . , the protein must obtain the correct ‘topology’ ) .\",\n \"The conventional model for this process suggests that topology is fixed when the first transmembrane domain of a protein is initially integrated into the membrane , while the ribosome is still building the protein .\",\n \"This model can explain most integral membrane proteins , which only have a single topology .\",\n \"However , it cannot explain the family of membrane proteins that have an almost equal chance of adopting one of two different topologies ( so-called ‘dual-topology proteins’ ) .\",\n \"Van Lehn et al . have now used computer modeling to simulate how a bacterial protein called EmrE ( which is a dual-topology protein ) integrates into the membrane via the translocon .\",\n \"The results reveal that a few transmembrane domains in EmrE do not fully integrate into the membrane while the ribosome is building the protein .\",\n \"Instead , these transmembrane domains slowly integrate after the ribosome has finished its job .\",\n \"These findings contradict the conventional model and suggest that some membrane proteins only become fully integrated after the protein-building process is complete .\",\n \"The next step in this work is to experimentally test predictions from the computer simulations .\"\n]"},"year":{"kind":"string","value":"2015"}}},{"rowIdx":4288,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["chromosomes and gene expression"],"string":"[\n \"chromosomes and gene expression\"\n]"},"title":{"kind":"string","value":"The tardigrade damage suppressor protein binds to nucleosomes and protects DNA from hydroxyl radicals"},"id":{"kind":"string","value":"elife-47682-v1"},"sections":{"kind":"list like","value":[["Tardigrades , which are also known as water bears or moss piglets , are small invertebrate animals that are found in marine , freshwater , and terrestrial habitats throughout the Earth ( reviewed in Guidetti et al . , 2012; Møbjerg et al . , 2011; Weronika and Łukasz , 2017 ) .","They are typically about 0 . 1 to 1 mm in length , and comprise a head segment in addition to four body segments that each contains two legs with claws .","Terrestrial tardigrades require a thin film of water to remain active .","In the absence of water , they undergo anhydrobiosis into a dormant dehydrated state from which they can be rehydrated to an active form .","In the anhydrobiotic state , tardigrades are resistant to extreme conditions of heat , cold , vacuum , pressure , radiation , and chemical treatments .","Remarkably , they have been found to survive exposure to the vacuum and radiation of outer space ( Jönsson et al . , 2008 ) .","Thus , the unique properties of tardigrades have led to considerable interest in these animals .","The analysis of their singular features should lead to significant new biological insights .","The molecular analysis of tardigrades has been advanced by the sequencing of the genomes of Ramazzottius varieornatus ( Hashimoto et al . , 2016 ) and Hypsibius exemplaris ( Yoshida et al . , 2017 ) .","[Note: the strain of tardigrades that was designated as Hypsibius dujardini in Yoshida et al . ( 2017 ) has since been found to be a new species that is now termed Hypsibius exemplaris ( GĄsiorek et al . , 2018 ) . We will use the new terminology in this paper . ] In R . varieornatus , the study of chromatin-associated factors revealed a tardigrade-specific protein , termed Dsup , for damage suppressor ( Hashimoto et al . , 2016; Hashimoto and Kunieda , 2017 ) .","Dsup is a highly charged and largely unstructured nuclear protein that binds to DNA .","Intriguingly , the expression of Dsup gene in human cells was observed to decrease DNA fragmentation induced by X-ray irradiation or treatment with hydrogen peroxide .","Moreover , Dsup-containing human cells exhibited higher viability after X-ray irradiation than control cells .","These findings suggest that Dsup is a unique protein that either directly or indirectly protects DNA .","In this work , we sought to examine the molecular function of Dsup .","It was previously shown that Dsup interacts with free DNA in an apparently nonspecific manner ( Hashimoto et al . , 2016 ) .","Because the natural form of DNA in the nucleus is chromatin , we investigated the binding of Dsup to nucleosomes .","These experiments revealed that R . varieornatus Dsup binds preferentially to nucleosomes relative to free DNA .","Then , to test the relevance of these findings to other tardigrades , we examined the properties of a Dsup-like protein from H . exemplaris , and found that this protein appears to be an ortholog of R . varieornatus Dsup .","Intriguingly , the Dsup proteins contain a region with sequence similarity to core consensus of the nucleosome-binding domain of vertebrate high mobility group N ( HMGN ) proteins , and the HMGN-like sequence in Dsup is important for its binding to nucleosomes .","Furthermore , in a purified biochemical system , both Dsup proteins are able to protect chromatin from cleavage by hydroxyl radicals , which are generated in cells by ionizing radiation as well as by treatment with hydrogen peroxide .","These studies thus reveal conserved functions by which Dsup maintains the integrity of chromosomal DNA under extreme conditions ."],["The R . varieornatus Dsup protein is a largely disordered and highly charged nuclear protein ( Figure 1A ) ( Hashimoto et al . , 2016; Hashimoto and Kunieda , 2017 ) .","To study its biochemical properties , we synthesized a FLAG- and His6-tagged version of the full-length protein in Escherichia coli and purified it to greater than 95% homogeneity by affinity chromatography ( Figure 1B ) .","We will refer to the R . varieornatus Dsup protein as ‘Rv Dsup’ .","Although Rv Dsup was observed to associate with free DNA ( Hashimoto et al . , 2016 ) , we tested the binding of Rv Dsup to nucleosomes , which are the natural form of DNA in the eukaryotic nucleus .","To this end , we reconstituted mononucleosomes with the 5S rDNA nucleosome positioning sequence from Xenopus borealis ( Rhodes , 1985; Hayes et al . , 1990; Fei et al . , 2018 ) .","We compared the binding of Rv Dsup to 147 bp DNA-containing mononucleosomes relative to the corresponding 147 bp free DNA by gel mobility shift analysis ( Figure 2A and Figure 2—figure supplement 1A ) .","These experiments revealed that Rv Dsup binds with a higher affinity to nucleosomes than to free DNA .","To test whether this effect is specific for the 5S rDNA sequence , we examined the binding of Rv Dsup to mononucleosomes and free DNA that contain the 601 nucleosome positioning sequence ( Lowary and Widom , 1998 ) .","As with the 5S rDNA nucleosomes , we observed more efficient binding of Rv Dsup to the 601 mononucleosomes than to the 601 free DNA ( Figure 2B and Figure 2—figure supplement 1B ) .","Thus , Rv Dsup binds preferentially to mononucleosomes relative to free DNA in a manner that appears to be independent of the specific DNA sequence .","Because the 147 bp DNA-containing mononucleosomes lack linker DNA that extends beyond the nucleosome core , we compared the binding of Rv Dsup to mononucleosomes that contain either 147 bp DNA or 181 bp DNA ( Figure 2C ) .","These experiments showed that the presence of linker DNA did not substantially alter the efficiency of Rv Dsup binding to nucleosomes .","It thus appears that Rv Dsup binds primarily to the nucleosome core rather than to the linker DNA .","In the gel shift experiments with Rv Dsup and mononucleosomes , there is typically a distinct major shifted band ( denoted by black dots in Figure 2 ) along with a minor faster migrating band .","This pattern can be seen particularly clearly in the titration of Rv Dsup with 147 bp 5S rDNA mononucleosomes in Figure 2C , and is suggestive of the binding of two molecules of Rv Dsup to the nucleosome .","With the 5S rDNA mononucleosomes and high concentrations of Rv Dsup , we also observed a slower migrating band ( Figure 2A ) , which may reflect additional Rv Dsup binding to the nucleosomes and/or higher-order aggregation of the Rv Dsup-nucleosome complexes .","In contrast , a distinct shifted band is not seen with Rv Dsup and free DNA .","To complement the studies with mononucleosomes , we tested whether Rv Dsup can be incorporated into extended periodic nucleosome arrays .","In these experiments , we assembled nucleosomes onto plasmid DNA by using a purified and defined ATP-dependent chromatin assembly system with the ACF motor protein and the dNLP core histone chaperone ( Fyodorov and Kadonaga , 2003; Khuong et al . , 2017 ) .","We also included separate reactions with histone H1 ( reviewed in Woodcock et al . , 2006; Happel and Doenecke , 2009; Kalashnikova et al . , 2016 ) as a positive control for a nucleosome-binding factor .","The presence of Rv Dsup does not inhibit the efficiency of ACF-mediated chromatin assembly , as measured by the DNA supercoiling assay ( Figure 3A ) .","Partial MNase digestion analysis showed that the assembly of chromatin with Rv Dsup results in a small but distinct increase in the nucleosome repeat length ( Figure 3B ) .","To analyze the binding of Rv Dsup to the nucleosome arrays , we extensively digested the ACF-assembled chromatin with MNase , and subjected the resulting mononucleosome species to nondenaturing gel electrophoresis ( Figure 3C ) .","This experiment revealed that Rv Dsup-bound mononucleosome particles are released from the chromatin upon extensive MNase digestion .","We then compared the Rv Dsup-mononucleosome particles that were generated via ACF assembly followed by MNase digestion , as in Figure 3C , with the Rv Dsup-mononucleosome particles formed by the addition of Rv Dsup to mononucleosomes , as in Figure 2A , and found that the differently prepared Rv Dsup-nucleosome particles migrate at approximately the same rate during nondenaturing gel electrophoresis ( Figure 3D ) .","It therefore appears that the interaction of Rv Dsup with nucleosomes in periodic arrays , as in Figure 3 , is similar to the binding of Rv Dsup to mononucleosome particles , as in Figure 2 .","Histone H1 is a major nucleosome-binding protein in animals , and it has been found to be present at approximately one molecule per nucleosome ( Bates and Thomas , 1981 ) ( reviewed in Woodcock et al . , 2006; Happel and Doenecke , 2009; Kalashnikova et al . , 2016 ) .","We were therefore interested in testing whether histone H1 and Rv Dsup can bind simultaneously to nucleosomes .","To this end , we carried out gel mobility shift analyses with 181 bp DNA mononucleosomes in the presence of varying concentrations of Rv Dsup and histone H1 ( Figure 4A ) .","( We used 181 bp DNA mononucleosomes to allow the binding of histone H1 to the linker DNA . )","These experiments revealed that the addition of Rv Dsup to H1-bound mononucleosomes results in the formation of a slower migrating Rv Dsup-H1-nucleosome complex .","Likewise , the addition of histone H1 to Rv Dsup-bound mononucleosomes yields Rv Dsup-H1-nucleosome complexes that migrate at the same rate as the species formed upon addition of Rv Dsup to H1-bound nucleosomes .","These results indicate that histone H1 and Rv Dsup can bind simultaneously to mononucleosomes .","We then examined whether the presence of both histone H1 and Rv Dsup results in a disruption of chromatin structure .","To address this question , we carried out ACF-mediated chromatin assembly reactions in the presence or absence of histone H1 and/or Rv Dsup .","These experiments revealed that the addition of both H1 and Rv Dsup did not alter the efficiency of chromatin assembly , as measured by the DNA supercoiling assay ( Figure 4B ) .","Furthermore , the presence of histone H1 and Rv Dsup did not disrupt the periodicity of the nucleosome arrays , as assessed with the partial MNase digestion assay ( Figure 4C ) .","Thus , histone H1 and Rv Dsup can bind simultaneously to nucleosomes , and this binding does not substantially alter or disrupt chromatin structure .","These findings are consistent with the observation that Rv Dsup is not deleterious to histone H1-containing human cells ( Hashimoto et al . , 2016 ) .","It was also important to assess whether Dsup is present in tardigrades other than R . varieornatus .","In this regard , we were interested in H . exemplaris , a tardigrade that was recently subjected to genome resequencing ( Yoshida et al . , 2017 ) .","R . varieornatus and H . exemplaris are limnoterrestrial tardigrades in the family Hypsibiidae , and both animals can undergo anhydrobiosis ( see , for example , Horikawa et al . , 2008; Kondo et al . , 2015; Wright , 1989 ) .","Dsup was initially not identified in H . exemplaris ( Yoshida et al . , 2017 ) , but further analysis revealed a Dsup-like protein in this organism ( Hashimoto and Kunieda , 2017 ) .","The H . exemplaris Dsup-like protein was found to have 26 . 4% amino acid identity with Rv Dsup as well as hydrophobicity and charge profiles that are similar to those of Rv Dsup .","To investigate the evolutionary relationship between these proteins , we examined the chromosomal regions that encompass the Dsup-related genes in R . varieornatus and H . exemplaris .","This analysis revealed that the Dsup-related genes in both organisms are flanked by the same neighboring genes ( Figure 5A ) .","Hence , based on the similarity of the proteins , including the closely related nuclear localization signals near the C-termini ( Hashimoto and Kunieda , 2017 ) , and the similarity of the gene arrangements ( Figure 5A ) , it appeared likely that the H . exemplaris Dsup-like protein and R . varieornatus Dsup are homologs .","It remained to be determined , however , whether the two proteins have related biochemical functions .","To this end , we synthesized and purified the H . exemplaris Dsup-like protein , which we will refer to as ‘He Dsup’ ( Figure 5B ) .","We then tested the binding of He Dsup to mononucleosomes and to free DNA , and found that He Dsup binds preferentially to nucleosomes relative to free DNA ( Figure 5C ) , as does Rv Dsup ( Figure 2 ) .","In addition , we directly compared the binding of He Dsup and Rv Dsup to mononucleosomes , and observed that each of the proteins shifts mononucleosomes to approximately the same position on the gel ( Figure 5D ) .","The slower migration of the Rv Dsup-nucleosome complexes relative to that of the He Dsup-nucleosome complexes is consistent with the larger size of Rv Dsup ( 445 amino acid residues ) compared to He Dsup ( 328 amino acid residues ) .","Therefore , in the analysis of Rv Dsup and He Dsup , the relationship between the proteins ( Hashimoto and Kunieda , 2017 ) , the similarity of the gene arrangements ( Figure 5A ) , and the conserved biochemical function ( Figures 2 , 5C and D ) lead to the conclusion that the two proteins are orthologs .","An intriguing property of Dsup is its ability to protect DNA in human cells from degradation that is induced either by X-ray irradiation or by treatment with hydrogen peroxide ( Hashimoto et al . , 2016 ) .","Because ionizing radiation and hydrogen peroxide both generate hydroxyl radicals as a major reactive oxygen species in cells ( Halliwell and Gutteridge , 1992; Stadtman , 1993; Riley , 1994 ) , we investigated whether purified Dsup affects hydroxyl radical-mediated DNA cleavage in a purified and defined biochemical system .","To this end , we carried out hydroxyl radical-mediated DNA cleavage reactions in the absence or presence of Dsup .","In these experiments , we generated hydroxyl radicals by the Udenfriend modification of the Fenton reaction ( Udenfriend et al . , 1954; Tullius et al . , 1987 ) .","Each of the hydroxyl radical reactions was carried out under the same conditions .","In the absence of Dsup , the 3 . 3 kb plasmid DNA was mostly degraded to DNA fragments ranging from 100 to 1000 nt ( Figure 6A ) .","Notably , the extent of cleavage of free plasmid DNA was nearly the same as the amount of cleavage of chromatin .","Thus , the presence of approximately one nucleosome per 200 bp DNA only slightly protects the DNA from hydroxyl radicals .","These results are consistent with the previous observation that hydroxyl radicals can cleave DNA in nucleosomes ( Hayes et al . , 1990 ) .","Upon addition of Rv Dsup , we observed substantial protection of free DNA as well as chromatin from hydroxyl radicals ( Figure 6A ) .","The Dsup-mediated protection was stronger with chromatin than with free DNA , possibly because the specific binding of Dsup to nucleosomes creates a highly resistant structure .","In the presence of 4 molecules of Rv Dsup per 200 bp DNA ( approximately 4 Dsup molecules per nucleosome ) in chromatin , there was a considerable amount of full-length linear DNA remaining after hydroxyl radical-mediated cleavage .","These results indicate that Rv Dsup is able to protect chromatin from the damaging effects of hydroxyl radicals .","Because hydroxyl radicals react primarily with hydrogen atoms that are exposed in the minor groove of DNA ( Balasubramanian et al . , 1998 ) , it appears likely that Dsup blocks access to the minor groove .","We additionally tested whether He Dsup and human TFIIB ( a nuclear protein that does not bind to nucleosomes ) can protect chromatin from hydroxyl radical-mediated DNA cleavage ( Figure 6B ) .","These experiments revealed that He Dsup , like Rv Dsup , is able to protect nucleosomal DNA from hydroxyl radical-mediated cleavage , and further support the conclusion that He Dsup is an ortholog of Rv Dsup .","As seen in Figure 6B , we consistently observed that Rv Dsup provides slightly more protection against hydroxyl radicals than He Dsup .","In contrast to Dsup , TFIIB did not confer any detectable protection of chromatin from hydroxyl radical-mediated DNA cleavage .","Thus , these findings collectively indicate that Dsup binds to nucleosomes and protects DNA from cleavage by hydroxyl radicals ( Figure 6C ) .","The homology between Rv Dsup and He Dsup led us to examine whether any of their conserved regions are related to protein sequences in organisms other than tardigrades .","This analysis led to the identification of sequence similarity between a segment in the C-terminal region of the Dsup proteins and a conserved sequence in the nucleosome-binding domain of HMGN proteins ( Figure 7A ) .","The HMGN proteins are abundant nucleosome-binding proteins that have been found only in vertebrates ( for reviews , see: Kugler et al . , 2012; González-Romero et al . , 2015 ) .","They bind to two high affinity sites on nucleosomes in a manner that is independent of the DNA sequence .","The nucleosome-binding domain of the HMGN proteins has been identified , and it contains a conserved core sequence , RRSARLSA , which is critical for the binding of HMGN proteins to nucleosomes ( Ueda et al . , 2008 ) .","Strikingly , both Dsup proteins contain a sequence that is similar to the RRSARLSA consensus ( Figure 7A ) .","To examine whether the HMGN-like sequence in the Dsup proteins is important for their binding to nucleosomes , we generated two mutant versions of Rv Dsup ( Figure 7A ) .","The M1 mutant Dsup is a C-terminal deletion that removes the HMGN-like region , and the M2 mutant Dsup contains three R to E substitution mutations in the HMGN-like region .","We purified the mutant Dsup proteins ( Figure 7B ) and then tested their ability to bind to mononucleosomes by gel mobility shift analysis ( Figure 7C ) .","These experiments revealed that the M1 mutant Dsup is nearly completely defective for binding to nucleosomes .","Hence , the C-terminal region of Rv Dsup ( from amino acid residues 360 to 445 ) is essential for nucleosome binding .","In addition , the M2 mutant Dsup exhibits reduced binding to nucleosomes .","Thus , the HMGN-like sequence in Dsup is important for its binding to nucleosomes .","We further investigated the ability of the mutant Dsup proteins to protect chromatin from hydroxyl radicals .","These experiments revealed that the nucleosome-binding-deficient M1 mutant Dsup is nearly completely defective for protection of chromatin against hydroxyl radicals .","We also observed that the M2 mutant Dsup is partially defective for protection against hydroxyl radicals .","These data support the model that the binding of Dsup to nucleosomes is required to protect chromatin from hydroxyl radical-mediated DNA damage ."],["Here we have found that Dsup , a tardigrade-specific factor , is a nucleosome-binding protein that protects chromosomal DNA from hydroxyl radical-mediated cleavage .","These findings provide a molecular explanation for the ability of Dsup to protect DNA in human cells from degradation by ionizing radiation or by treatment with hydrogen peroxide ( Hashimoto et al . , 2016 ) .","More generally , these results suggest that Dsup protects the tardigrade genome from hydroxyl radical-mediated damage under diverse conditions .","For instance , protection of the tardigrade genome from hydroxyl radicals may contribute to survival after extended periods in the anhydrobiotic state ( Wełnicz et al . , 2011 ) .","Notably , Dsup and histone H1 can bind simultaneously to nucleosomes ( Figure 4A ) .","Both R . varieornatus and H . exemplaris appear to contain histone H1 , and it is thus likely that Dsup normally acts in conjunction with H1 .","In this regard , it is also notable that Dsup functions in histone H1-containing human cells ( Hashimoto et al . , 2016 ) .","Rv Dsup and He Dsup are orthologs .","In addition to their sequence similarity and related gene arrangements ( Figure 5A ) , both Rv Dsup and He Dsup are nucleosome-binding proteins that protect chromatin from hydroxyl radicals ( Figures 5 and 6 ) .","It had been previously suggested that Dsup is not present in H . exemplaris ( Yoshida et al . , 2017 ) .","If true , that would have implied that Dsup is not generally important for tardigrades .","Instead , the identification of Dsup as a chromatin-protective protein in two species suggests that it is a key factor that contributes to the unique properties of tardigrades .","Unexpectedly , the Dsup proteins contain a region that exhibits sequence similarity to the conserved core of the nucleosome-binding domain of HMGN proteins ( Figure 7A ) .","Moreover , the HMGN-like sequence in Rv Dsup is functionally important for its ability to bind to nucleosomes ( Figure 7C ) and to protect chromatin from hydroxyl radicals ( Figure 7D ) .","Because the HMGN proteins have been found only in vertebrates ( González-Romero et al . , 2015 ) , the origin of the HMGN-like sequences in the tardigrade Dsup proteins is , at present , a mystery .","It is possible but not likely ( see , for example , Doolittle , 1994 ) that the Dsup and HMGN proteins provide a very rare example of sequence convergence .","In the future , detailed functional and evolutionary analyses might reveal the relation between these proteins .","The Dsup proteins are highly charged and largely disordered ( Hashimoto et al . , 2016; Hashimoto and Kunieda , 2017 ) ( Figure 1A ) .","With respect to the latter , examination of the amino acid composition of the Dsup proteins reveals that they are enriched in serine , alanine , glycine , and lysine ( SAGK ) residues .","Rv Dsup contains more than 60% SAGK residues , and He Dsup has over 50% SAGK residues .","SAGK are disorder-promoting amino acids ( Dunker et al . , 2001 ) , and the SAGK residues may form a diffuse mass of protein that protects the chromosomal DNA from hydroxyl radical-mediated cleavage in a variety of conditions ( Figure 6C ) .","This model is consistent with the observation that tardigrades can survive high doses of ionizing radiation in the active as well as anhydriobiotic states ( see , for example , Jönsson et al . , 2005; Horikawa et al . , 2006 ) .","In conclusion , these studies reveal molecular aspects of Dsup function and suggest how this intriguing protein may contribute to the unique biological properties of tardigrades .","A direct mechanism can be envisaged in which Dsup binds specifically to nucleosomes and protects the DNA from damaging agents such as hydroxyl radicals via coverage of the chromatin with its SAGK-rich disordered regions .","In the future , the analysis of Dsup will reveal new insights into the physical and molecular bases of its functions and might further lead to its practical applications .","For instance , it is possible that the DNA-protective properties of Dsup could be used to extend the longevity of cells .","Thus , this unique protein from an extreme organism could be a new and powerful reagent in biological research ."],["The cDNA sequence for Rv Dsup was synthesized with an N-terminal His6 tag and a C-terminal FLAG tag ( Integrated DNA Technologies; San Diego , CA ) .","The resulting DNA fragment was subcloned into pET21b to give pET21b-His6-Dsup-FLAG .","For expression , freshly transformed Escherichia coli strain BL21 ( DE3 ) was grown in LB medium ( 2 L volume; 40 µg/mL ampicillin ) at 37°C to A600 nm of approximately 0 . 6 to 0 . 8 .","The synthesis of Dsup was induced by the addition of IPTG to 0 . 4 mM final concentration , and the culture was incubated at 18°C for 16 to 18 hr .","The bacteria were collected by centrifugation ( Fiberlite F9−4×1000y rotor; 6 , 000 rpm; 10 min; 18°C ) .","Until stated otherwise , the next series of operations were carried out at room temperature ( 22°C ) .","The pellet was resuspended in 20 mL of Denaturing Binding Buffer [20 mM sodium phosphate , pH 7 . 8 , containing 8 M urea , 0 . 5 M NaCl , 5 mM 2-mercaptoethanol , 0 . 4 mM PMSF] .","The cells were lysed with 3 × 15 strokes in a Wheaton Dounce homogenizer ( B pestle ) , with a 10 min interval between each set of 15 strokes .","The mixture was incubated for 1 hr and then subjected to centrifugation ( Fiberlite F21S-8×50y rotor; 15 , 000 rpm; 20 min ) .","The supernatant was collected and stored , and the pellet was resuspended with 15 mL of Denaturing Binding Buffer .","The suspended pellet was dispersed with 15 strokes in a Wheaton Dounce homogenizer ( B pestle ) , incubated for 1 hr , and subjected to centrifugation ( Fiberlite F21S-8×50y rotor; 15 , 000 rpm; 20 min ) .","The second supernatant was combined with the first supernatant , and the resulting mixture was incubated with 3 mL of Ni-NTA agarose beads ( Qiagen; Germantown , MD ) for 2 hr on a rotating wheel at 4°C .","At room temperature ( 22°C ) , the mixture was transferred into a Bio-Rad Econo-Pac chromatography column , and the resin was washed with 2 × 10 mL of Denaturing Wash Buffer [20 mM sodium phosphate , pH 6 . 0 , containing 8 M urea , 0 . 5 M NaCl , 5 mM 2-mercaptoethanol , 0 . 4 mM PMSF] .","The protein was eluted with Denaturing Elution Buffer [20 mM sodium phosphate , pH 4 . 0 , containing 8 M urea , 0 . 5 M NaCl , 5 mM 2-mercaptoethanol , 0 . 4 mM PMSF] in 5 × 1 mL steps , with a 2 min interval between the addition of each mL of elution buffer .","Unless stated otherwise , all subsequent operations were performed at 4°C .","The eluate fractions were combined and dialyzed overnight in 4 L of PBS , and then incubated on a rotator wheel with 0 . 5 mL of anti-FLAG M2 Affinity Gel ( Sigma-Aldrich; St . Louis , MO ) for 3 to 4 hr .","The beads were washed with 3 × 0 . 5 mL of Buffer A [20 mM Tris-HCl , pH 7 . 9 , containing 150 mM NaCl , 2 mM MgCl2 , 0 . 2 mM EDTA , 15% ( v/v ) glycerol , 1 mM DTT] , and protein was eluted with 4 × 0 . 1 mL portions of Buffer B [Buffer A containing 0 . 4 mg/mL FLAG peptide ( Sigma-Aldrich; St . Louis , MO ) and 0 . 5 mg/mL recombinant human insulin] .","The protein fractions were frozen in liquid nitrogen and stored at −80°C .","The Rv Dsup that was used in Figures 1–4 was purified by this method .","The nondenaturing method works very well for the purification of wild-type and mutant Dsup proteins from R . varieornatus and H . exemplaris .","Here , we describe the synthesis and purification of He Dsup .","The E . coli codon-optimized cDNA sequence for He Dsup was synthesized with a C-terminal FLAG tag ( Integrated DNA Technologies; San Diego , CA ) and inserted as an NcoI-XhoI fragment into pET21b-His6-Nco ( Kassavetis et al . , 1998 ) .","The resulting plasmid was transformed into Rosetta pLysS ( MilliporeSigma ) .","The cells were grown at 37°C in LB medium ( 0 . 5 L; 80 and 34 µg/mL , ampicillin and chloramphenicol , respectively ) to an A600 nm of 0 . 8 , induced with IPTG to 0 . 5 mM final concentration , grown for an additional 2 hr , and harvested by centrifugation ( Fiberlite F14S-6×250y rotor; 10 , 000 rpm; 15 min; 4°C ) .","Unless stated otherwise , the remaining operations were carried out at 4°C .","The cells ( 1 . 8 g ) were resuspended in 12 . 6 mL Lysis Buffer [40 mM potassium phosphate , pH 7 . 6 , 0 . 01% ( v/v ) NP-40 ( Pierce ) , 0 . 1 mM EDTA , 580 mM NaCl , 10% ( v/v ) glycerol , 10 mM 2-mercaptoethanol , 1 µg/mL leupeptin , 1 µg/mL pepstatin , 0 . 5 mM PMSF , and 300 µg/mL lysozyme] , incubated for 30 min on ice , sonicated ( Branson Sonifier 450 with a 0 . 25 inch microtip; 20% output; 10 cycles of sonication of 13 s each; kept below 6°C by chilling in an ice-ethanol bath ) , and clarified by centrifugation ( Fiberlite F21S-8×50y rotor; 18 , 000 rpm; 30 min ) .","Dsup was purified from the soluble lysate supernatant fraction on a 0 . 5 mL Ni-NTA agarose column that was washed with 8 mL Buffer E [40 mM potassium phosphate , pH 7 . 6 , 20 mM imidazole , 500 mM NaCl , 10% ( v/v ) glycerol , 10 mM 2-mercaptoethanol , 0 . 5 mM PMSF] , pre-eluted with 300 µL Buffer E containing 200 mM imidazole , and eluted with 700 µL of the same buffer ( yielding 5 . 8 mg/mL He Dsup and 3 . 6 mg/mL Rv Dsup ) .","The samples were frozen in liquid nitrogen and stored at −80°C .","For further purification , a portion of each sample ( approximately 0 . 1 mg Dsup ) was loaded onto a 300 µL anti-FLAG M2 agarose column that was pre-equilibrated in Buffer A2 [20 mM Tris-HCl , pH 7 . 9 , containing 150 mM NaCl , 2 mM MgCl2 , 0 . 2 mM EDTA , 15% ( v/v ) glycerol , 1 mM 2-mercaptoethanol] .","The column was left undisturbed for 5 min and washed with 2 mL Buffer A2 .","The Dsup was eluted by the addition of 250 µL ( to give the pre-elution fraction ) followed by 300 µL ( elution fraction ) Buffer A2 containing 300 µg/mL 3XFLAG peptide ( ApexBio Technology ) .","The samples were frozen in liquid nitrogen and stored at −80°C .","The preparations of He Dsup and Rv Dsup in Figures 6 and 7 were prepared in this manner .","The method of preparation described in this paragraph is the most efficient means of obtaining high yields of pure Dsup proteins .","Alternate denaturing method .","We also purified Dsup from the pellet of the bacterial cell lysate ( prepared as described in the preceding paragraph ) as follows .","The pellet was resuspended in Buffer PG [40 mM potassium phosphate , pH 7 . 6 , 20 mM imidazole , 6 M guanidine HCl , 10% ( v/v ) glycerol , 10 mM 2-mercaptoethanol , 1 µg/mL leupeptin , 1 µg/mL pepstatin , 0 . 5 mM PMSF] at room temperature ( 22°C ) , sonicated ( Branson Sonifier 450 with a 0 . 25 inch microtip; 20% output; 4 cycles of sonication of 10 s each; kept below 6°C by chilling in an ice bath ) , and clarified by centrifugation ( Fiberlite F21S-8×50y rotor; 18 , 000 rpm; 15 min; 4°C ) .","The supernatant was loaded onto a 1 mL Ni-NTA agarose column , which was then washed with 11 mL Buffer E containing 6 M urea .","The Dsup was renatured on the column with sequential 1 . 2 mL washes of Buffer E containing 5 . 0 , 4 . 5 , 4 . 0 , 3 . 5 , 3 . 0 , 2 . 5 , 2 . 0 , 1 . 5 , 1 . 0 and 0 M urea .","The column was washed with 0 . 8 mL Buffer E containing 200 mM imidazole to give the pre-elution fraction , and protein was eluted with 1 . 3 mL of the same buffer to give the elution fraction ( containing approximately 1 mg/mL Dsup ) .","A portion of the elution fraction ( approximately 0 . 1 mg Dsup ) was loaded onto a 300 µL anti-FLAG M2 agarose column that was pre-equilibrated in Buffer A2 [20 mM Tris-HCl , pH 7 . 9 , containing 150 mM NaCl , 2 mM MgCl2 , 0 . 2 mM EDTA , 15% ( v/v ) glycerol , 1 mM 2-mercaptoethanol] .","The column was left undisturbed for 5 min and washed with 2 mL buffer A2 .","The Dsup was eluted by the addition of 250 µL ( to give the pre-elution fraction ) followed by 300 µL ( elution fraction; 100 µg yield ) Buffer A2 containing 300 µg/mL 3XFLAG peptide ( ApexBio Technology ) .","The protein fractions were frozen in liquid nitrogen and stored at −80°C .","The Dsup proteins that were used in the gel mobility shift assays in Figures 5 , 6 and 7 were prepared by the method described in this section .","It might be noted that Dsup proteins are hydrophilic and highly-charged proteins that bind weakly to SDS and thus migrate slowly on SDS-polyacrylamide gels relative to ‘average’ proteins such as the molecular mass markers .","To confirm the authenticity of the Dsup proteins , we subjected wild-type R . varieornatus Dsup and wild-type H . exemplaris Dsup to LC-ESI-TOF-MS ( liquid chromatography-electrospray ionization time-of-flight mass spectrometry ) .","For each of the two proteins , the observed mass was within one mass unit of the predicted ( calculated ) mass .","Thus , the Dsup proteins appear to be authentic .","Mononucleosomes were reconstituted by using the salt dialysis method ( Stein , 1989; Fei et al . , 2015 ) with purified recombinant Drosophila core histones ( Levenstein and Kadonaga , 2002; Khuong et al . , 2017 ) and the following DNA fragments .","The 147 bp Xenopus borealis 5S rDNA fragment corresponds to the nucleosome positioning sequence that was mapped in Fei et al . ( 2018 ) .","The sequence of the longer 181 bp X . borealis 5S rDNA fragment ( with the central 147 bp positioning sequence indicated by lower case type ) is as follows: CAGGCTGTCAAGGCCGGgcttgttttcctgcctgggggaaaagaccctggcatggggaggagctgggccccccccagaaggcagcacaaggggaggaaaagtcagccttgtgctcgcctacggccataccaccctgaaagtgcccgatatcgtctgatctcggaAGCCAAGCAGGGTCGGG .","The 147 bp synthetic 601 nucleosome positioning sequence is described in Lowary and Widom ( 1998 ) .","For nucleosome reconstitution , the DNA fragments were amplified by PCR and purified by 1 . 25% agarose gel electrophoresis and gel extraction ( QIAquick Gel Extraction Kit; Qiagen; Germantown , MD ) .","After reconstitution , the quality of the nucleosomes was assessed by nondenaturing 6% polyacrylamide gel electrophoresis .","If necessary , the histone:DNA ratios in the reconstitution reactions were adjusted to achieve the efficient conversion of the DNA into mononucleosomes .","If multiple nucleosomal species ( due to the presence of mixture of differently positioned nucleosomes ) were observed , the samples were heated at 58°C for 10 min to facilitate the movement of the differently positioned nucleosomes to the most stable location .","The resulting mononucleosomes were stored at 4°C prior to use in the gel mobility shift experiments .","Mononucleosomes ( approximately 0 . 3 µM stock concentration; 60 nM final concentration ) were combined with the indicated amounts of purified Dsup protein in HEG buffer [25 mM Hepes ( K+ ) , pH 7 . 6 , 0 . 1 mM EDTA , 10% ( v/v ) glycerol] containing 100 mM KCl , and 100 ng bovine serum albumin in a total volume of 15 µL .","The samples were incubated at 30°C for 30 min , and then loaded onto a nondenaturing 4 . 5% polyacrylamide gel that had been pre-equilibrated at 4°C and pre-run at 3 . 5 V/cm for 30 min .","The gels were run at 4°C for 30 min at 3 . 5 V/cm and then for 2 hr at 8 V/cm .","The nucleosomes were visualized by staining of the DNA with ethidium bromide .","In experiments that involved testing Dsup and histone H1 , the reaction medium additionally included 0 . 05% ( v/v ) NP-40 ( Pierce ) and 2 mM MgCl2 .","The histone H1 was purified from Drosophila embryos by the method of Croston et al . ( 1991 ) .","To ensure reproducibility of the data , each of the reported experiments was performed a minimum of two independent times with different preparations ( i . e . , biologically distinct samples ) of Dsup .","The ATP-dependent assembly of periodic nucleosome arrays with the ACF motor protein and the dNLP core histone chaperone was carried out essentially as described in Fyodorov and Kadonaga ( 2003 ) and Khuong et al . ( 2017 ) .","A standard reaction included purified Drosophila core histones ( 0 . 35 µg ) , purified Drosophila ACF ( 10 nM ) , purified Drosophila dNLP ( 1 . 8 µg ) , 2 mM ATP , purified Drosophila topoisomerase I ( ND423 ) ( 1 nM ) , and pGIE-0 plasmid DNA ( 0 . 35 µg ) ( Pazin et al . , 1994 ) in a total volume of 70 µL .","Purified Dsup and histone H1 were included where indicated .","The final KCl concentration was 100 mM for samples that were analyzed by the DNA supercoiling and partial MNase assays and 50 mM for samples that were subjected to extensive ( limit ) MNase digestion .","The assembly reactions were incubated at 27°C for 90 min .","The partial MNase , extensive MNase , and DNA supercoiling assays were performed as described by Fyodorov and Kadonaga ( 2003 ) and Khuong et al . ( 2017 ) .","To ensure reproducibility of the data , each of the reported experiments was performed a minimum of two independent times with different preparations ( i . e . , biologically distinct samples ) of Dsup .","Nucleosomes were reconstituted onto plasmid pGIE-0 ( 3269 bp ) by the salt dialysis method ( Stein , 1989; Fei et al . , 2015 ) with 16 . 4 pmol Drosophila core histone octamers per pmol pGIE-0 ( average of one nucleosome per 200 bp DNA ) .","The final dialysis buffer was Buffer TEN [10 mM Tris-HCl , pH 8 . 0 , 1 mM EDTA , 0 . 01% ( v/v ) NP-40 ( Pierce ) ] containing 50 mM NaCl .","Proteins were diluted into Buffer TEN containing 50 mM NaCl prior to use .","For each series of reactions with a particular factor ( such as Rv Dsup , He Dsup , or human TFIIB ) , the protein and its corresponding storage buffer were diluted in parallel and then used in a manner that ensured that each reaction was carried out under identical buffer conditions .","We also note that the highest concentration of glycerol in any of the reactions was 0 . 014% ( v/v ) .","At this concentration of glycerol , we did not detect quenching of the hydroxyl radical cleavage reactions by the buffer medium .","Hydroxyl radical-mediated cleavage of DNA was carried out essentially as described by Tullius et al . ( 1987 ) .","Briefly , nucleosomal pGIE-0 ( 162 fmol ) along with the indicated amount of added protein were incubated at room temperature ( 22°C ) for 10 min in 90 µL Buffer TEN containing 50 mM NaCl .","Then , in each tube , three separate droplets of 60 mM ascorbic acid ( 3 . 3 µL ) , 100 µM Fe ( II ) and 200 µM EDTA ( 3 . 3 µL ) , and 0 . 3% ( v/v ) H2O2 ( 3 . 3 µL ) were deposited onto different locations on the tube wall ( suspended above the chromatin sample at the bottom of the tube ) , mixed together , and vortexed into the 90 µL binding reaction .","The reaction was stopped 2 min later by the addition of 10 µL of 200 mM thiourea .","The DNA was extracted with phenol-CHCl3-isoamyl alcohol , precipitated with ethanol , and resuspended in 10 µL of 50 mM NaOH-1 mM EDTA and 2 µL of Purple Loading Dye ( New England Biolabs ) .","Alkaline agarose gel electrophoresis was performed as described by Shin and Day ( 1995 ) , and the DNA was stained with GelRed ( Biotium ) in 50 mM Tris-HCl , pH 6 . 8 .","To ensure reproducibility of the data , each of the reported experiments was performed a minimum of two independent times with different preparations ( i . e . , biologically distinct samples ) of Dsup ."]],"string":"[\n [\n \"Tardigrades , which are also known as water bears or moss piglets , are small invertebrate animals that are found in marine , freshwater , and terrestrial habitats throughout the Earth ( reviewed in Guidetti et al . , 2012; Møbjerg et al . , 2011; Weronika and Łukasz , 2017 ) .\",\n \"They are typically about 0 . 1 to 1 mm in length , and comprise a head segment in addition to four body segments that each contains two legs with claws .\",\n \"Terrestrial tardigrades require a thin film of water to remain active .\",\n \"In the absence of water , they undergo anhydrobiosis into a dormant dehydrated state from which they can be rehydrated to an active form .\",\n \"In the anhydrobiotic state , tardigrades are resistant to extreme conditions of heat , cold , vacuum , pressure , radiation , and chemical treatments .\",\n \"Remarkably , they have been found to survive exposure to the vacuum and radiation of outer space ( Jönsson et al . , 2008 ) .\",\n \"Thus , the unique properties of tardigrades have led to considerable interest in these animals .\",\n \"The analysis of their singular features should lead to significant new biological insights .\",\n \"The molecular analysis of tardigrades has been advanced by the sequencing of the genomes of Ramazzottius varieornatus ( Hashimoto et al . , 2016 ) and Hypsibius exemplaris ( Yoshida et al . , 2017 ) .\",\n \"[Note: the strain of tardigrades that was designated as Hypsibius dujardini in Yoshida et al . ( 2017 ) has since been found to be a new species that is now termed Hypsibius exemplaris ( GĄsiorek et al . , 2018 ) . We will use the new terminology in this paper . ] In R . varieornatus , the study of chromatin-associated factors revealed a tardigrade-specific protein , termed Dsup , for damage suppressor ( Hashimoto et al . , 2016; Hashimoto and Kunieda , 2017 ) .\",\n \"Dsup is a highly charged and largely unstructured nuclear protein that binds to DNA .\",\n \"Intriguingly , the expression of Dsup gene in human cells was observed to decrease DNA fragmentation induced by X-ray irradiation or treatment with hydrogen peroxide .\",\n \"Moreover , Dsup-containing human cells exhibited higher viability after X-ray irradiation than control cells .\",\n \"These findings suggest that Dsup is a unique protein that either directly or indirectly protects DNA .\",\n \"In this work , we sought to examine the molecular function of Dsup .\",\n \"It was previously shown that Dsup interacts with free DNA in an apparently nonspecific manner ( Hashimoto et al . , 2016 ) .\",\n \"Because the natural form of DNA in the nucleus is chromatin , we investigated the binding of Dsup to nucleosomes .\",\n \"These experiments revealed that R . varieornatus Dsup binds preferentially to nucleosomes relative to free DNA .\",\n \"Then , to test the relevance of these findings to other tardigrades , we examined the properties of a Dsup-like protein from H . exemplaris , and found that this protein appears to be an ortholog of R . varieornatus Dsup .\",\n \"Intriguingly , the Dsup proteins contain a region with sequence similarity to core consensus of the nucleosome-binding domain of vertebrate high mobility group N ( HMGN ) proteins , and the HMGN-like sequence in Dsup is important for its binding to nucleosomes .\",\n \"Furthermore , in a purified biochemical system , both Dsup proteins are able to protect chromatin from cleavage by hydroxyl radicals , which are generated in cells by ionizing radiation as well as by treatment with hydrogen peroxide .\",\n \"These studies thus reveal conserved functions by which Dsup maintains the integrity of chromosomal DNA under extreme conditions .\"\n ],\n [\n \"The R . varieornatus Dsup protein is a largely disordered and highly charged nuclear protein ( Figure 1A ) ( Hashimoto et al . , 2016; Hashimoto and Kunieda , 2017 ) .\",\n \"To study its biochemical properties , we synthesized a FLAG- and His6-tagged version of the full-length protein in Escherichia coli and purified it to greater than 95% homogeneity by affinity chromatography ( Figure 1B ) .\",\n \"We will refer to the R . varieornatus Dsup protein as ‘Rv Dsup’ .\",\n \"Although Rv Dsup was observed to associate with free DNA ( Hashimoto et al . , 2016 ) , we tested the binding of Rv Dsup to nucleosomes , which are the natural form of DNA in the eukaryotic nucleus .\",\n \"To this end , we reconstituted mononucleosomes with the 5S rDNA nucleosome positioning sequence from Xenopus borealis ( Rhodes , 1985; Hayes et al . , 1990; Fei et al . , 2018 ) .\",\n \"We compared the binding of Rv Dsup to 147 bp DNA-containing mononucleosomes relative to the corresponding 147 bp free DNA by gel mobility shift analysis ( Figure 2A and Figure 2—figure supplement 1A ) .\",\n \"These experiments revealed that Rv Dsup binds with a higher affinity to nucleosomes than to free DNA .\",\n \"To test whether this effect is specific for the 5S rDNA sequence , we examined the binding of Rv Dsup to mononucleosomes and free DNA that contain the 601 nucleosome positioning sequence ( Lowary and Widom , 1998 ) .\",\n \"As with the 5S rDNA nucleosomes , we observed more efficient binding of Rv Dsup to the 601 mononucleosomes than to the 601 free DNA ( Figure 2B and Figure 2—figure supplement 1B ) .\",\n \"Thus , Rv Dsup binds preferentially to mononucleosomes relative to free DNA in a manner that appears to be independent of the specific DNA sequence .\",\n \"Because the 147 bp DNA-containing mononucleosomes lack linker DNA that extends beyond the nucleosome core , we compared the binding of Rv Dsup to mononucleosomes that contain either 147 bp DNA or 181 bp DNA ( Figure 2C ) .\",\n \"These experiments showed that the presence of linker DNA did not substantially alter the efficiency of Rv Dsup binding to nucleosomes .\",\n \"It thus appears that Rv Dsup binds primarily to the nucleosome core rather than to the linker DNA .\",\n \"In the gel shift experiments with Rv Dsup and mononucleosomes , there is typically a distinct major shifted band ( denoted by black dots in Figure 2 ) along with a minor faster migrating band .\",\n \"This pattern can be seen particularly clearly in the titration of Rv Dsup with 147 bp 5S rDNA mononucleosomes in Figure 2C , and is suggestive of the binding of two molecules of Rv Dsup to the nucleosome .\",\n \"With the 5S rDNA mononucleosomes and high concentrations of Rv Dsup , we also observed a slower migrating band ( Figure 2A ) , which may reflect additional Rv Dsup binding to the nucleosomes and/or higher-order aggregation of the Rv Dsup-nucleosome complexes .\",\n \"In contrast , a distinct shifted band is not seen with Rv Dsup and free DNA .\",\n \"To complement the studies with mononucleosomes , we tested whether Rv Dsup can be incorporated into extended periodic nucleosome arrays .\",\n \"In these experiments , we assembled nucleosomes onto plasmid DNA by using a purified and defined ATP-dependent chromatin assembly system with the ACF motor protein and the dNLP core histone chaperone ( Fyodorov and Kadonaga , 2003; Khuong et al . , 2017 ) .\",\n \"We also included separate reactions with histone H1 ( reviewed in Woodcock et al . , 2006; Happel and Doenecke , 2009; Kalashnikova et al . , 2016 ) as a positive control for a nucleosome-binding factor .\",\n \"The presence of Rv Dsup does not inhibit the efficiency of ACF-mediated chromatin assembly , as measured by the DNA supercoiling assay ( Figure 3A ) .\",\n \"Partial MNase digestion analysis showed that the assembly of chromatin with Rv Dsup results in a small but distinct increase in the nucleosome repeat length ( Figure 3B ) .\",\n \"To analyze the binding of Rv Dsup to the nucleosome arrays , we extensively digested the ACF-assembled chromatin with MNase , and subjected the resulting mononucleosome species to nondenaturing gel electrophoresis ( Figure 3C ) .\",\n \"This experiment revealed that Rv Dsup-bound mononucleosome particles are released from the chromatin upon extensive MNase digestion .\",\n \"We then compared the Rv Dsup-mononucleosome particles that were generated via ACF assembly followed by MNase digestion , as in Figure 3C , with the Rv Dsup-mononucleosome particles formed by the addition of Rv Dsup to mononucleosomes , as in Figure 2A , and found that the differently prepared Rv Dsup-nucleosome particles migrate at approximately the same rate during nondenaturing gel electrophoresis ( Figure 3D ) .\",\n \"It therefore appears that the interaction of Rv Dsup with nucleosomes in periodic arrays , as in Figure 3 , is similar to the binding of Rv Dsup to mononucleosome particles , as in Figure 2 .\",\n \"Histone H1 is a major nucleosome-binding protein in animals , and it has been found to be present at approximately one molecule per nucleosome ( Bates and Thomas , 1981 ) ( reviewed in Woodcock et al . , 2006; Happel and Doenecke , 2009; Kalashnikova et al . , 2016 ) .\",\n \"We were therefore interested in testing whether histone H1 and Rv Dsup can bind simultaneously to nucleosomes .\",\n \"To this end , we carried out gel mobility shift analyses with 181 bp DNA mononucleosomes in the presence of varying concentrations of Rv Dsup and histone H1 ( Figure 4A ) .\",\n \"( We used 181 bp DNA mononucleosomes to allow the binding of histone H1 to the linker DNA . )\",\n \"These experiments revealed that the addition of Rv Dsup to H1-bound mononucleosomes results in the formation of a slower migrating Rv Dsup-H1-nucleosome complex .\",\n \"Likewise , the addition of histone H1 to Rv Dsup-bound mononucleosomes yields Rv Dsup-H1-nucleosome complexes that migrate at the same rate as the species formed upon addition of Rv Dsup to H1-bound nucleosomes .\",\n \"These results indicate that histone H1 and Rv Dsup can bind simultaneously to mononucleosomes .\",\n \"We then examined whether the presence of both histone H1 and Rv Dsup results in a disruption of chromatin structure .\",\n \"To address this question , we carried out ACF-mediated chromatin assembly reactions in the presence or absence of histone H1 and/or Rv Dsup .\",\n \"These experiments revealed that the addition of both H1 and Rv Dsup did not alter the efficiency of chromatin assembly , as measured by the DNA supercoiling assay ( Figure 4B ) .\",\n \"Furthermore , the presence of histone H1 and Rv Dsup did not disrupt the periodicity of the nucleosome arrays , as assessed with the partial MNase digestion assay ( Figure 4C ) .\",\n \"Thus , histone H1 and Rv Dsup can bind simultaneously to nucleosomes , and this binding does not substantially alter or disrupt chromatin structure .\",\n \"These findings are consistent with the observation that Rv Dsup is not deleterious to histone H1-containing human cells ( Hashimoto et al . , 2016 ) .\",\n \"It was also important to assess whether Dsup is present in tardigrades other than R . varieornatus .\",\n \"In this regard , we were interested in H . exemplaris , a tardigrade that was recently subjected to genome resequencing ( Yoshida et al . , 2017 ) .\",\n \"R . varieornatus and H . exemplaris are limnoterrestrial tardigrades in the family Hypsibiidae , and both animals can undergo anhydrobiosis ( see , for example , Horikawa et al . , 2008; Kondo et al . , 2015; Wright , 1989 ) .\",\n \"Dsup was initially not identified in H . exemplaris ( Yoshida et al . , 2017 ) , but further analysis revealed a Dsup-like protein in this organism ( Hashimoto and Kunieda , 2017 ) .\",\n \"The H . exemplaris Dsup-like protein was found to have 26 . 4% amino acid identity with Rv Dsup as well as hydrophobicity and charge profiles that are similar to those of Rv Dsup .\",\n \"To investigate the evolutionary relationship between these proteins , we examined the chromosomal regions that encompass the Dsup-related genes in R . varieornatus and H . exemplaris .\",\n \"This analysis revealed that the Dsup-related genes in both organisms are flanked by the same neighboring genes ( Figure 5A ) .\",\n \"Hence , based on the similarity of the proteins , including the closely related nuclear localization signals near the C-termini ( Hashimoto and Kunieda , 2017 ) , and the similarity of the gene arrangements ( Figure 5A ) , it appeared likely that the H . exemplaris Dsup-like protein and R . varieornatus Dsup are homologs .\",\n \"It remained to be determined , however , whether the two proteins have related biochemical functions .\",\n \"To this end , we synthesized and purified the H . exemplaris Dsup-like protein , which we will refer to as ‘He Dsup’ ( Figure 5B ) .\",\n \"We then tested the binding of He Dsup to mononucleosomes and to free DNA , and found that He Dsup binds preferentially to nucleosomes relative to free DNA ( Figure 5C ) , as does Rv Dsup ( Figure 2 ) .\",\n \"In addition , we directly compared the binding of He Dsup and Rv Dsup to mononucleosomes , and observed that each of the proteins shifts mononucleosomes to approximately the same position on the gel ( Figure 5D ) .\",\n \"The slower migration of the Rv Dsup-nucleosome complexes relative to that of the He Dsup-nucleosome complexes is consistent with the larger size of Rv Dsup ( 445 amino acid residues ) compared to He Dsup ( 328 amino acid residues ) .\",\n \"Therefore , in the analysis of Rv Dsup and He Dsup , the relationship between the proteins ( Hashimoto and Kunieda , 2017 ) , the similarity of the gene arrangements ( Figure 5A ) , and the conserved biochemical function ( Figures 2 , 5C and D ) lead to the conclusion that the two proteins are orthologs .\",\n \"An intriguing property of Dsup is its ability to protect DNA in human cells from degradation that is induced either by X-ray irradiation or by treatment with hydrogen peroxide ( Hashimoto et al . , 2016 ) .\",\n \"Because ionizing radiation and hydrogen peroxide both generate hydroxyl radicals as a major reactive oxygen species in cells ( Halliwell and Gutteridge , 1992; Stadtman , 1993; Riley , 1994 ) , we investigated whether purified Dsup affects hydroxyl radical-mediated DNA cleavage in a purified and defined biochemical system .\",\n \"To this end , we carried out hydroxyl radical-mediated DNA cleavage reactions in the absence or presence of Dsup .\",\n \"In these experiments , we generated hydroxyl radicals by the Udenfriend modification of the Fenton reaction ( Udenfriend et al . , 1954; Tullius et al . , 1987 ) .\",\n \"Each of the hydroxyl radical reactions was carried out under the same conditions .\",\n \"In the absence of Dsup , the 3 . 3 kb plasmid DNA was mostly degraded to DNA fragments ranging from 100 to 1000 nt ( Figure 6A ) .\",\n \"Notably , the extent of cleavage of free plasmid DNA was nearly the same as the amount of cleavage of chromatin .\",\n \"Thus , the presence of approximately one nucleosome per 200 bp DNA only slightly protects the DNA from hydroxyl radicals .\",\n \"These results are consistent with the previous observation that hydroxyl radicals can cleave DNA in nucleosomes ( Hayes et al . , 1990 ) .\",\n \"Upon addition of Rv Dsup , we observed substantial protection of free DNA as well as chromatin from hydroxyl radicals ( Figure 6A ) .\",\n \"The Dsup-mediated protection was stronger with chromatin than with free DNA , possibly because the specific binding of Dsup to nucleosomes creates a highly resistant structure .\",\n \"In the presence of 4 molecules of Rv Dsup per 200 bp DNA ( approximately 4 Dsup molecules per nucleosome ) in chromatin , there was a considerable amount of full-length linear DNA remaining after hydroxyl radical-mediated cleavage .\",\n \"These results indicate that Rv Dsup is able to protect chromatin from the damaging effects of hydroxyl radicals .\",\n \"Because hydroxyl radicals react primarily with hydrogen atoms that are exposed in the minor groove of DNA ( Balasubramanian et al . , 1998 ) , it appears likely that Dsup blocks access to the minor groove .\",\n \"We additionally tested whether He Dsup and human TFIIB ( a nuclear protein that does not bind to nucleosomes ) can protect chromatin from hydroxyl radical-mediated DNA cleavage ( Figure 6B ) .\",\n \"These experiments revealed that He Dsup , like Rv Dsup , is able to protect nucleosomal DNA from hydroxyl radical-mediated cleavage , and further support the conclusion that He Dsup is an ortholog of Rv Dsup .\",\n \"As seen in Figure 6B , we consistently observed that Rv Dsup provides slightly more protection against hydroxyl radicals than He Dsup .\",\n \"In contrast to Dsup , TFIIB did not confer any detectable protection of chromatin from hydroxyl radical-mediated DNA cleavage .\",\n \"Thus , these findings collectively indicate that Dsup binds to nucleosomes and protects DNA from cleavage by hydroxyl radicals ( Figure 6C ) .\",\n \"The homology between Rv Dsup and He Dsup led us to examine whether any of their conserved regions are related to protein sequences in organisms other than tardigrades .\",\n \"This analysis led to the identification of sequence similarity between a segment in the C-terminal region of the Dsup proteins and a conserved sequence in the nucleosome-binding domain of HMGN proteins ( Figure 7A ) .\",\n \"The HMGN proteins are abundant nucleosome-binding proteins that have been found only in vertebrates ( for reviews , see: Kugler et al . , 2012; González-Romero et al . , 2015 ) .\",\n \"They bind to two high affinity sites on nucleosomes in a manner that is independent of the DNA sequence .\",\n \"The nucleosome-binding domain of the HMGN proteins has been identified , and it contains a conserved core sequence , RRSARLSA , which is critical for the binding of HMGN proteins to nucleosomes ( Ueda et al . , 2008 ) .\",\n \"Strikingly , both Dsup proteins contain a sequence that is similar to the RRSARLSA consensus ( Figure 7A ) .\",\n \"To examine whether the HMGN-like sequence in the Dsup proteins is important for their binding to nucleosomes , we generated two mutant versions of Rv Dsup ( Figure 7A ) .\",\n \"The M1 mutant Dsup is a C-terminal deletion that removes the HMGN-like region , and the M2 mutant Dsup contains three R to E substitution mutations in the HMGN-like region .\",\n \"We purified the mutant Dsup proteins ( Figure 7B ) and then tested their ability to bind to mononucleosomes by gel mobility shift analysis ( Figure 7C ) .\",\n \"These experiments revealed that the M1 mutant Dsup is nearly completely defective for binding to nucleosomes .\",\n \"Hence , the C-terminal region of Rv Dsup ( from amino acid residues 360 to 445 ) is essential for nucleosome binding .\",\n \"In addition , the M2 mutant Dsup exhibits reduced binding to nucleosomes .\",\n \"Thus , the HMGN-like sequence in Dsup is important for its binding to nucleosomes .\",\n \"We further investigated the ability of the mutant Dsup proteins to protect chromatin from hydroxyl radicals .\",\n \"These experiments revealed that the nucleosome-binding-deficient M1 mutant Dsup is nearly completely defective for protection of chromatin against hydroxyl radicals .\",\n \"We also observed that the M2 mutant Dsup is partially defective for protection against hydroxyl radicals .\",\n \"These data support the model that the binding of Dsup to nucleosomes is required to protect chromatin from hydroxyl radical-mediated DNA damage .\"\n ],\n [\n \"Here we have found that Dsup , a tardigrade-specific factor , is a nucleosome-binding protein that protects chromosomal DNA from hydroxyl radical-mediated cleavage .\",\n \"These findings provide a molecular explanation for the ability of Dsup to protect DNA in human cells from degradation by ionizing radiation or by treatment with hydrogen peroxide ( Hashimoto et al . , 2016 ) .\",\n \"More generally , these results suggest that Dsup protects the tardigrade genome from hydroxyl radical-mediated damage under diverse conditions .\",\n \"For instance , protection of the tardigrade genome from hydroxyl radicals may contribute to survival after extended periods in the anhydrobiotic state ( Wełnicz et al . , 2011 ) .\",\n \"Notably , Dsup and histone H1 can bind simultaneously to nucleosomes ( Figure 4A ) .\",\n \"Both R . varieornatus and H . exemplaris appear to contain histone H1 , and it is thus likely that Dsup normally acts in conjunction with H1 .\",\n \"In this regard , it is also notable that Dsup functions in histone H1-containing human cells ( Hashimoto et al . , 2016 ) .\",\n \"Rv Dsup and He Dsup are orthologs .\",\n \"In addition to their sequence similarity and related gene arrangements ( Figure 5A ) , both Rv Dsup and He Dsup are nucleosome-binding proteins that protect chromatin from hydroxyl radicals ( Figures 5 and 6 ) .\",\n \"It had been previously suggested that Dsup is not present in H . exemplaris ( Yoshida et al . , 2017 ) .\",\n \"If true , that would have implied that Dsup is not generally important for tardigrades .\",\n \"Instead , the identification of Dsup as a chromatin-protective protein in two species suggests that it is a key factor that contributes to the unique properties of tardigrades .\",\n \"Unexpectedly , the Dsup proteins contain a region that exhibits sequence similarity to the conserved core of the nucleosome-binding domain of HMGN proteins ( Figure 7A ) .\",\n \"Moreover , the HMGN-like sequence in Rv Dsup is functionally important for its ability to bind to nucleosomes ( Figure 7C ) and to protect chromatin from hydroxyl radicals ( Figure 7D ) .\",\n \"Because the HMGN proteins have been found only in vertebrates ( González-Romero et al . , 2015 ) , the origin of the HMGN-like sequences in the tardigrade Dsup proteins is , at present , a mystery .\",\n \"It is possible but not likely ( see , for example , Doolittle , 1994 ) that the Dsup and HMGN proteins provide a very rare example of sequence convergence .\",\n \"In the future , detailed functional and evolutionary analyses might reveal the relation between these proteins .\",\n \"The Dsup proteins are highly charged and largely disordered ( Hashimoto et al . , 2016; Hashimoto and Kunieda , 2017 ) ( Figure 1A ) .\",\n \"With respect to the latter , examination of the amino acid composition of the Dsup proteins reveals that they are enriched in serine , alanine , glycine , and lysine ( SAGK ) residues .\",\n \"Rv Dsup contains more than 60% SAGK residues , and He Dsup has over 50% SAGK residues .\",\n \"SAGK are disorder-promoting amino acids ( Dunker et al . , 2001 ) , and the SAGK residues may form a diffuse mass of protein that protects the chromosomal DNA from hydroxyl radical-mediated cleavage in a variety of conditions ( Figure 6C ) .\",\n \"This model is consistent with the observation that tardigrades can survive high doses of ionizing radiation in the active as well as anhydriobiotic states ( see , for example , Jönsson et al . , 2005; Horikawa et al . , 2006 ) .\",\n \"In conclusion , these studies reveal molecular aspects of Dsup function and suggest how this intriguing protein may contribute to the unique biological properties of tardigrades .\",\n \"A direct mechanism can be envisaged in which Dsup binds specifically to nucleosomes and protects the DNA from damaging agents such as hydroxyl radicals via coverage of the chromatin with its SAGK-rich disordered regions .\",\n \"In the future , the analysis of Dsup will reveal new insights into the physical and molecular bases of its functions and might further lead to its practical applications .\",\n \"For instance , it is possible that the DNA-protective properties of Dsup could be used to extend the longevity of cells .\",\n \"Thus , this unique protein from an extreme organism could be a new and powerful reagent in biological research .\"\n ],\n [\n \"The cDNA sequence for Rv Dsup was synthesized with an N-terminal His6 tag and a C-terminal FLAG tag ( Integrated DNA Technologies; San Diego , CA ) .\",\n \"The resulting DNA fragment was subcloned into pET21b to give pET21b-His6-Dsup-FLAG .\",\n \"For expression , freshly transformed Escherichia coli strain BL21 ( DE3 ) was grown in LB medium ( 2 L volume; 40 µg/mL ampicillin ) at 37°C to A600 nm of approximately 0 . 6 to 0 . 8 .\",\n \"The synthesis of Dsup was induced by the addition of IPTG to 0 . 4 mM final concentration , and the culture was incubated at 18°C for 16 to 18 hr .\",\n \"The bacteria were collected by centrifugation ( Fiberlite F9−4×1000y rotor; 6 , 000 rpm; 10 min; 18°C ) .\",\n \"Until stated otherwise , the next series of operations were carried out at room temperature ( 22°C ) .\",\n \"The pellet was resuspended in 20 mL of Denaturing Binding Buffer [20 mM sodium phosphate , pH 7 . 8 , containing 8 M urea , 0 . 5 M NaCl , 5 mM 2-mercaptoethanol , 0 . 4 mM PMSF] .\",\n \"The cells were lysed with 3 × 15 strokes in a Wheaton Dounce homogenizer ( B pestle ) , with a 10 min interval between each set of 15 strokes .\",\n \"The mixture was incubated for 1 hr and then subjected to centrifugation ( Fiberlite F21S-8×50y rotor; 15 , 000 rpm; 20 min ) .\",\n \"The supernatant was collected and stored , and the pellet was resuspended with 15 mL of Denaturing Binding Buffer .\",\n \"The suspended pellet was dispersed with 15 strokes in a Wheaton Dounce homogenizer ( B pestle ) , incubated for 1 hr , and subjected to centrifugation ( Fiberlite F21S-8×50y rotor; 15 , 000 rpm; 20 min ) .\",\n \"The second supernatant was combined with the first supernatant , and the resulting mixture was incubated with 3 mL of Ni-NTA agarose beads ( Qiagen; Germantown , MD ) for 2 hr on a rotating wheel at 4°C .\",\n \"At room temperature ( 22°C ) , the mixture was transferred into a Bio-Rad Econo-Pac chromatography column , and the resin was washed with 2 × 10 mL of Denaturing Wash Buffer [20 mM sodium phosphate , pH 6 . 0 , containing 8 M urea , 0 . 5 M NaCl , 5 mM 2-mercaptoethanol , 0 . 4 mM PMSF] .\",\n \"The protein was eluted with Denaturing Elution Buffer [20 mM sodium phosphate , pH 4 . 0 , containing 8 M urea , 0 . 5 M NaCl , 5 mM 2-mercaptoethanol , 0 . 4 mM PMSF] in 5 × 1 mL steps , with a 2 min interval between the addition of each mL of elution buffer .\",\n \"Unless stated otherwise , all subsequent operations were performed at 4°C .\",\n \"The eluate fractions were combined and dialyzed overnight in 4 L of PBS , and then incubated on a rotator wheel with 0 . 5 mL of anti-FLAG M2 Affinity Gel ( Sigma-Aldrich; St . Louis , MO ) for 3 to 4 hr .\",\n \"The beads were washed with 3 × 0 . 5 mL of Buffer A [20 mM Tris-HCl , pH 7 . 9 , containing 150 mM NaCl , 2 mM MgCl2 , 0 . 2 mM EDTA , 15% ( v/v ) glycerol , 1 mM DTT] , and protein was eluted with 4 × 0 . 1 mL portions of Buffer B [Buffer A containing 0 . 4 mg/mL FLAG peptide ( Sigma-Aldrich; St . Louis , MO ) and 0 . 5 mg/mL recombinant human insulin] .\",\n \"The protein fractions were frozen in liquid nitrogen and stored at −80°C .\",\n \"The Rv Dsup that was used in Figures 1–4 was purified by this method .\",\n \"The nondenaturing method works very well for the purification of wild-type and mutant Dsup proteins from R . varieornatus and H . exemplaris .\",\n \"Here , we describe the synthesis and purification of He Dsup .\",\n \"The E . coli codon-optimized cDNA sequence for He Dsup was synthesized with a C-terminal FLAG tag ( Integrated DNA Technologies; San Diego , CA ) and inserted as an NcoI-XhoI fragment into pET21b-His6-Nco ( Kassavetis et al . , 1998 ) .\",\n \"The resulting plasmid was transformed into Rosetta pLysS ( MilliporeSigma ) .\",\n \"The cells were grown at 37°C in LB medium ( 0 . 5 L; 80 and 34 µg/mL , ampicillin and chloramphenicol , respectively ) to an A600 nm of 0 . 8 , induced with IPTG to 0 . 5 mM final concentration , grown for an additional 2 hr , and harvested by centrifugation ( Fiberlite F14S-6×250y rotor; 10 , 000 rpm; 15 min; 4°C ) .\",\n \"Unless stated otherwise , the remaining operations were carried out at 4°C .\",\n \"The cells ( 1 . 8 g ) were resuspended in 12 . 6 mL Lysis Buffer [40 mM potassium phosphate , pH 7 . 6 , 0 . 01% ( v/v ) NP-40 ( Pierce ) , 0 . 1 mM EDTA , 580 mM NaCl , 10% ( v/v ) glycerol , 10 mM 2-mercaptoethanol , 1 µg/mL leupeptin , 1 µg/mL pepstatin , 0 . 5 mM PMSF , and 300 µg/mL lysozyme] , incubated for 30 min on ice , sonicated ( Branson Sonifier 450 with a 0 . 25 inch microtip; 20% output; 10 cycles of sonication of 13 s each; kept below 6°C by chilling in an ice-ethanol bath ) , and clarified by centrifugation ( Fiberlite F21S-8×50y rotor; 18 , 000 rpm; 30 min ) .\",\n \"Dsup was purified from the soluble lysate supernatant fraction on a 0 . 5 mL Ni-NTA agarose column that was washed with 8 mL Buffer E [40 mM potassium phosphate , pH 7 . 6 , 20 mM imidazole , 500 mM NaCl , 10% ( v/v ) glycerol , 10 mM 2-mercaptoethanol , 0 . 5 mM PMSF] , pre-eluted with 300 µL Buffer E containing 200 mM imidazole , and eluted with 700 µL of the same buffer ( yielding 5 . 8 mg/mL He Dsup and 3 . 6 mg/mL Rv Dsup ) .\",\n \"The samples were frozen in liquid nitrogen and stored at −80°C .\",\n \"For further purification , a portion of each sample ( approximately 0 . 1 mg Dsup ) was loaded onto a 300 µL anti-FLAG M2 agarose column that was pre-equilibrated in Buffer A2 [20 mM Tris-HCl , pH 7 . 9 , containing 150 mM NaCl , 2 mM MgCl2 , 0 . 2 mM EDTA , 15% ( v/v ) glycerol , 1 mM 2-mercaptoethanol] .\",\n \"The column was left undisturbed for 5 min and washed with 2 mL Buffer A2 .\",\n \"The Dsup was eluted by the addition of 250 µL ( to give the pre-elution fraction ) followed by 300 µL ( elution fraction ) Buffer A2 containing 300 µg/mL 3XFLAG peptide ( ApexBio Technology ) .\",\n \"The samples were frozen in liquid nitrogen and stored at −80°C .\",\n \"The preparations of He Dsup and Rv Dsup in Figures 6 and 7 were prepared in this manner .\",\n \"The method of preparation described in this paragraph is the most efficient means of obtaining high yields of pure Dsup proteins .\",\n \"Alternate denaturing method .\",\n \"We also purified Dsup from the pellet of the bacterial cell lysate ( prepared as described in the preceding paragraph ) as follows .\",\n \"The pellet was resuspended in Buffer PG [40 mM potassium phosphate , pH 7 . 6 , 20 mM imidazole , 6 M guanidine HCl , 10% ( v/v ) glycerol , 10 mM 2-mercaptoethanol , 1 µg/mL leupeptin , 1 µg/mL pepstatin , 0 . 5 mM PMSF] at room temperature ( 22°C ) , sonicated ( Branson Sonifier 450 with a 0 . 25 inch microtip; 20% output; 4 cycles of sonication of 10 s each; kept below 6°C by chilling in an ice bath ) , and clarified by centrifugation ( Fiberlite F21S-8×50y rotor; 18 , 000 rpm; 15 min; 4°C ) .\",\n \"The supernatant was loaded onto a 1 mL Ni-NTA agarose column , which was then washed with 11 mL Buffer E containing 6 M urea .\",\n \"The Dsup was renatured on the column with sequential 1 . 2 mL washes of Buffer E containing 5 . 0 , 4 . 5 , 4 . 0 , 3 . 5 , 3 . 0 , 2 . 5 , 2 . 0 , 1 . 5 , 1 . 0 and 0 M urea .\",\n \"The column was washed with 0 . 8 mL Buffer E containing 200 mM imidazole to give the pre-elution fraction , and protein was eluted with 1 . 3 mL of the same buffer to give the elution fraction ( containing approximately 1 mg/mL Dsup ) .\",\n \"A portion of the elution fraction ( approximately 0 . 1 mg Dsup ) was loaded onto a 300 µL anti-FLAG M2 agarose column that was pre-equilibrated in Buffer A2 [20 mM Tris-HCl , pH 7 . 9 , containing 150 mM NaCl , 2 mM MgCl2 , 0 . 2 mM EDTA , 15% ( v/v ) glycerol , 1 mM 2-mercaptoethanol] .\",\n \"The column was left undisturbed for 5 min and washed with 2 mL buffer A2 .\",\n \"The Dsup was eluted by the addition of 250 µL ( to give the pre-elution fraction ) followed by 300 µL ( elution fraction; 100 µg yield ) Buffer A2 containing 300 µg/mL 3XFLAG peptide ( ApexBio Technology ) .\",\n \"The protein fractions were frozen in liquid nitrogen and stored at −80°C .\",\n \"The Dsup proteins that were used in the gel mobility shift assays in Figures 5 , 6 and 7 were prepared by the method described in this section .\",\n \"It might be noted that Dsup proteins are hydrophilic and highly-charged proteins that bind weakly to SDS and thus migrate slowly on SDS-polyacrylamide gels relative to ‘average’ proteins such as the molecular mass markers .\",\n \"To confirm the authenticity of the Dsup proteins , we subjected wild-type R . varieornatus Dsup and wild-type H . exemplaris Dsup to LC-ESI-TOF-MS ( liquid chromatography-electrospray ionization time-of-flight mass spectrometry ) .\",\n \"For each of the two proteins , the observed mass was within one mass unit of the predicted ( calculated ) mass .\",\n \"Thus , the Dsup proteins appear to be authentic .\",\n \"Mononucleosomes were reconstituted by using the salt dialysis method ( Stein , 1989; Fei et al . , 2015 ) with purified recombinant Drosophila core histones ( Levenstein and Kadonaga , 2002; Khuong et al . , 2017 ) and the following DNA fragments .\",\n \"The 147 bp Xenopus borealis 5S rDNA fragment corresponds to the nucleosome positioning sequence that was mapped in Fei et al . ( 2018 ) .\",\n \"The sequence of the longer 181 bp X . borealis 5S rDNA fragment ( with the central 147 bp positioning sequence indicated by lower case type ) is as follows: CAGGCTGTCAAGGCCGGgcttgttttcctgcctgggggaaaagaccctggcatggggaggagctgggccccccccagaaggcagcacaaggggaggaaaagtcagccttgtgctcgcctacggccataccaccctgaaagtgcccgatatcgtctgatctcggaAGCCAAGCAGGGTCGGG .\",\n \"The 147 bp synthetic 601 nucleosome positioning sequence is described in Lowary and Widom ( 1998 ) .\",\n \"For nucleosome reconstitution , the DNA fragments were amplified by PCR and purified by 1 . 25% agarose gel electrophoresis and gel extraction ( QIAquick Gel Extraction Kit; Qiagen; Germantown , MD ) .\",\n \"After reconstitution , the quality of the nucleosomes was assessed by nondenaturing 6% polyacrylamide gel electrophoresis .\",\n \"If necessary , the histone:DNA ratios in the reconstitution reactions were adjusted to achieve the efficient conversion of the DNA into mononucleosomes .\",\n \"If multiple nucleosomal species ( due to the presence of mixture of differently positioned nucleosomes ) were observed , the samples were heated at 58°C for 10 min to facilitate the movement of the differently positioned nucleosomes to the most stable location .\",\n \"The resulting mononucleosomes were stored at 4°C prior to use in the gel mobility shift experiments .\",\n \"Mononucleosomes ( approximately 0 . 3 µM stock concentration; 60 nM final concentration ) were combined with the indicated amounts of purified Dsup protein in HEG buffer [25 mM Hepes ( K+ ) , pH 7 . 6 , 0 . 1 mM EDTA , 10% ( v/v ) glycerol] containing 100 mM KCl , and 100 ng bovine serum albumin in a total volume of 15 µL .\",\n \"The samples were incubated at 30°C for 30 min , and then loaded onto a nondenaturing 4 . 5% polyacrylamide gel that had been pre-equilibrated at 4°C and pre-run at 3 . 5 V/cm for 30 min .\",\n \"The gels were run at 4°C for 30 min at 3 . 5 V/cm and then for 2 hr at 8 V/cm .\",\n \"The nucleosomes were visualized by staining of the DNA with ethidium bromide .\",\n \"In experiments that involved testing Dsup and histone H1 , the reaction medium additionally included 0 . 05% ( v/v ) NP-40 ( Pierce ) and 2 mM MgCl2 .\",\n \"The histone H1 was purified from Drosophila embryos by the method of Croston et al . ( 1991 ) .\",\n \"To ensure reproducibility of the data , each of the reported experiments was performed a minimum of two independent times with different preparations ( i . e . , biologically distinct samples ) of Dsup .\",\n \"The ATP-dependent assembly of periodic nucleosome arrays with the ACF motor protein and the dNLP core histone chaperone was carried out essentially as described in Fyodorov and Kadonaga ( 2003 ) and Khuong et al . ( 2017 ) .\",\n \"A standard reaction included purified Drosophila core histones ( 0 . 35 µg ) , purified Drosophila ACF ( 10 nM ) , purified Drosophila dNLP ( 1 . 8 µg ) , 2 mM ATP , purified Drosophila topoisomerase I ( ND423 ) ( 1 nM ) , and pGIE-0 plasmid DNA ( 0 . 35 µg ) ( Pazin et al . , 1994 ) in a total volume of 70 µL .\",\n \"Purified Dsup and histone H1 were included where indicated .\",\n \"The final KCl concentration was 100 mM for samples that were analyzed by the DNA supercoiling and partial MNase assays and 50 mM for samples that were subjected to extensive ( limit ) MNase digestion .\",\n \"The assembly reactions were incubated at 27°C for 90 min .\",\n \"The partial MNase , extensive MNase , and DNA supercoiling assays were performed as described by Fyodorov and Kadonaga ( 2003 ) and Khuong et al . ( 2017 ) .\",\n \"To ensure reproducibility of the data , each of the reported experiments was performed a minimum of two independent times with different preparations ( i . e . , biologically distinct samples ) of Dsup .\",\n \"Nucleosomes were reconstituted onto plasmid pGIE-0 ( 3269 bp ) by the salt dialysis method ( Stein , 1989; Fei et al . , 2015 ) with 16 . 4 pmol Drosophila core histone octamers per pmol pGIE-0 ( average of one nucleosome per 200 bp DNA ) .\",\n \"The final dialysis buffer was Buffer TEN [10 mM Tris-HCl , pH 8 . 0 , 1 mM EDTA , 0 . 01% ( v/v ) NP-40 ( Pierce ) ] containing 50 mM NaCl .\",\n \"Proteins were diluted into Buffer TEN containing 50 mM NaCl prior to use .\",\n \"For each series of reactions with a particular factor ( such as Rv Dsup , He Dsup , or human TFIIB ) , the protein and its corresponding storage buffer were diluted in parallel and then used in a manner that ensured that each reaction was carried out under identical buffer conditions .\",\n \"We also note that the highest concentration of glycerol in any of the reactions was 0 . 014% ( v/v ) .\",\n \"At this concentration of glycerol , we did not detect quenching of the hydroxyl radical cleavage reactions by the buffer medium .\",\n \"Hydroxyl radical-mediated cleavage of DNA was carried out essentially as described by Tullius et al . ( 1987 ) .\",\n \"Briefly , nucleosomal pGIE-0 ( 162 fmol ) along with the indicated amount of added protein were incubated at room temperature ( 22°C ) for 10 min in 90 µL Buffer TEN containing 50 mM NaCl .\",\n \"Then , in each tube , three separate droplets of 60 mM ascorbic acid ( 3 . 3 µL ) , 100 µM Fe ( II ) and 200 µM EDTA ( 3 . 3 µL ) , and 0 . 3% ( v/v ) H2O2 ( 3 . 3 µL ) were deposited onto different locations on the tube wall ( suspended above the chromatin sample at the bottom of the tube ) , mixed together , and vortexed into the 90 µL binding reaction .\",\n \"The reaction was stopped 2 min later by the addition of 10 µL of 200 mM thiourea .\",\n \"The DNA was extracted with phenol-CHCl3-isoamyl alcohol , precipitated with ethanol , and resuspended in 10 µL of 50 mM NaOH-1 mM EDTA and 2 µL of Purple Loading Dye ( New England Biolabs ) .\",\n \"Alkaline agarose gel electrophoresis was performed as described by Shin and Day ( 1995 ) , and the DNA was stained with GelRed ( Biotium ) in 50 mM Tris-HCl , pH 6 . 8 .\",\n \"To ensure reproducibility of the data , each of the reported experiments was performed a minimum of two independent times with different preparations ( i . e . , biologically distinct samples ) of Dsup .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Tardigrades , also known as water bears , are animals that can survive extreme conditions .","The tardigrade Ramazzottius varieornatus contains a unique nuclear protein termed Dsup , for damage suppressor , which can increase the resistance of human cells to DNA damage under conditions , such as ionizing radiation or hydrogen peroxide treatment , that generate hydroxyl radicals .","Here we find that R . varieornatus Dsup is a nucleosome-binding protein that protects chromatin from hydroxyl radicals .","Moreover , a Dsup ortholog from the tardigrade Hypsibius exemplaris similarly binds to nucleosomes and protects DNA from hydroxyl radicals .","Strikingly , a conserved region in Dsup proteins exhibits sequence similarity to the nucleosome-binding domain of vertebrate HMGN proteins and is functionally important for nucleosome binding and hydroxyl radical protection .","These findings suggest that Dsup promotes the survival of tardigrades under diverse conditions by a direct mechanism that involves binding to nucleosomes and protecting chromosomal DNA from hydroxyl radicals ."],"string":"[\n \"Tardigrades , also known as water bears , are animals that can survive extreme conditions .\",\n \"The tardigrade Ramazzottius varieornatus contains a unique nuclear protein termed Dsup , for damage suppressor , which can increase the resistance of human cells to DNA damage under conditions , such as ionizing radiation or hydrogen peroxide treatment , that generate hydroxyl radicals .\",\n \"Here we find that R . varieornatus Dsup is a nucleosome-binding protein that protects chromatin from hydroxyl radicals .\",\n \"Moreover , a Dsup ortholog from the tardigrade Hypsibius exemplaris similarly binds to nucleosomes and protects DNA from hydroxyl radicals .\",\n \"Strikingly , a conserved region in Dsup proteins exhibits sequence similarity to the nucleosome-binding domain of vertebrate HMGN proteins and is functionally important for nucleosome binding and hydroxyl radical protection .\",\n \"These findings suggest that Dsup promotes the survival of tardigrades under diverse conditions by a direct mechanism that involves binding to nucleosomes and protecting chromosomal DNA from hydroxyl radicals .\"\n]"},"summary":{"kind":"list like","value":["Tardigrades , also known as water bears and moss piglets , are small animals found in many different environments on land and sea .","These animals have the remarkable ability to survive extremes including very low temperatures , high levels of radiation and exposure to chemicals that are harmful to other forms of life .","Tardigrades have even been found to survive the harsh conditions of outer space .","X-rays are a type of radiation naturally produced by lightning strikes and are also found in cosmic rays from outer space .","High doses of X-rays can cause genetic mutations that may lead to serious illness or death .","This is because when X-rays come into contact with water they split the water molecules to make particles known as hydroxyl radicals , which in turn damage the DNA inside cells .","The genomes of animals and plants are made of DNA , which is packaged into a structure called chromatin .","Previous studies identified a protein named Dsup in a tardigrade called Ramazzottius varieornatus that can protect human cells from damage by X-rays .","However , it was not known whether Dsup binds directly to chromatin or plays a more indirect role in protecting DNA .","Chavez , Cruz-Becerra , Fei , Kassavetis et al . used biochemical approaches to study Dsup .","Their experiments revealed that Dsup from R . varieornatus binds to chromatin to protect the DNA from damage by hydroxyl radicals , and that the Dsup protein in another tardigrade species also works in a similar way .","Further analysis showed that a region of Dsup that is needed to bind to chromatin is very similar to a region that had been previously found only in chromatin-binding proteins from humans and other vertebrates ( animals with backbones ) .","This connection between Dsup and vertebrate chromatin-binding proteins remains a mystery .","The new findings about tardigrade Dsup may help researchers develop animal cells that live longer under normal or extreme environmental conditions .","In this manner , Dsup could be used to expand the range of applications of cells in biotechnology .","It could also increase the effectiveness of current methods , such as the production of some pharmaceuticals , that depend upon the use of cultured cells ."],"string":"[\n \"Tardigrades , also known as water bears and moss piglets , are small animals found in many different environments on land and sea .\",\n \"These animals have the remarkable ability to survive extremes including very low temperatures , high levels of radiation and exposure to chemicals that are harmful to other forms of life .\",\n \"Tardigrades have even been found to survive the harsh conditions of outer space .\",\n \"X-rays are a type of radiation naturally produced by lightning strikes and are also found in cosmic rays from outer space .\",\n \"High doses of X-rays can cause genetic mutations that may lead to serious illness or death .\",\n \"This is because when X-rays come into contact with water they split the water molecules to make particles known as hydroxyl radicals , which in turn damage the DNA inside cells .\",\n \"The genomes of animals and plants are made of DNA , which is packaged into a structure called chromatin .\",\n \"Previous studies identified a protein named Dsup in a tardigrade called Ramazzottius varieornatus that can protect human cells from damage by X-rays .\",\n \"However , it was not known whether Dsup binds directly to chromatin or plays a more indirect role in protecting DNA .\",\n \"Chavez , Cruz-Becerra , Fei , Kassavetis et al . used biochemical approaches to study Dsup .\",\n \"Their experiments revealed that Dsup from R . varieornatus binds to chromatin to protect the DNA from damage by hydroxyl radicals , and that the Dsup protein in another tardigrade species also works in a similar way .\",\n \"Further analysis showed that a region of Dsup that is needed to bind to chromatin is very similar to a region that had been previously found only in chromatin-binding proteins from humans and other vertebrates ( animals with backbones ) .\",\n \"This connection between Dsup and vertebrate chromatin-binding proteins remains a mystery .\",\n \"The new findings about tardigrade Dsup may help researchers develop animal cells that live longer under normal or extreme environmental conditions .\",\n \"In this manner , Dsup could be used to expand the range of applications of cells in biotechnology .\",\n \"It could also increase the effectiveness of current methods , such as the production of some pharmaceuticals , that depend upon the use of cultured cells .\"\n]"},"year":{"kind":"string","value":"2019"}}},{"rowIdx":4289,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["neuroscience"],"string":"[\n \"neuroscience\"\n]"},"title":{"kind":"string","value":"Accelerated redevelopment of vocal skills is preceded by lasting reorganization of the song motor circuitry"},"id":{"kind":"string","value":"elife-43194-v2"},"sections":{"kind":"list like","value":[["Complex motor skills , such as singing or playing an instrument , are not inherently determined but need to be acquired through repetitive practice .","Many specialized skills are used only incidentally however , and skill performance degrades during intermittent periods of non-use .","This means that skills need to be re-acquired each time the need arises .","For example , trained human surgeons need to re-acquire their surgical proficiency after a period of absence ( Crewther et al . , 2016 ) , Capuchin monkeys need to acquire manipulative foraging skills to retrieve difficult-to-access , seasonally available food items ( Eadie , 2015 ) , and songbirds need to re-develop high-quality songs to attract a mate after wintering or long-distance migration ( Slagsvold , 1976 ) .","Whereas learning novel motor skills is generally a lengthy process , the short-term availability of suitable mates and food items imposes a considerable time pressure on the re-acquisition of such specialized skills .","Birdsong , an established model system for vocal motor learning ( Brainard and Doupe , 2013 ) , is characterized by a high degree of complex and rapid acoustic modulations .","The fine motor skills that are necessary to produce such complex , high quality vocalizations are thought to advertise an individual’s quality through performance-related characteristics such as song complexity and/or production rate ( Podos , 1997; Drăgănoiu et al . , 2002; Holveck and Riebel , 2007; Byers et al . , 2010 ) .","When juvenile songbirds are one to two months old they start singing noisy , unstructured ‘subsongs’ akin to human babbling ( Doupe and Kuhl , 1999 ) , which gradually develop into variable , but recognizable species-specific ‘plastic songs’ ( Marler and Peters , 1982a; Nottebohm et al . , 1986; Tchernichovski et al . , 2001; Mori et al . , 2018 ) .","With extensive vocal rehearsal the songs slowly consolidate into high performance ‘crystallized songs’ , a process that can take up more than five months in some songbird species ( Nottebohm et al . , 1986 ) .","For adult songbirds that annually need to re-acquire their songs this process of juvenile song development greatly exceeds the time that is available to attract a mate during the early breeding season .","Thus , adult songbirds must considerably speed-up song re-acquisition to quickly produce songs of adequate quality to impress conspecifics .","It is currently unclear how songbirds cope with the seasonally imposed time pressure on song development , and what mechanisms may be involved to ensure reproductive success under such time constraints in nature .","The seasonal development and degradation in vocal performance are highly correlated with changes in testosterone levels ( Nottebohm et al . , 1987; Smith et al . , 1997; Tramontin et al . , 2000; Voigt and Leitner , 2008 ) , and are often accompanied by gross anatomical and cytoarchitectural restructuring of the brain areas involved in song production ( Nottebohm , 1981; Gahr , 1990; Kirn et al . , 1994; Kafitz et al . , 1999; Thompson and Brenowitz , 2005; Balthazart et al . , 2008; Vellema et al . , 2014 ) .","Thus testosterone-regulated adaptations of the songbird brain may play an important role in optimizing song performance .","In this study we used adult female canaries ( Serinus canaria ) to investigate how a periodically acquired motor behavior attains a high performance level under developmental time pressure .","Although female canaries rarely sing spontaneously , and never produce high quality songs under natural conditions ( Pesch and Guttinger , 1985 ) , the systemic application of testosterone leads to the development of high-performance song with male-typical features ( Leonard , 1939; Shoemaker , 1939; Vallet et al . , 1996 ) .","This trait provides us with a well-defined behavioral baseline , and allows us to manipulate in detail the onset and offset of song development through repeated testosterone treatments .","Here we demonstrate that during a protracted testosterone treatment , adult female canaries gradually develop stable , species-typical songs through a process of song crystallization similar to what is known for naturally-raised juvenile male canaries ( Mori et al . , 2018 ) .","Re-treatment with testosterone several months after birds have stopped singing , leads to a rapid recurrence of song performance with song features that strongly resemble those after the first treatment .","We propose that once developed , vocal motor memories are retained for an extended period of time , enabling adult animals to recover song performance quickly at a later time , a process termed ‘savings’ ( Ebbinghaus , 2013 ) .","We further demonstrate that neurons in the songbird’s premotor nucleus HVC ( proper name ) , a central nucleus in the brain circuitry that controls song production ( Nottebohm et al . , 1976 ) , irreversibly loses a significant number of dendritic spines during the initial testosterone-induced development of vocal skills .","This state of reduced spine density is subsequently maintained over long periods in which testosterone levels are low and vocal skills are not used .","The observed lasting synaptic pruning could play an important role in the formation of vocal skill savings , enabling birds to rapidly re-acquire song performance within a restricted time period ."],["To study the development of vocal skills in adult female canaries we recorded and analyzed the entire song ontogeny in acoustically separated animals ( 5 . 6 ± 0 . 6 million song syllables per bird ) , while manipulating song output by consecutively implanting , removing , and re-implanting birds with testosterone implants .","Whereas no songs were observed prior to treatment , the systemic application of testosterone ( T1+ ) stimulated birds to start singing their first songs after three days ( 3 . 22 ± 0 . 46 days ) .","The phases of female song development were similar to those previously reported for male canaries ( Figure","1 ) ( Nottebohm et al . , 1986; Mori et al . , 2018; Weichel et al . , 1986 ) .","The first subsongs were produced in an irregular fashion and consisted of unstructured syllables with few distinctive features .","Songs gradually developed into plastic songs in which different phrases of repeated syllables could be clearly distinguished , albeit with variation between syllables of the same type .","During the following six months , female canary songs continued to develop , gradually crystallizing into the species-typical stable songs .","Although structurally similar to male canary songs , female songs had relatively small syllable repertoires ( 6 . 2 ± 1 . 1 syllable types ) , consistent with previous reports ( Vallet et al . , 1996; Fusani et al . , 2003; Hartog et al . , 2009 ) .","To investigate the birds’ abilities to re-acquire song performance after a period without vocal practice we withdrew testosterone ( T1- ) from the animals , completely abolishing singing behavior in approximately three days ( 3 . 14 ± 0 . 63 days ) .","After 2½ months of no song production birds were treated for a second time with testosterone ( T2+ ) , inducing song output after three days ( 3 . 14 ± 0 . 74 days ) .","No subsongs were observed after the second testosterone treatment , and all birds were able to produce plastic songs from the first day of singing .","We first compared the development of temporal song features during the first and second testosterone treatments ( Figure 2 , Figure 2—figure supplement 1 , Figure 2—figure supplement 2 ) .","Particularly the speed at which subsequent syllables are repeated , the syllable repetition rate ( SR ) , has been strongly associated with individual performance ( Podos , 1997; Podos , 1996 ) and has been shown to increase the attractiveness of the song to conspecifics ( Vallet et al . , 1998; Vallet and Kreutzer , 1995 ) .","We observed that during the first two weeks of testosterone-induced female song development , all syllables were typically produced at a similar rate ( SR: 10 . 3 ± 1 . 3 Hz ) .","While practicing the song , different syllables were gradually sung at different rates , becoming more distinct towards song crystallization ( Figure 2A ) .","Once crystallized , syllables could be distinguished as fast syllables ( SR: 21 . 7 ± 1 . 0 Hz ) , medium-speed syllables ( SR: 11 . 9 ± 1 . 1 Hz ) , and slow syllables ( SR: 4 . 9 ± 0 . 7 Hz ) .","A similar distribution of syllable rates redeveloped during a 2nd testosterone treatment .","The observed range of syllable rates was similar to what has previously been reported for wild-living male canaries ( Leitner et al . , 2001 ) .","To determine how fast this syllable rate pattern emerged during subsequent testosterone treatments we took crystallized song from the last week of the first testosterone treatment as a reference pattern , and calculated the correlation coefficient ( CC ) between this reference and each day of song development and re-development ( Figure 2B & C ) .","While syllable repetition rates gradually developed and stabilized over the course of 160 ± 8 days during the first testosterone treatment , stable rates were obtained more than seven times faster , within 22 ± 2 days , during the second treatment ( Figure 2D; paired t-test: p<0 . 001 ) .","In addition the maximum daily increase in similarity ( dmax ) was also significantly higher during song re-development than during initial song development ( Figure 2E; dmax: 0 . 021 ± 0 . 005 for T1+ , and 0 . 078 ± 0 . 012 for T2+; paired t-test: p<0 . 05 ) , indicating a much accelerated development of song performance in birds with previous singing experience .","Since syllable rate is a compound feature that is determined by both syllable duration and the interval between subsequent syllables , we analyzed syllable durations and pause durations separately .","Syllable durations developed slowly during the initial song acquisition ( T1+ ) , reaching stable values after 153 ± 19 days , while pause durations stabilized in 82 ± 13 days ( Figure 2—figure supplement 1 , Figure 2—figure supplement 2 ) .","Both temporal features re-emerged more quickly during song re-acquisition ( T2+ ) than during the first song developmental phase ( duration: 4 . 8 ± 1 . 3 days; paired t-test: p<0 . 001; pause: 10 . 7 ± 2 . 2 days; paired t-test: p<0 . 01; Figure 2—figure supplement 1 ) .","To determine the bird’s ability to recover spectral song patterns we analyzed the recurring patterns of frequency modulation ( FM ) , amplitude modulation ( AM ) , bandwidth ( BW ) , mean frequency ( MF ) and Wiener entropy ( E ) during song acquisition and re-acquisition .","Similar to temporal song features , spectral features were more quickly established during song re-acquisition ( T2+ ) than during the initial song development ( T1+ ) ( Figure 3 , Figure 3—figure supplement 1 , Figure 3—figure supplement 2 ) .","All spectral features developed gradually over more than 110 days before reaching stable values .","After a period in which the birds did not produce any song , testosterone-induced song re-acquisition led to a rapid stabilization of spectral patterns within 16 days ( Figure 3 , Figure 3—figure supplement 1; paired t test: p<0 . 01 ) .","Thus both temporal and spectral song features were acquired significantly faster by experienced birds that had developed singing skills once before .","Whereas song re-acquisition in female canaries resulted in a rapid recurrence of stable temporal and spectral patterns , not all song features re-developed in the same way .","Most notably , some song features demonstrated a clear deterioration in the absence of vocal practice , while other song features remained stable despite the absence of vocal practice ( Figure 3G & H ) .","Of the studied temporal song parameters , syllable durations did not show any deterioration in the period that birds did not sing ( T1- ) and thus also did not need to be re-acquired ( Figure 2—figure supplement 1A–C; Figure 2—figure supplement 2A ) .","Both the syllable rate and the pause duration between syllables did deteriorate however , and a short phase of re-development was required to recover the originally developed patterns ( Figure 2 , Figure 2—figure supplement 1D–F , Figure 2—figure supplement 2B ) .","The accelerated re-acquisition of syllable repetition rates thus appears to be driven by the need to re-optimize the timing between syllables rather than syllable lengths .","Of the spectral syllable features that were studied , the FM and AM patterns deteriorated when birds stopped singing ( T1- ) , but were re-acquired during a short developmental phase after a 2nd testosterone treatment ( T2+ ) ( Figure 3A–C , Figure 3—figure supplement 1A–C , Figure 3—figure supplement 2A & B ) .","BW , MF , and E did not need to be re-acquired , but maintained strong similarities with the initial acoustic patterns despite the absence of song production in the intermittent period ( T1- ) ( Figure 3D–F , Figure 3—figure supplement 1D–I , Figure 3—figure supplement 2C–E ) .","Thus song re-acquisition can be characterized by recalling a combination of sound features that immediately can be reproduced and sound features that require a short phase of redevelopment .","The songs that reappeared during the second testosterone treatment were strikingly similar to those that developed after the first treatment ( Figure 4 ) .","Visual inspection of the song spectrograms revealed no differences in syllable repertoire when comparing crystallized songs during the first and second testosterone treatment ( Figure 4A & B; 6 . 2 ± 1 . 1 syllable types for both treatments ) .","In addition both temporal and spectral song features demonstrated a strong similarity in their distribution patterns between subsequent testosterone treatments ( T1xT2 ) , with correlation coefficients above 0 . 82 for all studied song features ( Figure 4C ) .","These values were not significantly different from the correlation coefficients obtained by cross-correlating distribution patterns from consecutive days during the first treatment period ( T1xT1 ) for any of the measured song features ( Figure 4C ) , indicating that the song patterns that developed during the first and second testosterone treatment are similar in structure .","To determine the similarity of syllables produced during the 1st and 2nd testosterone treatments we calculated for each bird the Euclidean distance across all eight analyzed temporal and spectral features within and between syllable types ( Figure 4—figure supplement 1 ) .","The Euclidean distance between two syllables provides a measure of similarity where a syllable is represented as a point with coordinates that correspond to the eight analyzed sound features .","The distance between syllables with similar sound features and thus similar coordinates is low , while the distance between syllables with differing sound features is high .","The mean Euclidean distances between syllables of the same type across the 1st and 2nd testosterone treatment ( T1xT2 ) were not different from the Euclidean distances between syllables from subsequent days during the 1st testosterone treatment ( T1xT1: d = 0 . 73 ± 0 . 04 , T1xT2: d = 0 . 76 ± 0 . 06; Tukey’s HSD: p=0 . 99 ) .","The mean Euclidean distances between syllables of different types across the two treatment periods were significantly larger than those between syllables of the same type ( T1xT2ext: d = 2 . 12 ± 0 . 23; Tukey’s HSD: p<0 . 001 ) .","These results indicate that for a given syllable the sound features that redeveloped after the 2nd testosterone treatment fell within the range of variation observed during the 1st testosterone treatment for that same syllable type , but were clearly distinguishable from the sound features of other syllable types .","To investigate if the sequential structure of the song was different between the two testosterone treatments we analyzed the probability that specific phrase transitions occurred in the songs ( Figure 4—figure supplement 2 ) .","Because the syllable repertoire is relatively small in testosterone treated female canaries , the number of possible phrase transitions is also small .","Nevertheless , on average only 8 . 7% of all possible phrase transitions were used in the songs .","Thus whereas the song sequences are not fixed as in for example zebra finches , the song sequences are not random either , following a limited subset of possible transitions similar to what has been observed in male canaries ( Leitner et al . , 2001; Markowitz et al . , 2013 ) .","The percentage of phrase transitions that made up the songs during the 1st and 2nd testosterone treatments were not different ( T1+: 9 . 2 ± 4 . 5%; T2+: 8 . 3 ± 4 . 2%; paired t-test: p=0 . 59 ) , and the probability distribution of these transitions were visually similar between treatments ( Figure 4—figure supplement 2A & B ) .","The linearity , consistency , and entropy of song phrase transitions did not differ significantly between the songs from the 1st and 2nd treatment periods ( Figure 4—figure supplement 2C ) .","Together these data strongly suggest that testosterone-induced song re-acquisition in canaries is driven towards the previously acquired song pattern .","The finding that the re-acquisition of song performance progresses considerably faster than the initial acquisition and leads to the production of highly similar song patterns suggests that singing skills are retained during intermittent silent periods .","Motor learning and memory are generally considered to rely on the maturation and consolidation of the synaptic connections within the neural circuitry that drives the behavior in question ( Changeux and Danchin , 1976; Yu and Zuo , 2011; Hoshiba et al . , 2017 ) .","To investigate changes in synaptic connectivity during vocal motor development we quantified the number of neuronal dendritic spines in the forebrain motor nucleus HVC and the robust motor nucleus of the arcopallium ( RA ) before , during , and after the acquisition of vocal motor skills ( Figure 5 ) .","Excitatory projection neurons in the HVC of testosterone-treated , singing female canaries displayed a more than 30% reduction in dendritic spine density compared to untreated , non-singing control birds ( C: 0 . 95 µm−1 , T1+: 0 . 64 µm−1; Dunnett’s test: p<0 . 01 ) , indicating a significant synaptic pruning during the first acquisition of singing skills ( Figure 5B ) .","Compared to non-singing control birds , spine densities remained significantly lower in birds that stopped singing for 2 . 5 months after testosterone removal ( C: 0 . 95 µm−1 , T1-: 0 . 69 µm−1; Dunnett’s test: p<0 . 01 ) .","Thus , the synaptic pruning in HVC associated with testosterone-induced song acquisition is not reversed when birds stop singing , but instead the pruned state is maintained in periods in which the birds do not sing ( Figure 5B ) .","In contrast , we did not observe any changes in dendritic spine densities in RA across the different experimental periods ( Figure 5C; ANOVA: p=0 . 857 ) .","Strong differences were observed in the density of dendritic spines between different spinous neurites in HVC ( range: 0 . 15 to 2 . 14 spines/µm ) .","To investigate which neurite types were associated with the observed spine pruning we calculated the probability distribution of neuronal dendrites in HVC based on their spine density ( Figure 5D–F ) .","We observed a marked shift in the distribution of dendrites from more densely-spined dendrites in control birds towards less densely-spined dendrites in the testosterone treatment ( T1+ ) and removal groups ( T1- ) , reflecting a global decrease in spine density .","The strongest reduction was detected in the range of dendrites with more than 0 . 8 spines per µm , which halved in number in both testosterone-treated , singing birds ( T1+ ) , and testosterone-removed , non-singing birds ( T1- ) compared to non-singing controls ( C: 0 . 64 ± 0 . 06 , T1+: 0 . 28 ± 0 . 05 , T1-: 0 . 33 ± 0 . 06; ANOVA: p<0 . 01 ) .","The total density of both spinous and aspinous neurites in HVC did not significantly differ between the treatment groups ( ANOVA: p>0 . 36 ) , suggesting that changes in dendrite densities were not caused by specific cell death .","These data suggest that the observed vocal motor acquisition is accompanied by a lasting synaptic pruning specifically in nucleus HVC , providing a possible anatomical site for motor memory that could facilitate the observed rapid re-acquisition of previously established song patterns ."],["The ability to learn new skills enables one to adapt to changes in the surrounding environment .","Whereas learning new skills requires time and effort , the survival of many species relies on their capacity to utilize specialized skills in a timely fashion , capitalizing on the short-term availability of foods , mates , and other environmental factors ( Prendergast et al . , 2002 ) .","Such a capacity is biologically relevant for numerous skills in a multitude of species , ranging from periodic foraging skills in dolphins ( Patterson et al . , 2016 ) , bats ( Clarin et al . , 2013 ) , and monkeys ( Boinski and Fragaszy , 1989 ) to courtship displays in crabs ( Mowles et al . , 2017 ) and songbirds ( Ota et al . , 2015 ) .","As the use of these specialized skills is interrupted over longer periods , for example during long-distance migration or hibernation ( Slagsvold , 1976; Prendergast et al . , 2002 ) , such skills need to be re-acquired quickly to maximize survival and reproductive success .","In this study we establish the repeated , hormone-driven acquisition of vocal motor performance in female canaries as a model system for studying motor savings .","Using this model , we show that while the initial development of specialized motor skills can be a lengthy process , motor performance can quickly be re-acquired at a later time , even after extended periods of non-use ( Figure 2 and 3 ) .","The accelerated song re-acquisition observed in this study indicates that acquiring a motor skill for the first time follows a different developmental trajectory than re-acquiring the same skill at a later time .","This is both evident from the accelerated consolidation of song performance during song re-development ( Figure 2 and 3 ) , and the observation that birds skip the subsong phase of song development when re-acquiring their songs .","Our data further suggest that songbirds have the ability to retain previously developed singing skills through a process of selective pruning of the neural circuitry ( Figure 5 ) , enabling them to rapidly recover songs of adequate quality to attract a mate before breeding opportunities disappear .","Several song parameters were maintained across a period in which birds did not sing , while other song features demonstrated clear deterioration ( Figure 3G & H ) .","Birds were able to recover those deteriorated song features with renewed vocal practice .","At the peripheral level , song performance depends on the accurate control of the respiratory system , the sound producing organ ( the syrinx ) , and the upper vocal tract .","Interestingly , those song features that required a phase of re-development to be optimized seem primarily associated with motor constraints in syrinx function ( Podos , 1997; Geberzahn and Aubin , 2014 ) .","Online modulation of frequency and amplitude requires active contractile modulation of syringeal muscles ( Goller and Suthers , 1996; Elemans et al . , 2008 ) .","Syllable repetition and the inter-syllabic interval also depend on how rapidly syringeal abductor and adductor muscles can open and close the syringeal aperture .","The upper vocal tract may act as a tunable acoustic filter of the sounds produced by the syrinx ( Nowicki , 1987; Beckers et al . , 2003; Riede et al . , 2006 ) and play a more important role in establishing the bandwidth , entropy and mean frequency of song syllables .","Thus song parameters linked to vocal tract modulation may be preserved after birds stopped singing , while song parameters linked to contraction speed of syringeal muscles appear to deteriorate .","Whereas male canaries are known to annually re-organize their songs , even when deafened ( Mori et al . , 2018 ) , no such modifications were observed in our study .","It is not clear to what extent annual changes in male canary song structure are due to relearning or are driven by a selection process from a previously learned collection of syllable types during juvenile development ( Nottebohm et al . , 1986; Marler , 1997; Gardner et al . , 2005; Belzner et al . , 2009 ) .","Considering that the female canaries in our study were acoustically separated during the testosterone-induced development of song suggests that the emerging song patterns are either for a large part innate ( Mori et al . , 2018; Gardner et al . , 2005 ) , or are limited to a memorized collection of syllable types that they heard earlier in life ( Belzner et al . , 2009; Marler and Peters , 1981; Marler and Peters , 1982b; Prather et al . , 2010; Kiefer et al . , 2014 ) .","The initial song acquisition may thus rely on an innate or memorized auditory template , while the accelerated song re-acquisition may be driven by a motor memory trace of what the birds learned to produce during the first song acquisition phase .","The syllable repertoires of our female canaries were much lower than what is common for males .","Such a small vocabulary would limit the possibilities to re-arrange or replace syllables without compromising song complexity , and thus few modifications to female songs can be expected .","During juvenile brain development in humans , mammals , and birds , synaptic connections are initially overproduced , followed by protracted , activity-dependent synapse elimination during puberty ( Huttenlocher , 1990; Petanjek et al . , 2011; Rakic et al . , 1986; Zuo et al . , 2005; Nixdorf-Bergweiler et al . , 1995 ) .","By selectively stabilizing functional synapses while pruning away inactive synapses , brain circuits are thought to reduce neuronal plasticity , consolidating newly learned information and reducing unnecessary redundancy ( Changeux and Danchin , 1976 ) .","The lasting spine pruning observed in the forebrain song control area HVC ( Figure 5 ) could reflect such a structural modification facilitating the long-term memorization of vocal patterns .","Optical imaging studies have demonstrated a rapid accumulation and stabilization of neuronal dendritic spines in HVC during developmental song learning in zebra finches ( Roberts et al . , 2010 ) .","In the mouse cortex , morphological modifications to dendritic spines have been observed that lasted well beyond the learning experience that caused the spine modifications ( Hofer et al . , 2006; Yang et al . , 2009; Xu et al . , 2009; Hofer et al . , 2009 ) , strongly suggesting that long-term memory retention of previously acquired skills is facilitated by stably maintained synaptic connections .","Despite such learning-related circuit modifications , the large majority of dendritic spines in the mammalian brain are stably maintained in adulthood ( Petanjek et al . , 2011; Rakic et al . , 1986; Zuo et al . , 2005; Yang et al . , 2009; Xu et al . , 2009; Grutzendler et al . , 2002 ) .","In contrast we observed a strong spine reduction in fully adult birds ( Figure 5 ) , suggesting that the observed synaptic pruning in the adult canary HVC during song acquisition may reflect a developmentally delayed , activity-dependent consolidation of the neural motor circuitry .","A recent study in zebra finches has shown that singing prevention during juvenile development can also avert developmental spine pruning in the robust motor nucleus of the arcopallium ( RA ) ( Hayase et al . , 2018 ) .","In addition , auditory input during sensorimotor learning may play an important role in circuit consolidation , and auditory deprivation has been shown to delay the natural juvenile maturation of HVC in zebra finches ( Huang et al . , 2018 ) .","It is not known how long such developmental processes can be postponed , but we did not observe delayed synaptic pruning in RA upon song acquisition in adult female canaries ( Figure 5C ) , suggesting that the time-window for large-scale spine pruning in RA may be more limited than in HVC .","The developmentally delayed maturation of HVC ( Figure 5B ) is consistent with our observation that the delayed , testosterone-induced development of song performance in adult female canaries follows a juvenile male-like developmental trajectory ( Figure 1 ) .","Thus , in the absence of singing behavior , parts of the vocal motor circuit may remain plastic in adult female canaries , only consolidating once neural activity within the circuitry increases to drive testosterone-induced song output .","Testosterone-induced song development in adult female canaries has previously been associated with changes in other neural attributes in HVC as well , including an increase in volume , neuron numbers ( but not densities ) , increased vascularization , and changes in cellular structure ( Vellema et al . , 2014; Hartog et al . , 2009; Gahr and Garcia-Segura , 1996; Nottebohm , 1980; Rasika et al . , 1994; Louissaint et al . , 2002 ) .","Many of the observed modifications to the song system follow an annually changing pattern in seasonally breeding male songbirds ( Nottebohm et al . , 1986; Smith et al . , 1997; Nottebohm , 1981; Kirn et al . , 1994 ) , indicating that in males these modifications are not maintained outside the breeding season when birds no longer sing high performance song .","In addition , withdrawing testosterone from male Gambel’s white-crowned sparrows , another well-studied seasonal singer , resulted in a rapid regression in HVC size , neuron number , and neuron size ( Thompson et al . , 2007 ) , indicating that these neural modifications are reversible and unlikely to reflect long-term memorization of vocal patterns .","The lasting synaptic pruning that we observed in HVC is to our knowledge the first report of a song-related modification to the adult songbird brain that persists after the behavior itself is no longer expressed .","HVC is at a central position between the motor circuitry that controls song output and a forebrain-basal ganglia circuit , analogous to the mammalian cortico-basal ganglia ( CBG ) circuit , which feeds back on the motor circuitry for song production ( Brainard and Doupe , 2013; Nottebohm et al . , 1976 ) .","Firing patterns in HVC neurons that project to RA are closely associated with millisecond precision of syllable timing ( Yu and Margoliash , 1996; Hahnloser et al . , 2002; Amador et al . , 2013 ) , suggesting that HVC is directly involved in maintaining time-related features of song performance .","Burst patterns in RA neurons on the other hand are more closely associated with intrasyllabic structure ( Yu and Margoliash , 1996; Chi and Margoliash , 2001 ) .","However , slowing down brain processes by local cooling of HVC has also been shown to change the intrasyllabic structure ( Long and Fee , 2008; Alonso et al . , 2015 ) , indicating that both HVC and RA activity play a role in maintaining the fine temporal structure within syllables .","The acquisition of song syllables in zebra finches is accompanied by increased timing accuracy of inhibitory neural firing within the HVC microcircuitry , suggesting that precisely-timed inhibition may play an important role in shaping song-related premotor sequences ( Kosche et al . , 2015; Vallentin et al . , 2016 ) .","The lasting synaptic reorganization in HVC during song acquisition may be guided by such inhibitory activity and provide a neural scaffold for both intra and intersyllabic timing .","RA-projecting HVC neurons are continuously replaced in the adult songbird brain ( Alvarez-Buylla et al . , 1990 ) , providing a base for ongoing variability in syringeal muscle control .","A permanently maintained HVC network may guide dynamically changing circuits in HVC and RA during subsequent periods of singing , quickly driving song features towards previously acquired patterns .","Neurons from HVC that project to the avian basal ganglia Area X are not replaced in adulthood , providing a permanent subpopulation of HVC neurons ( Gahr , 1990; Alvarez-Buylla et al . , 1988 ) .","Although we could not specifically distinguish between neuron types in our study , we observed the strongest reduction of 50% in the number of densely-spined dendrites in HVC after birds developed song .","Previous studies have shown that those HVC neurons that project to Area X predominantly consist of neurons with densely-spined dendrites ( Kornfeld et al . , 2017; Nixdorf et al . , 1989; Kubota and Taniguchi , 1998; Benton et al . , 1998; Mooney , 2000; Mooney and Prather , 2005; Fortune and Margoliash , 1995 ) .","Thus our observed synaptic pruning may be related to a major reorganization within the HVC to Area X connection , a reorganization that could reflect the long-term memorization of vocal skills .","CBG-like circuits have been strongly implicated in the learning and long-term retention of motor skills in humans ( Albouy et al . , 2008; Debas et al . , 2010; Lehéricy et al . , 2006; Poldrack et al . , 2005; Coynel et al . , 2010 ) , mammals ( Barnes et al . , 2005; Pasupathy and Miller , 2005; Sheth et al . , 2011; Yin et al . , 2009 ) , and birds ( Bottjer et al . , 1984; Scharff and Nottebohm , 1991; Brainard and Doupe , 2000; Kao et al . , 2005; Olveczky et al . , 2005; Aronov et al . , 2008; Sober and Brainard , 2009; Andalman and Fee , 2009; Ölveczky et al . , 2011; Charlesworth et al . , 2012 ) .","Vocal exploration is driven by activity in the avian CBG circuit ( Kao et al . , 2005 ) , which may guide synaptic modifications in plastic motor circuits ( Mehaffey and Doupe , 2015 ) , even in adulthood .","Although selective elimination of X-projecting HVC neurons did not appear to alter the song structure of adult zebra finches ( Scharff et al . , 2000 ) , altered expression of FoxP2 in Area X of adult male zebra finches has been associated with changes in vocal performance ( Murugan et al . , 2013; Thompson et al . , 2013; Teramitsu and White , 2006 ) .","In addition neurotoxic lesions in Area X lead to changes in singing tempo and syllable sequencing in adult zebra finches ( Kubikova et al . , 2014 ) , suggesting that the HVC to Area X connection continues to play a role in the long-term maintenance of vocal sequences .","The mechanisms behind learning-related , selective stabilization and elimination of dendritic spines remain largely unknown .","Sex hormones may play an important role in the stabilization of such synaptic connections during the development of motor skills .","Many of the brain areas that are involved in song learning and production express high numbers of androgen receptors ( Arnold et al . , 1976; Balthazart et al . , 1992; Metzdorf et al . , 1999; Nastiuk and Clayton , 1995; Gahr and Metzdorf , 1997 ) , and X-projecting HVC neurons express estrogen receptors ( Gahr , 1990 ) that can be activated after conversion of testosterone into estradiol .","Furthermore , changes in neuronal dendritic growth and synaptic morphology in the songbird RA have been observed to accompany testosterone-induced song development in female canaries ( DeVoogd and Nottebohm , 1981; Devoogd et al . , 1985; Canady et al . , 1988 ) .","In addition , Frankl-Vilches et al . ( Frankl-Vilches et al . , 2015 ) recently observed that 85% of the seasonally and testosterone upregulated genes in the canary HVC are related to neuron differentiation , axon , dendrite and synapse organization .","In the brain of birds and mammals , sex hormones are known to upregulate the expression of brain-derived neurotrophic factor ( BDNF ) ( Dittrich et al . , 1999; Rasika et al . , 1999; Sohrabji et al . , 1995 ) , which in its turn contributes to the stabilization of synapses through the interaction with tyrosine receptor kinase B ( TrkB ) ( Koleske , 2013 ) .","Furthermore , BDNF has been shown to be essential during testosterone-induced song development in female canaries ( Hartog et al . , 2009 ) , providing a likely pathway by which sex hormones can consolidate motor skills by stabilizing synapses .","Testosterone levels and song consolidation are tightly linked , and it is difficult to determine if brain anatomical changes in our and other studies are caused by testosterone , singing activity or both .","Interestingly , local intracerebral testosterone implants close to nucleus RA did not cause anatomical changes in this androgen receptor-rich brain area ( Brenowitz and Lent , 2002 ) , suggesting that modifications in RA are driven by singing-related , activity-dependent input from HVC .","Whether or not testosterone , vocal practice , or both play a role in the observed synaptic pruning in HVC , the vocal circuitry demonstrated synaptic changes that persisted through several months in which testosterone levels were low and birds did not sing , potentially enabling the birds to quickly re-acquire their previously developed song patterns .","Sex hormones can be regulated by both environmental and social factors ( Goymann et al . , 2007; Oliveira , 2004; Wingfield et al . , 1990 ) , thereby providing a timed cue for the consolidation of behavioral output and the underlying brain circuitry .","Such a cue could facilitate the timely formation and recall of long-term memories of relevant sensory and motor information .","Our study suggests that lasting , hormone-driven synaptic pruning of related brain circuitries could form a basis for such long-term memories , enabling a quick recovery of previously acquired skills .","The ability to respond quickly to environmental and social cues through such primed hormone-sensitive control mechanisms would constitute an important adaptation to ensure competitive proficiency and maximize reproductive success under environmental restrictions .","The results obtained from this study may have implications not only across different animal groups , but may generally apply to different motor , sensory , and motivational systems that rely on a rapid recall of previously learned information ."],["For this study adult domesticated canaries ( Serinus canaria ) were either purchased from a local breeder in Antwerp or taken from the breeding colony of the Max Planck Institute for Ornithology , Seewiesen .","All animals were raised and kept under local natural daylight conditions , until the day length reached 11 hr in early March .","Birds were subsequently housed individually in sound attenuating chambers for song recordings .","Experimental procedures were conducted according to the guidelines of the Federation of European Animal Science Associations ( FELASA ) and approved by the Ethical Committee on animal experiments of the University of Antwerp .","To stimulate song production in naive female canaries , birds were subcutaneously implanted ( T1+ ) with 8 mm silastic tubes ( Dow Corning , Midland , MI; ID: 1 . 47 mm ) filled with crystalline testosterone ( Sigma-Aldrich Co . , St . Louis , MO ) .","During the hormone treatments a light cycle of 11 hr light , and 13 hr darkness was maintained to exclude photoperiodic effects on song production .","This light cycle was chosen because under natural conditions it corresponds to a phase of testosterone up-regulation and physiological sensitivity to hormone fluctuations in canaries ( Nottebohm et al . , 1987 ) .","After consolidation of song we removed the hormone implants ( T1- ) to end song production , and changed the daylight conditions to 8 hr light and 16 hr dark , inducing a molting phase of approximately 1 . 5 months .","Once the molt was finished , the day length was gradually returned to 11 hr over the following month .","Subsequently , the song-experienced birds received a second testosterone treatment ( T2+ ) , and were sacrificed 5 weeks after implantation .","Blood samples were collected from the birds’ right wing veins using heparinized haematocrit capillaries ( Brand , Wertheim , Germany ) .","Directly after blood collection , samples were centrifuged at 3000 RPM for 10 min to separate cells from plasma , and stored at −80°C for later analysis .","Testosterone concentrations in the blood plasma were determined by radioimmunoassay ( RIA ) after extraction and partial purification on diatomaceous earth ( glycol ) columns , following the procedures described previously ( Goymann et al . , 2001; Goymann et al . , 2008 ) .","Briefly , plasma samples were extracted with dichloromethane ( DCM ) after overnight equilibration of the plasma with 1500 dpm of 3H-labeled testosterone ( Perkin-Elmer , Rodgau , Germany ) .","After separation of the organic phase , the extracts were re-suspended in 2 × 250 µl 2% ethylacetate in isooctane and fractioned using columns of diatomaceous earth:propylene glycol:ethylene glycol ( 6:1 . 5:1 . 5 , w:v:v ) .","Collected testosterone fractions were dried , re-suspended in phosphate-buffered saline with 1% gelatin ( PBSG ) , and left to equilibrate overnight at 4°C .","Extraction of plasma testosterone with dichloromethane resulted in 86 ± 11% ( mean ± sd ) recovery .","Hormone samples were incubated with antisera against testosterone ( Esoterix Endocrinology , Calabasas Hills , CA ) , and after 30 min 3H-labeled steroids ( 13500 dpm ) were added and left to incubate for 20 hr at 4°C .","Free steroids were separated from the bound fractions by absorption on dextran-coated charcoal , centrifuged , and decanted into scintillation vials to be counted .","Testosterone concentrations in the plasma samples were measured in one assay .","The lower detection limit of the RIA was 0 . 33 pg/tube , and all measured plasma levels were above the lower detection limit .","The intra-assay variation of a chicken plasma pool as control sample at the beginning and end of the assay was 6 . 7% .","Pooled plasma levels of testosterone for all birds during the different consecutive hormone treatments are shown in Table 1 .","Mean detected hormone levels were within range of physiological plasma levels in nest-building male canaries ( range: 360–7970 pg/ml; n = 6 ) and previously reported values for other small passerines ( Wingfield and Hahn , 1994; Kempenaers et al . , 2008; Apfelbeck and Goymann , 2011 ) .","To monitor the entire song ontogeny during consecutive testosterone treatments randomly selected , naive female canaries ( n = 6 ) were kept individually in sound attenuating chambers during testosterone treatment ( T1+ ) , testosterone removal ( T1- ) and testosterone re-treatment ( T2+ ) , while song output was continuously recorded .","This approach resulted in a song database of 5 . 6 ± 0 . 6 million song syllables per bird , recorded over the course of approximately 1 year .","The vocal activity of each bird was recorded using Sound Analysis Pro 2 . 0 ( Tchernichovski et al . , 2000 ) .","Omnidirectional condenser microphones ( TC-20; Earthworks , Milford , NH ) connected to a multi-channel microphone preamplifier ( UA-1000; Roland , Los Angeles , CA ) were used to acquire and digitize all sounds produced within the sound attenuating boxes with a sampling frequency of 44 . 1 kHz .","The incoming signal was filtered online using an amplitude and Wiener entropy threshold to exclude background noises , and saved in 60 s waveform audio files ( 16-bit PCM format ) .","Vocalizations were segmented into individual syllables with the fully automated Feature Batch module in Sound Analysis Pro by applying an amplitude threshold to the sound wave .","Because the distance from perch to microphone ranged between 10 and 20 cm only , little variation in recorded amplitude levels can be expected .","The amplitude threshold was selected once manually to assure reliable segmentation , and was kept constant during the analyses of all sound phrases within birds .","To filter out non-song vocalizations , syllables were only included when produced within a song bout of at least 750 ms . A song bout was defined as a sequence of sounds traversing the amplitude threshold with an interval of no more than 100 ms . We used Sound Analysis Pro to measure duration ( d ) , pause duration ( i ) , syllable rate ( SR ) , frequency modulation ( FM ) , amplitude modulation ( AM ) , syllable bandwidth ( BW ) , mean frequency ( MF ) and Wiener entropy ( E ) for each syllable and stored these syllable features in MySQL 5 . 1 tables ( Oracle , Redwood Shores , CA ) .","Bandwidth was calculated for each syllable by subtracting the minimum peak frequency from the maximum peak frequency .","The syllable rate was defined for each syllable as: SRa = ( da +ia ) −1 , where da is the duration of syllable ‘a’ , and ia is the interval between the end of syllable ‘a’ and the beginning of the consecutive syllable within the same song bout , irrespective of syllable type .","For a detailed computational description of how Sound Analysis Pro calculates the different sound features we refer to the accompanying publication ( Tchernichovski et al . , 2000 ) and the online manual ( http://soundanalysispro . com ) .","To investigate the dynamic change of song features over the course of the experiment , daily histograms were obtained from the entire dataset by rounding individual values to the nearest integer , and plotting the number of times those integers occurred each day .","To determine the SRs that were most commonly used at the onset and crystallization of song development a non-linear exponential curve ( 1 ) was fitted through the peak values of each SR histogram .","( 1 ) Fx=a+b1+ec-xd Further developmental correlation plots for all temporal and spectral song features were produced by calculating the Pearson product-moment correlation coefficient ( CC ) between a 7 day average of the histogram pattern at the time of song stabilization and all other recorded days .","This calculation resulted in a value between −1 and 1 for each recorded day , and was used as a measure of similarity where one describes an absolute copy of the stable song pattern , and −1 describes the absolute inverse of the stable song pattern .","A non-linear exponential curve ( 1 ) was fitted through the CC values and song parameters were considered stable as soon as the daily increase in similarity dropped below 0 . 001 .","For each song feature we used the fit curve to determine the duration between testosterone implantation and feature stabilization , and to determine the maximum daily increase in similarity ( dmax ) .","Dmax could only be calculated for birds and song features that demonstrated a developmental increase in similarity , and was only used for statistical purposes if three or more birds demonstrated such an increase .","Mean CC plots combining all analyzed birds were calculated by averaging the raw ( non-fitted ) CC values that were calculated for each bird and for each day .","We aligned song development in the different birds based on the first day where we detected song .","Spectrograms of the original sound files were visually inspected on syllable-like sound structures to determine the syllable repertoire of each bird and the first occurrence of song after testosterone treatments .","To assess syllable similarity during consecutive testosterone treatments we calculated the Euclidean distance between each of 100 randomly selected syllables for each syllable type from stable songs during both the T1+ and T2+ periods .","The Euclidean distance between two syllables ‘a’ and ‘b’ with coordinates c = SR , d , i , FM , AM , BW , MF , E was defined as:da , b=∑c=1n ( b1-a1 ) 2 To give all analyzed temporal and spectral features equal weight in the analysis , each feature was normalized by dividing its value by its global median value from all syllables produced by all birds recorded in this study .","First , for each syllable type the mean Euclidean distance between 100 syllables of the same type during T1+ ( T1xT1 ) was calculated to determine how much baseline variation existed between syllables of the same type .","Lower values indicate a higher similarity between syllables .","Secondly , we calculated the mean Euclidean distance between 100 syllables of the same type from the T1+ and T2+ ( T1xT2 ) periods to determine if syllables produced during the 2nd testosterone treatment were similar to those produced during the 1st testosterone treatment .","Finally , we calculated the mean Euclidean distance between 100 syllables of the same type from the T1 +period with each of 100 syllables from all other types of syllables that a bird produced ( T1xT2ext ) to provide a measure of dissimilarity between syllables of different types .","All observed syllable types were included in this analysis with the exception of single transitional syllables connecting two song phrases .","To assess similarity of song syntax between subsequent testosterone treatments we determined the probability of song phrase transitions during the T1+ and T2+ periods for each bird .","On average 794 . 3 phrase transitions per bird were used for the analysis .","From the resulting transition probability distributions we calculated the sequence linearity , consistency and entropy as previously described ( Scharff and Nottebohm , 1991; Daou et al . , 2012 ) .","In short , sequence linearity is expressed as the number of syllable types divided by the number of transition types and addresses the way phrases are ordered in a song .","Sequence consistency is expressed as the sum of common phrase transitions divided by the sum of total transitions and addresses how often the same sequences are produced .","Common phrase transitions were defined as transitions that make up at least 5% of all phrase transitions .","The entropy ( S ) of the transition probability distribution with transition type Ti is a measure of the spread of that distribution and was calculated as:S=-∑i=1nTi log2 ( Ti ) To estimate spine densities and dendrite densities in motor nucleus HVC and RA , brains were collected from a control group of adult female canaries that did not receive any testosterone treatment ( C: n = 6 ) , a group of birds that was sacrificed five months after testosterone treatment ( T1+: n = 8 ) , and a group of birds that was sacrificed at least 2 . 5 months after testosterone withdrawal ( T1-: n = 6 ) .","All experimental birds were raised and kept in group aviaries together with other male and female canaries throughout the experiment to assure similar social and auditory conditions .","Birds were transferred to sound attenuating chambers for two weeks after testosterone treatment to assure that birds were singing , and prior to sacrifice .","Individuals were randomly allocated to the different treatment groups .","All birds were maintained on a light cycle of 11 hr light , and 13 hr darkness and were between 2 . 5 and 3 years old at the time of sacrifice .","Brains were processed for Golgi-Cox staining using the FD Rapid GolgiStain kit ( FD NeuroTechnologies , Columbia , MD ) .","After overnight fixation in a 4% formaldehyde solution in phosphate-buffered saline ( PBS; 10 mM; pH 7 . 4 ) , brains were stored in PBS with 0 . 05% sodium azide at 4°C until further processing .","Brain hemispheres were separated with a razor blade and left hemispheres were immersed for two weeks in an impregnation solution consisting of equal amounts of FD solutions A and B in total darkness at room temperature .","Following three days of incubation in FD solution C , brains were cut into 30 µm sagittal sections using a sliding microtome ( Leica Microsystems GmbH , Wetzlar , Germany ) , which were stored in PBS with 0 . 05% sodium azide at 4°C .","Sections with a regular interval of 180 µm were mounted on SuperFrost glass slides ( Menzel GmbH , Brauschweig , Germany ) , developed in a solution of two parts distilled water , one part FD solutions D , and one part FD solution E for ten minutes , and rinsed in distilled water before embedding in CC/Mount tissue mounting medium ( Sigma-Aldrich ) .","Slides were incubated at 70°C until the mounting medium had hardened , cleared in xylene , and further embedded in Roti-Histokitt II ( Carl Roth , Karlsruhe , Germany ) before coverslipping .","For each bird , z-stacks were obtained from three brain sections of nucleus HVC and two sections of nucleus RA with a Nikon Eclipse Ti microscope ( Nikon , Tokyo , Japan ) , equipped with a 60x oil immersion lens ( CFI Plan Apo VC 60x Oil ) .","The outline of HVC and RA in each section was delineated in ImageJ ( http://rsb . info . nih . gov/ij/ ) , and 100 µm2 non-overlapping regions of interests ( ROIs ) were randomly placed within the boundaries of the target nucleus prior to the quantifications .","Considering the relatively uniform distribution of X-projecting HVC neurons and RA-projecting HVC neurons ( Fortune and Margoliash , 1995 ) , this approach should result in a homogenous sampling across treatment groups .","Neuronal dendritic spine densities were estimated for each brain section by checking ROIs in random order until five ROIs were located that contained spinous dendrite segments ( 15 ROIs per animal for HVC and 10 ROIs for RA ) .","The individual dendrite segments within the ROIs were traced in ImageJ to measure the length , and the number of visible spines that originated from the traced segment was manually counted .","Spine densities were calculated as the number of visible spines per µm of dendrite .","All visible spine types were included in the quantifications , including filopodia , long , thin , stubby , mushroom and branched spines ( Risher et al . , 2014 ) .","For branched spines , each spine head was counted separately .","To further obtain a density measure of spinous dendrites we counted the total number of spinous dendrite segments in all ROIs examined , and divided the number of observed dendrite segments with the number of ROIs , giving an estimation of the number of spinous dendrites per ROI .","Neuronal dendrites were further binned into categories containing 0–0 . 2 , 0 . 2–0 . 4 , 0 . 4–0 . 6 , 0 . 6–0 . 8 , 0 . 8–1 . 0 , 1 . 0–1 . 2 , 1 . 2–1 . 4 , 1 . 4–1 . 6 , 1 . 6–1 . 8 spines per µm .","The probability distribution of neuronal dendrite segments was then obtained by dividing the observed number of spinous dendrites per ROI in each bin with the total number of spinous dendrites per ROI .","To obtain a density measure of aspinous neurites in HVC , the number of visible spine-less neurite segments were counted in ten new non-overlapping ROIs that were randomly placed within the boundaries of HVC .","We refer to these projections as neurites , because we were unable to distinguish between aspinous dendrites and axons in our tissues .","All quantifications were conducted by an experimenter that was blind to the experimental condition of the animals .","Statistical analyses were performed in SAS 9 . 3 ( SAS Institute , Cary , NC ) .","No sample sizes were calculated prior to the experiments .","A technical replication was defined as a repeated measurement of the same individual , and biological replications constitute the number of sampled individuals ( n ) .","Differences in spine and dendrite densities between treatment groups were tested with a one-way analysis of variance ( ANOVA ) with Dunnett’s correction for multiple comparisons against one control value ( untreated birds ) .","Changes in hormone levels were determined using a repeated one-way ANOVA with Dunnett’s correction .","Differences in syllable similarity and syllable rates of individual syllable types were tested with one-way ANOVAs , and when significant , Tukey's HSD test was used to analyze differences between time points .","Song pattern similarities between the 1st and 2nd hormone treatment were established by comparing the average CC from seven consecutive days of stable song during the 1st treatment with the average CC from seven consecutive days of stable song production during the 2nd treatment .","Paired-samples t-tests were used to compare CC’s , sequence parameters , and developmental time parameters between subsequent hormone treatments .","To determine song feature deterioration we calculated the difference between the average CC from the last 7 days of stable song production during the 1st testosterone treatment and the average CC from the first 2 days of song production during the 2nd treatment .","Feature recovery was determined by subtracting the average CC from the first 2 days of song production from the average CC from the last 7 days of stable song production during the 2nd testosterone treatment .","One-sample t-tests were used to determine if feature deterioration and recovery significantly exceeded zero .","Measurement values in the text are given as means ± SEM , unless stated otherwise .","No outliers or data were excluded from analysis ."]],"string":"[\n [\n \"Complex motor skills , such as singing or playing an instrument , are not inherently determined but need to be acquired through repetitive practice .\",\n \"Many specialized skills are used only incidentally however , and skill performance degrades during intermittent periods of non-use .\",\n \"This means that skills need to be re-acquired each time the need arises .\",\n \"For example , trained human surgeons need to re-acquire their surgical proficiency after a period of absence ( Crewther et al . , 2016 ) , Capuchin monkeys need to acquire manipulative foraging skills to retrieve difficult-to-access , seasonally available food items ( Eadie , 2015 ) , and songbirds need to re-develop high-quality songs to attract a mate after wintering or long-distance migration ( Slagsvold , 1976 ) .\",\n \"Whereas learning novel motor skills is generally a lengthy process , the short-term availability of suitable mates and food items imposes a considerable time pressure on the re-acquisition of such specialized skills .\",\n \"Birdsong , an established model system for vocal motor learning ( Brainard and Doupe , 2013 ) , is characterized by a high degree of complex and rapid acoustic modulations .\",\n \"The fine motor skills that are necessary to produce such complex , high quality vocalizations are thought to advertise an individual’s quality through performance-related characteristics such as song complexity and/or production rate ( Podos , 1997; Drăgănoiu et al . , 2002; Holveck and Riebel , 2007; Byers et al . , 2010 ) .\",\n \"When juvenile songbirds are one to two months old they start singing noisy , unstructured ‘subsongs’ akin to human babbling ( Doupe and Kuhl , 1999 ) , which gradually develop into variable , but recognizable species-specific ‘plastic songs’ ( Marler and Peters , 1982a; Nottebohm et al . , 1986; Tchernichovski et al . , 2001; Mori et al . , 2018 ) .\",\n \"With extensive vocal rehearsal the songs slowly consolidate into high performance ‘crystallized songs’ , a process that can take up more than five months in some songbird species ( Nottebohm et al . , 1986 ) .\",\n \"For adult songbirds that annually need to re-acquire their songs this process of juvenile song development greatly exceeds the time that is available to attract a mate during the early breeding season .\",\n \"Thus , adult songbirds must considerably speed-up song re-acquisition to quickly produce songs of adequate quality to impress conspecifics .\",\n \"It is currently unclear how songbirds cope with the seasonally imposed time pressure on song development , and what mechanisms may be involved to ensure reproductive success under such time constraints in nature .\",\n \"The seasonal development and degradation in vocal performance are highly correlated with changes in testosterone levels ( Nottebohm et al . , 1987; Smith et al . , 1997; Tramontin et al . , 2000; Voigt and Leitner , 2008 ) , and are often accompanied by gross anatomical and cytoarchitectural restructuring of the brain areas involved in song production ( Nottebohm , 1981; Gahr , 1990; Kirn et al . , 1994; Kafitz et al . , 1999; Thompson and Brenowitz , 2005; Balthazart et al . , 2008; Vellema et al . , 2014 ) .\",\n \"Thus testosterone-regulated adaptations of the songbird brain may play an important role in optimizing song performance .\",\n \"In this study we used adult female canaries ( Serinus canaria ) to investigate how a periodically acquired motor behavior attains a high performance level under developmental time pressure .\",\n \"Although female canaries rarely sing spontaneously , and never produce high quality songs under natural conditions ( Pesch and Guttinger , 1985 ) , the systemic application of testosterone leads to the development of high-performance song with male-typical features ( Leonard , 1939; Shoemaker , 1939; Vallet et al . , 1996 ) .\",\n \"This trait provides us with a well-defined behavioral baseline , and allows us to manipulate in detail the onset and offset of song development through repeated testosterone treatments .\",\n \"Here we demonstrate that during a protracted testosterone treatment , adult female canaries gradually develop stable , species-typical songs through a process of song crystallization similar to what is known for naturally-raised juvenile male canaries ( Mori et al . , 2018 ) .\",\n \"Re-treatment with testosterone several months after birds have stopped singing , leads to a rapid recurrence of song performance with song features that strongly resemble those after the first treatment .\",\n \"We propose that once developed , vocal motor memories are retained for an extended period of time , enabling adult animals to recover song performance quickly at a later time , a process termed ‘savings’ ( Ebbinghaus , 2013 ) .\",\n \"We further demonstrate that neurons in the songbird’s premotor nucleus HVC ( proper name ) , a central nucleus in the brain circuitry that controls song production ( Nottebohm et al . , 1976 ) , irreversibly loses a significant number of dendritic spines during the initial testosterone-induced development of vocal skills .\",\n \"This state of reduced spine density is subsequently maintained over long periods in which testosterone levels are low and vocal skills are not used .\",\n \"The observed lasting synaptic pruning could play an important role in the formation of vocal skill savings , enabling birds to rapidly re-acquire song performance within a restricted time period .\"\n ],\n [\n \"To study the development of vocal skills in adult female canaries we recorded and analyzed the entire song ontogeny in acoustically separated animals ( 5 . 6 ± 0 . 6 million song syllables per bird ) , while manipulating song output by consecutively implanting , removing , and re-implanting birds with testosterone implants .\",\n \"Whereas no songs were observed prior to treatment , the systemic application of testosterone ( T1+ ) stimulated birds to start singing their first songs after three days ( 3 . 22 ± 0 . 46 days ) .\",\n \"The phases of female song development were similar to those previously reported for male canaries ( Figure\",\n \"1 ) ( Nottebohm et al . , 1986; Mori et al . , 2018; Weichel et al . , 1986 ) .\",\n \"The first subsongs were produced in an irregular fashion and consisted of unstructured syllables with few distinctive features .\",\n \"Songs gradually developed into plastic songs in which different phrases of repeated syllables could be clearly distinguished , albeit with variation between syllables of the same type .\",\n \"During the following six months , female canary songs continued to develop , gradually crystallizing into the species-typical stable songs .\",\n \"Although structurally similar to male canary songs , female songs had relatively small syllable repertoires ( 6 . 2 ± 1 . 1 syllable types ) , consistent with previous reports ( Vallet et al . , 1996; Fusani et al . , 2003; Hartog et al . , 2009 ) .\",\n \"To investigate the birds’ abilities to re-acquire song performance after a period without vocal practice we withdrew testosterone ( T1- ) from the animals , completely abolishing singing behavior in approximately three days ( 3 . 14 ± 0 . 63 days ) .\",\n \"After 2½ months of no song production birds were treated for a second time with testosterone ( T2+ ) , inducing song output after three days ( 3 . 14 ± 0 . 74 days ) .\",\n \"No subsongs were observed after the second testosterone treatment , and all birds were able to produce plastic songs from the first day of singing .\",\n \"We first compared the development of temporal song features during the first and second testosterone treatments ( Figure 2 , Figure 2—figure supplement 1 , Figure 2—figure supplement 2 ) .\",\n \"Particularly the speed at which subsequent syllables are repeated , the syllable repetition rate ( SR ) , has been strongly associated with individual performance ( Podos , 1997; Podos , 1996 ) and has been shown to increase the attractiveness of the song to conspecifics ( Vallet et al . , 1998; Vallet and Kreutzer , 1995 ) .\",\n \"We observed that during the first two weeks of testosterone-induced female song development , all syllables were typically produced at a similar rate ( SR: 10 . 3 ± 1 . 3 Hz ) .\",\n \"While practicing the song , different syllables were gradually sung at different rates , becoming more distinct towards song crystallization ( Figure 2A ) .\",\n \"Once crystallized , syllables could be distinguished as fast syllables ( SR: 21 . 7 ± 1 . 0 Hz ) , medium-speed syllables ( SR: 11 . 9 ± 1 . 1 Hz ) , and slow syllables ( SR: 4 . 9 ± 0 . 7 Hz ) .\",\n \"A similar distribution of syllable rates redeveloped during a 2nd testosterone treatment .\",\n \"The observed range of syllable rates was similar to what has previously been reported for wild-living male canaries ( Leitner et al . , 2001 ) .\",\n \"To determine how fast this syllable rate pattern emerged during subsequent testosterone treatments we took crystallized song from the last week of the first testosterone treatment as a reference pattern , and calculated the correlation coefficient ( CC ) between this reference and each day of song development and re-development ( Figure 2B & C ) .\",\n \"While syllable repetition rates gradually developed and stabilized over the course of 160 ± 8 days during the first testosterone treatment , stable rates were obtained more than seven times faster , within 22 ± 2 days , during the second treatment ( Figure 2D; paired t-test: p<0 . 001 ) .\",\n \"In addition the maximum daily increase in similarity ( dmax ) was also significantly higher during song re-development than during initial song development ( Figure 2E; dmax: 0 . 021 ± 0 . 005 for T1+ , and 0 . 078 ± 0 . 012 for T2+; paired t-test: p<0 . 05 ) , indicating a much accelerated development of song performance in birds with previous singing experience .\",\n \"Since syllable rate is a compound feature that is determined by both syllable duration and the interval between subsequent syllables , we analyzed syllable durations and pause durations separately .\",\n \"Syllable durations developed slowly during the initial song acquisition ( T1+ ) , reaching stable values after 153 ± 19 days , while pause durations stabilized in 82 ± 13 days ( Figure 2—figure supplement 1 , Figure 2—figure supplement 2 ) .\",\n \"Both temporal features re-emerged more quickly during song re-acquisition ( T2+ ) than during the first song developmental phase ( duration: 4 . 8 ± 1 . 3 days; paired t-test: p<0 . 001; pause: 10 . 7 ± 2 . 2 days; paired t-test: p<0 . 01; Figure 2—figure supplement 1 ) .\",\n \"To determine the bird’s ability to recover spectral song patterns we analyzed the recurring patterns of frequency modulation ( FM ) , amplitude modulation ( AM ) , bandwidth ( BW ) , mean frequency ( MF ) and Wiener entropy ( E ) during song acquisition and re-acquisition .\",\n \"Similar to temporal song features , spectral features were more quickly established during song re-acquisition ( T2+ ) than during the initial song development ( T1+ ) ( Figure 3 , Figure 3—figure supplement 1 , Figure 3—figure supplement 2 ) .\",\n \"All spectral features developed gradually over more than 110 days before reaching stable values .\",\n \"After a period in which the birds did not produce any song , testosterone-induced song re-acquisition led to a rapid stabilization of spectral patterns within 16 days ( Figure 3 , Figure 3—figure supplement 1; paired t test: p<0 . 01 ) .\",\n \"Thus both temporal and spectral song features were acquired significantly faster by experienced birds that had developed singing skills once before .\",\n \"Whereas song re-acquisition in female canaries resulted in a rapid recurrence of stable temporal and spectral patterns , not all song features re-developed in the same way .\",\n \"Most notably , some song features demonstrated a clear deterioration in the absence of vocal practice , while other song features remained stable despite the absence of vocal practice ( Figure 3G & H ) .\",\n \"Of the studied temporal song parameters , syllable durations did not show any deterioration in the period that birds did not sing ( T1- ) and thus also did not need to be re-acquired ( Figure 2—figure supplement 1A–C; Figure 2—figure supplement 2A ) .\",\n \"Both the syllable rate and the pause duration between syllables did deteriorate however , and a short phase of re-development was required to recover the originally developed patterns ( Figure 2 , Figure 2—figure supplement 1D–F , Figure 2—figure supplement 2B ) .\",\n \"The accelerated re-acquisition of syllable repetition rates thus appears to be driven by the need to re-optimize the timing between syllables rather than syllable lengths .\",\n \"Of the spectral syllable features that were studied , the FM and AM patterns deteriorated when birds stopped singing ( T1- ) , but were re-acquired during a short developmental phase after a 2nd testosterone treatment ( T2+ ) ( Figure 3A–C , Figure 3—figure supplement 1A–C , Figure 3—figure supplement 2A & B ) .\",\n \"BW , MF , and E did not need to be re-acquired , but maintained strong similarities with the initial acoustic patterns despite the absence of song production in the intermittent period ( T1- ) ( Figure 3D–F , Figure 3—figure supplement 1D–I , Figure 3—figure supplement 2C–E ) .\",\n \"Thus song re-acquisition can be characterized by recalling a combination of sound features that immediately can be reproduced and sound features that require a short phase of redevelopment .\",\n \"The songs that reappeared during the second testosterone treatment were strikingly similar to those that developed after the first treatment ( Figure 4 ) .\",\n \"Visual inspection of the song spectrograms revealed no differences in syllable repertoire when comparing crystallized songs during the first and second testosterone treatment ( Figure 4A & B; 6 . 2 ± 1 . 1 syllable types for both treatments ) .\",\n \"In addition both temporal and spectral song features demonstrated a strong similarity in their distribution patterns between subsequent testosterone treatments ( T1xT2 ) , with correlation coefficients above 0 . 82 for all studied song features ( Figure 4C ) .\",\n \"These values were not significantly different from the correlation coefficients obtained by cross-correlating distribution patterns from consecutive days during the first treatment period ( T1xT1 ) for any of the measured song features ( Figure 4C ) , indicating that the song patterns that developed during the first and second testosterone treatment are similar in structure .\",\n \"To determine the similarity of syllables produced during the 1st and 2nd testosterone treatments we calculated for each bird the Euclidean distance across all eight analyzed temporal and spectral features within and between syllable types ( Figure 4—figure supplement 1 ) .\",\n \"The Euclidean distance between two syllables provides a measure of similarity where a syllable is represented as a point with coordinates that correspond to the eight analyzed sound features .\",\n \"The distance between syllables with similar sound features and thus similar coordinates is low , while the distance between syllables with differing sound features is high .\",\n \"The mean Euclidean distances between syllables of the same type across the 1st and 2nd testosterone treatment ( T1xT2 ) were not different from the Euclidean distances between syllables from subsequent days during the 1st testosterone treatment ( T1xT1: d = 0 . 73 ± 0 . 04 , T1xT2: d = 0 . 76 ± 0 . 06; Tukey’s HSD: p=0 . 99 ) .\",\n \"The mean Euclidean distances between syllables of different types across the two treatment periods were significantly larger than those between syllables of the same type ( T1xT2ext: d = 2 . 12 ± 0 . 23; Tukey’s HSD: p<0 . 001 ) .\",\n \"These results indicate that for a given syllable the sound features that redeveloped after the 2nd testosterone treatment fell within the range of variation observed during the 1st testosterone treatment for that same syllable type , but were clearly distinguishable from the sound features of other syllable types .\",\n \"To investigate if the sequential structure of the song was different between the two testosterone treatments we analyzed the probability that specific phrase transitions occurred in the songs ( Figure 4—figure supplement 2 ) .\",\n \"Because the syllable repertoire is relatively small in testosterone treated female canaries , the number of possible phrase transitions is also small .\",\n \"Nevertheless , on average only 8 . 7% of all possible phrase transitions were used in the songs .\",\n \"Thus whereas the song sequences are not fixed as in for example zebra finches , the song sequences are not random either , following a limited subset of possible transitions similar to what has been observed in male canaries ( Leitner et al . , 2001; Markowitz et al . , 2013 ) .\",\n \"The percentage of phrase transitions that made up the songs during the 1st and 2nd testosterone treatments were not different ( T1+: 9 . 2 ± 4 . 5%; T2+: 8 . 3 ± 4 . 2%; paired t-test: p=0 . 59 ) , and the probability distribution of these transitions were visually similar between treatments ( Figure 4—figure supplement 2A & B ) .\",\n \"The linearity , consistency , and entropy of song phrase transitions did not differ significantly between the songs from the 1st and 2nd treatment periods ( Figure 4—figure supplement 2C ) .\",\n \"Together these data strongly suggest that testosterone-induced song re-acquisition in canaries is driven towards the previously acquired song pattern .\",\n \"The finding that the re-acquisition of song performance progresses considerably faster than the initial acquisition and leads to the production of highly similar song patterns suggests that singing skills are retained during intermittent silent periods .\",\n \"Motor learning and memory are generally considered to rely on the maturation and consolidation of the synaptic connections within the neural circuitry that drives the behavior in question ( Changeux and Danchin , 1976; Yu and Zuo , 2011; Hoshiba et al . , 2017 ) .\",\n \"To investigate changes in synaptic connectivity during vocal motor development we quantified the number of neuronal dendritic spines in the forebrain motor nucleus HVC and the robust motor nucleus of the arcopallium ( RA ) before , during , and after the acquisition of vocal motor skills ( Figure 5 ) .\",\n \"Excitatory projection neurons in the HVC of testosterone-treated , singing female canaries displayed a more than 30% reduction in dendritic spine density compared to untreated , non-singing control birds ( C: 0 . 95 µm−1 , T1+: 0 . 64 µm−1; Dunnett’s test: p<0 . 01 ) , indicating a significant synaptic pruning during the first acquisition of singing skills ( Figure 5B ) .\",\n \"Compared to non-singing control birds , spine densities remained significantly lower in birds that stopped singing for 2 . 5 months after testosterone removal ( C: 0 . 95 µm−1 , T1-: 0 . 69 µm−1; Dunnett’s test: p<0 . 01 ) .\",\n \"Thus , the synaptic pruning in HVC associated with testosterone-induced song acquisition is not reversed when birds stop singing , but instead the pruned state is maintained in periods in which the birds do not sing ( Figure 5B ) .\",\n \"In contrast , we did not observe any changes in dendritic spine densities in RA across the different experimental periods ( Figure 5C; ANOVA: p=0 . 857 ) .\",\n \"Strong differences were observed in the density of dendritic spines between different spinous neurites in HVC ( range: 0 . 15 to 2 . 14 spines/µm ) .\",\n \"To investigate which neurite types were associated with the observed spine pruning we calculated the probability distribution of neuronal dendrites in HVC based on their spine density ( Figure 5D–F ) .\",\n \"We observed a marked shift in the distribution of dendrites from more densely-spined dendrites in control birds towards less densely-spined dendrites in the testosterone treatment ( T1+ ) and removal groups ( T1- ) , reflecting a global decrease in spine density .\",\n \"The strongest reduction was detected in the range of dendrites with more than 0 . 8 spines per µm , which halved in number in both testosterone-treated , singing birds ( T1+ ) , and testosterone-removed , non-singing birds ( T1- ) compared to non-singing controls ( C: 0 . 64 ± 0 . 06 , T1+: 0 . 28 ± 0 . 05 , T1-: 0 . 33 ± 0 . 06; ANOVA: p<0 . 01 ) .\",\n \"The total density of both spinous and aspinous neurites in HVC did not significantly differ between the treatment groups ( ANOVA: p>0 . 36 ) , suggesting that changes in dendrite densities were not caused by specific cell death .\",\n \"These data suggest that the observed vocal motor acquisition is accompanied by a lasting synaptic pruning specifically in nucleus HVC , providing a possible anatomical site for motor memory that could facilitate the observed rapid re-acquisition of previously established song patterns .\"\n ],\n [\n \"The ability to learn new skills enables one to adapt to changes in the surrounding environment .\",\n \"Whereas learning new skills requires time and effort , the survival of many species relies on their capacity to utilize specialized skills in a timely fashion , capitalizing on the short-term availability of foods , mates , and other environmental factors ( Prendergast et al . , 2002 ) .\",\n \"Such a capacity is biologically relevant for numerous skills in a multitude of species , ranging from periodic foraging skills in dolphins ( Patterson et al . , 2016 ) , bats ( Clarin et al . , 2013 ) , and monkeys ( Boinski and Fragaszy , 1989 ) to courtship displays in crabs ( Mowles et al . , 2017 ) and songbirds ( Ota et al . , 2015 ) .\",\n \"As the use of these specialized skills is interrupted over longer periods , for example during long-distance migration or hibernation ( Slagsvold , 1976; Prendergast et al . , 2002 ) , such skills need to be re-acquired quickly to maximize survival and reproductive success .\",\n \"In this study we establish the repeated , hormone-driven acquisition of vocal motor performance in female canaries as a model system for studying motor savings .\",\n \"Using this model , we show that while the initial development of specialized motor skills can be a lengthy process , motor performance can quickly be re-acquired at a later time , even after extended periods of non-use ( Figure 2 and 3 ) .\",\n \"The accelerated song re-acquisition observed in this study indicates that acquiring a motor skill for the first time follows a different developmental trajectory than re-acquiring the same skill at a later time .\",\n \"This is both evident from the accelerated consolidation of song performance during song re-development ( Figure 2 and 3 ) , and the observation that birds skip the subsong phase of song development when re-acquiring their songs .\",\n \"Our data further suggest that songbirds have the ability to retain previously developed singing skills through a process of selective pruning of the neural circuitry ( Figure 5 ) , enabling them to rapidly recover songs of adequate quality to attract a mate before breeding opportunities disappear .\",\n \"Several song parameters were maintained across a period in which birds did not sing , while other song features demonstrated clear deterioration ( Figure 3G & H ) .\",\n \"Birds were able to recover those deteriorated song features with renewed vocal practice .\",\n \"At the peripheral level , song performance depends on the accurate control of the respiratory system , the sound producing organ ( the syrinx ) , and the upper vocal tract .\",\n \"Interestingly , those song features that required a phase of re-development to be optimized seem primarily associated with motor constraints in syrinx function ( Podos , 1997; Geberzahn and Aubin , 2014 ) .\",\n \"Online modulation of frequency and amplitude requires active contractile modulation of syringeal muscles ( Goller and Suthers , 1996; Elemans et al . , 2008 ) .\",\n \"Syllable repetition and the inter-syllabic interval also depend on how rapidly syringeal abductor and adductor muscles can open and close the syringeal aperture .\",\n \"The upper vocal tract may act as a tunable acoustic filter of the sounds produced by the syrinx ( Nowicki , 1987; Beckers et al . , 2003; Riede et al . , 2006 ) and play a more important role in establishing the bandwidth , entropy and mean frequency of song syllables .\",\n \"Thus song parameters linked to vocal tract modulation may be preserved after birds stopped singing , while song parameters linked to contraction speed of syringeal muscles appear to deteriorate .\",\n \"Whereas male canaries are known to annually re-organize their songs , even when deafened ( Mori et al . , 2018 ) , no such modifications were observed in our study .\",\n \"It is not clear to what extent annual changes in male canary song structure are due to relearning or are driven by a selection process from a previously learned collection of syllable types during juvenile development ( Nottebohm et al . , 1986; Marler , 1997; Gardner et al . , 2005; Belzner et al . , 2009 ) .\",\n \"Considering that the female canaries in our study were acoustically separated during the testosterone-induced development of song suggests that the emerging song patterns are either for a large part innate ( Mori et al . , 2018; Gardner et al . , 2005 ) , or are limited to a memorized collection of syllable types that they heard earlier in life ( Belzner et al . , 2009; Marler and Peters , 1981; Marler and Peters , 1982b; Prather et al . , 2010; Kiefer et al . , 2014 ) .\",\n \"The initial song acquisition may thus rely on an innate or memorized auditory template , while the accelerated song re-acquisition may be driven by a motor memory trace of what the birds learned to produce during the first song acquisition phase .\",\n \"The syllable repertoires of our female canaries were much lower than what is common for males .\",\n \"Such a small vocabulary would limit the possibilities to re-arrange or replace syllables without compromising song complexity , and thus few modifications to female songs can be expected .\",\n \"During juvenile brain development in humans , mammals , and birds , synaptic connections are initially overproduced , followed by protracted , activity-dependent synapse elimination during puberty ( Huttenlocher , 1990; Petanjek et al . , 2011; Rakic et al . , 1986; Zuo et al . , 2005; Nixdorf-Bergweiler et al . , 1995 ) .\",\n \"By selectively stabilizing functional synapses while pruning away inactive synapses , brain circuits are thought to reduce neuronal plasticity , consolidating newly learned information and reducing unnecessary redundancy ( Changeux and Danchin , 1976 ) .\",\n \"The lasting spine pruning observed in the forebrain song control area HVC ( Figure 5 ) could reflect such a structural modification facilitating the long-term memorization of vocal patterns .\",\n \"Optical imaging studies have demonstrated a rapid accumulation and stabilization of neuronal dendritic spines in HVC during developmental song learning in zebra finches ( Roberts et al . , 2010 ) .\",\n \"In the mouse cortex , morphological modifications to dendritic spines have been observed that lasted well beyond the learning experience that caused the spine modifications ( Hofer et al . , 2006; Yang et al . , 2009; Xu et al . , 2009; Hofer et al . , 2009 ) , strongly suggesting that long-term memory retention of previously acquired skills is facilitated by stably maintained synaptic connections .\",\n \"Despite such learning-related circuit modifications , the large majority of dendritic spines in the mammalian brain are stably maintained in adulthood ( Petanjek et al . , 2011; Rakic et al . , 1986; Zuo et al . , 2005; Yang et al . , 2009; Xu et al . , 2009; Grutzendler et al . , 2002 ) .\",\n \"In contrast we observed a strong spine reduction in fully adult birds ( Figure 5 ) , suggesting that the observed synaptic pruning in the adult canary HVC during song acquisition may reflect a developmentally delayed , activity-dependent consolidation of the neural motor circuitry .\",\n \"A recent study in zebra finches has shown that singing prevention during juvenile development can also avert developmental spine pruning in the robust motor nucleus of the arcopallium ( RA ) ( Hayase et al . , 2018 ) .\",\n \"In addition , auditory input during sensorimotor learning may play an important role in circuit consolidation , and auditory deprivation has been shown to delay the natural juvenile maturation of HVC in zebra finches ( Huang et al . , 2018 ) .\",\n \"It is not known how long such developmental processes can be postponed , but we did not observe delayed synaptic pruning in RA upon song acquisition in adult female canaries ( Figure 5C ) , suggesting that the time-window for large-scale spine pruning in RA may be more limited than in HVC .\",\n \"The developmentally delayed maturation of HVC ( Figure 5B ) is consistent with our observation that the delayed , testosterone-induced development of song performance in adult female canaries follows a juvenile male-like developmental trajectory ( Figure 1 ) .\",\n \"Thus , in the absence of singing behavior , parts of the vocal motor circuit may remain plastic in adult female canaries , only consolidating once neural activity within the circuitry increases to drive testosterone-induced song output .\",\n \"Testosterone-induced song development in adult female canaries has previously been associated with changes in other neural attributes in HVC as well , including an increase in volume , neuron numbers ( but not densities ) , increased vascularization , and changes in cellular structure ( Vellema et al . , 2014; Hartog et al . , 2009; Gahr and Garcia-Segura , 1996; Nottebohm , 1980; Rasika et al . , 1994; Louissaint et al . , 2002 ) .\",\n \"Many of the observed modifications to the song system follow an annually changing pattern in seasonally breeding male songbirds ( Nottebohm et al . , 1986; Smith et al . , 1997; Nottebohm , 1981; Kirn et al . , 1994 ) , indicating that in males these modifications are not maintained outside the breeding season when birds no longer sing high performance song .\",\n \"In addition , withdrawing testosterone from male Gambel’s white-crowned sparrows , another well-studied seasonal singer , resulted in a rapid regression in HVC size , neuron number , and neuron size ( Thompson et al . , 2007 ) , indicating that these neural modifications are reversible and unlikely to reflect long-term memorization of vocal patterns .\",\n \"The lasting synaptic pruning that we observed in HVC is to our knowledge the first report of a song-related modification to the adult songbird brain that persists after the behavior itself is no longer expressed .\",\n \"HVC is at a central position between the motor circuitry that controls song output and a forebrain-basal ganglia circuit , analogous to the mammalian cortico-basal ganglia ( CBG ) circuit , which feeds back on the motor circuitry for song production ( Brainard and Doupe , 2013; Nottebohm et al . , 1976 ) .\",\n \"Firing patterns in HVC neurons that project to RA are closely associated with millisecond precision of syllable timing ( Yu and Margoliash , 1996; Hahnloser et al . , 2002; Amador et al . , 2013 ) , suggesting that HVC is directly involved in maintaining time-related features of song performance .\",\n \"Burst patterns in RA neurons on the other hand are more closely associated with intrasyllabic structure ( Yu and Margoliash , 1996; Chi and Margoliash , 2001 ) .\",\n \"However , slowing down brain processes by local cooling of HVC has also been shown to change the intrasyllabic structure ( Long and Fee , 2008; Alonso et al . , 2015 ) , indicating that both HVC and RA activity play a role in maintaining the fine temporal structure within syllables .\",\n \"The acquisition of song syllables in zebra finches is accompanied by increased timing accuracy of inhibitory neural firing within the HVC microcircuitry , suggesting that precisely-timed inhibition may play an important role in shaping song-related premotor sequences ( Kosche et al . , 2015; Vallentin et al . , 2016 ) .\",\n \"The lasting synaptic reorganization in HVC during song acquisition may be guided by such inhibitory activity and provide a neural scaffold for both intra and intersyllabic timing .\",\n \"RA-projecting HVC neurons are continuously replaced in the adult songbird brain ( Alvarez-Buylla et al . , 1990 ) , providing a base for ongoing variability in syringeal muscle control .\",\n \"A permanently maintained HVC network may guide dynamically changing circuits in HVC and RA during subsequent periods of singing , quickly driving song features towards previously acquired patterns .\",\n \"Neurons from HVC that project to the avian basal ganglia Area X are not replaced in adulthood , providing a permanent subpopulation of HVC neurons ( Gahr , 1990; Alvarez-Buylla et al . , 1988 ) .\",\n \"Although we could not specifically distinguish between neuron types in our study , we observed the strongest reduction of 50% in the number of densely-spined dendrites in HVC after birds developed song .\",\n \"Previous studies have shown that those HVC neurons that project to Area X predominantly consist of neurons with densely-spined dendrites ( Kornfeld et al . , 2017; Nixdorf et al . , 1989; Kubota and Taniguchi , 1998; Benton et al . , 1998; Mooney , 2000; Mooney and Prather , 2005; Fortune and Margoliash , 1995 ) .\",\n \"Thus our observed synaptic pruning may be related to a major reorganization within the HVC to Area X connection , a reorganization that could reflect the long-term memorization of vocal skills .\",\n \"CBG-like circuits have been strongly implicated in the learning and long-term retention of motor skills in humans ( Albouy et al . , 2008; Debas et al . , 2010; Lehéricy et al . , 2006; Poldrack et al . , 2005; Coynel et al . , 2010 ) , mammals ( Barnes et al . , 2005; Pasupathy and Miller , 2005; Sheth et al . , 2011; Yin et al . , 2009 ) , and birds ( Bottjer et al . , 1984; Scharff and Nottebohm , 1991; Brainard and Doupe , 2000; Kao et al . , 2005; Olveczky et al . , 2005; Aronov et al . , 2008; Sober and Brainard , 2009; Andalman and Fee , 2009; Ölveczky et al . , 2011; Charlesworth et al . , 2012 ) .\",\n \"Vocal exploration is driven by activity in the avian CBG circuit ( Kao et al . , 2005 ) , which may guide synaptic modifications in plastic motor circuits ( Mehaffey and Doupe , 2015 ) , even in adulthood .\",\n \"Although selective elimination of X-projecting HVC neurons did not appear to alter the song structure of adult zebra finches ( Scharff et al . , 2000 ) , altered expression of FoxP2 in Area X of adult male zebra finches has been associated with changes in vocal performance ( Murugan et al . , 2013; Thompson et al . , 2013; Teramitsu and White , 2006 ) .\",\n \"In addition neurotoxic lesions in Area X lead to changes in singing tempo and syllable sequencing in adult zebra finches ( Kubikova et al . , 2014 ) , suggesting that the HVC to Area X connection continues to play a role in the long-term maintenance of vocal sequences .\",\n \"The mechanisms behind learning-related , selective stabilization and elimination of dendritic spines remain largely unknown .\",\n \"Sex hormones may play an important role in the stabilization of such synaptic connections during the development of motor skills .\",\n \"Many of the brain areas that are involved in song learning and production express high numbers of androgen receptors ( Arnold et al . , 1976; Balthazart et al . , 1992; Metzdorf et al . , 1999; Nastiuk and Clayton , 1995; Gahr and Metzdorf , 1997 ) , and X-projecting HVC neurons express estrogen receptors ( Gahr , 1990 ) that can be activated after conversion of testosterone into estradiol .\",\n \"Furthermore , changes in neuronal dendritic growth and synaptic morphology in the songbird RA have been observed to accompany testosterone-induced song development in female canaries ( DeVoogd and Nottebohm , 1981; Devoogd et al . , 1985; Canady et al . , 1988 ) .\",\n \"In addition , Frankl-Vilches et al . ( Frankl-Vilches et al . , 2015 ) recently observed that 85% of the seasonally and testosterone upregulated genes in the canary HVC are related to neuron differentiation , axon , dendrite and synapse organization .\",\n \"In the brain of birds and mammals , sex hormones are known to upregulate the expression of brain-derived neurotrophic factor ( BDNF ) ( Dittrich et al . , 1999; Rasika et al . , 1999; Sohrabji et al . , 1995 ) , which in its turn contributes to the stabilization of synapses through the interaction with tyrosine receptor kinase B ( TrkB ) ( Koleske , 2013 ) .\",\n \"Furthermore , BDNF has been shown to be essential during testosterone-induced song development in female canaries ( Hartog et al . , 2009 ) , providing a likely pathway by which sex hormones can consolidate motor skills by stabilizing synapses .\",\n \"Testosterone levels and song consolidation are tightly linked , and it is difficult to determine if brain anatomical changes in our and other studies are caused by testosterone , singing activity or both .\",\n \"Interestingly , local intracerebral testosterone implants close to nucleus RA did not cause anatomical changes in this androgen receptor-rich brain area ( Brenowitz and Lent , 2002 ) , suggesting that modifications in RA are driven by singing-related , activity-dependent input from HVC .\",\n \"Whether or not testosterone , vocal practice , or both play a role in the observed synaptic pruning in HVC , the vocal circuitry demonstrated synaptic changes that persisted through several months in which testosterone levels were low and birds did not sing , potentially enabling the birds to quickly re-acquire their previously developed song patterns .\",\n \"Sex hormones can be regulated by both environmental and social factors ( Goymann et al . , 2007; Oliveira , 2004; Wingfield et al . , 1990 ) , thereby providing a timed cue for the consolidation of behavioral output and the underlying brain circuitry .\",\n \"Such a cue could facilitate the timely formation and recall of long-term memories of relevant sensory and motor information .\",\n \"Our study suggests that lasting , hormone-driven synaptic pruning of related brain circuitries could form a basis for such long-term memories , enabling a quick recovery of previously acquired skills .\",\n \"The ability to respond quickly to environmental and social cues through such primed hormone-sensitive control mechanisms would constitute an important adaptation to ensure competitive proficiency and maximize reproductive success under environmental restrictions .\",\n \"The results obtained from this study may have implications not only across different animal groups , but may generally apply to different motor , sensory , and motivational systems that rely on a rapid recall of previously learned information .\"\n ],\n [\n \"For this study adult domesticated canaries ( Serinus canaria ) were either purchased from a local breeder in Antwerp or taken from the breeding colony of the Max Planck Institute for Ornithology , Seewiesen .\",\n \"All animals were raised and kept under local natural daylight conditions , until the day length reached 11 hr in early March .\",\n \"Birds were subsequently housed individually in sound attenuating chambers for song recordings .\",\n \"Experimental procedures were conducted according to the guidelines of the Federation of European Animal Science Associations ( FELASA ) and approved by the Ethical Committee on animal experiments of the University of Antwerp .\",\n \"To stimulate song production in naive female canaries , birds were subcutaneously implanted ( T1+ ) with 8 mm silastic tubes ( Dow Corning , Midland , MI; ID: 1 . 47 mm ) filled with crystalline testosterone ( Sigma-Aldrich Co . , St . Louis , MO ) .\",\n \"During the hormone treatments a light cycle of 11 hr light , and 13 hr darkness was maintained to exclude photoperiodic effects on song production .\",\n \"This light cycle was chosen because under natural conditions it corresponds to a phase of testosterone up-regulation and physiological sensitivity to hormone fluctuations in canaries ( Nottebohm et al . , 1987 ) .\",\n \"After consolidation of song we removed the hormone implants ( T1- ) to end song production , and changed the daylight conditions to 8 hr light and 16 hr dark , inducing a molting phase of approximately 1 . 5 months .\",\n \"Once the molt was finished , the day length was gradually returned to 11 hr over the following month .\",\n \"Subsequently , the song-experienced birds received a second testosterone treatment ( T2+ ) , and were sacrificed 5 weeks after implantation .\",\n \"Blood samples were collected from the birds’ right wing veins using heparinized haematocrit capillaries ( Brand , Wertheim , Germany ) .\",\n \"Directly after blood collection , samples were centrifuged at 3000 RPM for 10 min to separate cells from plasma , and stored at −80°C for later analysis .\",\n \"Testosterone concentrations in the blood plasma were determined by radioimmunoassay ( RIA ) after extraction and partial purification on diatomaceous earth ( glycol ) columns , following the procedures described previously ( Goymann et al . , 2001; Goymann et al . , 2008 ) .\",\n \"Briefly , plasma samples were extracted with dichloromethane ( DCM ) after overnight equilibration of the plasma with 1500 dpm of 3H-labeled testosterone ( Perkin-Elmer , Rodgau , Germany ) .\",\n \"After separation of the organic phase , the extracts were re-suspended in 2 × 250 µl 2% ethylacetate in isooctane and fractioned using columns of diatomaceous earth:propylene glycol:ethylene glycol ( 6:1 . 5:1 . 5 , w:v:v ) .\",\n \"Collected testosterone fractions were dried , re-suspended in phosphate-buffered saline with 1% gelatin ( PBSG ) , and left to equilibrate overnight at 4°C .\",\n \"Extraction of plasma testosterone with dichloromethane resulted in 86 ± 11% ( mean ± sd ) recovery .\",\n \"Hormone samples were incubated with antisera against testosterone ( Esoterix Endocrinology , Calabasas Hills , CA ) , and after 30 min 3H-labeled steroids ( 13500 dpm ) were added and left to incubate for 20 hr at 4°C .\",\n \"Free steroids were separated from the bound fractions by absorption on dextran-coated charcoal , centrifuged , and decanted into scintillation vials to be counted .\",\n \"Testosterone concentrations in the plasma samples were measured in one assay .\",\n \"The lower detection limit of the RIA was 0 . 33 pg/tube , and all measured plasma levels were above the lower detection limit .\",\n \"The intra-assay variation of a chicken plasma pool as control sample at the beginning and end of the assay was 6 . 7% .\",\n \"Pooled plasma levels of testosterone for all birds during the different consecutive hormone treatments are shown in Table 1 .\",\n \"Mean detected hormone levels were within range of physiological plasma levels in nest-building male canaries ( range: 360–7970 pg/ml; n = 6 ) and previously reported values for other small passerines ( Wingfield and Hahn , 1994; Kempenaers et al . , 2008; Apfelbeck and Goymann , 2011 ) .\",\n \"To monitor the entire song ontogeny during consecutive testosterone treatments randomly selected , naive female canaries ( n = 6 ) were kept individually in sound attenuating chambers during testosterone treatment ( T1+ ) , testosterone removal ( T1- ) and testosterone re-treatment ( T2+ ) , while song output was continuously recorded .\",\n \"This approach resulted in a song database of 5 . 6 ± 0 . 6 million song syllables per bird , recorded over the course of approximately 1 year .\",\n \"The vocal activity of each bird was recorded using Sound Analysis Pro 2 . 0 ( Tchernichovski et al . , 2000 ) .\",\n \"Omnidirectional condenser microphones ( TC-20; Earthworks , Milford , NH ) connected to a multi-channel microphone preamplifier ( UA-1000; Roland , Los Angeles , CA ) were used to acquire and digitize all sounds produced within the sound attenuating boxes with a sampling frequency of 44 . 1 kHz .\",\n \"The incoming signal was filtered online using an amplitude and Wiener entropy threshold to exclude background noises , and saved in 60 s waveform audio files ( 16-bit PCM format ) .\",\n \"Vocalizations were segmented into individual syllables with the fully automated Feature Batch module in Sound Analysis Pro by applying an amplitude threshold to the sound wave .\",\n \"Because the distance from perch to microphone ranged between 10 and 20 cm only , little variation in recorded amplitude levels can be expected .\",\n \"The amplitude threshold was selected once manually to assure reliable segmentation , and was kept constant during the analyses of all sound phrases within birds .\",\n \"To filter out non-song vocalizations , syllables were only included when produced within a song bout of at least 750 ms . A song bout was defined as a sequence of sounds traversing the amplitude threshold with an interval of no more than 100 ms . We used Sound Analysis Pro to measure duration ( d ) , pause duration ( i ) , syllable rate ( SR ) , frequency modulation ( FM ) , amplitude modulation ( AM ) , syllable bandwidth ( BW ) , mean frequency ( MF ) and Wiener entropy ( E ) for each syllable and stored these syllable features in MySQL 5 . 1 tables ( Oracle , Redwood Shores , CA ) .\",\n \"Bandwidth was calculated for each syllable by subtracting the minimum peak frequency from the maximum peak frequency .\",\n \"The syllable rate was defined for each syllable as: SRa = ( da +ia ) −1 , where da is the duration of syllable ‘a’ , and ia is the interval between the end of syllable ‘a’ and the beginning of the consecutive syllable within the same song bout , irrespective of syllable type .\",\n \"For a detailed computational description of how Sound Analysis Pro calculates the different sound features we refer to the accompanying publication ( Tchernichovski et al . , 2000 ) and the online manual ( http://soundanalysispro . com ) .\",\n \"To investigate the dynamic change of song features over the course of the experiment , daily histograms were obtained from the entire dataset by rounding individual values to the nearest integer , and plotting the number of times those integers occurred each day .\",\n \"To determine the SRs that were most commonly used at the onset and crystallization of song development a non-linear exponential curve ( 1 ) was fitted through the peak values of each SR histogram .\",\n \"( 1 ) Fx=a+b1+ec-xd Further developmental correlation plots for all temporal and spectral song features were produced by calculating the Pearson product-moment correlation coefficient ( CC ) between a 7 day average of the histogram pattern at the time of song stabilization and all other recorded days .\",\n \"This calculation resulted in a value between −1 and 1 for each recorded day , and was used as a measure of similarity where one describes an absolute copy of the stable song pattern , and −1 describes the absolute inverse of the stable song pattern .\",\n \"A non-linear exponential curve ( 1 ) was fitted through the CC values and song parameters were considered stable as soon as the daily increase in similarity dropped below 0 . 001 .\",\n \"For each song feature we used the fit curve to determine the duration between testosterone implantation and feature stabilization , and to determine the maximum daily increase in similarity ( dmax ) .\",\n \"Dmax could only be calculated for birds and song features that demonstrated a developmental increase in similarity , and was only used for statistical purposes if three or more birds demonstrated such an increase .\",\n \"Mean CC plots combining all analyzed birds were calculated by averaging the raw ( non-fitted ) CC values that were calculated for each bird and for each day .\",\n \"We aligned song development in the different birds based on the first day where we detected song .\",\n \"Spectrograms of the original sound files were visually inspected on syllable-like sound structures to determine the syllable repertoire of each bird and the first occurrence of song after testosterone treatments .\",\n \"To assess syllable similarity during consecutive testosterone treatments we calculated the Euclidean distance between each of 100 randomly selected syllables for each syllable type from stable songs during both the T1+ and T2+ periods .\",\n \"The Euclidean distance between two syllables ‘a’ and ‘b’ with coordinates c = SR , d , i , FM , AM , BW , MF , E was defined as:da , b=∑c=1n ( b1-a1 ) 2 To give all analyzed temporal and spectral features equal weight in the analysis , each feature was normalized by dividing its value by its global median value from all syllables produced by all birds recorded in this study .\",\n \"First , for each syllable type the mean Euclidean distance between 100 syllables of the same type during T1+ ( T1xT1 ) was calculated to determine how much baseline variation existed between syllables of the same type .\",\n \"Lower values indicate a higher similarity between syllables .\",\n \"Secondly , we calculated the mean Euclidean distance between 100 syllables of the same type from the T1+ and T2+ ( T1xT2 ) periods to determine if syllables produced during the 2nd testosterone treatment were similar to those produced during the 1st testosterone treatment .\",\n \"Finally , we calculated the mean Euclidean distance between 100 syllables of the same type from the T1 +period with each of 100 syllables from all other types of syllables that a bird produced ( T1xT2ext ) to provide a measure of dissimilarity between syllables of different types .\",\n \"All observed syllable types were included in this analysis with the exception of single transitional syllables connecting two song phrases .\",\n \"To assess similarity of song syntax between subsequent testosterone treatments we determined the probability of song phrase transitions during the T1+ and T2+ periods for each bird .\",\n \"On average 794 . 3 phrase transitions per bird were used for the analysis .\",\n \"From the resulting transition probability distributions we calculated the sequence linearity , consistency and entropy as previously described ( Scharff and Nottebohm , 1991; Daou et al . , 2012 ) .\",\n \"In short , sequence linearity is expressed as the number of syllable types divided by the number of transition types and addresses the way phrases are ordered in a song .\",\n \"Sequence consistency is expressed as the sum of common phrase transitions divided by the sum of total transitions and addresses how often the same sequences are produced .\",\n \"Common phrase transitions were defined as transitions that make up at least 5% of all phrase transitions .\",\n \"The entropy ( S ) of the transition probability distribution with transition type Ti is a measure of the spread of that distribution and was calculated as:S=-∑i=1nTi log2 ( Ti ) To estimate spine densities and dendrite densities in motor nucleus HVC and RA , brains were collected from a control group of adult female canaries that did not receive any testosterone treatment ( C: n = 6 ) , a group of birds that was sacrificed five months after testosterone treatment ( T1+: n = 8 ) , and a group of birds that was sacrificed at least 2 . 5 months after testosterone withdrawal ( T1-: n = 6 ) .\",\n \"All experimental birds were raised and kept in group aviaries together with other male and female canaries throughout the experiment to assure similar social and auditory conditions .\",\n \"Birds were transferred to sound attenuating chambers for two weeks after testosterone treatment to assure that birds were singing , and prior to sacrifice .\",\n \"Individuals were randomly allocated to the different treatment groups .\",\n \"All birds were maintained on a light cycle of 11 hr light , and 13 hr darkness and were between 2 . 5 and 3 years old at the time of sacrifice .\",\n \"Brains were processed for Golgi-Cox staining using the FD Rapid GolgiStain kit ( FD NeuroTechnologies , Columbia , MD ) .\",\n \"After overnight fixation in a 4% formaldehyde solution in phosphate-buffered saline ( PBS; 10 mM; pH 7 . 4 ) , brains were stored in PBS with 0 . 05% sodium azide at 4°C until further processing .\",\n \"Brain hemispheres were separated with a razor blade and left hemispheres were immersed for two weeks in an impregnation solution consisting of equal amounts of FD solutions A and B in total darkness at room temperature .\",\n \"Following three days of incubation in FD solution C , brains were cut into 30 µm sagittal sections using a sliding microtome ( Leica Microsystems GmbH , Wetzlar , Germany ) , which were stored in PBS with 0 . 05% sodium azide at 4°C .\",\n \"Sections with a regular interval of 180 µm were mounted on SuperFrost glass slides ( Menzel GmbH , Brauschweig , Germany ) , developed in a solution of two parts distilled water , one part FD solutions D , and one part FD solution E for ten minutes , and rinsed in distilled water before embedding in CC/Mount tissue mounting medium ( Sigma-Aldrich ) .\",\n \"Slides were incubated at 70°C until the mounting medium had hardened , cleared in xylene , and further embedded in Roti-Histokitt II ( Carl Roth , Karlsruhe , Germany ) before coverslipping .\",\n \"For each bird , z-stacks were obtained from three brain sections of nucleus HVC and two sections of nucleus RA with a Nikon Eclipse Ti microscope ( Nikon , Tokyo , Japan ) , equipped with a 60x oil immersion lens ( CFI Plan Apo VC 60x Oil ) .\",\n \"The outline of HVC and RA in each section was delineated in ImageJ ( http://rsb . info . nih . gov/ij/ ) , and 100 µm2 non-overlapping regions of interests ( ROIs ) were randomly placed within the boundaries of the target nucleus prior to the quantifications .\",\n \"Considering the relatively uniform distribution of X-projecting HVC neurons and RA-projecting HVC neurons ( Fortune and Margoliash , 1995 ) , this approach should result in a homogenous sampling across treatment groups .\",\n \"Neuronal dendritic spine densities were estimated for each brain section by checking ROIs in random order until five ROIs were located that contained spinous dendrite segments ( 15 ROIs per animal for HVC and 10 ROIs for RA ) .\",\n \"The individual dendrite segments within the ROIs were traced in ImageJ to measure the length , and the number of visible spines that originated from the traced segment was manually counted .\",\n \"Spine densities were calculated as the number of visible spines per µm of dendrite .\",\n \"All visible spine types were included in the quantifications , including filopodia , long , thin , stubby , mushroom and branched spines ( Risher et al . , 2014 ) .\",\n \"For branched spines , each spine head was counted separately .\",\n \"To further obtain a density measure of spinous dendrites we counted the total number of spinous dendrite segments in all ROIs examined , and divided the number of observed dendrite segments with the number of ROIs , giving an estimation of the number of spinous dendrites per ROI .\",\n \"Neuronal dendrites were further binned into categories containing 0–0 . 2 , 0 . 2–0 . 4 , 0 . 4–0 . 6 , 0 . 6–0 . 8 , 0 . 8–1 . 0 , 1 . 0–1 . 2 , 1 . 2–1 . 4 , 1 . 4–1 . 6 , 1 . 6–1 . 8 spines per µm .\",\n \"The probability distribution of neuronal dendrite segments was then obtained by dividing the observed number of spinous dendrites per ROI in each bin with the total number of spinous dendrites per ROI .\",\n \"To obtain a density measure of aspinous neurites in HVC , the number of visible spine-less neurite segments were counted in ten new non-overlapping ROIs that were randomly placed within the boundaries of HVC .\",\n \"We refer to these projections as neurites , because we were unable to distinguish between aspinous dendrites and axons in our tissues .\",\n \"All quantifications were conducted by an experimenter that was blind to the experimental condition of the animals .\",\n \"Statistical analyses were performed in SAS 9 . 3 ( SAS Institute , Cary , NC ) .\",\n \"No sample sizes were calculated prior to the experiments .\",\n \"A technical replication was defined as a repeated measurement of the same individual , and biological replications constitute the number of sampled individuals ( n ) .\",\n \"Differences in spine and dendrite densities between treatment groups were tested with a one-way analysis of variance ( ANOVA ) with Dunnett’s correction for multiple comparisons against one control value ( untreated birds ) .\",\n \"Changes in hormone levels were determined using a repeated one-way ANOVA with Dunnett’s correction .\",\n \"Differences in syllable similarity and syllable rates of individual syllable types were tested with one-way ANOVAs , and when significant , Tukey's HSD test was used to analyze differences between time points .\",\n \"Song pattern similarities between the 1st and 2nd hormone treatment were established by comparing the average CC from seven consecutive days of stable song during the 1st treatment with the average CC from seven consecutive days of stable song production during the 2nd treatment .\",\n \"Paired-samples t-tests were used to compare CC’s , sequence parameters , and developmental time parameters between subsequent hormone treatments .\",\n \"To determine song feature deterioration we calculated the difference between the average CC from the last 7 days of stable song production during the 1st testosterone treatment and the average CC from the first 2 days of song production during the 2nd treatment .\",\n \"Feature recovery was determined by subtracting the average CC from the first 2 days of song production from the average CC from the last 7 days of stable song production during the 2nd testosterone treatment .\",\n \"One-sample t-tests were used to determine if feature deterioration and recovery significantly exceeded zero .\",\n \"Measurement values in the text are given as means ± SEM , unless stated otherwise .\",\n \"No outliers or data were excluded from analysis .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Complex motor skills take considerable time and practice to learn .","Without continued practice the level of skill performance quickly degrades , posing a problem for the timely utilization of skilled motor behaviors .","Here we quantified the recurring development of vocal motor skills and the accompanying changes in synaptic connectivity in the brain of a songbird , while manipulating skill performance by consecutively administrating and withdrawing testosterone .","We demonstrate that a songbird with prior singing experience can significantly accelerate the re-acquisition of vocal performance .","We further demonstrate that an increase in vocal performance is accompanied by a pronounced synaptic pruning in the forebrain vocal motor area HVC , a reduction that is not reversed when birds stop singing .","These results provide evidence that lasting synaptic changes in the motor circuitry are associated with the savings of motor skills , enabling a rapid recovery of motor performance under environmental time constraints ."],"string":"[\n \"Complex motor skills take considerable time and practice to learn .\",\n \"Without continued practice the level of skill performance quickly degrades , posing a problem for the timely utilization of skilled motor behaviors .\",\n \"Here we quantified the recurring development of vocal motor skills and the accompanying changes in synaptic connectivity in the brain of a songbird , while manipulating skill performance by consecutively administrating and withdrawing testosterone .\",\n \"We demonstrate that a songbird with prior singing experience can significantly accelerate the re-acquisition of vocal performance .\",\n \"We further demonstrate that an increase in vocal performance is accompanied by a pronounced synaptic pruning in the forebrain vocal motor area HVC , a reduction that is not reversed when birds stop singing .\",\n \"These results provide evidence that lasting synaptic changes in the motor circuitry are associated with the savings of motor skills , enabling a rapid recovery of motor performance under environmental time constraints .\"\n]"},"summary":{"kind":"list like","value":["Developing a complex skill , for example learning how to play the violin , takes considerable time and effort .","If you then abandon the violin for months or years , your ability to play will deteriorate over time .","However , when you do pick up the violin again you will be able to recover your proficiency much faster than you did when learning for the first time .","It therefore seems that while first learning a complex motor skill , some sort of memory is formed and stored .","Learning and memory in general rely on the patterns of connections , called synapses , among neurons in the brain .","Early in development , neurons make too many of these connections .","This network is then refined over time as unneeded connections are discarded .","However , the structural changes to the network that make it easier to re-acquire a skill were not well understood .","Canaries are a useful example of this sort of learning .","Young males learn complex songs that are critical for their ability to attract a mate , and can quickly re-acquire their songs at the beginning of each mating season .","Female canaries do not normally sing but will develop songs if they are implanted with a testosterone-releasing device .","When the implant is removed , they stop singing .","To investigate how songbirds re-acquire songs , Vellema et al . gave female canaries a testosterone implant , and the birds gradually developed songs .","The implant was then removed , and for two and a half months the birds did not sing .","Vellema et al . then gave the canaries a second testosterone treatment .","The canaries rapidly began to sing songs that were strikingly similar to the ones they sang during the initial learning period .","Examining the brains of these canaries revealed that major structural changes in brain connections occurred while the canaries first developed their songs .","After the initial period of testosterone exposure , the female canaries had fewer synapses in a brain region that is associated with learning and producing motor tasks .","Importantly , this reorganization of the brain circuitry was irreversible .","These findings provide fundamental insights into how we learn and maintain new motor skills .","Similar rapid re-learning phenomena are observed in other areas of neuroscience , and future research can explore whether the mechanism described here extends to these areas .","Possibly , this mechanism could also illuminate how we can regain skills lost after trauma or injury ."],"string":"[\n \"Developing a complex skill , for example learning how to play the violin , takes considerable time and effort .\",\n \"If you then abandon the violin for months or years , your ability to play will deteriorate over time .\",\n \"However , when you do pick up the violin again you will be able to recover your proficiency much faster than you did when learning for the first time .\",\n \"It therefore seems that while first learning a complex motor skill , some sort of memory is formed and stored .\",\n \"Learning and memory in general rely on the patterns of connections , called synapses , among neurons in the brain .\",\n \"Early in development , neurons make too many of these connections .\",\n \"This network is then refined over time as unneeded connections are discarded .\",\n \"However , the structural changes to the network that make it easier to re-acquire a skill were not well understood .\",\n \"Canaries are a useful example of this sort of learning .\",\n \"Young males learn complex songs that are critical for their ability to attract a mate , and can quickly re-acquire their songs at the beginning of each mating season .\",\n \"Female canaries do not normally sing but will develop songs if they are implanted with a testosterone-releasing device .\",\n \"When the implant is removed , they stop singing .\",\n \"To investigate how songbirds re-acquire songs , Vellema et al . gave female canaries a testosterone implant , and the birds gradually developed songs .\",\n \"The implant was then removed , and for two and a half months the birds did not sing .\",\n \"Vellema et al . then gave the canaries a second testosterone treatment .\",\n \"The canaries rapidly began to sing songs that were strikingly similar to the ones they sang during the initial learning period .\",\n \"Examining the brains of these canaries revealed that major structural changes in brain connections occurred while the canaries first developed their songs .\",\n \"After the initial period of testosterone exposure , the female canaries had fewer synapses in a brain region that is associated with learning and producing motor tasks .\",\n \"Importantly , this reorganization of the brain circuitry was irreversible .\",\n \"These findings provide fundamental insights into how we learn and maintain new motor skills .\",\n \"Similar rapid re-learning phenomena are observed in other areas of neuroscience , and future research can explore whether the mechanism described here extends to these areas .\",\n \"Possibly , this mechanism could also illuminate how we can regain skills lost after trauma or injury .\"\n]"},"year":{"kind":"string","value":"2019"}}},{"rowIdx":4290,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["stem cells and regenerative medicine","research communication"],"string":"[\n \"stem cells and regenerative medicine\",\n \"research communication\"\n]"},"title":{"kind":"string","value":"Sox9+ messenger cells orchestrate large-scale skeletal regeneration in the mammalian rib"},"id":{"kind":"string","value":"elife-40715-v1"},"sections":{"kind":"list like","value":[["Whereas amphibians regenerate large portions of their skeletons after injury or amputation , natural large-scale skeletal repair in mammals is more limited .","A notable exception is the rib .","Craniofacial surgeons often extract large segments of rib cartilage and bone for autologous repair of skeletal defects in other parts of the body , and in post-operative visits have noted extensive regeneration at the donor rib site ( Kawanabe and Nagata , 2006; Munro and Guyuron , 1981; Srour et al . , 2015 ) .","To better understand the unique regenerative potential of the rib , we have recently developed analogous large-scale rib cartilage and bone regeneration models in the mouse ( Srour et al . , 2015; Tripuraneni et al . , 2015 ) .","In most mammalian bones , healing involves the formation of a callus using a mixture of direct ossification and endochondral ossification via a cartilage callus intermediate ( Gerstenfeld et al . , 2003; Hall , 2014; Marsell and Einhorn , 2011 ) .","The identity and regulation of the adult skeletal progenitors that build the callus remain incompletely understood .","While markers for a number of different skeletal stem cells have been reported ( Balani et al . , 2017; Bianco and Robey , 2015; He et al . , 2017; Matthews et al . , 2014; Park et al . , 2012; Ransom et al . , 2016; Shi et al . , 2017 ) , their relative roles during bone repair are less clear , particularly in cases of large-scale bone regeneration such as in the rib .","During skeletal repair , studies have shown that cells from the periosteum , a heterogeneous connective tissue sheath covering the bone , are major contributors to the callus ( Colnot , 2009; Duchamp de Lageneste et al . , 2018; Murao et al . , 2013 ) .","Accordingly , we have found that the periosteum is essential for regeneration of the rib bone ( Tripuraneni et al . , 2015 ) .","During normal bone homeostasis , periosteal stem cells generate bone-producing osteoblasts but not cartilage-producing chondrocytes ( Roberts et al . , 2015 ) .","How injury stimulates periosteal stem cells to generate chondrocytes is unclear .","Although some fractures can heal in the absence of a cartilage callus , for example when the fracture is rigidly stabilized ( Thompson et al . , 2002 ) , the formation of a large cartilage callus appears to be required in large-scale bone regeneration .","Unfortunately little is known about the specific periosteal progenitor population that drives the formation of the cartilage callus nor the signaling pathways required .","Recent studies have used Cre-based lineage tracing experiments to show that cells marked by expression of Gremlin1 ( Worthley et al . , 2015 ) , Axin2 ( Ransom et al . , 2016 ) , Gli1 ( Shi et al . , 2017 ) , Act2a ( Matthews et al . , 2016 ) , Periostin ( Duchamp de Lageneste et al . , 2018 ) and Sox9 ( Balani et al . , 2017; He et al . , 2017 ) can be found in the periosteum and contribute to the fracture callus during repair .","Other than participation , the specific role of any of these progenitor population remains unclear .","In this study , we therefore focus on the role of one subpopulation within the periosteum and its specific role in driving callus formation and bone regeneration .","As Sox9 has a well-known function in promoting chondrogenesis during embryonic development ( Akiyama et al . , 2002; Lefebvre et al . , 1997 ) , we postulated that Sox9-expressing cells in the adult periosteum may possess a potent ability to form or promote cartilage in response to bone injury .","In addition , a requirement for Sox9-expressing cells in repair or homeostasis has not yet been established .","The signaling pathways required for generating a cartilage callus from the periosteum in response to injury are also not well-characterized .","The formation of the cartilage callus in fractures has been thought to involve a recapitulation of endochondral bone development ( Bahney et al . , 2014; Maes et al . , 2010; Marsell and Einhorn , 2011; Yang et al . , 2014; Zhou et al . , 2014 ) .","One of the most well-known signaling pathways during endochondral bone development is the Hh pathway , activation of which promotes chondrogenic proliferation and osteocyte maturation ( Long et al . , 2001; Shi et al . , 2017; St-Jacques et al . , 1999 ) .","Studies have investigated Hh signaling during fracture repair , but due to conflicting results it remains unclear if Hh signaling has the same role in bone repair as it does during bone development .","Genetic ablation of the obligate Hh co-receptor Smo in mice , using two different ubiquitously inducible Cre lines , resulted in reduced bone formation during fracture repair , yet was not reported to disrupt initial cartilage callus formation ( Baht et al . , 2014; Wang et al . , 2010 ) .","Forced activation of Hh signaling throughout the mouse during fracture repair , using an inducible constitutively active Smo allele , resulted in increased bone formation ( Baht et al . , 2014 ) , similar to what was seen upon engraftment of cells overexpressing Hh or treatment with an Hh agonist ( Edwards et al . , 2005; Huang et al . , 2014; Zou et al . , 2014 ) .","However , on which cell types Hh acts upon , and whether it regulates the decision to build the cartilage callus and/or other aspects of bone repair in mammals , has remained unknown .","In this study we examine the role of the Sox9-expressing periosteal subpopulation ( referred to as Sox9+ cells hereafter ) during large-scale rib repair .","This subpopulation appears to be a key player in orchestrating the formation of a large repair callus in the rib that consists of an unusual hybrid osteochondral cell type with properties of both chondrocytes and osteoblasts .","Loss of Smo in Sox9+ periosteal cells prior to injury results in a near-complete failure of cartilage callus formation and bone regeneration .","This Sox9+ subpopulation must be able to respond to Hh signaling in order to initiate this process , indicating that Hh signaling’s role in bone repair is distinct from its role in bone development .","Additionally , since Sox9+ periosteal cells contribute to only a minority of callus cells , we suggest that Sox9+ periosteal cells act as ‘messenger’ cells and orchestrate repair by inducing the differentiation of neighboring callus cells through non-autonomous signals .","Overall our results indicate that bone regeneration does not fully recapitulate bone development , and that the periosteum consists of subpopulations that may have different roles/responses during repair ."],["Like appendicular long bones , the bony portion of the rib develops via an endochondral process including growth plates at either end and a central hollow bone marrow cavity .","Both human and murine rib bones display remarkable regenerative potential ( Srour et al . , 2015; Tripuraneni et al . , 2015 ) , however the cellular basis for such large-scale repair remains unknown .","To better understand the cellular sequence of events during regeneration , we analyzed 3 mm rib bone defects at sequential time points up to 10 weeks post-resection ( wpr ) ( Figure 1A ) .","Histology at 5 days post-resection ( dpr ) revealed cells with a mesenchymal-like morphology filling the entire resected region ( Figure 1B ) .","We then observed formation of a substantial alcian-blue positive callus spanning most of the defect by 1 wpr ( Figure 1A ) , with many of these cells displaying a cartilage-like morphology at 10 dpr ( Figure 1C ) .","Histology revealed increasing bone formation by 10 and 14 dpr ( Figure 1C , D ) , with extensive alizarin-positive mineralization across the defect at 4 wpr and full remodeling to the pre-injury organization by 10 wpr ( Figure 1A ) .","Next , we used double fluorescent RNA in situ hybridization ( RNA-ISH ) to characterize the molecular identity of cells during the regeneration process .","At 5 dpr , mesenchymal cells within the lesion co-expressed Sox9 , a master regulator of the chondrocyte lineage ( Akiyama et al . , 2002; Lefebvre et al . , 1997 ) , and Runx2 , a master regulator of the osteoblast lineage ( Lian and Stein , 2003 ) ( Figure 1B ) .","While co-expression of chondrocyte and osteoblast markers has been observed during early bone development , normally as differentiation proceeds , a cell will express only chondrocyte or only osteoblast markers .","Surprisingly , we observed co-expression of genes associated with chondrocyte and osteoblast differentiation within single cells throughout the repair process .","For example , we see co-expression of Sox9 with the major osteoblast collagen gene Col1a1 ( Figure 1—figure supplement 1A ) .","As the callus matures bone and cartilage markers continue to be co-expressed at high levels .","The major chondrocyte collagen gene Col2a1 was observed to be co-expressed with the late osteoblast marker Bglap ( also known as Osteocalcin ) ( Figure 1C and Figure 1—figure supplement 1C ) , and with Col1a1 at both 10 and 14 dpr ( Figure 1D and Figure 1—figure supplement 1B ) .","We observed co-expression of chondrocyte and osteoblast markers in cells of both cartilage and osteoblast morphology , including cells on the surface of newly formed trabecular bone ( Figure 1C , D , Figure 1—figure supplement 1C ) .","Similar co-expression results were obtained using mice double transgenic for a reporter of hypertrophic chondrocytes ( Col10a1-mCherry ) and osteoblasts ( Col1 ( 2 . 3 ) -GFP ) as well as using double immunofluorescence for COL1 and COL2 protein ( Figure 1E , Figure 1—figure supplement 1D ) .","In contrast , we did not observe co-expression of Col2a1 and Col1a1 in the rib growth plate under the same assay conditions ( Figure 1F ) .","Single colorimetric RNA-ISH assays of near-adjacent sections at 7 dpr confirmed co-expression of Sox9 , Runx2 , Col2a1 , Col1a1 , Bglap , and the hypertrophic cartilage collagen gene Col10a1 in cells of cartilage morphology within the callus ( Figure 1—figure supplement 2 ) .","Together , these findings indicate that , in marked contrast to the growth plate , cells within the rib repair callus co-express cartilage and bone markers while displaying either a chondrocyte or osteoblast morphology .","We therefore refer to these cells as ‘hybrid’ osteochondral skeletal cells .","We next examined the source of the skeletal cells that mediate repair .","These cells are likely derived from the periosteum , since removal of the periosteum along with the bone , results in a failure of cartilage callus formation ( Figure 2—figure supplement 1 ) and subsequent bone repair ( Tripuraneni et al . , 2015 ) .","Sox9 is a master regulatory gene of chondrogenesis , and a previous study indicated that Sox9+ cells in the femur periosteum can contribute to callus formation after fracture ( He et al . , 2017 ) .","We therefore examined whether Sox9-expressing periosteal cells contribute to the repair callus during rib regeneration .","To do so , we administered three consecutive daily doses of tamoxifen to Sox9-CreERT2; ROSA26-loxP-stop-loxP-tdTomato mice , followed by a 4-day chase to allow clearance of residual tamoxifen ( Figure 2A , “Pre” regimen ) .","In the absence of injury , we observed that Sox9-expressing cells were predominantly found in the periosteum and but only constituted 6 ± 0 . 3% of the population , with almost no labeled cells seen in the marrow compartment ( Figure 2B ) .","After rib resection , we found that 22 ± 1 . 3% of callus cells were tdTomato+ by 10 dpr with many of these cells having a typical chondrocyte morphology ( Figure 2C ) .","At 14 dpr ( Figure 2C ) , we observed tdTomato expression in both chondrocyte-like cells , as well as osteoblast-like cells lining new trabecular bone ( as defined by near-adjacent H&E sections ) , with contribution to osteoblast-like cells also evident by 21 dpr ( Figure 2—figure supplement 2A ) .","We observed fewer tdTomato+ cells at 14 dpr suggesting that as the callus matures , Sox9+ lineage cells are remodeled out and replaced by non-Sox9+ lineage cells .","Pre-existing -expressing cells , thus contribute to only a minority of the cells that form the initial repair callus , including only a subset of the callus cells with hybrid osteochondral characteristics .","Most of the cells in the rib callus are therefore derived from a lineage that did not express Sox9 prior to injury .","In addition , similar to reports by He et al . ( He et al . , 2017 ) we found that also after femur fracture , our “Pre” tamoxifen regimen revealed contribution of pre-existing Sox9+ cells to the callus ( Figure 2—figure supplement 3A ) .","We further found that some of the cells within the femur fracture callus also co-expressed Col1a1 and Col2a1 strongly , suggesting a similar hybrid identity to that observed in the rib callus ( Figure 2—figure supplement 3B ) .","Since only a minority of the callus cells are derived from the Sox9-expressing periosteal subpopulation , as opposed to the majority of the callus cells that expresses Sox9 mRNA at 5 dpr ( Figure 1B ) , we instead applied tamoxifen at the time of injury plus 2 days following ( “Post” regimen , Figure 2D ) .","This regimen will capture both the subpopulation that expresses Sox9 prior to injury as well as any cells that turn on Sox9 in response to injury .","Consistent with detection of endogenous Sox9 mRNA expression in early callus cells ( e . g . Figure 1B ) , we found that this later tamoxifen regimen labeled the majority of callus cells by 10 dpr ( 85 . 5 ± 2 . 8% ) and most of the osteoblast-like cells lining newly forming trabecular bone at 14 ( Figure 2E ) and 28 dpr .","Further , typical of osteocytes , some of the tdTomato+ cells at 28 dpr were found embedded in bone ( Figure 2—figure supplement 2B ) .","Thus , depending on when tamoxifen is administered ( Pre vs . Post ) , either a minority or majority of the callus cells are labelled .","Since we hypothesized that the Hh pathway may be important for large-scale repair , we first determined the expression of Hh ligands and Ptch1 after rib resection .","As expected , based on the expression of Ihh in the growth plate , callus cells with cartilage morphology expressed Ihh and Ptch1 .","Interestingly we also detected the related ligand , Shh , in these cells ( Figure 3A and C ) .","At earlier stages Ihh expression was undetectable until 7 dpr and then only at very low levels in developing chondrocytes .","However , Shh expression was readily detectable at 5 dpr and 7dpr similar to reports by others ( Matsumoto et al . , 2016 ) although at lower levels than found in cells with cartilage morphology ( Figure 3A and C ) .","Ptch1 , a read-out of the Hh pathway , was also detected in tdTomato+ periosteal cells a 3 dpr , supporting the idea that these cells are responsive to a Hh signal prior to entering the callus ( Figure 3—figure supplement 1A ) .","We then tested the requirement for Hh signaling in large-scale bone regeneration by deleting Smo , the required Hh co-receptor , using the Sox9-CreERT2 transgene and employing either the Pre or Post tamoxifen treatment regimens .","Using Smo RNA-ISH , we observed that the Post tamoxifen treatment resulted in deletion of Smo broadly ( Figure 3B ) as predicted given that Cre is active in most cells ( Figure 2D ) .","Pre tamoxifen treatment resulted in deletion of Smo in the Sox9-positive lineage subpopulation which were marked by including the tdTomato reporter in the cross ( Figure 3B ) .","This was expected , since deletion of Smo occurred prior to surgery and thus only a minority of the cells in the repair callus are expected to be null for Smo ( Figure 2C ) .","We then examined the callus at 7 dpr while there is a mix of both immature and differentiating cells .","In control Pre tamoxifen treatment animals , tdTomato+ cells could be found scattered across the developing callus and were not preferentially found in clusters of differentiating chondrocytes suggesting that Sox9+ cells do not differentiate ahead of other cells in the callus ( Figure 3—figure supplement 1B ) .","In addition , in regions of the control callus where cells appeared immature and in Pre tamoxifen treatment calluses , there was no strong correlation between the tdTomato trace and the expression of Shh , or read-outs of Hh signaling such as Ptch1 or Gli2 ( Pak and Segal , 2016 ) suggesting that in contrast to our earlier observations in the periosteum , that at these later stages , tdTomato cells are not strongly responding to a Hh signal ( Figure 3C and Figure 3—figure supplement 1C ) .","Furthermore , in cells neighboring Tdtomato+ cells , we did not see any enrichment of Ptch1 or Gli2 expression suggesting that they were not receiving a potent Hh signal that could be released by Tdtomato+ cells .","We then investigated the consequence of Smo deletion on cartilage callus formation .","Surprisingly , Smo deletion using either the Pre or Post tamoxifen treatment resulted in a similarly near-complete loss of the cartilage callus at both 7 and 10 dpr ( Figure 4A , Figure 4—source data 1 ) , despite the Pre treatment only deleting Smo in a small subset of callus progenitors ( Figures 2C and 3B ) .","After Smo deletion , although cells did not form a mature callus , they still co-expressed Sox9 and Runx2 at 5 dpr ( Figure 4—figure supplement 1A ) .","At 10 dpr , in contrast to co-expression of Col2a1 and Col1a1 throughout the control callus , the few Col1a1-expressing cells that formed after Smo deletion lacked high levels of Col2a1 and vice versa , demonstrating a lack of hybrid osteochondral cells ( Figure 3B ) .","The lack of callus formation and hybrid cells following Smo deletion was not reflected by altered numbers of pHH3+ proliferative or TUNEL+ apoptotic cells at either 7 or 10 dpr , and lineage tracing with the tdTomato reporter revealed similar numbers of cells within the resection site at 10 dpr ( Figure 4C , Figure 4—source data 2 , Figure 4D , Figure 4—source data 3 , Figure 4—figure supplement 1B , Figure 4-figure supplement-source data 1 ) .","Thus , Hh signaling is likely not required for the early proliferative expansion in response to injury or for cell survival .","Instead , we conclude that Hh signaling is required in Sox9+ cells for promoting the differentiation of non-Sox9 lineage cells into hybrid osteochondral cells of the callus .","When the Sox9+ Pre population is null for Smo , non-Sox9 lineage cells are still unable to differentiate ( despite expressing Ptch1 ) resulting in the near complete failure of callus formation .","Thus , this minority Sox9+ periosteal lineage population plays a critical initiating role in callus differentiation .","We next assessed the consequence of defective callus formation on subsequent regeneration of rib bone in Smo-deleted animals .","In contrast to control animals showing robust Col1a1 and Col2a1 co-expression in cells lining new trabecular bone at 14 dpr , deletion of Smo using either the Pre or Post regimens resulted in a near complete absence of co-expressing cells .","Instead , only small numbers of Col2a1-only cells were observed , primarily near the cut ends of the bone , while cells with osteoblast morphology expressed predominantly only Col1a1 ( Figure 5A ) .","Analysis of H and E-stained histological sections confirmed a marked decrease in bone formation at 14 dpr , despite substantial mesenchyme still observed in the resection site , with the magnitude of the bone defect similar in both regimens ( Figure 5B ) .","We found that ‘Late’ removal of Smo ( injection of tamoxifen at 3–5 dpr targeting the whole callus ) only caused a slight delay in repair with a fully bridged callus evident at 14 dpr ( Figure 5—figure supplement 1 ) suggesting that the decrease in bone formation seen in both the Pre and Post regimens is largely related to reduced cartilage callus formation .","Whole-mount staining with alizarin red and alcian blue confirmed a failure of bone union at 4 and 6 wpr in both Pre and Post conditions ( Figure 5C ) .","These findings demonstrate that the Sox9+ subpopulation requires Hh signaling not only to build the repair callus but that a substantial cartilage callus may be needed to efficiently regenerate bone ."],["We find that building an extensive callus of a unique type of hybrid osteochondral skeletal cell is essential for successfully bridging large gaps in adult mammalian rib bone .","During this process , Hh signaling plays a critical role , distinct from that in the developing growth plate , in promoting the ultimate differentiation of these hybrid osteochondral skeletal cells .","Further , we provide genetic evidence that Hh signaling acts upon a rare periosteal subpopulation of Sox9-expressing cells , that behave as messenger cells .","These Sox9+ periosteal cells then stimulate , through a yet to be determined signal , their neighboring non-Sox9-lineage cells , which constitute a majority of the callus , to differentiate and build new callus and bone ( summarized in Figure 6 ) .","We observe high-level co-expression of genes typically associated with cartilage ( Sox9 , Col2a1 , Col10a1 ) or bone ( Col1a1 , Bglap ) in callus cells during murine rib bone regeneration .","This is in marked contrast to the largely exclusive expression of cartilage versus bone genes in the developing growth plates , although hypertrophic chondrocytes do express low levels of many bone-associated genes ( Gerstenfeld and Shapiro , 1996 ) .","The trajectory of hybrid cells is also fundamentally different from the subpopulation that is proposed to ‘transdifferentiate’ from chondrocytes to osteoblasts in the growth plate ( Bahney et al . , 2014; Shimomura et al . , 1975; von der Mark and von der Mark , 1977; Yang et al . , 2014; Zhou et al . , 2014 ) .","In the growth plate , these transdifferentiating cells express chondrocyte-associated genes first and then , through a process that remains mysterious , turn off chondrocyte-associated genes and turn on osteoblast-associated genes .","Here , we confirm a similar process of ‘first cartilage and then bone’ in the rib growth plate .","In contrast , in the regenerating rib , we observe that callus cells co-express cartilage- and bone-associated genes at the earliest stages .","We propose that they then maintain this hybrid osteochondral identity as they shift from making a cartilaginous and then a bone-like matrix .","We also observe a similarly hybrid osteochondral cell in the femur fracture callus , although further investigations will be required to determine how similar these cells are between the rib and femur callus .","Moreover , similar hybrid cells have been reported during zebrafish jawbone repair ( Paul et al . , 2016 ) suggesting that skeletal cells with dual chondrocyte/osteoblast properties may be critical for bone regeneration across vertebrate species .","Co-expression of bone and cartilage programs in the same cell is certainly not unique to the regenerating callus .","For example , Col1a1 and Col2a1 are also highly expressed in fibrocartilage cells , however this tissue is morphologically quite different in terms of its high fiber to cell ratio when compared to the regenerating rib callus ( Benjamin and Ralphs , 2004 ) .","Of note , a rare developmental skeletal type has been described , historically referred to as ‘chondroid bone’ or ‘secondary cartilage’ , that does share many features with the hybrid osteochondral cells we observe during regeneration ( Goret-Nicaise , 1984; Shibata and Yokohama-Tamaki , 2008 ) .","Therefore , it may be that these cells are not unique to regeneration , but rather are selectively utilized due to their ability to rapidly proliferate in a relatively avascular environment ( a property of cartilage [Dennis et al . , 2015] ) while directly producing mineralized matrix ( a property of bone ) .","Whereas some bone does form in our Smo-deleted mice , this is not sufficient to bridge the lesion and healing fails , resulting in a persistent non-union .","Cells within this bone do not display hybrid chondrocyte/osteoblast character , consistent with residual bone forming by direct ossification rather than ossification through a callus intermediate .","These findings suggest that during large-scale bone regeneration , Hh signaling in Sox9-expressing periosteal cells is selectively required for the formation of a hybrid osteochondral callus .","Hh signaling may play an important but more subtle role in osteocyte maturation as fractures still heal in the absence of Smo but with decreased or delayed bone formation ( Baht et al . , 2014; Wang et al . , 2010 ) .","Similarly , we see production of bone ( although delayed ) in our rib resection model with a Late KO of Smo ( Figure 5—figure supplement 1 ) .","In contrast to our rib model , however , loss of Smo in these femur/tibia fracture assays still resulted in the formation of a cartilage callus .","We do not know why these results contrast from ours where cartilage callus formation is dramatically compromised .","One possibility is that the efficacy of Smo removal in these fracture experiments was not efficient during cartilage callus stages , alternatively , there could be different requirements for Hh signaling depending on the type of injury ( resection vs . fracture ) .","Interestingly , the formation of a cartilage callus may not be as critical during fracture repair , as fractures can repair solely through direct ossification ( Colnot et al . , 2003 ) .","While in contrast , during large-scale repair , the process of direct ossification is not sufficient to build large pieces of bone and instead , building a hybrid osteochondral callus may be particularly important for bridging large bone gaps .","Hh signaling is known to promote chondrocyte proliferation and osteoblast differentiation in the developing growth plate ( Long et al . , 2004; Long et al . , 2001 ) .","Surprisingly , we found that loss of Hh signaling did not affect the early proliferation of callus cells or the differentiation of osteoblasts in the residual directly ossifying bone .","Instead , we provide evidence that Hh signaling has a distinct and essential role in promoting the differentiation of Sox9/Runx2 expressing progenitors into the hybrid osteochondral skeletal cells that form the repair callus .","These results indicate that the regeneration of the rib bone , including its dependence on Hh signaling , does not simply the recapitulate developmental processes seen at the growth plate .","While the heterogeneity of the cells in the periosteum and their lineage relationships remain incompletely understood , we propose that Sox9+ periosteal cells or a subpopulation within them , play an essential instructive role for callus formation during large-scale bone regeneration that has not been previously described in the context of repair .","The Sox9+ periosteal subpopulation may be distinct from the previously described subpopulations that are Gli1+ , αSMA+ , Gremlin1+ , or Axin2+ , as RNA-seq analysis of the periosteum from an uninjured femur indicated that Gli1 , αSMA , Axin2 , and Gremlin1 are not highly expressed in the Sox9+ cells ( He et al . , 2017 ) .","Future studies tracing the lineage of these other populations and determining their dependence on Hh signaling will be needed to resolve whether Sox9+ cells represent a subset of one of these other more abundant populations , or alternatively a distinct progenitor subpopulation .","In addition , efforts to delineate potential differences that may exist in periosteal populations from different bones , may explain why some bones regenerate well , while others do not .","One of the most striking findings from our study is that Sox9+ cells are essential for efficient callus formation and rib bone regeneration , despite contributing to only a minority of cells within the callus and regenerated bone .","Based on this observation , we propose that the Sox9+ cells act as ‘messenger’ cells by releasing a yet to be determined signal that promotes the differentiation of neighboring cells Sox9-negative lineage cells .","One possibility is that Sox9+ progenitors differentiate early in response to the initial Hh signal ( potentially Shh , since strong expression is evident early ) and then propagate another wave of Hh signaling to neighboring Sox9-negative cells , similar to the role of Hh in the morphogenetic furrow during Drosophila eye development ( Domínguez and Hafen , 1997; Kumar , 2011; Ma et al . , 1993 ) .","The failure of Sox9+ cells to differentiate and thus express Shh and Ihh , could then lower the total Hh signal in the callus to below a critical threshold need for cartilage differentiation .","However , our results suggest that Sox9+ cells are not the first to differentiate and while they likely respond to a Hh signal early in the periosteum , they are likely not strong propagators of a further Hh signal as cells nearby Sox9+ lineage cells do not display a strong upregulation of Hh pathway read-outs as the callus differentiates .","While a response to Hh signaling may require more sensitive assays , we found the expression of both Ptch1 and Gli2 to be very low in undifferentiated cells at 7 dpr in both control and Pre Smo KO calluses Figure 3C and Figure 3—figure supplement 1 ) suggesting that they were not receiving a potent secondary Hh signal .","In addition , the expression of Shh was not particularly strong in the tdTomato+ cells at this stage ( Figure 3C ) .","Thus , we instead favor a hypothesis , that in response to Hh signaling Sox9+ cells emit another to-be-identified relay signal .","This signal then orchestrates repair by promoting the differentiation of neighboring cells from a non-Sox9+ lineage into mature matrix-producing hybrid osteochondral skeletal cells that bridge the lesion ( Figure 6 ) .","It is possible that Hh signaling may have an additional role in later stages of repair ( osteocyte differentiation ) , but our study supports a critical role early in large-scale rib repair .","Future investigations into factors produced by Sox9-lineage cells that promote callus formation may lead to better strategies of boosting bone repair in other parts of the body that do not heal as effectively ."],["All procedures were approved by the University of Southern California Institutional Animal Care and Use Committee ( Protocol #: 11256 , 20639 ) .","We used the following mouse lines: Sox9-CreERT2 ( Sox9tm1 ( cre/ERT2 ) Haak [Soeda et al . , 2010] ) , R26R-tdTomato ( B6;129S6-Gt ( ROSA ) 26Sortm9 ( CAG-tdTomato ) Hze/J; JAX 007905 ) , Smofl/fl ( Smotm2Amc/J; JAX 004526 , Col1a1 ( 2 . 3-GFP ) ( B6 . Cg-Tg; Col1a1*2 . 3-GFP ) 1Rowe/J;JAX 013134 ) , Col10a1-mCherry ( Maye et al . , 2011 ) .","To generate Sox9-CreERT2;tdTomato;Smofl/fl mice , Sox9-CreERT2;tdTomato males were crossed to Smofl/fl females and Sox9CreERT2;tdTomato;Smofl/+ offspring males were back-crossed to Smofl/fl females to generate Sox9-CreERT2;tdTomato;Smofl/fl offspring .","Both male and female mice between 6–8 weeks old were used for experiments .","Control mice were uninduced siblings or tamoxifen-induced Sox9-CreERT2;tdTomato mice .","Rib resections were performed as previously described ( Tripuraneni et al . , 2015 ) with the modification that a bone segment of 3 mm was removed and that post-operative pain was managed with buprenorphine SR ( ZooPharm ) at a dose of 0 . 5 uL/gram .","Rib repair was assessed after set healing time points: 0 dpr – 10 wpr .","To induce Cre recombination , 100 uL of a 20 mg/ml stock of Tamoxifen ( Sigma-Aldrich: T5648-1G , dissolved in corn oil at 60°C for 2 hr ) was used per injection .","Tamoxifen injections were administered intraperitoneally using a 25-gauge needle .","Two injection schedules were used:","1 ) three consecutive injections starting 7 days before resection ( 'Pre' regimen ) ,","2 ) four consecutive injections starting 1 day prior to surgery ( 'Post' regiment ) .","Uninduced controls were injected with corn oil only .","Femur fracture assays were carried out as previously described ( He et al . , 2017 ) .","Samples were fixed with 4% PFA overnight at room temperature , decalcified with 20% ETDA at pH 7 . 5 for 10–14 days , and then processed for paraffin embedding .","A microtome ( Shandon Finesse Me+: 77500102 ) was used to cut paraffin sections seven microns thick .","The sections were mounted on Superfrost Plus slides ( VWR , 48311–703 ) .","After deparaffinizing slides in Citrisolv and rehydrating , H and E or Safranin O staining was carried out using standard protocols .","Skeletal staining was performed on EtOH-fixed samples ( Rigueur and Lyons , 2014 ) .","To visualize native tdTomato fluorescence , samples were fixed in 4% PFA on ice for 30 min and placed in 30% sucrose overnight at room temperature .","The samples were embedded in OCT and flash frozen in an EtOH dry ice bath .","10 µM thick sections were cut using a Leica CM3050 S cryostat .","Tape ( cryofilm type 3C ( 16UF ) C-FUF303 ) was used to preserve the histology of the bone ( Kawamoto , 2003 ) .","OCT was removed with a 1xPBS wash before mounting .","Detection of pHH3 and Tdtomato proteins was carried out on paraffin sections .","Slides were de-waxed and cells were permeabilized with 0 . 1% Triton-X followed by antigen retrieval in 10 mM sodium citrate , 0 . 05% Tween 20 , pH of 6 . 0 in a 95°C water bath for 30 min .","Slides were blocked in 20% serum for 1 hr and then incubated with the primary antibodies overnight at 4°C ( anti-pHH3 , Millipore: 06–570 , 1:200; anti-mCherry which also detects tdTomato , Novus Biological: NBP2-25158SS , 1:200; anti-Col1 , Abcam: ab34710 , 1:250; anti-Col2 , Southern Biotech: 1320–01 , 1:200 ) .","Secondary antibodies used were: Alexa Fluor 568 goat anti-rabbit ( ThermoFisher: A-11011 , 1:250 ) , Alexa Fluor 568 goat anti-chicken ( Abcam: ab175477 , 1:500 ) , and Alexa Fluor 488 donkey anti-goat ( Abcam:ab150129 , 1:500 ) .","To detect apoptosis , the In Situ Cell Death Detection Kit , Fluorescein ( Sigma-Aldrich: 11684795910 ) was used as directed .","Fluorescent and colorimetric RNA in situ hybridization ( RNA-ISH ) was performed on 7 µm paraffin sections as previously described ( Paul et al . , 2016 ) .","Complementary DIG or FL labeled RNA probes were generated following kit instructions ( Sigma-Aldrich: 11277073910 and 11685619910 ) and were detected with Anti-Digoxigenin-POD ( Sigma-Aldrich: 11207733910 ) and Anti-Fluorescein-POD ( Sigma-Aldrich: 11426346910 ) .","For double fluorescent RNA-ISH the TSA Cyanine three and Fluorescein system from Perkin Elmer was used as directed ( NEL753001KT ) .","For colorimetric RNA-ISH Anti-Digoxigenin-AP ( Sigma-Aldrich: 11093274910 ) was used to detect the probes .","Probes were generated to the following sequences: Slides with fluorescence were mounted with Vectashield with DAPI ( Vector Laboratories: H1200 ) and were imaged with a Nikon AZ100 Macroscope and photographed ( Nikon Digital sight DS-Fi1 ) .","Fluorescent images were edited for contrast and color levels in Adobe Photoshop CS5 .","For quantification , all images were taken at the same magnification for each data set .","Student’s t-test was used to compare groups .","A probability value of 0 . 05 or less was marked as significant .","Each data point was plotted on the scatter plot and the mean was defined on the graph , unless the data set was normalized .","Statistical tests were performed using GraphPad .","To determine the amount of cartilage , a mid-sagittal section through both mutant and control sample was stained with Safranin O and quantified in ImageJ .","In brief , the image was thresholded for the Safranin O color ( orange ) and the area of the color was measured .","To determine bone area , samples were stained with H and E and analyzed with the BioQuant image analysis program .","The bone area was compared to the entire resected area .","The values were normalized within each time point .","Quantification of the number of cells expressing pHH3 was carried out using the analyze particle function in ImageJ with the repair callus defined as the area of interest .","The red channel was used to count the number of cells positive for pHH3 while the blue channel was used to count the total number of cells ( nuclei stained with DAPI ) .","The ratio of pHH3 positive cells to total cells was used to calculate the percentage of cells in mitosis .","This method was also used to quantify the number of tdTomato+ cells within the callus .","To quantify apoptosis , Adobe Photoshop CS5 was used to analyze the green channel .","The number of green pixels compared to the total number of blue pixels ( DAPI ) in the region of interest was calculated .","Analysis of pHH3 and TUNEL positivity was done on de-identified images by several laboratory members ."]],"string":"[\n [\n \"Whereas amphibians regenerate large portions of their skeletons after injury or amputation , natural large-scale skeletal repair in mammals is more limited .\",\n \"A notable exception is the rib .\",\n \"Craniofacial surgeons often extract large segments of rib cartilage and bone for autologous repair of skeletal defects in other parts of the body , and in post-operative visits have noted extensive regeneration at the donor rib site ( Kawanabe and Nagata , 2006; Munro and Guyuron , 1981; Srour et al . , 2015 ) .\",\n \"To better understand the unique regenerative potential of the rib , we have recently developed analogous large-scale rib cartilage and bone regeneration models in the mouse ( Srour et al . , 2015; Tripuraneni et al . , 2015 ) .\",\n \"In most mammalian bones , healing involves the formation of a callus using a mixture of direct ossification and endochondral ossification via a cartilage callus intermediate ( Gerstenfeld et al . , 2003; Hall , 2014; Marsell and Einhorn , 2011 ) .\",\n \"The identity and regulation of the adult skeletal progenitors that build the callus remain incompletely understood .\",\n \"While markers for a number of different skeletal stem cells have been reported ( Balani et al . , 2017; Bianco and Robey , 2015; He et al . , 2017; Matthews et al . , 2014; Park et al . , 2012; Ransom et al . , 2016; Shi et al . , 2017 ) , their relative roles during bone repair are less clear , particularly in cases of large-scale bone regeneration such as in the rib .\",\n \"During skeletal repair , studies have shown that cells from the periosteum , a heterogeneous connective tissue sheath covering the bone , are major contributors to the callus ( Colnot , 2009; Duchamp de Lageneste et al . , 2018; Murao et al . , 2013 ) .\",\n \"Accordingly , we have found that the periosteum is essential for regeneration of the rib bone ( Tripuraneni et al . , 2015 ) .\",\n \"During normal bone homeostasis , periosteal stem cells generate bone-producing osteoblasts but not cartilage-producing chondrocytes ( Roberts et al . , 2015 ) .\",\n \"How injury stimulates periosteal stem cells to generate chondrocytes is unclear .\",\n \"Although some fractures can heal in the absence of a cartilage callus , for example when the fracture is rigidly stabilized ( Thompson et al . , 2002 ) , the formation of a large cartilage callus appears to be required in large-scale bone regeneration .\",\n \"Unfortunately little is known about the specific periosteal progenitor population that drives the formation of the cartilage callus nor the signaling pathways required .\",\n \"Recent studies have used Cre-based lineage tracing experiments to show that cells marked by expression of Gremlin1 ( Worthley et al . , 2015 ) , Axin2 ( Ransom et al . , 2016 ) , Gli1 ( Shi et al . , 2017 ) , Act2a ( Matthews et al . , 2016 ) , Periostin ( Duchamp de Lageneste et al . , 2018 ) and Sox9 ( Balani et al . , 2017; He et al . , 2017 ) can be found in the periosteum and contribute to the fracture callus during repair .\",\n \"Other than participation , the specific role of any of these progenitor population remains unclear .\",\n \"In this study , we therefore focus on the role of one subpopulation within the periosteum and its specific role in driving callus formation and bone regeneration .\",\n \"As Sox9 has a well-known function in promoting chondrogenesis during embryonic development ( Akiyama et al . , 2002; Lefebvre et al . , 1997 ) , we postulated that Sox9-expressing cells in the adult periosteum may possess a potent ability to form or promote cartilage in response to bone injury .\",\n \"In addition , a requirement for Sox9-expressing cells in repair or homeostasis has not yet been established .\",\n \"The signaling pathways required for generating a cartilage callus from the periosteum in response to injury are also not well-characterized .\",\n \"The formation of the cartilage callus in fractures has been thought to involve a recapitulation of endochondral bone development ( Bahney et al . , 2014; Maes et al . , 2010; Marsell and Einhorn , 2011; Yang et al . , 2014; Zhou et al . , 2014 ) .\",\n \"One of the most well-known signaling pathways during endochondral bone development is the Hh pathway , activation of which promotes chondrogenic proliferation and osteocyte maturation ( Long et al . , 2001; Shi et al . , 2017; St-Jacques et al . , 1999 ) .\",\n \"Studies have investigated Hh signaling during fracture repair , but due to conflicting results it remains unclear if Hh signaling has the same role in bone repair as it does during bone development .\",\n \"Genetic ablation of the obligate Hh co-receptor Smo in mice , using two different ubiquitously inducible Cre lines , resulted in reduced bone formation during fracture repair , yet was not reported to disrupt initial cartilage callus formation ( Baht et al . , 2014; Wang et al . , 2010 ) .\",\n \"Forced activation of Hh signaling throughout the mouse during fracture repair , using an inducible constitutively active Smo allele , resulted in increased bone formation ( Baht et al . , 2014 ) , similar to what was seen upon engraftment of cells overexpressing Hh or treatment with an Hh agonist ( Edwards et al . , 2005; Huang et al . , 2014; Zou et al . , 2014 ) .\",\n \"However , on which cell types Hh acts upon , and whether it regulates the decision to build the cartilage callus and/or other aspects of bone repair in mammals , has remained unknown .\",\n \"In this study we examine the role of the Sox9-expressing periosteal subpopulation ( referred to as Sox9+ cells hereafter ) during large-scale rib repair .\",\n \"This subpopulation appears to be a key player in orchestrating the formation of a large repair callus in the rib that consists of an unusual hybrid osteochondral cell type with properties of both chondrocytes and osteoblasts .\",\n \"Loss of Smo in Sox9+ periosteal cells prior to injury results in a near-complete failure of cartilage callus formation and bone regeneration .\",\n \"This Sox9+ subpopulation must be able to respond to Hh signaling in order to initiate this process , indicating that Hh signaling’s role in bone repair is distinct from its role in bone development .\",\n \"Additionally , since Sox9+ periosteal cells contribute to only a minority of callus cells , we suggest that Sox9+ periosteal cells act as ‘messenger’ cells and orchestrate repair by inducing the differentiation of neighboring callus cells through non-autonomous signals .\",\n \"Overall our results indicate that bone regeneration does not fully recapitulate bone development , and that the periosteum consists of subpopulations that may have different roles/responses during repair .\"\n ],\n [\n \"Like appendicular long bones , the bony portion of the rib develops via an endochondral process including growth plates at either end and a central hollow bone marrow cavity .\",\n \"Both human and murine rib bones display remarkable regenerative potential ( Srour et al . , 2015; Tripuraneni et al . , 2015 ) , however the cellular basis for such large-scale repair remains unknown .\",\n \"To better understand the cellular sequence of events during regeneration , we analyzed 3 mm rib bone defects at sequential time points up to 10 weeks post-resection ( wpr ) ( Figure 1A ) .\",\n \"Histology at 5 days post-resection ( dpr ) revealed cells with a mesenchymal-like morphology filling the entire resected region ( Figure 1B ) .\",\n \"We then observed formation of a substantial alcian-blue positive callus spanning most of the defect by 1 wpr ( Figure 1A ) , with many of these cells displaying a cartilage-like morphology at 10 dpr ( Figure 1C ) .\",\n \"Histology revealed increasing bone formation by 10 and 14 dpr ( Figure 1C , D ) , with extensive alizarin-positive mineralization across the defect at 4 wpr and full remodeling to the pre-injury organization by 10 wpr ( Figure 1A ) .\",\n \"Next , we used double fluorescent RNA in situ hybridization ( RNA-ISH ) to characterize the molecular identity of cells during the regeneration process .\",\n \"At 5 dpr , mesenchymal cells within the lesion co-expressed Sox9 , a master regulator of the chondrocyte lineage ( Akiyama et al . , 2002; Lefebvre et al . , 1997 ) , and Runx2 , a master regulator of the osteoblast lineage ( Lian and Stein , 2003 ) ( Figure 1B ) .\",\n \"While co-expression of chondrocyte and osteoblast markers has been observed during early bone development , normally as differentiation proceeds , a cell will express only chondrocyte or only osteoblast markers .\",\n \"Surprisingly , we observed co-expression of genes associated with chondrocyte and osteoblast differentiation within single cells throughout the repair process .\",\n \"For example , we see co-expression of Sox9 with the major osteoblast collagen gene Col1a1 ( Figure 1—figure supplement 1A ) .\",\n \"As the callus matures bone and cartilage markers continue to be co-expressed at high levels .\",\n \"The major chondrocyte collagen gene Col2a1 was observed to be co-expressed with the late osteoblast marker Bglap ( also known as Osteocalcin ) ( Figure 1C and Figure 1—figure supplement 1C ) , and with Col1a1 at both 10 and 14 dpr ( Figure 1D and Figure 1—figure supplement 1B ) .\",\n \"We observed co-expression of chondrocyte and osteoblast markers in cells of both cartilage and osteoblast morphology , including cells on the surface of newly formed trabecular bone ( Figure 1C , D , Figure 1—figure supplement 1C ) .\",\n \"Similar co-expression results were obtained using mice double transgenic for a reporter of hypertrophic chondrocytes ( Col10a1-mCherry ) and osteoblasts ( Col1 ( 2 . 3 ) -GFP ) as well as using double immunofluorescence for COL1 and COL2 protein ( Figure 1E , Figure 1—figure supplement 1D ) .\",\n \"In contrast , we did not observe co-expression of Col2a1 and Col1a1 in the rib growth plate under the same assay conditions ( Figure 1F ) .\",\n \"Single colorimetric RNA-ISH assays of near-adjacent sections at 7 dpr confirmed co-expression of Sox9 , Runx2 , Col2a1 , Col1a1 , Bglap , and the hypertrophic cartilage collagen gene Col10a1 in cells of cartilage morphology within the callus ( Figure 1—figure supplement 2 ) .\",\n \"Together , these findings indicate that , in marked contrast to the growth plate , cells within the rib repair callus co-express cartilage and bone markers while displaying either a chondrocyte or osteoblast morphology .\",\n \"We therefore refer to these cells as ‘hybrid’ osteochondral skeletal cells .\",\n \"We next examined the source of the skeletal cells that mediate repair .\",\n \"These cells are likely derived from the periosteum , since removal of the periosteum along with the bone , results in a failure of cartilage callus formation ( Figure 2—figure supplement 1 ) and subsequent bone repair ( Tripuraneni et al . , 2015 ) .\",\n \"Sox9 is a master regulatory gene of chondrogenesis , and a previous study indicated that Sox9+ cells in the femur periosteum can contribute to callus formation after fracture ( He et al . , 2017 ) .\",\n \"We therefore examined whether Sox9-expressing periosteal cells contribute to the repair callus during rib regeneration .\",\n \"To do so , we administered three consecutive daily doses of tamoxifen to Sox9-CreERT2; ROSA26-loxP-stop-loxP-tdTomato mice , followed by a 4-day chase to allow clearance of residual tamoxifen ( Figure 2A , “Pre” regimen ) .\",\n \"In the absence of injury , we observed that Sox9-expressing cells were predominantly found in the periosteum and but only constituted 6 ± 0 . 3% of the population , with almost no labeled cells seen in the marrow compartment ( Figure 2B ) .\",\n \"After rib resection , we found that 22 ± 1 . 3% of callus cells were tdTomato+ by 10 dpr with many of these cells having a typical chondrocyte morphology ( Figure 2C ) .\",\n \"At 14 dpr ( Figure 2C ) , we observed tdTomato expression in both chondrocyte-like cells , as well as osteoblast-like cells lining new trabecular bone ( as defined by near-adjacent H&E sections ) , with contribution to osteoblast-like cells also evident by 21 dpr ( Figure 2—figure supplement 2A ) .\",\n \"We observed fewer tdTomato+ cells at 14 dpr suggesting that as the callus matures , Sox9+ lineage cells are remodeled out and replaced by non-Sox9+ lineage cells .\",\n \"Pre-existing -expressing cells , thus contribute to only a minority of the cells that form the initial repair callus , including only a subset of the callus cells with hybrid osteochondral characteristics .\",\n \"Most of the cells in the rib callus are therefore derived from a lineage that did not express Sox9 prior to injury .\",\n \"In addition , similar to reports by He et al . ( He et al . , 2017 ) we found that also after femur fracture , our “Pre” tamoxifen regimen revealed contribution of pre-existing Sox9+ cells to the callus ( Figure 2—figure supplement 3A ) .\",\n \"We further found that some of the cells within the femur fracture callus also co-expressed Col1a1 and Col2a1 strongly , suggesting a similar hybrid identity to that observed in the rib callus ( Figure 2—figure supplement 3B ) .\",\n \"Since only a minority of the callus cells are derived from the Sox9-expressing periosteal subpopulation , as opposed to the majority of the callus cells that expresses Sox9 mRNA at 5 dpr ( Figure 1B ) , we instead applied tamoxifen at the time of injury plus 2 days following ( “Post” regimen , Figure 2D ) .\",\n \"This regimen will capture both the subpopulation that expresses Sox9 prior to injury as well as any cells that turn on Sox9 in response to injury .\",\n \"Consistent with detection of endogenous Sox9 mRNA expression in early callus cells ( e . g . Figure 1B ) , we found that this later tamoxifen regimen labeled the majority of callus cells by 10 dpr ( 85 . 5 ± 2 . 8% ) and most of the osteoblast-like cells lining newly forming trabecular bone at 14 ( Figure 2E ) and 28 dpr .\",\n \"Further , typical of osteocytes , some of the tdTomato+ cells at 28 dpr were found embedded in bone ( Figure 2—figure supplement 2B ) .\",\n \"Thus , depending on when tamoxifen is administered ( Pre vs . Post ) , either a minority or majority of the callus cells are labelled .\",\n \"Since we hypothesized that the Hh pathway may be important for large-scale repair , we first determined the expression of Hh ligands and Ptch1 after rib resection .\",\n \"As expected , based on the expression of Ihh in the growth plate , callus cells with cartilage morphology expressed Ihh and Ptch1 .\",\n \"Interestingly we also detected the related ligand , Shh , in these cells ( Figure 3A and C ) .\",\n \"At earlier stages Ihh expression was undetectable until 7 dpr and then only at very low levels in developing chondrocytes .\",\n \"However , Shh expression was readily detectable at 5 dpr and 7dpr similar to reports by others ( Matsumoto et al . , 2016 ) although at lower levels than found in cells with cartilage morphology ( Figure 3A and C ) .\",\n \"Ptch1 , a read-out of the Hh pathway , was also detected in tdTomato+ periosteal cells a 3 dpr , supporting the idea that these cells are responsive to a Hh signal prior to entering the callus ( Figure 3—figure supplement 1A ) .\",\n \"We then tested the requirement for Hh signaling in large-scale bone regeneration by deleting Smo , the required Hh co-receptor , using the Sox9-CreERT2 transgene and employing either the Pre or Post tamoxifen treatment regimens .\",\n \"Using Smo RNA-ISH , we observed that the Post tamoxifen treatment resulted in deletion of Smo broadly ( Figure 3B ) as predicted given that Cre is active in most cells ( Figure 2D ) .\",\n \"Pre tamoxifen treatment resulted in deletion of Smo in the Sox9-positive lineage subpopulation which were marked by including the tdTomato reporter in the cross ( Figure 3B ) .\",\n \"This was expected , since deletion of Smo occurred prior to surgery and thus only a minority of the cells in the repair callus are expected to be null for Smo ( Figure 2C ) .\",\n \"We then examined the callus at 7 dpr while there is a mix of both immature and differentiating cells .\",\n \"In control Pre tamoxifen treatment animals , tdTomato+ cells could be found scattered across the developing callus and were not preferentially found in clusters of differentiating chondrocytes suggesting that Sox9+ cells do not differentiate ahead of other cells in the callus ( Figure 3—figure supplement 1B ) .\",\n \"In addition , in regions of the control callus where cells appeared immature and in Pre tamoxifen treatment calluses , there was no strong correlation between the tdTomato trace and the expression of Shh , or read-outs of Hh signaling such as Ptch1 or Gli2 ( Pak and Segal , 2016 ) suggesting that in contrast to our earlier observations in the periosteum , that at these later stages , tdTomato cells are not strongly responding to a Hh signal ( Figure 3C and Figure 3—figure supplement 1C ) .\",\n \"Furthermore , in cells neighboring Tdtomato+ cells , we did not see any enrichment of Ptch1 or Gli2 expression suggesting that they were not receiving a potent Hh signal that could be released by Tdtomato+ cells .\",\n \"We then investigated the consequence of Smo deletion on cartilage callus formation .\",\n \"Surprisingly , Smo deletion using either the Pre or Post tamoxifen treatment resulted in a similarly near-complete loss of the cartilage callus at both 7 and 10 dpr ( Figure 4A , Figure 4—source data 1 ) , despite the Pre treatment only deleting Smo in a small subset of callus progenitors ( Figures 2C and 3B ) .\",\n \"After Smo deletion , although cells did not form a mature callus , they still co-expressed Sox9 and Runx2 at 5 dpr ( Figure 4—figure supplement 1A ) .\",\n \"At 10 dpr , in contrast to co-expression of Col2a1 and Col1a1 throughout the control callus , the few Col1a1-expressing cells that formed after Smo deletion lacked high levels of Col2a1 and vice versa , demonstrating a lack of hybrid osteochondral cells ( Figure 3B ) .\",\n \"The lack of callus formation and hybrid cells following Smo deletion was not reflected by altered numbers of pHH3+ proliferative or TUNEL+ apoptotic cells at either 7 or 10 dpr , and lineage tracing with the tdTomato reporter revealed similar numbers of cells within the resection site at 10 dpr ( Figure 4C , Figure 4—source data 2 , Figure 4D , Figure 4—source data 3 , Figure 4—figure supplement 1B , Figure 4-figure supplement-source data 1 ) .\",\n \"Thus , Hh signaling is likely not required for the early proliferative expansion in response to injury or for cell survival .\",\n \"Instead , we conclude that Hh signaling is required in Sox9+ cells for promoting the differentiation of non-Sox9 lineage cells into hybrid osteochondral cells of the callus .\",\n \"When the Sox9+ Pre population is null for Smo , non-Sox9 lineage cells are still unable to differentiate ( despite expressing Ptch1 ) resulting in the near complete failure of callus formation .\",\n \"Thus , this minority Sox9+ periosteal lineage population plays a critical initiating role in callus differentiation .\",\n \"We next assessed the consequence of defective callus formation on subsequent regeneration of rib bone in Smo-deleted animals .\",\n \"In contrast to control animals showing robust Col1a1 and Col2a1 co-expression in cells lining new trabecular bone at 14 dpr , deletion of Smo using either the Pre or Post regimens resulted in a near complete absence of co-expressing cells .\",\n \"Instead , only small numbers of Col2a1-only cells were observed , primarily near the cut ends of the bone , while cells with osteoblast morphology expressed predominantly only Col1a1 ( Figure 5A ) .\",\n \"Analysis of H and E-stained histological sections confirmed a marked decrease in bone formation at 14 dpr , despite substantial mesenchyme still observed in the resection site , with the magnitude of the bone defect similar in both regimens ( Figure 5B ) .\",\n \"We found that ‘Late’ removal of Smo ( injection of tamoxifen at 3–5 dpr targeting the whole callus ) only caused a slight delay in repair with a fully bridged callus evident at 14 dpr ( Figure 5—figure supplement 1 ) suggesting that the decrease in bone formation seen in both the Pre and Post regimens is largely related to reduced cartilage callus formation .\",\n \"Whole-mount staining with alizarin red and alcian blue confirmed a failure of bone union at 4 and 6 wpr in both Pre and Post conditions ( Figure 5C ) .\",\n \"These findings demonstrate that the Sox9+ subpopulation requires Hh signaling not only to build the repair callus but that a substantial cartilage callus may be needed to efficiently regenerate bone .\"\n ],\n [\n \"We find that building an extensive callus of a unique type of hybrid osteochondral skeletal cell is essential for successfully bridging large gaps in adult mammalian rib bone .\",\n \"During this process , Hh signaling plays a critical role , distinct from that in the developing growth plate , in promoting the ultimate differentiation of these hybrid osteochondral skeletal cells .\",\n \"Further , we provide genetic evidence that Hh signaling acts upon a rare periosteal subpopulation of Sox9-expressing cells , that behave as messenger cells .\",\n \"These Sox9+ periosteal cells then stimulate , through a yet to be determined signal , their neighboring non-Sox9-lineage cells , which constitute a majority of the callus , to differentiate and build new callus and bone ( summarized in Figure 6 ) .\",\n \"We observe high-level co-expression of genes typically associated with cartilage ( Sox9 , Col2a1 , Col10a1 ) or bone ( Col1a1 , Bglap ) in callus cells during murine rib bone regeneration .\",\n \"This is in marked contrast to the largely exclusive expression of cartilage versus bone genes in the developing growth plates , although hypertrophic chondrocytes do express low levels of many bone-associated genes ( Gerstenfeld and Shapiro , 1996 ) .\",\n \"The trajectory of hybrid cells is also fundamentally different from the subpopulation that is proposed to ‘transdifferentiate’ from chondrocytes to osteoblasts in the growth plate ( Bahney et al . , 2014; Shimomura et al . , 1975; von der Mark and von der Mark , 1977; Yang et al . , 2014; Zhou et al . , 2014 ) .\",\n \"In the growth plate , these transdifferentiating cells express chondrocyte-associated genes first and then , through a process that remains mysterious , turn off chondrocyte-associated genes and turn on osteoblast-associated genes .\",\n \"Here , we confirm a similar process of ‘first cartilage and then bone’ in the rib growth plate .\",\n \"In contrast , in the regenerating rib , we observe that callus cells co-express cartilage- and bone-associated genes at the earliest stages .\",\n \"We propose that they then maintain this hybrid osteochondral identity as they shift from making a cartilaginous and then a bone-like matrix .\",\n \"We also observe a similarly hybrid osteochondral cell in the femur fracture callus , although further investigations will be required to determine how similar these cells are between the rib and femur callus .\",\n \"Moreover , similar hybrid cells have been reported during zebrafish jawbone repair ( Paul et al . , 2016 ) suggesting that skeletal cells with dual chondrocyte/osteoblast properties may be critical for bone regeneration across vertebrate species .\",\n \"Co-expression of bone and cartilage programs in the same cell is certainly not unique to the regenerating callus .\",\n \"For example , Col1a1 and Col2a1 are also highly expressed in fibrocartilage cells , however this tissue is morphologically quite different in terms of its high fiber to cell ratio when compared to the regenerating rib callus ( Benjamin and Ralphs , 2004 ) .\",\n \"Of note , a rare developmental skeletal type has been described , historically referred to as ‘chondroid bone’ or ‘secondary cartilage’ , that does share many features with the hybrid osteochondral cells we observe during regeneration ( Goret-Nicaise , 1984; Shibata and Yokohama-Tamaki , 2008 ) .\",\n \"Therefore , it may be that these cells are not unique to regeneration , but rather are selectively utilized due to their ability to rapidly proliferate in a relatively avascular environment ( a property of cartilage [Dennis et al . , 2015] ) while directly producing mineralized matrix ( a property of bone ) .\",\n \"Whereas some bone does form in our Smo-deleted mice , this is not sufficient to bridge the lesion and healing fails , resulting in a persistent non-union .\",\n \"Cells within this bone do not display hybrid chondrocyte/osteoblast character , consistent with residual bone forming by direct ossification rather than ossification through a callus intermediate .\",\n \"These findings suggest that during large-scale bone regeneration , Hh signaling in Sox9-expressing periosteal cells is selectively required for the formation of a hybrid osteochondral callus .\",\n \"Hh signaling may play an important but more subtle role in osteocyte maturation as fractures still heal in the absence of Smo but with decreased or delayed bone formation ( Baht et al . , 2014; Wang et al . , 2010 ) .\",\n \"Similarly , we see production of bone ( although delayed ) in our rib resection model with a Late KO of Smo ( Figure 5—figure supplement 1 ) .\",\n \"In contrast to our rib model , however , loss of Smo in these femur/tibia fracture assays still resulted in the formation of a cartilage callus .\",\n \"We do not know why these results contrast from ours where cartilage callus formation is dramatically compromised .\",\n \"One possibility is that the efficacy of Smo removal in these fracture experiments was not efficient during cartilage callus stages , alternatively , there could be different requirements for Hh signaling depending on the type of injury ( resection vs . fracture ) .\",\n \"Interestingly , the formation of a cartilage callus may not be as critical during fracture repair , as fractures can repair solely through direct ossification ( Colnot et al . , 2003 ) .\",\n \"While in contrast , during large-scale repair , the process of direct ossification is not sufficient to build large pieces of bone and instead , building a hybrid osteochondral callus may be particularly important for bridging large bone gaps .\",\n \"Hh signaling is known to promote chondrocyte proliferation and osteoblast differentiation in the developing growth plate ( Long et al . , 2004; Long et al . , 2001 ) .\",\n \"Surprisingly , we found that loss of Hh signaling did not affect the early proliferation of callus cells or the differentiation of osteoblasts in the residual directly ossifying bone .\",\n \"Instead , we provide evidence that Hh signaling has a distinct and essential role in promoting the differentiation of Sox9/Runx2 expressing progenitors into the hybrid osteochondral skeletal cells that form the repair callus .\",\n \"These results indicate that the regeneration of the rib bone , including its dependence on Hh signaling , does not simply the recapitulate developmental processes seen at the growth plate .\",\n \"While the heterogeneity of the cells in the periosteum and their lineage relationships remain incompletely understood , we propose that Sox9+ periosteal cells or a subpopulation within them , play an essential instructive role for callus formation during large-scale bone regeneration that has not been previously described in the context of repair .\",\n \"The Sox9+ periosteal subpopulation may be distinct from the previously described subpopulations that are Gli1+ , αSMA+ , Gremlin1+ , or Axin2+ , as RNA-seq analysis of the periosteum from an uninjured femur indicated that Gli1 , αSMA , Axin2 , and Gremlin1 are not highly expressed in the Sox9+ cells ( He et al . , 2017 ) .\",\n \"Future studies tracing the lineage of these other populations and determining their dependence on Hh signaling will be needed to resolve whether Sox9+ cells represent a subset of one of these other more abundant populations , or alternatively a distinct progenitor subpopulation .\",\n \"In addition , efforts to delineate potential differences that may exist in periosteal populations from different bones , may explain why some bones regenerate well , while others do not .\",\n \"One of the most striking findings from our study is that Sox9+ cells are essential for efficient callus formation and rib bone regeneration , despite contributing to only a minority of cells within the callus and regenerated bone .\",\n \"Based on this observation , we propose that the Sox9+ cells act as ‘messenger’ cells by releasing a yet to be determined signal that promotes the differentiation of neighboring cells Sox9-negative lineage cells .\",\n \"One possibility is that Sox9+ progenitors differentiate early in response to the initial Hh signal ( potentially Shh , since strong expression is evident early ) and then propagate another wave of Hh signaling to neighboring Sox9-negative cells , similar to the role of Hh in the morphogenetic furrow during Drosophila eye development ( Domínguez and Hafen , 1997; Kumar , 2011; Ma et al . , 1993 ) .\",\n \"The failure of Sox9+ cells to differentiate and thus express Shh and Ihh , could then lower the total Hh signal in the callus to below a critical threshold need for cartilage differentiation .\",\n \"However , our results suggest that Sox9+ cells are not the first to differentiate and while they likely respond to a Hh signal early in the periosteum , they are likely not strong propagators of a further Hh signal as cells nearby Sox9+ lineage cells do not display a strong upregulation of Hh pathway read-outs as the callus differentiates .\",\n \"While a response to Hh signaling may require more sensitive assays , we found the expression of both Ptch1 and Gli2 to be very low in undifferentiated cells at 7 dpr in both control and Pre Smo KO calluses Figure 3C and Figure 3—figure supplement 1 ) suggesting that they were not receiving a potent secondary Hh signal .\",\n \"In addition , the expression of Shh was not particularly strong in the tdTomato+ cells at this stage ( Figure 3C ) .\",\n \"Thus , we instead favor a hypothesis , that in response to Hh signaling Sox9+ cells emit another to-be-identified relay signal .\",\n \"This signal then orchestrates repair by promoting the differentiation of neighboring cells from a non-Sox9+ lineage into mature matrix-producing hybrid osteochondral skeletal cells that bridge the lesion ( Figure 6 ) .\",\n \"It is possible that Hh signaling may have an additional role in later stages of repair ( osteocyte differentiation ) , but our study supports a critical role early in large-scale rib repair .\",\n \"Future investigations into factors produced by Sox9-lineage cells that promote callus formation may lead to better strategies of boosting bone repair in other parts of the body that do not heal as effectively .\"\n ],\n [\n \"All procedures were approved by the University of Southern California Institutional Animal Care and Use Committee ( Protocol #: 11256 , 20639 ) .\",\n \"We used the following mouse lines: Sox9-CreERT2 ( Sox9tm1 ( cre/ERT2 ) Haak [Soeda et al . , 2010] ) , R26R-tdTomato ( B6;129S6-Gt ( ROSA ) 26Sortm9 ( CAG-tdTomato ) Hze/J; JAX 007905 ) , Smofl/fl ( Smotm2Amc/J; JAX 004526 , Col1a1 ( 2 . 3-GFP ) ( B6 . Cg-Tg; Col1a1*2 . 3-GFP ) 1Rowe/J;JAX 013134 ) , Col10a1-mCherry ( Maye et al . , 2011 ) .\",\n \"To generate Sox9-CreERT2;tdTomato;Smofl/fl mice , Sox9-CreERT2;tdTomato males were crossed to Smofl/fl females and Sox9CreERT2;tdTomato;Smofl/+ offspring males were back-crossed to Smofl/fl females to generate Sox9-CreERT2;tdTomato;Smofl/fl offspring .\",\n \"Both male and female mice between 6–8 weeks old were used for experiments .\",\n \"Control mice were uninduced siblings or tamoxifen-induced Sox9-CreERT2;tdTomato mice .\",\n \"Rib resections were performed as previously described ( Tripuraneni et al . , 2015 ) with the modification that a bone segment of 3 mm was removed and that post-operative pain was managed with buprenorphine SR ( ZooPharm ) at a dose of 0 . 5 uL/gram .\",\n \"Rib repair was assessed after set healing time points: 0 dpr – 10 wpr .\",\n \"To induce Cre recombination , 100 uL of a 20 mg/ml stock of Tamoxifen ( Sigma-Aldrich: T5648-1G , dissolved in corn oil at 60°C for 2 hr ) was used per injection .\",\n \"Tamoxifen injections were administered intraperitoneally using a 25-gauge needle .\",\n \"Two injection schedules were used:\",\n \"1 ) three consecutive injections starting 7 days before resection ( 'Pre' regimen ) ,\",\n \"2 ) four consecutive injections starting 1 day prior to surgery ( 'Post' regiment ) .\",\n \"Uninduced controls were injected with corn oil only .\",\n \"Femur fracture assays were carried out as previously described ( He et al . , 2017 ) .\",\n \"Samples were fixed with 4% PFA overnight at room temperature , decalcified with 20% ETDA at pH 7 . 5 for 10–14 days , and then processed for paraffin embedding .\",\n \"A microtome ( Shandon Finesse Me+: 77500102 ) was used to cut paraffin sections seven microns thick .\",\n \"The sections were mounted on Superfrost Plus slides ( VWR , 48311–703 ) .\",\n \"After deparaffinizing slides in Citrisolv and rehydrating , H and E or Safranin O staining was carried out using standard protocols .\",\n \"Skeletal staining was performed on EtOH-fixed samples ( Rigueur and Lyons , 2014 ) .\",\n \"To visualize native tdTomato fluorescence , samples were fixed in 4% PFA on ice for 30 min and placed in 30% sucrose overnight at room temperature .\",\n \"The samples were embedded in OCT and flash frozen in an EtOH dry ice bath .\",\n \"10 µM thick sections were cut using a Leica CM3050 S cryostat .\",\n \"Tape ( cryofilm type 3C ( 16UF ) C-FUF303 ) was used to preserve the histology of the bone ( Kawamoto , 2003 ) .\",\n \"OCT was removed with a 1xPBS wash before mounting .\",\n \"Detection of pHH3 and Tdtomato proteins was carried out on paraffin sections .\",\n \"Slides were de-waxed and cells were permeabilized with 0 . 1% Triton-X followed by antigen retrieval in 10 mM sodium citrate , 0 . 05% Tween 20 , pH of 6 . 0 in a 95°C water bath for 30 min .\",\n \"Slides were blocked in 20% serum for 1 hr and then incubated with the primary antibodies overnight at 4°C ( anti-pHH3 , Millipore: 06–570 , 1:200; anti-mCherry which also detects tdTomato , Novus Biological: NBP2-25158SS , 1:200; anti-Col1 , Abcam: ab34710 , 1:250; anti-Col2 , Southern Biotech: 1320–01 , 1:200 ) .\",\n \"Secondary antibodies used were: Alexa Fluor 568 goat anti-rabbit ( ThermoFisher: A-11011 , 1:250 ) , Alexa Fluor 568 goat anti-chicken ( Abcam: ab175477 , 1:500 ) , and Alexa Fluor 488 donkey anti-goat ( Abcam:ab150129 , 1:500 ) .\",\n \"To detect apoptosis , the In Situ Cell Death Detection Kit , Fluorescein ( Sigma-Aldrich: 11684795910 ) was used as directed .\",\n \"Fluorescent and colorimetric RNA in situ hybridization ( RNA-ISH ) was performed on 7 µm paraffin sections as previously described ( Paul et al . , 2016 ) .\",\n \"Complementary DIG or FL labeled RNA probes were generated following kit instructions ( Sigma-Aldrich: 11277073910 and 11685619910 ) and were detected with Anti-Digoxigenin-POD ( Sigma-Aldrich: 11207733910 ) and Anti-Fluorescein-POD ( Sigma-Aldrich: 11426346910 ) .\",\n \"For double fluorescent RNA-ISH the TSA Cyanine three and Fluorescein system from Perkin Elmer was used as directed ( NEL753001KT ) .\",\n \"For colorimetric RNA-ISH Anti-Digoxigenin-AP ( Sigma-Aldrich: 11093274910 ) was used to detect the probes .\",\n \"Probes were generated to the following sequences: Slides with fluorescence were mounted with Vectashield with DAPI ( Vector Laboratories: H1200 ) and were imaged with a Nikon AZ100 Macroscope and photographed ( Nikon Digital sight DS-Fi1 ) .\",\n \"Fluorescent images were edited for contrast and color levels in Adobe Photoshop CS5 .\",\n \"For quantification , all images were taken at the same magnification for each data set .\",\n \"Student’s t-test was used to compare groups .\",\n \"A probability value of 0 . 05 or less was marked as significant .\",\n \"Each data point was plotted on the scatter plot and the mean was defined on the graph , unless the data set was normalized .\",\n \"Statistical tests were performed using GraphPad .\",\n \"To determine the amount of cartilage , a mid-sagittal section through both mutant and control sample was stained with Safranin O and quantified in ImageJ .\",\n \"In brief , the image was thresholded for the Safranin O color ( orange ) and the area of the color was measured .\",\n \"To determine bone area , samples were stained with H and E and analyzed with the BioQuant image analysis program .\",\n \"The bone area was compared to the entire resected area .\",\n \"The values were normalized within each time point .\",\n \"Quantification of the number of cells expressing pHH3 was carried out using the analyze particle function in ImageJ with the repair callus defined as the area of interest .\",\n \"The red channel was used to count the number of cells positive for pHH3 while the blue channel was used to count the total number of cells ( nuclei stained with DAPI ) .\",\n \"The ratio of pHH3 positive cells to total cells was used to calculate the percentage of cells in mitosis .\",\n \"This method was also used to quantify the number of tdTomato+ cells within the callus .\",\n \"To quantify apoptosis , Adobe Photoshop CS5 was used to analyze the green channel .\",\n \"The number of green pixels compared to the total number of blue pixels ( DAPI ) in the region of interest was calculated .\",\n \"Analysis of pHH3 and TUNEL positivity was done on de-identified images by several laboratory members .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Most bones in mammals display a limited capacity for natural large-scale repair .","The ribs are a notable exception , yet the source of their remarkable regenerative ability remains unknown .","Here , we identify a Sox9-expressing periosteal subpopulation that orchestrates large-scale regeneration of murine rib bones .","Deletion of the obligate Hedgehog co-receptor , Smoothened , in Sox9-expressing cells prior to injury results in a near-complete loss of callus formation and rib bone regeneration .","In contrast to its role in development , Hedgehog signaling is dispensable for the proliferative expansion of callus cells in response to injury .","Instead , Sox9-positive lineage cells require Hh signaling to stimulate neighboring cells to differentiate via an unknown signal into a skeletal cell type with dual chondrocyte/osteoblast properties .","This type of callus cell may be critical for bridging large bone injuries .","Thus despite contributing to only a subset of callus cells , Sox9-positive progenitors play a major role in orchestrating large-scale bone regeneration .","Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review .","The Reviewing Editor's assessment is that all the issues have been addressed ( see decision letter ) ."],"string":"[\n \"Most bones in mammals display a limited capacity for natural large-scale repair .\",\n \"The ribs are a notable exception , yet the source of their remarkable regenerative ability remains unknown .\",\n \"Here , we identify a Sox9-expressing periosteal subpopulation that orchestrates large-scale regeneration of murine rib bones .\",\n \"Deletion of the obligate Hedgehog co-receptor , Smoothened , in Sox9-expressing cells prior to injury results in a near-complete loss of callus formation and rib bone regeneration .\",\n \"In contrast to its role in development , Hedgehog signaling is dispensable for the proliferative expansion of callus cells in response to injury .\",\n \"Instead , Sox9-positive lineage cells require Hh signaling to stimulate neighboring cells to differentiate via an unknown signal into a skeletal cell type with dual chondrocyte/osteoblast properties .\",\n \"This type of callus cell may be critical for bridging large bone injuries .\",\n \"Thus despite contributing to only a subset of callus cells , Sox9-positive progenitors play a major role in orchestrating large-scale bone regeneration .\",\n \"Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review .\",\n \"The Reviewing Editor's assessment is that all the issues have been addressed ( see decision letter ) .\"\n]"},"summary":{"kind":"list like","value":["Fractures to major bones often heal slowly or incompletely , especially in older people , and large bone injuries do not repair naturally .","By comparison , rib bones show an unusual capacity to regrow and repair themselves even when a large portion is damaged .","Previous research suggests that the connective tissue around the ribs helps to support and co-ordinate bone healing .","Yet it is currently not clear why ribs have a greater capacity to repair these large injuries compared to other bones .","Kuwahara et al . have now examined bone repair by studying how ribs heal in mice .","The experiments show that around 6% of the connective tissue cells are critical for large-scale repair .","Kuwahara et al . named these cells messenger cells .","These cells detect the presence of a signal molecule called Hedgehog ( Hh ) , however , if these cells lose the ability to respond to the Hh molecules , the ribs do not heal properly .","Further examination revealed that these messenger cells co-ordinate repair by encouraging other cells to build a special kind of bone-healing tissue with hybrid properties of both cartilage and bone .","Further research could now examine how messenger cells co-ordinate healing and if their properties could be adapted to help repair other bones .","Ultimately , understanding how messenger cells work may even provide insights into new ways to repair and regenerate other tissues and organs too ."],"string":"[\n \"Fractures to major bones often heal slowly or incompletely , especially in older people , and large bone injuries do not repair naturally .\",\n \"By comparison , rib bones show an unusual capacity to regrow and repair themselves even when a large portion is damaged .\",\n \"Previous research suggests that the connective tissue around the ribs helps to support and co-ordinate bone healing .\",\n \"Yet it is currently not clear why ribs have a greater capacity to repair these large injuries compared to other bones .\",\n \"Kuwahara et al . have now examined bone repair by studying how ribs heal in mice .\",\n \"The experiments show that around 6% of the connective tissue cells are critical for large-scale repair .\",\n \"Kuwahara et al . named these cells messenger cells .\",\n \"These cells detect the presence of a signal molecule called Hedgehog ( Hh ) , however , if these cells lose the ability to respond to the Hh molecules , the ribs do not heal properly .\",\n \"Further examination revealed that these messenger cells co-ordinate repair by encouraging other cells to build a special kind of bone-healing tissue with hybrid properties of both cartilage and bone .\",\n \"Further research could now examine how messenger cells co-ordinate healing and if their properties could be adapted to help repair other bones .\",\n \"Ultimately , understanding how messenger cells work may even provide insights into new ways to repair and regenerate other tissues and organs too .\"\n]"},"year":{"kind":"string","value":"2019"}}},{"rowIdx":4291,"cells":{"headings":{"kind":"list like","value":["Introduction","Results and discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results and discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["plant biology","structural biology and molecular biophysics"],"string":"[\n \"plant biology\",\n \"structural biology and molecular biophysics\"\n]"},"title":{"kind":"string","value":"The structure of plant photosystem I super-complex at 2.8 Å resolution"},"id":{"kind":"string","value":"elife-07433-v2"},"sections":{"kind":"list like","value":[["Oxygenic photosynthesis , in which the conversion of sunlight into chemical energy by plants , green algae , and cyanobacteria , occurs , underpins the survival of virtually all life forms .","By producing oxygen and assimilating carbon dioxide into organic matter , this process determines , to a large extent , the composition of our atmosphere and provides essential food and fuel .","Light photons are captured by pigments in very large membrane–bound complexes and the excitation energy is utilized to form NADPH and ATP .","Two reaction centers , photosystem II ( PSII ) and photosystem I ( PSI ) , drive this electron transport chain .","PSII oxidizes water to produce oxygen and reduce membrane-embedded quinones .","The reduced quinones are then utilized by the cytochrome b6f complex to produce a proton gradient across the membrane and to reduce the small copper protein plastocyanin ( PC ) , the electron donor of PSI .","After an additional photon is absorbed by any of the 200 antenna pigments of PSI , its energy migrates through this large network of connected pigments and eventually oxidizes P700 , a special chlorophyll pair located at the center of PSI .","The electron removed from P700 by this oxidation event migrates along an internal electron transport chain and finally reduces ferredoxin ( Fd ) , the final electron acceptor of PSI .","Reduced Fd is utilized by several cellular pathways , mainly the reduction of NADP to NADPH .","This NADPH and ATP generated by the thylakoid ATP-synthase complex powers the Calvin cycle to produce carbohydrates .","Oxygenic photosynthesis evolved over 3 billion years ago in cyanobacteria ( Blankenship , 1992; Barber , 2004; Nelson , 2013 ) .","Later , approximately 1 . 5 billion years ago , the first photosynthetic eukaryotes appeared , eventually evolving into land plants roughly 0 . 5 billion years ago .","The basic building blocks of photosynthesis are remarkably conserved .","The architectures of both photosystems have been determined by numerous techniques , but X-ray crystallography has provided the most detailed structural information for the four large membrane complexes catalyzing oxygenic photosynthesis ( Nelson and Ben-Shem , 2004; Nelson and Yocum , 2006; Croce and van Amerongen , 2013; Nelson and Junge , 2015 ) .","Structures at the highest resolution has been obtained for thermophilic cyanobacteria , but representative structures from eukaryotic chloroplasts , especially plants , are scarce ( Jordan et al . , 2001; Ben-Shem et al . , 2003; Kurisu et al . , 2003; Stroebel et al . , 2003; Ferreira et al . , 2004; Loll et al . , 2005; Amunts et al . , 2007 , 2010; Umena et al . , 2011 ) .","Here , we report the structure of plant PSI super-complex at high resolution ."],["The crystal structure of plant PSI was first reported at 4 . 4 Å resolution ( Ben-Shem et al . , 2003 ) and has been improved up to 3 . 3 Å resolution in the last decade ( PDB 2WSC ) .","This PSI preparation was limited to pea plants from the variety Alaska , and good crystals were hard to come by ( Amunts et al . , 2007 , 2010 ) .","Therefore , we screened for new robust crystals that are abundant , stable , and much more uniform .","The new crystals could be obtained from several pea plants varieties , a large proportion of them diffracted to 3 Å with several yielding higher resolutions .","In contrast to the P21 symmetry of the previous crystal , the current crystal belonged to higher symmetry space group P212121 .","The organization of the PSI unit within the new crystal was also markedly different .","In the P21 crystal the PSI-LHCI complex was organized as parallel layers in which the iron-sulfur clusters FX , FA and FB face the adjacent P700 ( Figure 1A ) .","The complexes inside the new crystal lattice were serially arranged in a crissed-crossed manner in which the polarity of each PSI unit contrasts another ( Figure 1B ) .","Consequently , the current crystals generated no net voltage ( data not shown ) , whereas a voltage of up to 50 V was recorded ( Toporik et al . , 2012 ) upon illumination of dried P21 crystals placed on electron conductive material . 10 . 7554/eLife . 07433 . 003Figure 1 . Comparison of two PSI-LHCI crystal lattices .","( A ) The previous PSI-LHCI crystal in the P21 space group with a layered arrangement of the complex .","This arrangement is capable of generating extremely high voltages upon illumination .","( B ) The new crystal lattice in the P212121 space group .","Iron sulfur clusters are colored in red and the pigments of the internal electron transport chain in magenta ( chlorophylls ) and blue ( quinones ) .","PSI-LHCI complexes are arranged in a crissed-crossed manner from left to right . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 003 The extreme size and complexity of the PSI assembly was a major obstacle for accurate and bias-free modeling .","The best way to eliminate model bias in X-ray crystallography is to utilize experimentally measured phase information .","Using the new , highly stable crystal form of PSI we were able to measure the weak native anomalous signal from the iron , sulfur , and phosphate atoms in the complex .","Starting with a minimal model containing only the three natively bound iron-sulfur clusters , the entire structure was eventually re-built with more than 35 , 000 atoms ( Figure 2 and Figure 2—figure supplement 1 , see ‘Material and methods’ section for details ) . 10 . 7554/eLife . 07433 . 004Figure 2 . Overall structure and organization of the plant PSI-LHCI supercomplex .","( A ) A view from the stromal side of the membrane of PSI-LHCI with the PsaL subunit pointing up .","The PsaF and PsaJ subunits connecting in the middle of LHCI are colored in magenta and green , respectively .","The three subunits of the stromal ridge , PsaC , PsaD , and PsaE , can be seen in the middle of the complex , colored cyan , pink , and blue , respectively .","The two iron-sulfur clusters of PsaC can be distinguished as yellow and orange clusters in the middle of the complex .","( B ) Pigment organization in PSI-LHCI .","The central pigments of the internal electron transport chain are colored red , chlorophylls of the core antenna green , chlorophyll a in LHCI in cyan , and chlorophyll b in magenta .","Carotenoids , which are distributed throughout the complex , are colored in blue and lipids in key connecting points and conserved positions in the core , in orange . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 00410 . 7554/eLife . 07433 . 005Figure 2—figure supplement 1 . Phasing substructure and electron density maps .","( A ) Positions of the native sulfurs , phosphates ( both in yellow ) , and iron atoms ( orange ) identified in the PSI-LHCI complex based on the measured anomalous signal .","( B ) Electron density maps .","The 2F − Fc map in blue , contoured at 1 . 3 rmsd , and an anomalous difference map in red , contoured at 2 rmsd around the phosphate atom of lipid 7001 bound by PsaA .","Residues 577:580 from PsaA are also shown . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 005 The structure of plant PSI includes 12 core subunits bound with four light-harvesting proteins comprising the LHCI antenna complex .","The entire complex contains 214 prosthetic groups , including 156 chlorophylls ( nine assigned as chlorophyll b ) , 32 carotenes , and 14 lipids , many of them located at key contact points of the complex .","The core photosynthetic reaction centers have remained virtually unchanged over the entire 2 billion years of their evolution ( Jordan et al . , 2001; Ben-Shem et al . , 2003; Amunts et al . , 2010 ) .","Instead , the evolution of PSI is marked by the loss and gain of whole subunits from the complex ( Scheller et al . , 2001; Nelson , 2011; Nelson and Junge , 2015 ) .","Compared to our previous model ( PDB 2WSC ) , the root-mean-square deviation ( rmsd ) between the plant and cyanobacterial core ( PDB 1JB0 ) decreased from 1 . 1 Å to 0 . 55 Å .","The majority of the changes made in the core subunits involved the configuration of extramembrane loops , which now closely resemble the cyanobacterial configuration .","The exceptions to this role are found at the anchor points of LHCI to the core ( discussed below ) and at the interfaces between plant-specific subunits , such as the PsaH–PsaL interaction ( Figure 3A ) .","The dramatic change from trimer to monomeric organization that occurred in eukaryotes , was triggered by the addition of the PsaH subunit ( Ben-Shem et al . , 2003 ) .","A new configuration for PsaH shows that this subunit binds four other core subunits .","Starting from the stromal side of the membrane , the N-terminus is tightly tucked between the N-terminus of PsaD and a eukaryotic-specific loop in the PsaL subunit .","PsaH then enters the membrane surrounding PsaL to prevent PSI trimerization and associates with PsaI and PsaB via mostly hydrophobic interactions ( Figure 3A ) . 10 . 7554/eLife . 07433 . 006Figure 3 . PsaH connects the state II and the PC binding sites .","( A ) PsaL is presented in blue , and the path of the PsaH subunit ( red ) travels through its extended , eukaryotic specific , loop .","The new PsaH chlorophyll ( green , marked as H1 ) connects to the PsaL-coordinated chlorophyll and carotenoid ( green and pink ) , as well as an additional chlorophyll trimer ( middle left , marked as PsaA trimer ) , which is proposed to connect PSI to LHCII in state II .","( B ) A luminal view of the PSI surface at the PC binding site .","The two hydrophobic helices are colored in grey ( PsaA ) and light blue ( PsaB ) .","The luminal PsaA loop is highlighted on the background of the entire PsaA subunit .","All the ligands ( with the exception of P700 ) are not shown .","( C and D )","Side by side view of the cyanobacterial ( C ) and plant ( D ) PC binding sites showing the extended PsaF helices that limit access from the membrane plane and the N terminus of PsaH ( red ) , which parallels the configuration of the PsaA luminal loop .","The approximate location of PC is indicated in blue . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 00610 . 7554/eLife . 07433 . 007Figure 3—figure supplement 1 . The proposed configuration of the state II super complex .","( A ) monomeric LHCII is shown in magenta near the PsaL subunit , juxtaposed to PsaK .","The region blown up in B is circled black .","( B ) This arrangement brings sites 5 and 12 from Lhcb next to the PsaA-coordinated chlorophyll trimer . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 007 Eukaryotes can modulate the distribution of excitation energy transfer between their two photosystems via a mechanism called state transitions ( Lunde et al . , 2000; Bellafiore et al . , 2005; Rochaix , 2011; Rochaix et al . , 2012 ) .","Under state II conditions , PSI associates with a mobile pool of the LHCII antennae , which increases its absorbance cross-section ( Kargul et al . , 2005; de Bianchi et al . , 2010 ) .","Genetic studies suggest that PsaH , PsaL , and PsaK play important roles in this process ( Scheller et al . , 2001; Zhang and Scheller , 2004 ) .","Electron microscopy studies have identified the binding site of the additional antennae complexes along the PsaL/PsaH-PsaK side ( Kargul et al . , 2005; Kouril et al . , 2005 ) .","A new chlorophyll bound by PsaH was identified at the current resolution .","This new chlorophyll , together with pigments bound by PsaL , probably participates in energy transfer into the core ( Figure 3A ) , suggesting that PsaH is not simply a ‘landing pad’ for LHCII , but is also important for energy transfer into the core during state II .","Additional pigments bound by PsaA in close proximity to subunit PsaK ( the structure of which is now almost completely defined ) provided the first accurate description of this binding site and suggest a mechanism for energy transfer into the core antenna through the PsaK side ( Figure 3—figure supplement 1 ) .","On the luminal side of the membrane , the new position of the N-terminus of PsaH suggests that this subunit also directly contributes to PC binding .","One of the distinguishing characteristics of the eukaryotic PSI is the stable complex it forms with its electron donor PC , which results in a thousand-fold acceleration of the electron transfer rate ( Bottin and Mathis , 1985 ) .","Three elements make up the PC binding site: the first is a positive patch located along the helix-turn-helix N-terminal domain of PsaF ( Hippler et al . , 1997 , 1998; Ben-Shem et al . , 2003 ) .","The second element is a hydrophobic patch composed of two parallel helices in PsaA and PsaB ( Figure 2B ) ( Sommer et al . , 2006; Kuhlgert et al . , 2012 ) .","The third conserved feature of the PC binding site is a PsaA luminal loop protruding from the generally flat luminal surface of PSI .","This loop is found in both plants and cyanobacteria PSI; the only exception being sequences from marine Prochlorococcus and their phages ( Mazor et al . , 2012 ) .","As seen in Figure 3C , D , the binding site of the cyanobacterial and plant complexes are similar .","However , it is clear that the plant binding site is buried deeper in the complex , this is achieved by the extension of the PsaF N-terminal and by the new position of the N-terminus of PsaH , which forms a loop mirroring the conformation of the conserved luminal PsaA loop , suggesting a direct role for PsaH in PC binding .","The PSI core is a highly efficient hub onto which diverse antennae systems connect such as phycobilisomes and IsiA-like assemblies in cyanobacteria and red algae and LHC type antennae in eukaryotes ( Berera et al . , 2009; Engelken et al . , 2010; Nelson and Junge , 2015; Wahadoszamen et al . , 2015 ) .","Remarkably , the core pigment organization is conserved across kingdoms despite this diversity in connected antenna ( Amunts et al . , 2007; Busch and Hippler , 2011; Croce and van Amerongen , 2013 ) , which suggests that the connection points between the core and the antennae are conserved .","The existence of red-absorbing pigments ( or ‘red traps’ ) is a general property of PSI ( Morosinotto et al . , 2005; Wientjes et al . , 2012 ) .","These pigments affect the rate of trapping in PSI and can affect the path of excitation migration in the complex .","Most of the eukaryotic red pigments have been shown to reside at LHCI .","However , red pigments may be lost from the core complex during the isolation of LHCI .","The first high-resolution PSI structure from thermophilic cyanobacteria revealed the organization of the core antenna ( Jordan et al . , 2001 ) .","A stacked chlorophyll trimer supported by an extended loop in PsaB was the best candidate for one of the strong red absorbers in this complex ( Jordan et al . , 2001 ) .","PsaB sequences from eukaryotes and many cyanobacteria lack this extended loop , resulting in this chlorophyll trimer being lost , as has been shown in the plant and mesophilic PSI structures ( Amunts et al . , 2010; Mazor et al . , 2014 ) .","At the current resolution , we observed new core chlorophyll bound between PsaG and Lhca1 and a newly discovered lipid ( Figure 4A ) .","This new chlorophyll restores the stacked chlorophyll trimer independent of the shortened PsaB loop and is responsible for one of the connection points between the core complex and the LHCI antenna , with a Mg–Mg distance of 12 . 5 Å between it and chlorophyll 1010 in Lhca1 ( the nomenclature for LHCII is used to describe Lhcas [Standfuss et al . , 2005] ) .","On the stromal side of the membrane , an additional chlorophyll trimer first discovered in Synechocystis is also responsible for an antenna attachment point with a Mg–Mg distance of 13 . 7 Å between the core chlorophyll A40 and chlorophyll 1005 in Lhca1 ( Figure 4—figure supplement 1 ) .","We suggest that chlorophyll trimers located at the periphery of the core antenna are extremely important for antenna attachment and are probably general attachment points to the core that are utilized not only by eukaryotes , but also by other antenna systems in cyanobacteria . 10 . 7554/eLife . 07433 . 008Figure 4 . Antenna connections in PSI-LHCI .","( A ) The configuration of the PsaG-Lhca1 pigment connection shown from the luminal side of the membrane .","The new stacked chlorophyll trimer ( numbered B1231 , B1232 and G1003 ) is shown .","The N-terminus of PsaG ( dark red ) supports one of the chlorophylls making up this trimer .","The entire trimer is connected with chlorophyll 10 ( numbered 1010 ) in Lhca1 ( blue ) .","( B ) The second LHCI-PSI connection between Lhca1 and PsaF ( magenta ) on the luminal side of the membrane is bound by a lipid ( orange ) .","( C ) The Lhca3 ( red ) -PsaA connection .","Two chlorophyll pairs mediate this interaction .","At the lumen face , 13 . 7 Å separate chlorophyll 3010 from chlorophyll 1114 .","On the stromal side , chlorophyll 3005 and chlorophyll 1108 are 16 . 5 Å apart .","( D ) Lhca2 ( blue ) —PsaJ ( green ) connecting chlorophylls . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 00810 . 7554/eLife . 07433 . 009Figure 4—figure supplement 1 . LhcA1 connects to PSI through chlorophyll trimers . The second chlorophyll trimer connecting Lhca1 to the core on the stromal side of the membrane bound by a short PsaB helix ( teal ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 00910 . 7554/eLife . 07433 . 010Figure 4—figure supplement 2 . PsaG occupy roughly an equivalent position compared to previous PSI-LHCI structures ( PsaG from 2WSC in cyan , PsaG from the current structure in red ) .","However the polarity of the transmembrane helices is reversed , as a result the N-terminus which was protruding outward from the complex now faces LHCI .","Three chlorophylls , a carotene and a lipid are coordinated by this subunit in the current structure . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 010 In contrast to the previous plant structures , which included a small pool of ‘Gap chlorophylls’ , only six pigment pairs connect LHCI to the core antenna in the current structure .","Lhca1 is the main connector for excitation transfer , harboring three chlorophylls that are within 14 Å of reaction center pigments ( Figure 4A , B and Figure 4—figure supplement 1 ) .","This close proximity ensures efficient and fast energy transfer .","Surprisingly , Lhca3 is also one of the main connection points with two such pairs ( Figure 4C ) .","The final excitonic connection between PSI to LHCI is located between chlorophyll J1302 bound by PsaJ and chlorophyll 2010 ( A chlorophyll b molecule ) .","The Mg–Mg distance of this pair is quite large ( 17 . 6 Å ) however , since the gap between LHCI and PSI allows for some movement , this distance can change to provide an efficient link between the core and LHCI ( Figure 4D ) .","To summarize , the extremely fast and efficient energy transfer processes that typify PSI-LHCI occurs through only six pairs of pigment molecules located at three sites .","These sites connect to the core antennae at the PsaG and PsaK poles through Lhca1 and Lhca3 , with Lhca2 playing a relatively minor role .","Our structure includes the fully modeled LHCI belt with nearly complete structures of all four Lhca proteins .","These structures reveal the essential features of the specific interactions between: each Lhca protein and the core; the red chlorophyll assembly present in Lhca4 and Lhca3; and a previously unknown pigment binding site , which is the probable site for the recently discovered non-photochemical quenching ( NPQ ) at the luminal gap region of LHCI ( de Bianchi et al . , 2010; Ballottari et al . , 2014 ) .","The LHCI belt is located on the PsaF side of the PSI core .","On the stromal face of the membrane , the four conserved N-terminal domains connect each Lhca subunit to its neighbor through interaction with an Lhca-specific loop ( loop 23 ) ( Figure 5A ) that follows immediately after the second transmembrane helix and supports a new chlorophyll site ( numbered","16 ) in Lhca4 and Lhca2 ( Figure 6A , B ) .","Lhca1 , which interacts with PsaG in the core complex , completely lacks this loop and instead contains a short , positively charged linker in this region ( Figure 5A ) .","One of the major changes in the structure of LHCI is the reversal in the polarity of PsaG .","While PsaG occupies roughly an equivalent position compared to previous PSI-LHCI structures , the first transmembrane helix contacts Lhca1 while the second one contacts PsaB ( Figure 4—figure supplement 2 ) .","Connections between each Lhca and the core are mediated by small regions immediately preceding the first transmembrane helix and are stabilized by salt bridges between negatively charged residues positioned at the membrane entrance point and a conserved arginine located at a helix turn below them ( Figure 5A ) .","Additional Lhca–Lhca interactions are provided by a short C-terminal segment on the luminal side that binds helix 2 as it exits the membrane , mainly via hydrophobic interactions ( Figure 5—figure supplement 1 ) . 10 . 7554/eLife . 07433 . 011Figure 5 . Lhcas connect to the PSI core through conserved structural elements .","( A ) A view from the stromal side of the membrane of LHCI .","Each Lhca connects to the next one through its conserved N-terminal domain ( marked N ) .","All connections to the core are mediated by the short region preceding the entrance of the first transmembrane helix into the membrane ( marked by a circle ) .","The extended loop ( L23 ) is conserved in all Lhcas except Lhca1 .","( B ) Electrostatic interactions determine the specificity of the Lhca4/5 binding site to PsaF .","The conserved E84-R209 interaction occurs within the PsaF ( magenta ) domain , which is partially membrane-buried , and Lhca4 ( raspberry ) .","( C ) Superposition of Lhca3 ( red ) and LHCII ( cyan ) .","Lhca3 interacts with the core via contacts with PsaA and PsaK through short extension ( red circle ) or deletion ( green circle ) in the otherwise highly conserved N terminus .","The extension of loop 23 is also seen at the bottom left corner of the image . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 01110 . 7554/eLife . 07433 . 012Figure 5—figure supplement 1 . Luminal side connections between the core PSI and LHCI . Short sequences immediately preceding the insertion point of the second transmembrane helix in the LHC fold ( marked by a red spot ) interact with the C-terminal sequences of the adjacent subunit ( marked with a blue spot ) .","Numbers indicate the sequence range shown in bold for the respective Lhca subunit . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 01210 . 7554/eLife . 07433 . 013Figure 6 . The interface within the Lhca heterodimers provides a mechanism for NPQ .","( A and B )","Structure of the Lhca1/4 ( A ) and Lhca2/3 ( B ) heterodimer as viewed from the membrane plane extrinsic to the PSI-LHCI complex .","The conserved connecting carotenoid in position L3 ( pink ) serves a structural role , connecting the two subunits of the dimer by linking chlorophyll 6 ( bottom right ) to chlorophyll 9 ( upper left corner ) .","The position of the new chlorophyll site bound by loop 23 of Lhca2 ( numbered 2016 ) is also shown .","( C and D )","A new chlorophyll site connects the pigments of the heterodimer .","Chlorophyll 17 ( numbered 4017 or 3017 ) is shown as viewed from the luminal side of the membrane .","The connections between the red pigment cluster on the right and position 9 in the adjacent subunit are marked with grey lines .","The new carotenoid site , L5 , is shown in C . This site lines the gap between LHCI and PSI and is therefore easily interchangeable .","The distance between L5 and of chlorophylls 17 π systems is 3 . 7 Å .","( E ) Lhca3 is colored in red .","A short helix segment from the first transmembrane helix is shown as sticks .","Chlorophyll 3005 is part of the red-absorbing dimer .","LHCII ( PDB: 2BHW ) was superimposed on the structure and shown in faint grey .","Chlorophyll 612 is shown as a ring .","The coordinating side chain was changed from histidine 68 in LHCII to asparagine 109 in Lhca3 .","Another important change is the leucine to glycine modification at position 105 , which allows chlorophyll 3005 to alter its ring orientation by approximately 12° .","( F ) The same site in Lhca4 shown in a similar fashion with Lhca4 colored in raspberry .","The same basic coordination is observed , though the ring tilt is smaller because of the alanine occupying position 95 . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 01310 . 7554/eLife . 07433 . 014Figure 6—figure supplement 1 . Identification of chlorophyll b molecules . Shown is the chlorophyll b occupying position 10 from Lhca2 .","Simulated annealing omit maps were calculated on a model which lacked the formyl-7 group ( shown in the image ) .","All b factors of the model were reset to the Wilson B factor , followed by a single round of simulated annealing and refinement using phenix . refine .","The difference map ( green ) is contoured at 2 . 5 rmsd and the 2Fo − Fc map ( blue ) is contoured at 1 . 2 rmsd . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 014 Genetic analysis in plants has revealed that each subunit has a specific binding site and , with the exception of the Lhca4-Lhca5 pair , the various Lhcas are not interchangeable ( Lucinski et al . , 2006; Wientjes et al . , 2009 ) .","The specific binding sites are identified in the current structure .","At the PsaG pole of LHCI , helix C of Lhca1 interacts with the first transmembrane helix of PsaG .","Additional protein–protein interactions occurred between a stromal loop of PsaB ( aa 307–320 ) and the N-terminus of Lhca1 ( Figure 5A ) .","The next contact point between LHCI and the core occurs between the N-terminus of Lhca4 and the C-terminus of PsaF , the conformation of which is almost identical to the conformation found in cyanobacteria .","This interaction consists of hydrophobic patches surrounding charged residues in the membrane-buried regions of both proteins .","This binding site should be shared between Lhca4 and Lhca5 and indeed , arginine 209 of PsaF and glutamate 84 of Lhca4 form electrostatic interactions in the middle of the hydrophobic binding site ( Figure 5B ) .","This residue is conserved in Lhca5 as well , but absent from Lhca1-3 sequences , explaining the specificity of the interaction .","The PsaF-Lhca4 interaction in the current structure does not include any pigments .","Energy transfer from Lhca4 to the core probably occurs through the Lhca1-PsaB pigment cluster or the Lhca2-PsaJ clusters .","Lhca2 is connected to the core almost exclusively through interactions with the N-terminus of PsaJ on the stromal side of the membrane ( Figure 5—figure supplement 1 ) .","The main contact point of Lhca3 is in the N-terminus of PsaA , which contacts a small patch just before helix 1 enters the membrane as in all other Lhcas ( Figure 5C ) .","The structure shows that this site is the main determinant of Lhca binding to the core .","In contrast to the previous PSI-LHCI model , Lhca3 follows the general fold of LHCII ( rmsd 0 . 8 Å between the two apoproteins ) .","Departures from the LHCII fold are seen in key contact points where small loops were extended or deleted from the otherwise conserved N-terminus domain to facilitate protein–protein interactions with the core ( Figure 5C ) .","All four Lhcas are remarkably similar to each other and to LHCII .","Most of the differences between them can be explained by their interaction partner , such as the loss of loop 23 in Lhca1 due to its binding to PsaG .","Two key differences in pigment organization were found between lhcas and other lhcs .","The extended loop 23 supports a new chlorophyll-binding site ( numbered 16 ) , common to Lhca4 and Lhca2 ( Figure 6A , B ) and an additional chlorophyll site , coordinated by transmembrane helix two , connects the two partners of each heterodimer .","Chlorophyll b pigments are bound by the different Lhca proteins to various extents and serve as antennae pigments .","Four binding sites for chlorophyll b were detected in the high resolution structure of LHCII ( Liu et al . , 2004; Standfuss et al . , 2005 ) as well as in the structure of CP29 ( Pan et al . , 2011 ) .","We were able to assign nine chlorophyll b sites in LHCI based on electron density maps ( Figure 6—figure supplement 1 ) .","All Lhca proteins contain a glutamine to glutamate change ( similarly to CP29 ) in equivalent positions to position 131 of LHCII , this change makes the binding of Chlorophyll b less likely at three sites ( 10 , 12 and 13 ) .","In agreement with this change we find that site 12 is occupied by chlorophyll a in all Lhcas and sites 10 and 13 are occupied differentially .","The distribution of chlorophyll b sites in LHCI is markedly uneven , with three sites located in Lhca2 ( 2010 , 2011 and 2013 ) , two sites found at Lhca1 ( site 1009 and 1010 ) , three sites at Lhca4 ( 4010 , 4011 and 4013 ) and a single site on Lhca3 ( site 3011 ) .","These findings are consistent with mutational data which identified more chlorophyll b sites on Lhca2 then on Lhca3 ( Castelletti et al . , 2003 ) .","Site 13 on Lhca1 , 2 and 4 is probably a mixed site , which can accommodate both chlorophyll a and chlorophyll b .","The high number of chlorophyll b pigments in Lhca2 and the fact that its closest connection to the core is mediated by chlorophyll b ( site 2010 , shown in Figure 4D ) is consistent with our suggestion that Lhca1 and Lhca3 are the main junctions for excitation energy transfer from LHCI to the PSI core .","The dipole orientations of all but two chlorophylls were identified in the current structure .","The most significant changes in LHCI compared to LHCII exist in two sites , 2009 and 4009 , where 90° rotations are observed .","Such a rotation is expected to impact the energy transfer processes within these LHCI subunits but the significance of this change cannot be ascertained from the rotation alone .","Each of the two Lhca1/4 Lhca2/3 heterodimers contains a blue- and red-absorbing subunit .","The structures of Lhca3 and Lhca4 clearly show a coordinating asparagine residue unique to this site ( Arnoux et al . , 2009 ) .","The configurations of the pigments themselves differed , with the red pigments of Lhca3 and Lhca4 tilted approximately 12° relative to their orientation in LHCII , Lhca1 , and Lhca2 due to a second change from a bulky leucine residue at position 64 of LHCII to glycine and alanine in Lhca3 and Lhca4 , respectively ( Figure 6E , F ) .","Additional connections that stabilize LHCI are formed by three luteins at position L3 .","These luteins bridge the chlorophylls at site 6 in Lhca1 , Lhca3 , and Lhca4 to PsaG , Lhca2 , and Lhca1 ( Figure 6A , B ) .","Photosynthetic eukaryotes respond to high light conditions by decreasing the efficiency of energy transfer from the antennae in a process called NPQ .","First discovered in the PSII complex , NPQ was recently reported in PSI .","NPQ sites are located on LHC proteins bound by the hydroxylated carotenoid zeaxanthin , which quenches harmful chlorophyll triplets ( Standfuss et al . , 2005 ) .","The main excitonic connections in the two Lhca dimers appear to be intimately linked to NPQ .","A new chlorophyll site ( numbered","17 ) coordinated by a histidine residue ( histidine 150 in Lhca4 and histidine 170 in lhca3 ) is located at the heterodimer interface of both Lhca1/4 and Lhca2/3 .","Site 17 links the putative red chlorophyll pair with site 9 of the adjacent complex .","In the Lhca1/4 interface , a new carotenoid site ( numbered 4505 or L5 ) forms co-planar π systems ( plane to plane distance 3 . 7 Å ) with the ring of chlorophyll 4017 , positioning it in a configuration that should provide efficient photo-protection from chlorophyll triplets ( Figure 6C , D ) .","We propose that this is also the site of NPQ in LHCI , and this fits well with the experimental observation ( Standfuss et al . , 2005 ) that the zeaxanthin responsible for NPQ is located near the red pair in the PSI-LHCI luminal gap region .","In response to lumen acidification , zeaxanthin is synthesized from violaxanthin by violaxanthin deepoxidase ( VDE ) as part of the xanthophyll cycle .","Both the activity and location of VDE are regulated by pH changes ( Ballottari et al . , 2014 ) .","At low pH , VDE is activated and binds the luminal side of the membrane , gaining access to its substrate .","On the luminal side , a large gap ( 25 Å ) separates LHCI from the core .","This gap stems from the fact that most of the connections between LHCI and PSI are located at the stromal side ( seen in Figure 7C , also compare Figure 5A and Figure 5—figure supplement 1 ) , leaving the luminal side open . 10 . 7554/eLife . 07433 . 015Figure 7 . The surface electrostatic potential of PSI-LHCI .","( A ) Luminal view of the electrostatic potential ( red for negative and blue for positive charges ) generated from PSI-LHCI apoproteins .","The gap region is shown as an open cavity ( the ligands occupying this cavity were omitted from the calculation ) lined with negative patches .","The plastocyanin binding site can be distinguished as a blue patch generated by the positively charged luminal double helix domain of PsaF .","( B ) The stromal electrostatic surface of PSI-LHCI showing the putative ferredoxin binding site as a basic patch on the left side of the stromal ridge .","( C ) A side view of the stromal gap shows the large opening facing the lumen .","Lhca1 and PsaG are omitted to reveal the internal cavity .","This surface was drown around all ligands identified in the gap with only chlorophyll 4017 and carotene 4505 omitted .","( D ) We propose that the negative patches lining the gap play a role in preventing the accessibility of VDE to gap pigments during regular growth .","While at high light conditions , lumen acidification partially neutralizes these negative charges , allowing VDE activity on gap xanthophylls . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 015 The complete modeling of LHCI side chains shows that the luminal side of PSI-LHCI contains patches of negative potential distributed on both sides of the PSI-LHCI gap ( Figure 7 ) .","We suggest that the negative charge characteristic of the luminal side of PSI-LHCI is an important feature regulating the accessibility of VDE to the PSI-LHCI gap .","Under low light conditions , the relatively high luminal pH will result in strong repulsive forces between these negative patches and the acidic domain of VDE , preventing VDE from binding to PSI .","Under high light , acidification of lumen , which is known to trigger VDE activity , will result in partial neutralization of these patches and allow VDE access to the PSI-LHCI gap regions ( Arnoux et al . , 2009 ) ( Figure 7D ) .","Carotene L4505 is ideally positioned to be exchanged easily via this mechanism ( Figure 7C ) .","Ligands , which are easily exchangeable , are particularly labile and may disassociate from the complex during its purification , accounting for a lack of a second carotene bound at the analogous position in Lhca3 .","In the current structure , position L5 is occupied with zeaxanthin; however , the pigment assignment cannot be definite , even at the current resolution .","In this work we presented the most complete plant PSI-LHCI structure obtained thus far , revealing the locations of and interactions among its protein subunits and more than 200 non-covalently bound photochemical cofactors .","Using the new crystal structure , we examined the network of contacts among the protein subunits from a structural perspective , which provide the basis for elucidating the functional organization of the complex ."],["Pea seeds ( Pisum sativum var . Kalvadon ) were washed with running water for 6–8 hr and seeded in a vermiculite tray .","Plants were germinated in the dark at 22°C for 5 days in shaded sunlight or the dark .","After germination , the plants were grown for an additional 10 days under cool-white fluorescent light at a photon flux density of 90–130 μE m−2 s−1 in a 14 hr light/10 hr dark cycle at 22°C .","Approximately 200 g of leaves were ground for 25 s in a blender with 1000 ml of ice-cold solution containing 0 . 3 M sucrose , 15 mM NaCl , 30 mM Tricine-NaOH ( pH 8 ) , 1 mM PMSF , 15 μM leupeptin , and 1 μM pepstatin A . The slurry was filtered through eight layers of cheesecloth and chloroplasts pelleted by centrifugation at 1000 g for 9 min .","The pellet was suspended in 500 ml of hypotonic medium ( 10 mM Tricine-NaOH , pH 8 ) to disrupt the chloroplasts .","Thylakoids were collected by centrifugation at 12 , 000 g for 10 min and resuspended in 500 ml of buffer containing 10 mM Tricine-NaOH ( pH 8 ) and 150 mM NaCl .","The thylakoid membranes were then pelleted at 8 , 000 g for 10 min and resuspended in a minimal volume of STN2 buffer ( 0 . 4 M sucrose , 20 mM Tricine-NaOH , pH 8 ) .","The thylakoid concentration was adjusted to 3 mg of chlorophyll per ml and 0 . 4% n-dodecyl-α-D-maltoside ( DDM ) added .","This concentration of detergent selectively extracts the ATP synthase , b6f , and PSII complexes .","After 5 min incubation on ice , the detergent- treated thylakoid membranes were collected by ultra-centrifugation at 200 , 000 g for 30 min .","The pellet was resuspended in a minimal volume of STN2 buffer , adjusted to 3 mg chlorophyll per ml , and stored at −80°C .","Frozen thylakoid membranes ( 20–25 ml ) containing 3 . 0 mg of chl/ml were thawed in cold water and solubilized with 1 . 5% DDM .","Insolubilized material was removed by ultracentrifugation at 120 , 000 g for 15 min .","The supernatant was applied to a DEAE-cellulose column ( DE-52 , Whatman , Inc . , 1 . 5 × 18 cm ) pre-equilibrated with 15 mM Tricine-Tris ( pH 8 . 0 ) containing 0 . 25% DDM .","The column was washed with the same buffer and PSI eluted with a 0–230 mM tetraethylammonium chloride linear gradient ( 75 ml in each chamber ) in 15 mM Tricine-Tris ( pH 8 . 0 ) containing 0 . 25% DDM .","Dark green fractions containing PSI were precipitated by 10% PEG6000 ( Hampton Research , Aliso Viejo , CA ) , followed by centrifugation at 5 , 000 g for 6 min .","The pellet was dissolved in 15 mM Tricine-Tris ( pH 8 . 0 ) and 0 . 05% DDM .","The green solution was applied to a 10–35% sucrose gradient containing the same buffer and centrifuged using the SW-40 rotor ( Beckman Coulter , Fullerton , CA ) at 37 , 000 rpm ( 170 , 000 g ) for 16 hr .","The wide green band containing PSI was collected and loaded onto a second DEAE-cellulose column ( 0 . 5 × 4 cm ) pre-equilibrated with 15 mM Tricine-Tris ( pH 8 . 0 ) and 0 . 05% DDM , mainly to concentrate it for a second sucrose gradient .","PSI was eluted with 230 mM tetraethylammonium chloride .","The collected dark green fraction was applied to a 10–35% sucrose gradient and centrifuged at 57 , 000 rpm ( 330 , 000 g ) for 4 hr using an SW-60 rotor ( Beckman Coulter ) .","Purified PSI appeared as a dark band in the middle of the tube , but only the central part of the band was collected .","The material was precipitated with 15% PEG1500 and 100 mM tetraethylammonium chloride and centrifuged at 10 , 000 g for 4 min .","The pellet was dissolved in a solution containing 2 mM Tricine ( pH 8 . 75 ) and 0 . 02% n-dodecyl-β-D-thiomaltoside ( DTM , Glycon Biochemicals , Luckenwalde , Germany ) and adjusted to a chlorophyll concentration of 2 . 5 mg/ml .","Crystallization was performed manually in 24-well plates using the sitting drop variant of the vapor-diffusion technique at 4°C ( Charles Super Company , Natick , MA ) .","Aliquots ( 6–8 μl ) of PSI solution were mixed with equal volumes of reservoir solution ( 50 mM di-potassium phosphate , 50 mM Tris [pH 8] , 12–17% PEG400 , 1% glycerol , 2 mM L-glutathione , and 0 . 03% octyl glucose neopentyl glycol ) and equilibrated against 0 . 5 ml of reservoir solution .","Dark green rectangular crystals appeared after 3 days at the higher PEG concentrations , but the best diffracting crystals appeared after 1 month at the lower PEG concentrations .","For cryogenic protection , the crystals were moved to a solution containing 50 mM di-potassium phosphate , 50 mM Tris ( pH 8 ) , 20% PEG400 , 2% glycerol , and 2 mM L-glutathione .","After a brief incubation the crystals were soaked sequentially in the same buffer containing 5% and 10% glycerol and immediately frozen in liquid nitrogen .","X-ray diffraction data were collected at the European Synchrotron Radiation Facility ( ID23- 2 , ID23-1 , ID-29 and MASSIF-3 ) , the Swiss Light Source ( PXI , PXII , and PXIII ) , and BESSYII .","Images were collected at 0 . 1° oscillation using full beam at exposures of 0 . 1–0 . 05 s .","The large size of the crystals helped mitigate the effect of radiation damage , yet only 60°–90° of data were collected from each crystal using constant translation of the crystal in the beam .","Images were integrated using XDS ( Kabsch , 2010 ) and scaled with XSCALE or AIMLESS ( Evans and Murshudov , 2013 ) .","Measurements were carried out at the peak of the iron fluorescence scan .","Individual datasets generally had I/SIGMA values of ∼1 at 3 . 1 Å with CC1/2 values of ∼0 . 5 calculated by XDS .","To obtain a more accurate measure of weak reflections , we combined several datasets measured under similar conditions .","This combination was possible because of the consistency of the new crystals .","The most effective method for combining datasets was to simply choose the sets with the strongest statistics at lower resolutions , and these crystals were also very similar to each other in terms of their unit cell dimensions .","The unified datasets contained 40–100 independent measurements of each reflection with I/SIGMA of ∼1 . 5 at 2 . 8 Å and CC1/2 of ∼0 . 4 at the same resolution ( Table 1 ) .","The CCanomalous calculated by XDS or AIMLESS was ∼0 . 3 at 6 Å .","Some individual datasets had measurable anomalous signals ( CCanomalous > 0 . 3 ) to 4 . 5 Å , but the final quality of phases was similar , as judged by the figure of merit for substructures from PHASER runs ( typically ∼0 . 6 [McCoy et al . , 2007] ) and visual inspection of the maps . 10 . 7554/eLife . 07433 . 016Table 1 . Data collection and refinement statisticsDOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 016Data collectionBeamlineBESSY PX14 . 1SLS PXI−X06SAESRF ID 23-2Wavelength ( Å ) 1 . 731 . 740 . 873Resolution ( Å ) 40−2 . 8 ( 2 . 85–2 . 8 ) 40−2 . 8 ( 2 . 85−2 . 8 ) 50−3 ( 3 . 1−3 ) Space groupP212121P212121P21Unit cell dimensionsa , b , c ( Å ) 188 . 7 , 200 . 8 , 212 . 4188 . 6 , 201 . 3 , 212 . 7120 . 6 , 189 . 2 , 129 . 7α , β , γ90 , 90 , 9090 , 90 , 9090 , 91 . 1 , 90Measured reflections7 , 831 , 30214 , 841 , 310724 , 010Unique reflections198 , 911200 , 218116 , 039Rpim ( % ) 0 . 051 ( 1 . 276 ) 0 . 030 ( 0 . 180 ) 0 . 099 ( 0 . 812 ) 10 . 5 ( 1 . 2 ) 13 . 4 ( 1 . 4 ) 5 . 8 ( 1 . 3 ) Completeness ( % ) 99 . 9 ( 98 . 4 ) 99 . 9 ( 98 . 4 ) 99 . 9 ( 99 . 2 ) Redundancy39 . 9 ( 37 ) 74 . 1 ( 33 . 4 ) 6 . 2 ( 5 . 4 ) Refinement statisticsResolution ( Å ) 40−2 . 840−2 . 850−3Rwork/Rfree25 . 6/26 . 524/25 . 225 . 8/29 . 3No .","of chains161617No .","of ligands214214197Average B-factor ( Å2 ) 98 . 711296 . 6R . M .","S deviationsBond angles1 . 922 . 4Bond lengths0 . 0040 . 0050 . 011Ramachandran statisticsFavoured region %90 . 290 . 286 . 6Allowed region %7 . 17 . 18 . 7Outlier region %2 . 72 . 74 . 7Data collection , scaling and merging statistics were calculated using XDS , AIMLESS and PHENIX XTRIAGE .","Refinement statistics are from PHENIX .","The crystals were initially solved by molecular replacement ( MR ) with a partial model containing only the reaction center subunits with chlorophylls modeled as rings using PHASER .","This solution was used to place the three iron-sulfur clusters , which were subsequently used as the initial substructure for locating additional sites .","These initial runs located approximately 40 sites of the 100 that were eventually modeled .","Phases were improved using DM ( Cowtan , 1994 ) , and the resulting maps showed most of the transmembrane helices of the reaction center with 11 transmembrane helices of LHCI ( missing helix 2 of Lhca3 ) .","To improve the phases , information from the visible parts of the reaction center was incorporated as a partial MR solution .","This model included the 22 transmembrane helices of PsaA and PsaB , the PsaC subunit , and chlorophyll rings , omitting all of the loops from the proteins .","From the maps generated in this step , we proceeded to build the model using Coot ( Emsley et al . , 2010 ) and used the modified phases as restraints during refinement in either PHENIX ( Adams et al . , 2010 ) or REFMAC ( Murshudov et al . , 1997 ) .","At various points during the refinement process , the phases were recalculated with the newly modeled sites using PHASER and including the improved model ( Read and McCoy , 2011 ) .","Final runs identified 99 sites , 89 of them present in the model .","The final model refined to an R-free of 25 . 2% with 3% Ramachandran outliers , a considerable decrease from previous values ( Table 1 ) .","A complete model for the lhca subunits of LHCI was obtained , as well as extensions and modifications to some of the other PSI subunits ( Table 2 ) .","Images were created using Pymol and electrostatic surfaces calculated using APBS ( Baker et al . , 2001 ) . 10 . 7554/eLife . 07433 . 017Table 2 . Amino acid changes between PSI-LHCI structuresDOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 017SubunitNumber of amino acidsModeled amino acidsNumber of changes4Y282WSCPsaA7587417291PsaB7347327321PsaC8180811PsaD*1561401355PsaE*9268659PsaF*15415015418PsaG*98959515PsaH*95846911PsaJ*4241425PsaK*134798510PsaL*16816016121Lhca1*20419316512Lhca22562061763Lhca324221016216Lhca42521971663The number of modeled amino acids in each subunit is shown and compared to the most recent PSI-LHCI structure ( 2WSC ) .","Insertions , deletions and extensions are counted as a single change .","Since the genome sequence of Pisum Sativum is not completely known ( genes with no DNA data are marked with an * ) we relied on high-throughput mRNA sequence data for verification ( Franssen et al . , 2011 ) ."]],"string":"[\n [\n \"Oxygenic photosynthesis , in which the conversion of sunlight into chemical energy by plants , green algae , and cyanobacteria , occurs , underpins the survival of virtually all life forms .\",\n \"By producing oxygen and assimilating carbon dioxide into organic matter , this process determines , to a large extent , the composition of our atmosphere and provides essential food and fuel .\",\n \"Light photons are captured by pigments in very large membrane–bound complexes and the excitation energy is utilized to form NADPH and ATP .\",\n \"Two reaction centers , photosystem II ( PSII ) and photosystem I ( PSI ) , drive this electron transport chain .\",\n \"PSII oxidizes water to produce oxygen and reduce membrane-embedded quinones .\",\n \"The reduced quinones are then utilized by the cytochrome b6f complex to produce a proton gradient across the membrane and to reduce the small copper protein plastocyanin ( PC ) , the electron donor of PSI .\",\n \"After an additional photon is absorbed by any of the 200 antenna pigments of PSI , its energy migrates through this large network of connected pigments and eventually oxidizes P700 , a special chlorophyll pair located at the center of PSI .\",\n \"The electron removed from P700 by this oxidation event migrates along an internal electron transport chain and finally reduces ferredoxin ( Fd ) , the final electron acceptor of PSI .\",\n \"Reduced Fd is utilized by several cellular pathways , mainly the reduction of NADP to NADPH .\",\n \"This NADPH and ATP generated by the thylakoid ATP-synthase complex powers the Calvin cycle to produce carbohydrates .\",\n \"Oxygenic photosynthesis evolved over 3 billion years ago in cyanobacteria ( Blankenship , 1992; Barber , 2004; Nelson , 2013 ) .\",\n \"Later , approximately 1 . 5 billion years ago , the first photosynthetic eukaryotes appeared , eventually evolving into land plants roughly 0 . 5 billion years ago .\",\n \"The basic building blocks of photosynthesis are remarkably conserved .\",\n \"The architectures of both photosystems have been determined by numerous techniques , but X-ray crystallography has provided the most detailed structural information for the four large membrane complexes catalyzing oxygenic photosynthesis ( Nelson and Ben-Shem , 2004; Nelson and Yocum , 2006; Croce and van Amerongen , 2013; Nelson and Junge , 2015 ) .\",\n \"Structures at the highest resolution has been obtained for thermophilic cyanobacteria , but representative structures from eukaryotic chloroplasts , especially plants , are scarce ( Jordan et al . , 2001; Ben-Shem et al . , 2003; Kurisu et al . , 2003; Stroebel et al . , 2003; Ferreira et al . , 2004; Loll et al . , 2005; Amunts et al . , 2007 , 2010; Umena et al . , 2011 ) .\",\n \"Here , we report the structure of plant PSI super-complex at high resolution .\"\n ],\n [\n \"The crystal structure of plant PSI was first reported at 4 . 4 Å resolution ( Ben-Shem et al . , 2003 ) and has been improved up to 3 . 3 Å resolution in the last decade ( PDB 2WSC ) .\",\n \"This PSI preparation was limited to pea plants from the variety Alaska , and good crystals were hard to come by ( Amunts et al . , 2007 , 2010 ) .\",\n \"Therefore , we screened for new robust crystals that are abundant , stable , and much more uniform .\",\n \"The new crystals could be obtained from several pea plants varieties , a large proportion of them diffracted to 3 Å with several yielding higher resolutions .\",\n \"In contrast to the P21 symmetry of the previous crystal , the current crystal belonged to higher symmetry space group P212121 .\",\n \"The organization of the PSI unit within the new crystal was also markedly different .\",\n \"In the P21 crystal the PSI-LHCI complex was organized as parallel layers in which the iron-sulfur clusters FX , FA and FB face the adjacent P700 ( Figure 1A ) .\",\n \"The complexes inside the new crystal lattice were serially arranged in a crissed-crossed manner in which the polarity of each PSI unit contrasts another ( Figure 1B ) .\",\n \"Consequently , the current crystals generated no net voltage ( data not shown ) , whereas a voltage of up to 50 V was recorded ( Toporik et al . , 2012 ) upon illumination of dried P21 crystals placed on electron conductive material . 10 . 7554/eLife . 07433 . 003Figure 1 . Comparison of two PSI-LHCI crystal lattices .\",\n \"( A ) The previous PSI-LHCI crystal in the P21 space group with a layered arrangement of the complex .\",\n \"This arrangement is capable of generating extremely high voltages upon illumination .\",\n \"( B ) The new crystal lattice in the P212121 space group .\",\n \"Iron sulfur clusters are colored in red and the pigments of the internal electron transport chain in magenta ( chlorophylls ) and blue ( quinones ) .\",\n \"PSI-LHCI complexes are arranged in a crissed-crossed manner from left to right . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 003 The extreme size and complexity of the PSI assembly was a major obstacle for accurate and bias-free modeling .\",\n \"The best way to eliminate model bias in X-ray crystallography is to utilize experimentally measured phase information .\",\n \"Using the new , highly stable crystal form of PSI we were able to measure the weak native anomalous signal from the iron , sulfur , and phosphate atoms in the complex .\",\n \"Starting with a minimal model containing only the three natively bound iron-sulfur clusters , the entire structure was eventually re-built with more than 35 , 000 atoms ( Figure 2 and Figure 2—figure supplement 1 , see ‘Material and methods’ section for details ) . 10 . 7554/eLife . 07433 . 004Figure 2 . Overall structure and organization of the plant PSI-LHCI supercomplex .\",\n \"( A ) A view from the stromal side of the membrane of PSI-LHCI with the PsaL subunit pointing up .\",\n \"The PsaF and PsaJ subunits connecting in the middle of LHCI are colored in magenta and green , respectively .\",\n \"The three subunits of the stromal ridge , PsaC , PsaD , and PsaE , can be seen in the middle of the complex , colored cyan , pink , and blue , respectively .\",\n \"The two iron-sulfur clusters of PsaC can be distinguished as yellow and orange clusters in the middle of the complex .\",\n \"( B ) Pigment organization in PSI-LHCI .\",\n \"The central pigments of the internal electron transport chain are colored red , chlorophylls of the core antenna green , chlorophyll a in LHCI in cyan , and chlorophyll b in magenta .\",\n \"Carotenoids , which are distributed throughout the complex , are colored in blue and lipids in key connecting points and conserved positions in the core , in orange . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 00410 . 7554/eLife . 07433 . 005Figure 2—figure supplement 1 . Phasing substructure and electron density maps .\",\n \"( A ) Positions of the native sulfurs , phosphates ( both in yellow ) , and iron atoms ( orange ) identified in the PSI-LHCI complex based on the measured anomalous signal .\",\n \"( B ) Electron density maps .\",\n \"The 2F − Fc map in blue , contoured at 1 . 3 rmsd , and an anomalous difference map in red , contoured at 2 rmsd around the phosphate atom of lipid 7001 bound by PsaA .\",\n \"Residues 577:580 from PsaA are also shown . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 005 The structure of plant PSI includes 12 core subunits bound with four light-harvesting proteins comprising the LHCI antenna complex .\",\n \"The entire complex contains 214 prosthetic groups , including 156 chlorophylls ( nine assigned as chlorophyll b ) , 32 carotenes , and 14 lipids , many of them located at key contact points of the complex .\",\n \"The core photosynthetic reaction centers have remained virtually unchanged over the entire 2 billion years of their evolution ( Jordan et al . , 2001; Ben-Shem et al . , 2003; Amunts et al . , 2010 ) .\",\n \"Instead , the evolution of PSI is marked by the loss and gain of whole subunits from the complex ( Scheller et al . , 2001; Nelson , 2011; Nelson and Junge , 2015 ) .\",\n \"Compared to our previous model ( PDB 2WSC ) , the root-mean-square deviation ( rmsd ) between the plant and cyanobacterial core ( PDB 1JB0 ) decreased from 1 . 1 Å to 0 . 55 Å .\",\n \"The majority of the changes made in the core subunits involved the configuration of extramembrane loops , which now closely resemble the cyanobacterial configuration .\",\n \"The exceptions to this role are found at the anchor points of LHCI to the core ( discussed below ) and at the interfaces between plant-specific subunits , such as the PsaH–PsaL interaction ( Figure 3A ) .\",\n \"The dramatic change from trimer to monomeric organization that occurred in eukaryotes , was triggered by the addition of the PsaH subunit ( Ben-Shem et al . , 2003 ) .\",\n \"A new configuration for PsaH shows that this subunit binds four other core subunits .\",\n \"Starting from the stromal side of the membrane , the N-terminus is tightly tucked between the N-terminus of PsaD and a eukaryotic-specific loop in the PsaL subunit .\",\n \"PsaH then enters the membrane surrounding PsaL to prevent PSI trimerization and associates with PsaI and PsaB via mostly hydrophobic interactions ( Figure 3A ) . 10 . 7554/eLife . 07433 . 006Figure 3 . PsaH connects the state II and the PC binding sites .\",\n \"( A ) PsaL is presented in blue , and the path of the PsaH subunit ( red ) travels through its extended , eukaryotic specific , loop .\",\n \"The new PsaH chlorophyll ( green , marked as H1 ) connects to the PsaL-coordinated chlorophyll and carotenoid ( green and pink ) , as well as an additional chlorophyll trimer ( middle left , marked as PsaA trimer ) , which is proposed to connect PSI to LHCII in state II .\",\n \"( B ) A luminal view of the PSI surface at the PC binding site .\",\n \"The two hydrophobic helices are colored in grey ( PsaA ) and light blue ( PsaB ) .\",\n \"The luminal PsaA loop is highlighted on the background of the entire PsaA subunit .\",\n \"All the ligands ( with the exception of P700 ) are not shown .\",\n \"( C and D )\",\n \"Side by side view of the cyanobacterial ( C ) and plant ( D ) PC binding sites showing the extended PsaF helices that limit access from the membrane plane and the N terminus of PsaH ( red ) , which parallels the configuration of the PsaA luminal loop .\",\n \"The approximate location of PC is indicated in blue . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 00610 . 7554/eLife . 07433 . 007Figure 3—figure supplement 1 . The proposed configuration of the state II super complex .\",\n \"( A ) monomeric LHCII is shown in magenta near the PsaL subunit , juxtaposed to PsaK .\",\n \"The region blown up in B is circled black .\",\n \"( B ) This arrangement brings sites 5 and 12 from Lhcb next to the PsaA-coordinated chlorophyll trimer . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 007 Eukaryotes can modulate the distribution of excitation energy transfer between their two photosystems via a mechanism called state transitions ( Lunde et al . , 2000; Bellafiore et al . , 2005; Rochaix , 2011; Rochaix et al . , 2012 ) .\",\n \"Under state II conditions , PSI associates with a mobile pool of the LHCII antennae , which increases its absorbance cross-section ( Kargul et al . , 2005; de Bianchi et al . , 2010 ) .\",\n \"Genetic studies suggest that PsaH , PsaL , and PsaK play important roles in this process ( Scheller et al . , 2001; Zhang and Scheller , 2004 ) .\",\n \"Electron microscopy studies have identified the binding site of the additional antennae complexes along the PsaL/PsaH-PsaK side ( Kargul et al . , 2005; Kouril et al . , 2005 ) .\",\n \"A new chlorophyll bound by PsaH was identified at the current resolution .\",\n \"This new chlorophyll , together with pigments bound by PsaL , probably participates in energy transfer into the core ( Figure 3A ) , suggesting that PsaH is not simply a ‘landing pad’ for LHCII , but is also important for energy transfer into the core during state II .\",\n \"Additional pigments bound by PsaA in close proximity to subunit PsaK ( the structure of which is now almost completely defined ) provided the first accurate description of this binding site and suggest a mechanism for energy transfer into the core antenna through the PsaK side ( Figure 3—figure supplement 1 ) .\",\n \"On the luminal side of the membrane , the new position of the N-terminus of PsaH suggests that this subunit also directly contributes to PC binding .\",\n \"One of the distinguishing characteristics of the eukaryotic PSI is the stable complex it forms with its electron donor PC , which results in a thousand-fold acceleration of the electron transfer rate ( Bottin and Mathis , 1985 ) .\",\n \"Three elements make up the PC binding site: the first is a positive patch located along the helix-turn-helix N-terminal domain of PsaF ( Hippler et al . , 1997 , 1998; Ben-Shem et al . , 2003 ) .\",\n \"The second element is a hydrophobic patch composed of two parallel helices in PsaA and PsaB ( Figure 2B ) ( Sommer et al . , 2006; Kuhlgert et al . , 2012 ) .\",\n \"The third conserved feature of the PC binding site is a PsaA luminal loop protruding from the generally flat luminal surface of PSI .\",\n \"This loop is found in both plants and cyanobacteria PSI; the only exception being sequences from marine Prochlorococcus and their phages ( Mazor et al . , 2012 ) .\",\n \"As seen in Figure 3C , D , the binding site of the cyanobacterial and plant complexes are similar .\",\n \"However , it is clear that the plant binding site is buried deeper in the complex , this is achieved by the extension of the PsaF N-terminal and by the new position of the N-terminus of PsaH , which forms a loop mirroring the conformation of the conserved luminal PsaA loop , suggesting a direct role for PsaH in PC binding .\",\n \"The PSI core is a highly efficient hub onto which diverse antennae systems connect such as phycobilisomes and IsiA-like assemblies in cyanobacteria and red algae and LHC type antennae in eukaryotes ( Berera et al . , 2009; Engelken et al . , 2010; Nelson and Junge , 2015; Wahadoszamen et al . , 2015 ) .\",\n \"Remarkably , the core pigment organization is conserved across kingdoms despite this diversity in connected antenna ( Amunts et al . , 2007; Busch and Hippler , 2011; Croce and van Amerongen , 2013 ) , which suggests that the connection points between the core and the antennae are conserved .\",\n \"The existence of red-absorbing pigments ( or ‘red traps’ ) is a general property of PSI ( Morosinotto et al . , 2005; Wientjes et al . , 2012 ) .\",\n \"These pigments affect the rate of trapping in PSI and can affect the path of excitation migration in the complex .\",\n \"Most of the eukaryotic red pigments have been shown to reside at LHCI .\",\n \"However , red pigments may be lost from the core complex during the isolation of LHCI .\",\n \"The first high-resolution PSI structure from thermophilic cyanobacteria revealed the organization of the core antenna ( Jordan et al . , 2001 ) .\",\n \"A stacked chlorophyll trimer supported by an extended loop in PsaB was the best candidate for one of the strong red absorbers in this complex ( Jordan et al . , 2001 ) .\",\n \"PsaB sequences from eukaryotes and many cyanobacteria lack this extended loop , resulting in this chlorophyll trimer being lost , as has been shown in the plant and mesophilic PSI structures ( Amunts et al . , 2010; Mazor et al . , 2014 ) .\",\n \"At the current resolution , we observed new core chlorophyll bound between PsaG and Lhca1 and a newly discovered lipid ( Figure 4A ) .\",\n \"This new chlorophyll restores the stacked chlorophyll trimer independent of the shortened PsaB loop and is responsible for one of the connection points between the core complex and the LHCI antenna , with a Mg–Mg distance of 12 . 5 Å between it and chlorophyll 1010 in Lhca1 ( the nomenclature for LHCII is used to describe Lhcas [Standfuss et al . , 2005] ) .\",\n \"On the stromal side of the membrane , an additional chlorophyll trimer first discovered in Synechocystis is also responsible for an antenna attachment point with a Mg–Mg distance of 13 . 7 Å between the core chlorophyll A40 and chlorophyll 1005 in Lhca1 ( Figure 4—figure supplement 1 ) .\",\n \"We suggest that chlorophyll trimers located at the periphery of the core antenna are extremely important for antenna attachment and are probably general attachment points to the core that are utilized not only by eukaryotes , but also by other antenna systems in cyanobacteria . 10 . 7554/eLife . 07433 . 008Figure 4 . Antenna connections in PSI-LHCI .\",\n \"( A ) The configuration of the PsaG-Lhca1 pigment connection shown from the luminal side of the membrane .\",\n \"The new stacked chlorophyll trimer ( numbered B1231 , B1232 and G1003 ) is shown .\",\n \"The N-terminus of PsaG ( dark red ) supports one of the chlorophylls making up this trimer .\",\n \"The entire trimer is connected with chlorophyll 10 ( numbered 1010 ) in Lhca1 ( blue ) .\",\n \"( B ) The second LHCI-PSI connection between Lhca1 and PsaF ( magenta ) on the luminal side of the membrane is bound by a lipid ( orange ) .\",\n \"( C ) The Lhca3 ( red ) -PsaA connection .\",\n \"Two chlorophyll pairs mediate this interaction .\",\n \"At the lumen face , 13 . 7 Å separate chlorophyll 3010 from chlorophyll 1114 .\",\n \"On the stromal side , chlorophyll 3005 and chlorophyll 1108 are 16 . 5 Å apart .\",\n \"( D ) Lhca2 ( blue ) —PsaJ ( green ) connecting chlorophylls . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 00810 . 7554/eLife . 07433 . 009Figure 4—figure supplement 1 . LhcA1 connects to PSI through chlorophyll trimers . The second chlorophyll trimer connecting Lhca1 to the core on the stromal side of the membrane bound by a short PsaB helix ( teal ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 00910 . 7554/eLife . 07433 . 010Figure 4—figure supplement 2 . PsaG occupy roughly an equivalent position compared to previous PSI-LHCI structures ( PsaG from 2WSC in cyan , PsaG from the current structure in red ) .\",\n \"However the polarity of the transmembrane helices is reversed , as a result the N-terminus which was protruding outward from the complex now faces LHCI .\",\n \"Three chlorophylls , a carotene and a lipid are coordinated by this subunit in the current structure . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 010 In contrast to the previous plant structures , which included a small pool of ‘Gap chlorophylls’ , only six pigment pairs connect LHCI to the core antenna in the current structure .\",\n \"Lhca1 is the main connector for excitation transfer , harboring three chlorophylls that are within 14 Å of reaction center pigments ( Figure 4A , B and Figure 4—figure supplement 1 ) .\",\n \"This close proximity ensures efficient and fast energy transfer .\",\n \"Surprisingly , Lhca3 is also one of the main connection points with two such pairs ( Figure 4C ) .\",\n \"The final excitonic connection between PSI to LHCI is located between chlorophyll J1302 bound by PsaJ and chlorophyll 2010 ( A chlorophyll b molecule ) .\",\n \"The Mg–Mg distance of this pair is quite large ( 17 . 6 Å ) however , since the gap between LHCI and PSI allows for some movement , this distance can change to provide an efficient link between the core and LHCI ( Figure 4D ) .\",\n \"To summarize , the extremely fast and efficient energy transfer processes that typify PSI-LHCI occurs through only six pairs of pigment molecules located at three sites .\",\n \"These sites connect to the core antennae at the PsaG and PsaK poles through Lhca1 and Lhca3 , with Lhca2 playing a relatively minor role .\",\n \"Our structure includes the fully modeled LHCI belt with nearly complete structures of all four Lhca proteins .\",\n \"These structures reveal the essential features of the specific interactions between: each Lhca protein and the core; the red chlorophyll assembly present in Lhca4 and Lhca3; and a previously unknown pigment binding site , which is the probable site for the recently discovered non-photochemical quenching ( NPQ ) at the luminal gap region of LHCI ( de Bianchi et al . , 2010; Ballottari et al . , 2014 ) .\",\n \"The LHCI belt is located on the PsaF side of the PSI core .\",\n \"On the stromal face of the membrane , the four conserved N-terminal domains connect each Lhca subunit to its neighbor through interaction with an Lhca-specific loop ( loop 23 ) ( Figure 5A ) that follows immediately after the second transmembrane helix and supports a new chlorophyll site ( numbered\",\n \"16 ) in Lhca4 and Lhca2 ( Figure 6A , B ) .\",\n \"Lhca1 , which interacts with PsaG in the core complex , completely lacks this loop and instead contains a short , positively charged linker in this region ( Figure 5A ) .\",\n \"One of the major changes in the structure of LHCI is the reversal in the polarity of PsaG .\",\n \"While PsaG occupies roughly an equivalent position compared to previous PSI-LHCI structures , the first transmembrane helix contacts Lhca1 while the second one contacts PsaB ( Figure 4—figure supplement 2 ) .\",\n \"Connections between each Lhca and the core are mediated by small regions immediately preceding the first transmembrane helix and are stabilized by salt bridges between negatively charged residues positioned at the membrane entrance point and a conserved arginine located at a helix turn below them ( Figure 5A ) .\",\n \"Additional Lhca–Lhca interactions are provided by a short C-terminal segment on the luminal side that binds helix 2 as it exits the membrane , mainly via hydrophobic interactions ( Figure 5—figure supplement 1 ) . 10 . 7554/eLife . 07433 . 011Figure 5 . Lhcas connect to the PSI core through conserved structural elements .\",\n \"( A ) A view from the stromal side of the membrane of LHCI .\",\n \"Each Lhca connects to the next one through its conserved N-terminal domain ( marked N ) .\",\n \"All connections to the core are mediated by the short region preceding the entrance of the first transmembrane helix into the membrane ( marked by a circle ) .\",\n \"The extended loop ( L23 ) is conserved in all Lhcas except Lhca1 .\",\n \"( B ) Electrostatic interactions determine the specificity of the Lhca4/5 binding site to PsaF .\",\n \"The conserved E84-R209 interaction occurs within the PsaF ( magenta ) domain , which is partially membrane-buried , and Lhca4 ( raspberry ) .\",\n \"( C ) Superposition of Lhca3 ( red ) and LHCII ( cyan ) .\",\n \"Lhca3 interacts with the core via contacts with PsaA and PsaK through short extension ( red circle ) or deletion ( green circle ) in the otherwise highly conserved N terminus .\",\n \"The extension of loop 23 is also seen at the bottom left corner of the image . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 01110 . 7554/eLife . 07433 . 012Figure 5—figure supplement 1 . Luminal side connections between the core PSI and LHCI . Short sequences immediately preceding the insertion point of the second transmembrane helix in the LHC fold ( marked by a red spot ) interact with the C-terminal sequences of the adjacent subunit ( marked with a blue spot ) .\",\n \"Numbers indicate the sequence range shown in bold for the respective Lhca subunit . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 01210 . 7554/eLife . 07433 . 013Figure 6 . The interface within the Lhca heterodimers provides a mechanism for NPQ .\",\n \"( A and B )\",\n \"Structure of the Lhca1/4 ( A ) and Lhca2/3 ( B ) heterodimer as viewed from the membrane plane extrinsic to the PSI-LHCI complex .\",\n \"The conserved connecting carotenoid in position L3 ( pink ) serves a structural role , connecting the two subunits of the dimer by linking chlorophyll 6 ( bottom right ) to chlorophyll 9 ( upper left corner ) .\",\n \"The position of the new chlorophyll site bound by loop 23 of Lhca2 ( numbered 2016 ) is also shown .\",\n \"( C and D )\",\n \"A new chlorophyll site connects the pigments of the heterodimer .\",\n \"Chlorophyll 17 ( numbered 4017 or 3017 ) is shown as viewed from the luminal side of the membrane .\",\n \"The connections between the red pigment cluster on the right and position 9 in the adjacent subunit are marked with grey lines .\",\n \"The new carotenoid site , L5 , is shown in C . This site lines the gap between LHCI and PSI and is therefore easily interchangeable .\",\n \"The distance between L5 and of chlorophylls 17 π systems is 3 . 7 Å .\",\n \"( E ) Lhca3 is colored in red .\",\n \"A short helix segment from the first transmembrane helix is shown as sticks .\",\n \"Chlorophyll 3005 is part of the red-absorbing dimer .\",\n \"LHCII ( PDB: 2BHW ) was superimposed on the structure and shown in faint grey .\",\n \"Chlorophyll 612 is shown as a ring .\",\n \"The coordinating side chain was changed from histidine 68 in LHCII to asparagine 109 in Lhca3 .\",\n \"Another important change is the leucine to glycine modification at position 105 , which allows chlorophyll 3005 to alter its ring orientation by approximately 12° .\",\n \"( F ) The same site in Lhca4 shown in a similar fashion with Lhca4 colored in raspberry .\",\n \"The same basic coordination is observed , though the ring tilt is smaller because of the alanine occupying position 95 . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 01310 . 7554/eLife . 07433 . 014Figure 6—figure supplement 1 . Identification of chlorophyll b molecules . Shown is the chlorophyll b occupying position 10 from Lhca2 .\",\n \"Simulated annealing omit maps were calculated on a model which lacked the formyl-7 group ( shown in the image ) .\",\n \"All b factors of the model were reset to the Wilson B factor , followed by a single round of simulated annealing and refinement using phenix . refine .\",\n \"The difference map ( green ) is contoured at 2 . 5 rmsd and the 2Fo − Fc map ( blue ) is contoured at 1 . 2 rmsd . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 014 Genetic analysis in plants has revealed that each subunit has a specific binding site and , with the exception of the Lhca4-Lhca5 pair , the various Lhcas are not interchangeable ( Lucinski et al . , 2006; Wientjes et al . , 2009 ) .\",\n \"The specific binding sites are identified in the current structure .\",\n \"At the PsaG pole of LHCI , helix C of Lhca1 interacts with the first transmembrane helix of PsaG .\",\n \"Additional protein–protein interactions occurred between a stromal loop of PsaB ( aa 307–320 ) and the N-terminus of Lhca1 ( Figure 5A ) .\",\n \"The next contact point between LHCI and the core occurs between the N-terminus of Lhca4 and the C-terminus of PsaF , the conformation of which is almost identical to the conformation found in cyanobacteria .\",\n \"This interaction consists of hydrophobic patches surrounding charged residues in the membrane-buried regions of both proteins .\",\n \"This binding site should be shared between Lhca4 and Lhca5 and indeed , arginine 209 of PsaF and glutamate 84 of Lhca4 form electrostatic interactions in the middle of the hydrophobic binding site ( Figure 5B ) .\",\n \"This residue is conserved in Lhca5 as well , but absent from Lhca1-3 sequences , explaining the specificity of the interaction .\",\n \"The PsaF-Lhca4 interaction in the current structure does not include any pigments .\",\n \"Energy transfer from Lhca4 to the core probably occurs through the Lhca1-PsaB pigment cluster or the Lhca2-PsaJ clusters .\",\n \"Lhca2 is connected to the core almost exclusively through interactions with the N-terminus of PsaJ on the stromal side of the membrane ( Figure 5—figure supplement 1 ) .\",\n \"The main contact point of Lhca3 is in the N-terminus of PsaA , which contacts a small patch just before helix 1 enters the membrane as in all other Lhcas ( Figure 5C ) .\",\n \"The structure shows that this site is the main determinant of Lhca binding to the core .\",\n \"In contrast to the previous PSI-LHCI model , Lhca3 follows the general fold of LHCII ( rmsd 0 . 8 Å between the two apoproteins ) .\",\n \"Departures from the LHCII fold are seen in key contact points where small loops were extended or deleted from the otherwise conserved N-terminus domain to facilitate protein–protein interactions with the core ( Figure 5C ) .\",\n \"All four Lhcas are remarkably similar to each other and to LHCII .\",\n \"Most of the differences between them can be explained by their interaction partner , such as the loss of loop 23 in Lhca1 due to its binding to PsaG .\",\n \"Two key differences in pigment organization were found between lhcas and other lhcs .\",\n \"The extended loop 23 supports a new chlorophyll-binding site ( numbered 16 ) , common to Lhca4 and Lhca2 ( Figure 6A , B ) and an additional chlorophyll site , coordinated by transmembrane helix two , connects the two partners of each heterodimer .\",\n \"Chlorophyll b pigments are bound by the different Lhca proteins to various extents and serve as antennae pigments .\",\n \"Four binding sites for chlorophyll b were detected in the high resolution structure of LHCII ( Liu et al . , 2004; Standfuss et al . , 2005 ) as well as in the structure of CP29 ( Pan et al . , 2011 ) .\",\n \"We were able to assign nine chlorophyll b sites in LHCI based on electron density maps ( Figure 6—figure supplement 1 ) .\",\n \"All Lhca proteins contain a glutamine to glutamate change ( similarly to CP29 ) in equivalent positions to position 131 of LHCII , this change makes the binding of Chlorophyll b less likely at three sites ( 10 , 12 and 13 ) .\",\n \"In agreement with this change we find that site 12 is occupied by chlorophyll a in all Lhcas and sites 10 and 13 are occupied differentially .\",\n \"The distribution of chlorophyll b sites in LHCI is markedly uneven , with three sites located in Lhca2 ( 2010 , 2011 and 2013 ) , two sites found at Lhca1 ( site 1009 and 1010 ) , three sites at Lhca4 ( 4010 , 4011 and 4013 ) and a single site on Lhca3 ( site 3011 ) .\",\n \"These findings are consistent with mutational data which identified more chlorophyll b sites on Lhca2 then on Lhca3 ( Castelletti et al . , 2003 ) .\",\n \"Site 13 on Lhca1 , 2 and 4 is probably a mixed site , which can accommodate both chlorophyll a and chlorophyll b .\",\n \"The high number of chlorophyll b pigments in Lhca2 and the fact that its closest connection to the core is mediated by chlorophyll b ( site 2010 , shown in Figure 4D ) is consistent with our suggestion that Lhca1 and Lhca3 are the main junctions for excitation energy transfer from LHCI to the PSI core .\",\n \"The dipole orientations of all but two chlorophylls were identified in the current structure .\",\n \"The most significant changes in LHCI compared to LHCII exist in two sites , 2009 and 4009 , where 90° rotations are observed .\",\n \"Such a rotation is expected to impact the energy transfer processes within these LHCI subunits but the significance of this change cannot be ascertained from the rotation alone .\",\n \"Each of the two Lhca1/4 Lhca2/3 heterodimers contains a blue- and red-absorbing subunit .\",\n \"The structures of Lhca3 and Lhca4 clearly show a coordinating asparagine residue unique to this site ( Arnoux et al . , 2009 ) .\",\n \"The configurations of the pigments themselves differed , with the red pigments of Lhca3 and Lhca4 tilted approximately 12° relative to their orientation in LHCII , Lhca1 , and Lhca2 due to a second change from a bulky leucine residue at position 64 of LHCII to glycine and alanine in Lhca3 and Lhca4 , respectively ( Figure 6E , F ) .\",\n \"Additional connections that stabilize LHCI are formed by three luteins at position L3 .\",\n \"These luteins bridge the chlorophylls at site 6 in Lhca1 , Lhca3 , and Lhca4 to PsaG , Lhca2 , and Lhca1 ( Figure 6A , B ) .\",\n \"Photosynthetic eukaryotes respond to high light conditions by decreasing the efficiency of energy transfer from the antennae in a process called NPQ .\",\n \"First discovered in the PSII complex , NPQ was recently reported in PSI .\",\n \"NPQ sites are located on LHC proteins bound by the hydroxylated carotenoid zeaxanthin , which quenches harmful chlorophyll triplets ( Standfuss et al . , 2005 ) .\",\n \"The main excitonic connections in the two Lhca dimers appear to be intimately linked to NPQ .\",\n \"A new chlorophyll site ( numbered\",\n \"17 ) coordinated by a histidine residue ( histidine 150 in Lhca4 and histidine 170 in lhca3 ) is located at the heterodimer interface of both Lhca1/4 and Lhca2/3 .\",\n \"Site 17 links the putative red chlorophyll pair with site 9 of the adjacent complex .\",\n \"In the Lhca1/4 interface , a new carotenoid site ( numbered 4505 or L5 ) forms co-planar π systems ( plane to plane distance 3 . 7 Å ) with the ring of chlorophyll 4017 , positioning it in a configuration that should provide efficient photo-protection from chlorophyll triplets ( Figure 6C , D ) .\",\n \"We propose that this is also the site of NPQ in LHCI , and this fits well with the experimental observation ( Standfuss et al . , 2005 ) that the zeaxanthin responsible for NPQ is located near the red pair in the PSI-LHCI luminal gap region .\",\n \"In response to lumen acidification , zeaxanthin is synthesized from violaxanthin by violaxanthin deepoxidase ( VDE ) as part of the xanthophyll cycle .\",\n \"Both the activity and location of VDE are regulated by pH changes ( Ballottari et al . , 2014 ) .\",\n \"At low pH , VDE is activated and binds the luminal side of the membrane , gaining access to its substrate .\",\n \"On the luminal side , a large gap ( 25 Å ) separates LHCI from the core .\",\n \"This gap stems from the fact that most of the connections between LHCI and PSI are located at the stromal side ( seen in Figure 7C , also compare Figure 5A and Figure 5—figure supplement 1 ) , leaving the luminal side open . 10 . 7554/eLife . 07433 . 015Figure 7 . The surface electrostatic potential of PSI-LHCI .\",\n \"( A ) Luminal view of the electrostatic potential ( red for negative and blue for positive charges ) generated from PSI-LHCI apoproteins .\",\n \"The gap region is shown as an open cavity ( the ligands occupying this cavity were omitted from the calculation ) lined with negative patches .\",\n \"The plastocyanin binding site can be distinguished as a blue patch generated by the positively charged luminal double helix domain of PsaF .\",\n \"( B ) The stromal electrostatic surface of PSI-LHCI showing the putative ferredoxin binding site as a basic patch on the left side of the stromal ridge .\",\n \"( C ) A side view of the stromal gap shows the large opening facing the lumen .\",\n \"Lhca1 and PsaG are omitted to reveal the internal cavity .\",\n \"This surface was drown around all ligands identified in the gap with only chlorophyll 4017 and carotene 4505 omitted .\",\n \"( D ) We propose that the negative patches lining the gap play a role in preventing the accessibility of VDE to gap pigments during regular growth .\",\n \"While at high light conditions , lumen acidification partially neutralizes these negative charges , allowing VDE activity on gap xanthophylls . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 015 The complete modeling of LHCI side chains shows that the luminal side of PSI-LHCI contains patches of negative potential distributed on both sides of the PSI-LHCI gap ( Figure 7 ) .\",\n \"We suggest that the negative charge characteristic of the luminal side of PSI-LHCI is an important feature regulating the accessibility of VDE to the PSI-LHCI gap .\",\n \"Under low light conditions , the relatively high luminal pH will result in strong repulsive forces between these negative patches and the acidic domain of VDE , preventing VDE from binding to PSI .\",\n \"Under high light , acidification of lumen , which is known to trigger VDE activity , will result in partial neutralization of these patches and allow VDE access to the PSI-LHCI gap regions ( Arnoux et al . , 2009 ) ( Figure 7D ) .\",\n \"Carotene L4505 is ideally positioned to be exchanged easily via this mechanism ( Figure 7C ) .\",\n \"Ligands , which are easily exchangeable , are particularly labile and may disassociate from the complex during its purification , accounting for a lack of a second carotene bound at the analogous position in Lhca3 .\",\n \"In the current structure , position L5 is occupied with zeaxanthin; however , the pigment assignment cannot be definite , even at the current resolution .\",\n \"In this work we presented the most complete plant PSI-LHCI structure obtained thus far , revealing the locations of and interactions among its protein subunits and more than 200 non-covalently bound photochemical cofactors .\",\n \"Using the new crystal structure , we examined the network of contacts among the protein subunits from a structural perspective , which provide the basis for elucidating the functional organization of the complex .\"\n ],\n [\n \"Pea seeds ( Pisum sativum var . Kalvadon ) were washed with running water for 6–8 hr and seeded in a vermiculite tray .\",\n \"Plants were germinated in the dark at 22°C for 5 days in shaded sunlight or the dark .\",\n \"After germination , the plants were grown for an additional 10 days under cool-white fluorescent light at a photon flux density of 90–130 μE m−2 s−1 in a 14 hr light/10 hr dark cycle at 22°C .\",\n \"Approximately 200 g of leaves were ground for 25 s in a blender with 1000 ml of ice-cold solution containing 0 . 3 M sucrose , 15 mM NaCl , 30 mM Tricine-NaOH ( pH 8 ) , 1 mM PMSF , 15 μM leupeptin , and 1 μM pepstatin A . The slurry was filtered through eight layers of cheesecloth and chloroplasts pelleted by centrifugation at 1000 g for 9 min .\",\n \"The pellet was suspended in 500 ml of hypotonic medium ( 10 mM Tricine-NaOH , pH 8 ) to disrupt the chloroplasts .\",\n \"Thylakoids were collected by centrifugation at 12 , 000 g for 10 min and resuspended in 500 ml of buffer containing 10 mM Tricine-NaOH ( pH 8 ) and 150 mM NaCl .\",\n \"The thylakoid membranes were then pelleted at 8 , 000 g for 10 min and resuspended in a minimal volume of STN2 buffer ( 0 . 4 M sucrose , 20 mM Tricine-NaOH , pH 8 ) .\",\n \"The thylakoid concentration was adjusted to 3 mg of chlorophyll per ml and 0 . 4% n-dodecyl-α-D-maltoside ( DDM ) added .\",\n \"This concentration of detergent selectively extracts the ATP synthase , b6f , and PSII complexes .\",\n \"After 5 min incubation on ice , the detergent- treated thylakoid membranes were collected by ultra-centrifugation at 200 , 000 g for 30 min .\",\n \"The pellet was resuspended in a minimal volume of STN2 buffer , adjusted to 3 mg chlorophyll per ml , and stored at −80°C .\",\n \"Frozen thylakoid membranes ( 20–25 ml ) containing 3 . 0 mg of chl/ml were thawed in cold water and solubilized with 1 . 5% DDM .\",\n \"Insolubilized material was removed by ultracentrifugation at 120 , 000 g for 15 min .\",\n \"The supernatant was applied to a DEAE-cellulose column ( DE-52 , Whatman , Inc . , 1 . 5 × 18 cm ) pre-equilibrated with 15 mM Tricine-Tris ( pH 8 . 0 ) containing 0 . 25% DDM .\",\n \"The column was washed with the same buffer and PSI eluted with a 0–230 mM tetraethylammonium chloride linear gradient ( 75 ml in each chamber ) in 15 mM Tricine-Tris ( pH 8 . 0 ) containing 0 . 25% DDM .\",\n \"Dark green fractions containing PSI were precipitated by 10% PEG6000 ( Hampton Research , Aliso Viejo , CA ) , followed by centrifugation at 5 , 000 g for 6 min .\",\n \"The pellet was dissolved in 15 mM Tricine-Tris ( pH 8 . 0 ) and 0 . 05% DDM .\",\n \"The green solution was applied to a 10–35% sucrose gradient containing the same buffer and centrifuged using the SW-40 rotor ( Beckman Coulter , Fullerton , CA ) at 37 , 000 rpm ( 170 , 000 g ) for 16 hr .\",\n \"The wide green band containing PSI was collected and loaded onto a second DEAE-cellulose column ( 0 . 5 × 4 cm ) pre-equilibrated with 15 mM Tricine-Tris ( pH 8 . 0 ) and 0 . 05% DDM , mainly to concentrate it for a second sucrose gradient .\",\n \"PSI was eluted with 230 mM tetraethylammonium chloride .\",\n \"The collected dark green fraction was applied to a 10–35% sucrose gradient and centrifuged at 57 , 000 rpm ( 330 , 000 g ) for 4 hr using an SW-60 rotor ( Beckman Coulter ) .\",\n \"Purified PSI appeared as a dark band in the middle of the tube , but only the central part of the band was collected .\",\n \"The material was precipitated with 15% PEG1500 and 100 mM tetraethylammonium chloride and centrifuged at 10 , 000 g for 4 min .\",\n \"The pellet was dissolved in a solution containing 2 mM Tricine ( pH 8 . 75 ) and 0 . 02% n-dodecyl-β-D-thiomaltoside ( DTM , Glycon Biochemicals , Luckenwalde , Germany ) and adjusted to a chlorophyll concentration of 2 . 5 mg/ml .\",\n \"Crystallization was performed manually in 24-well plates using the sitting drop variant of the vapor-diffusion technique at 4°C ( Charles Super Company , Natick , MA ) .\",\n \"Aliquots ( 6–8 μl ) of PSI solution were mixed with equal volumes of reservoir solution ( 50 mM di-potassium phosphate , 50 mM Tris [pH 8] , 12–17% PEG400 , 1% glycerol , 2 mM L-glutathione , and 0 . 03% octyl glucose neopentyl glycol ) and equilibrated against 0 . 5 ml of reservoir solution .\",\n \"Dark green rectangular crystals appeared after 3 days at the higher PEG concentrations , but the best diffracting crystals appeared after 1 month at the lower PEG concentrations .\",\n \"For cryogenic protection , the crystals were moved to a solution containing 50 mM di-potassium phosphate , 50 mM Tris ( pH 8 ) , 20% PEG400 , 2% glycerol , and 2 mM L-glutathione .\",\n \"After a brief incubation the crystals were soaked sequentially in the same buffer containing 5% and 10% glycerol and immediately frozen in liquid nitrogen .\",\n \"X-ray diffraction data were collected at the European Synchrotron Radiation Facility ( ID23- 2 , ID23-1 , ID-29 and MASSIF-3 ) , the Swiss Light Source ( PXI , PXII , and PXIII ) , and BESSYII .\",\n \"Images were collected at 0 . 1° oscillation using full beam at exposures of 0 . 1–0 . 05 s .\",\n \"The large size of the crystals helped mitigate the effect of radiation damage , yet only 60°–90° of data were collected from each crystal using constant translation of the crystal in the beam .\",\n \"Images were integrated using XDS ( Kabsch , 2010 ) and scaled with XSCALE or AIMLESS ( Evans and Murshudov , 2013 ) .\",\n \"Measurements were carried out at the peak of the iron fluorescence scan .\",\n \"Individual datasets generally had I/SIGMA values of ∼1 at 3 . 1 Å with CC1/2 values of ∼0 . 5 calculated by XDS .\",\n \"To obtain a more accurate measure of weak reflections , we combined several datasets measured under similar conditions .\",\n \"This combination was possible because of the consistency of the new crystals .\",\n \"The most effective method for combining datasets was to simply choose the sets with the strongest statistics at lower resolutions , and these crystals were also very similar to each other in terms of their unit cell dimensions .\",\n \"The unified datasets contained 40–100 independent measurements of each reflection with I/SIGMA of ∼1 . 5 at 2 . 8 Å and CC1/2 of ∼0 . 4 at the same resolution ( Table 1 ) .\",\n \"The CCanomalous calculated by XDS or AIMLESS was ∼0 . 3 at 6 Å .\",\n \"Some individual datasets had measurable anomalous signals ( CCanomalous > 0 . 3 ) to 4 . 5 Å , but the final quality of phases was similar , as judged by the figure of merit for substructures from PHASER runs ( typically ∼0 . 6 [McCoy et al . , 2007] ) and visual inspection of the maps . 10 . 7554/eLife . 07433 . 016Table 1 . Data collection and refinement statisticsDOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 016Data collectionBeamlineBESSY PX14 . 1SLS PXI−X06SAESRF ID 23-2Wavelength ( Å ) 1 . 731 . 740 . 873Resolution ( Å ) 40−2 . 8 ( 2 . 85–2 . 8 ) 40−2 . 8 ( 2 . 85−2 . 8 ) 50−3 ( 3 . 1−3 ) Space groupP212121P212121P21Unit cell dimensionsa , b , c ( Å ) 188 . 7 , 200 . 8 , 212 . 4188 . 6 , 201 . 3 , 212 . 7120 . 6 , 189 . 2 , 129 . 7α , β , γ90 , 90 , 9090 , 90 , 9090 , 91 . 1 , 90Measured reflections7 , 831 , 30214 , 841 , 310724 , 010Unique reflections198 , 911200 , 218116 , 039Rpim ( % ) 0 . 051 ( 1 . 276 ) 0 . 030 ( 0 . 180 ) 0 . 099 ( 0 . 812 ) 10 . 5 ( 1 . 2 ) 13 . 4 ( 1 . 4 ) 5 . 8 ( 1 . 3 ) Completeness ( % ) 99 . 9 ( 98 . 4 ) 99 . 9 ( 98 . 4 ) 99 . 9 ( 99 . 2 ) Redundancy39 . 9 ( 37 ) 74 . 1 ( 33 . 4 ) 6 . 2 ( 5 . 4 ) Refinement statisticsResolution ( Å ) 40−2 . 840−2 . 850−3Rwork/Rfree25 . 6/26 . 524/25 . 225 . 8/29 . 3No .\",\n \"of chains161617No .\",\n \"of ligands214214197Average B-factor ( Å2 ) 98 . 711296 . 6R . M .\",\n \"S deviationsBond angles1 . 922 . 4Bond lengths0 . 0040 . 0050 . 011Ramachandran statisticsFavoured region %90 . 290 . 286 . 6Allowed region %7 . 17 . 18 . 7Outlier region %2 . 72 . 74 . 7Data collection , scaling and merging statistics were calculated using XDS , AIMLESS and PHENIX XTRIAGE .\",\n \"Refinement statistics are from PHENIX .\",\n \"The crystals were initially solved by molecular replacement ( MR ) with a partial model containing only the reaction center subunits with chlorophylls modeled as rings using PHASER .\",\n \"This solution was used to place the three iron-sulfur clusters , which were subsequently used as the initial substructure for locating additional sites .\",\n \"These initial runs located approximately 40 sites of the 100 that were eventually modeled .\",\n \"Phases were improved using DM ( Cowtan , 1994 ) , and the resulting maps showed most of the transmembrane helices of the reaction center with 11 transmembrane helices of LHCI ( missing helix 2 of Lhca3 ) .\",\n \"To improve the phases , information from the visible parts of the reaction center was incorporated as a partial MR solution .\",\n \"This model included the 22 transmembrane helices of PsaA and PsaB , the PsaC subunit , and chlorophyll rings , omitting all of the loops from the proteins .\",\n \"From the maps generated in this step , we proceeded to build the model using Coot ( Emsley et al . , 2010 ) and used the modified phases as restraints during refinement in either PHENIX ( Adams et al . , 2010 ) or REFMAC ( Murshudov et al . , 1997 ) .\",\n \"At various points during the refinement process , the phases were recalculated with the newly modeled sites using PHASER and including the improved model ( Read and McCoy , 2011 ) .\",\n \"Final runs identified 99 sites , 89 of them present in the model .\",\n \"The final model refined to an R-free of 25 . 2% with 3% Ramachandran outliers , a considerable decrease from previous values ( Table 1 ) .\",\n \"A complete model for the lhca subunits of LHCI was obtained , as well as extensions and modifications to some of the other PSI subunits ( Table 2 ) .\",\n \"Images were created using Pymol and electrostatic surfaces calculated using APBS ( Baker et al . , 2001 ) . 10 . 7554/eLife . 07433 . 017Table 2 . Amino acid changes between PSI-LHCI structuresDOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 017SubunitNumber of amino acidsModeled amino acidsNumber of changes4Y282WSCPsaA7587417291PsaB7347327321PsaC8180811PsaD*1561401355PsaE*9268659PsaF*15415015418PsaG*98959515PsaH*95846911PsaJ*4241425PsaK*134798510PsaL*16816016121Lhca1*20419316512Lhca22562061763Lhca324221016216Lhca42521971663The number of modeled amino acids in each subunit is shown and compared to the most recent PSI-LHCI structure ( 2WSC ) .\",\n \"Insertions , deletions and extensions are counted as a single change .\",\n \"Since the genome sequence of Pisum Sativum is not completely known ( genes with no DNA data are marked with an * ) we relied on high-throughput mRNA sequence data for verification ( Franssen et al . , 2011 ) .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Most life forms on Earth are supported by solar energy harnessed by oxygenic photosynthesis .","In eukaryotes , photosynthesis is achieved by large membrane-embedded super-complexes , containing reaction centers and connected antennae .","Here , we report the structure of the higher plant PSI-LHCI super-complex determined at 2 . 8 Å resolution .","The structure includes 16 subunits and more than 200 prosthetic groups , which are mostly light harvesting pigments .","The complete structures of the four LhcA subunits of LHCI include 52 chlorophyll a and 9 chlorophyll b molecules , as well as 10 carotenoids and 4 lipids .","The structure of PSI-LHCI includes detailed protein pigments and pigment–pigment interactions , essential for the mechanism of excitation energy transfer and its modulation in one of nature's most efficient photochemical machines ."],"string":"[\n \"Most life forms on Earth are supported by solar energy harnessed by oxygenic photosynthesis .\",\n \"In eukaryotes , photosynthesis is achieved by large membrane-embedded super-complexes , containing reaction centers and connected antennae .\",\n \"Here , we report the structure of the higher plant PSI-LHCI super-complex determined at 2 . 8 Å resolution .\",\n \"The structure includes 16 subunits and more than 200 prosthetic groups , which are mostly light harvesting pigments .\",\n \"The complete structures of the four LhcA subunits of LHCI include 52 chlorophyll a and 9 chlorophyll b molecules , as well as 10 carotenoids and 4 lipids .\",\n \"The structure of PSI-LHCI includes detailed protein pigments and pigment–pigment interactions , essential for the mechanism of excitation energy transfer and its modulation in one of nature's most efficient photochemical machines .\"\n]"},"summary":{"kind":"list like","value":["Most plants , green algae and some bacteria use a process called photosynthesis to convert energy from sunlight into the chemical energy they need to survive and grow .","With this energy , these organisms use carbon dioxide and water to create organic matter and release oxygen into the atmosphere .","Therefore , photosynthesis plays a major role in providing the basis for life on earth .","During photosynthesis , molecules of pigments known as chlorophyll and carotenoid capture the light energy .","These pigments are contained within large groups ( or ‘complexes’ ) of proteins that sit in membrane structures within cells .","Two of the protein complexes—called photosystem I and LHCI—interact with each other to form a ‘supercomplex’ that transfers energy to a small protein called ferredoxin .","To achieve this , the light energy captured by pigment molecules is transferred to other pigment molecules so that the energy is funneled towards the center of photosystem I . Mazor et al . used a technique called X-ray crystallography to create a very detailed three-dimensional model of photosystem I and LHCI from pea plants .","The model shows how the twelve proteins of photosystem I are arranged in relation to the four proteins of the LHCI complex .","The super-complex contains more than 200 other molecules , which are mostly chlorophylls and carotenoids .","Of these , 61 chlorophyll molecules and ten carotenoid molecules are found in LHCI .","The model also provides detailed information about how the pigments interact with each other and with the proteins in the supercomplex .","Mazor et al . 's detailed model may help us to understand how these interactions allow photosystem I to harvest light energy with almost 100% efficiency , and aid efforts to develop new technologies that harness light ."],"string":"[\n \"Most plants , green algae and some bacteria use a process called photosynthesis to convert energy from sunlight into the chemical energy they need to survive and grow .\",\n \"With this energy , these organisms use carbon dioxide and water to create organic matter and release oxygen into the atmosphere .\",\n \"Therefore , photosynthesis plays a major role in providing the basis for life on earth .\",\n \"During photosynthesis , molecules of pigments known as chlorophyll and carotenoid capture the light energy .\",\n \"These pigments are contained within large groups ( or ‘complexes’ ) of proteins that sit in membrane structures within cells .\",\n \"Two of the protein complexes—called photosystem I and LHCI—interact with each other to form a ‘supercomplex’ that transfers energy to a small protein called ferredoxin .\",\n \"To achieve this , the light energy captured by pigment molecules is transferred to other pigment molecules so that the energy is funneled towards the center of photosystem I . Mazor et al . used a technique called X-ray crystallography to create a very detailed three-dimensional model of photosystem I and LHCI from pea plants .\",\n \"The model shows how the twelve proteins of photosystem I are arranged in relation to the four proteins of the LHCI complex .\",\n \"The super-complex contains more than 200 other molecules , which are mostly chlorophylls and carotenoids .\",\n \"Of these , 61 chlorophyll molecules and ten carotenoid molecules are found in LHCI .\",\n \"The model also provides detailed information about how the pigments interact with each other and with the proteins in the supercomplex .\",\n \"Mazor et al . 's detailed model may help us to understand how these interactions allow photosystem I to harvest light energy with almost 100% efficiency , and aid efforts to develop new technologies that harness light .\"\n]"},"year":{"kind":"string","value":"2015"}}},{"rowIdx":4292,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["cell biology"],"string":"[\n \"cell biology\"\n]"},"title":{"kind":"string","value":"Cytoplasmic translocation of the retinoblastoma protein disrupts sarcomeric organization"},"id":{"kind":"string","value":"elife-01228-v1"},"sections":{"kind":"list like","value":[["Skeletal muscle degeneration , which is characterized by the progressive depletion of muscle strength , occurs in a variety of chronic diseases including advanced cancer , congestive heart failure , and AIDS ( Tisdale , 2002 ) .","The underlying intercellular mechanism is currently thought to be multifactorial .","Inflammatory cytokines , particularly TNF-α , have been shown to be key mediators of cancer-related skeletal muscle degeneration ( Tisdale , 2002; Seruga et al . , 2008 ) .","Elevated levels of TNF-α precede the onset of cancer-related skeletal muscle degeneration and act through several cancer-related signaling pathways such as the p53 and nuclear factor kappa B ( NF-κB ) pathways ( Guttridge et al . , 2000; Cai et al . , 2004; Schwarzkopf et al . , 2006 ) .","Retinoblastoma protein ( Rb ) prevents tumor formation by inducing differentiation , controlling cell-cycle progression , and maintaining genomic stability ( Burkhart and Sage , 2008 ) .","To date , numerous studies of Rb function have focused on the transcriptional regulation of E2F .","Rb forms a transcriptional repressor complex with two protein groups , E2F transcription factors and LXCXE motif-containing proteins ( Halaban , 2005; Burkhart and Sage , 2008 ) .","Rb activity is regulated by sequential phosphorylation on several serine and threonine residues , first by cyclin D/cyclin-dependent kinase 4 ( CDK4 ) and then by cyclin E/CDK2 complexes ( Halaban , 2005 ) .","This serial phosphorylation of Rb induces dissociation of the transcriptional repressor complex , allowing expression of E2F-target genes , which are required for many cellular processes .","Loss of Rb function in many cancer cells is frequently caused by aberrant CDK-mediated phosphorylation ( Chau and Wang , 2003; Burkhart and Sage , 2008 ) .","Consequently , selective CDK inhibition is considered a potentially useful approach for cancer treatment ( Malumbres and Barbacid , 2009 ) .","In addition , inactivation of Rb , which is induced by TNF-α treatment , has been shown to lead to various cellular behaviors including proliferation of vascular smooth muscle cells ( Rastogi et al . , 2012 ) and apoptosis of fibroblasts and aortic endothelial cells ( Chau and Wang , 2003; Rastogi et al . , 2012 ) .","Recently , a non-nuclear function of Rb has been reported; where Rb at the mitochondria participates in TNF-α-induced apoptosis ( Hilgendorf et al . , 2013 ) .","However , it is still unknown whether the Rb pathway is involved in cancer-related skeletal muscle degeneration mediated by TNF-α .","A sarcomere is the basic functional unit of striated muscle and consists of two sets of filaments: thick and thin ( Squire , 1997; Gautel , 2011 ) .","The thick filaments are composed of myosin proteins and the thin filaments are assembled from polymerized actin monomers , called filamentous actin ( F-actin ) .","The contractile activity of skeletal muscle is achieved through the actin and myosin filaments sliding past one another ( Squire , 1997 ) .","The motor function of striated muscle therefore , requires the well-ordered assembly of sarcomeres , which is closely tied to the highly organized actin cytoskeleton .","The formins are a large family of proteins and are characterized by the presence of the conserved formin homology 2 ( FH2 ) domain ( Kovar , 2006; Campellone and Welch , 2010 ) .","The FH2 domain promotes actin nucleation and polymerization , thereby producing long straight actin filaments and regulating cytoskeletal organization .","It has been reported that several members of the formin family serve as key regulators of actin dynamics during sarcomeric organization in striated muscle ( Taniguchi et al . , 2009; Kan et al . , 2012; Mi-Mi et al . , 2012 ) .","In this study , we present a potential mechanism underlying TNF-α-induced skeletal muscle degeneration .","We propose a novel function for Rb; where Rb disrupts sarcomeric organization in human skeletal muscle myotubes ( HSMMs ) following its phosphorylation and translocation into the cytoplasm .","Our study implicates the tumor suppressor protein in the regulation of cytoskeletal organization ."],["To gain insights into the role of Rb in cancer-related skeletal muscle degeneration , we first examined the phosphorylation kinetics and subcellular localization of Rb in TNF-α-treated HSMMs .","For this purpose , cells were pretreated with interferon-gamma ( IFN-γ , 100 ng/ml ) for 8 hr prior to initial TNF-α treatment in order to promote cellular sensitivity to the effects of TNF-α ( Tsujimoto et al . , 1986 ) .","During differentiation from myoblasts to myotubes ( from day 0 to day 4 ) , Rb shifted from a phosphorylated to an unphosphorylated state ( Figure 1A ) .","Moreover , following TNF-α treatment Rb phosphorylation on the CDK4-specific phosphorylation site , serine 780 ( Kitagawa et al . , 1996 ) , was induced ( Figure 1A ) .","This was not observed on threonine 821 , a CDK2-specific phosphorylation site ( Halaban , 2005 ) .","In untreated HSMMs , Rb was primarily present in the nucleus , but translocated to the cytoplasm after TNF-α treatment ( Figure 1B , C ) .","In addition , we found that phosphorylated Rb in TNF-α-treated HSMMs was predominantly located in the cytoplasm ( Figure 1D ) .","In accordance with the induction of Rb phosphorylation on a CDK4-specific phosphorylation site , TNF-α treatment led to an increase in the level of nuclear CDK4 ( Figure 1E ) .","The vast majority of nuclear Rb was in an unphosphorylated state , while cytoplasmic Rb that accumulated after TNF-α treatment was in a phosphorylated state ( Figure 1E ) .","We then tested whether TNF-α-induced cytoplasmic accumulation of Rb is caused by CDK4-mediated Rb phosphorylation .","When CDK4 was depleted by short hairpin RNA ( shRNA ) , cytoplasmic accumulation of Rb induced by TNF-α was decreased ( Figure 1F–H ) .","These results suggest that phosphorylation of Rb by CDK4 triggers its cytoplasmic translocation in HSMMs . 10 . 7554/eLife . 01228 . 003Figure 1 . TNF-α induces cytoplasmic translocation of Rb .","( A–E )","HSMMs were treated with TNF-α for 2 days .","( A ) CDK4-mediated phosphorylation of Rb is induced by TNF-α treatment .","At the indicated time points after differentiation stimuli , total cell lysates were prepared and immunoprecipitated with anti-Rb antibody , followed by immunoblotting .","TNF-α was added for the last 2 days of 6-day cultures .","The open and solid arrowheads indicate the position of phosphorylated and unphosphorylated Rb , respectively .","( B ) Cytoplasmic translocation of Rb is caused by TNF-α treatment .","Z-stack confocal images of Rb and 4′ , 6-diamidino-2-phenylindole ( DAPI ) -stained nuclei were obtained ( 50 slices at 0 . 3-μm intervals ) .","X-Y section images for Rb and DAPI-stained nuclei and X-Z section images for Rb along yellow lines in the X-Y section images are shown .","Scale bar , 20 μm .","( C ) Line plots denote the fluorescence intensities of Rb ( red lines ) and DAPI ( blue lines ) along the yellow lines in B . Intensity values were normalized by the maximum value of each plot .","a . u . , arbitrary units .","( D ) Phosphorylated Rb is localized in the cytoplasm .","Confocal images for Rb phosphorylated at S780 ( red ) and DAPI-stained nuclei ( blue ) .","Scale bar , 20 μm .","( E ) Nuclear CDK4 expression is increased after TNF-α treatment .","Cytoplasmic ( Cyto ) and nuclear ( Nuc ) lysates were analyzed by immunoblotting with the antibodies indicated .","Tubulin and TFIIB were used as loading controls for cytoplasmic and nuclear lysates , respectively .","The open and solid arrowheads indicate the position of phosphorylated and unphosphorylated Rb .","( F–H )","HSMMs were infected with adenoviruses expressing control non-target shRNA or shRNA against CDK4 at a MOI of 10 pfu/nucleus and then treated with TNF-α for 2 days .","( F ) Experimental design and reference time frame .","( G ) The cytoplasmic ( Cyto ) and nuclear ( Nuc ) lysates were analyzed by immunoblotting .","( H ) TNF-α-induced cytoplasmic translocation of Rb is prevented by CDK4 depletion .","The cytoplasmic and nuclear lysates were subjected to immunoprecipitation and probed by immunoblotting .","The open and solid arrowheads indicate the position of phosphorylated and unphosphorylated Rb , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 003 In human skeletal muscle myoblasts , Rb was mainly in a phosphorylated state ( Figure 1A , day 0 ) , although the localization of S780-phosphorylated Rb was confined to the nucleus ( Figure 2A ) .","Lamina-associated polypeptide ( LAP ) 2α , a binding partner of nucleoplasmic A-type lamins , interacts with S780-phosphorylated Rb in mitotic myoblasts ( Markiewicz et al . , 2005 ) and is known to play an important role in nuclear tethering of Rb ( Markiewicz et al . , 2002 ) .","Indeed , when LAP2α was depleted by shRNA in human myoblasts , Rb localized to the cytoplasm as well as the nucleus ( Figure 2B ) .","Given the level of LAP2α decreased during muscle differentiation ( Markiewicz et al . , 2005 ) ( Figure 2C ) , these results suggest that low-level LAP2α expression in HSMMs facilitates the cytoplasmic translocation of Rb .","In many types of cancer cells , Rb exists predominantly in a phosphorylated state , but is primarily localized in the nucleus .","It has been reported that LAP2α is an E2F-target gene and its expression is enhanced in cancer cells ( Parise et al . , 2006; Ward et al . , 2011 ) .","Overexpressed LAP2α may therefore serve to tether Rb to the nucleus in cancer cells and the cytoplasmic translocation of Rb may be triggered in cells that express low levels of LAP2α , such as terminally differentiated cells . 10 . 7554/eLife . 01228 . 004Figure 2 . Loss of LAP2α affects the cytoplasmic translocation of Rb .","( A ) Phosphorylated Rb is localized in the nucleus in human skeletal muscle myoblasts .","Confocal images for Rb phosphorylated at S780 and DAPI-stained nuclei .","Scale bar , 20 μm .","( B ) Rb is translocated to the cytoplasm by LAP2α depletion .","Human skeletal muscle myoblasts were transfected with an mCherry-HA-Rb expression plasmid together with an shRNA expression plasmid against eGFP or LAP2α .","Confocal images for mCherry , LAP2α and DAPI-stained nuclei .","The arrowheads indicate transfected cells .","Scale bar , 20 μm .","( C ) LAP2α expression is decreased in HSMMs .","Total cell lysates from human skeletal muscle myoblasts and HSMMs were subjected to immunoblotting . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 004 We next tested whether the motor function of skeletal muscle is affected under this condition .","In response to electric pulse stimulation ( EPS ) , which evokes a contractile reaction in myotubes ( Fujita et al . , 2007 ) , approximately 55% of HSMMs displayed beating , an index of contractile reaction ( Figure 3A; Video 1 ) , while it was hardly observed after TNF-α treatment ( Figure 3A; Video 2 ) .","The beating was not induced in mitotic myoblasts or prematurely differentiated myoblasts implying that the sarcomeric structure , which is essential for the contractile activity of muscle cells ( Squire , 1997 ) , is organized in HSMMs .","To evaluate the effect of TNF-α on the periodic assembly of sarcomeres , we examined the distribution of α-actinin , a major component of Z-disks , as Z-disks define the lateral borders of individual sarcomeres ( Gautel , 2011 ) .","In untreated HSMMs , α-actinin was observed at evenly spaced intervals , whereas in TNF-α-treated HSMMs the peak-to-peak distance in line plots of α-actinin intensity along the myofibril was larger and inconsistent ( Figure 3B–D ) .","Furthermore , when the repeating pattern of α-actinin distribution was analyzed using an autocorrelation image processing technique , peak values were much lower in TNF-α-treated HSMMs ( Figure 3E ) .","These results indicate that the periodic assembly of sarcomeres is disrupted by TNF-α treatment . 10 . 7554/eLife . 01228 . 005Figure 3 . TNF-α disrupts sarcomeric organization .","( A–E )","HSMMs were treated with TNF-α for 2 days .","( A ) Contractile activity of HSMMs is impaired by TNF-α treatment .","EPS was applied to HSMMs .","The percentage of beating cells from a total of 100 HSMMs is shown .","Results are presented as mean ± SD from three independent experiments .","*p<0 . 002 , determined by the Student’s t-test .","( B–E )","Sarcomeric organization of HSMMs .","Confocal images for α-actinin ( B ) and merged images of F-actin ( green ) and α-actinin ( red ) ( C ) are shown .","Scale bar , 10 μm in B and 5 μm in C . ( D ) Line plots of α-actinin fluorescence intensity along individual myofibrils ( denoted by yellow lines in C ) .","Intensity values were normalized by the maximum value for each fibril .","a . u . , arbitrary units .","( E ) Autocorrelation analyses of the α-actinin distribution .","Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 00510 . 7554/eLife . 01228 . 006Video 1 . Image of live beating HSMMs in response to EPS . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 00610 . 7554/eLife . 01228 . 007Video 2 . Image of live beating TNF-α-treated HSMMs in response to EPS . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 007 In striated muscle cells , anti-parallel actin filaments spanning the sarcomeres are crosslinked to the Z-disks ( Sparrow and Schock , 2009 ) .","The peak-to-peak distance in line plots of α-actinin intensity therefore reflects a counterbalance between tensile forces generated by the actin filaments .","Sarcomeric disorganization represents an imbalance in the tensile forces , which may be caused by defective actin filament formation .","The close association of accurate sarcomeric organization with actin polymerization was demonstrated by treatment of the cells with the actin polymerization inhibitor cytochalasin D . After 30 min of treatment , the periodic arrangement of α-actinin was not well ordered ( Figure 4A–C ) and was strongly disordered by 60 min ( Figure 4D ) . 10 . 7554/eLife . 01228 . 008Figure 4 . Inhibition of actin polymerization disorganizes sarcomeric assembly .","( A–D )","HSMMs were treated with 2 μM cytochalasin D ( Cyto D ) for 30 min ( A–C ) or 60 min ( D ) at room temperature .","( A ) The periodic arrangement of α-actinin is not well ordered after 30 min of Cyto D-treatment .","Merged images of F-actin ( green ) and α-actinin ( red ) .","Scale bar , 5 μm .","( B ) Line plots of α-actinin fluorescence intensity along individual myofibrils ( denoted by yellow lines in A ) .","Intensity values were normalized by the maximum value for each fibril .","a . u . , arbitrary units .","( C ) Autocorrelation analyses of the α-actinin distribution .","Scale bar , 5 μm .","( D ) The lateral periodicity in the α-actinin distribution is strongly disordered by 60 min of treatment .","Merged images of F-actin ( green ) and α-actinin ( red ) .","Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 008 We then evaluated the role of Rb in the negative regulation of sarcomeric organization using Rb knockdown HSMMs ( Figure 5A , B ) .","Rb is known to be required for myogenic differentiation but not for the maintenance of the terminally differentiated state in myotubes ( Huh et al . , 2004 ) .","In accordance with this , sarcomeric organization was not affected when Rb was depleted by shRNA ( Figure 5C ) .","After TNF-α treatment , the periodic assembly of sarcomeres was disrupted in control shRNA-expressing HSMMs , but TNF-α failed to induce the sarcomeric disorganization in sh-Rb-expressing HSMMs ( Figure 5D–F ) .","Next , we directly examined whether cytoplasmic Rb is capable of disrupting sarcomeric organization .","To this end , HSMMs were infected with adenoviruses expressing a heterologous nuclear exporting signal ( NES ) -fused form of Rb , which was tagged with monomeric red fluorescent protein mCherry and influenza hemaglutinin ( HA ) at its amino-terminus ( mCherry-HA-NES Rb ) .","Given a small population of endogenous Rb was localized in the cytoplasm after TNF-α treatment , we expressed NES Rb at a comparable level to TNF-α-induced cytoplasmic Rb ( Figure 6A , asterisk vs the open arrowhead ) .","Under this condition , sarcomeric organization was not well ordered as in TNF-α-treated HSMMs ( Figure 6B–D ) .","When we expressed higher levels of NES Rb in HSMMs ( Figure 6E , F ) , the periodic arrangement of α-actinin was significantly disordered when compared to mCherry-HA-Rb-expressing cells ( Figure 6G ) .","Taken together , these results suggest that Rb is involved in TNF-α-induced sarcomeric disorganization and TNF-α-induced cytoplasmic Rb may have a pivotal role in this process . 10 . 7554/eLife . 01228 . 009Figure 5 . Rb contributes to TNF-α-induced sarcomeric disorganization .","( A–F )","HSMMs were infected with adenoviruses expressing control non-target shRNA or shRNA against Rb at a MOI of 10 pfu/nucleus and then treated with TNF-α for 2 days .","( A and B )","Depletion of Rb protein was verified by immunoblotting ( A ) and epifluorescence microscopy ( B ) .","The open and solid arrowheads indicate the position of phosphorylated and unphosphorylated Rb , respectively .","Scale bar , 20 μm .","( C ) Confocal images for F-actin and α-actinin .","Scale bar , 10 μm .","( D and E )","TNF-α-induced sarcomeric disorganization is attenuated by Rb depletion .","Confocal images for α-actinin ( D ) and merged images of F-actin ( green ) and α-actinin ( red ) ( E ) .","Scale bar , 10 μm in D and 5 μm in E . ( F ) Autocorrelation analyses of the α-actinin distribution .","Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 00910 . 7554/eLife . 01228 . 010Figure 6 . Sarcomeric organization is impaired by cytoplasmic Rb .","( A–G )","HSMMs were infected with adenoviruses expressing mCherry-HA-Rb or mCherry-HA-NES Rb at a MOI of 10 pfu/nucleus ( A–D ) or 50 pfu/nucleus ( E–G ) for 4 days .","( A ) The expression of exogenous Rb proteins .","Cytoplasmic ( Cyto ) and nuclear ( Nuc ) lysates were prepared from infected HSMMs in parallel with TNF-α-treated HSMMs .","The lysates were subjected to immunoprecipitation and probed by immunoblotting .","The open and solid arrowheads indicate the position of phosphorylated and unphosphorylated Rb .","Asterisk indicates the position of exogenous Rb .","( B ) Sarcomeric structure is not well ordered in NES Rb-expressing HSMMs .","Merged images of F-actin ( green ) and α-actinin ( red ) .","Scale bar , 5 μm .","( C ) Line plots of α-actinin fluorescence intensity along individual myofibrils ( denoted by yellow lines in B ) .","Intensity values were normalized by the maximum value for each fibril .","a . u . , arbitrary units .","( D ) Autocorrelation analyses of the α-actinin distribution .","Scale bar , 5 μm .","( E and F )","The expression and distribution of exogenous Rb proteins were analyzed by immunoblotting ( E ) and confocal microscopy ( F ) .","The open and solid arrowheads indicate the position of exogenous and endogenous Rb , respectively .","Scale bar , 20 μm .","( G ) Sarcomeric structure is strongly disordered in NES Rb-expressing HSMMs .","Confocal images for F-actin and α-actinin .","Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 010 Similarly to TNF-α-treated HSMMs , the cytoplasmic translocation of Rb has been shown to be induced by its CDK-mediated phosphorylation in certain types of cancer cells ( Jiao et al . , 2008 ) , although the function of cytoplasmic Rb in the cancer cells has not been described thus far .","In the cancer cells , Rb is phosphorylated on CDK2 phosphorylation sites as well as CDK4 phosphorylation sites , whereas our data showed that Rb phosphorylation on the CDK2-specific phosphorylation site , threonine 821 , was not induced by TNF-α treatment ( Figure 1A , day 6 ) .","We therefore reasoned that the selective phosphorylation of Rb by specific CDKs affects the function of cytoplasmic Rb .","Given that it has been reported that threonine 821/threonine 826 phosphorylation disrupts Rb binding to LXCXE motif-containing proteins ( Knudsen and Wang , 1996; Dick and Rubin , 2013 ) , we examined whether the function of cytoplasmic Rb is achieved through its interaction with LXCXE motif-containing proteins .","We introduced mCherry-HA-NES Rb lacking exon 22 ( Rb Δexon 22 ) , which is known to be an LXCXE-binding deficient mutant ( Henley et al . , 2010 ) , into HSMMs and found that the inhibitory effect of NES Rb Δexon 22 on sarcomeric organization was less effective as compared to NES Rb-expressing HSMMs ( Figure 7A ) . 10 . 7554/eLife . 01228 . 011Figure 7 . The function of cytoplasmic Rb is rendered through its interaction with LXCXE motif-containing proteins .","( A ) Sarcomeric structure is not disordered in NES Rb Δexon 22-expressing HSMMs .","HSMMs were infected with an adenovirus expressing mCherry-HA-NES Rb Δexon 22 at a MOI of 50 pfu/nucleus for 4 days .","Confocal images for F-actin and α-actinin .","Scale bar , 10 μm .","( B ) mDia1 contains the LXCXE motif .","Sequence alignment of the LXCXE motif of mDia1 and other known Rb-binding proteins .","( C ) The primary structure of mDia1 and its mutants .","Scheme represents location of GBD and DAD of mDia1 .","Numbers denote amino acid positions in mDia1 isform2 .","The LXCXE motif is present in GBD ( amino acid positions 153 to 157 ) .","ΔGBD/ΔDAD , doubly deleted mDia1 lacking both GBD and DAD .","( D ) The LXCXE motif is required for the in vitro interaction between Rb and mDia1 .","Purified Flag-tagged mDia1 proteins were mixed with full-length recombinant Rb protein and immunoprecipitates were analyzed by immunoblotting .","( E ) Rb interacts with mDia1 after TNF-α treatment .","HSMMs were treated with TNF-α for 2 days .","The cytoplasmic lysates were subjected to immunoprecipitation and probed by immunoblotting .","The open arrowheads indicate the position of phosphorylated Rb .","( F ) Expression and purification of GST fusion Rb proteins .","The purity of bacterially expressed GST-Rb proteins was evaluated by SDS-PAGE , followed by CBB staining .","GST-Rb wild-type protein encompasses amino acids 379–928 .","GST-Rb Mut CDK2 contains serine/threonine to alanine substitutions at CDK2-specific phosphorylation sites ( S612 and T821 ) .","( G ) GST-Rb proteins were preincubated with CDK4/Cyclin D1 and CDK2/Cyclin E proteins in the presence or absence of ATP and then mixed with purified Flag-mDia1 protein .","The interaction between mDia1 and Rb proteins was analyzed by immunoprecipitation with anti-Flag antibody-agarose beads . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 01110 . 7554/eLife . 01228 . 012Figure 7—figure supplement 1 . Identification of NES Rb-binding protein . HSMMs were infected with adenoviruses expressing mCherry-HA-NES Rb WT or mCherry-HA-NES Rb Δexon 22 at a MOI of 50 pfu/nucleus for 4 days .","Total cell lysates from adenovirus-infected HSMMs were immunoprecipitated with anti-HA antibody-conjugated agarose beads .","The bound proteins were analyzed by SDS-PAGE and visualized by CBB staining .","Individual protein bands were excised from SDS-PAGE gel and protein samples were subjected to electrospray ionization mass spectrometric analysis .","The lists of proteins from the bands are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 012 Next , to explore how cytoplasmic Rb disorganizes sarcomeric assembly in HSMMs , we searched for the binding proteins of cytoplasmic Rb using mCherry-HA-NES Rb wild-type ( WT ) -expressing HSMMs .","Total cell lysates from adenovirus-infected HSMMs were immunoprecipitated with anti-HA antibody-conjugated agarose beads and the bound proteins were subjected to electrospray ionization mass spectrometric analysis ( Figure 7—figure supplement 1 ) .","Among the proteins identified , we focused on mammalian diaphanous-related formin 1 ( mDia1 ) , a potent actin nucleation factor ( Watanabe et al . , 1997 ) , as the LXCXE motif is contained in the GTPase binding domain ( GBD ) of mDia1 ( Figure 7B , C ) .","It is noteworthy that the band including mDia1 was hardly detected in mCherry-HA-NES Rb Δexon 22-expressing HSMMs ( Figure 7—figure supplement 1 , band 1 ) .","Data from purified wild-type and LXCXE motif-deleted mutant mDia1 proteins ( WT and ΔLXCXE ) ( Figure 7C ) showed that binding of mDia1 to Rb was abolished by deletion of the LXCXE motif ( Figure 7D ) .","Cytoplasmic Rb , which was phosphorylated and accumulated after TNF-α treatment , interacted with mDia1 ( Figure 7E ) .","We then examined whether the selective phosphorylation of Rb affects this interaction .","Unphosphorylated Rb protein containing the large pocket , an important domain for interactions with a variety of cellular proteins ( Burkhart and Sage , 2008 ) , bound to mDia1 in vitro and the binding level was strongly reduced according to its phosphorylation catalyzed by cyclin D/CDK4 and cyclin E/CDK2 complexes ( Figure 7F , G ) .","Although differences in the patterns of Rb phosphorylation by cyclin D/CDK4 and cyclin E/CDK2 have been reported , we failed to detect the selective phosphorylation of Rb by our in vitro phosphorylation system , which may be due to a supraphysiological activity of CDKs in vitro .","We then tested a mutant Rb protein bearing serine/threonine-to-alanine substitutions in the CDK2-specific phosphorylation sites , serine 612 and threonine 821 ( Mut CDK2 ) .","The mutant Rb exhibited substantial interaction with mDia1 even after phosphorylation ( Figure 7G ) , suggesting that phosphorylation of Rb on CDK4 phosphorylation sites alone does not impair its interaction with mDia1 and TNF-α-induced cytoplasmic Rb has the potential to interact with mDia1 .","Muscle wasting/atrophy accompanies cancer-related skeletal muscle degeneration ( Tisdale , 2002; Acharyya et al . , 2004 ) .","We therefore carried out immunohistological analysis on normal and atrophied tibialis anterior muscles excised from cancer patients and examined the localization of Rb in these muscles ( Figure 8A ) .","In normal muscles , Z-disks were regularly aligned and the sarcomere striation pattern was clearly observed ( Figure 8B , left ) .","In contrast , the Z-disks were misaligned and the sarcomeric banding pattern was not well organized in the atrophied muscles ( Figure 8B , right ) .","With regard to the localization of Rb , it was mainly located in the nucleus in normal muscle cells , but Rb could be observed in the cytoplasm , as well as the nucleus in atrophied muscle cells ( Figure 8C ) .","In both normal and atrophied skeletal muscles , the localization of mDia1 was mainly confined to the Z-disk ( Figure 8B ) , and cytoplasmic Rb observed in atrophied muscles colocalized with mDia1 ( Figure 8C ) . 10 . 7554/eLife . 01228 . 013Figure 8 . Cytoplasmic Rb colocalizes with mDia1 in atrophied skeletal muscle .","( A–C′ )","Normal and atrophied tibialis anterior muscles were surgically excised from cancer patients .","Cross sections ( A ) and longitudinal sections ( B–C′ ) were stained .","( A ) Hematoxylin and eosin-stained cryosections .","Scale bar , 400 μm .","( B–C′ )","Localization of Rb and mDia1 in normal and atrophied tibialis anterior muscles .","Confocal images of cryosections stained with anti-mDia1 and anti-α-actinin antibodies ( B ) or anti-Rb and anti-mDia1 antibodies ( C , magnified in C′ ) .","Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 013 We next tested whether inhibition of mDia1 activity is responsible for the TNF-α-induced disorganization of the sarcomere .","For this purpose , we generated a recombinant adenovirus expressing the constitutively active form of mDia1 ( Watanabe et al . , 1999 ) , which lacks the entire GBD and carboxy-terminal diaphanous autoregulatory domain ( DAD ) ( Figure 7C ) .","We introduced mDia1 ΔGBD/ΔDAD tagged with green fluorescent protein ( GFP-mDia1 ΔGBD/ΔDAD ) into HSMMs and treated them with TNF-α ( Figure 9A ) .","mDia1 ΔGBD/ΔDAD itself did not affect either α-actinin distribution or the contractile reaction of HSMMs ( Figure 9B , C ) .","After TNF-α treatment , however , the lateral alignment of α-actinin in GFP-mDia1 ΔGBD/ΔDAD-expressing HSMMs was well ordered as compared to that in control GFP-expressing HSMMs ( Figure 9D–F ) .","Accordingly , the percentage of beating cells decreased by TNF-α treatment was restored by the introduction of mDia1 ΔGBD/ΔDAD ( p<0 . 02 , determined by the Student’s t-test ) ( Figure 9C ) . 10 . 7554/eLife . 01228 . 014Figure 9 . TNF-α-induced sarcomeric disorganization is prevented by constitutively active mDia1 . ( A–F ) HSMMs were infected with adenoviruses expressing GFP or GFP-mDia1 ΔGBD/ΔDAD at a MOI of 1 pfu/nucleus and then treated with TNF-α for 2 days .","( A ) The expression was analyzed by immunoblotting .","( B ) Confocal images for α-actinin .","Scale bar , 5 μm .","( C ) TNF-α-induced contractile dysfunction is diminished by constitutively active mDia1 .","EPS was applied to HSMMs .","The percentage of beating cells from a total of 100 HSMMs is shown .","Results are presented as mean ± SD from three independent experiments .","*p<0 . 002; **p<0 . 02 , determined by the Student’s t-test .","( D and E )","Constitutively active mDia1 recovers TNF-α-induced sarcomeric disorganization .","Confocal images for α-actinin ( D ) and merged images of F-actin ( green ) and α-actinin ( red ) ( E ) .","Scale bar , 10 μm in D and 5 μm in E . ( F ) Autocorrelation analyses of the α-actinin distribution .","Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 014"],["In this study , we have elucidated a novel pathway of cancer-related skeletal muscle degeneration .","TNF-α induces CDK4 activation and the concomitant phosphorylation of Rb .","Subsequently , cytoplasmic translocation of Rb is triggered .","Cytoplasmic Rb disrupts sarcomeric organization , which may be caused by dysfunction of mDia1 .","The precise role of mDia1 in the regulation of sarcomeric organization is poorly understood ( Sparrow and Schock , 2009 ) , but the contribution of mDia1 to sarcomeric organization is supported .","When mDia1 was depleted by shRNA , the lateral periodicity in the distribution of α-actinin was markedly perturbed ( Figure 10A , B ) .","The importance of actin nucleation factors for the periodic assembly of sarcomeres is proposed by the recent observations that depletion of actin nucleation factors , such as Fhod3 and leiomodin , results in disordered α-actinin distribution in cardiomyocytes ( Chereau et al . , 2008; Taniguchi et al . , 2009; Iskratsch et al . , 2010 ) .","The notion that the degree of sarcomeric disorganization is dependent on the inhibition of actin polymerization is further supported by our data that show Z-disk alignment is more severely impaired by longer-term treatment of cytochalasin D ( Figure 4 ) . 10 . 7554/eLife . 01228 . 015Figure 10 . mDia1 is critical for sarcomeric organization .","( A and B )","HSMMs were infected with adenoviruses expressing control non-target shRNA or shRNA against mDia1 at a MOI of 5 pfu/nucleus for 4 days .","( A ) Depletion of mDia1 protein was verified by immunoblotting .","( B ) Confocal images for F-actin and α-actinin .","Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 015 Phosphorylation of Rb and the subsequent activation of E2F transcriptional activity enhance the expression of E2F-target genes , which include cell-cycle regulators ( e . g . , Tk1 and Dhfr ) .","Although Rb phosphorylated at serine 780 is unable to bind to E2F1 ( Kitagawa et al . , 1996 ) , quantitative PCR ( qPCR ) analysis did not reveal any statistically significant differences in the expression of Tk1 and Dhfr after TNF-α treatment ( Figure 11 ) .","During muscle differentiation , methylation of histone H3 lysine 9 and DNA methylation occur at several E2F-target gene promoters including Tk1 and Dhfr ( Ait-Si-Ali et al . , 2004; Blanchet et al . , 2011 ) .","These epigenetic changes may trigger the permanent silencing of E2F-target gene expression and prevent E2F1 from activating their expression after terminal differentiation . 10 . 7554/eLife . 01228 . 016Figure 11 . The expression of cell-cycle regulators and mitochondrial biogenesis factors after TNF-α treatment . HSMMs were treated with TNF-α for 2 days .","Quantification of the expression of cell-cycle regulators and mitochondrial biogenesis factors .","Results are presented as mean ± SD from three independent experiments .","*p<0 . 02 , determined by the Student’s t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 016 It has been reported that phosphorylation of Rb is induced in the skeletal muscles of mice under fasting conditions ( Blanchet et al . , 2011 ) , which leads to the activation of E2F1 transcriptional activity and increases the expression levels of mitochondrial biogenesis factors ( Ppargc1a and Tfam ) .","In contrast , although TNF-α treatment induced Rb phosphorylation , the expression of Ppargc1a and Tfam decreased after TNF-α treatment ( Remels et al . , 2010 ) ( Figure 11 ) .","These findings suggest that other TNF-α-induced signaling pathways , such as the NF-κB pathway , may affect the transcriptional activity of E2F1 ( Araki et al . , 2008 ) and/or regulation of the expression of mitochondrial biogenesis factors ( Remels et al . , 2010 ) .","The contractile activity of skeletal muscle is caused by the sliding of thick and thin filaments in the sarcomere , and this sliding action is intimately linked to the proper control of adenosine 5’-triphosphate ( ATP ) production ( Squire , 1997 ) .","Because mitochondria are responsible for cellular ATP production , incomplete restoration of the percentage of beating cells by mDia1 ΔGBD/ΔDAD might be ascribed to an impairment of mitochondrial function caused by TNF-α ( Figure 9C ) .","In cancer patients , the circulating levels of TNF-α and IFN-γ would be lower than the amounts used in this study .","Monocytes/macrophages infiltrate in atrophied muscles and may produce considerable amounts of these inflammatory cytokines .","It may therefore be possible that the local concentrations of TNF-α and IFN-γ are elevated in atrophied muscles , which then contributes to phosphorylation of Rb in the long-term .","TNF-α-induced skeletal muscle degeneration is achieved through multiple mechanisms .","The results presented in this study propose a novel non-nuclear function for Rb , which is independent of the transcriptional regulation of E2F and may be involved in TNF-α-induced skeletal muscle degeneration .","In the future , it would be interesting to clarify the role of mDia1 and determine how cytoplasmic Rb disrupts sarcomeric organization ."],["Early passage human skeletal myoblasts purchased from Lonza ( Basel , Switzerland ) were cultured according to the manufacturer’s instructions using Lonza-supplied growth medium ( SkGM-2 BulletKit ) .","Cells grown to approximately 80% confluence were induced to differentiate into multinucleated myotubes by switching to differentiation medium ( DMEM-F12 containing 2% horse serum ) .","Under these conditions , numerous myotubes could be detected on the fourth day .","For TNF-α treatment , HSMMs were cultured in fresh serum-free media containing TNF-α ( 100 ng/ml ) for 2 days .","TNF-α was repeatedly added every 24 hr .","Recombinant human TNF-α and IFN-γ were purchased from PeproTech ( Rocky Hill , NJ ) .","Cytochalasin D was obtained from Sigma ( St . Louis , MO ) .","Immunoprecipitations were performed using anti-Flag antibody- ( M2; Sigma ) and anti-HA antibody- ( 3F10; Roche , Indianapolis , IN ) conjugated agarose beads .","The anti-Rb antibody ( G3-245; BD Biosciences , San Jose , CA ) and anti-mDia1 antibody ( AP50 , a gift from S Narumiya ) ( Watanabe et al . , 1997 ) were used for immunoprecipitation .","The following antibodies were used for immunoblotting: anti-Rb ( G3-245 and ab6075; Abcam , Cambridge , UK ) , anti-phospho Rb-S780 ( 9307; Cell Signaling Technology , Danvers , MA ) , anti-phospho Rb-T821 ( a gift from K Tamai , CycLex ) , anti-CDK4 ( C-22; Santa Cruz Biotechnology , Santa Cruz , CA ) , anti-mDia1 ( AP50 and 51 [we used two kinds of mDia1 antibodies: one is AP50 which is mentioned above; the other is 51 , which is a clone name of BD antibody]; BD Transduction Laboratories , Franklin Lakes , NJ ) , anti-LAP2α ( ab5162; Abcam ) , anti-GFP ( 598; MBL , Nagoya , Japan ) , anti-α-Tubulin ( DM1A; Sigma ) , anti-TFIIB ( C-18; Santa Cruz Biotechnology ) , anti-Flag-Peroxidase ( M2; Sigma ) and anti-HA-Peroxidase ( 3F10; Roche ) .","The recombinant adenoviruses expressing mCherry-HA-Rb , mCherry-HA-NES Rb , mCherry-HA-NES Rb Δexon 22 , GFP and GFP-mDia1 ΔGBD/ΔDAD were generated using the ViraPower adenoviral expression system ( Invitrogen , Carlsbad , CA ) .","The recombinant adenovirus-mCherry-HA-NES Rb contained the NES of MAPKK ( NLVDLQKKLEELELDEQQ ) ( Fukuda et al . , 1996 ) between mCherry and Rb .","The recombinant adenoviruses expressing shRNAs were generated using the BLOCK-iT adenoviral RNAi expression system ( Invitrogen ) .","For shRNA-mediated gene silencing , the respective target sequences were as follows: CDK4 , 5′-CCTAGATTTCCTTCATGCCAA-3′ ( sh-CDK4 ) ; mDia1 , 5′-GCCCAGAATCTCTCAATCTTT-3′ ( sh-mDia1 ) ; Rb , 5′-CAGAGATCGTGTATTGAGATT-3′ ( sh-Rb ) ; the non-target control , 5′-CAACAAGATGAAGAGCACCAA-3′ ( sh-Control ) .","The recombinant adenoviruses were purified with AsEasy virus purification kits ( Agilent technologies , Palo Alto , CA ) and adenovirus infections were performed with ViraDuctin adenovirus transduction reagent ( Cell Biolabs , San Diego , CA ) .","Mammalian expression vectors that encode shRNAs against human LAP2α or enhanced GFP ( eGFP ) were constructed by cloning suitable oligonucleotide sequences ( human LAP2α , 5′-CAGAAGAGAATTGATCAGT-3′; eGFP , 5′-ACAACAGCCACAACGTCTA-3′ ) into the pSilencer 2 . 1-U6 Hygro vector ( Ambion , Austin , TX ) .","pXJ Flag-mDia1 was obtained from C Koh ( Xie et al . , 2008 ) .","Flag-mDia1 plasmids were transfected into 293T cells and total cell lysates were extracted .","The mDia1 proteins tagged with a Flag epitope at their amino-termini were captured on anti-Flag antibody-agarose beads and eluted by competition with free 1× Flag peptide ( Sigma ) in 50 mM Tris ( pH 7 . 4 ) and 50 mM NaCl .","The purity of the mDia1 proteins , wild-type and ΔLXCXE , was evaluated by SDS-polyacrylamide gel electrophoresis ( SDS-PAGE ) , followed by Coomassie Brilliant Blue staining ( CBB staining ) .","Full-length recombinant Rb protein was purchased from QED bioscience ( San Diego , CA ) .","The recombinant glutathione S-transferase ( GST ) fusion Rb proteins were produced in BL21 E . coli .","GST-Rb proteins were incubated with CDK4/Cyclin D1 ( Merck Millipore , Billerica , MA ) and CDK2/Cyclin E ( Merck Millipore ) proteins in the presence or absence of 100 μM ATP in the kinase buffer ( 20 mM Tris [pH 7 . 5] , 10 mM MgCl2 and 1 mM dithiothreitol [DTT] ) for 30 min at 30°C .","The cells were fixed with 4% paraformaldehyde ( PFA ) in phosphate-buffered saline ( PBS ) for 30 min , permeabilized with 0 . 2% ( vol/vol ) Triton X-100/PBS for 5 min , and then blocked with 1% bovine serum albumin ( BSA ) /PBS for 30 min at room temperature .","Subsequently , the cells were incubated with anti-Rb ( 1:200 , 9309; Cell Signaling Technology ) , anti-phospho Rb-S780 ( 1:300 , 13H9L5; Novex , Carlsbad , CA ) and anti-LAP2α ( 1:300 , ab5162; Abcam ) antibodies for 1 hr , and further incubated with Alexa Fluor 546-conjugated goat anti-mouse IgG , Alexa Fluor 546-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated goat anti-rabbit IgG antibodies ( Molecular Probes , Carlsbad , CA ) for 30 min .","DAPI was used for nuclear staining .","For double-staining for α-actinin and F-actin , cytoskeletal stabilizing buffer was used in lieu of PBS , as described previously ( Hirata et al . , 2008 ) .","The cells were incubated with anti-α-actinin antibody ( 1:100 , EA53; Sigma ) , followed by incubation with Alexa Fluor 488- or 546-conjugated goat anti-mouse IgG antibody and Alexa Fluor 546- or 633-phalloidin ( Molecular Probes ) .","Confocal images were taken using a PerkinElmer Spinning Disk microscope or a Nikon A1Rsi microscope , equipped with oil-immersion objectives ( 60× and 100× ) .","Epifluorescence images were taken using a Nikon A1Rsi microscope , equipped with an oil-immersion objective ( 60× ) and an electron multiplying charge-coupled device camera ( DU897; Andor technology , Belfast , UK ) .","Images were acquired with the Volocity software and the Nikon NIS-Elements imaging software .","To evaluate periodicity in the distribution of α-actinin , 256 × 256 pixel , corresponding to 12 × 12 μm , sample areas of fluorescence images of α-actinin were subjected to computing autocorrelation analyses as described previously ( Peterson et al . , 2004 ) using the ImageJ program ( NIH , version 10 . 2 ) .","Each autocorrelation image was then normalized by the value of the central peak in the image .","Features of periodicity of its distribution are represented as local peaks other than the central maxima .","Sample images of α-actinin and normalized autocorrelation images are shown .","Frozen blocks of human skeletal muscle were obtained from Asterand ( Detroit , MI ) , who acquired appropriate informed consent from patients under the Institutional Review Board ( IRB ) approval .","Subject characteristics are described in Table 1 . 10 . 7554/eLife . 01228 . 017Table 1 . Subject characteristicsDOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 017Biosample diagnosisNormalAtrophyGender and age , yearsFemale , 15Female , 14Cancer diagnosisOsteosarcomaSynovial sarcomaCancer locationShin boneSoft tissues of shinHeight , cm166159Weight , kg5050BMI , kg/m218 . 1419 . 78BMI , body mass index The sections were fixed in acetone for 10 min , permeabilized with 0 . 2% ( vol/vol ) Triton X-100/PBS for 30 min , and then blocked with CAS-Block ( Invitrogen ) for 10 min at room temperature .","Subsequently , the sections were incubated with anti-α-actinin , anti-Rb ( 1:100 , ab24; Abcam ) and anti-mDia1 ( 1:400 , ab11173; Abcam ) antibodies overnight at 4°C .","They were further incubated with appropriate fluorescence-labeled secondary antibodies for 1 hr at room temperature .","HSMMs grown on 4-well plates ( Nunc , Naperville , IL ) were pretreated with IFN-γ and then placed in a C-Dish electrode chamber ( IonOptix , Milton , MA ) after changing to fresh serum-free media .","EPS was applied to HSMMs using a C-Pace pulse generator ( IonOptix ) at 40 V/60 mm , 1 Hz , 10 ms for 2 days in parallel with TNF-α treatment .","The media were changed and TNF-α was repeatedly added every 24 hr .","Images of live beating cells were taken using Nikon Eclipse Ti-U microscope , equipped with a 20× objective lens .","Images were sequentially acquired with Nikon NIS-Elements imaging software at a frame rate of 15 fps .","Total RNA extraction , cDNA preparation , and real-time qPCR analyses were performed as described previously ( Kawauchi et al . , 2012 ) .","The primer sets used were: Tk1 , 5′-CATTAACCTGCCCACTGT-3′ forward and 5′-GATCACCAGGCACTTGTA-3′ reverse; Dhfr , 5′-TCATGGTTGGTTCGCTAA-3′ forward and 5′-TGAAGAGGTTGTGGTCATT-3′ reverse; Tfam , 5′-TGTAGAAGCCACGGTGTT-3′ forward and 5′-ACAACCATCAACTCTGAATACAAT-3′ reverse; Ppargc1a , 5′-TGAAGAGGCAAGAGACAGAATGA-3′ forward and 5′-CACACGCACACTCCATCAC-3′ reverse; B2m , 5′-GCATTCCTGAAGCTGACA-3′ forward and 5′-CGTGAGTAAACCTGAATCTTT-3′ reverse .","After normalization against B2m , data show mRNA expression levels relative to control expression levels for each experiment .","Mass spectrometry analysis was performed by Proteomics International ( Perth , Australia ) .","Protein samples were enzymatically digested to produce fragmented peptides and the resulting peptides were analyzed by electrospray ionization mass spectrometry using the Ultimate 3000 nano HPLC system ( Dionex , Sunnyvale , CA ) in combination with a 4000 Q TRAP mass spectrometer ( Applied Biosystems , Foster City , CA ) .","The spectra were analyzed by Mascot sequence matching software ( Matrix Science , Boston , MA ) ."]],"string":"[\n [\n \"Skeletal muscle degeneration , which is characterized by the progressive depletion of muscle strength , occurs in a variety of chronic diseases including advanced cancer , congestive heart failure , and AIDS ( Tisdale , 2002 ) .\",\n \"The underlying intercellular mechanism is currently thought to be multifactorial .\",\n \"Inflammatory cytokines , particularly TNF-α , have been shown to be key mediators of cancer-related skeletal muscle degeneration ( Tisdale , 2002; Seruga et al . , 2008 ) .\",\n \"Elevated levels of TNF-α precede the onset of cancer-related skeletal muscle degeneration and act through several cancer-related signaling pathways such as the p53 and nuclear factor kappa B ( NF-κB ) pathways ( Guttridge et al . , 2000; Cai et al . , 2004; Schwarzkopf et al . , 2006 ) .\",\n \"Retinoblastoma protein ( Rb ) prevents tumor formation by inducing differentiation , controlling cell-cycle progression , and maintaining genomic stability ( Burkhart and Sage , 2008 ) .\",\n \"To date , numerous studies of Rb function have focused on the transcriptional regulation of E2F .\",\n \"Rb forms a transcriptional repressor complex with two protein groups , E2F transcription factors and LXCXE motif-containing proteins ( Halaban , 2005; Burkhart and Sage , 2008 ) .\",\n \"Rb activity is regulated by sequential phosphorylation on several serine and threonine residues , first by cyclin D/cyclin-dependent kinase 4 ( CDK4 ) and then by cyclin E/CDK2 complexes ( Halaban , 2005 ) .\",\n \"This serial phosphorylation of Rb induces dissociation of the transcriptional repressor complex , allowing expression of E2F-target genes , which are required for many cellular processes .\",\n \"Loss of Rb function in many cancer cells is frequently caused by aberrant CDK-mediated phosphorylation ( Chau and Wang , 2003; Burkhart and Sage , 2008 ) .\",\n \"Consequently , selective CDK inhibition is considered a potentially useful approach for cancer treatment ( Malumbres and Barbacid , 2009 ) .\",\n \"In addition , inactivation of Rb , which is induced by TNF-α treatment , has been shown to lead to various cellular behaviors including proliferation of vascular smooth muscle cells ( Rastogi et al . , 2012 ) and apoptosis of fibroblasts and aortic endothelial cells ( Chau and Wang , 2003; Rastogi et al . , 2012 ) .\",\n \"Recently , a non-nuclear function of Rb has been reported; where Rb at the mitochondria participates in TNF-α-induced apoptosis ( Hilgendorf et al . , 2013 ) .\",\n \"However , it is still unknown whether the Rb pathway is involved in cancer-related skeletal muscle degeneration mediated by TNF-α .\",\n \"A sarcomere is the basic functional unit of striated muscle and consists of two sets of filaments: thick and thin ( Squire , 1997; Gautel , 2011 ) .\",\n \"The thick filaments are composed of myosin proteins and the thin filaments are assembled from polymerized actin monomers , called filamentous actin ( F-actin ) .\",\n \"The contractile activity of skeletal muscle is achieved through the actin and myosin filaments sliding past one another ( Squire , 1997 ) .\",\n \"The motor function of striated muscle therefore , requires the well-ordered assembly of sarcomeres , which is closely tied to the highly organized actin cytoskeleton .\",\n \"The formins are a large family of proteins and are characterized by the presence of the conserved formin homology 2 ( FH2 ) domain ( Kovar , 2006; Campellone and Welch , 2010 ) .\",\n \"The FH2 domain promotes actin nucleation and polymerization , thereby producing long straight actin filaments and regulating cytoskeletal organization .\",\n \"It has been reported that several members of the formin family serve as key regulators of actin dynamics during sarcomeric organization in striated muscle ( Taniguchi et al . , 2009; Kan et al . , 2012; Mi-Mi et al . , 2012 ) .\",\n \"In this study , we present a potential mechanism underlying TNF-α-induced skeletal muscle degeneration .\",\n \"We propose a novel function for Rb; where Rb disrupts sarcomeric organization in human skeletal muscle myotubes ( HSMMs ) following its phosphorylation and translocation into the cytoplasm .\",\n \"Our study implicates the tumor suppressor protein in the regulation of cytoskeletal organization .\"\n ],\n [\n \"To gain insights into the role of Rb in cancer-related skeletal muscle degeneration , we first examined the phosphorylation kinetics and subcellular localization of Rb in TNF-α-treated HSMMs .\",\n \"For this purpose , cells were pretreated with interferon-gamma ( IFN-γ , 100 ng/ml ) for 8 hr prior to initial TNF-α treatment in order to promote cellular sensitivity to the effects of TNF-α ( Tsujimoto et al . , 1986 ) .\",\n \"During differentiation from myoblasts to myotubes ( from day 0 to day 4 ) , Rb shifted from a phosphorylated to an unphosphorylated state ( Figure 1A ) .\",\n \"Moreover , following TNF-α treatment Rb phosphorylation on the CDK4-specific phosphorylation site , serine 780 ( Kitagawa et al . , 1996 ) , was induced ( Figure 1A ) .\",\n \"This was not observed on threonine 821 , a CDK2-specific phosphorylation site ( Halaban , 2005 ) .\",\n \"In untreated HSMMs , Rb was primarily present in the nucleus , but translocated to the cytoplasm after TNF-α treatment ( Figure 1B , C ) .\",\n \"In addition , we found that phosphorylated Rb in TNF-α-treated HSMMs was predominantly located in the cytoplasm ( Figure 1D ) .\",\n \"In accordance with the induction of Rb phosphorylation on a CDK4-specific phosphorylation site , TNF-α treatment led to an increase in the level of nuclear CDK4 ( Figure 1E ) .\",\n \"The vast majority of nuclear Rb was in an unphosphorylated state , while cytoplasmic Rb that accumulated after TNF-α treatment was in a phosphorylated state ( Figure 1E ) .\",\n \"We then tested whether TNF-α-induced cytoplasmic accumulation of Rb is caused by CDK4-mediated Rb phosphorylation .\",\n \"When CDK4 was depleted by short hairpin RNA ( shRNA ) , cytoplasmic accumulation of Rb induced by TNF-α was decreased ( Figure 1F–H ) .\",\n \"These results suggest that phosphorylation of Rb by CDK4 triggers its cytoplasmic translocation in HSMMs . 10 . 7554/eLife . 01228 . 003Figure 1 . TNF-α induces cytoplasmic translocation of Rb .\",\n \"( A–E )\",\n \"HSMMs were treated with TNF-α for 2 days .\",\n \"( A ) CDK4-mediated phosphorylation of Rb is induced by TNF-α treatment .\",\n \"At the indicated time points after differentiation stimuli , total cell lysates were prepared and immunoprecipitated with anti-Rb antibody , followed by immunoblotting .\",\n \"TNF-α was added for the last 2 days of 6-day cultures .\",\n \"The open and solid arrowheads indicate the position of phosphorylated and unphosphorylated Rb , respectively .\",\n \"( B ) Cytoplasmic translocation of Rb is caused by TNF-α treatment .\",\n \"Z-stack confocal images of Rb and 4′ , 6-diamidino-2-phenylindole ( DAPI ) -stained nuclei were obtained ( 50 slices at 0 . 3-μm intervals ) .\",\n \"X-Y section images for Rb and DAPI-stained nuclei and X-Z section images for Rb along yellow lines in the X-Y section images are shown .\",\n \"Scale bar , 20 μm .\",\n \"( C ) Line plots denote the fluorescence intensities of Rb ( red lines ) and DAPI ( blue lines ) along the yellow lines in B . Intensity values were normalized by the maximum value of each plot .\",\n \"a . u . , arbitrary units .\",\n \"( D ) Phosphorylated Rb is localized in the cytoplasm .\",\n \"Confocal images for Rb phosphorylated at S780 ( red ) and DAPI-stained nuclei ( blue ) .\",\n \"Scale bar , 20 μm .\",\n \"( E ) Nuclear CDK4 expression is increased after TNF-α treatment .\",\n \"Cytoplasmic ( Cyto ) and nuclear ( Nuc ) lysates were analyzed by immunoblotting with the antibodies indicated .\",\n \"Tubulin and TFIIB were used as loading controls for cytoplasmic and nuclear lysates , respectively .\",\n \"The open and solid arrowheads indicate the position of phosphorylated and unphosphorylated Rb .\",\n \"( F–H )\",\n \"HSMMs were infected with adenoviruses expressing control non-target shRNA or shRNA against CDK4 at a MOI of 10 pfu/nucleus and then treated with TNF-α for 2 days .\",\n \"( F ) Experimental design and reference time frame .\",\n \"( G ) The cytoplasmic ( Cyto ) and nuclear ( Nuc ) lysates were analyzed by immunoblotting .\",\n \"( H ) TNF-α-induced cytoplasmic translocation of Rb is prevented by CDK4 depletion .\",\n \"The cytoplasmic and nuclear lysates were subjected to immunoprecipitation and probed by immunoblotting .\",\n \"The open and solid arrowheads indicate the position of phosphorylated and unphosphorylated Rb , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 003 In human skeletal muscle myoblasts , Rb was mainly in a phosphorylated state ( Figure 1A , day 0 ) , although the localization of S780-phosphorylated Rb was confined to the nucleus ( Figure 2A ) .\",\n \"Lamina-associated polypeptide ( LAP ) 2α , a binding partner of nucleoplasmic A-type lamins , interacts with S780-phosphorylated Rb in mitotic myoblasts ( Markiewicz et al . , 2005 ) and is known to play an important role in nuclear tethering of Rb ( Markiewicz et al . , 2002 ) .\",\n \"Indeed , when LAP2α was depleted by shRNA in human myoblasts , Rb localized to the cytoplasm as well as the nucleus ( Figure 2B ) .\",\n \"Given the level of LAP2α decreased during muscle differentiation ( Markiewicz et al . , 2005 ) ( Figure 2C ) , these results suggest that low-level LAP2α expression in HSMMs facilitates the cytoplasmic translocation of Rb .\",\n \"In many types of cancer cells , Rb exists predominantly in a phosphorylated state , but is primarily localized in the nucleus .\",\n \"It has been reported that LAP2α is an E2F-target gene and its expression is enhanced in cancer cells ( Parise et al . , 2006; Ward et al . , 2011 ) .\",\n \"Overexpressed LAP2α may therefore serve to tether Rb to the nucleus in cancer cells and the cytoplasmic translocation of Rb may be triggered in cells that express low levels of LAP2α , such as terminally differentiated cells . 10 . 7554/eLife . 01228 . 004Figure 2 . Loss of LAP2α affects the cytoplasmic translocation of Rb .\",\n \"( A ) Phosphorylated Rb is localized in the nucleus in human skeletal muscle myoblasts .\",\n \"Confocal images for Rb phosphorylated at S780 and DAPI-stained nuclei .\",\n \"Scale bar , 20 μm .\",\n \"( B ) Rb is translocated to the cytoplasm by LAP2α depletion .\",\n \"Human skeletal muscle myoblasts were transfected with an mCherry-HA-Rb expression plasmid together with an shRNA expression plasmid against eGFP or LAP2α .\",\n \"Confocal images for mCherry , LAP2α and DAPI-stained nuclei .\",\n \"The arrowheads indicate transfected cells .\",\n \"Scale bar , 20 μm .\",\n \"( C ) LAP2α expression is decreased in HSMMs .\",\n \"Total cell lysates from human skeletal muscle myoblasts and HSMMs were subjected to immunoblotting . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 004 We next tested whether the motor function of skeletal muscle is affected under this condition .\",\n \"In response to electric pulse stimulation ( EPS ) , which evokes a contractile reaction in myotubes ( Fujita et al . , 2007 ) , approximately 55% of HSMMs displayed beating , an index of contractile reaction ( Figure 3A; Video 1 ) , while it was hardly observed after TNF-α treatment ( Figure 3A; Video 2 ) .\",\n \"The beating was not induced in mitotic myoblasts or prematurely differentiated myoblasts implying that the sarcomeric structure , which is essential for the contractile activity of muscle cells ( Squire , 1997 ) , is organized in HSMMs .\",\n \"To evaluate the effect of TNF-α on the periodic assembly of sarcomeres , we examined the distribution of α-actinin , a major component of Z-disks , as Z-disks define the lateral borders of individual sarcomeres ( Gautel , 2011 ) .\",\n \"In untreated HSMMs , α-actinin was observed at evenly spaced intervals , whereas in TNF-α-treated HSMMs the peak-to-peak distance in line plots of α-actinin intensity along the myofibril was larger and inconsistent ( Figure 3B–D ) .\",\n \"Furthermore , when the repeating pattern of α-actinin distribution was analyzed using an autocorrelation image processing technique , peak values were much lower in TNF-α-treated HSMMs ( Figure 3E ) .\",\n \"These results indicate that the periodic assembly of sarcomeres is disrupted by TNF-α treatment . 10 . 7554/eLife . 01228 . 005Figure 3 . TNF-α disrupts sarcomeric organization .\",\n \"( A–E )\",\n \"HSMMs were treated with TNF-α for 2 days .\",\n \"( A ) Contractile activity of HSMMs is impaired by TNF-α treatment .\",\n \"EPS was applied to HSMMs .\",\n \"The percentage of beating cells from a total of 100 HSMMs is shown .\",\n \"Results are presented as mean ± SD from three independent experiments .\",\n \"*p<0 . 002 , determined by the Student’s t-test .\",\n \"( B–E )\",\n \"Sarcomeric organization of HSMMs .\",\n \"Confocal images for α-actinin ( B ) and merged images of F-actin ( green ) and α-actinin ( red ) ( C ) are shown .\",\n \"Scale bar , 10 μm in B and 5 μm in C . ( D ) Line plots of α-actinin fluorescence intensity along individual myofibrils ( denoted by yellow lines in C ) .\",\n \"Intensity values were normalized by the maximum value for each fibril .\",\n \"a . u . , arbitrary units .\",\n \"( E ) Autocorrelation analyses of the α-actinin distribution .\",\n \"Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 00510 . 7554/eLife . 01228 . 006Video 1 . Image of live beating HSMMs in response to EPS . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 00610 . 7554/eLife . 01228 . 007Video 2 . Image of live beating TNF-α-treated HSMMs in response to EPS . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 007 In striated muscle cells , anti-parallel actin filaments spanning the sarcomeres are crosslinked to the Z-disks ( Sparrow and Schock , 2009 ) .\",\n \"The peak-to-peak distance in line plots of α-actinin intensity therefore reflects a counterbalance between tensile forces generated by the actin filaments .\",\n \"Sarcomeric disorganization represents an imbalance in the tensile forces , which may be caused by defective actin filament formation .\",\n \"The close association of accurate sarcomeric organization with actin polymerization was demonstrated by treatment of the cells with the actin polymerization inhibitor cytochalasin D . After 30 min of treatment , the periodic arrangement of α-actinin was not well ordered ( Figure 4A–C ) and was strongly disordered by 60 min ( Figure 4D ) . 10 . 7554/eLife . 01228 . 008Figure 4 . Inhibition of actin polymerization disorganizes sarcomeric assembly .\",\n \"( A–D )\",\n \"HSMMs were treated with 2 μM cytochalasin D ( Cyto D ) for 30 min ( A–C ) or 60 min ( D ) at room temperature .\",\n \"( A ) The periodic arrangement of α-actinin is not well ordered after 30 min of Cyto D-treatment .\",\n \"Merged images of F-actin ( green ) and α-actinin ( red ) .\",\n \"Scale bar , 5 μm .\",\n \"( B ) Line plots of α-actinin fluorescence intensity along individual myofibrils ( denoted by yellow lines in A ) .\",\n \"Intensity values were normalized by the maximum value for each fibril .\",\n \"a . u . , arbitrary units .\",\n \"( C ) Autocorrelation analyses of the α-actinin distribution .\",\n \"Scale bar , 5 μm .\",\n \"( D ) The lateral periodicity in the α-actinin distribution is strongly disordered by 60 min of treatment .\",\n \"Merged images of F-actin ( green ) and α-actinin ( red ) .\",\n \"Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 008 We then evaluated the role of Rb in the negative regulation of sarcomeric organization using Rb knockdown HSMMs ( Figure 5A , B ) .\",\n \"Rb is known to be required for myogenic differentiation but not for the maintenance of the terminally differentiated state in myotubes ( Huh et al . , 2004 ) .\",\n \"In accordance with this , sarcomeric organization was not affected when Rb was depleted by shRNA ( Figure 5C ) .\",\n \"After TNF-α treatment , the periodic assembly of sarcomeres was disrupted in control shRNA-expressing HSMMs , but TNF-α failed to induce the sarcomeric disorganization in sh-Rb-expressing HSMMs ( Figure 5D–F ) .\",\n \"Next , we directly examined whether cytoplasmic Rb is capable of disrupting sarcomeric organization .\",\n \"To this end , HSMMs were infected with adenoviruses expressing a heterologous nuclear exporting signal ( NES ) -fused form of Rb , which was tagged with monomeric red fluorescent protein mCherry and influenza hemaglutinin ( HA ) at its amino-terminus ( mCherry-HA-NES Rb ) .\",\n \"Given a small population of endogenous Rb was localized in the cytoplasm after TNF-α treatment , we expressed NES Rb at a comparable level to TNF-α-induced cytoplasmic Rb ( Figure 6A , asterisk vs the open arrowhead ) .\",\n \"Under this condition , sarcomeric organization was not well ordered as in TNF-α-treated HSMMs ( Figure 6B–D ) .\",\n \"When we expressed higher levels of NES Rb in HSMMs ( Figure 6E , F ) , the periodic arrangement of α-actinin was significantly disordered when compared to mCherry-HA-Rb-expressing cells ( Figure 6G ) .\",\n \"Taken together , these results suggest that Rb is involved in TNF-α-induced sarcomeric disorganization and TNF-α-induced cytoplasmic Rb may have a pivotal role in this process . 10 . 7554/eLife . 01228 . 009Figure 5 . Rb contributes to TNF-α-induced sarcomeric disorganization .\",\n \"( A–F )\",\n \"HSMMs were infected with adenoviruses expressing control non-target shRNA or shRNA against Rb at a MOI of 10 pfu/nucleus and then treated with TNF-α for 2 days .\",\n \"( A and B )\",\n \"Depletion of Rb protein was verified by immunoblotting ( A ) and epifluorescence microscopy ( B ) .\",\n \"The open and solid arrowheads indicate the position of phosphorylated and unphosphorylated Rb , respectively .\",\n \"Scale bar , 20 μm .\",\n \"( C ) Confocal images for F-actin and α-actinin .\",\n \"Scale bar , 10 μm .\",\n \"( D and E )\",\n \"TNF-α-induced sarcomeric disorganization is attenuated by Rb depletion .\",\n \"Confocal images for α-actinin ( D ) and merged images of F-actin ( green ) and α-actinin ( red ) ( E ) .\",\n \"Scale bar , 10 μm in D and 5 μm in E . ( F ) Autocorrelation analyses of the α-actinin distribution .\",\n \"Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 00910 . 7554/eLife . 01228 . 010Figure 6 . Sarcomeric organization is impaired by cytoplasmic Rb .\",\n \"( A–G )\",\n \"HSMMs were infected with adenoviruses expressing mCherry-HA-Rb or mCherry-HA-NES Rb at a MOI of 10 pfu/nucleus ( A–D ) or 50 pfu/nucleus ( E–G ) for 4 days .\",\n \"( A ) The expression of exogenous Rb proteins .\",\n \"Cytoplasmic ( Cyto ) and nuclear ( Nuc ) lysates were prepared from infected HSMMs in parallel with TNF-α-treated HSMMs .\",\n \"The lysates were subjected to immunoprecipitation and probed by immunoblotting .\",\n \"The open and solid arrowheads indicate the position of phosphorylated and unphosphorylated Rb .\",\n \"Asterisk indicates the position of exogenous Rb .\",\n \"( B ) Sarcomeric structure is not well ordered in NES Rb-expressing HSMMs .\",\n \"Merged images of F-actin ( green ) and α-actinin ( red ) .\",\n \"Scale bar , 5 μm .\",\n \"( C ) Line plots of α-actinin fluorescence intensity along individual myofibrils ( denoted by yellow lines in B ) .\",\n \"Intensity values were normalized by the maximum value for each fibril .\",\n \"a . u . , arbitrary units .\",\n \"( D ) Autocorrelation analyses of the α-actinin distribution .\",\n \"Scale bar , 5 μm .\",\n \"( E and F )\",\n \"The expression and distribution of exogenous Rb proteins were analyzed by immunoblotting ( E ) and confocal microscopy ( F ) .\",\n \"The open and solid arrowheads indicate the position of exogenous and endogenous Rb , respectively .\",\n \"Scale bar , 20 μm .\",\n \"( G ) Sarcomeric structure is strongly disordered in NES Rb-expressing HSMMs .\",\n \"Confocal images for F-actin and α-actinin .\",\n \"Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 010 Similarly to TNF-α-treated HSMMs , the cytoplasmic translocation of Rb has been shown to be induced by its CDK-mediated phosphorylation in certain types of cancer cells ( Jiao et al . , 2008 ) , although the function of cytoplasmic Rb in the cancer cells has not been described thus far .\",\n \"In the cancer cells , Rb is phosphorylated on CDK2 phosphorylation sites as well as CDK4 phosphorylation sites , whereas our data showed that Rb phosphorylation on the CDK2-specific phosphorylation site , threonine 821 , was not induced by TNF-α treatment ( Figure 1A , day 6 ) .\",\n \"We therefore reasoned that the selective phosphorylation of Rb by specific CDKs affects the function of cytoplasmic Rb .\",\n \"Given that it has been reported that threonine 821/threonine 826 phosphorylation disrupts Rb binding to LXCXE motif-containing proteins ( Knudsen and Wang , 1996; Dick and Rubin , 2013 ) , we examined whether the function of cytoplasmic Rb is achieved through its interaction with LXCXE motif-containing proteins .\",\n \"We introduced mCherry-HA-NES Rb lacking exon 22 ( Rb Δexon 22 ) , which is known to be an LXCXE-binding deficient mutant ( Henley et al . , 2010 ) , into HSMMs and found that the inhibitory effect of NES Rb Δexon 22 on sarcomeric organization was less effective as compared to NES Rb-expressing HSMMs ( Figure 7A ) . 10 . 7554/eLife . 01228 . 011Figure 7 . The function of cytoplasmic Rb is rendered through its interaction with LXCXE motif-containing proteins .\",\n \"( A ) Sarcomeric structure is not disordered in NES Rb Δexon 22-expressing HSMMs .\",\n \"HSMMs were infected with an adenovirus expressing mCherry-HA-NES Rb Δexon 22 at a MOI of 50 pfu/nucleus for 4 days .\",\n \"Confocal images for F-actin and α-actinin .\",\n \"Scale bar , 10 μm .\",\n \"( B ) mDia1 contains the LXCXE motif .\",\n \"Sequence alignment of the LXCXE motif of mDia1 and other known Rb-binding proteins .\",\n \"( C ) The primary structure of mDia1 and its mutants .\",\n \"Scheme represents location of GBD and DAD of mDia1 .\",\n \"Numbers denote amino acid positions in mDia1 isform2 .\",\n \"The LXCXE motif is present in GBD ( amino acid positions 153 to 157 ) .\",\n \"ΔGBD/ΔDAD , doubly deleted mDia1 lacking both GBD and DAD .\",\n \"( D ) The LXCXE motif is required for the in vitro interaction between Rb and mDia1 .\",\n \"Purified Flag-tagged mDia1 proteins were mixed with full-length recombinant Rb protein and immunoprecipitates were analyzed by immunoblotting .\",\n \"( E ) Rb interacts with mDia1 after TNF-α treatment .\",\n \"HSMMs were treated with TNF-α for 2 days .\",\n \"The cytoplasmic lysates were subjected to immunoprecipitation and probed by immunoblotting .\",\n \"The open arrowheads indicate the position of phosphorylated Rb .\",\n \"( F ) Expression and purification of GST fusion Rb proteins .\",\n \"The purity of bacterially expressed GST-Rb proteins was evaluated by SDS-PAGE , followed by CBB staining .\",\n \"GST-Rb wild-type protein encompasses amino acids 379–928 .\",\n \"GST-Rb Mut CDK2 contains serine/threonine to alanine substitutions at CDK2-specific phosphorylation sites ( S612 and T821 ) .\",\n \"( G ) GST-Rb proteins were preincubated with CDK4/Cyclin D1 and CDK2/Cyclin E proteins in the presence or absence of ATP and then mixed with purified Flag-mDia1 protein .\",\n \"The interaction between mDia1 and Rb proteins was analyzed by immunoprecipitation with anti-Flag antibody-agarose beads . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 01110 . 7554/eLife . 01228 . 012Figure 7—figure supplement 1 . Identification of NES Rb-binding protein . HSMMs were infected with adenoviruses expressing mCherry-HA-NES Rb WT or mCherry-HA-NES Rb Δexon 22 at a MOI of 50 pfu/nucleus for 4 days .\",\n \"Total cell lysates from adenovirus-infected HSMMs were immunoprecipitated with anti-HA antibody-conjugated agarose beads .\",\n \"The bound proteins were analyzed by SDS-PAGE and visualized by CBB staining .\",\n \"Individual protein bands were excised from SDS-PAGE gel and protein samples were subjected to electrospray ionization mass spectrometric analysis .\",\n \"The lists of proteins from the bands are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 012 Next , to explore how cytoplasmic Rb disorganizes sarcomeric assembly in HSMMs , we searched for the binding proteins of cytoplasmic Rb using mCherry-HA-NES Rb wild-type ( WT ) -expressing HSMMs .\",\n \"Total cell lysates from adenovirus-infected HSMMs were immunoprecipitated with anti-HA antibody-conjugated agarose beads and the bound proteins were subjected to electrospray ionization mass spectrometric analysis ( Figure 7—figure supplement 1 ) .\",\n \"Among the proteins identified , we focused on mammalian diaphanous-related formin 1 ( mDia1 ) , a potent actin nucleation factor ( Watanabe et al . , 1997 ) , as the LXCXE motif is contained in the GTPase binding domain ( GBD ) of mDia1 ( Figure 7B , C ) .\",\n \"It is noteworthy that the band including mDia1 was hardly detected in mCherry-HA-NES Rb Δexon 22-expressing HSMMs ( Figure 7—figure supplement 1 , band 1 ) .\",\n \"Data from purified wild-type and LXCXE motif-deleted mutant mDia1 proteins ( WT and ΔLXCXE ) ( Figure 7C ) showed that binding of mDia1 to Rb was abolished by deletion of the LXCXE motif ( Figure 7D ) .\",\n \"Cytoplasmic Rb , which was phosphorylated and accumulated after TNF-α treatment , interacted with mDia1 ( Figure 7E ) .\",\n \"We then examined whether the selective phosphorylation of Rb affects this interaction .\",\n \"Unphosphorylated Rb protein containing the large pocket , an important domain for interactions with a variety of cellular proteins ( Burkhart and Sage , 2008 ) , bound to mDia1 in vitro and the binding level was strongly reduced according to its phosphorylation catalyzed by cyclin D/CDK4 and cyclin E/CDK2 complexes ( Figure 7F , G ) .\",\n \"Although differences in the patterns of Rb phosphorylation by cyclin D/CDK4 and cyclin E/CDK2 have been reported , we failed to detect the selective phosphorylation of Rb by our in vitro phosphorylation system , which may be due to a supraphysiological activity of CDKs in vitro .\",\n \"We then tested a mutant Rb protein bearing serine/threonine-to-alanine substitutions in the CDK2-specific phosphorylation sites , serine 612 and threonine 821 ( Mut CDK2 ) .\",\n \"The mutant Rb exhibited substantial interaction with mDia1 even after phosphorylation ( Figure 7G ) , suggesting that phosphorylation of Rb on CDK4 phosphorylation sites alone does not impair its interaction with mDia1 and TNF-α-induced cytoplasmic Rb has the potential to interact with mDia1 .\",\n \"Muscle wasting/atrophy accompanies cancer-related skeletal muscle degeneration ( Tisdale , 2002; Acharyya et al . , 2004 ) .\",\n \"We therefore carried out immunohistological analysis on normal and atrophied tibialis anterior muscles excised from cancer patients and examined the localization of Rb in these muscles ( Figure 8A ) .\",\n \"In normal muscles , Z-disks were regularly aligned and the sarcomere striation pattern was clearly observed ( Figure 8B , left ) .\",\n \"In contrast , the Z-disks were misaligned and the sarcomeric banding pattern was not well organized in the atrophied muscles ( Figure 8B , right ) .\",\n \"With regard to the localization of Rb , it was mainly located in the nucleus in normal muscle cells , but Rb could be observed in the cytoplasm , as well as the nucleus in atrophied muscle cells ( Figure 8C ) .\",\n \"In both normal and atrophied skeletal muscles , the localization of mDia1 was mainly confined to the Z-disk ( Figure 8B ) , and cytoplasmic Rb observed in atrophied muscles colocalized with mDia1 ( Figure 8C ) . 10 . 7554/eLife . 01228 . 013Figure 8 . Cytoplasmic Rb colocalizes with mDia1 in atrophied skeletal muscle .\",\n \"( A–C′ )\",\n \"Normal and atrophied tibialis anterior muscles were surgically excised from cancer patients .\",\n \"Cross sections ( A ) and longitudinal sections ( B–C′ ) were stained .\",\n \"( A ) Hematoxylin and eosin-stained cryosections .\",\n \"Scale bar , 400 μm .\",\n \"( B–C′ )\",\n \"Localization of Rb and mDia1 in normal and atrophied tibialis anterior muscles .\",\n \"Confocal images of cryosections stained with anti-mDia1 and anti-α-actinin antibodies ( B ) or anti-Rb and anti-mDia1 antibodies ( C , magnified in C′ ) .\",\n \"Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 013 We next tested whether inhibition of mDia1 activity is responsible for the TNF-α-induced disorganization of the sarcomere .\",\n \"For this purpose , we generated a recombinant adenovirus expressing the constitutively active form of mDia1 ( Watanabe et al . , 1999 ) , which lacks the entire GBD and carboxy-terminal diaphanous autoregulatory domain ( DAD ) ( Figure 7C ) .\",\n \"We introduced mDia1 ΔGBD/ΔDAD tagged with green fluorescent protein ( GFP-mDia1 ΔGBD/ΔDAD ) into HSMMs and treated them with TNF-α ( Figure 9A ) .\",\n \"mDia1 ΔGBD/ΔDAD itself did not affect either α-actinin distribution or the contractile reaction of HSMMs ( Figure 9B , C ) .\",\n \"After TNF-α treatment , however , the lateral alignment of α-actinin in GFP-mDia1 ΔGBD/ΔDAD-expressing HSMMs was well ordered as compared to that in control GFP-expressing HSMMs ( Figure 9D–F ) .\",\n \"Accordingly , the percentage of beating cells decreased by TNF-α treatment was restored by the introduction of mDia1 ΔGBD/ΔDAD ( p<0 . 02 , determined by the Student’s t-test ) ( Figure 9C ) . 10 . 7554/eLife . 01228 . 014Figure 9 . TNF-α-induced sarcomeric disorganization is prevented by constitutively active mDia1 . ( A–F ) HSMMs were infected with adenoviruses expressing GFP or GFP-mDia1 ΔGBD/ΔDAD at a MOI of 1 pfu/nucleus and then treated with TNF-α for 2 days .\",\n \"( A ) The expression was analyzed by immunoblotting .\",\n \"( B ) Confocal images for α-actinin .\",\n \"Scale bar , 5 μm .\",\n \"( C ) TNF-α-induced contractile dysfunction is diminished by constitutively active mDia1 .\",\n \"EPS was applied to HSMMs .\",\n \"The percentage of beating cells from a total of 100 HSMMs is shown .\",\n \"Results are presented as mean ± SD from three independent experiments .\",\n \"*p<0 . 002; **p<0 . 02 , determined by the Student’s t-test .\",\n \"( D and E )\",\n \"Constitutively active mDia1 recovers TNF-α-induced sarcomeric disorganization .\",\n \"Confocal images for α-actinin ( D ) and merged images of F-actin ( green ) and α-actinin ( red ) ( E ) .\",\n \"Scale bar , 10 μm in D and 5 μm in E . ( F ) Autocorrelation analyses of the α-actinin distribution .\",\n \"Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 014\"\n ],\n [\n \"In this study , we have elucidated a novel pathway of cancer-related skeletal muscle degeneration .\",\n \"TNF-α induces CDK4 activation and the concomitant phosphorylation of Rb .\",\n \"Subsequently , cytoplasmic translocation of Rb is triggered .\",\n \"Cytoplasmic Rb disrupts sarcomeric organization , which may be caused by dysfunction of mDia1 .\",\n \"The precise role of mDia1 in the regulation of sarcomeric organization is poorly understood ( Sparrow and Schock , 2009 ) , but the contribution of mDia1 to sarcomeric organization is supported .\",\n \"When mDia1 was depleted by shRNA , the lateral periodicity in the distribution of α-actinin was markedly perturbed ( Figure 10A , B ) .\",\n \"The importance of actin nucleation factors for the periodic assembly of sarcomeres is proposed by the recent observations that depletion of actin nucleation factors , such as Fhod3 and leiomodin , results in disordered α-actinin distribution in cardiomyocytes ( Chereau et al . , 2008; Taniguchi et al . , 2009; Iskratsch et al . , 2010 ) .\",\n \"The notion that the degree of sarcomeric disorganization is dependent on the inhibition of actin polymerization is further supported by our data that show Z-disk alignment is more severely impaired by longer-term treatment of cytochalasin D ( Figure 4 ) . 10 . 7554/eLife . 01228 . 015Figure 10 . mDia1 is critical for sarcomeric organization .\",\n \"( A and B )\",\n \"HSMMs were infected with adenoviruses expressing control non-target shRNA or shRNA against mDia1 at a MOI of 5 pfu/nucleus for 4 days .\",\n \"( A ) Depletion of mDia1 protein was verified by immunoblotting .\",\n \"( B ) Confocal images for F-actin and α-actinin .\",\n \"Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 015 Phosphorylation of Rb and the subsequent activation of E2F transcriptional activity enhance the expression of E2F-target genes , which include cell-cycle regulators ( e . g . , Tk1 and Dhfr ) .\",\n \"Although Rb phosphorylated at serine 780 is unable to bind to E2F1 ( Kitagawa et al . , 1996 ) , quantitative PCR ( qPCR ) analysis did not reveal any statistically significant differences in the expression of Tk1 and Dhfr after TNF-α treatment ( Figure 11 ) .\",\n \"During muscle differentiation , methylation of histone H3 lysine 9 and DNA methylation occur at several E2F-target gene promoters including Tk1 and Dhfr ( Ait-Si-Ali et al . , 2004; Blanchet et al . , 2011 ) .\",\n \"These epigenetic changes may trigger the permanent silencing of E2F-target gene expression and prevent E2F1 from activating their expression after terminal differentiation . 10 . 7554/eLife . 01228 . 016Figure 11 . The expression of cell-cycle regulators and mitochondrial biogenesis factors after TNF-α treatment . HSMMs were treated with TNF-α for 2 days .\",\n \"Quantification of the expression of cell-cycle regulators and mitochondrial biogenesis factors .\",\n \"Results are presented as mean ± SD from three independent experiments .\",\n \"*p<0 . 02 , determined by the Student’s t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 016 It has been reported that phosphorylation of Rb is induced in the skeletal muscles of mice under fasting conditions ( Blanchet et al . , 2011 ) , which leads to the activation of E2F1 transcriptional activity and increases the expression levels of mitochondrial biogenesis factors ( Ppargc1a and Tfam ) .\",\n \"In contrast , although TNF-α treatment induced Rb phosphorylation , the expression of Ppargc1a and Tfam decreased after TNF-α treatment ( Remels et al . , 2010 ) ( Figure 11 ) .\",\n \"These findings suggest that other TNF-α-induced signaling pathways , such as the NF-κB pathway , may affect the transcriptional activity of E2F1 ( Araki et al . , 2008 ) and/or regulation of the expression of mitochondrial biogenesis factors ( Remels et al . , 2010 ) .\",\n \"The contractile activity of skeletal muscle is caused by the sliding of thick and thin filaments in the sarcomere , and this sliding action is intimately linked to the proper control of adenosine 5’-triphosphate ( ATP ) production ( Squire , 1997 ) .\",\n \"Because mitochondria are responsible for cellular ATP production , incomplete restoration of the percentage of beating cells by mDia1 ΔGBD/ΔDAD might be ascribed to an impairment of mitochondrial function caused by TNF-α ( Figure 9C ) .\",\n \"In cancer patients , the circulating levels of TNF-α and IFN-γ would be lower than the amounts used in this study .\",\n \"Monocytes/macrophages infiltrate in atrophied muscles and may produce considerable amounts of these inflammatory cytokines .\",\n \"It may therefore be possible that the local concentrations of TNF-α and IFN-γ are elevated in atrophied muscles , which then contributes to phosphorylation of Rb in the long-term .\",\n \"TNF-α-induced skeletal muscle degeneration is achieved through multiple mechanisms .\",\n \"The results presented in this study propose a novel non-nuclear function for Rb , which is independent of the transcriptional regulation of E2F and may be involved in TNF-α-induced skeletal muscle degeneration .\",\n \"In the future , it would be interesting to clarify the role of mDia1 and determine how cytoplasmic Rb disrupts sarcomeric organization .\"\n ],\n [\n \"Early passage human skeletal myoblasts purchased from Lonza ( Basel , Switzerland ) were cultured according to the manufacturer’s instructions using Lonza-supplied growth medium ( SkGM-2 BulletKit ) .\",\n \"Cells grown to approximately 80% confluence were induced to differentiate into multinucleated myotubes by switching to differentiation medium ( DMEM-F12 containing 2% horse serum ) .\",\n \"Under these conditions , numerous myotubes could be detected on the fourth day .\",\n \"For TNF-α treatment , HSMMs were cultured in fresh serum-free media containing TNF-α ( 100 ng/ml ) for 2 days .\",\n \"TNF-α was repeatedly added every 24 hr .\",\n \"Recombinant human TNF-α and IFN-γ were purchased from PeproTech ( Rocky Hill , NJ ) .\",\n \"Cytochalasin D was obtained from Sigma ( St . Louis , MO ) .\",\n \"Immunoprecipitations were performed using anti-Flag antibody- ( M2; Sigma ) and anti-HA antibody- ( 3F10; Roche , Indianapolis , IN ) conjugated agarose beads .\",\n \"The anti-Rb antibody ( G3-245; BD Biosciences , San Jose , CA ) and anti-mDia1 antibody ( AP50 , a gift from S Narumiya ) ( Watanabe et al . , 1997 ) were used for immunoprecipitation .\",\n \"The following antibodies were used for immunoblotting: anti-Rb ( G3-245 and ab6075; Abcam , Cambridge , UK ) , anti-phospho Rb-S780 ( 9307; Cell Signaling Technology , Danvers , MA ) , anti-phospho Rb-T821 ( a gift from K Tamai , CycLex ) , anti-CDK4 ( C-22; Santa Cruz Biotechnology , Santa Cruz , CA ) , anti-mDia1 ( AP50 and 51 [we used two kinds of mDia1 antibodies: one is AP50 which is mentioned above; the other is 51 , which is a clone name of BD antibody]; BD Transduction Laboratories , Franklin Lakes , NJ ) , anti-LAP2α ( ab5162; Abcam ) , anti-GFP ( 598; MBL , Nagoya , Japan ) , anti-α-Tubulin ( DM1A; Sigma ) , anti-TFIIB ( C-18; Santa Cruz Biotechnology ) , anti-Flag-Peroxidase ( M2; Sigma ) and anti-HA-Peroxidase ( 3F10; Roche ) .\",\n \"The recombinant adenoviruses expressing mCherry-HA-Rb , mCherry-HA-NES Rb , mCherry-HA-NES Rb Δexon 22 , GFP and GFP-mDia1 ΔGBD/ΔDAD were generated using the ViraPower adenoviral expression system ( Invitrogen , Carlsbad , CA ) .\",\n \"The recombinant adenovirus-mCherry-HA-NES Rb contained the NES of MAPKK ( NLVDLQKKLEELELDEQQ ) ( Fukuda et al . , 1996 ) between mCherry and Rb .\",\n \"The recombinant adenoviruses expressing shRNAs were generated using the BLOCK-iT adenoviral RNAi expression system ( Invitrogen ) .\",\n \"For shRNA-mediated gene silencing , the respective target sequences were as follows: CDK4 , 5′-CCTAGATTTCCTTCATGCCAA-3′ ( sh-CDK4 ) ; mDia1 , 5′-GCCCAGAATCTCTCAATCTTT-3′ ( sh-mDia1 ) ; Rb , 5′-CAGAGATCGTGTATTGAGATT-3′ ( sh-Rb ) ; the non-target control , 5′-CAACAAGATGAAGAGCACCAA-3′ ( sh-Control ) .\",\n \"The recombinant adenoviruses were purified with AsEasy virus purification kits ( Agilent technologies , Palo Alto , CA ) and adenovirus infections were performed with ViraDuctin adenovirus transduction reagent ( Cell Biolabs , San Diego , CA ) .\",\n \"Mammalian expression vectors that encode shRNAs against human LAP2α or enhanced GFP ( eGFP ) were constructed by cloning suitable oligonucleotide sequences ( human LAP2α , 5′-CAGAAGAGAATTGATCAGT-3′; eGFP , 5′-ACAACAGCCACAACGTCTA-3′ ) into the pSilencer 2 . 1-U6 Hygro vector ( Ambion , Austin , TX ) .\",\n \"pXJ Flag-mDia1 was obtained from C Koh ( Xie et al . , 2008 ) .\",\n \"Flag-mDia1 plasmids were transfected into 293T cells and total cell lysates were extracted .\",\n \"The mDia1 proteins tagged with a Flag epitope at their amino-termini were captured on anti-Flag antibody-agarose beads and eluted by competition with free 1× Flag peptide ( Sigma ) in 50 mM Tris ( pH 7 . 4 ) and 50 mM NaCl .\",\n \"The purity of the mDia1 proteins , wild-type and ΔLXCXE , was evaluated by SDS-polyacrylamide gel electrophoresis ( SDS-PAGE ) , followed by Coomassie Brilliant Blue staining ( CBB staining ) .\",\n \"Full-length recombinant Rb protein was purchased from QED bioscience ( San Diego , CA ) .\",\n \"The recombinant glutathione S-transferase ( GST ) fusion Rb proteins were produced in BL21 E . coli .\",\n \"GST-Rb proteins were incubated with CDK4/Cyclin D1 ( Merck Millipore , Billerica , MA ) and CDK2/Cyclin E ( Merck Millipore ) proteins in the presence or absence of 100 μM ATP in the kinase buffer ( 20 mM Tris [pH 7 . 5] , 10 mM MgCl2 and 1 mM dithiothreitol [DTT] ) for 30 min at 30°C .\",\n \"The cells were fixed with 4% paraformaldehyde ( PFA ) in phosphate-buffered saline ( PBS ) for 30 min , permeabilized with 0 . 2% ( vol/vol ) Triton X-100/PBS for 5 min , and then blocked with 1% bovine serum albumin ( BSA ) /PBS for 30 min at room temperature .\",\n \"Subsequently , the cells were incubated with anti-Rb ( 1:200 , 9309; Cell Signaling Technology ) , anti-phospho Rb-S780 ( 1:300 , 13H9L5; Novex , Carlsbad , CA ) and anti-LAP2α ( 1:300 , ab5162; Abcam ) antibodies for 1 hr , and further incubated with Alexa Fluor 546-conjugated goat anti-mouse IgG , Alexa Fluor 546-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated goat anti-rabbit IgG antibodies ( Molecular Probes , Carlsbad , CA ) for 30 min .\",\n \"DAPI was used for nuclear staining .\",\n \"For double-staining for α-actinin and F-actin , cytoskeletal stabilizing buffer was used in lieu of PBS , as described previously ( Hirata et al . , 2008 ) .\",\n \"The cells were incubated with anti-α-actinin antibody ( 1:100 , EA53; Sigma ) , followed by incubation with Alexa Fluor 488- or 546-conjugated goat anti-mouse IgG antibody and Alexa Fluor 546- or 633-phalloidin ( Molecular Probes ) .\",\n \"Confocal images were taken using a PerkinElmer Spinning Disk microscope or a Nikon A1Rsi microscope , equipped with oil-immersion objectives ( 60× and 100× ) .\",\n \"Epifluorescence images were taken using a Nikon A1Rsi microscope , equipped with an oil-immersion objective ( 60× ) and an electron multiplying charge-coupled device camera ( DU897; Andor technology , Belfast , UK ) .\",\n \"Images were acquired with the Volocity software and the Nikon NIS-Elements imaging software .\",\n \"To evaluate periodicity in the distribution of α-actinin , 256 × 256 pixel , corresponding to 12 × 12 μm , sample areas of fluorescence images of α-actinin were subjected to computing autocorrelation analyses as described previously ( Peterson et al . , 2004 ) using the ImageJ program ( NIH , version 10 . 2 ) .\",\n \"Each autocorrelation image was then normalized by the value of the central peak in the image .\",\n \"Features of periodicity of its distribution are represented as local peaks other than the central maxima .\",\n \"Sample images of α-actinin and normalized autocorrelation images are shown .\",\n \"Frozen blocks of human skeletal muscle were obtained from Asterand ( Detroit , MI ) , who acquired appropriate informed consent from patients under the Institutional Review Board ( IRB ) approval .\",\n \"Subject characteristics are described in Table 1 . 10 . 7554/eLife . 01228 . 017Table 1 . Subject characteristicsDOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 017Biosample diagnosisNormalAtrophyGender and age , yearsFemale , 15Female , 14Cancer diagnosisOsteosarcomaSynovial sarcomaCancer locationShin boneSoft tissues of shinHeight , cm166159Weight , kg5050BMI , kg/m218 . 1419 . 78BMI , body mass index The sections were fixed in acetone for 10 min , permeabilized with 0 . 2% ( vol/vol ) Triton X-100/PBS for 30 min , and then blocked with CAS-Block ( Invitrogen ) for 10 min at room temperature .\",\n \"Subsequently , the sections were incubated with anti-α-actinin , anti-Rb ( 1:100 , ab24; Abcam ) and anti-mDia1 ( 1:400 , ab11173; Abcam ) antibodies overnight at 4°C .\",\n \"They were further incubated with appropriate fluorescence-labeled secondary antibodies for 1 hr at room temperature .\",\n \"HSMMs grown on 4-well plates ( Nunc , Naperville , IL ) were pretreated with IFN-γ and then placed in a C-Dish electrode chamber ( IonOptix , Milton , MA ) after changing to fresh serum-free media .\",\n \"EPS was applied to HSMMs using a C-Pace pulse generator ( IonOptix ) at 40 V/60 mm , 1 Hz , 10 ms for 2 days in parallel with TNF-α treatment .\",\n \"The media were changed and TNF-α was repeatedly added every 24 hr .\",\n \"Images of live beating cells were taken using Nikon Eclipse Ti-U microscope , equipped with a 20× objective lens .\",\n \"Images were sequentially acquired with Nikon NIS-Elements imaging software at a frame rate of 15 fps .\",\n \"Total RNA extraction , cDNA preparation , and real-time qPCR analyses were performed as described previously ( Kawauchi et al . , 2012 ) .\",\n \"The primer sets used were: Tk1 , 5′-CATTAACCTGCCCACTGT-3′ forward and 5′-GATCACCAGGCACTTGTA-3′ reverse; Dhfr , 5′-TCATGGTTGGTTCGCTAA-3′ forward and 5′-TGAAGAGGTTGTGGTCATT-3′ reverse; Tfam , 5′-TGTAGAAGCCACGGTGTT-3′ forward and 5′-ACAACCATCAACTCTGAATACAAT-3′ reverse; Ppargc1a , 5′-TGAAGAGGCAAGAGACAGAATGA-3′ forward and 5′-CACACGCACACTCCATCAC-3′ reverse; B2m , 5′-GCATTCCTGAAGCTGACA-3′ forward and 5′-CGTGAGTAAACCTGAATCTTT-3′ reverse .\",\n \"After normalization against B2m , data show mRNA expression levels relative to control expression levels for each experiment .\",\n \"Mass spectrometry analysis was performed by Proteomics International ( Perth , Australia ) .\",\n \"Protein samples were enzymatically digested to produce fragmented peptides and the resulting peptides were analyzed by electrospray ionization mass spectrometry using the Ultimate 3000 nano HPLC system ( Dionex , Sunnyvale , CA ) in combination with a 4000 Q TRAP mass spectrometer ( Applied Biosystems , Foster City , CA ) .\",\n \"The spectra were analyzed by Mascot sequence matching software ( Matrix Science , Boston , MA ) .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Skeletal muscle degeneration is a complication arising from a variety of chronic diseases including advanced cancer .","Pro-inflammatory cytokine TNF-α plays a pivotal role in mediating cancer-related skeletal muscle degeneration .","Here , we show a novel function for retinoblastoma protein ( Rb ) , where Rb causes sarcomeric disorganization .","In human skeletal muscle myotubes ( HSMMs ) , up-regulation of cyclin-dependent kinase 4 ( CDK4 ) and concomitant phosphorylation of Rb was induced by TNF-α treatment , resulting in the translocation of phosphorylated Rb to the cytoplasm .","Moreover , induced expression of the nuclear exporting signal ( NES ) -fused form of Rb caused disruption of sarcomeric organization .","We identified mammalian diaphanous-related formin 1 ( mDia1 ) , a potent actin nucleation factor , as a binding partner of cytoplasmic Rb and found that mDia1 helps maintain the structural integrity of the sarcomere .","These results reveal a novel non-nuclear function for Rb and suggest a potential mechanism of TNF-α-induced disruption of sarcomeric organization ."],"string":"[\n \"Skeletal muscle degeneration is a complication arising from a variety of chronic diseases including advanced cancer .\",\n \"Pro-inflammatory cytokine TNF-α plays a pivotal role in mediating cancer-related skeletal muscle degeneration .\",\n \"Here , we show a novel function for retinoblastoma protein ( Rb ) , where Rb causes sarcomeric disorganization .\",\n \"In human skeletal muscle myotubes ( HSMMs ) , up-regulation of cyclin-dependent kinase 4 ( CDK4 ) and concomitant phosphorylation of Rb was induced by TNF-α treatment , resulting in the translocation of phosphorylated Rb to the cytoplasm .\",\n \"Moreover , induced expression of the nuclear exporting signal ( NES ) -fused form of Rb caused disruption of sarcomeric organization .\",\n \"We identified mammalian diaphanous-related formin 1 ( mDia1 ) , a potent actin nucleation factor , as a binding partner of cytoplasmic Rb and found that mDia1 helps maintain the structural integrity of the sarcomere .\",\n \"These results reveal a novel non-nuclear function for Rb and suggest a potential mechanism of TNF-α-induced disruption of sarcomeric organization .\"\n]"},"summary":{"kind":"list like","value":["Skeletal muscles , such as the biceps and calves , are one of three main muscle groups in the body , and a range of chronic diseases—including cancer , heart disease and AIDS—can cause wasting and a loss of strength in these muscles .","Many different cellular processes are known to be involved in the degeneration of skeletal muscle during illness .","For example , in people suffering from cancer , the immune response produces large numbers of molecules called inflammatory cytokines to combat the cancer cells , and these molecules are thought to have a role in the breakdown of skeletal muscle .","A cytokine called tumour necrosis factor alpha , or TNF-α for short , is thought to cause muscle damage , but the details of this process are not fully understood .","One possibility is that TNF-α interacts with a protein called Rb—short for retinoblastoma protein—that suppresses the proliferation of cells that leads to cancer .","However , if this protein is modified by a chemical process called phosphorylation , the Rb molecules will not be able to suppress the genes that lead to excessive cell growth .","The hyperphosphorylation of Rb has been observed in many cancer cells , and it has been shown that high levels of TNF-α in cells results in Rb not working properly , but it has not been clear if faulty Rb also leads to the breakdown of skeletal muscle .","Now Araki et al . provide evidence that the phosphorylation of Rb by TNF-α leads to skeletal muscle degeneration .","Araki et al . found that in muscle cells that contain high concentrations of TNF-α , the Rb molecules move from the nuclei of the cells , where they interact with genes , to the cytoplasm , where they disrupt the formation of structural fibres .","This means that Rb inhibits the ability of muscle cells to slide over one during contractions and relaxation , as happens in normal muscle tissue .","If confirmed by further experiments , these results could lead to the development of new approaches for the treatment of skeletal muscle degeneration ."],"string":"[\n \"Skeletal muscles , such as the biceps and calves , are one of three main muscle groups in the body , and a range of chronic diseases—including cancer , heart disease and AIDS—can cause wasting and a loss of strength in these muscles .\",\n \"Many different cellular processes are known to be involved in the degeneration of skeletal muscle during illness .\",\n \"For example , in people suffering from cancer , the immune response produces large numbers of molecules called inflammatory cytokines to combat the cancer cells , and these molecules are thought to have a role in the breakdown of skeletal muscle .\",\n \"A cytokine called tumour necrosis factor alpha , or TNF-α for short , is thought to cause muscle damage , but the details of this process are not fully understood .\",\n \"One possibility is that TNF-α interacts with a protein called Rb—short for retinoblastoma protein—that suppresses the proliferation of cells that leads to cancer .\",\n \"However , if this protein is modified by a chemical process called phosphorylation , the Rb molecules will not be able to suppress the genes that lead to excessive cell growth .\",\n \"The hyperphosphorylation of Rb has been observed in many cancer cells , and it has been shown that high levels of TNF-α in cells results in Rb not working properly , but it has not been clear if faulty Rb also leads to the breakdown of skeletal muscle .\",\n \"Now Araki et al . provide evidence that the phosphorylation of Rb by TNF-α leads to skeletal muscle degeneration .\",\n \"Araki et al . found that in muscle cells that contain high concentrations of TNF-α , the Rb molecules move from the nuclei of the cells , where they interact with genes , to the cytoplasm , where they disrupt the formation of structural fibres .\",\n \"This means that Rb inhibits the ability of muscle cells to slide over one during contractions and relaxation , as happens in normal muscle tissue .\",\n \"If confirmed by further experiments , these results could lead to the development of new approaches for the treatment of skeletal muscle degeneration .\"\n]"},"year":{"kind":"string","value":"2013"}}},{"rowIdx":4293,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["neuroscience"],"string":"[\n \"neuroscience\"\n]"},"title":{"kind":"string","value":"Neuronal reactivation during post-learning sleep consolidates long-term memory in Drosophila"},"id":{"kind":"string","value":"elife-42786-v2"},"sections":{"kind":"list like","value":[["Animals guide their behavior in part based on the memories of past learning experiences .","Temporally , these memories can be either short- or long-lasting; short-term memory ( STM ) shapes future animal behavior within seconds , minutes to hours after a learning experience , whereas long-term memory ( LTM ) holds the learned information for hours , days or even a lifetime .","STM is thought to rely on protein synthesis-independent covalent modifications and changes in weight of existing synaptic connections , whereas LTM is believed to reflect protein synthesis-dependent structural changes in specific synapses ( Kandel , 2001 ) .","Depending on the salience or duration of the learning experience , some STMs can be transformed into LTMs through the process of memory consolidation ( Dudai et al . , 2015 ) .","Numerous studies in vertebrates suggest that sleep has a beneficial effect on memory in various learning tasks ( Marshall and Born , 2007; Smith , 2001 ) .","Studies in humans and rodents have shown that an insufficient amount of sleep impairs cognitive functions such as memory formation and retention , while an ample amount of sleep after a learning experience supports memory storage ( Goel et al . , 2009; Diekelmann and Born , 2010 ) .","In addition , several studies have found that animals , including humans , exhibit an enhanced amount and quality of sleep after experiencing a novel or enriched environment ( Gais et al . , 2002; Smith and Wong , 1991; Walker and Stickgold , 2006 ) .","One hypothesis is that sleep plays an active role in consolidation of newly acquired memories into long-term storage , that are critical for the animal future actions .","To date , however , a mechanistic understanding of sleep function in this selective memory consolidation remains elusive .","Over the last two decades , studies of sleep and LTM have expanded from the almost exclusive studies in mammals to other animals , including insects .","Sleep is now well-documented in Drosophila .","Sleep in Drosophila exhibits almost all the hallmarks of sleep in mammals , including homeostatic regulation , diminished behavioral responsiveness , and the existence of different sleep stages as characterized by distinct electrophysiological signatures ( Dissel et al . , 2015a; Yap et al . , 2017; van Alphen et al . , 2013 ) .","Recently , a link between sleep and LTM has been established: sleep deprivation before and after learning impairs mnemonic performance ( Ganguly-Fitzgerald et al . , 2006; Seugnet et al . , 2008; Seugnet et al . , 2009 ) , whereas sleep induction enhances memory formation and retention in wild-type ( Donlea et al . , 2011 ) and restores memory in memory mutant flies ( Dissel et al . , 2015b ) .","Moreover , flies that were exposed to a socially enriched environment , including courtship learning , exhibit an increased amount of sleep ( Ganguly-Fitzgerald et al . , 2006 ) .","Courtship conditioning has been extensively exploited to study both memory and sleep in Drosophila .","Courtship conditioning is an ethologically relevant form of complex learning whereby male flies learn to associate the outcome of their own behavior with multisensory cues presented by females during courtship ( Ejima et al . , 2005 ) .","Naive Drosophila males eagerly court both virgin and mated females , which are generally receptive and unreceptive , respectively .","However , after being repeatedly rejected by mated females , males become less likely to court other mated females ( Ejima et al . , 2005; Keleman et al . , 2012 ) .","Like with other types of learning , the resulting memory can last from minutes to days depending on the training protocol .","We previously established that activity of the dopaminergic aSP13 neurons ( DAN-aSP13s ) is necessary and sufficient for STM acquisition via the dopamine receptor DopR1 in the γ neurons of the mushroom body known as Kenyon Cells ( γKCs ) ( Keleman et al . , 2012 ) , a neuropil in the Drosophila central brain critical for memory formation ( Heisenberg et al . , 1985 ) .","Moreover , we recently demonstrated that the activity of the same DAN-aSP13s is also essential for the consolidation of courtship STM to LTM .","This requirement is observed in a discrete time window after learning , and it is mediated via the dopamine receptor DopR1 in the γKCs ( Krüttner et al . , 2015 ) .","In this study , we examine the mechanisms of DAN-aSP13 post-learning activation .","We show here that DAN-aSP13s are activated during sleep after courtship experience that induces LTM .","We present evidence that the specific class of sleep-promoting neurons in the fan-shaped body ( FB ) , a neuropil previously implicated in sleep regulation ( Donlea et al . , 2011 ) , activates DANs-aSP13 in the discrete time window after learning to consolidate courtship LTM ."],["To investigate the role of sleep in post-learning activation of DAN-aSP13s , and hence LTM consolidation , we first asked whether DAN-aSP13s are active in freely behaving males after a prolonged experience with mated females , which induces LTM .","To monitor activity of DAN-aSP13s for several hours in unrestrained males we employed a luminescence-based transcriptional reporter of neuronal activity ( Guo et al . , 2017 ) .","Using specific DAN-aSP13s GAL4 driver ( Aso et al . , 2014a ) ( Figure 1—figure supplement 1A ) , we expressed luciferase exclusively in DAN-aSP13s under the control of multimerized binding sites for the neuronal activity regulated gene Lola and FLP recombinase target sites ( FRT ) for its activity dependent cell specific expression ( MB315B-GAL4>UAS-FLP , Lola ( FRT ) stop ( FRT ) LUC ) ( Chen et al . , 2016 ) .","We measured luminescence as a proxy for neuronal activity in males that had undergone prolonged training with mated females and in naive males without prior courtship experience .","As a control we used males after prolonged training with virgin females which does not induce memory when tested 24 hr later with mated females ( our unpublished results ) .","For training , single males were paired with a single recently-mated or virgin female for 6 hr .","Afterwards , trained , naïve and control males were individually transferred to a 96-well luminescence reading plate .","Luminescence measurements were taken every 15 min over 16 hr beginning at a common starting time for all males , at the hour seven from the onset of training .","Notably , flies that had undergone training with mated females displayed a gradual increase of the luminescence signal , which was significantly different from naïve males between 7–10 hr from the onset of training ( Figure 1A ) .","In contrast , control males that had undergone training with virgin females also displayed an increase in luminescence however , it was identical to that in naïve males ( Figure 1—figure supplement 1B ) .","That specific time window of DAN-aSP13 activation after onset of training with mated females was preserved in males that were trained in a later circadian time during the day ( Figure 1—figure supplement 1C ) .","Together , these data show that DAN-aSP13s are activated in a specific time window after a learning experience that leads to LTM .","It was previously shown that flies that had been subjected to social enrichment , including courtship experience , display an increased amount of day time sleep ( Ganguly-Fitzgerald et al . , 2006 ) .","To determine whether Drosophila males sleep in the time period when DAN-aSP13s are activated , we measured the amount of sleep in males after prolonged courtship training and in naïve males .","Sleep was analyzed with Drosophila Activity Monitors , and periods of inactivity lasting for at least 5 min were classified as sleep ( Shaw et al . , 2000; Hendricks et al . , 2000 ) .","Both trained and naive wild-type males showed a significant amount of day time sleep , particularly during hours 7–10 when DAN-aSP13s are active .","Importantly , males that had undergone training for LTM slept significantly more in this time window in comparison to naïve males ( Figure 1B ) .","Both groups displayed the same amount of sleep throughout the night .","Notably , enhancement of post-training sleep occurs between 7–10 hr from the start of training regardless of the circadian time of training .","Males that were trained for LTM in the afternoon instead of the usual morning session , displayed an increased amount of sleep between 7–10 hr after onset of training ( Figure 1—figure supplement 1D ) .","They also had a normal LTM when tested 24 hr later ( Figure 1—figure supplement 1E , Figure 1—figure supplement 2A ) .","To evaluate their memory , we used automated video analysis to derive a courtship index ( CI ) for each male; CI is defined as the percentage of time over a 10 min test period during which the male courts the female .","Memory is represented as a suppression index ( SI ) , which is the relative reduction in the median courtship indices of trained versus naïve males: SI = 100*[1-CItrain+/CItrain-] ( Keleman et al . , 2012 ) .","In contrast , males that were trained for 1 hr to induce STM did not exhibit an increased amount of sleep ( Figure 1—figure supplement 1F ) .","Together , these data suggest that only training that can induce LTM leads to sleep enhancement between 7–10 hr after the start of training .","To investigate whether the enhancement of post-training sleep is caused by learning or a prolonged intense activity such as courtship towards mated female , we monitored sleep pattern and courtship memory of mutants for the dopamine receptor DopR1 that do not form courtship memory due to an impairment in memory acquisition ( Keleman et al . , 2012 ) .","DopR1 mutant males , although underwent the same courtship experience and courted mated females as vigorously or more than the wild-type males during 6 hr training ( Figure 1—figure supplement 1G ) , neither displayed an increased amount of post-training sleep ( Figure 1C ) nor formed LTM ( Figure 1—figure supplement 1H , Figure 1—figure supplement 2B ) .","Together , these data show that learning is essential for the sleep enhancement in the specific time window after training .","To determine whether post-learning sleep is necessary for LTM consolidation , we deprived flies of sleep during specific time intervals after training and tested their memory 24 hr later .","Single wild-type males were trained with mated females for 6 hr and immediately after were exposed to intermittent gentle mechanical perturbation spanning 2 hr intervals .","Naïve males were sleep deprived during the same time periods .","Control males that were trained with mated females and allowed to sleep , and males that were sleep deprived after training between 9–11 and 10–12 hr had normal SIs of approximately 30–40% .","However , males that were deprived of sleep between 7–9 and 8–10 hr had SIs indistinguishable from 0 ( Figure 2A , Figure 2—figure supplement 2A ) .","Deprivation of sleep during the night did not impair LTM significantly , except for a small but statistically-significant effect between 14–16 hr ( Figure 2—figure supplement 1A , Figure 2—figure supplement 2E ) .","These results show that sleep deprivation between 7–9 hr after the start of training blocks LTM , and thus post-learning sleep is necessary for LTM consolidation .","Previously , we had shown that silencing of DAN-aSP13s in a discrete time window after training impairs LTM ( Krüttner et al . , 2015 ) .","To test whether the temporal effect of sleep deprivation mimics the effect of DAN-aSP13s silencing , we expressed specifically in DAN-aSP13s ( Figure 2—figure supplement 1B ) a temperature-sensitive inhibitory form of dynamin shibire ( shits ) ( Kitamoto , 2002 ) ( VT005526-LexA>LexAop-shits ) , which blocks synaptic transmission at 32°C but not at 22°C .","We silenced DAN-aSP13s at 2 hr intervals after a 6 hr training session .","Inhibition of DAN-aSP13s between 7–9 or 8–10 hr , but not between 9–12 hr , after the onset of training abolished LTM .","Control males , in which DAN-aSP13s remained functional throughout the assay , had a normal SI between 30–40% ( Figure 2B , Figure 2—figure supplement 2B ) .","These results show that sleep and DAN-aSP13 activity are essential for memory consolidation in the same time window after training .","To investigate whether sleep can consolidate STM into LTM , we tested whether artificial enhancement of sleep would lead to memory consolidation .","We trained males for 1 hr , which does not generate LTM ( Krüttner et al . , 2015 ) , and then induced sleep at various time intervals afterwards .","Since activation of the FB neurons induces sleep ( Donlea et al . , 2011 ) , we expressed the thermosensitive cation channel TrpA1 ( Viswanath et al . , 2003; Rosenzweig et al . , 2005 ) ( open at 30°C and closed at 20°C ) in FB neurons ( 104y-GAL4 > UAS-TrpA1 ) and monitored sleep for 24 hr .","Consistent with previous reports , males in which FB neurons were activated slept significantly more than males that were kept at 20°C and the empty-GAL4 control males ( pBDP-GAL4 > UAS-TrpA1 ) ( Figure 2—figure supplement 1C ) .","To induce sleep precisely with 2 hr temporal resolution after a 1 hr training session , we used the optogenetic activator CsChrimson ( Klapoetke et al . , 2014 ) .","We found that activating FB neurons ( 104y-GAL4 > UAS CsChrimson ) in the period between 5–7 hr after the onset of training led to a SI of ~ 30% , which is similar to that obtained after LTM training ( Figure 2C , Figure 2—figure supplement 2C ) .","In contrast , males activated at other time intervals or not activated at all failed to consolidate LTM and had SIs that were indistinguishable from 0 .","This temporal window was identical to that observed for DAN-aSP13s activation to consolidate LTM ( VT005526-LexA > LexAop-CsChrimson ) ( Figure 2D , Figure 2—figure supplement 2D ) .","When we activated FB neurons ( 104y-GAL4 > UAS-CsChrimson ) while silencing DAN-aSP13s ( VT005526-LexA > LexAop-Shits ) between 5–7 hr after the onset of training we did not observe LTM ( Figure 2C , Figure 2—figure supplement 2C ) .","Taken together , these data show that enhancement of sleep after a learning experience that induces STM can consolidate it to LTM , in a manner that requires activation of DAN-aSP13s .","This LTM consolidation is a specific effect of DAN-aSP13s activation after training since activation of DAN-aSP13s ( VT005526-LexA > LexAop-CsChrimson ) in naïve males between 5–7 hr when they normally display a significant amount of sleep does not induce courtship suppression towards mated females and thus ‘courtship LTM’ ( Figure 2—figure supplement 1D ) .","To test whether FB activation excites DAN-aSP13s , we optogenetically activated FB neurons ( 104y-GAL4 > UAS-Chrimson88 ) and monitored calcium levels in DANs located in the protocerebral anterior medial ( PAM ) cluster in explant brains ( R58E02-LexA > LexAop-GCamP6s ) ( Pfeiffer et al . , 2008; Chen et al . , 2013 ) .","The PAM cluster of DANs consists of multiple neuronal classes , including a class of DAN-aSP13s , which is thought to respond to rewarding stimuli and promote their avoidance ( Aso et al . , 2014b ) ( Figure 3—figure supplement 1A ) .","Since the expression pattern of 104y-GAL4 is not exclusive to the FB ( Donlea et al . , 2011 ) , to activate FB neurons selectively , we used a digital mirror device ( DMD ) to target the illumination activating Chrimson to specific layers in the FB ( Strother et al . , 2018 ) .","104y-FB neurons form three layers of projections: dorsal ( dFB ) , medial ( mFB ) and ventral ( vFB ) ( Donlea et al . , 2011 ) .","To test the targeted illumination , we expressed in 104y neurons both Chrimson88 and GCamP6s ( 104y-GAL4 > UAS-Chrimson88 > UAS-GCamP6s ) and selectively activated one of two layers of the FB ( dFB or vFB ) while monitoring calcium levels in the presence of tetradotoxin ( TTX ) .","TTX inhibits propagation of action potentials ( Boccaccio et al . , 1999 ) , and thus calcium signals should only be observed in the projections located in the targeted layer .","Upon activation of either dFB or vFB layers , a robust calcium response was observed only in the activated layer , implying that this local activation was highly restricted ( Figure 3A ) .","We next activated all FB layers using targeted illumination and monitored calcium responses in DAN-aSP13s ( 104y-GAL4 > UAS-Chrimson88 , R58E02-LexA > LexAop-GCamP6s ) .","Local activation of FB neurons elicited an excitatory response in DAN-aSP13s ( Figure 3B ) .","Given that the FB has a multilayered organization , we next aimed to identify the specific layer of the 104y-FB expression pattern that provides excitatory input to DAN-aSP13s .","We individually activated all three 104y-FB layers and monitored calcium levels in DAN-aSP13s .","Surprisingly , activation of the dFB layer , which has been recently implicated in homeostatic sleep regulation ( Donlea et al . , 2011 ) , resulted in a mixture of small inhibitory and excitatory responses ( Figure 3C ) , whereas activation of the vFB layer induced excitatory responses in DAN-aSP13s ( Figure 3D ) .","Activation of the mFB had no effect on DAN-aSP13 activity ( Figure 3—figure supplement 1B ) .","These data show that neurons that project to the vFB and potentially the dFB layers provide an excitatory input onto DAN-aSP13s and may thus have a role in LTM consolidation .","To identify the specific neurons in the 104y-GAL4 line that project to the different FB layers , we performed multicolor flip-out experiments ( Nern et al . , 2015 ) .","We identified two distinct neuronal populations that project to either the dFB or vFB layers ( Figure 4—figure supplement 1A ) .","Next , we searched the Janelia ( Jenett et al . , 2012 ) and Vienna Tiles ( VT ) ( Tirian and Dickson , 2017 ) collections for specific GAL4 driver lines with expression in these two cell types .","We identified one sparse GAL4 line with expression in the dFB neurons ( R23E10 ) and one in the vFB neurons ( VT036875 ) .","Since the expression pattern of the VT036875-GAL4 line also includes a class of DAN-β’1 neuron , we combined this line with PAM-GAL80 to restrict its expression to vFB neurons only ( VT036875-GAL4 , R58E02-GAL80 ) .","In addition , we generated a split line ( SS057264-GAL4 ) ( Pfeiffer et al . , 2010; Luan et al . , 2006 ) with the expression restricted to the vFB neurons only ( Figure 4—figure supplement 1B , C , D , E ) .","As previously reported , thermogenetic activation of dFB over a 24 hr time period resulted in an increased amount of sleep ( R23E10-GAL4 > UAS-TrpA1 ) ( Figure 4A ) .","Sleep increase was displayed mainly during the day time since males sleep almost continuously during night .","We found that activation of vFB ( VT036875-GAL4 > UAS-TrpA1; VT036875-GAL4 > UAS-TrpA1 , R58E02-GAL80; SS057264-GAL4 > UAS-TrpA1 ) also robustly induced day time sleep ( Figure 4B , C , D ) in comparison to the genetic control ( pBDP-GAL4 > UAS-TrpA1 ) ( Figure 4—figure supplement 1G ) .","Loss of night time sleep observed upon prolonged vFB activation with VT036875-GAL4 ( Figure 4B ) was likely caused by co-activation of wake promoting DAN-β’1 neurons ( Sitaraman et al . , 2015a ) ( Figure 4—figure supplement 1C ) .","Accordingly , males upon activation with the same line while excluding DAN-β’1 neurons displayed a higher level of both day and night sleep ( Figure 4C , Figure 4—figure supplement 1D ) .","Similarly , acute optogenetic activation of the dFB and vFB neurons with CsChrimson for 1 hr also significantly enhanced the amount of sleep ( R23E10-GAL4 > UAS -CsChrimson; VT036875-GAL4 > UAS-CsChrimson; VT036875-GAL4 > UAS-CsChrimson , R58E02-GAL80; SS057264-GAL4 > UAS-CsChrimson; 104y-GAL4 > UAS-CsChrimson ) in comparison to the genetic control ( pBDP-GAL4 > UAS-CsChrimson ) but to a lesser degree when DAN-β’1 neurons were co-activated ( Figure 4—figure supplement 1F ) .","To test whether these neurons provide an excitatory input on DAN-aSP13s , we activated either dFB ( R23E10-GAL4 > UAS-Chrimson88 ) or vFB ( VT036875-GAL4 > UAS-Chrimson88; VT036875-GAL4 > UAS-Chrimson88 , R58E02-GAL80; SS057264-GAL4 > UAS-Chrimson88 ) neurons and monitored calcium levels in DAN-aSP13s .","Since specific DANs innervate MB lobes in well-defined discrete areas , we used a broad PAM-DAN GAL4 driver ( R58E02-LexA > LexAop-GCamP6s ) .","To monitor activity specifically in DAN-aSP13s upon activation of FB neurons we focused on the region at the tip of the MBγ lobe ( Figure 3—figure supplement 1A ) .","Activation of dFB neurons did not elicit calcium changes in DAN-aSP13s ( Figure 4A ) however , activation of vFB neurons elicited a robust increase of calcium levels in DAN-aSP13s ( Figure 4B , C , D ) .","These data show that neurons in the vFB provide excitatory input onto DAN-aSP13s .","Consistent with the functional connectivity results , optogenetic activation of dFB neurons ( R23E10-GAL4 > UAS-CsChrimson ) between 5–7 hr after a 1 hr training period did not lead to LTM consolidation .","However , activation of either a combination of dFB and vFB neurons ( 104y-GAL4 > UAS-CsChrimson ) or just the vFB neurons ( VT036875-GAL4 > UAS-CsChrimson; VT036875-GAL4 > UAS-CsChrimson , R58E02-GAL80; SS057264-GAL4 > UAS-CsChrimson ) in that same time window fully consolidated STM to LTM ( Figure 5A , Figure 5—figure supplement 1A ) .","In addition , silencing of dFB neurons ( R23E10-GAL4 > UAS-Shits ) in the period between 7–10 hr after the onset of a 6 hr training period did not affect LTM persistence , while silencing of a combination of dFB and vFB or just the vFB neurons ( VT036875-GAL4 > UAS-Shits; SS057264-GAL4 > UAS-Shits and 104y-GAL4 > UAS-Shits ) in the same time window strongly impaired LTM consolidation ( Figure 5B , Figure 5—figure supplement 1B ) .","These results show that a class of sleep-promoting neurons in the vFB layer of the 104y-FB expression pattern is necessary and sufficient to consolidate LTM during post-learning sleep .","dFB neurons , although they promote sleep , appear to have no role in courtship LTM consolidation ."],["The activity of DAN-aSP13s , which is essential for courtship memory acquisition ( Keleman et al . , 2012 ) , is also necessary during a discrete post-learning time window for LTM consolidation ( Krüttner et al . , 2015 ) .","Because neuronal reactivation occurs during sleep in rodents ( Wilson and McNaughton , 1994; Smith , 2001 ) , we hypothesized that post-learning activation of DAN-aSP13s involves a sleep-dependent mechanism .","Using behavioral analysis and neuronal activity monitoring and perturbation approaches , we have shown here that DAN-aSP13s display an increased activity in freely behaving animals during sleep after a prolonged learning experience .","We demonstrated that this sleep is necessary for LTM consolidation , and it can be mediated by a specific class of sleep promoting neurons in the ventral layer of the FB ( vFB ) .","These vFB neurons consolidate courtship LTM in a discrete time window and provide an excitatory input to DAN-aSP13s .","Thus , the data we present here provide a causal link between sleep promoting neurons in the vFB , post-learning activation of dopaminergic neurons , and LTM consolidation .","Based on our data , we propose the following model for sleep-dependent consolidation of courtship LTM in Drosophila ( Figure 6 ) .","During a prolonged learning experience , γKCs and DAN-aSP13s are repeatedly activated by olfactory and behavioral cues presented by an unreceptive female , respectively .","Whereas prolonged wakefulness leads to an increase in homeostatic sleep drive in ellipsoid body neurons that in turn is conveyed to dFB ( Liu et al . , 2016 ) , we hypothesize that an extended learning experience generates a learning-dependent sleep drive that is transmitted to vFB .","In turn , the vFB neurons enhance sleep after learning and provide an excitatory input back on DAN-aSP13s .","We believe that one potential site of a learning-dependent sleep drive are the MB neurons since they have been implicated in both memory formation ( Heisenberg et al . , 1985 ) and sleep regulation ( Sitaraman et al . , 2015b ) .","Dopamine released upon DAN-aSP13 reactivation stimulates molecular processes in γKCs ( Krüttner et al . , 2015 ) that are different from those engaged during STM acquisition and involve protein synthesis that is essential for LTM formation and persistence .","Dopaminergic pathways are thought to convey information about whether an experience is rewarding or punishing and thus , worth remembering ( Gais et al . , 2002; Schultz , 2010 ) .","Post-learning neuronal activity of the dopaminergic hippocampal inputs from the Ventral Tagmental Area ( VTA ) has been implicated in the consolidation of fear memory in rodents .","Interestingly , this activity is critical during a discrete time window after learning ( Rossato et al . , 2009 ) .","Post-learning activity of the VTA dopamine neurons has been also implicated in the reactivation during sleep of the hippocampal cells involved earlier in encoding of the spatial experience ( Gomperts et al . , 2015 ) .","Here we show that post-learning activation of DAN-aSP13s mediates the consolidation of courtship LTM in Drosophila .","We propose that reactivation during sleep of the dopamine neurons that were previously active during memory acquisition ensures that spurious experiences are not admitted into LTM storage and thus only experiences that are either sufficiently salient or persistent become long-lasting memories .","Specifically , reactivation of DAN-aSP13s during post-learning sleep enhances reactivation of the γKCs and cognate MBON-M6 , which together with DAN-aSP13s form a recurrent circuit necessary for courtship memory acquisition ( Zhao et al . , 2018 ) .","We consider two hypotheses to account for this selectivity .","During sleep , vFB neurons might selectively reactivate only the relevant DANs , or alternatively , they might activate all DANs but only the relevant subset is able to consolidate LTM .","The selective-reactivation model would require some marker to distinguish which DANs were activated , whereas the selective-consolidation model would require a marker in the synapses of the γKCs that were earlier active during memory acquisition , for example translational regulator Orb2 , which regulates translation upon neuronal activity during LTM consolidation ( Krüttner et al . , 2015 ) .","Activity of dopaminergic neurons regulates sleep-wake states in animals , including flies ( Dzirasa et al . , 2006 ) .","Artificial activation of DAN-aSP13s has been shown to increase wakefulness ( Sitaraman et al . , 2015a ) .","In contrast , the data we present here imply that activation of vFB neurons , although they activate DAN-aSP13s , do not promote wakefulness .","These results suggest that activation of DAN-aSP13s by vFB neurons during sleep is qualitatively different from a direct optogenetic or thermogenetic activation used in previous studies .","One potential explanation is that post-learning sleep involves activation of the vFB circuit , which provides both excitatory stimulus to DAN-aSP13s and inhibitory input to motor neurons .","Another possibility is that post-learning sleep activates a subset od DAN-aSP13s that do not affect wakefulness .","We have identified here a class of sleep-promoting neurons in the ventral layer of FB that are distinct from the well-studied sleep-promoting neurons in the dorsal layer of FB , which regulate sleep homeostasis ( Donlea et al . , 2011; Donlea et al . , 2014; Pimentel et al . , 2016 ) .","Given that vFB neurons enhance sleep and activate DAN-aSP13s for LTM consolidation , whereas dFB neurons are neither necessary nor sufficient for LTM consolidation , we hypothesize that dFB and vFB neurons promote distinct components of sleep that have different functions .","Homeostatic sleep is thought to facilitate memory encoding by downscaling synaptic weights and clearing metabolites from the brain accumulated during wakefulness ( Bushey et al . , 2011; Xie et al . , 2013; Tononi and Cirelli , 2014 ) .","In contrast , the function of learning-dependent sleep might be to facilitate memory consolidation by strengthening synaptic connections that were engaged earlier during memory acquisition ( Frank , 2012 ) .","Thus , the co-operation of homeostatic- and experience-dependent sleep would facilitate optimal conditions for learning new information and , if appropriate , incorporating it into long-term storage .","Recent studies have implied that sleep in flies , as in humans and rodents , exhibits sleep stages characterized by distinct electrophysiological signatures ( Yap et al . , 2017; van Alphen et al . , 2013 ) .","Interestingly , the signature of sleep that is induced by activation of the dFB neurons seems to have a simpler oscillatory pattern , recorded by local field potentials , than sleep that is induced by activation of the FB neurons comprising both dFB and vFB neurons ( Yap et al . , 2017 ) .","Thus , these data support our hypothesis that dFB and vFB neurons promote sleep with different properties and likely different functions .","It is thought that sleep evolved in animals that are capable of complex learning which requires selective attention ( Kirszenblat and van Swinderen , 2015 ) .","Courtship learning is a multisensory form of learning that requires selective attention of a male to associate multiple learning cues presented by the mated female with the outcome of his own behavior ( Kirszenblat and van Swinderen , 2015 ) .","Accordingly , studies in bees have shown that sleep affects a complex form of learning such as spatial memory but has no role in the simple learning paradigm of proboscis extension ( Vorster and Born , 2015 ) .","Hence , it would be interesting to investigate whether post-learning sleep is involved in the consolidation of other types of memory in Drosophila , such as the well-studied Pavlovian olfactory associative learning whereby animals associate an individual learning cue with a behavioral contingency .","In this work , we establish a functional link between a novel class of sleep-promoting neurons in the FB , post-learning reactivation of dopaminergic neurons and consolidation of courtship LTM .","Moreover , our data suggest that sleep promoting vFB neurons mediate a learning-dependent regulation of sleep that is distinct from the homeostatic control which is facilitated by dFB neurons ( Liu et al . , 2016 ) .","Thus , we uncover a causal link between sleep-mediated neuronal reactivation and LTM consolidation in Drosophila .","In addition , we establish courtship LTM in Drosophila as a tractable model to investigate the mechanisms that link learning-dependent sleep , neuronal reactivation and LTM consolidation ."],["Flies for behavioral experiments were reared in vials with standard cornmeal food at 25°C , or as indicated , at 60% humidity in a 12 hr light:12 hr dark cycle .","Detailed information regarding specific strains and genotypes is provided in the Key resources table section .","Courtship conditioning was performed as described ( Siwicki and Ladewski , 2003 ) .","Briefly , solitary males aged for 5–6 days after eclosion were placed for training ( during day time as indicated ) in food chambers for 1 hr ( STM ) or 6 hr ( LTM ) either with ( trained ) or without ( naïve ) a single mated female .","After training each male was recovered , allowed to rest for 30 min or 24 hr and tested with a fresh mated female .","Tests were performed in 10 mm diameter chambers and videotaped for 10 min ( Prosilica GT cameras , Allied Vison Technologies ) .","Automated video analysis was used to derive a courtship index ( CI ) for each male , defined as the percentage of time over a 10 min test period during which the male courts the female .","Memory was calculated as a suppression index ( SI ) that is a relative reduction in the mean courtship indices of trained ( CI+ ) versus naïve ( CI- ) populations: SI = 100*[1-CItrain+/CItrain-] .","To monitor sleep over a 24 hr time period , male flies were individually inserted into 65 mm glass tubes containing standard fly food , loaded into TriKinetics Drosophila activity monitors ( DAM ) , and housed under 12 hr light:12 hr dark schedule ( Donelson et al . , 2012 ) .","Periods of inactivity lasting at least 5 min were classified as sleep .","Total 24 hr sleep quantity ( day time and night time sleep ) was extracted from DAM system as described ( Donelson et al . , 2012 ) .","To monitor sleep upon acute induction with csChrimson , individual males were reared at 25°C on retinal supplemented ( 0 . 1 mM ) cornmeal medium in darkness for 4–5 days after eclosion .","For sleep induction single males were placed in 10 mm diameter behavioral chambers in a temperature and illumination-controlled box and videotaped ( Prosilica GT cameras , Allied Vison Technologies ) .","The amount of sleep was scored manually with periods of inactivity lasting at least 5 min being considered as sleep .","For sleep deprivation , flies were subjected to intermittent mechanical perturbation method while housed in TriKinetics DAM monitors .","Flies received mechanical perturbations on a horizontal shaker with a total cycle of 15 s/min delivered in eight pulses of 1–3 s each occurring intermittently at random times .","A MATLAB script ( Kamyshev et al . , 1999 ) ( permutation test ) implemented in Keleman et al . ( 2012 ) was used for statistical comparison of SIs between two groups .","Briefly , the entire set of courtship indices for both naïve and trained flies were pooled and then randomly assorted into simulated naïve and trained groups of the same size as the original data .","A SI was calculated for each of 100 , 000 randomly permutated data sets , and P values were estimated for the null hypothesis that learning equals 0 ( H0: SI = 0 ) or for the null hypothesis that experimental and control males learn equally well ( H0: SI = SIc ) .","Luminescence assay for detecting neuronal activity in freely behaving adult flies was modified from Guo et al . ( 2017 ) .","Flies were starved on filter soaked in water overnight prior to training .","They were transferred to fresh chambers with filter paper containing 40 mM Luciferin ( GOLDBIO ) in a 2% sucrose solution .","For luminescence measurements after training , flies were placed into 96-well plates ( Greiner Bio-one ) with 40 mM Luciferin in a 2% sucrose solution .","CLARIOstar microplate reader ( BMG Labtech ) was used for luminescence detection .","Luminescence was measured every 15 min over 16 hr .","Relative Luminescence ( R . Luminescence ) was calculated by dividing the luminescence of the experienced and naïve groups by the luminescence of the genetic controls ( males with luciferase reporter only , fed with luciferin ) at every measurement time point .","Single housed male flies were reared at 25°C on retinal supplemented ( 0 . 1 mM ) cornmeal medium in darkness for 4–5 days after eclosion .","For sleep induction or memory consolidation specific classes of neurons were optogenetically activated in behavior chambers housed in a temperature and illumination-controlled box .","During activation , the behavior chamber was illuminated with 617 nm LEDs ( Red-Orange LUXEON Rebel LED—122 lm; Luxeon Star LEDs , Brantford , Ontario , Canada ) with a 3 mm thick diffuser between the LED and flies .","The LED was driven by a customized linear current controller .","20 HZ red light was used to activate neurons with intensity varying from 20 uW/mm2 to 70 uW/mm2 .","The LED board was cooled by a customized liquid cooling system to maintain the temperature in the chamber .","Single housed male flies were reared at 25°C on retinal supplemented ( 0 . 1 mM ) cornmeal medium in darkness for 4–5 days after eclosion .","Brains of the immobilized flies on ice were dissected out in saline ( 103 mM NaCl , 3 mM KCl , 2 mM CaCl2 , 4 mM MgCl2 , 26 mM NaHCO3 , 1 mM NaH2PO4 , 8 mM trehalose , 10 mM glucose , 5 mM TES , bubbled with 95% O2/5% CO2 ) and mounted anterior up on the cover slip in a Sylgard-lined dish in a 20°C saline bath .","Brains were imaged with a resonant scanning two-photon microscope with near-infrared excitation ( 920 nm , Spectra-Physics , INSIGHT DS DUAL ) and a 25x objective ( Nikon MRD77225 25XW ) .","The microscope was controlled by ScanImage 2015 . v3 ( Vidrio Technologies ) .","Images of brain volume were acquired with ~ 157 μm x 157 μm field of view at 512 × 512 pixels resolution .","Each frame and each volume was sampled by 42 frames with 3 μm per step , at approximately 1 Hz volume rate .","Times series of volume images were acquired and the excitation power for calcium imaging ~ 12 mW .","For the optogenetic activation , the light-gated ion channel Chrimson88 was activated with a 660 nm LED ( M660L3 Thorlabs ) coupled to a digital micromirror device ( DMD ) ( Texas Instruments DLPC300 Light Crafter ) and combined with the imaging light path using a FF757-DiO1 dichroic ( Semrock ) .","On the emission side , the primary dichroic was Di02-R635 ( Semrock ) , the detection arm dichroic was 565DCXR ( Chroma ) , and the emission filters were FF03-525/50 and FF01-625/90 ( Semrock ) .","Photostimulation light was delivered in a pulse train that consisted of six 8 s or 15 s pulses ( 100% duty cycle during each pulse ) with a 30 s inter-pulse interval .","The light intensity was ~ 0 . 6 mW/mm2 , as measured using Thorlabs S170C power sensor .","All image data were analyzed off-line .","Region of interest ( ROI ) of DAN-aSP13 at mushroom body γ5 compartment was manually defined in Fiji or CircuitCatcher ( a customized python program by Daniel Bushey ) and the average GCaMP signal intensity within the DAN-aSP13 ROI was taken as the calcium activity of DAN-aSP13 .","Time series calcium activity of DAN-aSP13 ( f ( t ) ) was extracted from the image data and then analyzed with customized Matlab ( MathWorks ) programs .","The normalized Calcium activity of DAN-aSP13 dF/F is defined as:dF/F= f ( t ) −F0F0 where the f ( t ) is the calcium signal intensity and the F0 is the mean F of the first 10 s of the image session before optogenetic activation .","dF/F traces of six stimulation were aligned to the LED onset and averaged to represent the DAN-aSP13 activity upon neuron activation .","To determine the connectivity from the activated neuron to DAN-aSP13 , the mean dF/F during 10 s pre-stimulation and the mean dF/F during stimulation were taken as baseline activity and stimulated activity in a fly .","Groups of baseline activities and stimulated activities of different flies were tested with student’s test or Wilcoxon rank sum test to determine if optogenetic activation had evoked significant calcium activity changes in DAN-aSP13 against the hypothesis that the baseline activity and stimulated activity were the same level .","p value smaller than 0 . 05 was taken as the criteria of connectivity ( either inhibitory or excitatory ) .","Correlation of determination ( r2 ) was used to measure the correlation between the time series calcium trace ( f ( t ) ) of each voxel and a predicted model .","Here we used the voltage driving the stimulation LED ( V ( t ) ) as the model .","The calcium trace of each voxel was firstly normalized to z-score , calculated with the following functionZ ( t ) = f ( t ) −μσ Where μ is the mean and σ is the standard deviation estimated of the f ( t ) .","Then by a linear fit of the model V ( t ) , we have the predicted calcium response Zp ( t ) .","Correlation coefficient ( r ) between Z ( t ) and Zp ( t ) was calculated by:r= cov ( Z ( t ) , Zp ( t ) ) σZ∗σZpwhere r= covZt , ZptσZ*σZp is the covariance of Z ( t ) and Zp ( t ) , σZ and σZp are the standard deviation of Z ( t ) and Zp ( t ) .","The correlation of determination ( R2 ) was simply the square of the r .","R2 is by definition in the range of 0 to 1 .","The higher the value is , the higher is the correlation ( both positive and negative ) between a voxel’s calcium trace and stimuli .","The r2 indexes of a whole brain volume are projected to a 16-bit image stack , which demonstrates the calcium response pattern evoked by stimuli .","Multicolor Flip-out ( MCFO ) was performed according to the protocol described in Nern et al . ( 2015 ) .","Immunostaining was performed according to a protocol described in Zhao et al . ( 2018 ) ."]],"string":"[\n [\n \"Animals guide their behavior in part based on the memories of past learning experiences .\",\n \"Temporally , these memories can be either short- or long-lasting; short-term memory ( STM ) shapes future animal behavior within seconds , minutes to hours after a learning experience , whereas long-term memory ( LTM ) holds the learned information for hours , days or even a lifetime .\",\n \"STM is thought to rely on protein synthesis-independent covalent modifications and changes in weight of existing synaptic connections , whereas LTM is believed to reflect protein synthesis-dependent structural changes in specific synapses ( Kandel , 2001 ) .\",\n \"Depending on the salience or duration of the learning experience , some STMs can be transformed into LTMs through the process of memory consolidation ( Dudai et al . , 2015 ) .\",\n \"Numerous studies in vertebrates suggest that sleep has a beneficial effect on memory in various learning tasks ( Marshall and Born , 2007; Smith , 2001 ) .\",\n \"Studies in humans and rodents have shown that an insufficient amount of sleep impairs cognitive functions such as memory formation and retention , while an ample amount of sleep after a learning experience supports memory storage ( Goel et al . , 2009; Diekelmann and Born , 2010 ) .\",\n \"In addition , several studies have found that animals , including humans , exhibit an enhanced amount and quality of sleep after experiencing a novel or enriched environment ( Gais et al . , 2002; Smith and Wong , 1991; Walker and Stickgold , 2006 ) .\",\n \"One hypothesis is that sleep plays an active role in consolidation of newly acquired memories into long-term storage , that are critical for the animal future actions .\",\n \"To date , however , a mechanistic understanding of sleep function in this selective memory consolidation remains elusive .\",\n \"Over the last two decades , studies of sleep and LTM have expanded from the almost exclusive studies in mammals to other animals , including insects .\",\n \"Sleep is now well-documented in Drosophila .\",\n \"Sleep in Drosophila exhibits almost all the hallmarks of sleep in mammals , including homeostatic regulation , diminished behavioral responsiveness , and the existence of different sleep stages as characterized by distinct electrophysiological signatures ( Dissel et al . , 2015a; Yap et al . , 2017; van Alphen et al . , 2013 ) .\",\n \"Recently , a link between sleep and LTM has been established: sleep deprivation before and after learning impairs mnemonic performance ( Ganguly-Fitzgerald et al . , 2006; Seugnet et al . , 2008; Seugnet et al . , 2009 ) , whereas sleep induction enhances memory formation and retention in wild-type ( Donlea et al . , 2011 ) and restores memory in memory mutant flies ( Dissel et al . , 2015b ) .\",\n \"Moreover , flies that were exposed to a socially enriched environment , including courtship learning , exhibit an increased amount of sleep ( Ganguly-Fitzgerald et al . , 2006 ) .\",\n \"Courtship conditioning has been extensively exploited to study both memory and sleep in Drosophila .\",\n \"Courtship conditioning is an ethologically relevant form of complex learning whereby male flies learn to associate the outcome of their own behavior with multisensory cues presented by females during courtship ( Ejima et al . , 2005 ) .\",\n \"Naive Drosophila males eagerly court both virgin and mated females , which are generally receptive and unreceptive , respectively .\",\n \"However , after being repeatedly rejected by mated females , males become less likely to court other mated females ( Ejima et al . , 2005; Keleman et al . , 2012 ) .\",\n \"Like with other types of learning , the resulting memory can last from minutes to days depending on the training protocol .\",\n \"We previously established that activity of the dopaminergic aSP13 neurons ( DAN-aSP13s ) is necessary and sufficient for STM acquisition via the dopamine receptor DopR1 in the γ neurons of the mushroom body known as Kenyon Cells ( γKCs ) ( Keleman et al . , 2012 ) , a neuropil in the Drosophila central brain critical for memory formation ( Heisenberg et al . , 1985 ) .\",\n \"Moreover , we recently demonstrated that the activity of the same DAN-aSP13s is also essential for the consolidation of courtship STM to LTM .\",\n \"This requirement is observed in a discrete time window after learning , and it is mediated via the dopamine receptor DopR1 in the γKCs ( Krüttner et al . , 2015 ) .\",\n \"In this study , we examine the mechanisms of DAN-aSP13 post-learning activation .\",\n \"We show here that DAN-aSP13s are activated during sleep after courtship experience that induces LTM .\",\n \"We present evidence that the specific class of sleep-promoting neurons in the fan-shaped body ( FB ) , a neuropil previously implicated in sleep regulation ( Donlea et al . , 2011 ) , activates DANs-aSP13 in the discrete time window after learning to consolidate courtship LTM .\"\n ],\n [\n \"To investigate the role of sleep in post-learning activation of DAN-aSP13s , and hence LTM consolidation , we first asked whether DAN-aSP13s are active in freely behaving males after a prolonged experience with mated females , which induces LTM .\",\n \"To monitor activity of DAN-aSP13s for several hours in unrestrained males we employed a luminescence-based transcriptional reporter of neuronal activity ( Guo et al . , 2017 ) .\",\n \"Using specific DAN-aSP13s GAL4 driver ( Aso et al . , 2014a ) ( Figure 1—figure supplement 1A ) , we expressed luciferase exclusively in DAN-aSP13s under the control of multimerized binding sites for the neuronal activity regulated gene Lola and FLP recombinase target sites ( FRT ) for its activity dependent cell specific expression ( MB315B-GAL4>UAS-FLP , Lola ( FRT ) stop ( FRT ) LUC ) ( Chen et al . , 2016 ) .\",\n \"We measured luminescence as a proxy for neuronal activity in males that had undergone prolonged training with mated females and in naive males without prior courtship experience .\",\n \"As a control we used males after prolonged training with virgin females which does not induce memory when tested 24 hr later with mated females ( our unpublished results ) .\",\n \"For training , single males were paired with a single recently-mated or virgin female for 6 hr .\",\n \"Afterwards , trained , naïve and control males were individually transferred to a 96-well luminescence reading plate .\",\n \"Luminescence measurements were taken every 15 min over 16 hr beginning at a common starting time for all males , at the hour seven from the onset of training .\",\n \"Notably , flies that had undergone training with mated females displayed a gradual increase of the luminescence signal , which was significantly different from naïve males between 7–10 hr from the onset of training ( Figure 1A ) .\",\n \"In contrast , control males that had undergone training with virgin females also displayed an increase in luminescence however , it was identical to that in naïve males ( Figure 1—figure supplement 1B ) .\",\n \"That specific time window of DAN-aSP13 activation after onset of training with mated females was preserved in males that were trained in a later circadian time during the day ( Figure 1—figure supplement 1C ) .\",\n \"Together , these data show that DAN-aSP13s are activated in a specific time window after a learning experience that leads to LTM .\",\n \"It was previously shown that flies that had been subjected to social enrichment , including courtship experience , display an increased amount of day time sleep ( Ganguly-Fitzgerald et al . , 2006 ) .\",\n \"To determine whether Drosophila males sleep in the time period when DAN-aSP13s are activated , we measured the amount of sleep in males after prolonged courtship training and in naïve males .\",\n \"Sleep was analyzed with Drosophila Activity Monitors , and periods of inactivity lasting for at least 5 min were classified as sleep ( Shaw et al . , 2000; Hendricks et al . , 2000 ) .\",\n \"Both trained and naive wild-type males showed a significant amount of day time sleep , particularly during hours 7–10 when DAN-aSP13s are active .\",\n \"Importantly , males that had undergone training for LTM slept significantly more in this time window in comparison to naïve males ( Figure 1B ) .\",\n \"Both groups displayed the same amount of sleep throughout the night .\",\n \"Notably , enhancement of post-training sleep occurs between 7–10 hr from the start of training regardless of the circadian time of training .\",\n \"Males that were trained for LTM in the afternoon instead of the usual morning session , displayed an increased amount of sleep between 7–10 hr after onset of training ( Figure 1—figure supplement 1D ) .\",\n \"They also had a normal LTM when tested 24 hr later ( Figure 1—figure supplement 1E , Figure 1—figure supplement 2A ) .\",\n \"To evaluate their memory , we used automated video analysis to derive a courtship index ( CI ) for each male; CI is defined as the percentage of time over a 10 min test period during which the male courts the female .\",\n \"Memory is represented as a suppression index ( SI ) , which is the relative reduction in the median courtship indices of trained versus naïve males: SI = 100*[1-CItrain+/CItrain-] ( Keleman et al . , 2012 ) .\",\n \"In contrast , males that were trained for 1 hr to induce STM did not exhibit an increased amount of sleep ( Figure 1—figure supplement 1F ) .\",\n \"Together , these data suggest that only training that can induce LTM leads to sleep enhancement between 7–10 hr after the start of training .\",\n \"To investigate whether the enhancement of post-training sleep is caused by learning or a prolonged intense activity such as courtship towards mated female , we monitored sleep pattern and courtship memory of mutants for the dopamine receptor DopR1 that do not form courtship memory due to an impairment in memory acquisition ( Keleman et al . , 2012 ) .\",\n \"DopR1 mutant males , although underwent the same courtship experience and courted mated females as vigorously or more than the wild-type males during 6 hr training ( Figure 1—figure supplement 1G ) , neither displayed an increased amount of post-training sleep ( Figure 1C ) nor formed LTM ( Figure 1—figure supplement 1H , Figure 1—figure supplement 2B ) .\",\n \"Together , these data show that learning is essential for the sleep enhancement in the specific time window after training .\",\n \"To determine whether post-learning sleep is necessary for LTM consolidation , we deprived flies of sleep during specific time intervals after training and tested their memory 24 hr later .\",\n \"Single wild-type males were trained with mated females for 6 hr and immediately after were exposed to intermittent gentle mechanical perturbation spanning 2 hr intervals .\",\n \"Naïve males were sleep deprived during the same time periods .\",\n \"Control males that were trained with mated females and allowed to sleep , and males that were sleep deprived after training between 9–11 and 10–12 hr had normal SIs of approximately 30–40% .\",\n \"However , males that were deprived of sleep between 7–9 and 8–10 hr had SIs indistinguishable from 0 ( Figure 2A , Figure 2—figure supplement 2A ) .\",\n \"Deprivation of sleep during the night did not impair LTM significantly , except for a small but statistically-significant effect between 14–16 hr ( Figure 2—figure supplement 1A , Figure 2—figure supplement 2E ) .\",\n \"These results show that sleep deprivation between 7–9 hr after the start of training blocks LTM , and thus post-learning sleep is necessary for LTM consolidation .\",\n \"Previously , we had shown that silencing of DAN-aSP13s in a discrete time window after training impairs LTM ( Krüttner et al . , 2015 ) .\",\n \"To test whether the temporal effect of sleep deprivation mimics the effect of DAN-aSP13s silencing , we expressed specifically in DAN-aSP13s ( Figure 2—figure supplement 1B ) a temperature-sensitive inhibitory form of dynamin shibire ( shits ) ( Kitamoto , 2002 ) ( VT005526-LexA>LexAop-shits ) , which blocks synaptic transmission at 32°C but not at 22°C .\",\n \"We silenced DAN-aSP13s at 2 hr intervals after a 6 hr training session .\",\n \"Inhibition of DAN-aSP13s between 7–9 or 8–10 hr , but not between 9–12 hr , after the onset of training abolished LTM .\",\n \"Control males , in which DAN-aSP13s remained functional throughout the assay , had a normal SI between 30–40% ( Figure 2B , Figure 2—figure supplement 2B ) .\",\n \"These results show that sleep and DAN-aSP13 activity are essential for memory consolidation in the same time window after training .\",\n \"To investigate whether sleep can consolidate STM into LTM , we tested whether artificial enhancement of sleep would lead to memory consolidation .\",\n \"We trained males for 1 hr , which does not generate LTM ( Krüttner et al . , 2015 ) , and then induced sleep at various time intervals afterwards .\",\n \"Since activation of the FB neurons induces sleep ( Donlea et al . , 2011 ) , we expressed the thermosensitive cation channel TrpA1 ( Viswanath et al . , 2003; Rosenzweig et al . , 2005 ) ( open at 30°C and closed at 20°C ) in FB neurons ( 104y-GAL4 > UAS-TrpA1 ) and monitored sleep for 24 hr .\",\n \"Consistent with previous reports , males in which FB neurons were activated slept significantly more than males that were kept at 20°C and the empty-GAL4 control males ( pBDP-GAL4 > UAS-TrpA1 ) ( Figure 2—figure supplement 1C ) .\",\n \"To induce sleep precisely with 2 hr temporal resolution after a 1 hr training session , we used the optogenetic activator CsChrimson ( Klapoetke et al . , 2014 ) .\",\n \"We found that activating FB neurons ( 104y-GAL4 > UAS CsChrimson ) in the period between 5–7 hr after the onset of training led to a SI of ~ 30% , which is similar to that obtained after LTM training ( Figure 2C , Figure 2—figure supplement 2C ) .\",\n \"In contrast , males activated at other time intervals or not activated at all failed to consolidate LTM and had SIs that were indistinguishable from 0 .\",\n \"This temporal window was identical to that observed for DAN-aSP13s activation to consolidate LTM ( VT005526-LexA > LexAop-CsChrimson ) ( Figure 2D , Figure 2—figure supplement 2D ) .\",\n \"When we activated FB neurons ( 104y-GAL4 > UAS-CsChrimson ) while silencing DAN-aSP13s ( VT005526-LexA > LexAop-Shits ) between 5–7 hr after the onset of training we did not observe LTM ( Figure 2C , Figure 2—figure supplement 2C ) .\",\n \"Taken together , these data show that enhancement of sleep after a learning experience that induces STM can consolidate it to LTM , in a manner that requires activation of DAN-aSP13s .\",\n \"This LTM consolidation is a specific effect of DAN-aSP13s activation after training since activation of DAN-aSP13s ( VT005526-LexA > LexAop-CsChrimson ) in naïve males between 5–7 hr when they normally display a significant amount of sleep does not induce courtship suppression towards mated females and thus ‘courtship LTM’ ( Figure 2—figure supplement 1D ) .\",\n \"To test whether FB activation excites DAN-aSP13s , we optogenetically activated FB neurons ( 104y-GAL4 > UAS-Chrimson88 ) and monitored calcium levels in DANs located in the protocerebral anterior medial ( PAM ) cluster in explant brains ( R58E02-LexA > LexAop-GCamP6s ) ( Pfeiffer et al . , 2008; Chen et al . , 2013 ) .\",\n \"The PAM cluster of DANs consists of multiple neuronal classes , including a class of DAN-aSP13s , which is thought to respond to rewarding stimuli and promote their avoidance ( Aso et al . , 2014b ) ( Figure 3—figure supplement 1A ) .\",\n \"Since the expression pattern of 104y-GAL4 is not exclusive to the FB ( Donlea et al . , 2011 ) , to activate FB neurons selectively , we used a digital mirror device ( DMD ) to target the illumination activating Chrimson to specific layers in the FB ( Strother et al . , 2018 ) .\",\n \"104y-FB neurons form three layers of projections: dorsal ( dFB ) , medial ( mFB ) and ventral ( vFB ) ( Donlea et al . , 2011 ) .\",\n \"To test the targeted illumination , we expressed in 104y neurons both Chrimson88 and GCamP6s ( 104y-GAL4 > UAS-Chrimson88 > UAS-GCamP6s ) and selectively activated one of two layers of the FB ( dFB or vFB ) while monitoring calcium levels in the presence of tetradotoxin ( TTX ) .\",\n \"TTX inhibits propagation of action potentials ( Boccaccio et al . , 1999 ) , and thus calcium signals should only be observed in the projections located in the targeted layer .\",\n \"Upon activation of either dFB or vFB layers , a robust calcium response was observed only in the activated layer , implying that this local activation was highly restricted ( Figure 3A ) .\",\n \"We next activated all FB layers using targeted illumination and monitored calcium responses in DAN-aSP13s ( 104y-GAL4 > UAS-Chrimson88 , R58E02-LexA > LexAop-GCamP6s ) .\",\n \"Local activation of FB neurons elicited an excitatory response in DAN-aSP13s ( Figure 3B ) .\",\n \"Given that the FB has a multilayered organization , we next aimed to identify the specific layer of the 104y-FB expression pattern that provides excitatory input to DAN-aSP13s .\",\n \"We individually activated all three 104y-FB layers and monitored calcium levels in DAN-aSP13s .\",\n \"Surprisingly , activation of the dFB layer , which has been recently implicated in homeostatic sleep regulation ( Donlea et al . , 2011 ) , resulted in a mixture of small inhibitory and excitatory responses ( Figure 3C ) , whereas activation of the vFB layer induced excitatory responses in DAN-aSP13s ( Figure 3D ) .\",\n \"Activation of the mFB had no effect on DAN-aSP13 activity ( Figure 3—figure supplement 1B ) .\",\n \"These data show that neurons that project to the vFB and potentially the dFB layers provide an excitatory input onto DAN-aSP13s and may thus have a role in LTM consolidation .\",\n \"To identify the specific neurons in the 104y-GAL4 line that project to the different FB layers , we performed multicolor flip-out experiments ( Nern et al . , 2015 ) .\",\n \"We identified two distinct neuronal populations that project to either the dFB or vFB layers ( Figure 4—figure supplement 1A ) .\",\n \"Next , we searched the Janelia ( Jenett et al . , 2012 ) and Vienna Tiles ( VT ) ( Tirian and Dickson , 2017 ) collections for specific GAL4 driver lines with expression in these two cell types .\",\n \"We identified one sparse GAL4 line with expression in the dFB neurons ( R23E10 ) and one in the vFB neurons ( VT036875 ) .\",\n \"Since the expression pattern of the VT036875-GAL4 line also includes a class of DAN-β’1 neuron , we combined this line with PAM-GAL80 to restrict its expression to vFB neurons only ( VT036875-GAL4 , R58E02-GAL80 ) .\",\n \"In addition , we generated a split line ( SS057264-GAL4 ) ( Pfeiffer et al . , 2010; Luan et al . , 2006 ) with the expression restricted to the vFB neurons only ( Figure 4—figure supplement 1B , C , D , E ) .\",\n \"As previously reported , thermogenetic activation of dFB over a 24 hr time period resulted in an increased amount of sleep ( R23E10-GAL4 > UAS-TrpA1 ) ( Figure 4A ) .\",\n \"Sleep increase was displayed mainly during the day time since males sleep almost continuously during night .\",\n \"We found that activation of vFB ( VT036875-GAL4 > UAS-TrpA1; VT036875-GAL4 > UAS-TrpA1 , R58E02-GAL80; SS057264-GAL4 > UAS-TrpA1 ) also robustly induced day time sleep ( Figure 4B , C , D ) in comparison to the genetic control ( pBDP-GAL4 > UAS-TrpA1 ) ( Figure 4—figure supplement 1G ) .\",\n \"Loss of night time sleep observed upon prolonged vFB activation with VT036875-GAL4 ( Figure 4B ) was likely caused by co-activation of wake promoting DAN-β’1 neurons ( Sitaraman et al . , 2015a ) ( Figure 4—figure supplement 1C ) .\",\n \"Accordingly , males upon activation with the same line while excluding DAN-β’1 neurons displayed a higher level of both day and night sleep ( Figure 4C , Figure 4—figure supplement 1D ) .\",\n \"Similarly , acute optogenetic activation of the dFB and vFB neurons with CsChrimson for 1 hr also significantly enhanced the amount of sleep ( R23E10-GAL4 > UAS -CsChrimson; VT036875-GAL4 > UAS-CsChrimson; VT036875-GAL4 > UAS-CsChrimson , R58E02-GAL80; SS057264-GAL4 > UAS-CsChrimson; 104y-GAL4 > UAS-CsChrimson ) in comparison to the genetic control ( pBDP-GAL4 > UAS-CsChrimson ) but to a lesser degree when DAN-β’1 neurons were co-activated ( Figure 4—figure supplement 1F ) .\",\n \"To test whether these neurons provide an excitatory input on DAN-aSP13s , we activated either dFB ( R23E10-GAL4 > UAS-Chrimson88 ) or vFB ( VT036875-GAL4 > UAS-Chrimson88; VT036875-GAL4 > UAS-Chrimson88 , R58E02-GAL80; SS057264-GAL4 > UAS-Chrimson88 ) neurons and monitored calcium levels in DAN-aSP13s .\",\n \"Since specific DANs innervate MB lobes in well-defined discrete areas , we used a broad PAM-DAN GAL4 driver ( R58E02-LexA > LexAop-GCamP6s ) .\",\n \"To monitor activity specifically in DAN-aSP13s upon activation of FB neurons we focused on the region at the tip of the MBγ lobe ( Figure 3—figure supplement 1A ) .\",\n \"Activation of dFB neurons did not elicit calcium changes in DAN-aSP13s ( Figure 4A ) however , activation of vFB neurons elicited a robust increase of calcium levels in DAN-aSP13s ( Figure 4B , C , D ) .\",\n \"These data show that neurons in the vFB provide excitatory input onto DAN-aSP13s .\",\n \"Consistent with the functional connectivity results , optogenetic activation of dFB neurons ( R23E10-GAL4 > UAS-CsChrimson ) between 5–7 hr after a 1 hr training period did not lead to LTM consolidation .\",\n \"However , activation of either a combination of dFB and vFB neurons ( 104y-GAL4 > UAS-CsChrimson ) or just the vFB neurons ( VT036875-GAL4 > UAS-CsChrimson; VT036875-GAL4 > UAS-CsChrimson , R58E02-GAL80; SS057264-GAL4 > UAS-CsChrimson ) in that same time window fully consolidated STM to LTM ( Figure 5A , Figure 5—figure supplement 1A ) .\",\n \"In addition , silencing of dFB neurons ( R23E10-GAL4 > UAS-Shits ) in the period between 7–10 hr after the onset of a 6 hr training period did not affect LTM persistence , while silencing of a combination of dFB and vFB or just the vFB neurons ( VT036875-GAL4 > UAS-Shits; SS057264-GAL4 > UAS-Shits and 104y-GAL4 > UAS-Shits ) in the same time window strongly impaired LTM consolidation ( Figure 5B , Figure 5—figure supplement 1B ) .\",\n \"These results show that a class of sleep-promoting neurons in the vFB layer of the 104y-FB expression pattern is necessary and sufficient to consolidate LTM during post-learning sleep .\",\n \"dFB neurons , although they promote sleep , appear to have no role in courtship LTM consolidation .\"\n ],\n [\n \"The activity of DAN-aSP13s , which is essential for courtship memory acquisition ( Keleman et al . , 2012 ) , is also necessary during a discrete post-learning time window for LTM consolidation ( Krüttner et al . , 2015 ) .\",\n \"Because neuronal reactivation occurs during sleep in rodents ( Wilson and McNaughton , 1994; Smith , 2001 ) , we hypothesized that post-learning activation of DAN-aSP13s involves a sleep-dependent mechanism .\",\n \"Using behavioral analysis and neuronal activity monitoring and perturbation approaches , we have shown here that DAN-aSP13s display an increased activity in freely behaving animals during sleep after a prolonged learning experience .\",\n \"We demonstrated that this sleep is necessary for LTM consolidation , and it can be mediated by a specific class of sleep promoting neurons in the ventral layer of the FB ( vFB ) .\",\n \"These vFB neurons consolidate courtship LTM in a discrete time window and provide an excitatory input to DAN-aSP13s .\",\n \"Thus , the data we present here provide a causal link between sleep promoting neurons in the vFB , post-learning activation of dopaminergic neurons , and LTM consolidation .\",\n \"Based on our data , we propose the following model for sleep-dependent consolidation of courtship LTM in Drosophila ( Figure 6 ) .\",\n \"During a prolonged learning experience , γKCs and DAN-aSP13s are repeatedly activated by olfactory and behavioral cues presented by an unreceptive female , respectively .\",\n \"Whereas prolonged wakefulness leads to an increase in homeostatic sleep drive in ellipsoid body neurons that in turn is conveyed to dFB ( Liu et al . , 2016 ) , we hypothesize that an extended learning experience generates a learning-dependent sleep drive that is transmitted to vFB .\",\n \"In turn , the vFB neurons enhance sleep after learning and provide an excitatory input back on DAN-aSP13s .\",\n \"We believe that one potential site of a learning-dependent sleep drive are the MB neurons since they have been implicated in both memory formation ( Heisenberg et al . , 1985 ) and sleep regulation ( Sitaraman et al . , 2015b ) .\",\n \"Dopamine released upon DAN-aSP13 reactivation stimulates molecular processes in γKCs ( Krüttner et al . , 2015 ) that are different from those engaged during STM acquisition and involve protein synthesis that is essential for LTM formation and persistence .\",\n \"Dopaminergic pathways are thought to convey information about whether an experience is rewarding or punishing and thus , worth remembering ( Gais et al . , 2002; Schultz , 2010 ) .\",\n \"Post-learning neuronal activity of the dopaminergic hippocampal inputs from the Ventral Tagmental Area ( VTA ) has been implicated in the consolidation of fear memory in rodents .\",\n \"Interestingly , this activity is critical during a discrete time window after learning ( Rossato et al . , 2009 ) .\",\n \"Post-learning activity of the VTA dopamine neurons has been also implicated in the reactivation during sleep of the hippocampal cells involved earlier in encoding of the spatial experience ( Gomperts et al . , 2015 ) .\",\n \"Here we show that post-learning activation of DAN-aSP13s mediates the consolidation of courtship LTM in Drosophila .\",\n \"We propose that reactivation during sleep of the dopamine neurons that were previously active during memory acquisition ensures that spurious experiences are not admitted into LTM storage and thus only experiences that are either sufficiently salient or persistent become long-lasting memories .\",\n \"Specifically , reactivation of DAN-aSP13s during post-learning sleep enhances reactivation of the γKCs and cognate MBON-M6 , which together with DAN-aSP13s form a recurrent circuit necessary for courtship memory acquisition ( Zhao et al . , 2018 ) .\",\n \"We consider two hypotheses to account for this selectivity .\",\n \"During sleep , vFB neurons might selectively reactivate only the relevant DANs , or alternatively , they might activate all DANs but only the relevant subset is able to consolidate LTM .\",\n \"The selective-reactivation model would require some marker to distinguish which DANs were activated , whereas the selective-consolidation model would require a marker in the synapses of the γKCs that were earlier active during memory acquisition , for example translational regulator Orb2 , which regulates translation upon neuronal activity during LTM consolidation ( Krüttner et al . , 2015 ) .\",\n \"Activity of dopaminergic neurons regulates sleep-wake states in animals , including flies ( Dzirasa et al . , 2006 ) .\",\n \"Artificial activation of DAN-aSP13s has been shown to increase wakefulness ( Sitaraman et al . , 2015a ) .\",\n \"In contrast , the data we present here imply that activation of vFB neurons , although they activate DAN-aSP13s , do not promote wakefulness .\",\n \"These results suggest that activation of DAN-aSP13s by vFB neurons during sleep is qualitatively different from a direct optogenetic or thermogenetic activation used in previous studies .\",\n \"One potential explanation is that post-learning sleep involves activation of the vFB circuit , which provides both excitatory stimulus to DAN-aSP13s and inhibitory input to motor neurons .\",\n \"Another possibility is that post-learning sleep activates a subset od DAN-aSP13s that do not affect wakefulness .\",\n \"We have identified here a class of sleep-promoting neurons in the ventral layer of FB that are distinct from the well-studied sleep-promoting neurons in the dorsal layer of FB , which regulate sleep homeostasis ( Donlea et al . , 2011; Donlea et al . , 2014; Pimentel et al . , 2016 ) .\",\n \"Given that vFB neurons enhance sleep and activate DAN-aSP13s for LTM consolidation , whereas dFB neurons are neither necessary nor sufficient for LTM consolidation , we hypothesize that dFB and vFB neurons promote distinct components of sleep that have different functions .\",\n \"Homeostatic sleep is thought to facilitate memory encoding by downscaling synaptic weights and clearing metabolites from the brain accumulated during wakefulness ( Bushey et al . , 2011; Xie et al . , 2013; Tononi and Cirelli , 2014 ) .\",\n \"In contrast , the function of learning-dependent sleep might be to facilitate memory consolidation by strengthening synaptic connections that were engaged earlier during memory acquisition ( Frank , 2012 ) .\",\n \"Thus , the co-operation of homeostatic- and experience-dependent sleep would facilitate optimal conditions for learning new information and , if appropriate , incorporating it into long-term storage .\",\n \"Recent studies have implied that sleep in flies , as in humans and rodents , exhibits sleep stages characterized by distinct electrophysiological signatures ( Yap et al . , 2017; van Alphen et al . , 2013 ) .\",\n \"Interestingly , the signature of sleep that is induced by activation of the dFB neurons seems to have a simpler oscillatory pattern , recorded by local field potentials , than sleep that is induced by activation of the FB neurons comprising both dFB and vFB neurons ( Yap et al . , 2017 ) .\",\n \"Thus , these data support our hypothesis that dFB and vFB neurons promote sleep with different properties and likely different functions .\",\n \"It is thought that sleep evolved in animals that are capable of complex learning which requires selective attention ( Kirszenblat and van Swinderen , 2015 ) .\",\n \"Courtship learning is a multisensory form of learning that requires selective attention of a male to associate multiple learning cues presented by the mated female with the outcome of his own behavior ( Kirszenblat and van Swinderen , 2015 ) .\",\n \"Accordingly , studies in bees have shown that sleep affects a complex form of learning such as spatial memory but has no role in the simple learning paradigm of proboscis extension ( Vorster and Born , 2015 ) .\",\n \"Hence , it would be interesting to investigate whether post-learning sleep is involved in the consolidation of other types of memory in Drosophila , such as the well-studied Pavlovian olfactory associative learning whereby animals associate an individual learning cue with a behavioral contingency .\",\n \"In this work , we establish a functional link between a novel class of sleep-promoting neurons in the FB , post-learning reactivation of dopaminergic neurons and consolidation of courtship LTM .\",\n \"Moreover , our data suggest that sleep promoting vFB neurons mediate a learning-dependent regulation of sleep that is distinct from the homeostatic control which is facilitated by dFB neurons ( Liu et al . , 2016 ) .\",\n \"Thus , we uncover a causal link between sleep-mediated neuronal reactivation and LTM consolidation in Drosophila .\",\n \"In addition , we establish courtship LTM in Drosophila as a tractable model to investigate the mechanisms that link learning-dependent sleep , neuronal reactivation and LTM consolidation .\"\n ],\n [\n \"Flies for behavioral experiments were reared in vials with standard cornmeal food at 25°C , or as indicated , at 60% humidity in a 12 hr light:12 hr dark cycle .\",\n \"Detailed information regarding specific strains and genotypes is provided in the Key resources table section .\",\n \"Courtship conditioning was performed as described ( Siwicki and Ladewski , 2003 ) .\",\n \"Briefly , solitary males aged for 5–6 days after eclosion were placed for training ( during day time as indicated ) in food chambers for 1 hr ( STM ) or 6 hr ( LTM ) either with ( trained ) or without ( naïve ) a single mated female .\",\n \"After training each male was recovered , allowed to rest for 30 min or 24 hr and tested with a fresh mated female .\",\n \"Tests were performed in 10 mm diameter chambers and videotaped for 10 min ( Prosilica GT cameras , Allied Vison Technologies ) .\",\n \"Automated video analysis was used to derive a courtship index ( CI ) for each male , defined as the percentage of time over a 10 min test period during which the male courts the female .\",\n \"Memory was calculated as a suppression index ( SI ) that is a relative reduction in the mean courtship indices of trained ( CI+ ) versus naïve ( CI- ) populations: SI = 100*[1-CItrain+/CItrain-] .\",\n \"To monitor sleep over a 24 hr time period , male flies were individually inserted into 65 mm glass tubes containing standard fly food , loaded into TriKinetics Drosophila activity monitors ( DAM ) , and housed under 12 hr light:12 hr dark schedule ( Donelson et al . , 2012 ) .\",\n \"Periods of inactivity lasting at least 5 min were classified as sleep .\",\n \"Total 24 hr sleep quantity ( day time and night time sleep ) was extracted from DAM system as described ( Donelson et al . , 2012 ) .\",\n \"To monitor sleep upon acute induction with csChrimson , individual males were reared at 25°C on retinal supplemented ( 0 . 1 mM ) cornmeal medium in darkness for 4–5 days after eclosion .\",\n \"For sleep induction single males were placed in 10 mm diameter behavioral chambers in a temperature and illumination-controlled box and videotaped ( Prosilica GT cameras , Allied Vison Technologies ) .\",\n \"The amount of sleep was scored manually with periods of inactivity lasting at least 5 min being considered as sleep .\",\n \"For sleep deprivation , flies were subjected to intermittent mechanical perturbation method while housed in TriKinetics DAM monitors .\",\n \"Flies received mechanical perturbations on a horizontal shaker with a total cycle of 15 s/min delivered in eight pulses of 1–3 s each occurring intermittently at random times .\",\n \"A MATLAB script ( Kamyshev et al . , 1999 ) ( permutation test ) implemented in Keleman et al . ( 2012 ) was used for statistical comparison of SIs between two groups .\",\n \"Briefly , the entire set of courtship indices for both naïve and trained flies were pooled and then randomly assorted into simulated naïve and trained groups of the same size as the original data .\",\n \"A SI was calculated for each of 100 , 000 randomly permutated data sets , and P values were estimated for the null hypothesis that learning equals 0 ( H0: SI = 0 ) or for the null hypothesis that experimental and control males learn equally well ( H0: SI = SIc ) .\",\n \"Luminescence assay for detecting neuronal activity in freely behaving adult flies was modified from Guo et al . ( 2017 ) .\",\n \"Flies were starved on filter soaked in water overnight prior to training .\",\n \"They were transferred to fresh chambers with filter paper containing 40 mM Luciferin ( GOLDBIO ) in a 2% sucrose solution .\",\n \"For luminescence measurements after training , flies were placed into 96-well plates ( Greiner Bio-one ) with 40 mM Luciferin in a 2% sucrose solution .\",\n \"CLARIOstar microplate reader ( BMG Labtech ) was used for luminescence detection .\",\n \"Luminescence was measured every 15 min over 16 hr .\",\n \"Relative Luminescence ( R . Luminescence ) was calculated by dividing the luminescence of the experienced and naïve groups by the luminescence of the genetic controls ( males with luciferase reporter only , fed with luciferin ) at every measurement time point .\",\n \"Single housed male flies were reared at 25°C on retinal supplemented ( 0 . 1 mM ) cornmeal medium in darkness for 4–5 days after eclosion .\",\n \"For sleep induction or memory consolidation specific classes of neurons were optogenetically activated in behavior chambers housed in a temperature and illumination-controlled box .\",\n \"During activation , the behavior chamber was illuminated with 617 nm LEDs ( Red-Orange LUXEON Rebel LED—122 lm; Luxeon Star LEDs , Brantford , Ontario , Canada ) with a 3 mm thick diffuser between the LED and flies .\",\n \"The LED was driven by a customized linear current controller .\",\n \"20 HZ red light was used to activate neurons with intensity varying from 20 uW/mm2 to 70 uW/mm2 .\",\n \"The LED board was cooled by a customized liquid cooling system to maintain the temperature in the chamber .\",\n \"Single housed male flies were reared at 25°C on retinal supplemented ( 0 . 1 mM ) cornmeal medium in darkness for 4–5 days after eclosion .\",\n \"Brains of the immobilized flies on ice were dissected out in saline ( 103 mM NaCl , 3 mM KCl , 2 mM CaCl2 , 4 mM MgCl2 , 26 mM NaHCO3 , 1 mM NaH2PO4 , 8 mM trehalose , 10 mM glucose , 5 mM TES , bubbled with 95% O2/5% CO2 ) and mounted anterior up on the cover slip in a Sylgard-lined dish in a 20°C saline bath .\",\n \"Brains were imaged with a resonant scanning two-photon microscope with near-infrared excitation ( 920 nm , Spectra-Physics , INSIGHT DS DUAL ) and a 25x objective ( Nikon MRD77225 25XW ) .\",\n \"The microscope was controlled by ScanImage 2015 . v3 ( Vidrio Technologies ) .\",\n \"Images of brain volume were acquired with ~ 157 μm x 157 μm field of view at 512 × 512 pixels resolution .\",\n \"Each frame and each volume was sampled by 42 frames with 3 μm per step , at approximately 1 Hz volume rate .\",\n \"Times series of volume images were acquired and the excitation power for calcium imaging ~ 12 mW .\",\n \"For the optogenetic activation , the light-gated ion channel Chrimson88 was activated with a 660 nm LED ( M660L3 Thorlabs ) coupled to a digital micromirror device ( DMD ) ( Texas Instruments DLPC300 Light Crafter ) and combined with the imaging light path using a FF757-DiO1 dichroic ( Semrock ) .\",\n \"On the emission side , the primary dichroic was Di02-R635 ( Semrock ) , the detection arm dichroic was 565DCXR ( Chroma ) , and the emission filters were FF03-525/50 and FF01-625/90 ( Semrock ) .\",\n \"Photostimulation light was delivered in a pulse train that consisted of six 8 s or 15 s pulses ( 100% duty cycle during each pulse ) with a 30 s inter-pulse interval .\",\n \"The light intensity was ~ 0 . 6 mW/mm2 , as measured using Thorlabs S170C power sensor .\",\n \"All image data were analyzed off-line .\",\n \"Region of interest ( ROI ) of DAN-aSP13 at mushroom body γ5 compartment was manually defined in Fiji or CircuitCatcher ( a customized python program by Daniel Bushey ) and the average GCaMP signal intensity within the DAN-aSP13 ROI was taken as the calcium activity of DAN-aSP13 .\",\n \"Time series calcium activity of DAN-aSP13 ( f ( t ) ) was extracted from the image data and then analyzed with customized Matlab ( MathWorks ) programs .\",\n \"The normalized Calcium activity of DAN-aSP13 dF/F is defined as:dF/F= f ( t ) −F0F0 where the f ( t ) is the calcium signal intensity and the F0 is the mean F of the first 10 s of the image session before optogenetic activation .\",\n \"dF/F traces of six stimulation were aligned to the LED onset and averaged to represent the DAN-aSP13 activity upon neuron activation .\",\n \"To determine the connectivity from the activated neuron to DAN-aSP13 , the mean dF/F during 10 s pre-stimulation and the mean dF/F during stimulation were taken as baseline activity and stimulated activity in a fly .\",\n \"Groups of baseline activities and stimulated activities of different flies were tested with student’s test or Wilcoxon rank sum test to determine if optogenetic activation had evoked significant calcium activity changes in DAN-aSP13 against the hypothesis that the baseline activity and stimulated activity were the same level .\",\n \"p value smaller than 0 . 05 was taken as the criteria of connectivity ( either inhibitory or excitatory ) .\",\n \"Correlation of determination ( r2 ) was used to measure the correlation between the time series calcium trace ( f ( t ) ) of each voxel and a predicted model .\",\n \"Here we used the voltage driving the stimulation LED ( V ( t ) ) as the model .\",\n \"The calcium trace of each voxel was firstly normalized to z-score , calculated with the following functionZ ( t ) = f ( t ) −μσ Where μ is the mean and σ is the standard deviation estimated of the f ( t ) .\",\n \"Then by a linear fit of the model V ( t ) , we have the predicted calcium response Zp ( t ) .\",\n \"Correlation coefficient ( r ) between Z ( t ) and Zp ( t ) was calculated by:r= cov ( Z ( t ) , Zp ( t ) ) σZ∗σZpwhere r= covZt , ZptσZ*σZp is the covariance of Z ( t ) and Zp ( t ) , σZ and σZp are the standard deviation of Z ( t ) and Zp ( t ) .\",\n \"The correlation of determination ( R2 ) was simply the square of the r .\",\n \"R2 is by definition in the range of 0 to 1 .\",\n \"The higher the value is , the higher is the correlation ( both positive and negative ) between a voxel’s calcium trace and stimuli .\",\n \"The r2 indexes of a whole brain volume are projected to a 16-bit image stack , which demonstrates the calcium response pattern evoked by stimuli .\",\n \"Multicolor Flip-out ( MCFO ) was performed according to the protocol described in Nern et al . ( 2015 ) .\",\n \"Immunostaining was performed according to a protocol described in Zhao et al . ( 2018 ) .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Animals consolidate some , but not all , learning experiences into long-term memory .","Across the animal kingdom , sleep has been found to have a beneficial effect on the consolidation of recently formed memories into long-term storage .","However , the underlying mechanisms of sleep dependent memory consolidation are poorly understood .","Here , we show that consolidation of courtship long-term memory in Drosophila is mediated by reactivation during sleep of dopaminergic neurons that were earlier involved in memory acquisition .","We identify specific fan-shaped body neurons that induce sleep after the learning experience and activate dopaminergic neurons for memory consolidation .","Thus , we provide a direct link between sleep , neuronal reactivation of dopaminergic neurons , and memory consolidation ."],"string":"[\n \"Animals consolidate some , but not all , learning experiences into long-term memory .\",\n \"Across the animal kingdom , sleep has been found to have a beneficial effect on the consolidation of recently formed memories into long-term storage .\",\n \"However , the underlying mechanisms of sleep dependent memory consolidation are poorly understood .\",\n \"Here , we show that consolidation of courtship long-term memory in Drosophila is mediated by reactivation during sleep of dopaminergic neurons that were earlier involved in memory acquisition .\",\n \"We identify specific fan-shaped body neurons that induce sleep after the learning experience and activate dopaminergic neurons for memory consolidation .\",\n \"Thus , we provide a direct link between sleep , neuronal reactivation of dopaminergic neurons , and memory consolidation .\"\n]"},"summary":{"kind":"list like","value":["Why do some memories fade after only a few seconds , whereas others last a lifetime ?","Studies suggest that part of the explanation has to do with sleep .","Experiments in rodents show that neural circuits that are active during learning become active again when an animal sleeps .","This process of reactivation , which may be akin to dreaming , helps strengthen specific memories and move them into long-term storage .","But the complexity of the mammalian brain has made it difficult to pin down the underlying mechanisms .","One possible solution is to study the mechanisms in a simpler brain with fewer neurons , such as that of the fruit fly Drosophila .","Dag , Lei et al . have now used molecular genetic tools to explore how sleep supports a specific type of learning in male fruit flies , called courtship learning .","Female fruit flies that have recently mated will reject the courtship efforts of other males .","A male fly that experiences repeated rejections therefore learns to avoid mated females in future .","This type of memory can last for at least a day – a long time in the life of a fly .","Dag , Lei et al . show that males that experience repeated rejections subsequently spend more time asleep than control males .","Preventing this sleep hinders the males from learning from their experience .","But how does this process work ?","During sleep , specific dopamine neurons that were active during the learning episode become active once again .","Blocking this reactivation prevents the flies from learning from their rejections .","By contrast , artificially activating the dopamine neurons enables flies with only limited experience of rejection to learn to avoid mated females .","Dag , Lei et al . show that neurons called vFB cells control this process .","The vFB neurons both induce sleep and reactivate the memory-inducing dopamine neurons .","These findings in fruit flies thus reveal a direct causal link between sleep , reactivation of memory traces , and persistence of memories .","They also show that fruit flies are a valid model for exploring the neural and molecular mechanisms connecting sleep and long-term memory ."],"string":"[\n \"Why do some memories fade after only a few seconds , whereas others last a lifetime ?\",\n \"Studies suggest that part of the explanation has to do with sleep .\",\n \"Experiments in rodents show that neural circuits that are active during learning become active again when an animal sleeps .\",\n \"This process of reactivation , which may be akin to dreaming , helps strengthen specific memories and move them into long-term storage .\",\n \"But the complexity of the mammalian brain has made it difficult to pin down the underlying mechanisms .\",\n \"One possible solution is to study the mechanisms in a simpler brain with fewer neurons , such as that of the fruit fly Drosophila .\",\n \"Dag , Lei et al . have now used molecular genetic tools to explore how sleep supports a specific type of learning in male fruit flies , called courtship learning .\",\n \"Female fruit flies that have recently mated will reject the courtship efforts of other males .\",\n \"A male fly that experiences repeated rejections therefore learns to avoid mated females in future .\",\n \"This type of memory can last for at least a day – a long time in the life of a fly .\",\n \"Dag , Lei et al . show that males that experience repeated rejections subsequently spend more time asleep than control males .\",\n \"Preventing this sleep hinders the males from learning from their experience .\",\n \"But how does this process work ?\",\n \"During sleep , specific dopamine neurons that were active during the learning episode become active once again .\",\n \"Blocking this reactivation prevents the flies from learning from their rejections .\",\n \"By contrast , artificially activating the dopamine neurons enables flies with only limited experience of rejection to learn to avoid mated females .\",\n \"Dag , Lei et al . show that neurons called vFB cells control this process .\",\n \"The vFB neurons both induce sleep and reactivate the memory-inducing dopamine neurons .\",\n \"These findings in fruit flies thus reveal a direct causal link between sleep , reactivation of memory traces , and persistence of memories .\",\n \"They also show that fruit flies are a valid model for exploring the neural and molecular mechanisms connecting sleep and long-term memory .\"\n]"},"year":{"kind":"string","value":"2019"}}},{"rowIdx":4294,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["neuroscience"],"string":"[\n \"neuroscience\"\n]"},"title":{"kind":"string","value":"A longitudinal study of Caenorhabditis elegans larvae reveals a novel locomotion switch, regulated by Gαs signaling"},"id":{"kind":"string","value":"elife-00782-v1"},"sections":{"kind":"list like","value":[["The life cycle of the nematode Caenorhabditis elegans is comprised of the embryonic stage , four larval stages termed L1–L4 , and adulthood .","Each larval stage lasts 8–12 hr at 20°C , and under standard conditions , and ends with a molt , when epidermal cells synthesize a new cuticle and the old one is shed .","Although C . elegans larvae are mostly continuously motile , each molt is accompanied by a 2–3 hr period of behavioral quiescence ( Singh and Sulston , 1978 ) , referred to as ‘lethargus’ .","Thus , the larval stages can be divided into motile intermolt sub-stages ( L1int–L4int ) and their corresponding lethargus sub-stages ( L1leth-L4leth ) .","However , behavior during distinct developmental sub-stages ( Singh and Sulston , 1978 ) has not previously been examined in detail .","Specifically , both the modulation of body posture and locomotion on developmental timescales remain largely unexplored .","C . elegans move directionally by propagating dorsoventral undulations along their body .","Upon receiving input from interneurons , motoneurons provide output to an array of longitudinal body-wall muscles in order to propagate body bends ( White et al . , 1986; Stetina et al . , 2006; Karbowski et al . , 2006; Stephens et al . , 2008 ) .","Head/neck motoneurons and muscles independently control head movements , such as exploratory head swings ( White et al . , 1986; Stetina et al . , 2006; Pirri et al . , 2009; Maguire et al . , 2011 ) .","The gsa-1 gene encodes a Gαs subunit , which activates the adenylyl cyclase ACY-1 , and the GSA-1 ( Gαs ) pathway has been previously shown to affect locomotion .","Activation ACY-1 by GSA-1 leads to the production of cyclic adenosine monophosphate ( cAMP ) .","The binding of cAMP to the protein kinase A ( PKA ) regulatory subunit KIN-2 dissociates it from the inactive holoenzyme , releasing the PKA catalytic subunit , KIN-1 .","Increased PKA activity enhances signaling at the neuromuscular junction , as well as increases cAMP response element ( CRE ) mediated transcription in C . elegans neurons ( Kimura et al . , 2002 ) .","Thus , upregulating this signaling pathway has been reported to result in hyperkinetic phenotypes , typically described as a nonspecific increase in the rate of locomotion ( Schade et al . , 2005; Reynolds et al . , 2005; Raizen et al . , 2008; Perez-Mansilla and Nurrish , 2009 ) , as well as in reduced quiescence during lethargus ( Raizen et al . , 2008; Belfer and Raizen , 2013 ) .","Here we present the first detailed analysis of locomotion patterns during developmentally relevant timescales , that is , periods in which significant developmental changes occur .","We have analyzed the initiation , propagation and eventual demise of individual dorsoventral body bends over a 14-hr period , from the mid-L4int stage to the mid-young-adult stage .","We found that some locomotion patterns undergo a gradual modulation , while others display abrupt switching .","In particular , two behavioral states , active wakefulness and quiet wakefulness , were observed during the mid- to late- L4int stage .","Active wakefulness was dominated by forward locomotion ( propagation of body-bends from the anterior to the posterior ) , but included intervals of backward locomotion and ‘dwelling’ ( non-directional dynamics of body bends ) .","In contrast , quiet wakefulness was dominated by dwelling behavior , although it included intervals of directed locomotion .","In individual animals , active wakefulness was observed to persist for epochs of 1–100 min .","Moreover , the switching between active and quiet wakefulness was abrupt , suggesting that they are distinct behavioral states .","The process underlying these states exhibited the signature of ergodicity breaking , characteristic of a scale-free switch , as opposed to having a simple rate constant that governs the dynamics of switching .","We further show that the transitions between behavioral states were regulated by the GSA-1 ( Gαs ) pathway: increased Gαs signaling stabilized the active wakefulness state both within and outside of lethargus , while decreased Gαs signaling destabilized this state , but only outside of lethargus ."],["To assay the modulation of behavior during development , we developed PyCelegans: a high-speed , modular image-processing tool for analyzing posture and locomotion of C . elegans on high performance computing resources .","The function we used for processing a single frame was limited to tracking a single animal .","However , the modular design of PyCelegans can accommodate multi-animal tracking once an appropriate substitute for this function is implemented , without further changes .","The rate of data capture for recording throughout a larval developmental stage at a sufficiently high temporal resolution can exceed 3 , 000 , 000 images per day .","For a dataset of this magnitude , the required post-processing is the rate-limiting step of the experiment .","Using PyCelegans , the rate-limiting component of the analysis scaled linearly with the number of available processing-cores .","By using 256 cores we achieved a speed-up of two orders of magnitude relative to previous implementations .","For proof of principle , analyses have been run on up to 1024 processors .","The number of processors that could be utilized , for example , from publically available clusters , is in the tens of thousands for a single dataset .","The objective of the image-processing portion of PyCelegans is to identify the head , tail , and body of the animal and to compute secondary properties based on this identification ( e . g . , the body midline , perimeter , and orientation ) .","Tertiary properties can then be computed from the resulting raw data .","Our analysis of posture and locomotion was based on dividing the midline of the body of each animal into 20 segments and measuring the local angles between them ( Figure 1A–B ) .","This method extended previous analyses based on body curvature as a function of time and body coordinate ( Fang-Yen et al . , 2010; Vidal-Gadea et al . , 2011 ) : we tagged individual body-bends , followed them from initiation to eventual demise , and recorded their origin , velocity , amplitude , and life-time .","The identification of the midlines and individual body-bends enabled measurements of local properties as a function of the body-coordinate ( e . g . , quiescence of individual body-segments , Figure 1C ) , as well as global behavioral patterns such as mean curvature and growth rate ( Figure 1D ) or modes of locomotion ( Figure 2 ) .","For instance , a 2% growth of body-length per hour was observed outside of lethargus , but during lethargus a 5% shortening of the body-length was observed ( Figure 2D ) .","This shortening may be due to continuous growth in body-volume at a period where adding surface area to the ( old ) cuticle was restricted .","PyCelegans enabled us to conduct such high-resolution measurements over extended periods for the first time . 10 . 7554/eLife . 00782 . 003Figure 1 . Measurements of angles associated with body-segments: the midline of the body of the worm was fitted to a spline and divided into 20 equal segments . The local angle at each of the 18 inner segments , θi , was defined as the angle between its two flanking segments , si−1 and si+1 .","( A ) Body-bends propagating from the head ( left arrow ) to the tail ( right arrow ) during forward locomotion in a wild-type mid L4 larva .","For the purpose of demonstration , the local angles of only eight segments along the body were plotted .","The color map corresponds to angles from the most anterior ( brown ) , through the middle ( orange and yellow ) , to the most posterior ( grey ) body-regions .","Each bend appears as a peak or a trough in the local angle .","A successive bend is initiated at the head ∼4 s before its predecessor reaches the tail .","( B ) Angles at the same eight body segments during early L4 lethargus .","When the animal is quiescent the local angles remain constant or relax slowly .","( C ) The fraction of time , calculated from a 10 min running window , that individual body segments ( as denoted on the worm schematic ) are quiescent .","Body-segments that are further removed from the head of the animal exhibit more quiescence than more anterior segments , such that the overall quiescent behavior associated with L4 lethargus reflects the quiescence of the head and the neck .","( D ) The length of the body ( top ) and the mean angle along its midline between the mid L4 and mid young adult stages .","Shaded area denotes the L4 lethargus period .","The reduction in body-length during lethargus may be a signature of growth in the volume of the body at a time when expanding the surface area of the body is constrained .","Panels ( C and D ) depict N = 37 animals , mean ± SEM .","Standard errors are illustrated as shadowed areas surrounding the plotted averages . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 00310 . 7554/eLife . 00782 . 004Figure 2 . Modulation of locomotion between the mid-L4 and mid-young-adult stages in wild-type animals .","( A ) The locomotion behavior of each animal at each time-point was determined to belong to one of four characteristic classes: forward ( Fwd ) , dwelling ( Dwl ) , quiescence ( Qsc ) or backward ( Bck ) .","The average fractions of time out of a 10-min running window in which animals exhibited each of the four characteristic behaviors were plotted .","( B ) The frequency of generation of body-bends ( top ) and the velocity of bends that propagated from the anterior to the posterior of the body , that is , during forward locomotion .","During lethargus , rearward propagating bends were rare , and a meaningful average could not be obtained .","Panels ( A and B ) depict N = 37 animals , mean ± SEM .","Standard errors are illustrated as shadowed areas surrounding the plotted averages .","( C and D )","The modulation of locomotion in single animals .","The bars above each plot indicate epochs of active wakefulness ( dark brown ) or quiet wakefulness ( light grey ) .","In all panels the shaded rectangular area indicates the period of L4 lethargus . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 004 Although the term lethargus , describing reduced activity associated with molting in a variety of nematode species , was already in use about a century ago ( Veglia , 1915; Sommerville , 1960; Singh and Sulston , 1978 ) , the detailed dynamics of this behavior have only recently been studied ( Raizen et al . , 2008; Iwanir et al . , 2013; Belfer and Raizen , 2013 ) .","In C . elegans , head/neck and body motor circuits govern the generation and propagation of body-bends ( White et al . , 1976 , 1986; McIntire et al . , 1993; Stetina et al . , 2006; Chen et al . , 2007; Wen et al . , 2012 ) , and the body motoneuron network exhibits an iterative pattern of six interconnected modules along the anterior-posterior axis ( Haspel and O’Donovan , 2011 ) .","It was thus possible that different body regions would exhibit distinct temporal dynamics of quiescence .","To identify such patterns , we divided the midline of the animal to 20 equi-length segments , and quantified the fraction of time spent in quiescence by the individual angles between pairs of segments , during the period from mid L4int to the mid young adult stage ( Figure 1C ) .","While an abrupt increase in the fraction of quiescence at the onset of lethargus occurred in all of the individual body-regions , most of them exhibited elevated quiescence during the preceding 2-hr period .","Interestingly , the most anterior region was the least quiescent throughout the measurement .","Subsequent regions exhibited successively increasing quiescence , with the mid-body being the most quiescent .","As a result of this ordering , the temporal dynamics of quiescence of the entire animal mirrored that of the head/neck region .","We thus concluded that the activity of the head/neck motoneuron circuits determined the outcome of previously reported measurements of whole-animal quiescence ( Raizen et al . , 2008; Singh et al . , 2011; Bringmann , 2011; Iwanir et al . , 2013; Belfer and Raizen , 2013 ) .","Although behavioral quiescence provides the most prominent example of modulation of posture and locomotion during development ( Raizen et al . , 2008; Singh et al . , 2011; Iwanir et al . 2013; Ghosh and Emmons 2008; Schwarz et al . , 2012 ) , additional instances of modulation could occur outside of lethargus .","To identify such phenomena , we focused on four categories of locomotion behavior: forward and backward locomotion were defined by the appropriate directional propagation of body-bends along the anterior-posterior axis , dwelling was defined as non-directional propagation of body-bends , and quiescence was defined as sub-threshold changes in body angles ( see ‘Materials and methods’ ) .","Aligning behavioral data by the onset of lethargus for each 10 hr measurement and averaging between animals , we observed a decrease in the percentage of time spent in forward locomotion during the 4 hr preceding L4leth and a corresponding increase in the percentage of time spent in dwelling ( Figure 2A ) .","During the first 1–2 hr following L4leth termination , wild-type behavior was characterized by a high , decreasing fraction of dwelling , and a low , increasing fraction of forward locomotion .","Backward locomotion was modulated more weakly during the period of the measurement .","In agreement with our previous findings using complementary methods ( Iwanir et al . , 2013 ) we found that the overall curvature , quantified as the mean absolute angle , was modulated during lethargus , and that the overall growth rate of the body length outside of lethargus was 2% per hour ( Figure 1D ) .","In addition , the propagation velocity of anterior-to-posterior body-bends and the frequency of generation of bends were gradually downregulated during late L4int , and upregulated during the early young adult stage ( Figure 2B ) .","Although the frequency of bend generation was naturally limited by how quickly bends propagated away from their point of origin , there was no a priori restriction on how low this frequency could be .","The similarity between the dynamics of bend generation frequency and of bend velocity suggested that the former could serve as an adequate proxy for the latter throughout the measurement period .","The gradual modulation of the average behavioral dynamics shown in Figure 2A could result from a progressive modulation in individual animals or from abrupt but asynchronous events .","To distinguish between these possibilities , we examined the behavior of the individual animals in our dataset .","We found that the forward velocities of individual animals decreased gradually ( data not shown ) .","In contrast , the fraction of time spent in forward locomotion and dwelling alternated between high and low values , giving rise to the definition of two behavioral states: active wakefulness , characterized by a high proportion of forward locomotion , and quiet wakefulness , characterized by a high proportion of dwelling ( Figure 2C–D ) .","Transitions between active and quiet wakefulness were in the form of rapid behavioral switches , suggesting that they represented two distinct behavioral states .","In order to further characterize the observed behavioral dynamics we measured the durations of the epochs of active wakefulness prior to the onset of L4leth .","The simplest model , a two-state Markov chain with constant rates of transitions into and out of the active wakefulness state , would yield Poisson statistics , that is , an exponential distribution of epoch durations .","The histogram of epochs longer than 3 min was thus fit to exponential ( N","( t ) ∼ e−t/τ ) and power-law ( N","( t ) ∼ t− ( 1+α ) ) distributions , and the Akaike information criteria ( AIC ) were calculated in order to compare the two models ( Burnham and Anderson , 2002; Burnham and Anderson , 2004 ) .","Interestingly , the power law fit ( Figure 3A , AIC = −3 . 1 ) was strongly favored over the exponential fit ( AIC = 13 . 1 ) : the Akaike weight was 0 . 9997 , indicating that the probability that the power-law model better described the data was 99 . 97% .","The exponent obtained from the power law fit was − ( 1+α ) = −1 . 83 ± 0 . 31 ( 95% confidence intervals , R = 0 . 95 ) .","These results indicated that a simple , Poissonian , model would not adequately describe the observed transitions between active and quiet wakefulness .","Rather , the long tail of the power-law distribution suggested that the active wakefulness state was stabilized during L4int . 10 . 7554/eLife . 00782 . 005Figure 3 . The dynamics of the active wakefulness state during the three hours prior to L4 lethargus in wild-type animals .","( A ) A histogram of the durations of epochs of active wakefulness plotted on a log-log scale .","Epoch durations longer than 3 min exhibited a power-law distribution with an exponent – ( 1+α ) = −1 . 83±0 . 31 .","( B ) Two displacements along the y-axis of a sample trace of the fraction of forward locomotion , Δy1 ( between filled circles ) and Δy2 ( between empty circles ) .","Both displacements correspond to an identical time interval , Δt .","The time-averaged mean square displacement ( TMSD ) is calculated in two steps:","( i ) using a sliding window to calculate the mean squared displacements along traces of each of the individual animals ( Golding and Cox , 2006 ) ;","( ii ) averaging the results obtained from the previous step for all animals .","( C ) The TMSD plotted on a log-log scale as a function of the time-interval , Δt .","The TMSD was calculated for the subset of N = 20 animals where data 3 hr prior to the onset of L4leth was available ( N = 20 ) .","The TMSD exhibited power-law growth with the exponent ( 1−α ) = 0 . 32 ± 0 . 03 , consistent with a value of α ≈ 0 . 7 .","Inset: for the purpose of illustration , the TMSD for a two-state Markov chain with a comparable mean duration of epochs is shown to reach its saturation value at Δt ≈ 400 s ( vertical dashed line ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 005 An additional quantity that can be used to detect the signature of an underlying non-Poissonian process is the time-averaged mean squared displacement ( TMSD ) : the mean squared difference between the values of the process at two time-points that are Δt apart , as illustrated in Figure 3B .","In simple cases such as a two-state Markov chain , when the TMSD is plotted as a function of Δt it saturates at a constant value at long measurement times ( Figure 3C , inset ) .","In contrast , in processes with no underlying timescale , that is , where the duration of the intervals follow a power-law distribution , the TMSD increases as the time interval increases , exhibiting its own , related , power-law long-term behavior ( S ( Δt ) ∼ Δt ( 1−α ) ) with an exponent 1−α , where α is determined by the durations of the epochs ( Stefani et al . , 2009; Burov et al . , 2010 ) .","We thus measured the TMSD for the fraction of time spent in active wakefulness during the 3 hr prior to L4leth .","As shown in Figure 3C , the resulting long-term behavior was characterized by the exponent 1−α = 0 . 32 ± 0 . 03 ( 95% confidence intervals , R = 0 . 96 ) , consistent with a value of α ≈ 0 . 7 .","These findings support the hypothesis of a mechanism for stabilizing the active wakefulness state during L4int , resulting in the observed distribution of epoch durations .","Both the loss of function mutation of the kin-2 gene—encoding a negative regulatory subunit of the cAMP-dependent protein kinase ( PKA ) KIN-1—and the gain of function mutation of the adenylyl cyclase gene acy-1 were inferred to result in increased activity of the holoenzyme ( Schade et al . , 2005; Reynolds et al . , 2005; Charlie et al . , 2006 ) .","Correspondingly , both mutations result in hyperkinetic behavior outside of lethargus and reduced quiescence during lethargus ( Schade et al . , 2005; Reynolds et al . , 2005; Charlie et al . , 2006; Perez-Mansilla and Nurrish , 2009; Belfer and Raizen , 2013 ) .","We thus assayed the dynamics of locomotion of these mutants from mid L4int to the mid young adult stage .","In particular , we asked whether both their velocity and the overall structure of the active and quiet wakefulness states were different from wild-type and from each other .","We found that in both cases active wakefulness persisted from mid L4int until the onset of L4leth , and resumed at the end of L4leth ( Figure 4A–B , D–E ) .","Correspondingly , as compared to wild-type , these mutants spent a significantly reduced amount of time in quiet wakefulness before and after lethargus ( Figures 4 and 7 ) , and exhibited an approximately two-fold increase in peak velocity during L4int and milder downregulation of velocity in anticipation of L4leth ( Figure 4C , F ) .","In addition , consistent with previous reports ( Belfer and Raizen , 2013 ) , during bouts of non-quiescent behavior in lethargus ( ‘motion bouts’ ) we observed a large increase in the prevalence of forward locomotion in kin-2 and acy-1 ( gf ) mutants .","Taken together , these results suggest that increased PKA activity stabilizes active wakefulness . 10 . 7554/eLife . 00782 . 006Figure 4 . Increased Gαs signaling stabilizes active wakefulness outside of L4leth . The effects of increased Gαs signaling on locomotion were assayed using animals mutant in two genes: a loss of function mutation of kin-2 , encoding a negative regulatory subunit of PKA , and a gain of function mutation of acy-1 , encoding an adenylyl cyclase .","( A and D )","Locomotion dynamics of a single animal between the mid L4 and the mid young adult stages .","In contrast to wild-type behavior , forward locomotion was also prominently observed during L4leth ( see also Figures 7 and 8 ) .","( B and E )","The average fractions of time out of a 10-min running window in which animals exhibited each of the four characteristic types of locomotion .","( C and F )","The frequency of generation of body-bends of mutant ( dark grey ) and wild-type ( light grey , data from Figure 2B ) animals .","Panels ( B–F ) depict Nkin-2 = 16 animals , Nacy-1 ( gf ) = 17 animals , mean ± SEM .","Standard errors are illustrated as shadowed areas surrounding the plotted averages . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 006 Animals carrying a null mutation of kin-1 or acy-1 die as embryos or first stage larvae respectively .","However , animals carrying a partial loss of function allele of the acy-1 gene appear superficially wild-type .","We assayed these mutants to determine whether the partial loss of function of the cyclase would confer the opposite phenotype from that of the gain of function , and found this to be the case outside of lethargus but not during lethargus .","During the last 4 hr of L4int quiet wakefulness was significantly exaggerated and the active wakefulness state was fragmented—animals displayed a larger number of switches out of brief epochs of active wakefulness ( Figure 5 ) .","Similarly , after the termination of L4leth , quiet wakefulness was exaggerated and active wakefulness was reduced .","However , during L4leth the dynamics of quiescence and locomotion of the mutants were similar to wild-type ( Figures 7 and 8 ) .","These results are consistent with the idea that PKA activity stabilizes the active wakefulness state .","They further suggest that during lethargus , when active wakefulness is strongly suppressed , the native role of PKA signaling in modulating locomotion and quiescence may be minor . 10 . 7554/eLife . 00782 . 007Figure 5 . Decreased Gαs signaling destabilizes active wakefulness outside of L4leth . The effects of decreased Gαs signaling on locomotion were assayed using a loss of function mutation of the adenylyl cyclase gene , acy-1 .","( A ) Locomotion dynamics of a single animal between the mid L4 and the mid young adult stages ( see also Figures 7and 8 ) .","( B ) The average fractions of time out of a 10-min running window in which animals exhibited each of the four characteristic types of locomotion .","( C ) The frequency of generation of body-bends of mutant ( dark grey ) and wild-type ( light grey , data from Figure 2B ) animals .","Panels ( B and C ) depict Nacy-1 ( lf ) =15 animals , mean ± SEM .","Standard errors are illustrated as shadowed areas surrounding the plotted averages . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 007 Tomosyn , a target of PKA ( Baba et al . , 2005 ) , was shown to negatively regulate both unc-13-dependent synaptic transmitter release and unc-31-dependent neuropeptide release in C . elegans ( Gracheva et al . , 2006; Gracheva et al . , 2007a , 2007b ) .","Since hyper-activation of PKA could bypass the requirement for UNC-31 in the docking of dense core vesicles ( DCVs ) , the GSA-1 ( Gαs ) and the UNC-31/CAPS pathways were suggested to converge to control DCV release ( Schade et al . , 2005; Reynolds et al . , 2005; Charlie et al . , 2006; Zhou et al . , 2007; Perez-Mansilla and Nurrish , 2009 ) .","We could not analyze the behavior of unc-13 mutants due to their severe locomotion defects , but in order to assess whether the modulation of locomotion during L4int depended on the release of DCVs , we assayed unc-31 mutants ( Figure 6 ) .","The resulting phenotype was similar to that of acy-1 ( lf ) mutants .","As compared to wild-type animals , outside of lethargus unc-31 mutants exhibited increased occupation of the quiet wakefulness state , a fragmented active wakefulness state , and overall lower velocities .","In contrast , during L4leth , the dynamics of quiescence and locomotion of unc-31 mutants were similar to wild-type .","Unlike acy-1 ( lf ) mutants , the mutation in the unc-31 gene resulted in elevated levels of backward locomotion outside of lethargus and a decrease in directed locomotion during motion bouts in lethargus ( Figures 7 and 8 ) .","Taken together with the acy-1 ( lf ) phenotype , these results support a model in which PKA activity stabilizes active wakefulness during L4int ( at least in part ) by regulating DCV exocytosis , but only plays a minor role in regulating locomotion during lethargus . 10 . 7554/eLife . 00782 . 008Figure 6 . Decreased neuropeptide release ( in animals mutant for the gene encoding UNC-31/CAPS ) , or decreased CREB activity ( in animals mutant for the gene encoding the C . elegans CREB ortholog , CRH-1 ) , destabilize active wakefulness outside of L4leth .","Both UNC-31/CAPS and CRH-1 act downstream of PKA .","( A and D )","Locomotion dynamics of a single animal between the mid L4 and the mid young adult stages ( see also Figures 7 and 8 ) .","( B and E )","The average fractions of time out of a 10-min running window in which animals exhibited each of the four characteristic types of locomotion .","( C and F )","The frequency of generation of body-bends of mutant ( dark grey ) and wild-type ( light grey , data from Figure 2B ) animals .","Panels ( B–F ) depict Nunc-31 = 19 , Ncrh-1 = 16 animals , mean ± SEM .","Standard errors are illustrated as shadowed areas surrounding the plotted averages . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 00810 . 7554/eLife . 00782 . 009Figure 7 . Comparisons of mutant and wild-type behavior before , during and after lethargus . Panels ( A–D ) aggregate the data collected during the 2–4 hr prior to the onset of L4leth , the 0–2 hr prior to the onset of L4leth , L4leth , and the 0–2 hr after the termination of L4leth .","Each column summarizes the data for a given type of locomotion ( Forward , Dwelling , Backward , and Quiescence ) for all genotypes .","Asterisks denote behavioral patters that were significantly different from wild-type ( p<0 . 05 ) .","Each bar depicts mean ± SEM , and the sizes of the datasets are the same as in Figures 2 , 4–6 . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 00910 . 7554/eLife . 00782 . 010Figure 8 . Forward and backward locomotion during motion bouts , that is , bouts of non-quiescent behavior during lethargus .","( A ) The percentage of the duration of individual motion bouts that was spent in directed locomotion ( forward—left , backward—right ) on each of the genetic backgrounds .","( B ) Venn diagrams depicting the fraction of motion bouts ( out of the total number of motion bouts that were longer than 10 s ) where any forward or backward locomotion was detected .","Increased Gαs signaling resulted in a larger fraction of motion bouts that included directed motion , and a larger percentage of the total duration of individual bouts that was spent in directed locomotion .","Partially decreased Gαs signaling resulted in wild-type-like motion bouts .","Strongly decreased neuropeptide release resulted in a mild reduction in forward locomotion and a mild increase in backward locomotion during motion bouts .","Loss of CREB function increased the fraction of motion bouts that included directed locomotion , as compared with wild-type .","Asterisks denote behavioral patters that were significantly different from wild-type ( p<0 . 05 ) .","( C–D )","The effects of Gαs signaling on the duration of bouts and the fraction of time spent in quiescence during two time intervals: t = 10–40 min ( C ) , and t = 105–250 min ( D ) , where t = 0 corresponds to the onset of L4leth . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 010 Another major target of PKA is the cAMP response element-binding protein ( CREB ) , a transcription factor that , after phosphorylation by PKA , induces gene expression through promoters containing the cAMP-response element ( CRE ) enhancer ( Mayr and Montminy , 2001 ) .","Although the C . elegans ortholog of CREB , CRH-1 , was primarily implicated in long-term habituation and memory ( Kimura et al . , 2002; Kauffman et al . , 2010; Nishida et al . , 2011; Timbers and Rankin , 2011 ) it was possible that it could also have a role in regulating larval locomotion .","To address this question , we assayed crh-1 mutants ( Figure 6 ) .","Outside of lethargus , crh-1 mutants exhibited more poorly-defined global locomotion states , although a clear active wakefulness state was observed in 4 out of 16 animals 5–6 hr prior to L4leth .","Gradual transitions between directed locomotion and dwelling were commonly observed during this period , in contrast to the abrupt wild-type switching .","In addition , the mutants exhibited lower overall velocities and increased backward locomotion .","During L4leth , the dynamics of quiescence of crh-1 mutants were similar to wild-type ( Figures 6 and 7 ) .","However , the mutants did show significantly increased levels of directed ( forward and backward ) locomotion during motion bouts as compared to wild-type ( Figures 6 and 8B ) .","The levels of directed motion during lethargus of crh-1 mutants were similar to those of kin-2 and acy-1 ( gf ) mutants , possibly indicating a disruption to lethargus behavior .","We concluded that two of the downstream targets of PKA , namely neuropeptide release and CREB , acted coherently to stabilize active wakefulness during L4int , while playing a minor role in regulating quiescence and locomotion during lethargus ."],["Changes to neural circuits induced by experience or development can occur on timescales of hours to months .","Neuromodulators such as biogenic amines or neuropeptides often act on such long timescales , modifying the output of neural circuits by altering the activity of neurons and affecting synaptic connections ( Bargmann , 2012; Marder , 2012 ) .","Yet there are few techniques for tracking long-term physiological and behavioral dynamics and mostly offer limited resolution ( Clark et al . , 2010; Fonio et al . , 2012; Hart , 2006 ) .","Counter-intuitively , despite the simplicity of invertebrate models , detailed longitudinal studies designed to follow their behavioral trends across development are rare as well .","In order to study these processes , we have established a novel high-throughput assay , and obtained the first analysis of C . elegans locomotion on developmental timescales .","Since data analysis is the bottleneck of prolonged , detailed studies of behavior , we developed PyCelegans , a suite of tools that leverages high performance parallel computing to speed up the computational analysis in our workflow .","Accordingly , bottlenecks were shifted back to experimental elements , permitting increased throughput .","PyCelegans leverages only open source , freely available tools and libraries .","These utilities ( Python , NumPy , SciPy , mpi4py ) are top tier open source scientific computing tools , among the most widely used by researchers developing their own analyses .","As such , it is virtually guaranteed that they will be available at any dedicated research computing facility .","Moreover , the suite is easily extendable—additional analyses and features are simple to incorporate within the existing framework .","Quiet wakefulness was previously reported in diverse species such as various mammals , birds and reptiles , and can comprise a large percentage of the waking state ( Ruckebusch , 1972; Flanigan , 1973; Campbell and Tobler , 1984 ) .","However , to the best of our knowledge , quiet wakefulness was not previously reported in nematodes .","During its larval development the nematode C . elegans is required to integrate newly differentiated neurons into its neural circuits ( Sulston , 1976; Sulston and Horvitz , 1977; White et al . , 1986 ) and to reshape the connections of existing cells ( White et al . , 1978; Thomas et al . , 1990 ) .","At the same time , the animal grows , develops new organs , and undergoes four molts .","Maintaining the functionality of essential motor programs such as feeding , locomotion and defecation concurrently with these anatomical and physiological changes imposes constraints on the developing animal; modulation of behavioral patterns may contribute towards satisfying these constraints .","In particular , 2–4 hr prior to lethargus , the seam cells exhibit increased synthetic activity and begin to deposit the new cuticle ( Singh and Sulston , 1978; Monsalve et al . , 2011 ) .","The timing of quiet wakefulness that we observed coincided with the timing of this activity .","Thus , it is possible that this state may assist with maintaining an appropriate energy balance .","Our analysis revealed a novel behavioral switch that toggled between the active and quiet wakefulness states prior to L4leth .","The distribution of durations of the epochs of active wakefulness supported a model in which this state was actively stabilized and ergodicity was weakly broken ( Stefani et al . , 2009; Burov et al . , 2010 ) .","We note that the global locomotion states that we report are distinct from the cGMP-dependent short intervals of continuous roaming or dwelling behavior , previously described in adults ( Fujiwara et al . , 2002 ) .","We have shown that upregulating or downregulating Gαs signaling stabilized or destabilized active wakefulness respectively .","Downregulated neuropeptide release in unc-31 mutants , a gene encoding the calcium-dependent activator protein for secretion ( CAPS ) required for the priming and docking of DCVs ( Speese et al . , 2007; Lin et al . , 2010 ) , affected behavior similarly to downregulated Gαs signaling .","These findings extend the previously reported effects of PKA signaling on locomotion and quiescence ( Schade et al . , 2005; Reynolds et al . , 2005; Charlie et al . , 2006; Raizen et al . , 2008; Perez-Mansilla and Nurrish , 2009; Belfer and Raizen , 2013 ) .","Moreover , PKA was shown to catalyze the phosphorylation of tomosyn , a highly-conserved syntaxin-binding protein .","Phosphorylation of tomosyn by PKA reduced its interaction with syntaxin and enhanced the formation of the SNARE complex ( Baba et al . , 2005 ) .","In C . elegans , tomosyn ( TOM-1 ) was shown to negatively regulate synaptic transmitter release and UNC-31/CAPS-dependent neuropeptide release ( Gracheva et al . , 2007a ) , and KIN-1 alleviated the suppression of both processes by phosphorylating TOM-1 ( J Richmond , personal communications , February 2013 ) .","Interestingly , a mutation in the tom-1 gene suppressed behavioral deficits and DCV accumulation in unc-31 mutants ( Gracheva et al . , 2007a ) .","Similarly , enhanced PKA activity was shown to be sufficient for bypassing the requirement for UNC-31 for the release of DCVs ( Zhou et al . , 2007; Perez-Mansilla and Nurrish , 2009 ) .","Taken together , these findings support a model in which KIN-1 and TOM-1 regulate locomotion .","Upregulating Gαs signaling resulted in enhanced active wakefulness outside of lethargus and enhanced directed locomotion during L4leth , mirroring previous findings in Drosophila melanogaster and in C . elegans ( Joiner et al . , 2006; Belfer and Raizen , 2013 ) .","These results were also consistent with a previous observation that acute activation of the photoactivated adenylyl cyclase PACα in cholinergic neurons evoked an enhanced activity response ( Weissenberger et al . , 2011 ) .","Downregulating Gαs signaling resulted in a clear locomotion phenotype outside of but not during L4leth .","However , loss of CREB function resulted in an enhancement of directed locomotion , as well as changes in the dynamics of bouts of motion and quiescence ( Figure 8C , D ) , during lethargus .","In addition , crh-1 mutants exhibited strong locomotion defects outside of lethargus .","Studies of the effects of cAMP signaling on sleep in D . melanogaster and in mice have demonstrated that CREB , when activated by PKA , promotes the duration of wakefulness ( Hendricks et al . , 2001; Graves et al . , 2003; Shaw and Franken , 2003 ) .","The mild loss of function phenotypes of acy-1 ( lf ) and unc-31 during lethargus may be explained by a floor effect , while the implications of the crh-1 phenotype remain to be understood .","Interestingly , a recent study reported that adenylyl cyclase activity increases the intensity of nighttime sleep in Drosophila ( van Alphen et al . , 2013 ) .","Taken together , our analyses indicated that both PKA-dependent pathways acted in concert to regulate active wakefulness during L4int and the early young adult stages .","Finally , the approach to quantifying locomotion presented here provides several distinct advantages .","Visible phenotypes were invaluable for uncovering molecular pathways that regulated behavior , but were largely based on crude classifications such as hypo- or hyper-kinesis or a focus on arbitrarily defined features .","It was recently shown that the space of shapes adopted by the nematode C . elegans is low dimensional , and that these dimensions ( ‘eigenworms’ ) can provide a quantitative description of behavior ( Stephens et al . , 2008 ) .","This exquisitely sensitive analysis revealed the underlying simplicity of complex locomotion dynamics as well as subtle behavioral phenotypes that had been previously undetectable ( Stephens et al . , 2010 , 2011; Brown et al . , 2013 ) .","However , this analysis does not provide an intuitive interpretation of the detected phenotypes that may directly relate to the underlying neuronal activity .","Tracking of individual body-bends opens the door to a heuristic description of C . elegans locomotion , composed of intuitive building blocks .","As in the case of the eigenworms , one underlying assumption of this analysis is that C . elegans does not maintain a ‘mental map’ of its environment , nor does it keep track of its own acceleration in the laboratory frame of reference .","Rather , it responds to external and internal cues solely by altering its own body-posture .","Specifically , the primary task of the motor neurons during directional motion is to generate body-bends and propagate them along the body in the appropriate direction ( Gray et al . , 2005; Stetina et al . , 2006; Stephens et al . , 2008; Haspel and O’Donovan , 2011; Boyle et al . , 2011; Wen et al . , 2012 ) .","It follows that a natural , heuristic model for describing directional locomotion can be constructed using the rates of generation , propagation , and decay of body-bends .","Tracking each body-bend from initiation to eventual demise thus provides direct experimental measurements of the basic building blocks of nematode locomotion ."],["C . elegans strains were maintained and grown according to standard protocols ( Brenner , 1974 ) .","The wild-type strain used was C . elegans variety Bristol , strain N2 .","The following mutant strains were obtained from the Caenorhabditis Genetics Center: KG518 acy-1 ( ce2 ) III ( gf ) , KG532 kin- 2 ( ce179 ) X , CB169 unc-31 ( e169 ) IV , KP1182 acy- 1 ( nu329 ) III ( lf ) and YT17 crh-1 ( tz2 ) III .","Animals were synchronized by restricting the duration of egg-laying ( placing gravid adults on plates for 6 hr , removing the parents , and selecting L4s 3 days later ) , grown at 20°C on standard NGM plates , and fed OP50 bacteria until their mid L4int larva stage .","They were then transferred to individual 0 . 8 × 3 . 7 mm ‘artificial dirt’ chambers ( Lockery et al . , 2008 ) .","The chambers , containing an overnight OP50 Escherichia coli culture that was concentrated 10-fold and suspended in NGM liquid ( Singh et al . , 2011; Iwanir et al . , 2013 ) , were sealed with a coverslip held in place with VALAP ( a mix of equal parts of vaseline , lanolin and paraffin wax ) at the corners and submerged , facedown , in NGM buffer inside a petri dish to prevent drying .","Each observation chamber contained a single animal .","Behavior was recorded for 10 hr at a rate of 10 frames per second using a 5 megapixels CCD camera ( Prosilica GC2450 , 2448 × 2050 pixels , Allied Vision Technologies , Stadtroda , Germany ) .","We recorded at a magnification of 4 . 2X , resulting in a resolution of 1 . 54 × 1 . 54 μm2/pixel; the body length of a typical L4leth larva was approximately 500 pixels ( 750 μm ) .","The contrast at the edges of the animals was maximized , typically by setting background levels to 230–250 on a greyscale of 0–255 , and by focusing slightly away from the middle plane of the pharynx .","We developed a suite of tools , called PyCelegans , for image analysis on high performance parallel computing resources .","Similar to previously described methods ( Husson et al . , 2013 ) , PyCelegans identifies the body of the animal in each frame and calculates the coordinates of 100 points along its midline , ordered from head to tail .","The rate of segmentation failure , where the animals could not be properly identified , was typically 5% of all frames .","Frames in which the animal was not identified were not included in the dataset , but their timing was accounted for when time-stamping subsequent frames .","Each midline was divided into 20 equal segments and the local angle at each of the inner 18 segments was calculated as shown in Figure 1A .","The raw angle dynamics data was smoothed with a Gaussian filter with a width of 5 frames ( 0 . 5 s ) .","Body-bends were defined as positions of local spatial maxima or minima of the angles ( Figure 1A ) .","The position of each bend was tracked from the time of its initiation until it decayed ( typically at the head or the tail ) or was interrupted by 10 consecutive missing frames .","Forward locomotion was defined by the propagation of bends in the anterior-posterior direction that persisted for at least three consecutive midline segments .","Backward locomotion was defined analogously .","Quiescence of an individual segment was defined as an interval in which the rate of change of the corresponding angle did not exceed a threshold of 0 . 01 radians/sec .","Neither missing frames in which the inferred change in angle was below a threshold of 0 . 3 radians , nor motion occurring in isolated single frames , were considered to interrupt a bout of quiescence .","At each time point , the whole-animal behavior was classified as forward , backward or dwelling by applying a majority rule to the dynamics of the individual body-bends .","Dwelling occurred when the number of bends propagating in both directions was equal ( typically zero ) .","Whole-animal quiescence was defined as the state where the most anterior angle ( between the head and neck segments ) and at least 16 of the remaining 17 angles were quiescent .","The fraction of time spent in each behavior was calculated with a running-average window of 10 min for Figures 1 and 2 and 4–6 min for Figure 3 .","The onset/termination of lethargus was defined as the first/last point of increasing/decreasing whole-animal quiescence fraction that was followed by 20 consecutive minutes of the quiescence fraction remaining above/below a threshold of 5% .","Source code , documentation , and a sample dataset are available to download from GitHub ( https://github . com/labello/pycelegans ) .","Mutant behavior was compared to wild-type in Figures 7 and 8 using a one-way analysis of variance ( ANOVA ) with Bonferroni adjustments to compensate for multiple comparisons .","Exponential and power-law models for the data presented in Figure 3 were compared by calculating Akaike information criteria ( AIC ) ( Burnham and Anderson , 2002; Burnham and Anderson , 2004 ) ."]],"string":"[\n [\n \"The life cycle of the nematode Caenorhabditis elegans is comprised of the embryonic stage , four larval stages termed L1–L4 , and adulthood .\",\n \"Each larval stage lasts 8–12 hr at 20°C , and under standard conditions , and ends with a molt , when epidermal cells synthesize a new cuticle and the old one is shed .\",\n \"Although C . elegans larvae are mostly continuously motile , each molt is accompanied by a 2–3 hr period of behavioral quiescence ( Singh and Sulston , 1978 ) , referred to as ‘lethargus’ .\",\n \"Thus , the larval stages can be divided into motile intermolt sub-stages ( L1int–L4int ) and their corresponding lethargus sub-stages ( L1leth-L4leth ) .\",\n \"However , behavior during distinct developmental sub-stages ( Singh and Sulston , 1978 ) has not previously been examined in detail .\",\n \"Specifically , both the modulation of body posture and locomotion on developmental timescales remain largely unexplored .\",\n \"C . elegans move directionally by propagating dorsoventral undulations along their body .\",\n \"Upon receiving input from interneurons , motoneurons provide output to an array of longitudinal body-wall muscles in order to propagate body bends ( White et al . , 1986; Stetina et al . , 2006; Karbowski et al . , 2006; Stephens et al . , 2008 ) .\",\n \"Head/neck motoneurons and muscles independently control head movements , such as exploratory head swings ( White et al . , 1986; Stetina et al . , 2006; Pirri et al . , 2009; Maguire et al . , 2011 ) .\",\n \"The gsa-1 gene encodes a Gαs subunit , which activates the adenylyl cyclase ACY-1 , and the GSA-1 ( Gαs ) pathway has been previously shown to affect locomotion .\",\n \"Activation ACY-1 by GSA-1 leads to the production of cyclic adenosine monophosphate ( cAMP ) .\",\n \"The binding of cAMP to the protein kinase A ( PKA ) regulatory subunit KIN-2 dissociates it from the inactive holoenzyme , releasing the PKA catalytic subunit , KIN-1 .\",\n \"Increased PKA activity enhances signaling at the neuromuscular junction , as well as increases cAMP response element ( CRE ) mediated transcription in C . elegans neurons ( Kimura et al . , 2002 ) .\",\n \"Thus , upregulating this signaling pathway has been reported to result in hyperkinetic phenotypes , typically described as a nonspecific increase in the rate of locomotion ( Schade et al . , 2005; Reynolds et al . , 2005; Raizen et al . , 2008; Perez-Mansilla and Nurrish , 2009 ) , as well as in reduced quiescence during lethargus ( Raizen et al . , 2008; Belfer and Raizen , 2013 ) .\",\n \"Here we present the first detailed analysis of locomotion patterns during developmentally relevant timescales , that is , periods in which significant developmental changes occur .\",\n \"We have analyzed the initiation , propagation and eventual demise of individual dorsoventral body bends over a 14-hr period , from the mid-L4int stage to the mid-young-adult stage .\",\n \"We found that some locomotion patterns undergo a gradual modulation , while others display abrupt switching .\",\n \"In particular , two behavioral states , active wakefulness and quiet wakefulness , were observed during the mid- to late- L4int stage .\",\n \"Active wakefulness was dominated by forward locomotion ( propagation of body-bends from the anterior to the posterior ) , but included intervals of backward locomotion and ‘dwelling’ ( non-directional dynamics of body bends ) .\",\n \"In contrast , quiet wakefulness was dominated by dwelling behavior , although it included intervals of directed locomotion .\",\n \"In individual animals , active wakefulness was observed to persist for epochs of 1–100 min .\",\n \"Moreover , the switching between active and quiet wakefulness was abrupt , suggesting that they are distinct behavioral states .\",\n \"The process underlying these states exhibited the signature of ergodicity breaking , characteristic of a scale-free switch , as opposed to having a simple rate constant that governs the dynamics of switching .\",\n \"We further show that the transitions between behavioral states were regulated by the GSA-1 ( Gαs ) pathway: increased Gαs signaling stabilized the active wakefulness state both within and outside of lethargus , while decreased Gαs signaling destabilized this state , but only outside of lethargus .\"\n ],\n [\n \"To assay the modulation of behavior during development , we developed PyCelegans: a high-speed , modular image-processing tool for analyzing posture and locomotion of C . elegans on high performance computing resources .\",\n \"The function we used for processing a single frame was limited to tracking a single animal .\",\n \"However , the modular design of PyCelegans can accommodate multi-animal tracking once an appropriate substitute for this function is implemented , without further changes .\",\n \"The rate of data capture for recording throughout a larval developmental stage at a sufficiently high temporal resolution can exceed 3 , 000 , 000 images per day .\",\n \"For a dataset of this magnitude , the required post-processing is the rate-limiting step of the experiment .\",\n \"Using PyCelegans , the rate-limiting component of the analysis scaled linearly with the number of available processing-cores .\",\n \"By using 256 cores we achieved a speed-up of two orders of magnitude relative to previous implementations .\",\n \"For proof of principle , analyses have been run on up to 1024 processors .\",\n \"The number of processors that could be utilized , for example , from publically available clusters , is in the tens of thousands for a single dataset .\",\n \"The objective of the image-processing portion of PyCelegans is to identify the head , tail , and body of the animal and to compute secondary properties based on this identification ( e . g . , the body midline , perimeter , and orientation ) .\",\n \"Tertiary properties can then be computed from the resulting raw data .\",\n \"Our analysis of posture and locomotion was based on dividing the midline of the body of each animal into 20 segments and measuring the local angles between them ( Figure 1A–B ) .\",\n \"This method extended previous analyses based on body curvature as a function of time and body coordinate ( Fang-Yen et al . , 2010; Vidal-Gadea et al . , 2011 ) : we tagged individual body-bends , followed them from initiation to eventual demise , and recorded their origin , velocity , amplitude , and life-time .\",\n \"The identification of the midlines and individual body-bends enabled measurements of local properties as a function of the body-coordinate ( e . g . , quiescence of individual body-segments , Figure 1C ) , as well as global behavioral patterns such as mean curvature and growth rate ( Figure 1D ) or modes of locomotion ( Figure 2 ) .\",\n \"For instance , a 2% growth of body-length per hour was observed outside of lethargus , but during lethargus a 5% shortening of the body-length was observed ( Figure 2D ) .\",\n \"This shortening may be due to continuous growth in body-volume at a period where adding surface area to the ( old ) cuticle was restricted .\",\n \"PyCelegans enabled us to conduct such high-resolution measurements over extended periods for the first time . 10 . 7554/eLife . 00782 . 003Figure 1 . Measurements of angles associated with body-segments: the midline of the body of the worm was fitted to a spline and divided into 20 equal segments . The local angle at each of the 18 inner segments , θi , was defined as the angle between its two flanking segments , si−1 and si+1 .\",\n \"( A ) Body-bends propagating from the head ( left arrow ) to the tail ( right arrow ) during forward locomotion in a wild-type mid L4 larva .\",\n \"For the purpose of demonstration , the local angles of only eight segments along the body were plotted .\",\n \"The color map corresponds to angles from the most anterior ( brown ) , through the middle ( orange and yellow ) , to the most posterior ( grey ) body-regions .\",\n \"Each bend appears as a peak or a trough in the local angle .\",\n \"A successive bend is initiated at the head ∼4 s before its predecessor reaches the tail .\",\n \"( B ) Angles at the same eight body segments during early L4 lethargus .\",\n \"When the animal is quiescent the local angles remain constant or relax slowly .\",\n \"( C ) The fraction of time , calculated from a 10 min running window , that individual body segments ( as denoted on the worm schematic ) are quiescent .\",\n \"Body-segments that are further removed from the head of the animal exhibit more quiescence than more anterior segments , such that the overall quiescent behavior associated with L4 lethargus reflects the quiescence of the head and the neck .\",\n \"( D ) The length of the body ( top ) and the mean angle along its midline between the mid L4 and mid young adult stages .\",\n \"Shaded area denotes the L4 lethargus period .\",\n \"The reduction in body-length during lethargus may be a signature of growth in the volume of the body at a time when expanding the surface area of the body is constrained .\",\n \"Panels ( C and D ) depict N = 37 animals , mean ± SEM .\",\n \"Standard errors are illustrated as shadowed areas surrounding the plotted averages . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 00310 . 7554/eLife . 00782 . 004Figure 2 . Modulation of locomotion between the mid-L4 and mid-young-adult stages in wild-type animals .\",\n \"( A ) The locomotion behavior of each animal at each time-point was determined to belong to one of four characteristic classes: forward ( Fwd ) , dwelling ( Dwl ) , quiescence ( Qsc ) or backward ( Bck ) .\",\n \"The average fractions of time out of a 10-min running window in which animals exhibited each of the four characteristic behaviors were plotted .\",\n \"( B ) The frequency of generation of body-bends ( top ) and the velocity of bends that propagated from the anterior to the posterior of the body , that is , during forward locomotion .\",\n \"During lethargus , rearward propagating bends were rare , and a meaningful average could not be obtained .\",\n \"Panels ( A and B ) depict N = 37 animals , mean ± SEM .\",\n \"Standard errors are illustrated as shadowed areas surrounding the plotted averages .\",\n \"( C and D )\",\n \"The modulation of locomotion in single animals .\",\n \"The bars above each plot indicate epochs of active wakefulness ( dark brown ) or quiet wakefulness ( light grey ) .\",\n \"In all panels the shaded rectangular area indicates the period of L4 lethargus . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 004 Although the term lethargus , describing reduced activity associated with molting in a variety of nematode species , was already in use about a century ago ( Veglia , 1915; Sommerville , 1960; Singh and Sulston , 1978 ) , the detailed dynamics of this behavior have only recently been studied ( Raizen et al . , 2008; Iwanir et al . , 2013; Belfer and Raizen , 2013 ) .\",\n \"In C . elegans , head/neck and body motor circuits govern the generation and propagation of body-bends ( White et al . , 1976 , 1986; McIntire et al . , 1993; Stetina et al . , 2006; Chen et al . , 2007; Wen et al . , 2012 ) , and the body motoneuron network exhibits an iterative pattern of six interconnected modules along the anterior-posterior axis ( Haspel and O’Donovan , 2011 ) .\",\n \"It was thus possible that different body regions would exhibit distinct temporal dynamics of quiescence .\",\n \"To identify such patterns , we divided the midline of the animal to 20 equi-length segments , and quantified the fraction of time spent in quiescence by the individual angles between pairs of segments , during the period from mid L4int to the mid young adult stage ( Figure 1C ) .\",\n \"While an abrupt increase in the fraction of quiescence at the onset of lethargus occurred in all of the individual body-regions , most of them exhibited elevated quiescence during the preceding 2-hr period .\",\n \"Interestingly , the most anterior region was the least quiescent throughout the measurement .\",\n \"Subsequent regions exhibited successively increasing quiescence , with the mid-body being the most quiescent .\",\n \"As a result of this ordering , the temporal dynamics of quiescence of the entire animal mirrored that of the head/neck region .\",\n \"We thus concluded that the activity of the head/neck motoneuron circuits determined the outcome of previously reported measurements of whole-animal quiescence ( Raizen et al . , 2008; Singh et al . , 2011; Bringmann , 2011; Iwanir et al . , 2013; Belfer and Raizen , 2013 ) .\",\n \"Although behavioral quiescence provides the most prominent example of modulation of posture and locomotion during development ( Raizen et al . , 2008; Singh et al . , 2011; Iwanir et al . 2013; Ghosh and Emmons 2008; Schwarz et al . , 2012 ) , additional instances of modulation could occur outside of lethargus .\",\n \"To identify such phenomena , we focused on four categories of locomotion behavior: forward and backward locomotion were defined by the appropriate directional propagation of body-bends along the anterior-posterior axis , dwelling was defined as non-directional propagation of body-bends , and quiescence was defined as sub-threshold changes in body angles ( see ‘Materials and methods’ ) .\",\n \"Aligning behavioral data by the onset of lethargus for each 10 hr measurement and averaging between animals , we observed a decrease in the percentage of time spent in forward locomotion during the 4 hr preceding L4leth and a corresponding increase in the percentage of time spent in dwelling ( Figure 2A ) .\",\n \"During the first 1–2 hr following L4leth termination , wild-type behavior was characterized by a high , decreasing fraction of dwelling , and a low , increasing fraction of forward locomotion .\",\n \"Backward locomotion was modulated more weakly during the period of the measurement .\",\n \"In agreement with our previous findings using complementary methods ( Iwanir et al . , 2013 ) we found that the overall curvature , quantified as the mean absolute angle , was modulated during lethargus , and that the overall growth rate of the body length outside of lethargus was 2% per hour ( Figure 1D ) .\",\n \"In addition , the propagation velocity of anterior-to-posterior body-bends and the frequency of generation of bends were gradually downregulated during late L4int , and upregulated during the early young adult stage ( Figure 2B ) .\",\n \"Although the frequency of bend generation was naturally limited by how quickly bends propagated away from their point of origin , there was no a priori restriction on how low this frequency could be .\",\n \"The similarity between the dynamics of bend generation frequency and of bend velocity suggested that the former could serve as an adequate proxy for the latter throughout the measurement period .\",\n \"The gradual modulation of the average behavioral dynamics shown in Figure 2A could result from a progressive modulation in individual animals or from abrupt but asynchronous events .\",\n \"To distinguish between these possibilities , we examined the behavior of the individual animals in our dataset .\",\n \"We found that the forward velocities of individual animals decreased gradually ( data not shown ) .\",\n \"In contrast , the fraction of time spent in forward locomotion and dwelling alternated between high and low values , giving rise to the definition of two behavioral states: active wakefulness , characterized by a high proportion of forward locomotion , and quiet wakefulness , characterized by a high proportion of dwelling ( Figure 2C–D ) .\",\n \"Transitions between active and quiet wakefulness were in the form of rapid behavioral switches , suggesting that they represented two distinct behavioral states .\",\n \"In order to further characterize the observed behavioral dynamics we measured the durations of the epochs of active wakefulness prior to the onset of L4leth .\",\n \"The simplest model , a two-state Markov chain with constant rates of transitions into and out of the active wakefulness state , would yield Poisson statistics , that is , an exponential distribution of epoch durations .\",\n \"The histogram of epochs longer than 3 min was thus fit to exponential ( N\",\n \"( t ) ∼ e−t/τ ) and power-law ( N\",\n \"( t ) ∼ t− ( 1+α ) ) distributions , and the Akaike information criteria ( AIC ) were calculated in order to compare the two models ( Burnham and Anderson , 2002; Burnham and Anderson , 2004 ) .\",\n \"Interestingly , the power law fit ( Figure 3A , AIC = −3 . 1 ) was strongly favored over the exponential fit ( AIC = 13 . 1 ) : the Akaike weight was 0 . 9997 , indicating that the probability that the power-law model better described the data was 99 . 97% .\",\n \"The exponent obtained from the power law fit was − ( 1+α ) = −1 . 83 ± 0 . 31 ( 95% confidence intervals , R = 0 . 95 ) .\",\n \"These results indicated that a simple , Poissonian , model would not adequately describe the observed transitions between active and quiet wakefulness .\",\n \"Rather , the long tail of the power-law distribution suggested that the active wakefulness state was stabilized during L4int . 10 . 7554/eLife . 00782 . 005Figure 3 . The dynamics of the active wakefulness state during the three hours prior to L4 lethargus in wild-type animals .\",\n \"( A ) A histogram of the durations of epochs of active wakefulness plotted on a log-log scale .\",\n \"Epoch durations longer than 3 min exhibited a power-law distribution with an exponent – ( 1+α ) = −1 . 83±0 . 31 .\",\n \"( B ) Two displacements along the y-axis of a sample trace of the fraction of forward locomotion , Δy1 ( between filled circles ) and Δy2 ( between empty circles ) .\",\n \"Both displacements correspond to an identical time interval , Δt .\",\n \"The time-averaged mean square displacement ( TMSD ) is calculated in two steps:\",\n \"( i ) using a sliding window to calculate the mean squared displacements along traces of each of the individual animals ( Golding and Cox , 2006 ) ;\",\n \"( ii ) averaging the results obtained from the previous step for all animals .\",\n \"( C ) The TMSD plotted on a log-log scale as a function of the time-interval , Δt .\",\n \"The TMSD was calculated for the subset of N = 20 animals where data 3 hr prior to the onset of L4leth was available ( N = 20 ) .\",\n \"The TMSD exhibited power-law growth with the exponent ( 1−α ) = 0 . 32 ± 0 . 03 , consistent with a value of α ≈ 0 . 7 .\",\n \"Inset: for the purpose of illustration , the TMSD for a two-state Markov chain with a comparable mean duration of epochs is shown to reach its saturation value at Δt ≈ 400 s ( vertical dashed line ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 005 An additional quantity that can be used to detect the signature of an underlying non-Poissonian process is the time-averaged mean squared displacement ( TMSD ) : the mean squared difference between the values of the process at two time-points that are Δt apart , as illustrated in Figure 3B .\",\n \"In simple cases such as a two-state Markov chain , when the TMSD is plotted as a function of Δt it saturates at a constant value at long measurement times ( Figure 3C , inset ) .\",\n \"In contrast , in processes with no underlying timescale , that is , where the duration of the intervals follow a power-law distribution , the TMSD increases as the time interval increases , exhibiting its own , related , power-law long-term behavior ( S ( Δt ) ∼ Δt ( 1−α ) ) with an exponent 1−α , where α is determined by the durations of the epochs ( Stefani et al . , 2009; Burov et al . , 2010 ) .\",\n \"We thus measured the TMSD for the fraction of time spent in active wakefulness during the 3 hr prior to L4leth .\",\n \"As shown in Figure 3C , the resulting long-term behavior was characterized by the exponent 1−α = 0 . 32 ± 0 . 03 ( 95% confidence intervals , R = 0 . 96 ) , consistent with a value of α ≈ 0 . 7 .\",\n \"These findings support the hypothesis of a mechanism for stabilizing the active wakefulness state during L4int , resulting in the observed distribution of epoch durations .\",\n \"Both the loss of function mutation of the kin-2 gene—encoding a negative regulatory subunit of the cAMP-dependent protein kinase ( PKA ) KIN-1—and the gain of function mutation of the adenylyl cyclase gene acy-1 were inferred to result in increased activity of the holoenzyme ( Schade et al . , 2005; Reynolds et al . , 2005; Charlie et al . , 2006 ) .\",\n \"Correspondingly , both mutations result in hyperkinetic behavior outside of lethargus and reduced quiescence during lethargus ( Schade et al . , 2005; Reynolds et al . , 2005; Charlie et al . , 2006; Perez-Mansilla and Nurrish , 2009; Belfer and Raizen , 2013 ) .\",\n \"We thus assayed the dynamics of locomotion of these mutants from mid L4int to the mid young adult stage .\",\n \"In particular , we asked whether both their velocity and the overall structure of the active and quiet wakefulness states were different from wild-type and from each other .\",\n \"We found that in both cases active wakefulness persisted from mid L4int until the onset of L4leth , and resumed at the end of L4leth ( Figure 4A–B , D–E ) .\",\n \"Correspondingly , as compared to wild-type , these mutants spent a significantly reduced amount of time in quiet wakefulness before and after lethargus ( Figures 4 and 7 ) , and exhibited an approximately two-fold increase in peak velocity during L4int and milder downregulation of velocity in anticipation of L4leth ( Figure 4C , F ) .\",\n \"In addition , consistent with previous reports ( Belfer and Raizen , 2013 ) , during bouts of non-quiescent behavior in lethargus ( ‘motion bouts’ ) we observed a large increase in the prevalence of forward locomotion in kin-2 and acy-1 ( gf ) mutants .\",\n \"Taken together , these results suggest that increased PKA activity stabilizes active wakefulness . 10 . 7554/eLife . 00782 . 006Figure 4 . Increased Gαs signaling stabilizes active wakefulness outside of L4leth . The effects of increased Gαs signaling on locomotion were assayed using animals mutant in two genes: a loss of function mutation of kin-2 , encoding a negative regulatory subunit of PKA , and a gain of function mutation of acy-1 , encoding an adenylyl cyclase .\",\n \"( A and D )\",\n \"Locomotion dynamics of a single animal between the mid L4 and the mid young adult stages .\",\n \"In contrast to wild-type behavior , forward locomotion was also prominently observed during L4leth ( see also Figures 7 and 8 ) .\",\n \"( B and E )\",\n \"The average fractions of time out of a 10-min running window in which animals exhibited each of the four characteristic types of locomotion .\",\n \"( C and F )\",\n \"The frequency of generation of body-bends of mutant ( dark grey ) and wild-type ( light grey , data from Figure 2B ) animals .\",\n \"Panels ( B–F ) depict Nkin-2 = 16 animals , Nacy-1 ( gf ) = 17 animals , mean ± SEM .\",\n \"Standard errors are illustrated as shadowed areas surrounding the plotted averages . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 006 Animals carrying a null mutation of kin-1 or acy-1 die as embryos or first stage larvae respectively .\",\n \"However , animals carrying a partial loss of function allele of the acy-1 gene appear superficially wild-type .\",\n \"We assayed these mutants to determine whether the partial loss of function of the cyclase would confer the opposite phenotype from that of the gain of function , and found this to be the case outside of lethargus but not during lethargus .\",\n \"During the last 4 hr of L4int quiet wakefulness was significantly exaggerated and the active wakefulness state was fragmented—animals displayed a larger number of switches out of brief epochs of active wakefulness ( Figure 5 ) .\",\n \"Similarly , after the termination of L4leth , quiet wakefulness was exaggerated and active wakefulness was reduced .\",\n \"However , during L4leth the dynamics of quiescence and locomotion of the mutants were similar to wild-type ( Figures 7 and 8 ) .\",\n \"These results are consistent with the idea that PKA activity stabilizes the active wakefulness state .\",\n \"They further suggest that during lethargus , when active wakefulness is strongly suppressed , the native role of PKA signaling in modulating locomotion and quiescence may be minor . 10 . 7554/eLife . 00782 . 007Figure 5 . Decreased Gαs signaling destabilizes active wakefulness outside of L4leth . The effects of decreased Gαs signaling on locomotion were assayed using a loss of function mutation of the adenylyl cyclase gene , acy-1 .\",\n \"( A ) Locomotion dynamics of a single animal between the mid L4 and the mid young adult stages ( see also Figures 7and 8 ) .\",\n \"( B ) The average fractions of time out of a 10-min running window in which animals exhibited each of the four characteristic types of locomotion .\",\n \"( C ) The frequency of generation of body-bends of mutant ( dark grey ) and wild-type ( light grey , data from Figure 2B ) animals .\",\n \"Panels ( B and C ) depict Nacy-1 ( lf ) =15 animals , mean ± SEM .\",\n \"Standard errors are illustrated as shadowed areas surrounding the plotted averages . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 007 Tomosyn , a target of PKA ( Baba et al . , 2005 ) , was shown to negatively regulate both unc-13-dependent synaptic transmitter release and unc-31-dependent neuropeptide release in C . elegans ( Gracheva et al . , 2006; Gracheva et al . , 2007a , 2007b ) .\",\n \"Since hyper-activation of PKA could bypass the requirement for UNC-31 in the docking of dense core vesicles ( DCVs ) , the GSA-1 ( Gαs ) and the UNC-31/CAPS pathways were suggested to converge to control DCV release ( Schade et al . , 2005; Reynolds et al . , 2005; Charlie et al . , 2006; Zhou et al . , 2007; Perez-Mansilla and Nurrish , 2009 ) .\",\n \"We could not analyze the behavior of unc-13 mutants due to their severe locomotion defects , but in order to assess whether the modulation of locomotion during L4int depended on the release of DCVs , we assayed unc-31 mutants ( Figure 6 ) .\",\n \"The resulting phenotype was similar to that of acy-1 ( lf ) mutants .\",\n \"As compared to wild-type animals , outside of lethargus unc-31 mutants exhibited increased occupation of the quiet wakefulness state , a fragmented active wakefulness state , and overall lower velocities .\",\n \"In contrast , during L4leth , the dynamics of quiescence and locomotion of unc-31 mutants were similar to wild-type .\",\n \"Unlike acy-1 ( lf ) mutants , the mutation in the unc-31 gene resulted in elevated levels of backward locomotion outside of lethargus and a decrease in directed locomotion during motion bouts in lethargus ( Figures 7 and 8 ) .\",\n \"Taken together with the acy-1 ( lf ) phenotype , these results support a model in which PKA activity stabilizes active wakefulness during L4int ( at least in part ) by regulating DCV exocytosis , but only plays a minor role in regulating locomotion during lethargus . 10 . 7554/eLife . 00782 . 008Figure 6 . Decreased neuropeptide release ( in animals mutant for the gene encoding UNC-31/CAPS ) , or decreased CREB activity ( in animals mutant for the gene encoding the C . elegans CREB ortholog , CRH-1 ) , destabilize active wakefulness outside of L4leth .\",\n \"Both UNC-31/CAPS and CRH-1 act downstream of PKA .\",\n \"( A and D )\",\n \"Locomotion dynamics of a single animal between the mid L4 and the mid young adult stages ( see also Figures 7 and 8 ) .\",\n \"( B and E )\",\n \"The average fractions of time out of a 10-min running window in which animals exhibited each of the four characteristic types of locomotion .\",\n \"( C and F )\",\n \"The frequency of generation of body-bends of mutant ( dark grey ) and wild-type ( light grey , data from Figure 2B ) animals .\",\n \"Panels ( B–F ) depict Nunc-31 = 19 , Ncrh-1 = 16 animals , mean ± SEM .\",\n \"Standard errors are illustrated as shadowed areas surrounding the plotted averages . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 00810 . 7554/eLife . 00782 . 009Figure 7 . Comparisons of mutant and wild-type behavior before , during and after lethargus . Panels ( A–D ) aggregate the data collected during the 2–4 hr prior to the onset of L4leth , the 0–2 hr prior to the onset of L4leth , L4leth , and the 0–2 hr after the termination of L4leth .\",\n \"Each column summarizes the data for a given type of locomotion ( Forward , Dwelling , Backward , and Quiescence ) for all genotypes .\",\n \"Asterisks denote behavioral patters that were significantly different from wild-type ( p<0 . 05 ) .\",\n \"Each bar depicts mean ± SEM , and the sizes of the datasets are the same as in Figures 2 , 4–6 . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 00910 . 7554/eLife . 00782 . 010Figure 8 . Forward and backward locomotion during motion bouts , that is , bouts of non-quiescent behavior during lethargus .\",\n \"( A ) The percentage of the duration of individual motion bouts that was spent in directed locomotion ( forward—left , backward—right ) on each of the genetic backgrounds .\",\n \"( B ) Venn diagrams depicting the fraction of motion bouts ( out of the total number of motion bouts that were longer than 10 s ) where any forward or backward locomotion was detected .\",\n \"Increased Gαs signaling resulted in a larger fraction of motion bouts that included directed motion , and a larger percentage of the total duration of individual bouts that was spent in directed locomotion .\",\n \"Partially decreased Gαs signaling resulted in wild-type-like motion bouts .\",\n \"Strongly decreased neuropeptide release resulted in a mild reduction in forward locomotion and a mild increase in backward locomotion during motion bouts .\",\n \"Loss of CREB function increased the fraction of motion bouts that included directed locomotion , as compared with wild-type .\",\n \"Asterisks denote behavioral patters that were significantly different from wild-type ( p<0 . 05 ) .\",\n \"( C–D )\",\n \"The effects of Gαs signaling on the duration of bouts and the fraction of time spent in quiescence during two time intervals: t = 10–40 min ( C ) , and t = 105–250 min ( D ) , where t = 0 corresponds to the onset of L4leth . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 010 Another major target of PKA is the cAMP response element-binding protein ( CREB ) , a transcription factor that , after phosphorylation by PKA , induces gene expression through promoters containing the cAMP-response element ( CRE ) enhancer ( Mayr and Montminy , 2001 ) .\",\n \"Although the C . elegans ortholog of CREB , CRH-1 , was primarily implicated in long-term habituation and memory ( Kimura et al . , 2002; Kauffman et al . , 2010; Nishida et al . , 2011; Timbers and Rankin , 2011 ) it was possible that it could also have a role in regulating larval locomotion .\",\n \"To address this question , we assayed crh-1 mutants ( Figure 6 ) .\",\n \"Outside of lethargus , crh-1 mutants exhibited more poorly-defined global locomotion states , although a clear active wakefulness state was observed in 4 out of 16 animals 5–6 hr prior to L4leth .\",\n \"Gradual transitions between directed locomotion and dwelling were commonly observed during this period , in contrast to the abrupt wild-type switching .\",\n \"In addition , the mutants exhibited lower overall velocities and increased backward locomotion .\",\n \"During L4leth , the dynamics of quiescence of crh-1 mutants were similar to wild-type ( Figures 6 and 7 ) .\",\n \"However , the mutants did show significantly increased levels of directed ( forward and backward ) locomotion during motion bouts as compared to wild-type ( Figures 6 and 8B ) .\",\n \"The levels of directed motion during lethargus of crh-1 mutants were similar to those of kin-2 and acy-1 ( gf ) mutants , possibly indicating a disruption to lethargus behavior .\",\n \"We concluded that two of the downstream targets of PKA , namely neuropeptide release and CREB , acted coherently to stabilize active wakefulness during L4int , while playing a minor role in regulating quiescence and locomotion during lethargus .\"\n ],\n [\n \"Changes to neural circuits induced by experience or development can occur on timescales of hours to months .\",\n \"Neuromodulators such as biogenic amines or neuropeptides often act on such long timescales , modifying the output of neural circuits by altering the activity of neurons and affecting synaptic connections ( Bargmann , 2012; Marder , 2012 ) .\",\n \"Yet there are few techniques for tracking long-term physiological and behavioral dynamics and mostly offer limited resolution ( Clark et al . , 2010; Fonio et al . , 2012; Hart , 2006 ) .\",\n \"Counter-intuitively , despite the simplicity of invertebrate models , detailed longitudinal studies designed to follow their behavioral trends across development are rare as well .\",\n \"In order to study these processes , we have established a novel high-throughput assay , and obtained the first analysis of C . elegans locomotion on developmental timescales .\",\n \"Since data analysis is the bottleneck of prolonged , detailed studies of behavior , we developed PyCelegans , a suite of tools that leverages high performance parallel computing to speed up the computational analysis in our workflow .\",\n \"Accordingly , bottlenecks were shifted back to experimental elements , permitting increased throughput .\",\n \"PyCelegans leverages only open source , freely available tools and libraries .\",\n \"These utilities ( Python , NumPy , SciPy , mpi4py ) are top tier open source scientific computing tools , among the most widely used by researchers developing their own analyses .\",\n \"As such , it is virtually guaranteed that they will be available at any dedicated research computing facility .\",\n \"Moreover , the suite is easily extendable—additional analyses and features are simple to incorporate within the existing framework .\",\n \"Quiet wakefulness was previously reported in diverse species such as various mammals , birds and reptiles , and can comprise a large percentage of the waking state ( Ruckebusch , 1972; Flanigan , 1973; Campbell and Tobler , 1984 ) .\",\n \"However , to the best of our knowledge , quiet wakefulness was not previously reported in nematodes .\",\n \"During its larval development the nematode C . elegans is required to integrate newly differentiated neurons into its neural circuits ( Sulston , 1976; Sulston and Horvitz , 1977; White et al . , 1986 ) and to reshape the connections of existing cells ( White et al . , 1978; Thomas et al . , 1990 ) .\",\n \"At the same time , the animal grows , develops new organs , and undergoes four molts .\",\n \"Maintaining the functionality of essential motor programs such as feeding , locomotion and defecation concurrently with these anatomical and physiological changes imposes constraints on the developing animal; modulation of behavioral patterns may contribute towards satisfying these constraints .\",\n \"In particular , 2–4 hr prior to lethargus , the seam cells exhibit increased synthetic activity and begin to deposit the new cuticle ( Singh and Sulston , 1978; Monsalve et al . , 2011 ) .\",\n \"The timing of quiet wakefulness that we observed coincided with the timing of this activity .\",\n \"Thus , it is possible that this state may assist with maintaining an appropriate energy balance .\",\n \"Our analysis revealed a novel behavioral switch that toggled between the active and quiet wakefulness states prior to L4leth .\",\n \"The distribution of durations of the epochs of active wakefulness supported a model in which this state was actively stabilized and ergodicity was weakly broken ( Stefani et al . , 2009; Burov et al . , 2010 ) .\",\n \"We note that the global locomotion states that we report are distinct from the cGMP-dependent short intervals of continuous roaming or dwelling behavior , previously described in adults ( Fujiwara et al . , 2002 ) .\",\n \"We have shown that upregulating or downregulating Gαs signaling stabilized or destabilized active wakefulness respectively .\",\n \"Downregulated neuropeptide release in unc-31 mutants , a gene encoding the calcium-dependent activator protein for secretion ( CAPS ) required for the priming and docking of DCVs ( Speese et al . , 2007; Lin et al . , 2010 ) , affected behavior similarly to downregulated Gαs signaling .\",\n \"These findings extend the previously reported effects of PKA signaling on locomotion and quiescence ( Schade et al . , 2005; Reynolds et al . , 2005; Charlie et al . , 2006; Raizen et al . , 2008; Perez-Mansilla and Nurrish , 2009; Belfer and Raizen , 2013 ) .\",\n \"Moreover , PKA was shown to catalyze the phosphorylation of tomosyn , a highly-conserved syntaxin-binding protein .\",\n \"Phosphorylation of tomosyn by PKA reduced its interaction with syntaxin and enhanced the formation of the SNARE complex ( Baba et al . , 2005 ) .\",\n \"In C . elegans , tomosyn ( TOM-1 ) was shown to negatively regulate synaptic transmitter release and UNC-31/CAPS-dependent neuropeptide release ( Gracheva et al . , 2007a ) , and KIN-1 alleviated the suppression of both processes by phosphorylating TOM-1 ( J Richmond , personal communications , February 2013 ) .\",\n \"Interestingly , a mutation in the tom-1 gene suppressed behavioral deficits and DCV accumulation in unc-31 mutants ( Gracheva et al . , 2007a ) .\",\n \"Similarly , enhanced PKA activity was shown to be sufficient for bypassing the requirement for UNC-31 for the release of DCVs ( Zhou et al . , 2007; Perez-Mansilla and Nurrish , 2009 ) .\",\n \"Taken together , these findings support a model in which KIN-1 and TOM-1 regulate locomotion .\",\n \"Upregulating Gαs signaling resulted in enhanced active wakefulness outside of lethargus and enhanced directed locomotion during L4leth , mirroring previous findings in Drosophila melanogaster and in C . elegans ( Joiner et al . , 2006; Belfer and Raizen , 2013 ) .\",\n \"These results were also consistent with a previous observation that acute activation of the photoactivated adenylyl cyclase PACα in cholinergic neurons evoked an enhanced activity response ( Weissenberger et al . , 2011 ) .\",\n \"Downregulating Gαs signaling resulted in a clear locomotion phenotype outside of but not during L4leth .\",\n \"However , loss of CREB function resulted in an enhancement of directed locomotion , as well as changes in the dynamics of bouts of motion and quiescence ( Figure 8C , D ) , during lethargus .\",\n \"In addition , crh-1 mutants exhibited strong locomotion defects outside of lethargus .\",\n \"Studies of the effects of cAMP signaling on sleep in D . melanogaster and in mice have demonstrated that CREB , when activated by PKA , promotes the duration of wakefulness ( Hendricks et al . , 2001; Graves et al . , 2003; Shaw and Franken , 2003 ) .\",\n \"The mild loss of function phenotypes of acy-1 ( lf ) and unc-31 during lethargus may be explained by a floor effect , while the implications of the crh-1 phenotype remain to be understood .\",\n \"Interestingly , a recent study reported that adenylyl cyclase activity increases the intensity of nighttime sleep in Drosophila ( van Alphen et al . , 2013 ) .\",\n \"Taken together , our analyses indicated that both PKA-dependent pathways acted in concert to regulate active wakefulness during L4int and the early young adult stages .\",\n \"Finally , the approach to quantifying locomotion presented here provides several distinct advantages .\",\n \"Visible phenotypes were invaluable for uncovering molecular pathways that regulated behavior , but were largely based on crude classifications such as hypo- or hyper-kinesis or a focus on arbitrarily defined features .\",\n \"It was recently shown that the space of shapes adopted by the nematode C . elegans is low dimensional , and that these dimensions ( ‘eigenworms’ ) can provide a quantitative description of behavior ( Stephens et al . , 2008 ) .\",\n \"This exquisitely sensitive analysis revealed the underlying simplicity of complex locomotion dynamics as well as subtle behavioral phenotypes that had been previously undetectable ( Stephens et al . , 2010 , 2011; Brown et al . , 2013 ) .\",\n \"However , this analysis does not provide an intuitive interpretation of the detected phenotypes that may directly relate to the underlying neuronal activity .\",\n \"Tracking of individual body-bends opens the door to a heuristic description of C . elegans locomotion , composed of intuitive building blocks .\",\n \"As in the case of the eigenworms , one underlying assumption of this analysis is that C . elegans does not maintain a ‘mental map’ of its environment , nor does it keep track of its own acceleration in the laboratory frame of reference .\",\n \"Rather , it responds to external and internal cues solely by altering its own body-posture .\",\n \"Specifically , the primary task of the motor neurons during directional motion is to generate body-bends and propagate them along the body in the appropriate direction ( Gray et al . , 2005; Stetina et al . , 2006; Stephens et al . , 2008; Haspel and O’Donovan , 2011; Boyle et al . , 2011; Wen et al . , 2012 ) .\",\n \"It follows that a natural , heuristic model for describing directional locomotion can be constructed using the rates of generation , propagation , and decay of body-bends .\",\n \"Tracking each body-bend from initiation to eventual demise thus provides direct experimental measurements of the basic building blocks of nematode locomotion .\"\n ],\n [\n \"C . elegans strains were maintained and grown according to standard protocols ( Brenner , 1974 ) .\",\n \"The wild-type strain used was C . elegans variety Bristol , strain N2 .\",\n \"The following mutant strains were obtained from the Caenorhabditis Genetics Center: KG518 acy-1 ( ce2 ) III ( gf ) , KG532 kin- 2 ( ce179 ) X , CB169 unc-31 ( e169 ) IV , KP1182 acy- 1 ( nu329 ) III ( lf ) and YT17 crh-1 ( tz2 ) III .\",\n \"Animals were synchronized by restricting the duration of egg-laying ( placing gravid adults on plates for 6 hr , removing the parents , and selecting L4s 3 days later ) , grown at 20°C on standard NGM plates , and fed OP50 bacteria until their mid L4int larva stage .\",\n \"They were then transferred to individual 0 . 8 × 3 . 7 mm ‘artificial dirt’ chambers ( Lockery et al . , 2008 ) .\",\n \"The chambers , containing an overnight OP50 Escherichia coli culture that was concentrated 10-fold and suspended in NGM liquid ( Singh et al . , 2011; Iwanir et al . , 2013 ) , were sealed with a coverslip held in place with VALAP ( a mix of equal parts of vaseline , lanolin and paraffin wax ) at the corners and submerged , facedown , in NGM buffer inside a petri dish to prevent drying .\",\n \"Each observation chamber contained a single animal .\",\n \"Behavior was recorded for 10 hr at a rate of 10 frames per second using a 5 megapixels CCD camera ( Prosilica GC2450 , 2448 × 2050 pixels , Allied Vision Technologies , Stadtroda , Germany ) .\",\n \"We recorded at a magnification of 4 . 2X , resulting in a resolution of 1 . 54 × 1 . 54 μm2/pixel; the body length of a typical L4leth larva was approximately 500 pixels ( 750 μm ) .\",\n \"The contrast at the edges of the animals was maximized , typically by setting background levels to 230–250 on a greyscale of 0–255 , and by focusing slightly away from the middle plane of the pharynx .\",\n \"We developed a suite of tools , called PyCelegans , for image analysis on high performance parallel computing resources .\",\n \"Similar to previously described methods ( Husson et al . , 2013 ) , PyCelegans identifies the body of the animal in each frame and calculates the coordinates of 100 points along its midline , ordered from head to tail .\",\n \"The rate of segmentation failure , where the animals could not be properly identified , was typically 5% of all frames .\",\n \"Frames in which the animal was not identified were not included in the dataset , but their timing was accounted for when time-stamping subsequent frames .\",\n \"Each midline was divided into 20 equal segments and the local angle at each of the inner 18 segments was calculated as shown in Figure 1A .\",\n \"The raw angle dynamics data was smoothed with a Gaussian filter with a width of 5 frames ( 0 . 5 s ) .\",\n \"Body-bends were defined as positions of local spatial maxima or minima of the angles ( Figure 1A ) .\",\n \"The position of each bend was tracked from the time of its initiation until it decayed ( typically at the head or the tail ) or was interrupted by 10 consecutive missing frames .\",\n \"Forward locomotion was defined by the propagation of bends in the anterior-posterior direction that persisted for at least three consecutive midline segments .\",\n \"Backward locomotion was defined analogously .\",\n \"Quiescence of an individual segment was defined as an interval in which the rate of change of the corresponding angle did not exceed a threshold of 0 . 01 radians/sec .\",\n \"Neither missing frames in which the inferred change in angle was below a threshold of 0 . 3 radians , nor motion occurring in isolated single frames , were considered to interrupt a bout of quiescence .\",\n \"At each time point , the whole-animal behavior was classified as forward , backward or dwelling by applying a majority rule to the dynamics of the individual body-bends .\",\n \"Dwelling occurred when the number of bends propagating in both directions was equal ( typically zero ) .\",\n \"Whole-animal quiescence was defined as the state where the most anterior angle ( between the head and neck segments ) and at least 16 of the remaining 17 angles were quiescent .\",\n \"The fraction of time spent in each behavior was calculated with a running-average window of 10 min for Figures 1 and 2 and 4–6 min for Figure 3 .\",\n \"The onset/termination of lethargus was defined as the first/last point of increasing/decreasing whole-animal quiescence fraction that was followed by 20 consecutive minutes of the quiescence fraction remaining above/below a threshold of 5% .\",\n \"Source code , documentation , and a sample dataset are available to download from GitHub ( https://github . com/labello/pycelegans ) .\",\n \"Mutant behavior was compared to wild-type in Figures 7 and 8 using a one-way analysis of variance ( ANOVA ) with Bonferroni adjustments to compensate for multiple comparisons .\",\n \"Exponential and power-law models for the data presented in Figure 3 were compared by calculating Akaike information criteria ( AIC ) ( Burnham and Anderson , 2002; Burnham and Anderson , 2004 ) .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Despite their simplicity , longitudinal studies of invertebrate models are rare .","We thus sought to characterize behavioral trends of Caenorhabditis elegans , from the mid fourth larval stage through the mid young adult stage .","We found that , outside of lethargus , animals exhibited abrupt switching between two distinct behavioral states: active wakefulness and quiet wakefulness .","The durations of epochs of active wakefulness exhibited non-Poisson statistics .","Increased Gαs signaling stabilized the active wakefulness state before , during and after lethargus .","In contrast , decreased Gαs signaling , decreased neuropeptide release , or decreased CREB activity destabilized active wakefulness outside of , but not during , lethargus .","Taken together , our findings support a model in which protein kinase A ( PKA ) stabilizes active wakefulness , at least in part through two of its downstream targets: neuropeptide release and CREB .","However , during lethargus , when active wakefulness is strongly suppressed , the native role of PKA signaling in modulating locomotion and quiescence may be minor ."],"string":"[\n \"Despite their simplicity , longitudinal studies of invertebrate models are rare .\",\n \"We thus sought to characterize behavioral trends of Caenorhabditis elegans , from the mid fourth larval stage through the mid young adult stage .\",\n \"We found that , outside of lethargus , animals exhibited abrupt switching between two distinct behavioral states: active wakefulness and quiet wakefulness .\",\n \"The durations of epochs of active wakefulness exhibited non-Poisson statistics .\",\n \"Increased Gαs signaling stabilized the active wakefulness state before , during and after lethargus .\",\n \"In contrast , decreased Gαs signaling , decreased neuropeptide release , or decreased CREB activity destabilized active wakefulness outside of , but not during , lethargus .\",\n \"Taken together , our findings support a model in which protein kinase A ( PKA ) stabilizes active wakefulness , at least in part through two of its downstream targets: neuropeptide release and CREB .\",\n \"However , during lethargus , when active wakefulness is strongly suppressed , the native role of PKA signaling in modulating locomotion and quiescence may be minor .\"\n]"},"summary":{"kind":"list like","value":["The roundworm C . elegans is a key model organism in neuroscience .","It has a simple nervous system , made up of just 302 neurons , and was the first multicellular organism to have its genome fully sequenced .","The lifecycle of C . elegans begins with an embryonic stage , followed by four larval stages and then adulthood , and worms can progress through this cycle in only three days .","However , relatively little is known about how the behaviour of the worms varies across these distinct developmental phases .","The body wall of C . elegans contains pairs of muscles that extend along its length , and when waves of muscle contraction travel along its body , the worm undergoes a sinusoidal pattern of movement .","A signalling cascade involving a molecule called protein kinase A is thought to help control these movements , and upregulation of this cascade has been shown to increase locomotion .","Now , Nagy et al . have analysed the movement of C . elegans during these different stages of development .","This involved developing an image processing tool that can analyze the position and posture of a worm’s body in each of three million ( or more ) images per day .","Using this tool , which is called PyCelegans , Nagy et al . identified two behavioral macro-states in one of the larval forms of C . elegans: these states , which can persist for hours , are referred to as active wakefulness and quiet wakefulness .","During periods of active wakefulness , the worms spent most ( but not all ) of their time moving forwards; during quiet wakefulness , they remained largely still .","The worms switched abruptly between these two states , and the transition seemed to be regulated by PKA signaling .","By using PyCelegans to compare locomotion in worms with mutations in genes encoding various components of this pathway , Nagy et al . showed that mutants with increased PKA activity spent more time in a state of active wakefulness , while the opposite was true for worms with mutations that reduced PKA activity .","In addition to providing new insights into the control of locomotion in C . elegans , this study has provided a new open-source PyCelegans suite of tools , which are available to be extended and adapted by other researchers for new uses ."],"string":"[\n \"The roundworm C . elegans is a key model organism in neuroscience .\",\n \"It has a simple nervous system , made up of just 302 neurons , and was the first multicellular organism to have its genome fully sequenced .\",\n \"The lifecycle of C . elegans begins with an embryonic stage , followed by four larval stages and then adulthood , and worms can progress through this cycle in only three days .\",\n \"However , relatively little is known about how the behaviour of the worms varies across these distinct developmental phases .\",\n \"The body wall of C . elegans contains pairs of muscles that extend along its length , and when waves of muscle contraction travel along its body , the worm undergoes a sinusoidal pattern of movement .\",\n \"A signalling cascade involving a molecule called protein kinase A is thought to help control these movements , and upregulation of this cascade has been shown to increase locomotion .\",\n \"Now , Nagy et al . have analysed the movement of C . elegans during these different stages of development .\",\n \"This involved developing an image processing tool that can analyze the position and posture of a worm’s body in each of three million ( or more ) images per day .\",\n \"Using this tool , which is called PyCelegans , Nagy et al . identified two behavioral macro-states in one of the larval forms of C . elegans: these states , which can persist for hours , are referred to as active wakefulness and quiet wakefulness .\",\n \"During periods of active wakefulness , the worms spent most ( but not all ) of their time moving forwards; during quiet wakefulness , they remained largely still .\",\n \"The worms switched abruptly between these two states , and the transition seemed to be regulated by PKA signaling .\",\n \"By using PyCelegans to compare locomotion in worms with mutations in genes encoding various components of this pathway , Nagy et al . showed that mutants with increased PKA activity spent more time in a state of active wakefulness , while the opposite was true for worms with mutations that reduced PKA activity .\",\n \"In addition to providing new insights into the control of locomotion in C . elegans , this study has provided a new open-source PyCelegans suite of tools , which are available to be extended and adapted by other researchers for new uses .\"\n]"},"year":{"kind":"string","value":"2013"}}},{"rowIdx":4295,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["cell biology"],"string":"[\n \"cell biology\"\n]"},"title":{"kind":"string","value":"SWELL1 regulates skeletal muscle cell size, intracellular signaling, adiposity and glucose metabolism"},"id":{"kind":"string","value":"elife-58941-v3"},"sections":{"kind":"list like","value":[["Maintenance of skeletal muscle mass and function is associated with improved metabolic health and is thought to protect against obesity and obesity-related diseases such as diabetes , nonalcoholic fatty liver disease , heart disease and osteoarthritis , and correlates with overall health in the aging population .","Skeletal muscle atrophy , the loss of skeletal muscle mass , is associated with cancer ( cachexia ) , heart failure , chronic corticosteroid use , paralysis or denervation ( disuse atrophy ) , chronic positive pressure ventilation ( diaphragm atrophy , inability to extubate ) , prolonged space flight ( Fitts et al . , 2001 ) or bed rest ( unloading ) and aging ( Schiaffino et al . , 2013 ) .","Each of these clinical scenarios also contribute to poor metabolic health and increase frailty .","Thus , a deeper understanding of the molecular mechanisms that regulate skeletal muscle maintenance , growth and function has important implications for human health .","Skeletal muscle growth is regulated by a multitude of stimuli , including growth factor signaling , cytokines , mechanical load , integrin signaling and hormones ( Schiaffino et al . , 2013 ) .","Activation of AKT-mTOR signaling downstream of insulin and IGF-1 is well established as a critical regulator of skeletal muscle differentiation and growth ( Schiaffino et al . , 2013 ) .","However , mechanical loading of muscle , as occurs with regular activity , exercise , and resistance training also induces mTOR-mediated skeletal muscle growth ( Ben-Sahra and Manning , 2017; Hornberger et al . , 2006; Yoon , 2017 ) , possibly via a growth-factor independent mechanosensory mechanism .","β1-integrin and focal adhesion kinase ( FAK ) signaling has been proposed as this mechanosensory system - sensing mechanical load on skeletal muscle and regulating hypertrophic signaling ( Carson and Wei , 2000; Schlaepfer et al . , 1999; Flück et al . , 1999; Klossner et al . , 2009 ) .","However , mechanosensitive ion channels , or ion channel complexes , may also regulate intracellular signaling , including PIEZO1 , TRPV4 and the recently identified LRRC8 channel complex that functionally encodes the volume-regulated anion current ( VRAC ) ( Syeda et al . , 2016; Voss et al . , 2014; Qiu et al . , 2014 ) .","Lrrc8a ( leucine-rich repeat containing protein 8A , also known as SWELL1 ) encodes a ~ 95 kDa membrane protein with four transmembrane domains and an intracellular , C-terminal leucine-rich repeat domain ( LRRD ) ( Osei-Owusu et al . , 2018 ) .","It was first described , in 2003 , as the site of a translocation mutation in a young woman with an immunodeficiency characterized by agammaglobulinemia and absent B-cells ( Sawada et al . , 2003; Kubota et al . , 2004 ) .","This phenotype led to studies linking LRRC8A to lymphocyte differentiation defects arising from impaired LRRC8A dependent GRB2-mediated PI3K-AKT signaling , in part based on data from a global Lrrc8a knock-out ( KO ) mouse ( Kumar et al . , 2014 ) .","Although LRRC8A was speculated in 2012 to form a hetero-hexameric ion channel complex with other LRRC8 family members ( Abascal and Zardoya , 2012 ) , it was not until 2014 that LRRC8A was experimentally identified as an essential component of the volume-regulated anion channel ( VRAC ) ( Voss et al . , 2014; Qiu et al . , 2014 ) , forming hetero-hexamers with LRRC8B-E ( Syeda et al . , 2016; Voss et al . , 2014 ) .","So , for about a decade , LRRC8A was considered a membrane protein that regulates PI3K-AKT-mediated lymphocyte function ( Sawada et al . , 2003; Kubota et al . , 2004 ) , putatively via non-ion channel , protein-protein interaction mediated signaling , and only later discovered to also form the long-studied VRAC ion channel signaling complex .","Indeed , since its first description >30 years ago ( Cahalan and Lewis , 1988; Hazama and Okada , 1988; Pedersen et al . , 2015 ) , VRAC has been associated with a multitude of complex physiological and pathophysiological functions , including cell proliferation , cell migration , angiogenesis , cell death and apoptosis ( Pedersen et al . , 2016; Eggermont et al . , 2001 ) ; however , the molecular mechanisms underlying these diverse functions had remained elusive without knowledge of the molecular identity of this ion channel complex .","We recently identified LRRC8A as a swell or stretch-activated volume sensor in adipocytes that regulates glucose uptake , lipid content , and adipocyte growth via a LRRC8A-GRB2-PI3K-AKT signaling pathway – providing a putative feed-forward amplifier to enhance adipocyte growth and insulin signaling during caloric excess ( Zhang et al . , 2017; Gunasekar et al . , 2019 ) .","Intriguingly , others have shown that mechanical stimuli applied by pulling on β1-integrins on cardiac muscle cells with magnetic beads activates a VRAC-like current , suggesting a putative connection between β1-integrin/focal adhesion kinase signaling and LRRC8A ( Browe and Baumgarten , 2003; Browe and Baumgarten , 2006 ) .","Taken together , these findings suggest that LRRC8A may connect β1-integrin-mediated mechano-transduction with insulin/IGF1-PI3K-AKT-mTOR signaling , which , in skeletal muscle is anticipated to regulate skeletal muscle differentiation , function and potentially also adiposity and systemic glucose metabolism ( Brüning et al . , 1998; Moller et al . , 1996; Kim et al . , 2000 ) .","In this study , we tested this hypothesis by examining LRRC8A dependent intracellular signaling and myotube differentiation in both C2C12 and primary skeletal muscle cells in vitro , and by performing skeletal muscle targeted LRRC8A loss-of-function experiments in vivo .","We find that myotube differentiation and insulin and stretch-mediated PI3K-AKT , ERK1/2 , mTOR signaling is strongly regulated by LRRC8A protein expression , and provide evidence that GRB2 signaling mediates these LRRC8A dependent effects .","Finally , using skeletal muscle Lrrc8a knock-out mice , we reveal the requirement of skeletal muscle LRRC8A for maintaining normal skeletal muscle cell size , muscle endurance , force generation , adiposity and glucose tolerance , under basal conditions and in the setting of overnutrition ."],["LRRC8A is the essential component of a hexameric ion channel signaling complex that encodes ICl , SWELL , or the volume-regulated anion current ( VRAC ) ( Voss et al . , 2014; Qiu et al . , 2014 ) .","While the LRRC8A complex has been shown to regulate cellular volume in response to application of non-physiological hypotonic extracellular solutions , the physiological function ( s ) of this ubiquitously expressed ion channel signaling complex remain unknown .","To determine the function of the LRRC8A channel complex in skeletal muscle , we genetically deleted Lrrc8a from C2C12 mouse myoblasts using CRISPR/cas9-mediated gene editing as described previously ( Zhang et al . , 2017; Kang et al . , 2018 ) , and from primary skeletal muscle cells isolated from Lrrc8a fl/fl mice transduced with adenoviral Cre-mCherry ( KO ) or mCherry alone ( WT control ) ( Zhang et al . , 2017 ) .","LRRC8A protein western blots confirmed robust Lrrc8a ablation in both Lrrc8a KO C2C212 myotubes and Lrrc8a KO primary skeletal myotubes ( Figure 1A ) .","Next , whole-cell patch clamp revealed that the hypotonically activated ( 210 mOsm ) outwardly rectifying current present in WT C2C12 myoblasts is abolished in Lrrc8a KO C2C12 myoblasts ( Figure 1B ) , confirming LRRC8A as also required for ICl , SWELL or VRAC in skeletal muscle myoblasts .","Remarkably , Lrrc8a ablation is associated with impaired myotube formation in both C2C12 myoblasts and in primary skeletal satellite cells ( Figure 1C ) , with an 58% and 45% reduction in myotube area in C2C12 and skeletal muscle myotubes , respectively , compared to WT .","As an alternative metric , myoblast fusion is also markedly reduced by 80% in Lrrc8a KO C2C12 compared to WT , as assessed by myotube fusion index ( number of nuclei inside myotubes/total number of nuclei; Figure 1C ) .","In order to further characterize the observed LRRC8A-dependent impairment in myotube formation in C2C12 and primary muscle cells , we performed genome-wide RNA sequencing ( RNA-seq ) of Lrrc8a KO C2C12 relative to control WT C2C12 myotubes .","These transcriptomic data revealed clear differences in the global transcriptional profile between WT and Lrrc8a KO C2C12 myotubes ( Figure 1D and Supplementary file 1 ) , with marked suppression of numerous skeletal muscle differentiation genes including Mef2a ( 0 . 2-fold ) , Myl2 ( 0 . 008-fold ) , Myl3 ( 0 . 01-fold ) , Myl4 ( 0 . 008-fold ) , Actc1 ( 0 . 005-fold ) , Tnnc2 ( 0 . 005-fold ) , Igf2 ( 0 . 01-fold ) ( Figure 1E ) .","Curiously , this suppression of myogenic differentiation is associated with marked induction of Ppargc1α ( PGC1α; 14-fold ) and Pparγ ( 3 . 7-fold ) .","PGC1α and Pparγ are positive regulators of skeletal muscle differentiation ( Haralampieva et al . , 2017; Ruas et al . , 2012; Singh et al . , 2007 ) , suggesting that the LRRC8A-dependent defect in skeletal muscle differentiation lies downstream of PGC1α and PPARγ .","To further define putative pathway dysregulation underlying disruptions in myogenesis observed in Lrrc8a KO C2C12 myotubes , we next performed pathway analysis on the transcriptome data .","We found that numerous signaling pathways essential for myogenic differentiation are disrupted , including insulin ( 2 × 10−3 ) , MAP kinase ( 5 × 10−4 ) , PI3K-AKT ( 1 × 10−4 ) , AMPK ( 6 × 10−5 ) , integrin ( 3 × 10−6 ) , mTOR ( 2 × 10−6 ) , integrin linked kinase ( 4 × 10−7 ) and IL-8 ( 1 × 10−7 ) signaling pathways ( Figure 1F and Supplementary file 2 ) .","Guided by the results of the pathway analysis , and the fact that skeletal myogenesis and maturation is regulated by insulin-PI3K-AKT-mTOR-MAPK ( Conejo et al . , 2001; Schiaffino et al . , 2013 ) , we directly examined a number of insulin-stimulated pathways in WT and Lrrc8a KO C2C12 myotubes , including insulin-stimulated AKT2-AS160 , FOXO1 and AMPK signaling .","Indeed , insulin-stimulated pAKT2 , pAS160 , pFOXO1 and pAMPK are abrogated in Lrrc8a KO myotubes compared to WT C2C12 myotubes ( Figure 2A and C ) .","Importantly , insulin-AKT-AS160 signaling is also diminished in Lrrc8a KO primary skeletal muscle myotubes compared to WT primary myotubes ( Figure 2B and D ) , consistent with the observed differentiation block ( Figure 1C ) .","This confirms that LRRC8A-dependent insulin-AKT and downstream signaling is not a feature specific to immortalized C2C12 myotubes but is also conserved in primary skeletal myotubes .","It is also notable that reduction in total AKT2 protein is associated with Lrrc8a ablation in both C2C12 and primary skeletal muscle cells , and this is consistent with threefold reduction in AKT2 mRNA expression observed in RNA sequencing data ( Figure 2E ) .","Moreover , transcription of a number of critical insulin signaling and glucose homeostatic genes are suppressed by Lrrc8a ablation , including GLUT4 ( Slc2a4 , 51-fold ) , FOXO3 ( 2-fold ) , FOXO4 ( 2 . 8-fold ) and FOXO6 ( 18-fold ) ( Figure 2E ) .","Indeed , FOXO signaling is thought to integrate insulin signaling with glucose metabolism ( Lee and Dong , 2017; Gross et al . , 2008 ) in a number of insulin sensitive tissues .","Collectively , these data indicate that impaired LRRC8A-dependent insulin-AKT-AS160-FOXO signaling is associated with the observed defect in myogenic differentiation upon Lrrc8a ablation in cultured skeletal myotubes , and also predict putative impairments in skeletal muscle glucose metabolism and oxidative metabolism .","We next asked if these LRRC8A-dependent effects on downstream skeletal myotube insulin signaling are due to impaired myotube differentiation , and associated impairments in insulin signaling , or if LRRC8A also regulates these signaling pathways in differentiated skeletal myotubes .","To test this , we first fully differentiated C2C12 myotubes , and then knocked down ( KD ) LRRC8A using Ad-shLRRC8A-mCherry post-differentiation and compared myotube size and insulin-stimulated signaling to Ad-shSCR-mCherry treated C2C12 myotubes ( Figure 2—figure supplement 1 ) .","Similar to Lrrc8a ablation prior to differentiation , shRNA-mediated LRRC8A KD of fully differentiated C2C12 myotubes mildly reduced myotube size ( Figure 2—figure supplement 1A ) , and significantly reduced insulin-stimulated pAKT2 , pAKT1 , pAS160 , pAMPK and pS6 ribosomal proteins ( Figure 2—figure supplement 1B and C ) , although in some cases , these differences were less marked than in C2C12 Lrrc8a KO myoblasts that were subsequently differentiated ( Figure 2 ) .","Also , the reductions in total AKT2 observed at both the mRNA ( Figure 2E , and Supplementary file","1 ) and protein levels ( Figure 2A ) in differentiated Lrrc8a KO C2C12 myoblasts are also observed upon LRRC8A KD in differentiated C2C12 myotubes ( Figure 2—figure supplement 1B and C ) , suggesting that total AKT2 expression is regulated by LRRC8A in fully differentiated myotubes .","As a complementary approach , we confirmed these findings in fully differentiated , primary skeletal myotubes isolated from Lrrc8afl/fl mice upon adenoviral transduction of Ad-CMV-Cre-mCherry ( KO ) compared to Ad-CMV-mCherry ( WT ) , and noted similar reductions in myotube size ( Figure 2—figure supplement 1D ) , and in basal levels of pAS160 , pAKT2 , AKT2 and pAKT1 signaling ( Figure 2—figure supplement 1E and F ) .","Taken together , these data indicate that impaired insulin signaling observed in LRRC8A-depleted myotubes is in part due to impaired myotube differentiation with secondary effects on insulin signaling , and in part due to a direct contribution of LRRC8A to intracellular signaling in fully differentiated myotubes .","To further validate LRRC8A-mediated effects on muscle differentiation and signaling , we re-expressed LRRC8A in Lrrc8a KO C2C12 myoblasts ( LRRC8A O/E ) and then examined myotube differentiation and basal activity of multiple intracellular signaling pathways by western blot , including pAKT1 , pAKT2 , pAS160 , p-p70S6K , pS6K and pERK1/2 as compared to WT and Lrrc8a KO C2C12 myotubes .","LRRC8A O/E to 2 . 12-fold WT levels fully rescues myotube development in Lrrc8a KO myotubes ( Figure 3A ) , as quantified by restoration of Lrrc8a KO myotube area to levels above WT ( Figure 3B ) .","This rescue of Lrrc8a KO myotube development upon LRRC8A O/E ( Figure 3A&B ) is associated with either restored ( pAS160 , AKT2 , pAKT1 , AKT1 , p70S6K ) or supra-normal ( pAKT2 , p-p70S6K , pS6K , pERK1/2 ) signaling ( Figure 3C&D ) compared to WT C2C12 myotubes .","These data demonstrate that LRRC8A protein expression level strongly regulates skeletal muscle insulin signaling and myogenic differentiation .","In a cellular context , there are numerous reports that VRAC and the LRRC8A complex that functionally encodes it is mechano-responsive ( Browe and Baumgarten , 2003; Browe and Baumgarten , 2006; Osei-Owusu et al . , 2018; Barakat et al . , 1999; Nakao et al . , 1999; Nilius and Droogmans , 2001; Romanenko et al . , 2002; Strange et al . , 2019 ) .","It is well established that mechanical stretch is an important regulator of skeletal muscle proliferation , differentiation and skeletal muscle hypertrophy and may be mediated by PI3K-AKT-MAPK signaling ( Schiaffino et al . , 2013; Ma et al . , 2017; Fu et al . , 2018 ) and integrin signaling pathways ( Carson and Wei , 2000; Schlaepfer et al . , 1999; Flück et al . , 1999; Klossner et al . , 2009 ) .","To determine if LRRC8A is also required for stretch-mediated AKT and MAP kinase signaling in skeletal myotubes , we subjected WT and Lrrc8a KO C2C12 myotubes to 0% or 5% equiaxial stretch using the FlexCell stretch system .","Mechanical stretch ( 5% ) is sufficient to stimulate PI3K-AKT2/AKT1-pAS160-MAPK ( ERK1/2 ) signaling in WT C2C12 in a LRRC8A-dependent manner ( Figure 4A and B ) .","These data position LRRC8A as a co-regulator of both insulin and stretch-mediated PI3K-AKT- pAS160-MAPK signaling .","It has been reported earlier in both lymphocyte and adipocytes that the LRRC8A complex interacts with Growth factor Receptor-Bound 2 ( GRB2 ) and regulates PI3K-AKT signaling ( Kumar et al . , 2014; Zhang et al . , 2017; Gunasekar et al . , 2019 ) , whereby GRB2 binds with IRS1/2 and negatively regulates insulin signaling ( Shan et al . , 2013 ) .","Indeed , GRB2 knock-down augments insulin-PI3K-MAPK signaling and induces myogenesis and myogenic differentiation genes ( Shan et al . , 2013; Liu et al . , 2009; Mitra and Thanabalu , 2017 ) .","To determine if LRRC8A and GRB2 interact in C2C12 myotubes , we overexpressed C-terminal 3XFlag tagged LRRC8A in C2C12 cells followed by immunoprecipitation ( IP ) with Flag antibody .","We observed significant GRB2 enrichment upon Flag IP from lysates of LRRC8A-3xFlag expressing C2C12 myotubes , consistent with a GRB2-LRRC8A interaction ( Figure 5A ) .","Based on the notion that LRRC8A titrates GRB2-mediated suppression of AKT/MAPK signaling , and that Lrrc8a ablation results in unrestrained GRB2-mediated AKT/MAPK inhibition ( Gunasekar et al . , 2019 ) , we next tested if GRB2 knock-down ( KD ) may rescue myogenic differentiation in Lrrc8a KO C2C12 myotubes .","shRNA-mediated GRB2 KD in Lrrc8a KO C2C12 myoblasts ( LRRC8A KO/shGRB2; Figure 5B ) stimulates myotube formation ( Figure 5C ) and increases myotube area ( Figure 5D ) , to levels equivalent to WT/shSCR ( Figure 5C and D ) .","Similarly , GRB2 KD in Lrrc8a KO C2C12 myotubes induces myogenic differentiation markers IGF1 , MyoHCl , MyoHClla and MyoHCIIb relative to both LRRC8A KO/shSCR and WT/shSCR ( Figure 5E and F ) .","These data are consistent with GRB2 suppression rescuing myotube differentiation in Lrrc8a KO C2C12 , and supports a model in which LRRC8A regulates myogenic differentiation by titrating GRB2-mediated signaling .","To examine the physiological consequences of Lrrc8a ablation in vivo , we generated skeletal muscle-specific Lrrc8a KO mice using Cre-LoxP technology by crossing Myf5-Cre mice with Lrrc8afl/fl mice ( Skm KO; Figure 6A ) , and confirmed robust skeletal muscle LRRC8A depletion in Skm KO gastrocnemius muscle , 12 . 3-fold lower than WT controls ( Figure 6B ) .","Remarkably , in contrast to the severe impairments in skeletal myogenesis observed in both Lrrc8a KO C2C12 and primary skeletal myotubes in vitro ( Figures 1 , 3 and 5 ) , Skm KO mice develop skeletal muscle mass comparable to WT littermates , based on Echo/MRI body composition ( Figure 6C ) and gross muscle weights ( Figure 6D ) , and are born at normal mendelian ratios ( Supplementary file 3 ) .","However , histological examination reveals a 27% reduction in skeletal myocyte cross-sectional area in Skm KO as compared to WT mice ( Figure 6E ) , suggesting a requirement for LRRC8A in skeletal muscle cell size regulation in vivo .","This is potentially due to reductions in myotube fusion , as observed in C2C12 and primary skeletal muscle cells in vitro ( Figure 1 ) , but occurring to a lesser degree in vivo .","These data indicate that the profound impairments in myogenesis observed in vitro may reflect a very early requirement for LRRC8A signaling in skeletal muscle development ( prior to LRRC8A protein elimination by Myf5-Cre-mediated LRRC8A recombination ) , or other fundamental differences in myogenic differentiation processes in vitro versus in vivo .","Since insulin signaling is an important regulator of skeletal muscle oxidative capacity and endurance ( Affourtit , 2016 ) , we next examined exercise tolerance on treadmill testing in Lrrc8afl/fl ( WT ) compared to Skm KO .","Skm KO mice exhibit a 14% reduced exercise capacity , compared to age and gender matched WT controls ( Figure 7A; Figure 7—Video 1 ) .","Hang-times on inversion testing are also reduced 29% in Skm KO compared to controls , further supporting reduced skeletal muscle endurance upon skeletal muscle LRRC8A depletion in vivo ( Figure 7B ) .","To determine if these reductions in muscle function in vivo are due to muscle-specific functional impairments , we next performed ex vivo experiments in which we isolated the soleus muscle from mice and performed twitch/train testing .","We observed that peak developed tetanic tension is 15% reduced in Skm KO soleus muscle compared to WT controls ( Figure 7C ) , suggesting a skeletal muscle autonomous mechanism , with no difference in time to fatigability ( TTF , Figure 7D ) or time to 50% decay ( Figure 7E ) .","To determine whether these LRRC8A dependent differences in muscle endurance and force were due to impaired oxidative capacity , we next measured oxygen consumption rate ( OCR ) and extracellular acidification rate ( ECAR ) in WT and Lrrc8a KO primary skeletal muscle cells , under basal and insulin-stimulated conditions ( Figure 7F ) .","Oxygen consumption of Lrrc8a KO primary myotubes are 26% lower than WT and , in contrast to WT cells , are unresponsive to insulin-stimulation ( Figure 7F ) , consistent with abrogation of insulin-AKT/ERK1/2 signaling upon skeletal muscle LRRC8A depletion .","These relative changes persist in the presence of Complex V and III inhibitors , Oligomycin and Antimycin A ( Figure 7F and G ) , suggesting that insulin-stimulated glycolytic pathways are primarily dysregulated upon LRRC8A depletion .","In contrast , FCCP , which maximally uncouples mitochondria , abolishes differences in oxygen consumption between WT and Lrrc8a KO primary muscle cells , suggesting no differences in functional mitochondrial content in Lrrc8a KO muscle .","Consistent with this finding , we observe no differences in muscle fiber-type based on Myosin Heavy Chain ( MHC ) Type 1 , MHC Type IIa , MHC Type IIx and MHC Type IIb in both superficial and deep tibialis anterior muscle in Skm KO as compared to WT mice ( Figure 7—figure supplement 1 ) , indicating the reductions in muscle force and endurance in Skm KO mice is not due to muscle fiber-type switching .","Indeed , it is increasingly appreciated that changes in muscle metabolic potential and morphology may occur independent from adaptive fiber-type switching as measured by a change in MHC expression ( Egan and Zierath , 2013 ) .","To examine an alternative mechanism , such as glycolysis , we measured extracellular acidification rate ( ECAR ) in WT and Lrrc8a KO primary myotubes .","Insulin-stimulated ECAR increases are abolished in Lrrc8a KO compared to WT cells , and these differences persist independent of electron transport chain modulators ( Figure 7H ) .","These data suggest that LRRC8A regulation of skeletal muscle cellular oxygen consumption occurs at the level of glucose metabolism - potentially via LRRC8A-dependent insulin-PI3K-AKT-AS160-GLUT4 signaling , glucose uptake and utilization .","These findings in primary skeletal muscle cells are supported by marked transcriptional suppression of numerous glycolytic genes: Aldoa , Eno3 , Pfkm , and Pgam2; and glucose and glycogen metabolism genes: Phka1 , Phka2 , Ppp1r3c and Gys1 , upon Lrrc8a ablation in C2C12 myotubes ( Figure 7—figure supplement 2 ) .","Guided by evidence of impaired insulin-PI3K-AKT-AS160-GLUT4 signaling observed in Lrrc8a KO C2C12 and primary myotubes , we next examined systemic glucose homeostasis and insulin sensitivity in WT and Skm KO mice by measuring glucose and insulin tolerance .","On a regular chow diet , there are no differences in either glucose tolerance or insulin tolerance ( Figure 8A ) between WT and Skm KO mice .","However , over the course of 16–24 weeks on chow diet Skm KO mice develop 29% increased adiposity based on body composition measurements ( Figure 8B ) compared to WT , with no significant difference in lean mass ( Figure 8C ) or in total body mass ( Figure 8C ) .","When Skm KO mice are raised on a high-fat-diet ( HFD ) for 16 weeks , there is no difference in adiposity observed ( Figure 8—figure supplement","1 ) compared to WT mice , but glucose tolerance is impaired ( Figure 8D ) and there is mild insulin resistance in HFD Skm KO mice as compared to WT ( Figure 8E ) .","Since Myf5 is also expressed in brown fat ( Seale et al . , 2008 ) , it is possible that these metabolic phenotypes arise from LRRC8A-mediated effects in brown fat and consequent changes in systemic metabolism .","To rule out this possibility , we repeated a subset of the above experiments in a skeletal muscle-targeted KO mouse generated by crossing the Myl1-Cre and Lrrc8afl/fl mice ( Myl1Cre/Lrrc8afl/fl ) , since Myl1-Cre is restricted to mature skeletal muscle ( Figure 8—figure supplement 2B ) , and excludes brown fat ( Bothe et al . , 2000 ) .","Similar to Myf5Cre/Lrrc8afl/fl mice , Myl1Cre/Lrrc8afl/fl mice fed a regular chow diet , have normal glucose tolerance ( Figure 8—figure supplement 2C ) , but exhibit 24% reduced exercise capacity on treadmill testing , as compared to WT ( Figure 8—figure supplement 2D ) .","Also , Myl1Cre/Lrrc8afl/fl mice develop increased visceral adiposity over time on regular chow , based on 24% increased epididymal adipose mass normalized to body mass ( Figure 8—figure supplement 2E ) , with no differences in inguinal adipose tissue , muscle mass ( Figure 8—figure supplement 2F ) , or total body mass ( Figure 8—figure supplement 2G ) .","These data suggest that impaired skeletal muscle glucose uptake in Skm KO ( Myl1Cre/Lrrc8afl/fl and Myf5Cre/Lrrc8afl/fl ) mice are compensated for by increased adipose glucose uptake and de novo lipogenesis , which contribute to preserved glucose tolerance , at the expense of increased adiposity in skeletal muscle-targeted Lrrc8a KO mice raised on a regular chow diet .","However , overnutrition-induced obesity , and the associated impairments in adipose and hepatic glucose disposal may uncover glucose intolerance and insulin resistance in skeletal muscle-targeted Lrrc8a KO mice ."],["Our data reveal that the LRRC8A channel complex regulates insulin/stretch-mediated AKT-AS160-GLUT4 , MAP kinase and mTOR signaling in differentiated myoblast cultures , with consequent effects on myogenic differentiation , insulin-stimulated glucose metabolism and oxygen consumption .","In vivo , skeletal muscle-targeted Lrrc8a KO mice have smaller skeletal muscle cells , impaired muscle endurance , and force generation , and are predisposed to adiposity , glucose intolerance and insulin resistance .","Insulin/stretch-mediated PI3K-AKT , mTOR signaling are well known to be important regulators of myogenic differentiation ( Rotwein and Wilson , 2009; Héron-Milhavet et al . , 2008 ) , metabolism and muscle function ( Schiaffino et al . , 2013 ) suggesting impaired LRRC8A-AKT-mTOR signaling may underlie the defect in myogenic differentiation .","Indeed , consistent with our previous findings and proposed model in adipocytes , in which LRRC8A mediates the interaction of GRB2 with IRS1 to regulate insulin-AKT signaling ( Zhang et al . , 2017; Gunasekar et al . , 2019 ) , LRRC8A also associates with GRB2 in skeletal myotubes , and GRB2 knock-down rescues impaired myogenic differentiation in Lrrc8a KO muscle cells .","Thus , our working model for LRRC8A-mediated regulation of insuln-PI3K-AKT and downstream signaling in adipocytes ( Gunasekar et al . , 2019 ) appears to be conserved in skeletal myotubes .","The in vitro phenotype that we observe in CRISPR/cas9-mediated Lrrc8a KO C2C12 myotubes and in Lrrc8a KO primary myotubes is consistent with the observation of Chen et al . , 2019 that used siRNA-mediated LRRC8A knock-down to demonstrate that the LRRC8A channel complex is required for myogenic differentiation .","However , the ability of both GRB2 KD and LRRC8A O/E to rescue myogenic differentiation and augment insulin-AKT , MAP kinase and mTOR signaling in Lrrc8a KO myotubes implicates non-canonical , non-conductive signaling mechanisms .","Based on our work and also previous studies ( Voss et al . , 2014; Qiu et al . , 2014 ) , LRRC8A O/E does not increase ICl , SWELL/VRAC to supranormal levels at the plasma membrane .","However , pAKT , pERK1/2 and mTOR levels are augmented by twofold to threefold above endogenous levels , upon twofold LRRC8A O/E in C2C12 myotubes .","These data suggest that alternative/non-canonical signaling mechanisms underlie LRRC8A signaling , as opposed to canonical/conductive signaling mechanisms .","Another finding that warrants further study is the requirement for LRRC8A in stretch-induced AKT and MAP kinase signaling in C2C12 myotubes upon static stretching .","Mechanical stretch is known to regulate myoblast proliferation and differentiation and myofiber hypertrophy via PI3K-AKT-MAPK signaling ( Schiaffino et al . , 2013; Ma et al . , 2017; Fu et al . , 2018 ) .","Also , there are numerous reports that VRAC , and presumably LRRC8A , is mechano-responsive ( Browe and Baumgarten , 2003; Browe and Baumgarten , 2006; Osei-Owusu et al . , 2018; Barakat et al . , 1999; Nakao et al . , 1999; Nilius and Droogmans , 2001; Romanenko et al . , 2002; Strange et al . , 2019 ) .","Therefore , it may not be surprising that LRRC8A complexes co-regulate both insulin and stretch-mediated PI3K-AKT , ERK1/2 signaling in skeletal myotubes - potentially integrating mechanical and hormonal stimuli to tune downstream signaling .","Indeed , the concept of mechano-tuning insulin signaling has been proposed and demonstrated in other cell systems ( Chen and Chalfie , 2014; Kim et al . , 2018 ) which implicate integrin signaling ( Kim et al . , 2018 ) as the mechano-sensory mechanism .","Curiously , it has been reported that VRAC can be activated in cardiac muscle cells by applying mechanical tension to β1-integrins ( Browe and Baumgarten , 2003; Browe and Baumgarten , 2006 ) , supporting the notion that , in striated muscle , integrin-LRRC8A may indeed participate in mechano-tuning insulin-AKT and downstream signaling .","Further studies to more fully delineate the putative molecular mechanisms are necessary .","It is also notable that the profound myogenic differentiation block observed upon Lrrc8a ablation in both C2C12 myotubes and primary myotubes in vitro is significantly milder in vivo , where only a 30% reduction in skeletal myocyte cross-sectional area is observed , with no change in total muscle mass , or lean content , in Skm KO mice .","This discordance in phenotype may reflect fundamental differences in the biology of skeletal muscle differentiation in vitro versus the in vivo milieu .","Alternatively , it may be that , although early , the time interval between Myf5-Cre expression early in myogenesis , and ultimate reductions in LRRC8A protein ( potentially ~3 days ) may extend beyond the critical period during which LRRC8A is required for myogenic differentiation .","Directly testing this hypothesis would require examining mice expressing Cre-recombinase at the precursor stage , in skeletal muscle satellite cells , such as Pax3 or Pax7 promoters ( Relaix et al . , 2006; Buckingham et al . , 2006 ) - these experiments are currently underway .","Although overall muscle development is grossly intact in both LRRC8A skeletal muscle KO ( Myl1Cre/Lrrc8afl/fl and Myf5Cre/Lrrc8afl/fl ) mice , there is a consistent reduction in exercise capacity , muscle endurance and force generation , and a propensity for increased adiposity over time compared to age and gender matched controls .","The impaired exercise capacity observed in skeletal muscle Lrrc8a KO mice are consistent with some level of insulin resistance , as in db/db mice ( Ostler et al . , 2014 ) and in humans ( Reusch et al . , 2013 ) , and may be due to impaired skeletal muscle glycolysis and oxygen consumption in LRRC8A-depleted skeletal muscle .","Furthermore , the increased gonadal adiposity , with preserved glucose and insulin tolerance , observed in LRRC8A skeletal muscle KO ( Myl1Cre/Lrrc8afl/fl and Myf5Cre/Lrrc8afl/fl ) mice phenocopy both skeletal muscle-specific insulin receptor KO mice ( MIRKO ) ( Brüning et al . , 1998 ) and transgenic mice expressing a skeletal muscle dominant-negative insulin receptor mutant ( Moller et al . , 1996 ) , wherein skeletal muscle-specific insulin resistance is compensated for by re-distribution of glucose from skeletal muscle to adipose tissue , to promote adiposity ( Kim et al . , 2000 ) .","In the case of LRRC8A skeletal muscle KO mice , overnutrition and HFD feeding unmasks this underlying mild insulin resistance and glucose intolerance , as adipose-tissue insulin resistance also begins to set in .","Recent findings from skeletal muscle specific AKT1/AKT2 double KO mice indicate that these effects may not attributable to solely to muscle AKT signaling ( Jaiswal et al . , 2019 ) , but potentially involve other insulin-sensitive signaling pathways .","In summary , we show that LRRC8A regulates myogenic differentiation and insulin-PI3K-AKT-AS160 , ERK1/2 , and mTOR signaling in myotubes via GRB2-mediated signaling .","In vivo , LRRC8A is required for maintaining normal exercise capacity , muscle endurance , adiposity under basal conditions , and systemic glycemia in the setting of overnutrition .","These findings contribute further to our understanding of LRRC8A channel complexes in the regulation of systemic metabolism ."],["The Institutional Animal Care and Use Committee of the Washington University in St . Louis and the University of Iowa approved all experimental procedures involving animals .","All the mice were housed in temperature , humidity , and light-controlled room and allowed free access to water and food .","Male and female Lrrc8a fl/fl ( WT ) , Myl1Cre/Lrrc8a fl/fl , Myf5Cre/Lrrc8a fl/fl ( skeletal muscle targeted Lrrc8a KO ) , were generated and used in these studies .","Myl1Cre ( JAX# 24713 ) and Myf5Cre ( JAX# 007893 ) mice were purchased from Jackson labs .","For high-fat diet ( HFD ) studies , we used Research Diets Inc ( Cat # D12492 ) ( 60 kcal% fat ) regimen starting at 14 weeks of age .","Lrrc8afl/fl mice were generated as previously described ( Zhang et al . , 2017 ) .","Briefly , Lrrc8a intronic sequences were obtained from Ensembl Transcript ID ENSMUST00000139454 .","All CRISPR/Cas9 sites were identified using ZiFit Targeter Version 4 . 2 ( http://zifit . partners . org/ZiFiT/ ) .","Pairs of oligonucleotides corresponding to the chosen CRISPR-Cas9 target sites were designed , synthesized , annealed , and cloned into the pX330-U6-Chimeric_BB-CBh-hSpCas9 construct ( Addgene plasmid # 42230 ) , following the protocol detailed in Cong et al . , 2013 .","CRISPR-Cas9 reagents and ssODNs were injected into the pronuclei of F1 mixed C57/129 mouse strain embryos at an injection solution concentration of 5 ng/μl and 75–100 ng/μl , respectively .","Correctly targeted mice were screened by PCR across the predicted loxP insertion sites on either side of Exon","3 . These mice were then backcrossed >8 generations into a C57BL/6 background .","Rabbit polyclonal anti-LRRC8A antibody was generated against the epitope QRTKSRIEQGIVDRSE ( Pacific Antibodies ) .","All other primary antibodies were purchased from Cells Signaling: anti-β-actin ( #8457 s ) , p-AKT1 ( #9018 s ) , Akt1 ( #2938 s ) , pAKT2 ( #8599 s ) , Akt2 ( #3063 s ) , p-AS160 ( #4288 s ) , AS160 ( #2670 s ) , AMPKα ( #5831 s ) , pAMPKα ( #2535 s ) , FoxO1 ( #2880 s ) and pFoxO1 ( #9464 s ) , p70 S6 Kinase ( #9202 s ) , p-p70 S6 Kinase ( #9205 s ) , pS6 Ribosomal ( #5364 s ) , GAPDH ( #5174 s ) , pErk1/2 ( #9101 s ) , Total Erk1/2 ( #9102 s ) .","Purified mouse anti-Grb2 was purchased from BD ( 610111 s ) .","Purified anti-flag mouse antibody was purchased from sigma .","Rabbit IgG Santa Cruz ( sc-2027 ) .","All primary antibodies were used at 1:1000 dilution , except for anti-flag at 1:2000 dilution .","All secondary antibody ( anti-rabbit-HRP and anti-mouse-HRP ) were used at 1:10 , 000 dilution .","Adenovirus type five with Ad5-CMV-mCherry ( 1 × 1010 PFU/ml ) , Ad5-CMV-Cre-mCherry ( 3 × 1010 PFU/ml ) were obtained from the University of Iowa viral vector core facility .","Adenovirus type five with Ad5-CMVmCherry-U6-hLRRC8A-shRNA , 2 . 2 × 1010 PFU/ml , ( AD3535 ) , ( Vector Biolabs ) .","Scrambled non-targeting control ( shSCR: Ad5-U6-scramble-mCherry , 1 × 1010 PFU/ml ) , ( Vector biolabs #3086 ) .","Ad5-CAG-LoxP-stop-LoxP-3XFlag- LRRC8A ( 1 × 1010 PFU/ml ) were obtained from Vector Biolabs .","Ad5-U6-shGRB2-GFP ( 1 × 109 PFU/ml ) and Ad5-U6-shSCR-GFP ( 1 × 1010 PFU/ml ) were obtained from Vector Biolabs .","All recordings were performed in the whole-cell configuration at room temperature , as previously described ( Zhang et al . , 2017; Kang et al . , 2018 ) .","Briefly , currents were measured with either an Axopatch 200B amplifier or a MultiClamp 700B amplifier ( Molecular Devices ) paired to a Digidata 1550 digitizer , using pClamp 10 . 4 software .","The intracellular solution contained ( in mM ) : 120 L-aspartic acid , 20 CsCl , 1 MgCl2 , 5 EGTA , 10 HEPES , 5 MgATP , 120 CsOH , 0 . 1 GTP , pH 7 . 2 with CsOH .","The extracellular solution for hypotonic stimulation contained ( in mM ) : 90 NaCl , 2 CsCl , 1 MgCl2 , 1 CaCl2 , 10 HEPES , five glucose , five mannitol , pH 7 . 4 with NaOH ( 210 mOsm/kg ) .","The isotonic extracellular solution contained the same composition as above except for mannitol concentration of 105 ( 300 mOsm/kg ) .","The osmolarity was checked by a vapor pressure osmometer 5500 ( Wescor ) .","Currents were filtered at 10 kHz and sampled at 100 μs interval .","The patch pipettes were pulled from borosilicate glass capillary tubes ( WPI ) using a P-87 micropipette puller ( Sutter Instruments ) .","The pipette resistance was ~4–6 MΩ when the patch pipette was filled with intracellular solution .","The holding potential was 0 mV .","Voltage ramps from −100 to +100 mV ( at 0 . 4 mV/ms ) were applied every 4 s .","Satellite cell isolation and differentiation were performed as described previously with minor modifications ( Hindi et al . , 2017 ) .","Briefly , gastrocnemius and quadriceps muscles were excised from Lrrc8aflfl mice ( 8–10 weeks old ) and washed twice with 1XPBS supplemented with 1% penicillin-streptomycin and fungizone ( 300 µl/100 ml ) .","Muscle tissue was incubated in DMEM-F12 media supplemented with collagenase II ( 2 mg/ml ) , 1% penicillin-streptomycin and fungizone ( 300 ul/100 ml ) and incubated at shaker for 90 min at 37°C .","Tissue was washed with 1X PBS and incubated again with DMEM-F12 media supplemented with collagenase II ( 1 mg/ml ) , dispase ( 0 . 5 mg/ml ) , 1% penicillin-streptomycin and fungizone ( 300 µl/100 ml ) in a shaker for 30 min at 37°C .","Subsequently , the tissue was minced and passed through a cell strainer ( 70 µm ) , and after centrifugation; satellite cells were plated on BD Matrigel‐coated dishes .","Cells were stimulated to differentiate into myoblasts in DMEM‐F12 , 20% fetal bovine serum ( FBS ) , 40 ng/ml basic fibroblast growth factor ( bfgf , R and D Systems , 233‐FB/CF ) , 1X non‐essential amino acids , 0 . 14 mM β‐mercaptoethanol , 1X penicillin/streptomycin , and Fungizone .","Myoblasts were maintained with 10 ng/ml bfgf and then differentiated in DMEM‐F12 , 2% FBS , 1X insulin–transferrin–selenium , when 80% confluency was reached .","WT C2C12 and Lrrc8a KO C2C12 cell line were cultured at 37°C , 5% CO2 Dulbecco’s modified Eagle’s medium ( DMEM; GIBCO ) supplemented with 10% fetal bovine serum ( FBS; Atlanta Bio selected ) and antibiotics 1% penicillin-streptomycin ( Gibco , USA ) .","Cells were grown to 80% confluency and then transferred to differentiation media DMEM supplemented with antibiotics and 2% horse serum ( HS; GIBCO ) to induce differentiation .","The differentiation media was changed every two days .","Cells were allowed to differentiate into myotubes for up to 6 days .","Subsequently , myotube images were taken for quantification of myotube surface area and fusion index .","After differentiation ( Day 7 ) , cells were imaged with Olympus IX73 microscope ( 10X objective , Olympus , Japan ) .","For each experimental condition , 5–6 bright field images were captured randomly from six-well plate .","Myotube surface area was quantified manually with ImageJ software .","The morphometric quantification was carried out by an independent observer who was blinded to the experimental conditions .","For fusion index , differentiated myotube growing on coverslip were washed with 1X PBS and fixed with 2% PFA .","After washing with 1XPBS three times , cells were permeabilized with 0 . 1% TritonX100 for 5 min at room temperature and subsequently blocking was done with 5% goat serum for 30 min .","Cells were stained with DAPI ( 1 µM ) for 15 min and after washing with 1X PBS , coverslip were mounted on slides with ProLong Diamond anti-fading agent .","Cells were imaged with Olympus IX73 microscope ( 10X objective , Olympus , Japan ) with bright field and DAPI filter .","Fusion index ( number of nuclei incorporated within the myotube/total number of nuclei present in that view field ) were analyzed by ImageJ .","RNA quality was assessed by Agilent BioAnalyzer 2100 by the University of Iowa Institute of Human Genetics , Genomics Division .","RNA integrity numbers greater than eight were accepted for RNAseq library preparation .","RNA libraries of 150 bp PolyA-enriched RNA were generated , and sequencing was performed on a HiSeq 4000 genome sequencing platform ( Illumina ) .","Sequencing results were uploaded and analyzed with BaseSpace ( Illumina ) .","Sequences were trimmed to 125 bp using FASTQ Toolkit ( Version 2 . 2 . 0 ) and aligned to Mus musculus mmp10 genome using RNA-Seq Alignment ( Version 1 . 1 . 0 ) .","Transcripts were assembled and differential gene expression was determined using Cufflinks Assembly and DE ( Version 2 . 1 . 0 ) .","Ingenuity Pathway Analysis ( QIAGEN ) was used to analyze significantly regulated genes which were filtered using cutoffs of >1 . 5 fragments per kilobase per million reads , >1 . 5 fold changes in gene expression , and a false discovery rate of <0 . 05 .","Heatmaps were generated to visualize significantly regulated genes .","For insulin stimulation , differentiated C2C12 myotubes were incubated in serum free media for 6 hr and stimulated with 0 and 10 nM insulin for 15 min; while differentiated primary myotubes were incubated in serum free media for 4 hr and stimulated with 0 and 10 nM insulin for 2 hr .","To examine intracellular signaling upon LRRC8A overexpression ( LRRC8A O/E ) , we overexpressed LRRC8A-3xFlag by transducing C2C12 myotubes with Ad5-CAG-LoxP-stop-LoxP-LRRC8A-3xFlag ( MOI 50–60 ) and Ad5-CMV-Cre-mCherry ( MOI 50–60 ) and polybrene ( 4 µg/ml ) in DMEM ( 2% FBS and 1% penicillin-streptomycin ) for 36 hr .","Ad5-CMV-Cre-mCherry alone with polybrene ( 4 µg/ml ) ( MOI 50–60 ) was transduced in WT C2C12 or Lrrc8a KO C2C12 as controls .","Viral transduction efficiency ( 60–70% ) was confirmed by mCherry fluorescence .","Cells were allowed to differentiate further in differentiation media up to 6 days .","Myotube images were taken before collecting lysates for further signaling studies .","GRB2 knock-down was achieved by transducing myotubes with Ad5-U6- shSCR-GFP ( Control , MOI 50–60 ) or Ad5-U6- sh LRRC8A-GFP ( GRB2 KD , MOI 50–60 ) in DMEM ( 2% FBS and 1% penicillin-streptomycin ) supplemented with polybrene ( 4 µg/ml ) for 24 hr .","Cells were allowed to differentiate further in differentiation media up to 6 days .","Differentiated myotube images were taken for myotube surface area quantification before collecting the cells for RNA isolation .","WT C2C12 cells were cultured in differentiation media for 6 days to form myotubes .","Subsequently myotubes were transduced either with Ad5-U6-scramble-mCherry ( sh-SCR ) or Ad5-mCherry-U6-shLRRC8A ( sh LRRC8A ) shRNA ( KD ) ( MOI 50–60 ) for 2 days and grown for 1 additional day prior to analysis .","Myotubes were then incubated in serum free media for 6 hr and stimulated with 0 and 10 nM insulin for 15 min .","Primary skeletal muscle cells isolated from Lrrc8a fl/fl mice were grown in differentiation media for 3 . 5 days .","To generate Lrrc8a KO primary myotubes , cells were transduced with either Ad5-CMV-CMV-mCherry ( WT ) or Ad5-CMV-Cre-mCherry ( KO ) ( MOI 50–60 ) for 2 days and then grown for 1 additional day prior to analysis .","Myotubes were then harvested to measure intracellular signaling under basal culture conditions .","C2C12 myotubes were plated in each well of a six-well BioFlex culture plate .","Cells were allowed to differentiate up to 6 days in differentiation media , and then placed into a Flexcell Jr .","Tension System ( FX-6000T ) and incubated at 37°C with 5% CO2 .","C2C12 myotubes on flexible membrane were subjected to either no tension or to static stretch of 5% for 15 min .","Cells were lysed and protein isolated for subsequent western blots .","Cells were washed with ice cold 1X PBS and lysed in ice-cold lysis buffer ( 150 mM NaCl , 20 mM HEPES , 1% NP-40 , 5 mM EDTA , pH 7 . 5 ) with added proteinase/phosphatase inhibitor ( Roche ) .","The cell lysate was further sonicated ( 20% pulse frequency for 20 s ) and centrifuged at 14 , 000 rpm for 20 min at 4°C .","The supernatant was collected and estimated for protein concentration using DC protein assay kit ( Bio-Rad ) .","For immunoblotting , an appropriate volume of 4 x Laemmli ( Bio-rad ) sample loading buffer was added to the sample ( 10–20 μg of protein ) , then heated at 90°C for 5 min before loading onto 4–20% gel ( Bio-Rad ) .","Proteins were separated using running buffer ( Bio-Rad ) for 2 hr at 110 V . Proteins were transferred to PVDF membrane ( Bio-Rad ) and membrane blocked in 5% ( w/v ) BSA or 5% ( w/v ) milk in TBST buffer ( 0 . 2 M Tris , 1 . 37 M NaCl , 0 . 2% Tween-20 , pH 7 . 4 ) at room temperature for 1 hr .","Blots were incubated with primary antibodies at 4°C overnight , followed by secondary antibody ( Bio-Rad , Goat-anti-mouse #170–5047 , Goat-anti-rabbit #170–6515 , all used at 1:10 , 000 ) at room temperature for one hour .","Membranes were washed three times and imaged by chemiluminescence ( Pierce ) by using a Chemidoc imaging system ( BioRad ) .","The images were further analyzed for band intensities using ImageJ software .","β-Actin or GAPDH levels were quantified for equal protein loading .","C2C12 myotubes were plated on 10 cm dishes in complete media and grown to 80% confluency .","For LRRC8A-3xFlag overexpression , Ad5-CAG-LoxP-stop-LoxP-3XFlag- LRRC8A ( MOI 50–60 ) and Ad5-CMV-Cre-mCherry ( MOI 50–60 ) along with polybrene ( 4 ug/ml ) were added to cells in DMEM media ( 2% FBS and 1% penicillin-streptomycin ) allowed to grow for 36 hr .","Cells were then switched to differentiation media for up to 6 days .","After that myotubes were harvested in ice-cold lysis buffer ( 150 mM NaCL , 20 mM HEPES , 1% NP-40 , 5 mM EDTA , pH 7 . 5 ) with added protease/phosphatase inhibitor ( Roche ) and kept on ice with gentle agitation for 15 min to allow complete lysis .","Lysated were incubated with anti-Flag antibody ( Sigma #F3165 ) or control rabbit IgG ( Santa Cruz sc-2027 ) rotating end over end overnight at 4°C .","Protein G sepharose beads ( GE ) were added for 4 hr and then samples were centrifuged at 10 , 000 g for 3 min and washed three times with RIPA buffer and re-suspended in laemmli buffer ( Bio-Rad ) , boiled for 5 min , separated by SDS-PAGE gel followed by the western blot protocol .","Differentiated cells were solubilized in TRIzol and the total RNA was isolated using PureLink RNA kit ( Life Technologies ) and column DNase digestion kit ( Life Technologies ) .","The cDNA synthesis , qRT-PCR reaction and quantification were carried out as described previously ( Zhang et al . , 2017 ) .","All experiments were performed in triplicate and GAPDH were used as internal standard to normalize the data .","All primers used for qRT-PCR are listed in Supplementary file","4 . Mice were euthanized and gastrocnemius muscle excised and washed with 1X PBS .","Muscles tissue were minced with surgical blade and kept in 8 vol of ice cold homogenization buffer ( 20 mM Tris , 137 mM NaCl , 2 . 7 mM KCl , 1 mM MgCl2 , 1% Triton X-100 , 10% ( w/v ) glycerol , 1 mM EDTA , 1 mM dithiothreitol , pH 7 . 8 ) supplemented with protease/phosphatase inhibitor ( Roche ) .","Tissues were homogenized on ice with a Dounce homogenizer ( 40–50 passes ) and incubated for overnight at 4°C with continuous rotation .","Tissue lysate was further sonicated in 20 s cycle intervals for 2–3 times and centrifuged at 14 , 000 rpm for 20 min at 4°C .","The supernatant was collected for protein concentration estimation using DC protein assay kit ( Bio-Rad ) .","Due to the high content of contractile protein in this preparation , coomassie gel staining was performed to demonstrate equal protein loading , and for quantification normalization of Western blots .","Mice were anesthetized with isoflurane followed by cervical dislocation .","Tibialis anterior ( TA ) muscle was carefully excised and gently immersed into the tissue-tek O . C . T medium placed on wooden cork .","Orientation of the tissue maintained while embedding in the medium .","Subsequently , wooden cork with tissue gently immersed into the liquid N2 pre-chilled isopentane bath for 10–14 s and store at −80°C .","Tissue sectioning ( 10 µm ) were done with Leica cryostat and all sections collected on positively charged microscope slide for H and E staining as described earlier ( Bonetto et al . , 2015 ) .","Briefly , TA sectioned slides were stained for 2 min in hematoxylin , 1 min in eosin and then dehydrated with ethanol and xylenes .","Subsequently , slides were mounted with coverslip and image were taken with EVOS cell imaging microscope ( 10X objective ) .","For quantification of fiber cross-sectional area , images were processed using ImageJ software to enhance contrast and smooth/sharpen cell boundaries and clearly demarcate muscle fiber cross sectional area .","All measurements were performed with an independent observer who was blinded to the identity of the slides .","Skeletal muscle fiber-type was quantified in WT and Skm KO tibialis anterior ( TA ) muscle as previously described ( Biltz et al . , 2020 ) .","Briefly , sections were immunostained against myosin heavy chain isoforms ( type I , type IIa , and type IIb BA‐F8 , SC‐71 and BF‐F3 , respectively , Developmental Studies Hybridoma Bank , Iowa City , IA ) and against laminin ( ab11575; Abcam , Cambridge , UK ) to quantify fiber types and areas .","Fiber types were identified on four non‐overlapping 20 × images in controlled locations: two from the superficial region and two from the deep .","Unstained fibers were assumed to be of type IIx .","Fiber areas were detected with a custom ImageJ macro using the automated Huang threshold and particle analyzer .","Regions of interest with a circularity of less than 0 . 5 were excluded as being out of the axial plane of that fiber .","Mouse treadmill exercise protocols were adapted from Dougherty et al . , 2016 .","Briefly , mice were first acclimated with the motorized treadmill ( Columbus Instruments Exer3/6 Treadmill Columbus , OH ) for 3 days by running 10–15 min ( with 3 min interval ) for three consecutive days at 7 m/min , with the electric shocking grid ( frequency 1 Hz ) installed in each lane .","During the treadmill testing , mice ran with a gradual increase in speed ( 5 . 5 m/minute to 22 m/minute ) and inclination ( 0o-15o ) at time intervals of 3 min each .","The total running distance for each mouse was recorded at the end of the experiment .","The predefined criteria for removing the mouse from the treadmill and recording the distance travelled was: continuous shock for 5 s or receiving 5–6 shocks within a time interval of 15 s .","These mice were promptly removed from the treadmill and total duration and distance were recorded for further analysis .","Mouse inversion test was performed using a wire-grid screen apparatus elevated to 50 cm .","Mice were stabilized on the screen inclined at 60o , with the mouse head facing towards the base of the screen .","The screen was slowly pivoted to 0o ( horizontal ) , such that the mouse was fully inverted and hanging upside down from the screen .","Soft bedding was placed underneath the screen to protect mouse from any injury , were they to fall .","The inversion test for each mouse was repeated two times with an interval of 45 min ( resting period ) .","The hang time for each mouse was repeated three times with an interval of 5 min .","The maximum hanging time limit for each mouse was set for 3 min .","Soleus muscle was carefully dissected and transferred to a specialized muscle stimulation system ( 1500A , Aurora Scientific , Aurora , ON , Canada ) where physiology tests were run in a blinded fashion .","Muscle was immersed in a Ringer solution ( in mM ) ( NaCl 137 , KCI 5 , CaCl2 2 , NaH2PO4 1 , NaHCO3 24 , MgSO4 1 , glucose 11 and curare 0 . 015 ) maintained at 37°C .","The distal tendon was secured with silk suture to the arm of a dual mode ergometer ( 300C-LR , Aurora Scientific , Aurora , ON , Canada ) and the proximal tendon secured to a stationary post .","Muscles were stimulated with an electrical stimulator ( 701C , Aurora Scientific , Aurora , ON , Canada ) using parallel platinum plate electrodes extending along the muscle .","Muscle slack length was set by increasing muscle length until passive force was detectable above the noise of the transducer and fiber length was measured through a micrometer reticule in the eyepiece of a dissecting microscope .","Optimal muscle length was then determined by incrementally increasing the length of the muscle by 10% of slack fiber length until the isometric tetanic force plateaued .","At this optimum length , force was recorded during a twitch contraction and isometric tetanic contraction ( 300 ms train of 0 . 3 ms pulses at 225 Hz ) .","The muscle was then fatigued with a bout of repeated tetanic contractions every 10 s until force dropped below 50% of peak .","At this point , the muscle was cut from the sutures and weighed .","This weight , along with peak fiber length and muscle density ( 1 . 056 g/cm3 ) , was used to calculate the physiological cross-sectional area ( PCSA ) and convert to specific force ( tension ) .","The experimental data were analyzed and quantified using Matlab ( Mathworks ) , and presented as peak tetanic tension ( Tetanic Tension ) – peak of the force recording during the tetanic contraction , normalized to PCSA; Time to fatigue ( TTF ) – time for the tetanic tension to fall below 50% of the peak value during the fatigue test; Half relaxation time ( HRT ) – half the time between force peak and return to baseline during the twitch contraction .","Cellular respiration was quantified in primary myotubes using the XF24 extracellular flux ( XF ) bioanalyzer ( Agilent Technologies/Seahorse Bioscience , North Billerica , MA , USA ) .","Primary skeletal muscle cells isolated from Lrrc8a fl/fl mice were plated on BD Matrigel‐coated plate at a density of 20 × 103 per well .","After 24 hr , cells were incubated in Ad5-CMV-mCherry or Ad5-CMV-Cre-mCherry ( MOI 90–100 ) in DMEM-F12 media ( 2% FBS and 1% penicillin-streptomycin ) for 24 hr .","Cells were then switched to differentiation media for another 3 days .","For insulin-stimulation , cells were incubated in serum free media for 4 hr and stimulated with 0 and 10 nM insulin for 2 hr .","Subsequently , medium was changed to XF‐DMEM , and kept in a non‐CO2 incubator for 60 min .","The basal oxygen consumption rate ( OCR ) was measured in XF‐DMEM .","Subsequently , oxygen consumption was measured after addition of each of the following compounds: oligomycin ( 1 μg/ml ) ( ATP-Linked OCR ) , carbonyl cyanide 4‐ ( trifluoromethoxy ) phenylhydrazone ( FCCP; 1 μM ) ( Maximal Capacity OCR ) and antimycin A ( 10 μM; Spare Capacity OCR ) ( Wende et al . , 2015 ) .","For the glycolysis stress test , prior to experimentation , cells were switched to glucose‐free XF‐DMEM and kept in a non‐CO2 incubator for 60 min .","Extracellular acidification rate ( ECAR ) was determined in XF‐DMEM followed by these additional conditions: glucose ( 10 mM ) , oligomycin ( 1 μM ) , and 2‐DG ( 100 mM ) .","Data for Seahorse experiments ( normalized to protein ) reflect the results of one Seahorse run/condition with six replicates .","Mouse body composition ( fat and lean mass ) was measured by nuclear magnetic resonance ( NMR; Echo-MRI 3-in-1 analyzer , EchoMRI , LLC ) .","For glucose tolerance test ( GTT ) , mice were fasted for 6 hr and intraperitoneal injection of glucose ( 1 g/kg body weight for lean mice and 0 . 75 g/kg of body weight for HFD mice ) administered .","Glucose level was monitored from tail-tip blood using a glucometer ( Bayer Healthcare LLC ) at the indicated times .","For insulin tolerance test ( ITT ) , mice were fasted for 4 hr and after an intra-peritoneal injection of insulin ( HumulinR , 1 U/kg for lean mice and 1 . 25 U/kg for HFD mice ) glucose level was measured by glucometer at the indicated times .","Data are represented as mean ± s . e . m . Two-tail paired or unpaired Student’s t-tests were used for comparison between two groups .","For three or more groups , data were analyzed by one-way analysis of variance and Tukey’s post hoc test .","For GTTs and ITTs , two-way analysis of variance ( Anova ) was used .","A p-value<0 . 05 was considered statistically significant .","* , ** and *** represents a p-value less than 0 . 05 , 0 . 01 and 0 . 001 respectively ."]],"string":"[\n [\n \"Maintenance of skeletal muscle mass and function is associated with improved metabolic health and is thought to protect against obesity and obesity-related diseases such as diabetes , nonalcoholic fatty liver disease , heart disease and osteoarthritis , and correlates with overall health in the aging population .\",\n \"Skeletal muscle atrophy , the loss of skeletal muscle mass , is associated with cancer ( cachexia ) , heart failure , chronic corticosteroid use , paralysis or denervation ( disuse atrophy ) , chronic positive pressure ventilation ( diaphragm atrophy , inability to extubate ) , prolonged space flight ( Fitts et al . , 2001 ) or bed rest ( unloading ) and aging ( Schiaffino et al . , 2013 ) .\",\n \"Each of these clinical scenarios also contribute to poor metabolic health and increase frailty .\",\n \"Thus , a deeper understanding of the molecular mechanisms that regulate skeletal muscle maintenance , growth and function has important implications for human health .\",\n \"Skeletal muscle growth is regulated by a multitude of stimuli , including growth factor signaling , cytokines , mechanical load , integrin signaling and hormones ( Schiaffino et al . , 2013 ) .\",\n \"Activation of AKT-mTOR signaling downstream of insulin and IGF-1 is well established as a critical regulator of skeletal muscle differentiation and growth ( Schiaffino et al . , 2013 ) .\",\n \"However , mechanical loading of muscle , as occurs with regular activity , exercise , and resistance training also induces mTOR-mediated skeletal muscle growth ( Ben-Sahra and Manning , 2017; Hornberger et al . , 2006; Yoon , 2017 ) , possibly via a growth-factor independent mechanosensory mechanism .\",\n \"β1-integrin and focal adhesion kinase ( FAK ) signaling has been proposed as this mechanosensory system - sensing mechanical load on skeletal muscle and regulating hypertrophic signaling ( Carson and Wei , 2000; Schlaepfer et al . , 1999; Flück et al . , 1999; Klossner et al . , 2009 ) .\",\n \"However , mechanosensitive ion channels , or ion channel complexes , may also regulate intracellular signaling , including PIEZO1 , TRPV4 and the recently identified LRRC8 channel complex that functionally encodes the volume-regulated anion current ( VRAC ) ( Syeda et al . , 2016; Voss et al . , 2014; Qiu et al . , 2014 ) .\",\n \"Lrrc8a ( leucine-rich repeat containing protein 8A , also known as SWELL1 ) encodes a ~ 95 kDa membrane protein with four transmembrane domains and an intracellular , C-terminal leucine-rich repeat domain ( LRRD ) ( Osei-Owusu et al . , 2018 ) .\",\n \"It was first described , in 2003 , as the site of a translocation mutation in a young woman with an immunodeficiency characterized by agammaglobulinemia and absent B-cells ( Sawada et al . , 2003; Kubota et al . , 2004 ) .\",\n \"This phenotype led to studies linking LRRC8A to lymphocyte differentiation defects arising from impaired LRRC8A dependent GRB2-mediated PI3K-AKT signaling , in part based on data from a global Lrrc8a knock-out ( KO ) mouse ( Kumar et al . , 2014 ) .\",\n \"Although LRRC8A was speculated in 2012 to form a hetero-hexameric ion channel complex with other LRRC8 family members ( Abascal and Zardoya , 2012 ) , it was not until 2014 that LRRC8A was experimentally identified as an essential component of the volume-regulated anion channel ( VRAC ) ( Voss et al . , 2014; Qiu et al . , 2014 ) , forming hetero-hexamers with LRRC8B-E ( Syeda et al . , 2016; Voss et al . , 2014 ) .\",\n \"So , for about a decade , LRRC8A was considered a membrane protein that regulates PI3K-AKT-mediated lymphocyte function ( Sawada et al . , 2003; Kubota et al . , 2004 ) , putatively via non-ion channel , protein-protein interaction mediated signaling , and only later discovered to also form the long-studied VRAC ion channel signaling complex .\",\n \"Indeed , since its first description >30 years ago ( Cahalan and Lewis , 1988; Hazama and Okada , 1988; Pedersen et al . , 2015 ) , VRAC has been associated with a multitude of complex physiological and pathophysiological functions , including cell proliferation , cell migration , angiogenesis , cell death and apoptosis ( Pedersen et al . , 2016; Eggermont et al . , 2001 ) ; however , the molecular mechanisms underlying these diverse functions had remained elusive without knowledge of the molecular identity of this ion channel complex .\",\n \"We recently identified LRRC8A as a swell or stretch-activated volume sensor in adipocytes that regulates glucose uptake , lipid content , and adipocyte growth via a LRRC8A-GRB2-PI3K-AKT signaling pathway – providing a putative feed-forward amplifier to enhance adipocyte growth and insulin signaling during caloric excess ( Zhang et al . , 2017; Gunasekar et al . , 2019 ) .\",\n \"Intriguingly , others have shown that mechanical stimuli applied by pulling on β1-integrins on cardiac muscle cells with magnetic beads activates a VRAC-like current , suggesting a putative connection between β1-integrin/focal adhesion kinase signaling and LRRC8A ( Browe and Baumgarten , 2003; Browe and Baumgarten , 2006 ) .\",\n \"Taken together , these findings suggest that LRRC8A may connect β1-integrin-mediated mechano-transduction with insulin/IGF1-PI3K-AKT-mTOR signaling , which , in skeletal muscle is anticipated to regulate skeletal muscle differentiation , function and potentially also adiposity and systemic glucose metabolism ( Brüning et al . , 1998; Moller et al . , 1996; Kim et al . , 2000 ) .\",\n \"In this study , we tested this hypothesis by examining LRRC8A dependent intracellular signaling and myotube differentiation in both C2C12 and primary skeletal muscle cells in vitro , and by performing skeletal muscle targeted LRRC8A loss-of-function experiments in vivo .\",\n \"We find that myotube differentiation and insulin and stretch-mediated PI3K-AKT , ERK1/2 , mTOR signaling is strongly regulated by LRRC8A protein expression , and provide evidence that GRB2 signaling mediates these LRRC8A dependent effects .\",\n \"Finally , using skeletal muscle Lrrc8a knock-out mice , we reveal the requirement of skeletal muscle LRRC8A for maintaining normal skeletal muscle cell size , muscle endurance , force generation , adiposity and glucose tolerance , under basal conditions and in the setting of overnutrition .\"\n ],\n [\n \"LRRC8A is the essential component of a hexameric ion channel signaling complex that encodes ICl , SWELL , or the volume-regulated anion current ( VRAC ) ( Voss et al . , 2014; Qiu et al . , 2014 ) .\",\n \"While the LRRC8A complex has been shown to regulate cellular volume in response to application of non-physiological hypotonic extracellular solutions , the physiological function ( s ) of this ubiquitously expressed ion channel signaling complex remain unknown .\",\n \"To determine the function of the LRRC8A channel complex in skeletal muscle , we genetically deleted Lrrc8a from C2C12 mouse myoblasts using CRISPR/cas9-mediated gene editing as described previously ( Zhang et al . , 2017; Kang et al . , 2018 ) , and from primary skeletal muscle cells isolated from Lrrc8a fl/fl mice transduced with adenoviral Cre-mCherry ( KO ) or mCherry alone ( WT control ) ( Zhang et al . , 2017 ) .\",\n \"LRRC8A protein western blots confirmed robust Lrrc8a ablation in both Lrrc8a KO C2C212 myotubes and Lrrc8a KO primary skeletal myotubes ( Figure 1A ) .\",\n \"Next , whole-cell patch clamp revealed that the hypotonically activated ( 210 mOsm ) outwardly rectifying current present in WT C2C12 myoblasts is abolished in Lrrc8a KO C2C12 myoblasts ( Figure 1B ) , confirming LRRC8A as also required for ICl , SWELL or VRAC in skeletal muscle myoblasts .\",\n \"Remarkably , Lrrc8a ablation is associated with impaired myotube formation in both C2C12 myoblasts and in primary skeletal satellite cells ( Figure 1C ) , with an 58% and 45% reduction in myotube area in C2C12 and skeletal muscle myotubes , respectively , compared to WT .\",\n \"As an alternative metric , myoblast fusion is also markedly reduced by 80% in Lrrc8a KO C2C12 compared to WT , as assessed by myotube fusion index ( number of nuclei inside myotubes/total number of nuclei; Figure 1C ) .\",\n \"In order to further characterize the observed LRRC8A-dependent impairment in myotube formation in C2C12 and primary muscle cells , we performed genome-wide RNA sequencing ( RNA-seq ) of Lrrc8a KO C2C12 relative to control WT C2C12 myotubes .\",\n \"These transcriptomic data revealed clear differences in the global transcriptional profile between WT and Lrrc8a KO C2C12 myotubes ( Figure 1D and Supplementary file 1 ) , with marked suppression of numerous skeletal muscle differentiation genes including Mef2a ( 0 . 2-fold ) , Myl2 ( 0 . 008-fold ) , Myl3 ( 0 . 01-fold ) , Myl4 ( 0 . 008-fold ) , Actc1 ( 0 . 005-fold ) , Tnnc2 ( 0 . 005-fold ) , Igf2 ( 0 . 01-fold ) ( Figure 1E ) .\",\n \"Curiously , this suppression of myogenic differentiation is associated with marked induction of Ppargc1α ( PGC1α; 14-fold ) and Pparγ ( 3 . 7-fold ) .\",\n \"PGC1α and Pparγ are positive regulators of skeletal muscle differentiation ( Haralampieva et al . , 2017; Ruas et al . , 2012; Singh et al . , 2007 ) , suggesting that the LRRC8A-dependent defect in skeletal muscle differentiation lies downstream of PGC1α and PPARγ .\",\n \"To further define putative pathway dysregulation underlying disruptions in myogenesis observed in Lrrc8a KO C2C12 myotubes , we next performed pathway analysis on the transcriptome data .\",\n \"We found that numerous signaling pathways essential for myogenic differentiation are disrupted , including insulin ( 2 × 10−3 ) , MAP kinase ( 5 × 10−4 ) , PI3K-AKT ( 1 × 10−4 ) , AMPK ( 6 × 10−5 ) , integrin ( 3 × 10−6 ) , mTOR ( 2 × 10−6 ) , integrin linked kinase ( 4 × 10−7 ) and IL-8 ( 1 × 10−7 ) signaling pathways ( Figure 1F and Supplementary file 2 ) .\",\n \"Guided by the results of the pathway analysis , and the fact that skeletal myogenesis and maturation is regulated by insulin-PI3K-AKT-mTOR-MAPK ( Conejo et al . , 2001; Schiaffino et al . , 2013 ) , we directly examined a number of insulin-stimulated pathways in WT and Lrrc8a KO C2C12 myotubes , including insulin-stimulated AKT2-AS160 , FOXO1 and AMPK signaling .\",\n \"Indeed , insulin-stimulated pAKT2 , pAS160 , pFOXO1 and pAMPK are abrogated in Lrrc8a KO myotubes compared to WT C2C12 myotubes ( Figure 2A and C ) .\",\n \"Importantly , insulin-AKT-AS160 signaling is also diminished in Lrrc8a KO primary skeletal muscle myotubes compared to WT primary myotubes ( Figure 2B and D ) , consistent with the observed differentiation block ( Figure 1C ) .\",\n \"This confirms that LRRC8A-dependent insulin-AKT and downstream signaling is not a feature specific to immortalized C2C12 myotubes but is also conserved in primary skeletal myotubes .\",\n \"It is also notable that reduction in total AKT2 protein is associated with Lrrc8a ablation in both C2C12 and primary skeletal muscle cells , and this is consistent with threefold reduction in AKT2 mRNA expression observed in RNA sequencing data ( Figure 2E ) .\",\n \"Moreover , transcription of a number of critical insulin signaling and glucose homeostatic genes are suppressed by Lrrc8a ablation , including GLUT4 ( Slc2a4 , 51-fold ) , FOXO3 ( 2-fold ) , FOXO4 ( 2 . 8-fold ) and FOXO6 ( 18-fold ) ( Figure 2E ) .\",\n \"Indeed , FOXO signaling is thought to integrate insulin signaling with glucose metabolism ( Lee and Dong , 2017; Gross et al . , 2008 ) in a number of insulin sensitive tissues .\",\n \"Collectively , these data indicate that impaired LRRC8A-dependent insulin-AKT-AS160-FOXO signaling is associated with the observed defect in myogenic differentiation upon Lrrc8a ablation in cultured skeletal myotubes , and also predict putative impairments in skeletal muscle glucose metabolism and oxidative metabolism .\",\n \"We next asked if these LRRC8A-dependent effects on downstream skeletal myotube insulin signaling are due to impaired myotube differentiation , and associated impairments in insulin signaling , or if LRRC8A also regulates these signaling pathways in differentiated skeletal myotubes .\",\n \"To test this , we first fully differentiated C2C12 myotubes , and then knocked down ( KD ) LRRC8A using Ad-shLRRC8A-mCherry post-differentiation and compared myotube size and insulin-stimulated signaling to Ad-shSCR-mCherry treated C2C12 myotubes ( Figure 2—figure supplement 1 ) .\",\n \"Similar to Lrrc8a ablation prior to differentiation , shRNA-mediated LRRC8A KD of fully differentiated C2C12 myotubes mildly reduced myotube size ( Figure 2—figure supplement 1A ) , and significantly reduced insulin-stimulated pAKT2 , pAKT1 , pAS160 , pAMPK and pS6 ribosomal proteins ( Figure 2—figure supplement 1B and C ) , although in some cases , these differences were less marked than in C2C12 Lrrc8a KO myoblasts that were subsequently differentiated ( Figure 2 ) .\",\n \"Also , the reductions in total AKT2 observed at both the mRNA ( Figure 2E , and Supplementary file\",\n \"1 ) and protein levels ( Figure 2A ) in differentiated Lrrc8a KO C2C12 myoblasts are also observed upon LRRC8A KD in differentiated C2C12 myotubes ( Figure 2—figure supplement 1B and C ) , suggesting that total AKT2 expression is regulated by LRRC8A in fully differentiated myotubes .\",\n \"As a complementary approach , we confirmed these findings in fully differentiated , primary skeletal myotubes isolated from Lrrc8afl/fl mice upon adenoviral transduction of Ad-CMV-Cre-mCherry ( KO ) compared to Ad-CMV-mCherry ( WT ) , and noted similar reductions in myotube size ( Figure 2—figure supplement 1D ) , and in basal levels of pAS160 , pAKT2 , AKT2 and pAKT1 signaling ( Figure 2—figure supplement 1E and F ) .\",\n \"Taken together , these data indicate that impaired insulin signaling observed in LRRC8A-depleted myotubes is in part due to impaired myotube differentiation with secondary effects on insulin signaling , and in part due to a direct contribution of LRRC8A to intracellular signaling in fully differentiated myotubes .\",\n \"To further validate LRRC8A-mediated effects on muscle differentiation and signaling , we re-expressed LRRC8A in Lrrc8a KO C2C12 myoblasts ( LRRC8A O/E ) and then examined myotube differentiation and basal activity of multiple intracellular signaling pathways by western blot , including pAKT1 , pAKT2 , pAS160 , p-p70S6K , pS6K and pERK1/2 as compared to WT and Lrrc8a KO C2C12 myotubes .\",\n \"LRRC8A O/E to 2 . 12-fold WT levels fully rescues myotube development in Lrrc8a KO myotubes ( Figure 3A ) , as quantified by restoration of Lrrc8a KO myotube area to levels above WT ( Figure 3B ) .\",\n \"This rescue of Lrrc8a KO myotube development upon LRRC8A O/E ( Figure 3A&B ) is associated with either restored ( pAS160 , AKT2 , pAKT1 , AKT1 , p70S6K ) or supra-normal ( pAKT2 , p-p70S6K , pS6K , pERK1/2 ) signaling ( Figure 3C&D ) compared to WT C2C12 myotubes .\",\n \"These data demonstrate that LRRC8A protein expression level strongly regulates skeletal muscle insulin signaling and myogenic differentiation .\",\n \"In a cellular context , there are numerous reports that VRAC and the LRRC8A complex that functionally encodes it is mechano-responsive ( Browe and Baumgarten , 2003; Browe and Baumgarten , 2006; Osei-Owusu et al . , 2018; Barakat et al . , 1999; Nakao et al . , 1999; Nilius and Droogmans , 2001; Romanenko et al . , 2002; Strange et al . , 2019 ) .\",\n \"It is well established that mechanical stretch is an important regulator of skeletal muscle proliferation , differentiation and skeletal muscle hypertrophy and may be mediated by PI3K-AKT-MAPK signaling ( Schiaffino et al . , 2013; Ma et al . , 2017; Fu et al . , 2018 ) and integrin signaling pathways ( Carson and Wei , 2000; Schlaepfer et al . , 1999; Flück et al . , 1999; Klossner et al . , 2009 ) .\",\n \"To determine if LRRC8A is also required for stretch-mediated AKT and MAP kinase signaling in skeletal myotubes , we subjected WT and Lrrc8a KO C2C12 myotubes to 0% or 5% equiaxial stretch using the FlexCell stretch system .\",\n \"Mechanical stretch ( 5% ) is sufficient to stimulate PI3K-AKT2/AKT1-pAS160-MAPK ( ERK1/2 ) signaling in WT C2C12 in a LRRC8A-dependent manner ( Figure 4A and B ) .\",\n \"These data position LRRC8A as a co-regulator of both insulin and stretch-mediated PI3K-AKT- pAS160-MAPK signaling .\",\n \"It has been reported earlier in both lymphocyte and adipocytes that the LRRC8A complex interacts with Growth factor Receptor-Bound 2 ( GRB2 ) and regulates PI3K-AKT signaling ( Kumar et al . , 2014; Zhang et al . , 2017; Gunasekar et al . , 2019 ) , whereby GRB2 binds with IRS1/2 and negatively regulates insulin signaling ( Shan et al . , 2013 ) .\",\n \"Indeed , GRB2 knock-down augments insulin-PI3K-MAPK signaling and induces myogenesis and myogenic differentiation genes ( Shan et al . , 2013; Liu et al . , 2009; Mitra and Thanabalu , 2017 ) .\",\n \"To determine if LRRC8A and GRB2 interact in C2C12 myotubes , we overexpressed C-terminal 3XFlag tagged LRRC8A in C2C12 cells followed by immunoprecipitation ( IP ) with Flag antibody .\",\n \"We observed significant GRB2 enrichment upon Flag IP from lysates of LRRC8A-3xFlag expressing C2C12 myotubes , consistent with a GRB2-LRRC8A interaction ( Figure 5A ) .\",\n \"Based on the notion that LRRC8A titrates GRB2-mediated suppression of AKT/MAPK signaling , and that Lrrc8a ablation results in unrestrained GRB2-mediated AKT/MAPK inhibition ( Gunasekar et al . , 2019 ) , we next tested if GRB2 knock-down ( KD ) may rescue myogenic differentiation in Lrrc8a KO C2C12 myotubes .\",\n \"shRNA-mediated GRB2 KD in Lrrc8a KO C2C12 myoblasts ( LRRC8A KO/shGRB2; Figure 5B ) stimulates myotube formation ( Figure 5C ) and increases myotube area ( Figure 5D ) , to levels equivalent to WT/shSCR ( Figure 5C and D ) .\",\n \"Similarly , GRB2 KD in Lrrc8a KO C2C12 myotubes induces myogenic differentiation markers IGF1 , MyoHCl , MyoHClla and MyoHCIIb relative to both LRRC8A KO/shSCR and WT/shSCR ( Figure 5E and F ) .\",\n \"These data are consistent with GRB2 suppression rescuing myotube differentiation in Lrrc8a KO C2C12 , and supports a model in which LRRC8A regulates myogenic differentiation by titrating GRB2-mediated signaling .\",\n \"To examine the physiological consequences of Lrrc8a ablation in vivo , we generated skeletal muscle-specific Lrrc8a KO mice using Cre-LoxP technology by crossing Myf5-Cre mice with Lrrc8afl/fl mice ( Skm KO; Figure 6A ) , and confirmed robust skeletal muscle LRRC8A depletion in Skm KO gastrocnemius muscle , 12 . 3-fold lower than WT controls ( Figure 6B ) .\",\n \"Remarkably , in contrast to the severe impairments in skeletal myogenesis observed in both Lrrc8a KO C2C12 and primary skeletal myotubes in vitro ( Figures 1 , 3 and 5 ) , Skm KO mice develop skeletal muscle mass comparable to WT littermates , based on Echo/MRI body composition ( Figure 6C ) and gross muscle weights ( Figure 6D ) , and are born at normal mendelian ratios ( Supplementary file 3 ) .\",\n \"However , histological examination reveals a 27% reduction in skeletal myocyte cross-sectional area in Skm KO as compared to WT mice ( Figure 6E ) , suggesting a requirement for LRRC8A in skeletal muscle cell size regulation in vivo .\",\n \"This is potentially due to reductions in myotube fusion , as observed in C2C12 and primary skeletal muscle cells in vitro ( Figure 1 ) , but occurring to a lesser degree in vivo .\",\n \"These data indicate that the profound impairments in myogenesis observed in vitro may reflect a very early requirement for LRRC8A signaling in skeletal muscle development ( prior to LRRC8A protein elimination by Myf5-Cre-mediated LRRC8A recombination ) , or other fundamental differences in myogenic differentiation processes in vitro versus in vivo .\",\n \"Since insulin signaling is an important regulator of skeletal muscle oxidative capacity and endurance ( Affourtit , 2016 ) , we next examined exercise tolerance on treadmill testing in Lrrc8afl/fl ( WT ) compared to Skm KO .\",\n \"Skm KO mice exhibit a 14% reduced exercise capacity , compared to age and gender matched WT controls ( Figure 7A; Figure 7—Video 1 ) .\",\n \"Hang-times on inversion testing are also reduced 29% in Skm KO compared to controls , further supporting reduced skeletal muscle endurance upon skeletal muscle LRRC8A depletion in vivo ( Figure 7B ) .\",\n \"To determine if these reductions in muscle function in vivo are due to muscle-specific functional impairments , we next performed ex vivo experiments in which we isolated the soleus muscle from mice and performed twitch/train testing .\",\n \"We observed that peak developed tetanic tension is 15% reduced in Skm KO soleus muscle compared to WT controls ( Figure 7C ) , suggesting a skeletal muscle autonomous mechanism , with no difference in time to fatigability ( TTF , Figure 7D ) or time to 50% decay ( Figure 7E ) .\",\n \"To determine whether these LRRC8A dependent differences in muscle endurance and force were due to impaired oxidative capacity , we next measured oxygen consumption rate ( OCR ) and extracellular acidification rate ( ECAR ) in WT and Lrrc8a KO primary skeletal muscle cells , under basal and insulin-stimulated conditions ( Figure 7F ) .\",\n \"Oxygen consumption of Lrrc8a KO primary myotubes are 26% lower than WT and , in contrast to WT cells , are unresponsive to insulin-stimulation ( Figure 7F ) , consistent with abrogation of insulin-AKT/ERK1/2 signaling upon skeletal muscle LRRC8A depletion .\",\n \"These relative changes persist in the presence of Complex V and III inhibitors , Oligomycin and Antimycin A ( Figure 7F and G ) , suggesting that insulin-stimulated glycolytic pathways are primarily dysregulated upon LRRC8A depletion .\",\n \"In contrast , FCCP , which maximally uncouples mitochondria , abolishes differences in oxygen consumption between WT and Lrrc8a KO primary muscle cells , suggesting no differences in functional mitochondrial content in Lrrc8a KO muscle .\",\n \"Consistent with this finding , we observe no differences in muscle fiber-type based on Myosin Heavy Chain ( MHC ) Type 1 , MHC Type IIa , MHC Type IIx and MHC Type IIb in both superficial and deep tibialis anterior muscle in Skm KO as compared to WT mice ( Figure 7—figure supplement 1 ) , indicating the reductions in muscle force and endurance in Skm KO mice is not due to muscle fiber-type switching .\",\n \"Indeed , it is increasingly appreciated that changes in muscle metabolic potential and morphology may occur independent from adaptive fiber-type switching as measured by a change in MHC expression ( Egan and Zierath , 2013 ) .\",\n \"To examine an alternative mechanism , such as glycolysis , we measured extracellular acidification rate ( ECAR ) in WT and Lrrc8a KO primary myotubes .\",\n \"Insulin-stimulated ECAR increases are abolished in Lrrc8a KO compared to WT cells , and these differences persist independent of electron transport chain modulators ( Figure 7H ) .\",\n \"These data suggest that LRRC8A regulation of skeletal muscle cellular oxygen consumption occurs at the level of glucose metabolism - potentially via LRRC8A-dependent insulin-PI3K-AKT-AS160-GLUT4 signaling , glucose uptake and utilization .\",\n \"These findings in primary skeletal muscle cells are supported by marked transcriptional suppression of numerous glycolytic genes: Aldoa , Eno3 , Pfkm , and Pgam2; and glucose and glycogen metabolism genes: Phka1 , Phka2 , Ppp1r3c and Gys1 , upon Lrrc8a ablation in C2C12 myotubes ( Figure 7—figure supplement 2 ) .\",\n \"Guided by evidence of impaired insulin-PI3K-AKT-AS160-GLUT4 signaling observed in Lrrc8a KO C2C12 and primary myotubes , we next examined systemic glucose homeostasis and insulin sensitivity in WT and Skm KO mice by measuring glucose and insulin tolerance .\",\n \"On a regular chow diet , there are no differences in either glucose tolerance or insulin tolerance ( Figure 8A ) between WT and Skm KO mice .\",\n \"However , over the course of 16–24 weeks on chow diet Skm KO mice develop 29% increased adiposity based on body composition measurements ( Figure 8B ) compared to WT , with no significant difference in lean mass ( Figure 8C ) or in total body mass ( Figure 8C ) .\",\n \"When Skm KO mice are raised on a high-fat-diet ( HFD ) for 16 weeks , there is no difference in adiposity observed ( Figure 8—figure supplement\",\n \"1 ) compared to WT mice , but glucose tolerance is impaired ( Figure 8D ) and there is mild insulin resistance in HFD Skm KO mice as compared to WT ( Figure 8E ) .\",\n \"Since Myf5 is also expressed in brown fat ( Seale et al . , 2008 ) , it is possible that these metabolic phenotypes arise from LRRC8A-mediated effects in brown fat and consequent changes in systemic metabolism .\",\n \"To rule out this possibility , we repeated a subset of the above experiments in a skeletal muscle-targeted KO mouse generated by crossing the Myl1-Cre and Lrrc8afl/fl mice ( Myl1Cre/Lrrc8afl/fl ) , since Myl1-Cre is restricted to mature skeletal muscle ( Figure 8—figure supplement 2B ) , and excludes brown fat ( Bothe et al . , 2000 ) .\",\n \"Similar to Myf5Cre/Lrrc8afl/fl mice , Myl1Cre/Lrrc8afl/fl mice fed a regular chow diet , have normal glucose tolerance ( Figure 8—figure supplement 2C ) , but exhibit 24% reduced exercise capacity on treadmill testing , as compared to WT ( Figure 8—figure supplement 2D ) .\",\n \"Also , Myl1Cre/Lrrc8afl/fl mice develop increased visceral adiposity over time on regular chow , based on 24% increased epididymal adipose mass normalized to body mass ( Figure 8—figure supplement 2E ) , with no differences in inguinal adipose tissue , muscle mass ( Figure 8—figure supplement 2F ) , or total body mass ( Figure 8—figure supplement 2G ) .\",\n \"These data suggest that impaired skeletal muscle glucose uptake in Skm KO ( Myl1Cre/Lrrc8afl/fl and Myf5Cre/Lrrc8afl/fl ) mice are compensated for by increased adipose glucose uptake and de novo lipogenesis , which contribute to preserved glucose tolerance , at the expense of increased adiposity in skeletal muscle-targeted Lrrc8a KO mice raised on a regular chow diet .\",\n \"However , overnutrition-induced obesity , and the associated impairments in adipose and hepatic glucose disposal may uncover glucose intolerance and insulin resistance in skeletal muscle-targeted Lrrc8a KO mice .\"\n ],\n [\n \"Our data reveal that the LRRC8A channel complex regulates insulin/stretch-mediated AKT-AS160-GLUT4 , MAP kinase and mTOR signaling in differentiated myoblast cultures , with consequent effects on myogenic differentiation , insulin-stimulated glucose metabolism and oxygen consumption .\",\n \"In vivo , skeletal muscle-targeted Lrrc8a KO mice have smaller skeletal muscle cells , impaired muscle endurance , and force generation , and are predisposed to adiposity , glucose intolerance and insulin resistance .\",\n \"Insulin/stretch-mediated PI3K-AKT , mTOR signaling are well known to be important regulators of myogenic differentiation ( Rotwein and Wilson , 2009; Héron-Milhavet et al . , 2008 ) , metabolism and muscle function ( Schiaffino et al . , 2013 ) suggesting impaired LRRC8A-AKT-mTOR signaling may underlie the defect in myogenic differentiation .\",\n \"Indeed , consistent with our previous findings and proposed model in adipocytes , in which LRRC8A mediates the interaction of GRB2 with IRS1 to regulate insulin-AKT signaling ( Zhang et al . , 2017; Gunasekar et al . , 2019 ) , LRRC8A also associates with GRB2 in skeletal myotubes , and GRB2 knock-down rescues impaired myogenic differentiation in Lrrc8a KO muscle cells .\",\n \"Thus , our working model for LRRC8A-mediated regulation of insuln-PI3K-AKT and downstream signaling in adipocytes ( Gunasekar et al . , 2019 ) appears to be conserved in skeletal myotubes .\",\n \"The in vitro phenotype that we observe in CRISPR/cas9-mediated Lrrc8a KO C2C12 myotubes and in Lrrc8a KO primary myotubes is consistent with the observation of Chen et al . , 2019 that used siRNA-mediated LRRC8A knock-down to demonstrate that the LRRC8A channel complex is required for myogenic differentiation .\",\n \"However , the ability of both GRB2 KD and LRRC8A O/E to rescue myogenic differentiation and augment insulin-AKT , MAP kinase and mTOR signaling in Lrrc8a KO myotubes implicates non-canonical , non-conductive signaling mechanisms .\",\n \"Based on our work and also previous studies ( Voss et al . , 2014; Qiu et al . , 2014 ) , LRRC8A O/E does not increase ICl , SWELL/VRAC to supranormal levels at the plasma membrane .\",\n \"However , pAKT , pERK1/2 and mTOR levels are augmented by twofold to threefold above endogenous levels , upon twofold LRRC8A O/E in C2C12 myotubes .\",\n \"These data suggest that alternative/non-canonical signaling mechanisms underlie LRRC8A signaling , as opposed to canonical/conductive signaling mechanisms .\",\n \"Another finding that warrants further study is the requirement for LRRC8A in stretch-induced AKT and MAP kinase signaling in C2C12 myotubes upon static stretching .\",\n \"Mechanical stretch is known to regulate myoblast proliferation and differentiation and myofiber hypertrophy via PI3K-AKT-MAPK signaling ( Schiaffino et al . , 2013; Ma et al . , 2017; Fu et al . , 2018 ) .\",\n \"Also , there are numerous reports that VRAC , and presumably LRRC8A , is mechano-responsive ( Browe and Baumgarten , 2003; Browe and Baumgarten , 2006; Osei-Owusu et al . , 2018; Barakat et al . , 1999; Nakao et al . , 1999; Nilius and Droogmans , 2001; Romanenko et al . , 2002; Strange et al . , 2019 ) .\",\n \"Therefore , it may not be surprising that LRRC8A complexes co-regulate both insulin and stretch-mediated PI3K-AKT , ERK1/2 signaling in skeletal myotubes - potentially integrating mechanical and hormonal stimuli to tune downstream signaling .\",\n \"Indeed , the concept of mechano-tuning insulin signaling has been proposed and demonstrated in other cell systems ( Chen and Chalfie , 2014; Kim et al . , 2018 ) which implicate integrin signaling ( Kim et al . , 2018 ) as the mechano-sensory mechanism .\",\n \"Curiously , it has been reported that VRAC can be activated in cardiac muscle cells by applying mechanical tension to β1-integrins ( Browe and Baumgarten , 2003; Browe and Baumgarten , 2006 ) , supporting the notion that , in striated muscle , integrin-LRRC8A may indeed participate in mechano-tuning insulin-AKT and downstream signaling .\",\n \"Further studies to more fully delineate the putative molecular mechanisms are necessary .\",\n \"It is also notable that the profound myogenic differentiation block observed upon Lrrc8a ablation in both C2C12 myotubes and primary myotubes in vitro is significantly milder in vivo , where only a 30% reduction in skeletal myocyte cross-sectional area is observed , with no change in total muscle mass , or lean content , in Skm KO mice .\",\n \"This discordance in phenotype may reflect fundamental differences in the biology of skeletal muscle differentiation in vitro versus the in vivo milieu .\",\n \"Alternatively , it may be that , although early , the time interval between Myf5-Cre expression early in myogenesis , and ultimate reductions in LRRC8A protein ( potentially ~3 days ) may extend beyond the critical period during which LRRC8A is required for myogenic differentiation .\",\n \"Directly testing this hypothesis would require examining mice expressing Cre-recombinase at the precursor stage , in skeletal muscle satellite cells , such as Pax3 or Pax7 promoters ( Relaix et al . , 2006; Buckingham et al . , 2006 ) - these experiments are currently underway .\",\n \"Although overall muscle development is grossly intact in both LRRC8A skeletal muscle KO ( Myl1Cre/Lrrc8afl/fl and Myf5Cre/Lrrc8afl/fl ) mice , there is a consistent reduction in exercise capacity , muscle endurance and force generation , and a propensity for increased adiposity over time compared to age and gender matched controls .\",\n \"The impaired exercise capacity observed in skeletal muscle Lrrc8a KO mice are consistent with some level of insulin resistance , as in db/db mice ( Ostler et al . , 2014 ) and in humans ( Reusch et al . , 2013 ) , and may be due to impaired skeletal muscle glycolysis and oxygen consumption in LRRC8A-depleted skeletal muscle .\",\n \"Furthermore , the increased gonadal adiposity , with preserved glucose and insulin tolerance , observed in LRRC8A skeletal muscle KO ( Myl1Cre/Lrrc8afl/fl and Myf5Cre/Lrrc8afl/fl ) mice phenocopy both skeletal muscle-specific insulin receptor KO mice ( MIRKO ) ( Brüning et al . , 1998 ) and transgenic mice expressing a skeletal muscle dominant-negative insulin receptor mutant ( Moller et al . , 1996 ) , wherein skeletal muscle-specific insulin resistance is compensated for by re-distribution of glucose from skeletal muscle to adipose tissue , to promote adiposity ( Kim et al . , 2000 ) .\",\n \"In the case of LRRC8A skeletal muscle KO mice , overnutrition and HFD feeding unmasks this underlying mild insulin resistance and glucose intolerance , as adipose-tissue insulin resistance also begins to set in .\",\n \"Recent findings from skeletal muscle specific AKT1/AKT2 double KO mice indicate that these effects may not attributable to solely to muscle AKT signaling ( Jaiswal et al . , 2019 ) , but potentially involve other insulin-sensitive signaling pathways .\",\n \"In summary , we show that LRRC8A regulates myogenic differentiation and insulin-PI3K-AKT-AS160 , ERK1/2 , and mTOR signaling in myotubes via GRB2-mediated signaling .\",\n \"In vivo , LRRC8A is required for maintaining normal exercise capacity , muscle endurance , adiposity under basal conditions , and systemic glycemia in the setting of overnutrition .\",\n \"These findings contribute further to our understanding of LRRC8A channel complexes in the regulation of systemic metabolism .\"\n ],\n [\n \"The Institutional Animal Care and Use Committee of the Washington University in St . Louis and the University of Iowa approved all experimental procedures involving animals .\",\n \"All the mice were housed in temperature , humidity , and light-controlled room and allowed free access to water and food .\",\n \"Male and female Lrrc8a fl/fl ( WT ) , Myl1Cre/Lrrc8a fl/fl , Myf5Cre/Lrrc8a fl/fl ( skeletal muscle targeted Lrrc8a KO ) , were generated and used in these studies .\",\n \"Myl1Cre ( JAX# 24713 ) and Myf5Cre ( JAX# 007893 ) mice were purchased from Jackson labs .\",\n \"For high-fat diet ( HFD ) studies , we used Research Diets Inc ( Cat # D12492 ) ( 60 kcal% fat ) regimen starting at 14 weeks of age .\",\n \"Lrrc8afl/fl mice were generated as previously described ( Zhang et al . , 2017 ) .\",\n \"Briefly , Lrrc8a intronic sequences were obtained from Ensembl Transcript ID ENSMUST00000139454 .\",\n \"All CRISPR/Cas9 sites were identified using ZiFit Targeter Version 4 . 2 ( http://zifit . partners . org/ZiFiT/ ) .\",\n \"Pairs of oligonucleotides corresponding to the chosen CRISPR-Cas9 target sites were designed , synthesized , annealed , and cloned into the pX330-U6-Chimeric_BB-CBh-hSpCas9 construct ( Addgene plasmid # 42230 ) , following the protocol detailed in Cong et al . , 2013 .\",\n \"CRISPR-Cas9 reagents and ssODNs were injected into the pronuclei of F1 mixed C57/129 mouse strain embryos at an injection solution concentration of 5 ng/μl and 75–100 ng/μl , respectively .\",\n \"Correctly targeted mice were screened by PCR across the predicted loxP insertion sites on either side of Exon\",\n \"3 . These mice were then backcrossed >8 generations into a C57BL/6 background .\",\n \"Rabbit polyclonal anti-LRRC8A antibody was generated against the epitope QRTKSRIEQGIVDRSE ( Pacific Antibodies ) .\",\n \"All other primary antibodies were purchased from Cells Signaling: anti-β-actin ( #8457 s ) , p-AKT1 ( #9018 s ) , Akt1 ( #2938 s ) , pAKT2 ( #8599 s ) , Akt2 ( #3063 s ) , p-AS160 ( #4288 s ) , AS160 ( #2670 s ) , AMPKα ( #5831 s ) , pAMPKα ( #2535 s ) , FoxO1 ( #2880 s ) and pFoxO1 ( #9464 s ) , p70 S6 Kinase ( #9202 s ) , p-p70 S6 Kinase ( #9205 s ) , pS6 Ribosomal ( #5364 s ) , GAPDH ( #5174 s ) , pErk1/2 ( #9101 s ) , Total Erk1/2 ( #9102 s ) .\",\n \"Purified mouse anti-Grb2 was purchased from BD ( 610111 s ) .\",\n \"Purified anti-flag mouse antibody was purchased from sigma .\",\n \"Rabbit IgG Santa Cruz ( sc-2027 ) .\",\n \"All primary antibodies were used at 1:1000 dilution , except for anti-flag at 1:2000 dilution .\",\n \"All secondary antibody ( anti-rabbit-HRP and anti-mouse-HRP ) were used at 1:10 , 000 dilution .\",\n \"Adenovirus type five with Ad5-CMV-mCherry ( 1 × 1010 PFU/ml ) , Ad5-CMV-Cre-mCherry ( 3 × 1010 PFU/ml ) were obtained from the University of Iowa viral vector core facility .\",\n \"Adenovirus type five with Ad5-CMVmCherry-U6-hLRRC8A-shRNA , 2 . 2 × 1010 PFU/ml , ( AD3535 ) , ( Vector Biolabs ) .\",\n \"Scrambled non-targeting control ( shSCR: Ad5-U6-scramble-mCherry , 1 × 1010 PFU/ml ) , ( Vector biolabs #3086 ) .\",\n \"Ad5-CAG-LoxP-stop-LoxP-3XFlag- LRRC8A ( 1 × 1010 PFU/ml ) were obtained from Vector Biolabs .\",\n \"Ad5-U6-shGRB2-GFP ( 1 × 109 PFU/ml ) and Ad5-U6-shSCR-GFP ( 1 × 1010 PFU/ml ) were obtained from Vector Biolabs .\",\n \"All recordings were performed in the whole-cell configuration at room temperature , as previously described ( Zhang et al . , 2017; Kang et al . , 2018 ) .\",\n \"Briefly , currents were measured with either an Axopatch 200B amplifier or a MultiClamp 700B amplifier ( Molecular Devices ) paired to a Digidata 1550 digitizer , using pClamp 10 . 4 software .\",\n \"The intracellular solution contained ( in mM ) : 120 L-aspartic acid , 20 CsCl , 1 MgCl2 , 5 EGTA , 10 HEPES , 5 MgATP , 120 CsOH , 0 . 1 GTP , pH 7 . 2 with CsOH .\",\n \"The extracellular solution for hypotonic stimulation contained ( in mM ) : 90 NaCl , 2 CsCl , 1 MgCl2 , 1 CaCl2 , 10 HEPES , five glucose , five mannitol , pH 7 . 4 with NaOH ( 210 mOsm/kg ) .\",\n \"The isotonic extracellular solution contained the same composition as above except for mannitol concentration of 105 ( 300 mOsm/kg ) .\",\n \"The osmolarity was checked by a vapor pressure osmometer 5500 ( Wescor ) .\",\n \"Currents were filtered at 10 kHz and sampled at 100 μs interval .\",\n \"The patch pipettes were pulled from borosilicate glass capillary tubes ( WPI ) using a P-87 micropipette puller ( Sutter Instruments ) .\",\n \"The pipette resistance was ~4–6 MΩ when the patch pipette was filled with intracellular solution .\",\n \"The holding potential was 0 mV .\",\n \"Voltage ramps from −100 to +100 mV ( at 0 . 4 mV/ms ) were applied every 4 s .\",\n \"Satellite cell isolation and differentiation were performed as described previously with minor modifications ( Hindi et al . , 2017 ) .\",\n \"Briefly , gastrocnemius and quadriceps muscles were excised from Lrrc8aflfl mice ( 8–10 weeks old ) and washed twice with 1XPBS supplemented with 1% penicillin-streptomycin and fungizone ( 300 µl/100 ml ) .\",\n \"Muscle tissue was incubated in DMEM-F12 media supplemented with collagenase II ( 2 mg/ml ) , 1% penicillin-streptomycin and fungizone ( 300 ul/100 ml ) and incubated at shaker for 90 min at 37°C .\",\n \"Tissue was washed with 1X PBS and incubated again with DMEM-F12 media supplemented with collagenase II ( 1 mg/ml ) , dispase ( 0 . 5 mg/ml ) , 1% penicillin-streptomycin and fungizone ( 300 µl/100 ml ) in a shaker for 30 min at 37°C .\",\n \"Subsequently , the tissue was minced and passed through a cell strainer ( 70 µm ) , and after centrifugation; satellite cells were plated on BD Matrigel‐coated dishes .\",\n \"Cells were stimulated to differentiate into myoblasts in DMEM‐F12 , 20% fetal bovine serum ( FBS ) , 40 ng/ml basic fibroblast growth factor ( bfgf , R and D Systems , 233‐FB/CF ) , 1X non‐essential amino acids , 0 . 14 mM β‐mercaptoethanol , 1X penicillin/streptomycin , and Fungizone .\",\n \"Myoblasts were maintained with 10 ng/ml bfgf and then differentiated in DMEM‐F12 , 2% FBS , 1X insulin–transferrin–selenium , when 80% confluency was reached .\",\n \"WT C2C12 and Lrrc8a KO C2C12 cell line were cultured at 37°C , 5% CO2 Dulbecco’s modified Eagle’s medium ( DMEM; GIBCO ) supplemented with 10% fetal bovine serum ( FBS; Atlanta Bio selected ) and antibiotics 1% penicillin-streptomycin ( Gibco , USA ) .\",\n \"Cells were grown to 80% confluency and then transferred to differentiation media DMEM supplemented with antibiotics and 2% horse serum ( HS; GIBCO ) to induce differentiation .\",\n \"The differentiation media was changed every two days .\",\n \"Cells were allowed to differentiate into myotubes for up to 6 days .\",\n \"Subsequently , myotube images were taken for quantification of myotube surface area and fusion index .\",\n \"After differentiation ( Day 7 ) , cells were imaged with Olympus IX73 microscope ( 10X objective , Olympus , Japan ) .\",\n \"For each experimental condition , 5–6 bright field images were captured randomly from six-well plate .\",\n \"Myotube surface area was quantified manually with ImageJ software .\",\n \"The morphometric quantification was carried out by an independent observer who was blinded to the experimental conditions .\",\n \"For fusion index , differentiated myotube growing on coverslip were washed with 1X PBS and fixed with 2% PFA .\",\n \"After washing with 1XPBS three times , cells were permeabilized with 0 . 1% TritonX100 for 5 min at room temperature and subsequently blocking was done with 5% goat serum for 30 min .\",\n \"Cells were stained with DAPI ( 1 µM ) for 15 min and after washing with 1X PBS , coverslip were mounted on slides with ProLong Diamond anti-fading agent .\",\n \"Cells were imaged with Olympus IX73 microscope ( 10X objective , Olympus , Japan ) with bright field and DAPI filter .\",\n \"Fusion index ( number of nuclei incorporated within the myotube/total number of nuclei present in that view field ) were analyzed by ImageJ .\",\n \"RNA quality was assessed by Agilent BioAnalyzer 2100 by the University of Iowa Institute of Human Genetics , Genomics Division .\",\n \"RNA integrity numbers greater than eight were accepted for RNAseq library preparation .\",\n \"RNA libraries of 150 bp PolyA-enriched RNA were generated , and sequencing was performed on a HiSeq 4000 genome sequencing platform ( Illumina ) .\",\n \"Sequencing results were uploaded and analyzed with BaseSpace ( Illumina ) .\",\n \"Sequences were trimmed to 125 bp using FASTQ Toolkit ( Version 2 . 2 . 0 ) and aligned to Mus musculus mmp10 genome using RNA-Seq Alignment ( Version 1 . 1 . 0 ) .\",\n \"Transcripts were assembled and differential gene expression was determined using Cufflinks Assembly and DE ( Version 2 . 1 . 0 ) .\",\n \"Ingenuity Pathway Analysis ( QIAGEN ) was used to analyze significantly regulated genes which were filtered using cutoffs of >1 . 5 fragments per kilobase per million reads , >1 . 5 fold changes in gene expression , and a false discovery rate of <0 . 05 .\",\n \"Heatmaps were generated to visualize significantly regulated genes .\",\n \"For insulin stimulation , differentiated C2C12 myotubes were incubated in serum free media for 6 hr and stimulated with 0 and 10 nM insulin for 15 min; while differentiated primary myotubes were incubated in serum free media for 4 hr and stimulated with 0 and 10 nM insulin for 2 hr .\",\n \"To examine intracellular signaling upon LRRC8A overexpression ( LRRC8A O/E ) , we overexpressed LRRC8A-3xFlag by transducing C2C12 myotubes with Ad5-CAG-LoxP-stop-LoxP-LRRC8A-3xFlag ( MOI 50–60 ) and Ad5-CMV-Cre-mCherry ( MOI 50–60 ) and polybrene ( 4 µg/ml ) in DMEM ( 2% FBS and 1% penicillin-streptomycin ) for 36 hr .\",\n \"Ad5-CMV-Cre-mCherry alone with polybrene ( 4 µg/ml ) ( MOI 50–60 ) was transduced in WT C2C12 or Lrrc8a KO C2C12 as controls .\",\n \"Viral transduction efficiency ( 60–70% ) was confirmed by mCherry fluorescence .\",\n \"Cells were allowed to differentiate further in differentiation media up to 6 days .\",\n \"Myotube images were taken before collecting lysates for further signaling studies .\",\n \"GRB2 knock-down was achieved by transducing myotubes with Ad5-U6- shSCR-GFP ( Control , MOI 50–60 ) or Ad5-U6- sh LRRC8A-GFP ( GRB2 KD , MOI 50–60 ) in DMEM ( 2% FBS and 1% penicillin-streptomycin ) supplemented with polybrene ( 4 µg/ml ) for 24 hr .\",\n \"Cells were allowed to differentiate further in differentiation media up to 6 days .\",\n \"Differentiated myotube images were taken for myotube surface area quantification before collecting the cells for RNA isolation .\",\n \"WT C2C12 cells were cultured in differentiation media for 6 days to form myotubes .\",\n \"Subsequently myotubes were transduced either with Ad5-U6-scramble-mCherry ( sh-SCR ) or Ad5-mCherry-U6-shLRRC8A ( sh LRRC8A ) shRNA ( KD ) ( MOI 50–60 ) for 2 days and grown for 1 additional day prior to analysis .\",\n \"Myotubes were then incubated in serum free media for 6 hr and stimulated with 0 and 10 nM insulin for 15 min .\",\n \"Primary skeletal muscle cells isolated from Lrrc8a fl/fl mice were grown in differentiation media for 3 . 5 days .\",\n \"To generate Lrrc8a KO primary myotubes , cells were transduced with either Ad5-CMV-CMV-mCherry ( WT ) or Ad5-CMV-Cre-mCherry ( KO ) ( MOI 50–60 ) for 2 days and then grown for 1 additional day prior to analysis .\",\n \"Myotubes were then harvested to measure intracellular signaling under basal culture conditions .\",\n \"C2C12 myotubes were plated in each well of a six-well BioFlex culture plate .\",\n \"Cells were allowed to differentiate up to 6 days in differentiation media , and then placed into a Flexcell Jr .\",\n \"Tension System ( FX-6000T ) and incubated at 37°C with 5% CO2 .\",\n \"C2C12 myotubes on flexible membrane were subjected to either no tension or to static stretch of 5% for 15 min .\",\n \"Cells were lysed and protein isolated for subsequent western blots .\",\n \"Cells were washed with ice cold 1X PBS and lysed in ice-cold lysis buffer ( 150 mM NaCl , 20 mM HEPES , 1% NP-40 , 5 mM EDTA , pH 7 . 5 ) with added proteinase/phosphatase inhibitor ( Roche ) .\",\n \"The cell lysate was further sonicated ( 20% pulse frequency for 20 s ) and centrifuged at 14 , 000 rpm for 20 min at 4°C .\",\n \"The supernatant was collected and estimated for protein concentration using DC protein assay kit ( Bio-Rad ) .\",\n \"For immunoblotting , an appropriate volume of 4 x Laemmli ( Bio-rad ) sample loading buffer was added to the sample ( 10–20 μg of protein ) , then heated at 90°C for 5 min before loading onto 4–20% gel ( Bio-Rad ) .\",\n \"Proteins were separated using running buffer ( Bio-Rad ) for 2 hr at 110 V . Proteins were transferred to PVDF membrane ( Bio-Rad ) and membrane blocked in 5% ( w/v ) BSA or 5% ( w/v ) milk in TBST buffer ( 0 . 2 M Tris , 1 . 37 M NaCl , 0 . 2% Tween-20 , pH 7 . 4 ) at room temperature for 1 hr .\",\n \"Blots were incubated with primary antibodies at 4°C overnight , followed by secondary antibody ( Bio-Rad , Goat-anti-mouse #170–5047 , Goat-anti-rabbit #170–6515 , all used at 1:10 , 000 ) at room temperature for one hour .\",\n \"Membranes were washed three times and imaged by chemiluminescence ( Pierce ) by using a Chemidoc imaging system ( BioRad ) .\",\n \"The images were further analyzed for band intensities using ImageJ software .\",\n \"β-Actin or GAPDH levels were quantified for equal protein loading .\",\n \"C2C12 myotubes were plated on 10 cm dishes in complete media and grown to 80% confluency .\",\n \"For LRRC8A-3xFlag overexpression , Ad5-CAG-LoxP-stop-LoxP-3XFlag- LRRC8A ( MOI 50–60 ) and Ad5-CMV-Cre-mCherry ( MOI 50–60 ) along with polybrene ( 4 ug/ml ) were added to cells in DMEM media ( 2% FBS and 1% penicillin-streptomycin ) allowed to grow for 36 hr .\",\n \"Cells were then switched to differentiation media for up to 6 days .\",\n \"After that myotubes were harvested in ice-cold lysis buffer ( 150 mM NaCL , 20 mM HEPES , 1% NP-40 , 5 mM EDTA , pH 7 . 5 ) with added protease/phosphatase inhibitor ( Roche ) and kept on ice with gentle agitation for 15 min to allow complete lysis .\",\n \"Lysated were incubated with anti-Flag antibody ( Sigma #F3165 ) or control rabbit IgG ( Santa Cruz sc-2027 ) rotating end over end overnight at 4°C .\",\n \"Protein G sepharose beads ( GE ) were added for 4 hr and then samples were centrifuged at 10 , 000 g for 3 min and washed three times with RIPA buffer and re-suspended in laemmli buffer ( Bio-Rad ) , boiled for 5 min , separated by SDS-PAGE gel followed by the western blot protocol .\",\n \"Differentiated cells were solubilized in TRIzol and the total RNA was isolated using PureLink RNA kit ( Life Technologies ) and column DNase digestion kit ( Life Technologies ) .\",\n \"The cDNA synthesis , qRT-PCR reaction and quantification were carried out as described previously ( Zhang et al . , 2017 ) .\",\n \"All experiments were performed in triplicate and GAPDH were used as internal standard to normalize the data .\",\n \"All primers used for qRT-PCR are listed in Supplementary file\",\n \"4 . Mice were euthanized and gastrocnemius muscle excised and washed with 1X PBS .\",\n \"Muscles tissue were minced with surgical blade and kept in 8 vol of ice cold homogenization buffer ( 20 mM Tris , 137 mM NaCl , 2 . 7 mM KCl , 1 mM MgCl2 , 1% Triton X-100 , 10% ( w/v ) glycerol , 1 mM EDTA , 1 mM dithiothreitol , pH 7 . 8 ) supplemented with protease/phosphatase inhibitor ( Roche ) .\",\n \"Tissues were homogenized on ice with a Dounce homogenizer ( 40–50 passes ) and incubated for overnight at 4°C with continuous rotation .\",\n \"Tissue lysate was further sonicated in 20 s cycle intervals for 2–3 times and centrifuged at 14 , 000 rpm for 20 min at 4°C .\",\n \"The supernatant was collected for protein concentration estimation using DC protein assay kit ( Bio-Rad ) .\",\n \"Due to the high content of contractile protein in this preparation , coomassie gel staining was performed to demonstrate equal protein loading , and for quantification normalization of Western blots .\",\n \"Mice were anesthetized with isoflurane followed by cervical dislocation .\",\n \"Tibialis anterior ( TA ) muscle was carefully excised and gently immersed into the tissue-tek O . C . T medium placed on wooden cork .\",\n \"Orientation of the tissue maintained while embedding in the medium .\",\n \"Subsequently , wooden cork with tissue gently immersed into the liquid N2 pre-chilled isopentane bath for 10–14 s and store at −80°C .\",\n \"Tissue sectioning ( 10 µm ) were done with Leica cryostat and all sections collected on positively charged microscope slide for H and E staining as described earlier ( Bonetto et al . , 2015 ) .\",\n \"Briefly , TA sectioned slides were stained for 2 min in hematoxylin , 1 min in eosin and then dehydrated with ethanol and xylenes .\",\n \"Subsequently , slides were mounted with coverslip and image were taken with EVOS cell imaging microscope ( 10X objective ) .\",\n \"For quantification of fiber cross-sectional area , images were processed using ImageJ software to enhance contrast and smooth/sharpen cell boundaries and clearly demarcate muscle fiber cross sectional area .\",\n \"All measurements were performed with an independent observer who was blinded to the identity of the slides .\",\n \"Skeletal muscle fiber-type was quantified in WT and Skm KO tibialis anterior ( TA ) muscle as previously described ( Biltz et al . , 2020 ) .\",\n \"Briefly , sections were immunostained against myosin heavy chain isoforms ( type I , type IIa , and type IIb BA‐F8 , SC‐71 and BF‐F3 , respectively , Developmental Studies Hybridoma Bank , Iowa City , IA ) and against laminin ( ab11575; Abcam , Cambridge , UK ) to quantify fiber types and areas .\",\n \"Fiber types were identified on four non‐overlapping 20 × images in controlled locations: two from the superficial region and two from the deep .\",\n \"Unstained fibers were assumed to be of type IIx .\",\n \"Fiber areas were detected with a custom ImageJ macro using the automated Huang threshold and particle analyzer .\",\n \"Regions of interest with a circularity of less than 0 . 5 were excluded as being out of the axial plane of that fiber .\",\n \"Mouse treadmill exercise protocols were adapted from Dougherty et al . , 2016 .\",\n \"Briefly , mice were first acclimated with the motorized treadmill ( Columbus Instruments Exer3/6 Treadmill Columbus , OH ) for 3 days by running 10–15 min ( with 3 min interval ) for three consecutive days at 7 m/min , with the electric shocking grid ( frequency 1 Hz ) installed in each lane .\",\n \"During the treadmill testing , mice ran with a gradual increase in speed ( 5 . 5 m/minute to 22 m/minute ) and inclination ( 0o-15o ) at time intervals of 3 min each .\",\n \"The total running distance for each mouse was recorded at the end of the experiment .\",\n \"The predefined criteria for removing the mouse from the treadmill and recording the distance travelled was: continuous shock for 5 s or receiving 5–6 shocks within a time interval of 15 s .\",\n \"These mice were promptly removed from the treadmill and total duration and distance were recorded for further analysis .\",\n \"Mouse inversion test was performed using a wire-grid screen apparatus elevated to 50 cm .\",\n \"Mice were stabilized on the screen inclined at 60o , with the mouse head facing towards the base of the screen .\",\n \"The screen was slowly pivoted to 0o ( horizontal ) , such that the mouse was fully inverted and hanging upside down from the screen .\",\n \"Soft bedding was placed underneath the screen to protect mouse from any injury , were they to fall .\",\n \"The inversion test for each mouse was repeated two times with an interval of 45 min ( resting period ) .\",\n \"The hang time for each mouse was repeated three times with an interval of 5 min .\",\n \"The maximum hanging time limit for each mouse was set for 3 min .\",\n \"Soleus muscle was carefully dissected and transferred to a specialized muscle stimulation system ( 1500A , Aurora Scientific , Aurora , ON , Canada ) where physiology tests were run in a blinded fashion .\",\n \"Muscle was immersed in a Ringer solution ( in mM ) ( NaCl 137 , KCI 5 , CaCl2 2 , NaH2PO4 1 , NaHCO3 24 , MgSO4 1 , glucose 11 and curare 0 . 015 ) maintained at 37°C .\",\n \"The distal tendon was secured with silk suture to the arm of a dual mode ergometer ( 300C-LR , Aurora Scientific , Aurora , ON , Canada ) and the proximal tendon secured to a stationary post .\",\n \"Muscles were stimulated with an electrical stimulator ( 701C , Aurora Scientific , Aurora , ON , Canada ) using parallel platinum plate electrodes extending along the muscle .\",\n \"Muscle slack length was set by increasing muscle length until passive force was detectable above the noise of the transducer and fiber length was measured through a micrometer reticule in the eyepiece of a dissecting microscope .\",\n \"Optimal muscle length was then determined by incrementally increasing the length of the muscle by 10% of slack fiber length until the isometric tetanic force plateaued .\",\n \"At this optimum length , force was recorded during a twitch contraction and isometric tetanic contraction ( 300 ms train of 0 . 3 ms pulses at 225 Hz ) .\",\n \"The muscle was then fatigued with a bout of repeated tetanic contractions every 10 s until force dropped below 50% of peak .\",\n \"At this point , the muscle was cut from the sutures and weighed .\",\n \"This weight , along with peak fiber length and muscle density ( 1 . 056 g/cm3 ) , was used to calculate the physiological cross-sectional area ( PCSA ) and convert to specific force ( tension ) .\",\n \"The experimental data were analyzed and quantified using Matlab ( Mathworks ) , and presented as peak tetanic tension ( Tetanic Tension ) – peak of the force recording during the tetanic contraction , normalized to PCSA; Time to fatigue ( TTF ) – time for the tetanic tension to fall below 50% of the peak value during the fatigue test; Half relaxation time ( HRT ) – half the time between force peak and return to baseline during the twitch contraction .\",\n \"Cellular respiration was quantified in primary myotubes using the XF24 extracellular flux ( XF ) bioanalyzer ( Agilent Technologies/Seahorse Bioscience , North Billerica , MA , USA ) .\",\n \"Primary skeletal muscle cells isolated from Lrrc8a fl/fl mice were plated on BD Matrigel‐coated plate at a density of 20 × 103 per well .\",\n \"After 24 hr , cells were incubated in Ad5-CMV-mCherry or Ad5-CMV-Cre-mCherry ( MOI 90–100 ) in DMEM-F12 media ( 2% FBS and 1% penicillin-streptomycin ) for 24 hr .\",\n \"Cells were then switched to differentiation media for another 3 days .\",\n \"For insulin-stimulation , cells were incubated in serum free media for 4 hr and stimulated with 0 and 10 nM insulin for 2 hr .\",\n \"Subsequently , medium was changed to XF‐DMEM , and kept in a non‐CO2 incubator for 60 min .\",\n \"The basal oxygen consumption rate ( OCR ) was measured in XF‐DMEM .\",\n \"Subsequently , oxygen consumption was measured after addition of each of the following compounds: oligomycin ( 1 μg/ml ) ( ATP-Linked OCR ) , carbonyl cyanide 4‐ ( trifluoromethoxy ) phenylhydrazone ( FCCP; 1 μM ) ( Maximal Capacity OCR ) and antimycin A ( 10 μM; Spare Capacity OCR ) ( Wende et al . , 2015 ) .\",\n \"For the glycolysis stress test , prior to experimentation , cells were switched to glucose‐free XF‐DMEM and kept in a non‐CO2 incubator for 60 min .\",\n \"Extracellular acidification rate ( ECAR ) was determined in XF‐DMEM followed by these additional conditions: glucose ( 10 mM ) , oligomycin ( 1 μM ) , and 2‐DG ( 100 mM ) .\",\n \"Data for Seahorse experiments ( normalized to protein ) reflect the results of one Seahorse run/condition with six replicates .\",\n \"Mouse body composition ( fat and lean mass ) was measured by nuclear magnetic resonance ( NMR; Echo-MRI 3-in-1 analyzer , EchoMRI , LLC ) .\",\n \"For glucose tolerance test ( GTT ) , mice were fasted for 6 hr and intraperitoneal injection of glucose ( 1 g/kg body weight for lean mice and 0 . 75 g/kg of body weight for HFD mice ) administered .\",\n \"Glucose level was monitored from tail-tip blood using a glucometer ( Bayer Healthcare LLC ) at the indicated times .\",\n \"For insulin tolerance test ( ITT ) , mice were fasted for 4 hr and after an intra-peritoneal injection of insulin ( HumulinR , 1 U/kg for lean mice and 1 . 25 U/kg for HFD mice ) glucose level was measured by glucometer at the indicated times .\",\n \"Data are represented as mean ± s . e . m . Two-tail paired or unpaired Student’s t-tests were used for comparison between two groups .\",\n \"For three or more groups , data were analyzed by one-way analysis of variance and Tukey’s post hoc test .\",\n \"For GTTs and ITTs , two-way analysis of variance ( Anova ) was used .\",\n \"A p-value<0 . 05 was considered statistically significant .\",\n \"* , ** and *** represents a p-value less than 0 . 05 , 0 . 01 and 0 . 001 respectively .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Maintenance of skeletal muscle is beneficial in obesity and Type 2 diabetes .","Mechanical stimulation can regulate skeletal muscle differentiation , growth and metabolism; however , the molecular mechanosensor remains unknown .","Here , we show that SWELL1 ( Lrrc8a ) functionally encodes a swell-activated anion channel that regulates PI3K-AKT , ERK1/2 , mTOR signaling , muscle differentiation , myoblast fusion , cellular oxygen consumption , and glycolysis in skeletal muscle cells .","LRRC8A over-expression in Lrrc8a KO myotubes boosts PI3K-AKT-mTOR signaling to supra-normal levels and fully rescues myotube formation .","Skeletal muscle-targeted Lrrc8a KO mice have smaller myofibers , generate less force ex vivo , and exhibit reduced exercise endurance , associated with increased adiposity under basal conditions , and glucose intolerance and insulin resistance when raised on a high-fat diet , compared to wild-type ( WT ) mice .","These results reveal that the LRRC8 complex regulates insulin-PI3K-AKT-mTOR signaling in skeletal muscle to influence skeletal muscle differentiation in vitro and skeletal myofiber size , muscle function , adiposity and systemic metabolism in vivo ."],"string":"[\n \"Maintenance of skeletal muscle is beneficial in obesity and Type 2 diabetes .\",\n \"Mechanical stimulation can regulate skeletal muscle differentiation , growth and metabolism; however , the molecular mechanosensor remains unknown .\",\n \"Here , we show that SWELL1 ( Lrrc8a ) functionally encodes a swell-activated anion channel that regulates PI3K-AKT , ERK1/2 , mTOR signaling , muscle differentiation , myoblast fusion , cellular oxygen consumption , and glycolysis in skeletal muscle cells .\",\n \"LRRC8A over-expression in Lrrc8a KO myotubes boosts PI3K-AKT-mTOR signaling to supra-normal levels and fully rescues myotube formation .\",\n \"Skeletal muscle-targeted Lrrc8a KO mice have smaller myofibers , generate less force ex vivo , and exhibit reduced exercise endurance , associated with increased adiposity under basal conditions , and glucose intolerance and insulin resistance when raised on a high-fat diet , compared to wild-type ( WT ) mice .\",\n \"These results reveal that the LRRC8 complex regulates insulin-PI3K-AKT-mTOR signaling in skeletal muscle to influence skeletal muscle differentiation in vitro and skeletal myofiber size , muscle function , adiposity and systemic metabolism in vivo .\"\n]"},"summary":{"kind":"list like","value":["Skeletal muscles – the force-generating tissue attached to bones – must maintain their mass and health for the body to work properly .","It is therefore necessary to understand how an organism can regulate the way skeletal muscles form , grow and heal .","A multitude of factors can control how muscles form , including mechanical signals .","The molecules that can sense these mechanical stimuli , however , remain unknown .","Mechanoresponsive ion channels provide possible candidates for these molecular sensors .","These proteins are studded through the cell membranes , where they can respond to mechanical changes by opening and allowing the flow of ions in and out of a cell , or by changing interactions with other proteins .","The SWELL1 protein is a component of an ion channel known as VRAC , which potentially responds to mechanical stimuli .","This channel is associated with many biological processes such as cells multiplying , migrating , growing and dying , but it is still unclear how .","Here , Kumar et al . first tested whether SWELL1 controls how skeletal muscle precursors mature into their differentiated and functional form .","These experiments showed that SWELL1 regulates this differentiation process under the influence of the hormone insulin , as well as mechanical signals such as cell stretching .","In addition , this work revealed that SWELL1 relies on an adaptor molecule called GRB2 to relay these signals in the cell .","Next , Kumar et al . genetically engineered mice lacking SWELL1 only in skeletal muscle .","These animals had smaller muscle cells , as well as muscles that were weaker and less enduring .","When raised on a high-calorie diet , the mutant mice also got more obese and developed resistance to insulin , which is an important step driving obesity-induced diabetes .","Together , these findings show that SWELL1 helps to regulate the formation and function of muscle cells , and highlights how an ion channel participates in these processes .","Healthy muscles are key for overall wellbeing , as they also protect against obesity and obesity-related conditions such as type 2 diabetes or nonalcoholic fatty liver disease .","This suggests that targeting SWELL1 could prove advantageous in a clinical setting ."],"string":"[\n \"Skeletal muscles – the force-generating tissue attached to bones – must maintain their mass and health for the body to work properly .\",\n \"It is therefore necessary to understand how an organism can regulate the way skeletal muscles form , grow and heal .\",\n \"A multitude of factors can control how muscles form , including mechanical signals .\",\n \"The molecules that can sense these mechanical stimuli , however , remain unknown .\",\n \"Mechanoresponsive ion channels provide possible candidates for these molecular sensors .\",\n \"These proteins are studded through the cell membranes , where they can respond to mechanical changes by opening and allowing the flow of ions in and out of a cell , or by changing interactions with other proteins .\",\n \"The SWELL1 protein is a component of an ion channel known as VRAC , which potentially responds to mechanical stimuli .\",\n \"This channel is associated with many biological processes such as cells multiplying , migrating , growing and dying , but it is still unclear how .\",\n \"Here , Kumar et al . first tested whether SWELL1 controls how skeletal muscle precursors mature into their differentiated and functional form .\",\n \"These experiments showed that SWELL1 regulates this differentiation process under the influence of the hormone insulin , as well as mechanical signals such as cell stretching .\",\n \"In addition , this work revealed that SWELL1 relies on an adaptor molecule called GRB2 to relay these signals in the cell .\",\n \"Next , Kumar et al . genetically engineered mice lacking SWELL1 only in skeletal muscle .\",\n \"These animals had smaller muscle cells , as well as muscles that were weaker and less enduring .\",\n \"When raised on a high-calorie diet , the mutant mice also got more obese and developed resistance to insulin , which is an important step driving obesity-induced diabetes .\",\n \"Together , these findings show that SWELL1 helps to regulate the formation and function of muscle cells , and highlights how an ion channel participates in these processes .\",\n \"Healthy muscles are key for overall wellbeing , as they also protect against obesity and obesity-related conditions such as type 2 diabetes or nonalcoholic fatty liver disease .\",\n \"This suggests that targeting SWELL1 could prove advantageous in a clinical setting .\"\n]"},"year":{"kind":"string","value":"2020"}}},{"rowIdx":4296,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["chromosomes and gene expression","biochemistry and chemical biology"],"string":"[\n \"chromosomes and gene expression\",\n \"biochemistry and chemical biology\"\n]"},"title":{"kind":"string","value":"The insulin receptor cellular IRES confers resistance to eIF4A inhibition"},"id":{"kind":"string","value":"elife-00542-v1"},"sections":{"kind":"list like","value":[["During times of stress the cell changes its gene expression profile to better manage the cause of the stress .","Coordinate changes in both transcription and translation occur ( Sengupta et al . , 2010; Spriggs et al . , 2010 ) .","A central pathway that responds to stress stimuli by controlling both protein and RNA synthesis is the insulin and insulin-like receptor-signaling ( IIS ) pathway .","The fundamental molecular architecture of the IIS pathway is conserved from flies to man ( Figure 1 ) ( Oldham , 2011 ) .","When IIS signaling is high , the protein kinase AKT is activated ( Ruggero and Sonenberg , 2005 ) .","AKT directly phosphorylates the Foxo family of transcription factors and consequently prevents activated transcription of Foxo target genes ( Brunet et al . , 1999 ) .","AKT also stimulates the activation of the mechanistic target of rapamycin ( mTOR ) protein ( Zoncu et al . , 2011 ) . 10 . 7554/eLife . 00542 . 003Figure 1 . Simplified insulin/insulin-like growth factor signaling diagram .","( A ) When Insulin receptor or Insulin-like growth factor receptor is active signaling through AKT inhibits Foxo transcription factors and activates mTOR .","mTOR in turn inhibits 4E-BP and activates S6K .","S6K in turn inhibits Pdcd4 and activates eIF4B .","When insulin signaling is low inhibition of Foxo is relieved and Foxo activates the transcription of Insulin receptor and 4E-BP .","The broken line indicates the proposed activation of Pdcd4 by Foxo .","( B ) Alignment of human ( Hs top ) and Drosophila ( Dm bottom ) Pdcd4 proteins .","Conserved Akt and S6K phosphorylation sites are indicated by asterisk .","Conserved MA3 domains are indicated by shaded boxes .","Arrowheads indicate conserved acidic residues important for eIF4A binding in humans .","( C ) eIF4A interacts with Pdcd4 in Drosophila cells .","Cytoplasmic extracts from a saturated culture of S2 cells were subjected to immunoprecipitation with antisera directed against eIF4A or preimmune serum .","Pdcd4 was detected with antisera against Pdcd4 .","( D ) Mutant Pdcd4 binds less efficiently to eIF4A than wildtype .","Cytoplasmic extracts from cultures of S2 cells expression wild-type Myc-Pdcd4 or mutant Myc-Pdcd4 ( AA ) were subjected to immunoprecipitation with antisera directed against eIF4A .","Myc-Pdcd4 was detected with mouse monoclonal antibody to the Myc tag .","Immunoprecipitated eIF4A was detected with rabbit antisera .","( E ) Immobilized Drosophila Pdcd4 interacts with Drosophila eIF4A .","On the top is a cartoon of approach .","On the bottom is an immnoblot of proteins eluted from the affinity columns .","Position of the recombinant eIF4A is indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 00542 . 003 Activated mTOR stimulates general translation , in part , by influencing the activity of the translation initiation complex eIF4F .","The eIF4F complex consists of eIF4E , the 7-methyl-Guanosine-cap ( m7G ) binding protein , eIF4A , an RNA helicase , and eIF4G , a large scaffolding protein .","In addition , the RNA binding protein eIF4B can associate with eIF4F to stimulate the helicase activity of eIF4A ( Ma and Blenis , 2009; Sonenberg and Hinnebusch , 2009; Zoncu et al . , 2011 ) .","mTOR stimulates general translation in part by inactivating translational inhibitors .","mTOR phosphorylates and inactivates the translation repressor eIF4E binding protein ( 4E-BP ) ( Gingras et al . , 1999 ) allowing efficient formation of the eIF4F complex .","In addition , mTOR activates ribosomal protein S6 kinase ( S6K ) ( Sarbassov et al . , 2005 ) .","S6K stimulates the helicase eIF4A by activating eIF4B and inhibiting programmed cell death protein 4 ( Pdcd4 ) , a known eIF4A inhibitor ( Figure 1 ) ( Yang et al . , 2003; Raught et al . , 2004; Dorrello et al . , 2006 ) .","Thus under conditions of high signaling through AKT and mTOR , cap-dependent translation is stimulated .","In times of stress , low levels of signaling through the IIS pathway lead to activated Foxo and 4E-BP in addition to inactive S6K .","Foxo moves to the nucleus and controls the transcription of its target genes ( Salih and Brunet , 2008 ) .","4E-BP prevents formation of the translation initiation complex eIF4F , thereby inhibiting m7G-dependent translation , and S6K no longer stimulates eIF4A .","This in turn leads to lower levels of global protein synthesis .","Thus the IIS pathway controls gene expression with two different branches: transcription of Foxo target genes and m7G-cap-dependent translation through 4E-BP and S6K .","The IIS pathway in Drosophila contains a mechanism that functionally couples activated transcription to translation .","A portion of the system includes a signaling and gene expression feedback loop for direct genetic targets of Drosophila Foxo .","The insulin-like receptor ( INR ) and 4E-BP genes are conserved transcriptional targets of Foxo ( Figure 1 ) ( Puig et al . , 2003; Puig and Tjian , 2005; Marr et al . , 2007; Hu et al . , 2008 ) .","Paradoxically , the insulin receptor protein , as well as mRNA , is being synthesized and accumulating under the same conditions when 4E-BP activity and expression is induced and S6K is inhibited ( Marr et al . , 2007 ) .","Foxo activates the transcription of the Drosophila insulin receptor gene from three promoters .","Each promoter produces a transcript with a distinct 5′ untranslated region ( UTR ) but identical coding region ( Casas-Tinto et al . , 2007; Marr et al . , 2007 ) .","Transcripts derived from promoter 1 are by far the most abundant and ubiquitous form of INR transcript ( Casas-Tinto et al . , 2007; Marr et al . , 2007 ) .","The Drosophila INR 5′ UTRs contain an internal ribosome entry site ( IRES ) that allows the message to escape 4E-BP inhibition of cap-dependent translation .","This mechanism provides a functional coupling of transcription and translation in times of stress that allows amplification of insulin receptor expression ( Marr et al . , 2007 ) .","Because IRES containing transcripts can outcompete cap-dependent transcripts under these conditions their translation is actually stimulated ( Svitkin et al . , 2005; Marr et al . , 2007 ) .","This leads to an effective switch of the cellular translation machinery to targets of the IIS pathway .","Thus , Foxo targets impose a translational program by activation of genes that repress general translation while simultaneously activating targets that are immune to this translational control .","This provides these targets with a competitive advantage allowing them to utilize the translation machinery that is freed by the general inhibition .","Here we identify Drosophila Pdcd4 as an additional Foxo target further enhancing the coupling of transcription and translation regulation in the IIS pathway .","Since the IIS pathway targets translation initiation through control of both eIF4E and eIF4A , we wondered if the most abundant and ubiquitous Drosophila INR 5′ UTR would also provide resistance to inhibition of eIF4A activity .","To answer this question we used both an in vitro translation system and a cell based assay to investigate the eIF4A requirements for efficient translation of reporters containing the INR 5′ UTR from Drosophila .","Because mammalian systems show the same type of regulation , we also investigated the role of eIF4A inhibition in the murine insulin receptor and insulin-like growth factor receptors .","( Giraud et al . , 2001; Meng et al . , 2008; Spriggs et al . , 2009b ) .","We find , in both the Drosophila and mouse systems , that the 5′ UTRs of the mRNAs for these receptors provide resistance to both eIF4E and eIF4A inhibition .","Taken together , these results indicate that these cellular messages have some of the lowest requirement for eIF4F activity for translation identified to date ."],["A connection between Foxo activation and translation inhibition was identified when it was discovered that 4E-BP expression is controlled by Foxo in Drosophila and mouse cells ( Junger et al . , 2003; Puig et al . , 2003; Hu et al . , 2008 ) .","Since the IIS pathway is also known to control eIF4A activity through Pdcd4 ( Figure 1A ) , we tested if this gene is under direct Foxo control in Drosophila .","Blast analysis of human Pdcd4 with the Drosophila genome identifies a single homologous protein encoded by CG10990 .","Alignment of human PDCD4 and CG10990 indicate that important regions of the protein are conserved ( Figure 1B ) ( Cash and Andrews , 2012 ) .","The two MA3 domains , including the acidic residues shown to be important for the interaction with eIF4A are conserved ( Chang et al . , 2009; Waters et al . , 2011 ) .","In addition , the Akt and S6K phosphorylation sites are conserved ( Palamarchuk et al . , 2005; Dorrello et al . , 2006 ) .","To determine if the interaction with eIF4A is conserved , we immunoprecipitated Drosophila eIF4A from cytoplasmic extracts derived from a saturated culture of Drosophila S2 cells .","Associated Pdcd4 was detected by immunoblot .","Pdcd4 is co-precipitated with antisera against eIF4A but not with preimmune serum indicating that Pdcd4 and eIF4A interact in Drosophila cells ( Figure 1C ) .","We next created Myc-tagged expression constructs for Drosophila Pdcd4 , one wildtype construct and a construct containing mutations in conserved residues in the first MA3 domain ( E282A , D286A ) .","The analogous mutations in human PDCD4 destabilize the interaction with eIF4A ( Chang et al . , 2009; Waters et al . , 2011 ) .","The constructs were transfected into growing Drosophila S2 cells .","Subsequent immunoprecipitation of eIF4A from these cells reveals a decreased association of the mutant Pdcd4 with eIF4A ( Figure 1D ) .","This indicates the mutations induce the same destabilization of the eIF4A–Pdcd4 interaction in Drosophila cells .","Interestingly under these conditions we detect multiple forms of Pdcd4 by western blot , most likely phosphorylated forms of Pdcd4 , and only the fastest migrating species associates with eIF4A .","To determine if Drosophila Pdcd4 can interact directly with eIF4A we used an affinity chromatography assay using recombinant proteins purified from Escherichia coli .","A GST fusion to Drosophila Pdcd4 was immobilized on glutathione agarose and recombinant Drosophila eIF4A was passed over the column ( Figure 1D ) .","As a control GST was also immobilized on glutathione agarose .","The eIF4A bound to the immobilized GST-Pdcd4 but not to GST alone indicating that Drosophila Pdcd4 can interact directly with Drosophila eIF4A .","Taken together these data are consistent with the notion that CG10990 is the Drosophila homologue of human PDCD4 .","There are hints in the literature , based on microarray experiments , indicating this gene is induced in response to nutrient stress and might be controlled by Foxo ( Gershman et al . , 2007 ) .","In an effort to determine if Foxo binds to the Pdcd4 gene in nutrient stressed animals we reanalyzed the only publically available Foxo ChIP ( Chromatin immunoprecipitation ) dataset ( Teleman et al . , 2008 ) .","These experiments were performed on starved larva .","We find Foxo binds the Pdcd4 gene in both the promoter and intronic regions with enrichment values as high as 16-fold over background ( Figure 2A ) .","To corroborate this finding , we performed ChIP on genomic DNA from a cell line with an inducible Foxo cDNA gene that has been modified so the Foxo protein produced is constitutively active because it is immune to the negative regulation by insulin signaling ( FoxoCA ) ( Puig et al . , 2003; Gershman et al . , 2007 ) .","This allows us to induce Foxo under conditions of high nutrient signaling and remove possible crosstalk from upstream signaling pathways .","We tested the enrichment of genomic sequences by qPCR using primers to the Pdcd4 promoter region ( Figure 2A ) compared to a region in the first intron of CG15414 , a gene just downstream of 4E-BP .","We find FoxoCA binds to the promoter region of Pdcd4 at levels comparable to a well-defined direct target , 4E-BP ( Junger et al . , 2003; Puig et al . , 2003; Marr et al . , 2007 ) ( Figure 2B ) .","To determine the effect on mRNA production under these conditions we performed quantitative RT-qPCR on induced cells .","We find that the steady-state level of Pdcd4 mRNA is increased about threefold in cells expressing active Foxo ( Figure 2C ) .","To determine if the effect is due to mRNA stability changes or new transcription we assayed intron-containing pre-mRNAs by RT-qPCR .","Since most splicing is co-transcriptional in Drosophila this is a good assay for new RNA synthesis ( Khodor et al . , 2011 ) .","We find that Pdcd4 pre-mRNA is increased , indicating an increase in transcription of the gene .","The increased mRNA also leads to increased protein synthesis as determined by immuno-blot with antibodies directed against Drosophila Pdcd4 ( Figure 2D ) .","This is likely an underestimate of the effect since these experiments are all done under high serum and insulin conditions that should result in the rapid turnover of Pdcd4 protein ( Dorrello et al . , 2006 ) . 10 . 7554/eLife . 00542 . 004Figure 2 . Foxo activates Pdcd4 in Drosophila cells .","( A ) Reanalysis of ChIP-chip data from Teleman et al . ( 2008 ) .","Genomic Browser view of Foxo binding to the Pdcd4 locus in starved larva .","The data are plotted as the enrichment ( log2 ) over mock precipitated samples .","Primers used for ChIP and qPCR are indicated .","( B ) ChIP of Foxo at 4E-BP promoter and Pdcd4 locus in Drosophila S2 cells expressing constitutively active Foxo ( FoxoCA ) .","The data are plotted as fold enrichment over a background region 1 kb downstream of 4E-BP .","Uninduced samples are plotted in white , induced samples in black ( error bars indicate SD ) .","( C ) RT-qPCR of Pdcd4 mRNA and pre-mRNA in Drosophila S2 cells expressing FoxoCA .","Data are plotted as fold-induction ( error bars indicate SD ) .","( D ) Immunoblot of total protein from Drosophila S2 cells expressing FoxoCA .","Positions of Pdcd4 and tubulin are indicated .","( E ) 4E-BP , GstD1 , and Pdcd4 RNA levels in untreated and paraquat-treated animals .","The levels of RNA were normalized to RP49 and are plotted as fold-induction relative to untreated animals ( error bars indicate SEM ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 00542 . 004 We tested if Foxo controls Pdcd4 in adult flies subjected to stress .","Adult wildtype or Foxo null flies ( Slack et al . , 2011 ) were treated with paraquat to induce oxidative stress and assayed for expression of 4E-BP , Pdcd4 , and GstD1 by RT-qPCR .","Consistent with previous results ( Wang et al . , 2005 ) , 4E-BP is induced by paraquat in a Foxo dependent manner .","Like 4E-BP , we find that Pdcd4 is induced in response to paraquat in a Foxo dependent manner ( Figure 2E ) .","To determine if the effect was due to loss of general oxidative stress response or if it is Foxo specific we examined the induction of GstD1 , a gene controlled by Nrf2 in response to oxidative stress ( Misra et al . , 2011 ) .","We find that GstD1 is still responsive indicating that the effects at Pdcd4 and 4E-BP are Foxo-specific and not due to a loss of responsiveness in the mutant flies ( Figure 2E ) .","These results are consistent with the idea that in addition to controlling the cap-binding complex through 4E-BP , active Foxo can influence eIF4A through activation of the Pdcd4 gene in response to stress .","Given that Foxo is activating transcription of the INR gene while Pdcd4 is also active , we hypothesized that since the INR mRNA is translated efficiently under these conditions ( Marr et al . , 2007 ) it must be at least partially immune to diminished eIF4A activity .","To determine the effect of increased Pdcd4 on the translation of insulin receptor UTR containing RNAs we modified a dicistronic mRNA assay which we previously used to investigate the effects of 4E-BP on insulin receptor translation in Drosophila ( Marr et al . , 2007 ) .","In this assay a construct is used that produces a RNA in which the open reading frames of renilla luciferase and firefly luciferase are present on the same transcript ( Figure 3A ) .","Renilla luciferase levels are an indication of total message produced in the cell and firefly luciferase levels are an indication of IRES dependent translation .","As previously reported , insertion of the INR 5′ UTR between the ORFs promotes translation of the second ORF ( Marr et al . , 2007 ) .","The levels of IRES activity of the INR UTR are comparable to the well-characterized IRES from Hepatitis C Virus ( Figure 3B ) ( Tsukiyama-Kohara et al . , 1992 ) .","The INR UTR and the HCV IRES both produce more firefly signal than the empty vector ( Figure 3D ) . 10 . 7554/eLife . 00542 . 005Figure 3 . Drosophila insulin receptor 5′UTR provides resistance to Pdcd4 . ( A ) Diagram of dicistronic reporters .","Translation of the Firefly ORF requires internal ribosome entry .","Firefly to renilla activity ratio provides an indication of IRES activity .","( B ) The Drosophila Insulin receptor UTR provides IRES activity comparable to the activity of HCV .","( C ) Renilla activity of the reporters .","( D ) Firefly activity of the reporters .","( E ) Dicistronic reporter activities in the presence of 4E-BP or Pdcd4 expression .","4E-BP and Pdcd4 stimulate the IRES activity of the insulin receptor UTRs .","Mutation of the critical acidic residues of Pdcd4 prevents the stimulation .","( F ) Renilla activity of the reporters in the presence of expressed proteins .","( G ) Firefly activity of the reporters in the presence of the expressed proteins ( error bars indicate SEM ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 00542 . 005 To determine the effect of Pdcd4 on the INR UTR we expressed Pdcd4 in Drosophila S2 cells and measured the activity of the dicistronic reporter .","In order to more accurately determine the effects of the protein in question we modified the assay so the production of the dicistronic mRNA is inducible .","This allows accumulation of the experimental protein in the cell before the dicistronic mRNA is produced giving a more precise measure of the effects on the activity of the dicistronic message .","The levels of expression of the second ORF relative to the first ORF are an indication of the cap-independent translation potential of the insert .","To validate this assay we reproduced our previous results with 4E-BP ( Marr et al . , 2007 ) .","As reported previously , expression of 4E-BP stimulates the translation of the second open reading frame in a reporter containing the INR 5′UTR ( Figure 3E , G ) .","Expression of Pdcd4 also stimulates translation of the second ORF dependent on the INR 5′UTR similar to the effects seen with the HCV IRES ( Figure 3E , G ) .","These effects are specific .","Mutation of the key acidic residues ( Figure 1B ) in Pdcd4 shown to disrupt eIF4A binding in the human system ( Waters et al . , 2011 ) prevent the IRES stimulation .","To address the role of eIF4A in insulin receptor translation , we used a highly specific small molecule inhibitor of eIF4A , hippuristanol ( Bordeleau et al . , 2006; Lindqvist et al . , 2008 ) .","Hippuristanol is a potent translation inhibitor that works in eukaryotes from yeast to human ( Lindqvist et al . , 2008 ) .","Hippuristanol inhibits the ATPase activity and RNA binding of eIF4A ( Bordeleau et al . , 2006; Lindqvist et al . , 2008 ) .","The small molecule binds to the protein in conserved regions V and VI in eIF4A homologues ( Lindqvist et al . , 2008 ) .","Importantly , the effects on translation can be rescued by addition of either wild-type or mutant forms of eIF4A that are immune to hippuristanol indicating that the effects are highly specific for eIF4A ( Bordeleau et al . , 2006; Lindqvist et al . , 2008 ) .","This small molecule had been used previously in the Drosophila system to investigate eIF4A requirements ( Iwasaki et al . , 2009 ) .","We performed in vitro competitive translation experiments with capped and polyadenylated firefly luciferase reporters ( Figure 4A ) and a Drosophila embryo extract translation system that has not been treated with micrococcal nuclease ( Gebauer et al . , 1999; Marr et al . , 2007 ) in the presence of hippuristanol .","The RNA reporters contain the 5′ UTR from the Drosophila insulin receptor .","In addition we include two control RNAs .","One control RNA contains a non-specific UTR derived from plasmid sequences .","The other RNA contains the IRES from the Hepatitis C virus ( HCV ) ( Tsukiyama-Kohara et al . , 1992 ) .","This IRES does not require eIF4A activity and controls for non-specific effects on the extract ( Pestova et al . , 1998 ) .","Under the experimental conditions , translation of the first control RNA is strongly inhibited by hippuristanol while the reporter containing the HCV IRES is completely resistant to eIF4A inhibition ( Figure 4A ) .","This small molecule inhibitor exposed a greatly diminished role for eIF4A in the Drosophila INR UTR mediated translation ( Figure 4A ) .","At the low and moderate concentrations of hippuristanol , the Drosophila INR UTR reporter retains almost complete activity comparable to the HCV UTR .","Even at the highest concentration of hippuristanol tested , the reporter containing the INR UTR retains >50% of the original translation activity .","To determine the effect of Pdcd4 in this system we added recombinant Pdcd4 to the translation extract .","Consistent with the data using hippuristanol , we find Pdcd4 can inhibit the control RNA but not the Drosophila INR UTR or the HCV IRES ( Figure 4B ) .","These finding suggests that translation of the most abundant Drosophila INR transcript can tolerate inhibition of eIF4A . 10 . 7554/eLife . 00542 . 006Figure 4 . Drosophila insulin receptor 5′UTR provides resistance to eIF4a inhibition .","( A ) Top: Diagram of RNAs used in the in vitro translation assays .","Bottom: Titration of hippuristanol in in vitro translation assays .","The shade of the bars indicates the final concentration of hippuristanol in the assay .","The legend appears above the graph .","Data are plotted as the fraction activity of the carrier treated extracts ( error bars indicate SEM ) .","( B ) Top: Diagram of RNAs used in the in vitro translation assays .","Bottom: Activity of these RNAs in in vitro translation assays in the absence ( white bars ) and presence ( black bars ) of Drosophila Pdcd4 ( error bars indicate SEM ) .","( C ) Dicistronic RNA translation in vitro .","Top: Diagram of RNAs used in the in vitro translation assays .","Bottom: Firefly to renilla ratio in the absence ( white bars ) and presence ( black bars ) of hippuristanol .","( D ) Top: Renilla activity in the dicistronic assay .","Bottom: Firefly activity in the dicistronic assay .","Shading as in C . Percentage above the bars indicates activity after hippuristanol addition relative to carrier treated samples . DOI: http://dx . doi . org/10 . 7554/eLife . 00542 . 006 We used a dicistronic RNA in the in vitro translation assay to directly test the IRES activity under hippuristanol treatment ( Figure 4C top ) .","Dicistronic RNAs were synthesized in vitro using T7 RNA polymerase .","The RNA was capped and polyadenylated and used to program the same Drosophila embryo translation system described above .","The extracts were treated with either hippuristanol or carrier .","Consistent with the monocistronic assay , both the INR and the HCV IRES containing transcripts show increased relative translation of the second ORF upon eIF4A inhibition ( Figure 4C ) .","The increase is due both to a resistance of the second ORF to the inhibition and a decrease in the cap-dependent translation of the first ORF ( Figure 4D ) .","Under these conditions there is a small amount of cryptic translation of the second ORF in the control transcript .","However both the renilla and firefly activites respond the same to the hippuristanol treatment .","Because the molecular architecture of the IIS signaling pathway is conserved in mammals , we wondered if this level of regulation would be conserved in mammals .","To address this , we cloned the 5′ UTR from the longest mRNAs for the mouse insulin receptor ( mINR ) and the mouse insulin-like growth factor receptor-I ( IGFR ) and created firefly luciferase reporters under the control of these UTRs ( Figure 5A ) .","Previously , it was reported that INR and IGFR UTRs confer cap-independent translation activity in human and rat ( Giraud et al . , 2001; Spriggs et al . , 2009b ) .","To extend this finding to the mouse system and ensure that our competitive rabbit reticulocyte system was capable of supporting cap-independent translation , we assayed translation of the mINR and IGFR reporters in the presence of excess m7G cap along with our control RNA and the HCV IRES reporters described above ( Figure 5A ) .","While the control RNA is inhibited by excess cap , translation from the 5′ UTR of mINR and IGFR is not only resistant to excess m7G cap but the activity actually increases in the presence of excess m7G cap .","This observation is common with UTRs that contain an IRES ( Svitkin et al . , 2005 ) .","The increase in activity for both the mINR and IGFR UTRs exceeded the IRES activity of the HCV UTR .","This indicates that mINR UTR and IGFR UTR are capable of conferring cap-independent translation initiation . 10 . 7554/eLife . 00542 . 007Figure 5 . Mammalian insulin receptor and insulin-like growth factor receptor 5′UTR provide resistance to eIF4a inhibition .","( A ) Diagram of RNAs used in the in vitro translation assays .","( B ) In vitro Translation in the absence ( white bars ) and presence ( black bars ) of excess m7G analogue .","Data are plotted as the fraction activity of the mock treated extracts ( error bars indicate SEM ) .","( C ) Titration of hippuristanol in in vitro translation assays .","Data are plotted as the fraction activity of the mock treated extracts ( error bars indicate SEM ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 00542 . 007 To directly test the ability of these UTRs to allow internal ribosome entry we performed a dicistronic assay in mammalian cells .","The UTRs were subcloned into a plasmid construct between the renilla and firefly open reading frames controlled by the RSV LTR .","Both the mINR and the IGFR UTRs supported substantial firefly activity compared to the original vector .","This is apparent both in the firefly to renilla ratio and in the raw firefly activity units ( Figure 5B ) .","This combined with the in vitro translation assays strongly suggest that the mINR and IGFR UTRs can support cap-independent translation .","To test for conservation of resistance to eIF4A inhibition , hippuristanol was titrated into the rabbit reticulocyte translation system .","As expected , the activity of the control RNA is reduced to <10% of the mock treated extract , and the HCV IRES is completely resistant to hippuristanol ( Figure 5C ) .","In fact , the HCV IRES is stimulated fourfold by addition of hippuristanol under these conditions .","Both mINR and IGFR UTRs confer resistance to hippuristanol ( Figure 5C ) .","Even at the highest concentrations of the small molecule the mINR and mIGFR UTRs remain roughly 50% active indicating a decreased requirement for eIF4A relative to the control RNA ."],["Stress responses controlled through the IIS pathway result in changes in both mRNA synthesis and protein synthesis .","These processes are coordinated to ensure proper expression of the downstream targets .","Under the same low IIS signaling conditions that activate 4E-BP , Pdcd4 is stabilized resulting in the inhibition of the DEAD box helicase eIF4A .","Thus , under these conditions the eIF4F complex is repressed by two mechanisms .","At the same time , the Foxo family of transcription factors is active and increasing the synthesis of certain mRNAs , one of which is the INR transcript itself .","Previously we identified a connection between 4E-BP mediated inhibition of the cap-binding complex and INR mRNA translation in Drosophila ( Marr et al . , 2007 ) .","The INR message is immune to the 4E-BP translational repression and thus is preferentially translated under low signaling conditions coupling the increase in mRNA expression to an increase in protein synthesis .","In the work presented here we extend this observation of gene expression coordination of INR mRNA to the eIF4A branch of the IIS signaling pathway .","First , we show that active Foxo is capable of directly stimulating the transcription of Pdcd4 , analogous to the activation of 4E-BP seen previously under these same conditions ( Junger et al . , 2003; Puig et al . , 2003 ) .","This provides a mechanism for the pathways controlling Foxo to enhance the inhibition of eIF4A when stressed or when nutrients are low .","Second we show that the 5′ UTR of the most abundant Drosophila INR transcript provides resistance to eIF4A inhibition comparable to the resistance seen with the HCV IRES that does not require eIF4A .","A similar finding has been seen for the Drosophila reaper 5′ UTR ( Hernandez et al . , 2004; Iwasaki et al . , 2009 ) .","The data presented above also support the conservation of the cap-independent mechanism of translation initiation of the insulin receptor and insulin-like growth factor receptor mRNAs in mammals .","The mouse transcripts show resistance to hippuristanol under conditions that almost completely inhibit a control RNA indicating that the resistance to eIF4A inhibition is also conserved .","There are no easily recognizable conserved sequence elements between the Drosophila and mouse UTRs , but the mode of regulation is conserved suggesting an important role for this type of translational control .","This defines a functional characteristic of the insulin receptor and IGF receptor transcripts that is conserved across hundreds of millions of years of evolution ( from flies to mammals ) .","Taken together with previous work , these data indicate that the coupling of transcription to translation of insulin receptor mRNA mediated by Foxo targets can culminate in an activated translational response .","Our findings highlight a unique characteristic of the insulin receptor and IGF receptor UTRs that differentiates them from other cellular transcripts .","In addition to being immune to 4E-BP , these IRESes are resistant to eIF4A inhibition .","While viral IRESes are fairly common , cellular IRESes are rare and relatively unexplored .","Where it has been explored , most cellular IRESes have a strong requirement for eIF4A ( Thoma et al . , 2004; Spriggs et al . , 2009a ) .","The INR and IGFR UTRs seem to require neither eIF4E nor eIF4A .","In fact , these UTRs have the lowest identified requirement for eIF4F activity of any cellular transcript thus far .","In addition , they are immune to two of the most important types of translational control , namely 4E-BP control and eIF4A inhibition .","Both of these features make sense given the cellular environment when these mRNAs are to be translated .","The conserved resistance to eIF4E and eIF4A inhibition of the insulin receptor transcripts should make them capable of out-competing other cellular transcripts with greater need for eIF4A or eIF4E .","Using these exceptional characteristics , the insulin receptor mRNA could out-compete more abundant transcripts under times of stress or when nutrients are limiting and 4E-BP and Pdcd4 are active .","We focused on the insulin receptor UTRs as a mechanism for continued translation under conditions of general inhibition of protein synthesis as this is one of the initial components of the pathway identified as a direct Foxo target .","In more recent work other components of the pathway have been identified as transcriptionally controlled by Foxo .","If these targets are to be translated when Foxo is active they should also require mechanisms to escape 4E-BP and Pdcd4 inhibition .","It remains to be seen if they will use the same mechanism as the INR mRNA or another mechanism ."],["Wildtype Canton S flies are from the Bloomingtion Stock Center .","foxOΔ94 has been described ( Slack et al . , 2011 ) .","Chromatin immunoprecipitation using Foxo antibodies followed by tiling array analysis was performed previously on starved larva ( Teleman et al . , 2008 ) .","The raw .","CEL files for Foxo precipitated and mock precipitated arrays were downloaded from the Teleman lab web page ( http://www . dkfz . de/en/signal-transduction-cancer/pages/Data . html ) .","Triplicate samples were combined and the Foxo precipitated samples were compared to mock precipitated samples using using the Affymetrix Tiling analysis ( TAS ) software .","Combined mock arrays were used to set the background signal intensities for the ChIP arrays .","The Integrated Genome Browser ( Nicol et al . , 2009 ) was used to visualize the resultant profile .","Oligonucleotides were synthesized corresponding to annotated transcripts with the longest 5′ for both Insulin receptor ( corresponding to EST G430111A11 ) and IGF-1 receptor ( corresponding to ESTs CJ180736 and CJ173921 ) ( Supplementary file 1 ) .","The oligos were used to clone the UTRs from cDNA derived from NIH 3T3 cells by PCR .","Drosophila eIF4A and Pdcd4 were cloned into pET28 in frame with the 6x HIS tag from cDNA using PCR and standard cloning methods .","The plasmid was transformed into BL21* ( DE3 ) cells ( Life Technologies , Grand Island , NY ) containing pLacIRARE2 ( Novagen , EMD Millipore , Billerica , MA ) .","After induction with 1 mM IPTG overnight at 25°C , eIF4A or PDCD-4 was purified using HisPur Ni-NTA Resin ( Thermo Fisher Scientific , Rockford , IL ) according to manufacturer’s directions .","PDCD-4 was eluted from the resin using 500 mM imidazole in PBS .","eIF4A was eluted using 50 mM EDTA in 1X PBS .","Purified 6His-Pdcd4 and 6His eIF4A was used to make rabbit polyclonal antisera ( Cocalico Biologicals , Inc . , Reamstown , PA ) .","Full length Drosophila Pdcd4 was cloned into pGEX2TKN in frame with GST .","GST-PDCD-4 or GST alone was expressed in BL21* ( DE3 ) cells ( Invitrogen , Grand Island , NY ) containing pLacIRARE2 ( Novagen , EMD Millipore , Billerica , MA ) .","Expression was induced with 1 mM IPTG overnight at 25°C .","Cells were resuspended and lysed in 1X PBS with lysozyme .","GST or GST-PDCD4 was immobilized on glutathione sepharose 4B ( GE Healthcare Biosciences , Pittsburgh , PA ) .","Equal amounts of recombinant eIF4A were applied to 50 µl of resin containing either GST or GST-Pdcd4 .","The resin was incubated for 1 hr at 4°C .","The resin was poured into a small spin column ( Pierce , Thermo Fisher Scientific , Rockford , IL ) and washed with 100 column volumes wash buffer ( 20 mM Tris pH 7 . 5 , 100 mM NaCl , 1 mM DTT , 0 . 1 mM EDTA , 0 . 1 mg/ml BSA ) .","Bound proteins were eluted with wash buffer containing 10 mM reduced glutathione for 1 hr at 4°C .","Samples were separated by SDS-PAGE , transferred to nitrocellulose and recombinant eIF4A was detected with a monoclonal antibody directed against the 6x HIS tag on eIF4A ( A00186 GenScript , Piscataway , NJ ) and a fluorescent secondary antibody against mouse IgG using a Li-Cor Odyssey Infrared imaging system .","15 ml of a saturated culture of Drosophila S2 cells were harvested by centrifugation .","The cells were washed once with 1X PBS .","The cells were resuspended in two packed cell volumes hypotonic buffer ( 10 mM HEPES pH 7 . 4 , 10 mM KCl , 5 mM MgCl2 , 1 mM DTT , 1X protease inhibitors [Sigma-Aldrich Corp . , St . Louis , MO] ) .","Triton X-100 was added to 0 . 5% and the cells were left on ice 30 min .","Nuclei were pelleted 10 min at 6000×g .","Supernatant was transferred to a new tube .","10% of the sample was saved for input analysis .","Polyclonal rabbit antisera to Drosophila eIF4A was added to the remainder of the sample and the sample was incubated at 4°C overnight with constant mixing .","Samples were centrifuged 2 min at 23 , 000×g .","The supernatant was combined with 50 µl protein-A Sepharose ( GE healthcare ) and incubated at 4°C for 2 hr .","The mixture was poured into a small spin column ( Pierce , #89869 ) connected to a needle and washed with 5 ml 1XPBS 0 . 1% Triton X-100 followed by 1 ml 1X PBS .","Proteins were elute by addition of 2X SDS-PAGE buffer ( 62 . 5 mM Tris-HCl , pH 6 . 8 , 25% glycerol , 2% SDS , 0 . 01% Bromophenol Blue , 5% β-mercaptoethanol ) incubation at room temperature for 10 min and collected by centrifugation .","Samples were separated by SDS-PAGE .","The gel was transferred to nitrocellulose and probed with rabbit α-Drosophila Pdcd4 antisera directed against full length 6His-Pdcd4 ( 1:1000 ) .","To avoid detection of the IgG used for precipitation the immunoblot was developed with biotinylated protein-A ( 1:5000 ) ( Lal et al . , 2005 ) followed by fluorescent labeled streptavidin ( 1:5000 ) using a Li-Cor Odyssey Infrared imaging system .","For experiments with the mutant Pdcd4 , 10 ml of Drosophila S2 cells were plated at 1 × 106 cells/ml in Schneider’s media supplemented with 10% FBS in a 10-cm dish .","Constructs expressing wild-type myc-Pdcd4 or mutant myc-Pdcd4-282 A286A were transfected using effectene ( Qiagen Inc . , Valencia , CA ) .","4 days later the cells were harvested and immunoprecipitation of eIF4A was performed as described above except transfected Pdcd4 was detected with a monoclonal antibody directed against the myc tag ( 9E10 ) and a fluorescent secondary antibody against mouse IgG using a Li-Cor Odyssey Infrared imaging system .","Protein concentrations were determined using BCA assay ( Pierce ) and equal amounts of protein were loaded onto a 10% SDS-PAGE gel .","The gel was transferred to nitrocellulose and probed with rabbit α-Drosophila Pdcd4 antisera directed against full length 6His-Pdcd4 ( 1:500 ) and mouse α-tubulin antibody ( 1:1000 ) .","Transcription templates for monocistronic RNAs were created using PCR containing a template-specific forward primer with a T7 promoter incorporated and a vector specific reverse primer .","Dicistronic RNA templates were made by digesting the cellular reporter vectors downstream of the firefly luciferase coding sequence and utilizing a T7 promoter incorporated in the vector .","Templates were purified using column clean up protocol and eluted in 50 µl 10 mM Tris pH 8 . 0 ( Epoch Life Science , Missouri City , TX ) .","Templates were transcribed using T7 polymerase and subsequently purified using LiCl precipitation .","Transcripts were capped using vaccinia virus capping enzyme ( New England Biolabs , Ipswich , MA ) as recommended and purified using RNeasy column protocol ( Qiagen ) .","Transcripts were tailed using poly ( A ) polymerase ( New England Biolabs , Ipswich , MA ) and purified using RNeasy columns ( Qiagen ) .","Embryo translation extracts were prepared as described from 0- to 4-hr embryos ( Marr et al . , 2007 ) .","Extracts were left untreated ( no Micrococcal nuclease treatment ) to allow translation under competitive conditions .","Translation assays were performed in 6 µl of Drosophila embryo extract , 0 . 1 mM spermidine , 60 µm Amino Acids , 16 . 8 mM creatine phosphate , 800 ng of creatine kinase , 24 mM HEPES ( pH 7 . 4 ) , 0 . 4 mM Mg acetate , 30 mM K acetate , 1 µg of calf liver tRNA , and 100 ng of template RNA in a 10-µl reaction .","Hippuristanol was added to a final concentration of 2µM or otherwise indicated .","For assays containing PDCD-4 , protein was added to a final concentration of 320 nM and was preincubated for 15 min with extract before the addition of RNA templates .","Translation reactions were incubated at 27°C for 1 hr and luciferase activity was measured using 100 µl of luciferase substrate ( Promega Corp . , Madison , WI ) .","Firefly luciferase was measured by adding 100 µl of 75 mM HEPES pH 8 . 0 , 5 mM MgSO4 , 20 mM DTT , 100 µM EDTA , 530 µM ATP , 0 . 5 mM coenzyme A , and 0 . 5 mM D-luciferin and renilla was measured by adding 100 µl 25 mM Na4PPi , 10 mM NaOAc , 15 mM EDTA , 0 . 5 M Na2SO4 , 1 . 0 M NaCl , and 0 . 1 mM Coelenterazine , pH 5 . 0 .","All experiments were performed at least twice in triplicate .","Translation assays were performed in 6 µl of untreated rabbit reticulocyte extract ( no Micrococcal nuclease treatment to allow translation under competitive conditions ) .","( Green Hectares , McFarland , WI ) , 0 . 1 mM spermidine , 60 µm Amino Acids , 16 . 8 mM creatine phosphate , 800 ng of creatine kinase , 24 mM HEPES ( pH 7 . 4 ) , 0 . 4 mM Mg acetate , 30 mM K acetate , 1 µg of calf liver tRNA , and 100 ng of template RNA in a 10-µl reaction .","Translation reactions were incubated at 37°C for 30 min and luciferase activity was measured using 100 µl of luciferase substrate ( Promega ) .","For assays containing excess m7G cap , cap structure analogue ( New England Biolabs , #S1407S ) was added to a final concentration of 1 mM .","All experiments were performed at least twice in triplicate .","Drosophila S2 cells with a stable transfection of constitutively active Foxo under the control of the metallothionein A promoter were maintained in Schneider’s Insect Media supplemented with 10% fetal bovine serum ( Puig et al . , 2003 ) .","These cells were plated at 1 . 25 × 106 cell/ml , and expression was induced by addition of 500 µm CuSO4 for 16 hr .","During induction the media was supplemented with 1 µg/m bovine insulin .","For protein samples , cells were lysed in RIPA buffer ( PBS containing 10 mM EDTA , 1% Triton X-100 , 1% SDS , 1% deoxycholate , 1× complete protease inhibitor [Roche , Indianapolis , IN] , 10% glycerol ) .","Total RNA was extracted from mock-treated and induced cells using TRI Reagent according to manufacturer’s protocol ( Molecular Research Center , Inc . , Cincinnati , OH ) .","5 µg of total RNA were digested by DNaseI ( New England Biolabs ) .","First strand cDNA sythesis was done using a mix of oligo-dT and random hexamers with MMLV reverse transcriptase .","The final concentrations of the cDNA reaction were 50 mM Tris-HCl ( pH 8 . 3 ) , 50 mM KCl , 3 mM MgCl2 , 10 mM DTT , 400 µM dNTPs , 1–2 µg RNA , 500 ng primers , 200 units MMLV RT . cDNAs were diluted 1:10 in TE pH 8 .","qPCR was run using 5 µl cDNA , GoTaq qPCR Master Mix ( Promega ) , and primers at a final concentration of 100 nM in a 20-µl reaction .","qPCR was done using specific primers against Drosophila Pdcd4 , RP49 , GstD1 , and 4E-BP ( Supplementary file 1 ) .","Pdcd4 fold-expression was calculated as a fraction of RP49 and normalized to mock-treated expression levels .","All experiments were performed at least twice in triplicate .","7-day-old adult male flies were starved for 5 hr and transferred to vials containing 5% sucrose or 5% sucrose+5 mM paraquat .","After 24 hr flies were harvested and total RNA was extracted from mock-treated and paraquat-treated flies using TRI Reagent according to manufacturer’s protocol ( Molecular Research Center , Inc . ) .","Previously described dicistronic reporter constructs ( Marr et al . , 2007 ) were subcloned into a plasmid containing the metallothionein A promoter for metal inducible expression ( Marr et al . , 2006 ) .","The IRES sequence from HCV ( Kieft et al . , 1999 ) was subcloned into the inducible expression vector .","For expression in S2 cells , Pdcd4 was cloned under a minimal actin promoter .","For the Pdcd4 double mutant construct , amino acids 282 , and 286 ( Figure 1B ) were mutated to alanine by site directed mutagenesis .","S2 cells were maintained in Schneider’s media with 10% FBS and Penicillin/Streptomycin .","For transfection , cells were plated at 1 . 25×106 cells/ml in Schneider’s supplemented with 10% FBS and an additional 1 µg/ml bovine insulin .","DNA was transfected at a 4:1 ratio expression plasmid to reporter plasmid using effectene transfection reagent ( Qiagen ) following instructions for S2 cells .","Cells were induced with 500 µM CuSO4 36 hr after transfection .","Cells were lysed in passive lysis buffer ( Promega ) and assayed 36 hr after induction using a dual luciferase assay .","Firefly expression was measured in 75 mM HEPES pH 8 . 0 , 5 mM MgSO4 , 20 mM DTT , 100 µM EDTA , 530 µM ATP , 0 . 5 mM coenzyme A , and 0 . 5 mM D-luciferin .","Renilla expression was measured by addition of an equal volume of 25 mM Na4PPi , 10 mM NaOAc , 15 mM EDTA , 0 . 5 M Na2SO4 , 1 . 0 M NaCl , and 0 . 1 mM Coelenterazine .","All experiments were performed at least twice in triplicate .","Drosophila S2 cells expressing constitutively active Foxo were formaldehyde crosslinked .","Nuclei were isolated , lysed , and chromatin was sonicated to 500–1000 bp in length .","Chromatin was incubated with polyclonal sera against full length Foxo .","The chromatin/antibody mix was then incubated with protein A beads to isolate Foxo-bound chromatin from the sample .","Purified DNA was assayed by qPCR to determine enrichment for genomic sites bound by Foxo .","Enrichment is based on signal increase compared to a region of the genome in the first intron of CG15414 ( Supplementary file 1 ) .","The mouse insulin receptor and insulin-like growth factor one receptor 5′ UTRs were subcloned into the plasmid pGLRSVRF .","This plasmid contains the RSV LTR followed by the renilla luciferase open reading frame , the firefly luciferase open reading frame and the SV40 early polyadenylation signal .","The UTRs were cloned between the renilla and firefly open reading frames .","For transfection NIH3T3 cells were trypsinized and counted .","350 µl of cells at a concentration of 1 × 105 cells per milliliter were plated in each well of a 24-well plate .","Cells were transfected using Effectene ( Qiagen ) according to manufacturer’s instructions .","Cells were lysed in passive lysis buffer ( Promega ) and assayed 36 hr after induction using a dual luciferase assay ( Promega ) ."]],"string":"[\n [\n \"During times of stress the cell changes its gene expression profile to better manage the cause of the stress .\",\n \"Coordinate changes in both transcription and translation occur ( Sengupta et al . , 2010; Spriggs et al . , 2010 ) .\",\n \"A central pathway that responds to stress stimuli by controlling both protein and RNA synthesis is the insulin and insulin-like receptor-signaling ( IIS ) pathway .\",\n \"The fundamental molecular architecture of the IIS pathway is conserved from flies to man ( Figure 1 ) ( Oldham , 2011 ) .\",\n \"When IIS signaling is high , the protein kinase AKT is activated ( Ruggero and Sonenberg , 2005 ) .\",\n \"AKT directly phosphorylates the Foxo family of transcription factors and consequently prevents activated transcription of Foxo target genes ( Brunet et al . , 1999 ) .\",\n \"AKT also stimulates the activation of the mechanistic target of rapamycin ( mTOR ) protein ( Zoncu et al . , 2011 ) . 10 . 7554/eLife . 00542 . 003Figure 1 . Simplified insulin/insulin-like growth factor signaling diagram .\",\n \"( A ) When Insulin receptor or Insulin-like growth factor receptor is active signaling through AKT inhibits Foxo transcription factors and activates mTOR .\",\n \"mTOR in turn inhibits 4E-BP and activates S6K .\",\n \"S6K in turn inhibits Pdcd4 and activates eIF4B .\",\n \"When insulin signaling is low inhibition of Foxo is relieved and Foxo activates the transcription of Insulin receptor and 4E-BP .\",\n \"The broken line indicates the proposed activation of Pdcd4 by Foxo .\",\n \"( B ) Alignment of human ( Hs top ) and Drosophila ( Dm bottom ) Pdcd4 proteins .\",\n \"Conserved Akt and S6K phosphorylation sites are indicated by asterisk .\",\n \"Conserved MA3 domains are indicated by shaded boxes .\",\n \"Arrowheads indicate conserved acidic residues important for eIF4A binding in humans .\",\n \"( C ) eIF4A interacts with Pdcd4 in Drosophila cells .\",\n \"Cytoplasmic extracts from a saturated culture of S2 cells were subjected to immunoprecipitation with antisera directed against eIF4A or preimmune serum .\",\n \"Pdcd4 was detected with antisera against Pdcd4 .\",\n \"( D ) Mutant Pdcd4 binds less efficiently to eIF4A than wildtype .\",\n \"Cytoplasmic extracts from cultures of S2 cells expression wild-type Myc-Pdcd4 or mutant Myc-Pdcd4 ( AA ) were subjected to immunoprecipitation with antisera directed against eIF4A .\",\n \"Myc-Pdcd4 was detected with mouse monoclonal antibody to the Myc tag .\",\n \"Immunoprecipitated eIF4A was detected with rabbit antisera .\",\n \"( E ) Immobilized Drosophila Pdcd4 interacts with Drosophila eIF4A .\",\n \"On the top is a cartoon of approach .\",\n \"On the bottom is an immnoblot of proteins eluted from the affinity columns .\",\n \"Position of the recombinant eIF4A is indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 00542 . 003 Activated mTOR stimulates general translation , in part , by influencing the activity of the translation initiation complex eIF4F .\",\n \"The eIF4F complex consists of eIF4E , the 7-methyl-Guanosine-cap ( m7G ) binding protein , eIF4A , an RNA helicase , and eIF4G , a large scaffolding protein .\",\n \"In addition , the RNA binding protein eIF4B can associate with eIF4F to stimulate the helicase activity of eIF4A ( Ma and Blenis , 2009; Sonenberg and Hinnebusch , 2009; Zoncu et al . , 2011 ) .\",\n \"mTOR stimulates general translation in part by inactivating translational inhibitors .\",\n \"mTOR phosphorylates and inactivates the translation repressor eIF4E binding protein ( 4E-BP ) ( Gingras et al . , 1999 ) allowing efficient formation of the eIF4F complex .\",\n \"In addition , mTOR activates ribosomal protein S6 kinase ( S6K ) ( Sarbassov et al . , 2005 ) .\",\n \"S6K stimulates the helicase eIF4A by activating eIF4B and inhibiting programmed cell death protein 4 ( Pdcd4 ) , a known eIF4A inhibitor ( Figure 1 ) ( Yang et al . , 2003; Raught et al . , 2004; Dorrello et al . , 2006 ) .\",\n \"Thus under conditions of high signaling through AKT and mTOR , cap-dependent translation is stimulated .\",\n \"In times of stress , low levels of signaling through the IIS pathway lead to activated Foxo and 4E-BP in addition to inactive S6K .\",\n \"Foxo moves to the nucleus and controls the transcription of its target genes ( Salih and Brunet , 2008 ) .\",\n \"4E-BP prevents formation of the translation initiation complex eIF4F , thereby inhibiting m7G-dependent translation , and S6K no longer stimulates eIF4A .\",\n \"This in turn leads to lower levels of global protein synthesis .\",\n \"Thus the IIS pathway controls gene expression with two different branches: transcription of Foxo target genes and m7G-cap-dependent translation through 4E-BP and S6K .\",\n \"The IIS pathway in Drosophila contains a mechanism that functionally couples activated transcription to translation .\",\n \"A portion of the system includes a signaling and gene expression feedback loop for direct genetic targets of Drosophila Foxo .\",\n \"The insulin-like receptor ( INR ) and 4E-BP genes are conserved transcriptional targets of Foxo ( Figure 1 ) ( Puig et al . , 2003; Puig and Tjian , 2005; Marr et al . , 2007; Hu et al . , 2008 ) .\",\n \"Paradoxically , the insulin receptor protein , as well as mRNA , is being synthesized and accumulating under the same conditions when 4E-BP activity and expression is induced and S6K is inhibited ( Marr et al . , 2007 ) .\",\n \"Foxo activates the transcription of the Drosophila insulin receptor gene from three promoters .\",\n \"Each promoter produces a transcript with a distinct 5′ untranslated region ( UTR ) but identical coding region ( Casas-Tinto et al . , 2007; Marr et al . , 2007 ) .\",\n \"Transcripts derived from promoter 1 are by far the most abundant and ubiquitous form of INR transcript ( Casas-Tinto et al . , 2007; Marr et al . , 2007 ) .\",\n \"The Drosophila INR 5′ UTRs contain an internal ribosome entry site ( IRES ) that allows the message to escape 4E-BP inhibition of cap-dependent translation .\",\n \"This mechanism provides a functional coupling of transcription and translation in times of stress that allows amplification of insulin receptor expression ( Marr et al . , 2007 ) .\",\n \"Because IRES containing transcripts can outcompete cap-dependent transcripts under these conditions their translation is actually stimulated ( Svitkin et al . , 2005; Marr et al . , 2007 ) .\",\n \"This leads to an effective switch of the cellular translation machinery to targets of the IIS pathway .\",\n \"Thus , Foxo targets impose a translational program by activation of genes that repress general translation while simultaneously activating targets that are immune to this translational control .\",\n \"This provides these targets with a competitive advantage allowing them to utilize the translation machinery that is freed by the general inhibition .\",\n \"Here we identify Drosophila Pdcd4 as an additional Foxo target further enhancing the coupling of transcription and translation regulation in the IIS pathway .\",\n \"Since the IIS pathway targets translation initiation through control of both eIF4E and eIF4A , we wondered if the most abundant and ubiquitous Drosophila INR 5′ UTR would also provide resistance to inhibition of eIF4A activity .\",\n \"To answer this question we used both an in vitro translation system and a cell based assay to investigate the eIF4A requirements for efficient translation of reporters containing the INR 5′ UTR from Drosophila .\",\n \"Because mammalian systems show the same type of regulation , we also investigated the role of eIF4A inhibition in the murine insulin receptor and insulin-like growth factor receptors .\",\n \"( Giraud et al . , 2001; Meng et al . , 2008; Spriggs et al . , 2009b ) .\",\n \"We find , in both the Drosophila and mouse systems , that the 5′ UTRs of the mRNAs for these receptors provide resistance to both eIF4E and eIF4A inhibition .\",\n \"Taken together , these results indicate that these cellular messages have some of the lowest requirement for eIF4F activity for translation identified to date .\"\n ],\n [\n \"A connection between Foxo activation and translation inhibition was identified when it was discovered that 4E-BP expression is controlled by Foxo in Drosophila and mouse cells ( Junger et al . , 2003; Puig et al . , 2003; Hu et al . , 2008 ) .\",\n \"Since the IIS pathway is also known to control eIF4A activity through Pdcd4 ( Figure 1A ) , we tested if this gene is under direct Foxo control in Drosophila .\",\n \"Blast analysis of human Pdcd4 with the Drosophila genome identifies a single homologous protein encoded by CG10990 .\",\n \"Alignment of human PDCD4 and CG10990 indicate that important regions of the protein are conserved ( Figure 1B ) ( Cash and Andrews , 2012 ) .\",\n \"The two MA3 domains , including the acidic residues shown to be important for the interaction with eIF4A are conserved ( Chang et al . , 2009; Waters et al . , 2011 ) .\",\n \"In addition , the Akt and S6K phosphorylation sites are conserved ( Palamarchuk et al . , 2005; Dorrello et al . , 2006 ) .\",\n \"To determine if the interaction with eIF4A is conserved , we immunoprecipitated Drosophila eIF4A from cytoplasmic extracts derived from a saturated culture of Drosophila S2 cells .\",\n \"Associated Pdcd4 was detected by immunoblot .\",\n \"Pdcd4 is co-precipitated with antisera against eIF4A but not with preimmune serum indicating that Pdcd4 and eIF4A interact in Drosophila cells ( Figure 1C ) .\",\n \"We next created Myc-tagged expression constructs for Drosophila Pdcd4 , one wildtype construct and a construct containing mutations in conserved residues in the first MA3 domain ( E282A , D286A ) .\",\n \"The analogous mutations in human PDCD4 destabilize the interaction with eIF4A ( Chang et al . , 2009; Waters et al . , 2011 ) .\",\n \"The constructs were transfected into growing Drosophila S2 cells .\",\n \"Subsequent immunoprecipitation of eIF4A from these cells reveals a decreased association of the mutant Pdcd4 with eIF4A ( Figure 1D ) .\",\n \"This indicates the mutations induce the same destabilization of the eIF4A–Pdcd4 interaction in Drosophila cells .\",\n \"Interestingly under these conditions we detect multiple forms of Pdcd4 by western blot , most likely phosphorylated forms of Pdcd4 , and only the fastest migrating species associates with eIF4A .\",\n \"To determine if Drosophila Pdcd4 can interact directly with eIF4A we used an affinity chromatography assay using recombinant proteins purified from Escherichia coli .\",\n \"A GST fusion to Drosophila Pdcd4 was immobilized on glutathione agarose and recombinant Drosophila eIF4A was passed over the column ( Figure 1D ) .\",\n \"As a control GST was also immobilized on glutathione agarose .\",\n \"The eIF4A bound to the immobilized GST-Pdcd4 but not to GST alone indicating that Drosophila Pdcd4 can interact directly with Drosophila eIF4A .\",\n \"Taken together these data are consistent with the notion that CG10990 is the Drosophila homologue of human PDCD4 .\",\n \"There are hints in the literature , based on microarray experiments , indicating this gene is induced in response to nutrient stress and might be controlled by Foxo ( Gershman et al . , 2007 ) .\",\n \"In an effort to determine if Foxo binds to the Pdcd4 gene in nutrient stressed animals we reanalyzed the only publically available Foxo ChIP ( Chromatin immunoprecipitation ) dataset ( Teleman et al . , 2008 ) .\",\n \"These experiments were performed on starved larva .\",\n \"We find Foxo binds the Pdcd4 gene in both the promoter and intronic regions with enrichment values as high as 16-fold over background ( Figure 2A ) .\",\n \"To corroborate this finding , we performed ChIP on genomic DNA from a cell line with an inducible Foxo cDNA gene that has been modified so the Foxo protein produced is constitutively active because it is immune to the negative regulation by insulin signaling ( FoxoCA ) ( Puig et al . , 2003; Gershman et al . , 2007 ) .\",\n \"This allows us to induce Foxo under conditions of high nutrient signaling and remove possible crosstalk from upstream signaling pathways .\",\n \"We tested the enrichment of genomic sequences by qPCR using primers to the Pdcd4 promoter region ( Figure 2A ) compared to a region in the first intron of CG15414 , a gene just downstream of 4E-BP .\",\n \"We find FoxoCA binds to the promoter region of Pdcd4 at levels comparable to a well-defined direct target , 4E-BP ( Junger et al . , 2003; Puig et al . , 2003; Marr et al . , 2007 ) ( Figure 2B ) .\",\n \"To determine the effect on mRNA production under these conditions we performed quantitative RT-qPCR on induced cells .\",\n \"We find that the steady-state level of Pdcd4 mRNA is increased about threefold in cells expressing active Foxo ( Figure 2C ) .\",\n \"To determine if the effect is due to mRNA stability changes or new transcription we assayed intron-containing pre-mRNAs by RT-qPCR .\",\n \"Since most splicing is co-transcriptional in Drosophila this is a good assay for new RNA synthesis ( Khodor et al . , 2011 ) .\",\n \"We find that Pdcd4 pre-mRNA is increased , indicating an increase in transcription of the gene .\",\n \"The increased mRNA also leads to increased protein synthesis as determined by immuno-blot with antibodies directed against Drosophila Pdcd4 ( Figure 2D ) .\",\n \"This is likely an underestimate of the effect since these experiments are all done under high serum and insulin conditions that should result in the rapid turnover of Pdcd4 protein ( Dorrello et al . , 2006 ) . 10 . 7554/eLife . 00542 . 004Figure 2 . Foxo activates Pdcd4 in Drosophila cells .\",\n \"( A ) Reanalysis of ChIP-chip data from Teleman et al . ( 2008 ) .\",\n \"Genomic Browser view of Foxo binding to the Pdcd4 locus in starved larva .\",\n \"The data are plotted as the enrichment ( log2 ) over mock precipitated samples .\",\n \"Primers used for ChIP and qPCR are indicated .\",\n \"( B ) ChIP of Foxo at 4E-BP promoter and Pdcd4 locus in Drosophila S2 cells expressing constitutively active Foxo ( FoxoCA ) .\",\n \"The data are plotted as fold enrichment over a background region 1 kb downstream of 4E-BP .\",\n \"Uninduced samples are plotted in white , induced samples in black ( error bars indicate SD ) .\",\n \"( C ) RT-qPCR of Pdcd4 mRNA and pre-mRNA in Drosophila S2 cells expressing FoxoCA .\",\n \"Data are plotted as fold-induction ( error bars indicate SD ) .\",\n \"( D ) Immunoblot of total protein from Drosophila S2 cells expressing FoxoCA .\",\n \"Positions of Pdcd4 and tubulin are indicated .\",\n \"( E ) 4E-BP , GstD1 , and Pdcd4 RNA levels in untreated and paraquat-treated animals .\",\n \"The levels of RNA were normalized to RP49 and are plotted as fold-induction relative to untreated animals ( error bars indicate SEM ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 00542 . 004 We tested if Foxo controls Pdcd4 in adult flies subjected to stress .\",\n \"Adult wildtype or Foxo null flies ( Slack et al . , 2011 ) were treated with paraquat to induce oxidative stress and assayed for expression of 4E-BP , Pdcd4 , and GstD1 by RT-qPCR .\",\n \"Consistent with previous results ( Wang et al . , 2005 ) , 4E-BP is induced by paraquat in a Foxo dependent manner .\",\n \"Like 4E-BP , we find that Pdcd4 is induced in response to paraquat in a Foxo dependent manner ( Figure 2E ) .\",\n \"To determine if the effect was due to loss of general oxidative stress response or if it is Foxo specific we examined the induction of GstD1 , a gene controlled by Nrf2 in response to oxidative stress ( Misra et al . , 2011 ) .\",\n \"We find that GstD1 is still responsive indicating that the effects at Pdcd4 and 4E-BP are Foxo-specific and not due to a loss of responsiveness in the mutant flies ( Figure 2E ) .\",\n \"These results are consistent with the idea that in addition to controlling the cap-binding complex through 4E-BP , active Foxo can influence eIF4A through activation of the Pdcd4 gene in response to stress .\",\n \"Given that Foxo is activating transcription of the INR gene while Pdcd4 is also active , we hypothesized that since the INR mRNA is translated efficiently under these conditions ( Marr et al . , 2007 ) it must be at least partially immune to diminished eIF4A activity .\",\n \"To determine the effect of increased Pdcd4 on the translation of insulin receptor UTR containing RNAs we modified a dicistronic mRNA assay which we previously used to investigate the effects of 4E-BP on insulin receptor translation in Drosophila ( Marr et al . , 2007 ) .\",\n \"In this assay a construct is used that produces a RNA in which the open reading frames of renilla luciferase and firefly luciferase are present on the same transcript ( Figure 3A ) .\",\n \"Renilla luciferase levels are an indication of total message produced in the cell and firefly luciferase levels are an indication of IRES dependent translation .\",\n \"As previously reported , insertion of the INR 5′ UTR between the ORFs promotes translation of the second ORF ( Marr et al . , 2007 ) .\",\n \"The levels of IRES activity of the INR UTR are comparable to the well-characterized IRES from Hepatitis C Virus ( Figure 3B ) ( Tsukiyama-Kohara et al . , 1992 ) .\",\n \"The INR UTR and the HCV IRES both produce more firefly signal than the empty vector ( Figure 3D ) . 10 . 7554/eLife . 00542 . 005Figure 3 . Drosophila insulin receptor 5′UTR provides resistance to Pdcd4 . ( A ) Diagram of dicistronic reporters .\",\n \"Translation of the Firefly ORF requires internal ribosome entry .\",\n \"Firefly to renilla activity ratio provides an indication of IRES activity .\",\n \"( B ) The Drosophila Insulin receptor UTR provides IRES activity comparable to the activity of HCV .\",\n \"( C ) Renilla activity of the reporters .\",\n \"( D ) Firefly activity of the reporters .\",\n \"( E ) Dicistronic reporter activities in the presence of 4E-BP or Pdcd4 expression .\",\n \"4E-BP and Pdcd4 stimulate the IRES activity of the insulin receptor UTRs .\",\n \"Mutation of the critical acidic residues of Pdcd4 prevents the stimulation .\",\n \"( F ) Renilla activity of the reporters in the presence of expressed proteins .\",\n \"( G ) Firefly activity of the reporters in the presence of the expressed proteins ( error bars indicate SEM ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 00542 . 005 To determine the effect of Pdcd4 on the INR UTR we expressed Pdcd4 in Drosophila S2 cells and measured the activity of the dicistronic reporter .\",\n \"In order to more accurately determine the effects of the protein in question we modified the assay so the production of the dicistronic mRNA is inducible .\",\n \"This allows accumulation of the experimental protein in the cell before the dicistronic mRNA is produced giving a more precise measure of the effects on the activity of the dicistronic message .\",\n \"The levels of expression of the second ORF relative to the first ORF are an indication of the cap-independent translation potential of the insert .\",\n \"To validate this assay we reproduced our previous results with 4E-BP ( Marr et al . , 2007 ) .\",\n \"As reported previously , expression of 4E-BP stimulates the translation of the second open reading frame in a reporter containing the INR 5′UTR ( Figure 3E , G ) .\",\n \"Expression of Pdcd4 also stimulates translation of the second ORF dependent on the INR 5′UTR similar to the effects seen with the HCV IRES ( Figure 3E , G ) .\",\n \"These effects are specific .\",\n \"Mutation of the key acidic residues ( Figure 1B ) in Pdcd4 shown to disrupt eIF4A binding in the human system ( Waters et al . , 2011 ) prevent the IRES stimulation .\",\n \"To address the role of eIF4A in insulin receptor translation , we used a highly specific small molecule inhibitor of eIF4A , hippuristanol ( Bordeleau et al . , 2006; Lindqvist et al . , 2008 ) .\",\n \"Hippuristanol is a potent translation inhibitor that works in eukaryotes from yeast to human ( Lindqvist et al . , 2008 ) .\",\n \"Hippuristanol inhibits the ATPase activity and RNA binding of eIF4A ( Bordeleau et al . , 2006; Lindqvist et al . , 2008 ) .\",\n \"The small molecule binds to the protein in conserved regions V and VI in eIF4A homologues ( Lindqvist et al . , 2008 ) .\",\n \"Importantly , the effects on translation can be rescued by addition of either wild-type or mutant forms of eIF4A that are immune to hippuristanol indicating that the effects are highly specific for eIF4A ( Bordeleau et al . , 2006; Lindqvist et al . , 2008 ) .\",\n \"This small molecule had been used previously in the Drosophila system to investigate eIF4A requirements ( Iwasaki et al . , 2009 ) .\",\n \"We performed in vitro competitive translation experiments with capped and polyadenylated firefly luciferase reporters ( Figure 4A ) and a Drosophila embryo extract translation system that has not been treated with micrococcal nuclease ( Gebauer et al . , 1999; Marr et al . , 2007 ) in the presence of hippuristanol .\",\n \"The RNA reporters contain the 5′ UTR from the Drosophila insulin receptor .\",\n \"In addition we include two control RNAs .\",\n \"One control RNA contains a non-specific UTR derived from plasmid sequences .\",\n \"The other RNA contains the IRES from the Hepatitis C virus ( HCV ) ( Tsukiyama-Kohara et al . , 1992 ) .\",\n \"This IRES does not require eIF4A activity and controls for non-specific effects on the extract ( Pestova et al . , 1998 ) .\",\n \"Under the experimental conditions , translation of the first control RNA is strongly inhibited by hippuristanol while the reporter containing the HCV IRES is completely resistant to eIF4A inhibition ( Figure 4A ) .\",\n \"This small molecule inhibitor exposed a greatly diminished role for eIF4A in the Drosophila INR UTR mediated translation ( Figure 4A ) .\",\n \"At the low and moderate concentrations of hippuristanol , the Drosophila INR UTR reporter retains almost complete activity comparable to the HCV UTR .\",\n \"Even at the highest concentration of hippuristanol tested , the reporter containing the INR UTR retains >50% of the original translation activity .\",\n \"To determine the effect of Pdcd4 in this system we added recombinant Pdcd4 to the translation extract .\",\n \"Consistent with the data using hippuristanol , we find Pdcd4 can inhibit the control RNA but not the Drosophila INR UTR or the HCV IRES ( Figure 4B ) .\",\n \"These finding suggests that translation of the most abundant Drosophila INR transcript can tolerate inhibition of eIF4A . 10 . 7554/eLife . 00542 . 006Figure 4 . Drosophila insulin receptor 5′UTR provides resistance to eIF4a inhibition .\",\n \"( A ) Top: Diagram of RNAs used in the in vitro translation assays .\",\n \"Bottom: Titration of hippuristanol in in vitro translation assays .\",\n \"The shade of the bars indicates the final concentration of hippuristanol in the assay .\",\n \"The legend appears above the graph .\",\n \"Data are plotted as the fraction activity of the carrier treated extracts ( error bars indicate SEM ) .\",\n \"( B ) Top: Diagram of RNAs used in the in vitro translation assays .\",\n \"Bottom: Activity of these RNAs in in vitro translation assays in the absence ( white bars ) and presence ( black bars ) of Drosophila Pdcd4 ( error bars indicate SEM ) .\",\n \"( C ) Dicistronic RNA translation in vitro .\",\n \"Top: Diagram of RNAs used in the in vitro translation assays .\",\n \"Bottom: Firefly to renilla ratio in the absence ( white bars ) and presence ( black bars ) of hippuristanol .\",\n \"( D ) Top: Renilla activity in the dicistronic assay .\",\n \"Bottom: Firefly activity in the dicistronic assay .\",\n \"Shading as in C . Percentage above the bars indicates activity after hippuristanol addition relative to carrier treated samples . DOI: http://dx . doi . org/10 . 7554/eLife . 00542 . 006 We used a dicistronic RNA in the in vitro translation assay to directly test the IRES activity under hippuristanol treatment ( Figure 4C top ) .\",\n \"Dicistronic RNAs were synthesized in vitro using T7 RNA polymerase .\",\n \"The RNA was capped and polyadenylated and used to program the same Drosophila embryo translation system described above .\",\n \"The extracts were treated with either hippuristanol or carrier .\",\n \"Consistent with the monocistronic assay , both the INR and the HCV IRES containing transcripts show increased relative translation of the second ORF upon eIF4A inhibition ( Figure 4C ) .\",\n \"The increase is due both to a resistance of the second ORF to the inhibition and a decrease in the cap-dependent translation of the first ORF ( Figure 4D ) .\",\n \"Under these conditions there is a small amount of cryptic translation of the second ORF in the control transcript .\",\n \"However both the renilla and firefly activites respond the same to the hippuristanol treatment .\",\n \"Because the molecular architecture of the IIS signaling pathway is conserved in mammals , we wondered if this level of regulation would be conserved in mammals .\",\n \"To address this , we cloned the 5′ UTR from the longest mRNAs for the mouse insulin receptor ( mINR ) and the mouse insulin-like growth factor receptor-I ( IGFR ) and created firefly luciferase reporters under the control of these UTRs ( Figure 5A ) .\",\n \"Previously , it was reported that INR and IGFR UTRs confer cap-independent translation activity in human and rat ( Giraud et al . , 2001; Spriggs et al . , 2009b ) .\",\n \"To extend this finding to the mouse system and ensure that our competitive rabbit reticulocyte system was capable of supporting cap-independent translation , we assayed translation of the mINR and IGFR reporters in the presence of excess m7G cap along with our control RNA and the HCV IRES reporters described above ( Figure 5A ) .\",\n \"While the control RNA is inhibited by excess cap , translation from the 5′ UTR of mINR and IGFR is not only resistant to excess m7G cap but the activity actually increases in the presence of excess m7G cap .\",\n \"This observation is common with UTRs that contain an IRES ( Svitkin et al . , 2005 ) .\",\n \"The increase in activity for both the mINR and IGFR UTRs exceeded the IRES activity of the HCV UTR .\",\n \"This indicates that mINR UTR and IGFR UTR are capable of conferring cap-independent translation initiation . 10 . 7554/eLife . 00542 . 007Figure 5 . Mammalian insulin receptor and insulin-like growth factor receptor 5′UTR provide resistance to eIF4a inhibition .\",\n \"( A ) Diagram of RNAs used in the in vitro translation assays .\",\n \"( B ) In vitro Translation in the absence ( white bars ) and presence ( black bars ) of excess m7G analogue .\",\n \"Data are plotted as the fraction activity of the mock treated extracts ( error bars indicate SEM ) .\",\n \"( C ) Titration of hippuristanol in in vitro translation assays .\",\n \"Data are plotted as the fraction activity of the mock treated extracts ( error bars indicate SEM ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 00542 . 007 To directly test the ability of these UTRs to allow internal ribosome entry we performed a dicistronic assay in mammalian cells .\",\n \"The UTRs were subcloned into a plasmid construct between the renilla and firefly open reading frames controlled by the RSV LTR .\",\n \"Both the mINR and the IGFR UTRs supported substantial firefly activity compared to the original vector .\",\n \"This is apparent both in the firefly to renilla ratio and in the raw firefly activity units ( Figure 5B ) .\",\n \"This combined with the in vitro translation assays strongly suggest that the mINR and IGFR UTRs can support cap-independent translation .\",\n \"To test for conservation of resistance to eIF4A inhibition , hippuristanol was titrated into the rabbit reticulocyte translation system .\",\n \"As expected , the activity of the control RNA is reduced to <10% of the mock treated extract , and the HCV IRES is completely resistant to hippuristanol ( Figure 5C ) .\",\n \"In fact , the HCV IRES is stimulated fourfold by addition of hippuristanol under these conditions .\",\n \"Both mINR and IGFR UTRs confer resistance to hippuristanol ( Figure 5C ) .\",\n \"Even at the highest concentrations of the small molecule the mINR and mIGFR UTRs remain roughly 50% active indicating a decreased requirement for eIF4A relative to the control RNA .\"\n ],\n [\n \"Stress responses controlled through the IIS pathway result in changes in both mRNA synthesis and protein synthesis .\",\n \"These processes are coordinated to ensure proper expression of the downstream targets .\",\n \"Under the same low IIS signaling conditions that activate 4E-BP , Pdcd4 is stabilized resulting in the inhibition of the DEAD box helicase eIF4A .\",\n \"Thus , under these conditions the eIF4F complex is repressed by two mechanisms .\",\n \"At the same time , the Foxo family of transcription factors is active and increasing the synthesis of certain mRNAs , one of which is the INR transcript itself .\",\n \"Previously we identified a connection between 4E-BP mediated inhibition of the cap-binding complex and INR mRNA translation in Drosophila ( Marr et al . , 2007 ) .\",\n \"The INR message is immune to the 4E-BP translational repression and thus is preferentially translated under low signaling conditions coupling the increase in mRNA expression to an increase in protein synthesis .\",\n \"In the work presented here we extend this observation of gene expression coordination of INR mRNA to the eIF4A branch of the IIS signaling pathway .\",\n \"First , we show that active Foxo is capable of directly stimulating the transcription of Pdcd4 , analogous to the activation of 4E-BP seen previously under these same conditions ( Junger et al . , 2003; Puig et al . , 2003 ) .\",\n \"This provides a mechanism for the pathways controlling Foxo to enhance the inhibition of eIF4A when stressed or when nutrients are low .\",\n \"Second we show that the 5′ UTR of the most abundant Drosophila INR transcript provides resistance to eIF4A inhibition comparable to the resistance seen with the HCV IRES that does not require eIF4A .\",\n \"A similar finding has been seen for the Drosophila reaper 5′ UTR ( Hernandez et al . , 2004; Iwasaki et al . , 2009 ) .\",\n \"The data presented above also support the conservation of the cap-independent mechanism of translation initiation of the insulin receptor and insulin-like growth factor receptor mRNAs in mammals .\",\n \"The mouse transcripts show resistance to hippuristanol under conditions that almost completely inhibit a control RNA indicating that the resistance to eIF4A inhibition is also conserved .\",\n \"There are no easily recognizable conserved sequence elements between the Drosophila and mouse UTRs , but the mode of regulation is conserved suggesting an important role for this type of translational control .\",\n \"This defines a functional characteristic of the insulin receptor and IGF receptor transcripts that is conserved across hundreds of millions of years of evolution ( from flies to mammals ) .\",\n \"Taken together with previous work , these data indicate that the coupling of transcription to translation of insulin receptor mRNA mediated by Foxo targets can culminate in an activated translational response .\",\n \"Our findings highlight a unique characteristic of the insulin receptor and IGF receptor UTRs that differentiates them from other cellular transcripts .\",\n \"In addition to being immune to 4E-BP , these IRESes are resistant to eIF4A inhibition .\",\n \"While viral IRESes are fairly common , cellular IRESes are rare and relatively unexplored .\",\n \"Where it has been explored , most cellular IRESes have a strong requirement for eIF4A ( Thoma et al . , 2004; Spriggs et al . , 2009a ) .\",\n \"The INR and IGFR UTRs seem to require neither eIF4E nor eIF4A .\",\n \"In fact , these UTRs have the lowest identified requirement for eIF4F activity of any cellular transcript thus far .\",\n \"In addition , they are immune to two of the most important types of translational control , namely 4E-BP control and eIF4A inhibition .\",\n \"Both of these features make sense given the cellular environment when these mRNAs are to be translated .\",\n \"The conserved resistance to eIF4E and eIF4A inhibition of the insulin receptor transcripts should make them capable of out-competing other cellular transcripts with greater need for eIF4A or eIF4E .\",\n \"Using these exceptional characteristics , the insulin receptor mRNA could out-compete more abundant transcripts under times of stress or when nutrients are limiting and 4E-BP and Pdcd4 are active .\",\n \"We focused on the insulin receptor UTRs as a mechanism for continued translation under conditions of general inhibition of protein synthesis as this is one of the initial components of the pathway identified as a direct Foxo target .\",\n \"In more recent work other components of the pathway have been identified as transcriptionally controlled by Foxo .\",\n \"If these targets are to be translated when Foxo is active they should also require mechanisms to escape 4E-BP and Pdcd4 inhibition .\",\n \"It remains to be seen if they will use the same mechanism as the INR mRNA or another mechanism .\"\n ],\n [\n \"Wildtype Canton S flies are from the Bloomingtion Stock Center .\",\n \"foxOΔ94 has been described ( Slack et al . , 2011 ) .\",\n \"Chromatin immunoprecipitation using Foxo antibodies followed by tiling array analysis was performed previously on starved larva ( Teleman et al . , 2008 ) .\",\n \"The raw .\",\n \"CEL files for Foxo precipitated and mock precipitated arrays were downloaded from the Teleman lab web page ( http://www . dkfz . de/en/signal-transduction-cancer/pages/Data . html ) .\",\n \"Triplicate samples were combined and the Foxo precipitated samples were compared to mock precipitated samples using using the Affymetrix Tiling analysis ( TAS ) software .\",\n \"Combined mock arrays were used to set the background signal intensities for the ChIP arrays .\",\n \"The Integrated Genome Browser ( Nicol et al . , 2009 ) was used to visualize the resultant profile .\",\n \"Oligonucleotides were synthesized corresponding to annotated transcripts with the longest 5′ for both Insulin receptor ( corresponding to EST G430111A11 ) and IGF-1 receptor ( corresponding to ESTs CJ180736 and CJ173921 ) ( Supplementary file 1 ) .\",\n \"The oligos were used to clone the UTRs from cDNA derived from NIH 3T3 cells by PCR .\",\n \"Drosophila eIF4A and Pdcd4 were cloned into pET28 in frame with the 6x HIS tag from cDNA using PCR and standard cloning methods .\",\n \"The plasmid was transformed into BL21* ( DE3 ) cells ( Life Technologies , Grand Island , NY ) containing pLacIRARE2 ( Novagen , EMD Millipore , Billerica , MA ) .\",\n \"After induction with 1 mM IPTG overnight at 25°C , eIF4A or PDCD-4 was purified using HisPur Ni-NTA Resin ( Thermo Fisher Scientific , Rockford , IL ) according to manufacturer’s directions .\",\n \"PDCD-4 was eluted from the resin using 500 mM imidazole in PBS .\",\n \"eIF4A was eluted using 50 mM EDTA in 1X PBS .\",\n \"Purified 6His-Pdcd4 and 6His eIF4A was used to make rabbit polyclonal antisera ( Cocalico Biologicals , Inc . , Reamstown , PA ) .\",\n \"Full length Drosophila Pdcd4 was cloned into pGEX2TKN in frame with GST .\",\n \"GST-PDCD-4 or GST alone was expressed in BL21* ( DE3 ) cells ( Invitrogen , Grand Island , NY ) containing pLacIRARE2 ( Novagen , EMD Millipore , Billerica , MA ) .\",\n \"Expression was induced with 1 mM IPTG overnight at 25°C .\",\n \"Cells were resuspended and lysed in 1X PBS with lysozyme .\",\n \"GST or GST-PDCD4 was immobilized on glutathione sepharose 4B ( GE Healthcare Biosciences , Pittsburgh , PA ) .\",\n \"Equal amounts of recombinant eIF4A were applied to 50 µl of resin containing either GST or GST-Pdcd4 .\",\n \"The resin was incubated for 1 hr at 4°C .\",\n \"The resin was poured into a small spin column ( Pierce , Thermo Fisher Scientific , Rockford , IL ) and washed with 100 column volumes wash buffer ( 20 mM Tris pH 7 . 5 , 100 mM NaCl , 1 mM DTT , 0 . 1 mM EDTA , 0 . 1 mg/ml BSA ) .\",\n \"Bound proteins were eluted with wash buffer containing 10 mM reduced glutathione for 1 hr at 4°C .\",\n \"Samples were separated by SDS-PAGE , transferred to nitrocellulose and recombinant eIF4A was detected with a monoclonal antibody directed against the 6x HIS tag on eIF4A ( A00186 GenScript , Piscataway , NJ ) and a fluorescent secondary antibody against mouse IgG using a Li-Cor Odyssey Infrared imaging system .\",\n \"15 ml of a saturated culture of Drosophila S2 cells were harvested by centrifugation .\",\n \"The cells were washed once with 1X PBS .\",\n \"The cells were resuspended in two packed cell volumes hypotonic buffer ( 10 mM HEPES pH 7 . 4 , 10 mM KCl , 5 mM MgCl2 , 1 mM DTT , 1X protease inhibitors [Sigma-Aldrich Corp . , St . Louis , MO] ) .\",\n \"Triton X-100 was added to 0 . 5% and the cells were left on ice 30 min .\",\n \"Nuclei were pelleted 10 min at 6000×g .\",\n \"Supernatant was transferred to a new tube .\",\n \"10% of the sample was saved for input analysis .\",\n \"Polyclonal rabbit antisera to Drosophila eIF4A was added to the remainder of the sample and the sample was incubated at 4°C overnight with constant mixing .\",\n \"Samples were centrifuged 2 min at 23 , 000×g .\",\n \"The supernatant was combined with 50 µl protein-A Sepharose ( GE healthcare ) and incubated at 4°C for 2 hr .\",\n \"The mixture was poured into a small spin column ( Pierce , #89869 ) connected to a needle and washed with 5 ml 1XPBS 0 . 1% Triton X-100 followed by 1 ml 1X PBS .\",\n \"Proteins were elute by addition of 2X SDS-PAGE buffer ( 62 . 5 mM Tris-HCl , pH 6 . 8 , 25% glycerol , 2% SDS , 0 . 01% Bromophenol Blue , 5% β-mercaptoethanol ) incubation at room temperature for 10 min and collected by centrifugation .\",\n \"Samples were separated by SDS-PAGE .\",\n \"The gel was transferred to nitrocellulose and probed with rabbit α-Drosophila Pdcd4 antisera directed against full length 6His-Pdcd4 ( 1:1000 ) .\",\n \"To avoid detection of the IgG used for precipitation the immunoblot was developed with biotinylated protein-A ( 1:5000 ) ( Lal et al . , 2005 ) followed by fluorescent labeled streptavidin ( 1:5000 ) using a Li-Cor Odyssey Infrared imaging system .\",\n \"For experiments with the mutant Pdcd4 , 10 ml of Drosophila S2 cells were plated at 1 × 106 cells/ml in Schneider’s media supplemented with 10% FBS in a 10-cm dish .\",\n \"Constructs expressing wild-type myc-Pdcd4 or mutant myc-Pdcd4-282 A286A were transfected using effectene ( Qiagen Inc . , Valencia , CA ) .\",\n \"4 days later the cells were harvested and immunoprecipitation of eIF4A was performed as described above except transfected Pdcd4 was detected with a monoclonal antibody directed against the myc tag ( 9E10 ) and a fluorescent secondary antibody against mouse IgG using a Li-Cor Odyssey Infrared imaging system .\",\n \"Protein concentrations were determined using BCA assay ( Pierce ) and equal amounts of protein were loaded onto a 10% SDS-PAGE gel .\",\n \"The gel was transferred to nitrocellulose and probed with rabbit α-Drosophila Pdcd4 antisera directed against full length 6His-Pdcd4 ( 1:500 ) and mouse α-tubulin antibody ( 1:1000 ) .\",\n \"Transcription templates for monocistronic RNAs were created using PCR containing a template-specific forward primer with a T7 promoter incorporated and a vector specific reverse primer .\",\n \"Dicistronic RNA templates were made by digesting the cellular reporter vectors downstream of the firefly luciferase coding sequence and utilizing a T7 promoter incorporated in the vector .\",\n \"Templates were purified using column clean up protocol and eluted in 50 µl 10 mM Tris pH 8 . 0 ( Epoch Life Science , Missouri City , TX ) .\",\n \"Templates were transcribed using T7 polymerase and subsequently purified using LiCl precipitation .\",\n \"Transcripts were capped using vaccinia virus capping enzyme ( New England Biolabs , Ipswich , MA ) as recommended and purified using RNeasy column protocol ( Qiagen ) .\",\n \"Transcripts were tailed using poly ( A ) polymerase ( New England Biolabs , Ipswich , MA ) and purified using RNeasy columns ( Qiagen ) .\",\n \"Embryo translation extracts were prepared as described from 0- to 4-hr embryos ( Marr et al . , 2007 ) .\",\n \"Extracts were left untreated ( no Micrococcal nuclease treatment ) to allow translation under competitive conditions .\",\n \"Translation assays were performed in 6 µl of Drosophila embryo extract , 0 . 1 mM spermidine , 60 µm Amino Acids , 16 . 8 mM creatine phosphate , 800 ng of creatine kinase , 24 mM HEPES ( pH 7 . 4 ) , 0 . 4 mM Mg acetate , 30 mM K acetate , 1 µg of calf liver tRNA , and 100 ng of template RNA in a 10-µl reaction .\",\n \"Hippuristanol was added to a final concentration of 2µM or otherwise indicated .\",\n \"For assays containing PDCD-4 , protein was added to a final concentration of 320 nM and was preincubated for 15 min with extract before the addition of RNA templates .\",\n \"Translation reactions were incubated at 27°C for 1 hr and luciferase activity was measured using 100 µl of luciferase substrate ( Promega Corp . , Madison , WI ) .\",\n \"Firefly luciferase was measured by adding 100 µl of 75 mM HEPES pH 8 . 0 , 5 mM MgSO4 , 20 mM DTT , 100 µM EDTA , 530 µM ATP , 0 . 5 mM coenzyme A , and 0 . 5 mM D-luciferin and renilla was measured by adding 100 µl 25 mM Na4PPi , 10 mM NaOAc , 15 mM EDTA , 0 . 5 M Na2SO4 , 1 . 0 M NaCl , and 0 . 1 mM Coelenterazine , pH 5 . 0 .\",\n \"All experiments were performed at least twice in triplicate .\",\n \"Translation assays were performed in 6 µl of untreated rabbit reticulocyte extract ( no Micrococcal nuclease treatment to allow translation under competitive conditions ) .\",\n \"( Green Hectares , McFarland , WI ) , 0 . 1 mM spermidine , 60 µm Amino Acids , 16 . 8 mM creatine phosphate , 800 ng of creatine kinase , 24 mM HEPES ( pH 7 . 4 ) , 0 . 4 mM Mg acetate , 30 mM K acetate , 1 µg of calf liver tRNA , and 100 ng of template RNA in a 10-µl reaction .\",\n \"Translation reactions were incubated at 37°C for 30 min and luciferase activity was measured using 100 µl of luciferase substrate ( Promega ) .\",\n \"For assays containing excess m7G cap , cap structure analogue ( New England Biolabs , #S1407S ) was added to a final concentration of 1 mM .\",\n \"All experiments were performed at least twice in triplicate .\",\n \"Drosophila S2 cells with a stable transfection of constitutively active Foxo under the control of the metallothionein A promoter were maintained in Schneider’s Insect Media supplemented with 10% fetal bovine serum ( Puig et al . , 2003 ) .\",\n \"These cells were plated at 1 . 25 × 106 cell/ml , and expression was induced by addition of 500 µm CuSO4 for 16 hr .\",\n \"During induction the media was supplemented with 1 µg/m bovine insulin .\",\n \"For protein samples , cells were lysed in RIPA buffer ( PBS containing 10 mM EDTA , 1% Triton X-100 , 1% SDS , 1% deoxycholate , 1× complete protease inhibitor [Roche , Indianapolis , IN] , 10% glycerol ) .\",\n \"Total RNA was extracted from mock-treated and induced cells using TRI Reagent according to manufacturer’s protocol ( Molecular Research Center , Inc . , Cincinnati , OH ) .\",\n \"5 µg of total RNA were digested by DNaseI ( New England Biolabs ) .\",\n \"First strand cDNA sythesis was done using a mix of oligo-dT and random hexamers with MMLV reverse transcriptase .\",\n \"The final concentrations of the cDNA reaction were 50 mM Tris-HCl ( pH 8 . 3 ) , 50 mM KCl , 3 mM MgCl2 , 10 mM DTT , 400 µM dNTPs , 1–2 µg RNA , 500 ng primers , 200 units MMLV RT . cDNAs were diluted 1:10 in TE pH 8 .\",\n \"qPCR was run using 5 µl cDNA , GoTaq qPCR Master Mix ( Promega ) , and primers at a final concentration of 100 nM in a 20-µl reaction .\",\n \"qPCR was done using specific primers against Drosophila Pdcd4 , RP49 , GstD1 , and 4E-BP ( Supplementary file 1 ) .\",\n \"Pdcd4 fold-expression was calculated as a fraction of RP49 and normalized to mock-treated expression levels .\",\n \"All experiments were performed at least twice in triplicate .\",\n \"7-day-old adult male flies were starved for 5 hr and transferred to vials containing 5% sucrose or 5% sucrose+5 mM paraquat .\",\n \"After 24 hr flies were harvested and total RNA was extracted from mock-treated and paraquat-treated flies using TRI Reagent according to manufacturer’s protocol ( Molecular Research Center , Inc . ) .\",\n \"Previously described dicistronic reporter constructs ( Marr et al . , 2007 ) were subcloned into a plasmid containing the metallothionein A promoter for metal inducible expression ( Marr et al . , 2006 ) .\",\n \"The IRES sequence from HCV ( Kieft et al . , 1999 ) was subcloned into the inducible expression vector .\",\n \"For expression in S2 cells , Pdcd4 was cloned under a minimal actin promoter .\",\n \"For the Pdcd4 double mutant construct , amino acids 282 , and 286 ( Figure 1B ) were mutated to alanine by site directed mutagenesis .\",\n \"S2 cells were maintained in Schneider’s media with 10% FBS and Penicillin/Streptomycin .\",\n \"For transfection , cells were plated at 1 . 25×106 cells/ml in Schneider’s supplemented with 10% FBS and an additional 1 µg/ml bovine insulin .\",\n \"DNA was transfected at a 4:1 ratio expression plasmid to reporter plasmid using effectene transfection reagent ( Qiagen ) following instructions for S2 cells .\",\n \"Cells were induced with 500 µM CuSO4 36 hr after transfection .\",\n \"Cells were lysed in passive lysis buffer ( Promega ) and assayed 36 hr after induction using a dual luciferase assay .\",\n \"Firefly expression was measured in 75 mM HEPES pH 8 . 0 , 5 mM MgSO4 , 20 mM DTT , 100 µM EDTA , 530 µM ATP , 0 . 5 mM coenzyme A , and 0 . 5 mM D-luciferin .\",\n \"Renilla expression was measured by addition of an equal volume of 25 mM Na4PPi , 10 mM NaOAc , 15 mM EDTA , 0 . 5 M Na2SO4 , 1 . 0 M NaCl , and 0 . 1 mM Coelenterazine .\",\n \"All experiments were performed at least twice in triplicate .\",\n \"Drosophila S2 cells expressing constitutively active Foxo were formaldehyde crosslinked .\",\n \"Nuclei were isolated , lysed , and chromatin was sonicated to 500–1000 bp in length .\",\n \"Chromatin was incubated with polyclonal sera against full length Foxo .\",\n \"The chromatin/antibody mix was then incubated with protein A beads to isolate Foxo-bound chromatin from the sample .\",\n \"Purified DNA was assayed by qPCR to determine enrichment for genomic sites bound by Foxo .\",\n \"Enrichment is based on signal increase compared to a region of the genome in the first intron of CG15414 ( Supplementary file 1 ) .\",\n \"The mouse insulin receptor and insulin-like growth factor one receptor 5′ UTRs were subcloned into the plasmid pGLRSVRF .\",\n \"This plasmid contains the RSV LTR followed by the renilla luciferase open reading frame , the firefly luciferase open reading frame and the SV40 early polyadenylation signal .\",\n \"The UTRs were cloned between the renilla and firefly open reading frames .\",\n \"For transfection NIH3T3 cells were trypsinized and counted .\",\n \"350 µl of cells at a concentration of 1 × 105 cells per milliliter were plated in each well of a 24-well plate .\",\n \"Cells were transfected using Effectene ( Qiagen ) according to manufacturer’s instructions .\",\n \"Cells were lysed in passive lysis buffer ( Promega ) and assayed 36 hr after induction using a dual luciferase assay ( Promega ) .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Under conditions of stress , such as limited growth factor signaling , translation is inhibited by the action of 4E-BP and PDCD4 .","These proteins , through inhibition of eIF4E and eIF4A , respectively , impair cap-dependent translation .","Under stress conditions FOXO transcription factors activate 4E-BP expression amplifying the repression .","Here we show that Drosophila FOXO binds the PDCD4 promoter and stimulates the transcription of PDCD4 in response to stress .","We have shown previously that the 5′ UTR of the Drosophila insulin-like receptor ( dINR ) supports cap-independent translation that is resistant to 4E-BP .","Using hippuristanol , an eIF4A inhibitor , we find that translation of dINR UTR containing transcripts are also resistant to eIF4A inhibition .","In addition , the murine insulin receptor and insulin-like growth factor receptor 5′ UTRs support cap-independent translation and have a similar resistance to hippuristanol .","This resistance to inhibition of eIF4E and eIF4A indicates a conserved strategy to allow translation of growth factor receptors under stress conditions ."],"string":"[\n \"Under conditions of stress , such as limited growth factor signaling , translation is inhibited by the action of 4E-BP and PDCD4 .\",\n \"These proteins , through inhibition of eIF4E and eIF4A , respectively , impair cap-dependent translation .\",\n \"Under stress conditions FOXO transcription factors activate 4E-BP expression amplifying the repression .\",\n \"Here we show that Drosophila FOXO binds the PDCD4 promoter and stimulates the transcription of PDCD4 in response to stress .\",\n \"We have shown previously that the 5′ UTR of the Drosophila insulin-like receptor ( dINR ) supports cap-independent translation that is resistant to 4E-BP .\",\n \"Using hippuristanol , an eIF4A inhibitor , we find that translation of dINR UTR containing transcripts are also resistant to eIF4A inhibition .\",\n \"In addition , the murine insulin receptor and insulin-like growth factor receptor 5′ UTRs support cap-independent translation and have a similar resistance to hippuristanol .\",\n \"This resistance to inhibition of eIF4E and eIF4A indicates a conserved strategy to allow translation of growth factor receptors under stress conditions .\"\n]"},"summary":{"kind":"list like","value":["Protein synthesis in eukaryotes occurs in two stages: transcription of DNA into messenger RNA ( mRNA ) in the nucleus , and then translation of that mRNA into a protein by ribosomes in the cytoplasm .","These processes are regulated by a complex network of signaling pathways that enables cells to tailor protein synthesis to match current conditions .","This involves regulating the expression of the genes that code for these proteins .","When cells experience stressful events , such as a shortage of oxygen or nutrients , they reduce the synthesis of most proteins .","This response is regulated , in part , by a signaling pathway known as the insulin and insulin-like receptor pathway .","In particular , stressful events inhibit a protein complex called eIF4F , which normally initiates the translation of mRNA molecules by binding to a structure on one end of the mRNA called the 5′ cap .","Despite this general inhibition , the production of certain other proteins—including the insulin receptor itself—is actually increased in response to stress .","Olson et al . have carried out a series of experiments to explore how inhibition of the eIF4F protein complex influences the translation of the mRNA for the insulin receptor .","The eIF4F complex is made up of three proteins , including one that binds to the 5′ cap and a helicase that unwinds the RNA .","Previous work in the fruit fly Drosophila showed that translation of this mRNA can continue even if formation of the eIF4F complex is inhibited by targeting the cap binding protein .","Olsen et al . now show that translation of this mRNA is also independent of the helicase .","Instead , translation is maintained under these conditions because the insulin receptor mRNA contains a sequence called an internal ribosome entry site , which allows ribosomes to bind to the mRNA without the influence of the 5′ cap .","Olson et al . reveal the details of this regulatory pathway in Drosophila and show that similar mechanisms are at work in mammalian cells , suggesting this pathway represents a crucial regulatory process that has been conserved during evolution .","A key question for future research is whether other genes within the insulin and insulin-receptor like signaling pathway use this same trick to evade translational inhibitors ."],"string":"[\n \"Protein synthesis in eukaryotes occurs in two stages: transcription of DNA into messenger RNA ( mRNA ) in the nucleus , and then translation of that mRNA into a protein by ribosomes in the cytoplasm .\",\n \"These processes are regulated by a complex network of signaling pathways that enables cells to tailor protein synthesis to match current conditions .\",\n \"This involves regulating the expression of the genes that code for these proteins .\",\n \"When cells experience stressful events , such as a shortage of oxygen or nutrients , they reduce the synthesis of most proteins .\",\n \"This response is regulated , in part , by a signaling pathway known as the insulin and insulin-like receptor pathway .\",\n \"In particular , stressful events inhibit a protein complex called eIF4F , which normally initiates the translation of mRNA molecules by binding to a structure on one end of the mRNA called the 5′ cap .\",\n \"Despite this general inhibition , the production of certain other proteins—including the insulin receptor itself—is actually increased in response to stress .\",\n \"Olson et al . have carried out a series of experiments to explore how inhibition of the eIF4F protein complex influences the translation of the mRNA for the insulin receptor .\",\n \"The eIF4F complex is made up of three proteins , including one that binds to the 5′ cap and a helicase that unwinds the RNA .\",\n \"Previous work in the fruit fly Drosophila showed that translation of this mRNA can continue even if formation of the eIF4F complex is inhibited by targeting the cap binding protein .\",\n \"Olsen et al . now show that translation of this mRNA is also independent of the helicase .\",\n \"Instead , translation is maintained under these conditions because the insulin receptor mRNA contains a sequence called an internal ribosome entry site , which allows ribosomes to bind to the mRNA without the influence of the 5′ cap .\",\n \"Olson et al . reveal the details of this regulatory pathway in Drosophila and show that similar mechanisms are at work in mammalian cells , suggesting this pathway represents a crucial regulatory process that has been conserved during evolution .\",\n \"A key question for future research is whether other genes within the insulin and insulin-receptor like signaling pathway use this same trick to evade translational inhibitors .\"\n]"},"year":{"kind":"string","value":"2013"}}},{"rowIdx":4297,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["neuroscience","cancer biology"],"string":"[\n \"neuroscience\",\n \"cancer biology\"\n]"},"title":{"kind":"string","value":"Autophagy linked FYVE (Alfy/WDFY3) is required for establishing neuronal connectivity in the mammalian brain"},"id":{"kind":"string","value":"elife-14810-v1"},"sections":{"kind":"list like","value":[["The Autophagy linked FYVE domain protein ( Alfy ) [gene name , WD40 repeat and FYVE domain protein 3 ( Wdfy3 ) ] is a member of the Beige and Chediak-Higashi ( BEACH ) domain containing proteins , a family of proteins implicated in vesicle trafficking and membrane dynamics ( Isakson et al . , 2013; Simonsen et al . , 2004 ) .","As its name implies , Alfy has been implicated in the degradative pathway macroautophagy , by acting as a molecular scaffold between select cargo and core members of the mammalian autophagic machinery such as Atg5 , p62 and Atg8 homologs ( Clausen et al . , 2010; Filimonenko et al . , 2010; Lystad et al . , 2014; Simonsen et al . , 2004 ) .","In addition , its functional FYVE zinc finger domain at the COOH-terminus permits partial co-localization to phosphatidylinositol-3-monophosphate ( PtdIns3P ) , especially at autophagosome membranes ( Simonsen et al . , 2004 ) .","Wdfy3 is evolutionarily conserved and the most extensively studied homolog is Blue Cheese ( bchs ) in D . melanogaster ( Finley et al . , 2003 ) .","In the developing and adult fly central nervous system ( CNS ) , Bchs is abundantly expressed , with preferential accumulation in axon terminals and at the growth cone ( Finley et al . , 2003; Khodosh et al . , 2006 ) .","Adult bchs null flies have a shortened life span and show signs of adult onset neurodegeneration , including the accumulation of ubiquitinated aggregates ( Filimonenko et al . , 2010; Finley et al . , 2003; Khodosh et al . , 2006 ) .","Loss-of-function ( LoF ) mutations in bchs disrupt the axonal transport of endolysosomal vesicles ( Lim and Kraut , 2009 ) , however no defects in axon guidance have been reported in bchs null larva ( Khodosh et al . , 2006 ) .","Recently it has been reported that in vertebrates , genetically diminished levels of Alfy disrupts neurogenesis leading to altered forebrain morphology ( Orosco et al . , 2014 ) .","Furthermore , genetic screening has revealed a possible role for the human homolog WDFY3 as a genetic risk factor for intellectual and developmental disabilities ( IDD ) , microcephaly and neuropsychiatric disorders ( Bonnet et al . , 2010; Iossifov et al . , 2012; Kadir et al . , 2016 ) .","These findings raise the possibility that Alfy could have an important function in mammalian CNS development .","Here , we present two new mouse models that eliminate Alfy expression and identify an essential role for Alfy during murine development .","Constitutive elimination of Alfy leads to perinatal lethality , in conjunction with developmental brain wiring defects throughout the CNS , involving forebrain commissures , internal capsule , optic chiasm , spinal cord and longitudinal tracts such as the medial forebrain bundle .","In the ventral midbrain , dopaminergic cell populations retain an immature morphology and their axons aberrantly project into the hypothalamic region , forming an ectopic commissure near the optic chiasm .","Consistent with a failure of axon guidance mechanisms , localization of glial guidepost cells for callosal axons were disrupted , and sensitivity of Alfy knockout axons to the trophic effect of Netrin-1 was significantly diminished .","Moreover , Alfy is enriched in membrane fractions , suggesting that it may play a key role in membrane trafficking events to establish neural connectivity in the mammalian brain ."],["To characterize the role of Alfy in mouse , we initially determined when and where Alfy/Wdfy3 is expressed .","Multiplex , semi-quantitative RT-PCR revealed that Wdfy3 mRNA could be detected as early as embryonic day ( E ) 11 in CNS tissue , and remains detectable throughout gestation ( Figure 1A ) .","Similar analysis in adult tissue revealed that the Wdfy3 transcript is ubiquitously expressed , and that the highest concentration of Alfy was observed in the brain ( Figure 1—figure supplement 1 ) , confirming previous results ( Simonsen et al . , 2004 ) .","Wdfy3 transcript is detected throughout the both the perinatal and adult brain , as determined by in situ hybridization ( ISH ) ( Figure 1B and not shown ) .","Immunoblotting revealed that expression of the protein was present uniformly throughout the brain ( Figure 1C ) .","Using both primary neuronal and purified astroglial cultures , endogenous Alfy expression was detected in both cell types ( Figure 1—figure supplement 2 ) , supporting recent transcriptome analysis of the mouse cortex ( Zhang et al . , 2014 ) .","Therefore , we conclude that Alfy is a CNS-enriched protein that is present in various neuronal and non-neuronal cell types in the developing and adult brain . 10 . 7554/eLife . 14810 . 003Figure 1 . Alfy is highly expressed throughout the developing and adult mouse CNS .","( A ) ( Top ) RT-PCR demonstrates Alfy/Wdfy3 can be detected as early as embryonic day 11 ( E11 ) and remains abundant throughout gestation ( E19 ) .","( A ) Multiplex PCR for the 5’ region of the gene encoding Alfy , Wdfy3 with Class III β-tubulin ( βIII , top ) and Glial fibrillary acid protein ( Gfap , bottom ) .","( Btm )","Quantification of transcript levels relative to βIII ( black ) and Gfap ( gray ) , n = 4 , bars represent mean ± SEM .","( B ) In situ hybridization of sections from P0 mice reveals strong expression of Wdfy3 throughout the brain .","Low magnification images stitched together to reveal a complete sagittal brain section representative of Wdfy3 mRNA distribution in a wildtype mouse .","An abundance of mRNA is observed throughout the newborn CNS ( n = 4 ) , whereas no mRNA staining above background is observed in Alfy KO mice ( data not shown ) .","The ISH reflects how Alfy is most highly expressed in brain ( Figure 1—figure supplement 1 ) and this expression is both neuronal and glial ( Figure 1—figure supplement 2 ) .","Abbreviations: cerebellum ( cb ) , cerebral cortex ( ctx ) , hippocampus ( hp ) , hypothalamus ( ht ) , midbrain ( mb ) , olfactory bulb ( ob ) , striatum ( st ) , superior colliculus ( sc ) and thalamus ( th ) .","( C ) Immunblotting reveals that Alfy expression is maintained throughout the adult brain .","Lysates generated from different regions of the adult brain , including the brain stem ( bs ) were probed with an Alfy antibody raised against its N-terminus .","γ-tubulin is shown as a loading control ( n = 4 ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 00310 . 7554/eLife . 14810 . 004Figure 1—figure supplement 1 . Alfy/Wdfy3 expression is highest in the brain .","( A ) Semi-quantitative RT-PCR for Wdfy3 mRNA in different organs from adult mice .","Relative levels of Wdfy3 mRNA ( corrected for β-Actin ) across the different organs is quantified below ( n = 5 ) .","Bars represent mean ± SEM .","Wdfy3 RNA is most highly expressed in brain , although the transcript can be detected in all tissues examined .","( B ) Immunoblotting for Alfy from tissue samples collected from adult mice .","Relative protein levels were normalized using the cytoskeletal protein vinculin as shown below ( n = 6 ) .","Bars represent mean ± SEM .","In confirmation with previous results , Alfy protein is most abundant in brain . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 00410 . 7554/eLife . 14810 . 005Figure 1—figure supplement 2 . Alfy is expressed in neurons and astroglia .","( A ) Primary cortical cultures were grown for seven days with or without mitotic inhibitors .","Mitotic inhibitors were added to cultures after plating to prevent glial proliferation and generate cultures that are enriched for neurons .","After seven days , lysates were prepared from the cultures and western blots containing 5 µg of total protein were probed with antibodies against Alfy , the glial marker GFAP , and the neuronal specific tubulin , BIII .","As expected , mitotically inhibited cultures had a minimal GFAP expression but contained abundant BIII Tubulin .","Alfy was detected in mitotically inhibited cultures , implying neurons endogenously express Alfy .","The experiment was replicated twice , using two litters of mice ( Control n = 3; KO n = 3 ) .","( B ) Alfy was detected in lysates prepared from primary astroglia cultures that were passaged and allowed to differentiate for fourteen days .","Western blots containing 3 µg of total protein were probed with antibodies against Alfy and GFAP .","Results from two independent cultures are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 005 To investigate the consequence of the genetic deletion of Wdfy3 in mice , we generated and characterized two different Alfy deficient mouse lines: One using gene trap ( GT ) -mediated disruption and a second using a conditional strategy ( Figure 2 , Figure 2—figure supplement 1 ) .","Whereas several GT lines disrupting the Wdfy3 locus were found , one that contained a GT insertion within the first intron was predicted to completely abolish production of the full length transcript .","To confirm , mice heterozygous for this mutation ( Alfy GT Het ) were interbred .","Homozygous mice ( Alfy GT ) were born at close to the expected Mendelian ratios ( WT: 19% , n = 20; Alfy GT Het: 52% , n = 54; Alfy GT: 28% , n= 29 ) .","Primer pairs spanning the transcript indicated that the full length Wdfy3 transcript is not produced in Alfy GT mice ( Figure 2—figure supplement 1B ) , and antibodies directed against the NH3- or COOH-terminus of Alfy confirmed that full length Alfy protein was not detectable in brain lysates ( not shown and Figure 2B ) .","This demonstrates that the GT insertion successfully mutagenized the Wdfy3 locus , leading to the loss of Alfy expression . 10 . 7554/eLife . 14810 . 006Figure 2 . Two lines of knockout ( KO ) mice reveal Alfy is essential for postnatal survival .","( A , B )","Alfy GT mice .","( A ) The GT cassette introduces a splice acceptor site ( SA ) and causes the premature termination of transcription through the introduction of a poly-adenylation ( pA ) tail .","The translation start site for Wdfy3 is located in exon IV .","RT-PCR indicates that the gene trap insertion leads to a loss of Wdfy3 transcript ( Figure 2—figure supplement 1A , B ) .","( B ) Western blot of brain lysates probed with an antibody against the COOH-terminus of Alfy and βIII ( n = 5 ) .","Abbreviations: long terminal repeats ( LTR ) , neomycin ( NEO ) , Phosphoglycerate kinase-1 promoter and Bruton tyrosine kinase splice donor site ( PGK-BTK-SD ) .","( C , D )","Alfy KO mice .","( C ) A conditional ( flox ) Wdfy3 allele is created by insertion of two loxP sites ( red triangles ) to flank exon 5 , leading to its excision upon exposure to Cre and the creation of the smaller knockout ( KO ) ‘Δ’ allele .","The two forward and one reverse primers ( arrows ) used for genotyping are noted ( also see Figure 2—figure supplement 1C ) .","( D ) Immunoblotting detects Alfy in brain lysates from heterozygous ( Alfy Het , n = 6 ) but not in Alfy KO mice ( n = 7 ) .","( E–G )","Characterization of newborn Alfy GT mice .","( E ) Newborn Alfy GT and littermatecontrol ( Ctrl ) mice .","Arrows highlight that control pups have stomachs full of milk whereas their GT littermates do not .","( F ) Alfy GT pups are consistently smaller than control .","After genotyping , the weights of the animals were percentile ranked and binned into four groups .","Approximately 50% of Alfy GT mice ( 8/15 ) had birth weights in the lowest 25th percentile , whereas heterozygous and wildtype littermates made up 92% ( 12/13 ) of mice with birth weights in the 75th percentile or greater .","Data were collected from 10 litters of mice , n = 55 .","Similar differences were observed between the Alfy KO pups and their heterozygous littermates ( data not shown ) .","( G ) Alfy GT pups lack a righting reflex .","The amount of time it took each pup to perform the task was averaged over three trials and recorded as the latency to right .","Alfy GT failed to rotate from back to their bellies within 60 sec .","A single factor ANOVA revealed genotype had a significant effect on pup behavior ( n = 6 WT , 9 Alfy GT Het , 7 Alfy GT; F ( 2 , 17 ) = 20 . 90 , p < 0 . 001 ) .","Fisher’s PLSD test indicated a significant difference between Alfy GT and WT ( p < 0 . 001 ) or heterozygous ( p < 0 . 001 ) littermates .","Bars represent mean ± SEM .","Similar differences were observed between Alfy KO pups and their heterozygous littermates ( Figure 2—figure supplement 1D ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 00610 . 7554/eLife . 14810 . 007Figure 2—figure supplement 1 . Creation and characterization of Alfy GT and Alfy KO mice .","( A , B )","Creation of Alfy GT mice .","( A ) PCR based genotyping of wildtype ( WT ) , Alfy GT heterozygotes ( Alfy GT Het ) and Alfy GT mice .","( B ) Semi-quantitative RT-PCR of Alfy GT mice .","Both the 5’ and 3’ portions of the Wdfy3 mRNA transcript are undetectable in Alfy GT mice by RT-PCR .","β-actin is shown as control .","n = 2 WT , 3 Alfy GT Het , 3 Alfy GT ) ( C ) PCR genotyping of Alfy KO mice .","PCR based genotyping of the homozygous and heterozygous conditional Wdfy3 allele ( Wdfy3loxP/+ and Wdfy3loxP/loxP , respectively ) and WT , Alfy KO heterozygous ( Alfy KO Het ) and Alfy KO mice .","( D ) Mice are born with an inherent ability to right themselves from a supine to prone position .","To determine if the loss of Alfy affected this behavior , marked and warmed pups were placed on their backs on a flat surface and given 60 s to rotate to a prone position .","The amount of time it took each pup to perform the task was averaged over three trials and recorded as the latency to right .","Alfy KO mice were not able to rotate from their backs to their bellies within 60 s , similar to Alfy GT mice ( Figure 2 ) .","A single factor ANOVA revealed genotype had a significant effect on pup behavior ( n = 4/genotype; F ( 1 , 6 ) = 43 . 14 , p < 0 . 001 ) .","Bars represent mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 007 The second Alfy-deficient mouse line was produced using a conditional strategy ( Figure 2C ) .","Mice carrying a conditional Wdfy3 allele were crossed to HprtCre/+ females to generate a constitutive null '∆' allele ( Figure 2C , Figure 2—figure supplement 2C ) ( Tang et al . , 2002 ) .","Similar to Alfy heterozygous for the GT insertion , mice heterozygous for the '∆' allele ( Alfy Het ) were healthy and fertile .","Breeding of Wdfy3+/∆::HprtCre/+females with Wdfy3loxP/loxP::Hprt+/Y males generated mice heterozygous ( Alfy Het ) and homozygous ( Alfy KO ) for the null allele close to the expected Mendelian ratios ( Alfy Het: 56% , n = 40; Alfy KO: 44% , n = 32 ) .","Alfy protein also was not detected in Alfy KO mice ( Figure 2D ) .","Although neonatal Alfy mutant pups appeared grossly normal , were reactive , and showed no sign of cyanosis , they all died several hours after birth .","Unlike their littermates , Alfy mutant pups failed to thrive and consistently showed an absence of milk in their stomachs ( Figure 2E ) .","Despite being properly cleaned , live Alfy mutant pups were frequently the smallest pups within the litter ( Figure 2F ) and were selectively buried under nesting material , indicating that the dams were rejecting them due to diminished or uncoordinated movement ( Turgeon and Meloche , 2009 ) .","To test this hypothesis , we performed a righting reflex test ( Figure 2G , Figure 2—figure supplement 1D , Video 1 ) .","Littermates with at least one copy of Alfy were able to right themselves within the allotted test time .","In contrast , mice lacking Alfy consistently failed at this test , and demonstrated uncoordinated behavior . 10 . 7554/eLife . 14810 . 008Video 1 . Mice lacking Alfy have an abnormal righting reflex . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 008 Histological examination of the newborn ( P0 ) brain by Nissl staining revealed that brains from Alfy GT and Alfy KO both demonstrated striking abnormalities in the forebrain , midbrain and hindbrain , including visibly smaller brains and gross enlargement of the lateral ventricles ( Figure 3A , B and Figure 3—figure supplement 1A ) .","Concurrently , there was an apparent loss and disorganization of interhemispheric axonal tracts throughout the brain .","Brains of Alfy GT Het or Alfy Het mice were indistinguishable from wildtype brains ( Figure 3A , B ) . 10 . 7554/eLife . 14810 . 009Figure 3 . Commissures fail to cross the midline appropriately in Alfy GT and KO brains .","( A , B )","Nissl stained forebrain from newborn ( P0 ) control or Alfy mutant ( Alfy GT or Alfy KO ) mice .","Coronal sections are shown .","Alfy mutants have many abnormal features , including enlarged ventricles ( * ) , and apparent white matter abnormalities , including absence of a corpus callosum ( cc ) at the midline ( black arrow ) , undetectable anterior commissure ( ac , white arrow ) , and dysmorphic internal capsule ( rectangle ) .","More caudal sections can be found in Figure 3—figure supplement 1A .","n = 6 WT , 3 Alfy GT Het , 7 Alfy GT; n = 3 Alfy Het , 3 Alfy KO .","( C , D )","Immunostaining highlights axonal abnormalities in Alfy mutant mice .","Coronal sections are shown .","The three major forebrain commissures fail to cross the midline in Alfy mutant brains .","( C ) The cc , hippocampal commissure ( hc ) and ( D ) ac are absent in Alfy mutants .","‘*’ denotes midline and lack of axonal connectivity .","Boxed regions are shown enlarged below .","n = 5/genotype .","The habenular and posterior commissures can be found in Figure 3—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 00910 . 7554/eLife . 14810 . 010Figure 3—figure supplement 1 . Alfy mutant mice have aberrant and disorganized axon projections .","( A ) H&E staining of neonatal brains reveal altered brain morphology in Alfy GT mice .","Coronal sections are shown .","In the absence of Alfy , the cerebral cortex ( ctx ) , thalamus ( th ) and hippocampus ( hp ) are dysmorphic .","Additional abnormalities in the Alfy null mice include a reduced size of the thalamus .","Alfy heterozygotes are indistinguishable from WT littermates ( data not shown ) .","Wildtype mice are shown as control ( Ctrl ) .","n = 3/genotype .","Scale bar = 250 μm .","( B ) NF staining reveals aberrant and disorganized axonal projections of Alfy mutant brains .","Coronal sections are shown .","Alfy GT mice show reduced staining in the ctx and abnormal NF staining in the internal capsule ( ic ) and tracts within the hp .","The fasciculus retroflexus ( fr , square box ) , mammalothalamic tract ( mt , circle ) and cerebral peduncle ( arrow ) are evident in the Ctrl but present as aberrant , disorganized axonal tracts in Alfy GT ( dashed box ) .","The external capsule ( arrowhead ) is present in Ctrl and Alfy null brains .","n = 5/genotype .","Additional abbreviations: hypothalamus ( ht ) , periaqueductal gray ( pag ) , striatum ( st ) , and superior colliculus ( sc ) .","DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 01010 . 7554/eLife . 14810 . 011Figure 3—figure supplement 2 . Aberrant and disorganized projections of the habenular and posterior commisural axons .","( A ) Low-power magnification of Ctrl and Alfy GT coronal sections are presented in the top panels .","The arrow points to the habenular commissure ( hac ) , which is presented at higher magnification below .","( B ) The posterior commissure ( pc ) .","Both appeared morphologically abnormal in Alfy GT mice , although both crossed the midline .","n = 3/genotype .","Scale bar as indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 011 To examine the axonal defects further , P0 Alfy mutant brains were stained for neurofilament ( NF ) ( Figure 3C , D and Figure 3—figure supplement 2 ) .","When compared to control , NF labeling of Alfy null brains was greatly reduced , suggesting fewer axonal tracts .","A notable abnormality was the failure of the three major forebrain commissures to cross the midline .","Axons of the corpus callosum , hippocampal commissure ( Figure 3C ) , and anterior commissure ( Figure 3D ) failed to decussate , leading to a separation of the two hemispheres .","Two smaller , caudal commissures , the habenular and posterior commissures ( Figure 3—figure supplement 2 ) , were morphologically abnormal and reduced in size however a portion of their tracts crossed the midline .","We next examined the retinal axon decussation at the optic chiasm by anterograde-labeling with DiI ( Figure 4A–C ) .","Ipsilateral and contralateral projections were quantified by calculating the ipsilateral index ( Kuwajima et al . , 2012 ) .","At P0 , the ipsilateral projection in Alfy GT mice ( 1 . 40 ± 0 . 06 ) was 40% larger than that in wildtype ( 1 . 0 ± 0 . 08 ) or heterozygous mice ( 1 . 1 ± 0 . 06 ) ( Figure 4C ) .","This significant difference suggests that in Alfy mutants , contralateral axons less readily cross the optic chiasm midline .","These data reinforce the findings that Alfy is required to properly establish commissures throughout the developing mouse brain . 10 . 7554/eLife . 14810 . 012Figure 4 . Midline crossing defects extend to the optic chiasm in Alfy GT and KO brains , possibly due to disrupted localization of guidepost glial cells .","( A–C )","Retinal decussation defects are observed in Alfy mutant mice .","( A ) Whole mounts of P0 optic chiasm are unilaterally labeled with DiI at the optic disc .","The ipsilateral projection denoted in the white dashed box , which is shown enlarged below .","WT and GT heterozygous littermates were indistinguishable .","A heterozygous mouse is shown as a control ( Ctrl ) .","( B ) Schematic representation of how the ipsilateral index is calculated .","Pixel intensities of contralateral and ipsilateral optic tracts are measured within 500 x 500 µm2 .","( C ) The ipsilateral index of Alfy mutant mice is greater than both WT and heterozygous ( GT Het ) littermate controls , indicating the abnormally enlarged ipsilateral projection .","Bars represent mean ± SEM , and statistical analysis was determined using one-way ANOVA followed by the Tukey’s post hoc test .","Respective n-values per genotype are noted in the bars .","*p < 0 . 05 .","( D , E )","Immunostaining for GFAP in P0 forebrain reveals mislocalization of guidepost glial cells .","Coronal sections are shown .","( D ) The glial wedge ( gw ) , the indusium griseum ( ig ) and midline zipper ( mz ) glia populations of glial cells were in the expected locations in WT littermate brains ( Ctrl ) .","In Alfy GT brains , the ig was not detectable ( expected location denoted by '*' ) , and the gw and mz cells were disorganized .","n = 3/genotype .","( E ) Glial populations within the fimbria ( fi ) and dentate gyrus ( dg ) of the hippocampus were apparent in both genotypes .","Boxed areas are shown at higher magnification below .","n = 3/genotype .","The examination of other potential intrinsic alterations that may contribute to forebrain axonal connectivity can be found in Figure 4—figure supplements 1 and 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 01210 . 7554/eLife . 14810 . 013Figure 4—figure supplement 1 . The loss of Alfy leads to modest changes in proliferation but not cell death in the neocortex .","( A ) Loss of Alfy leads to a moderate but significant decrease in cortical plate thickness .","Measurements of P0 cortex reveal that the cortical plate/subplate ( CP/SP ) of Alfy GT brains ( n = 3 ) are modestly but significantly thinner than control ( Ctrl , n = 3 ) brains , ( t ( 4 ) = 5 . 30 , p < 0 . 01 ) .","The marginal zone ( MZ ) and intermediate zone ( IZ ) do not reach significance , ( t ( 4 ) = 1 . 67 , p = 0 . 17 and t ( 4 ) = 2 . 38 , p = 0 . 076 , respectively ) .","Bars denote mean ± St . Dev; * , p < 0 . 01 .","( B , C )","Loss of Alfy leads to a subtle defect in cell proliferation at E15 . 5 but not at E13 . 5 .","A single injection of BrdU was administered to time-pregnant dams at E13 . 5 or E15 . 5 and embryos were collected 1 hr post-injection .","( B ) A clear band of BrdU-positive cells ( red ) was detected in the subventricular zone and deep cortical layer in all three littermate genotypes ( WT: n = 2 , heterozygous: n = 3 , and Alfy GT n = 3; two litters ) .","Hoechst 33342 nuclear DNA stain is in blue .","( C ) Colorimetric BrdU was used to quantify the number of proliferative cells in the developing cortical plate .","At E13 . 5 , Quantification revealed no significant difference in the number of BrdU positive ( BrdU+ ) cells between Alfy KO and heterozygous littermate control ( Ctrl ) t ( 8 ) = 1 . 59 , p = 0 . 15 ( Ctrl: n = 5 , Alfy KO: n = 5; two litters ) .","At E15 . 5 , there was a modest , but significant reduction in the absence of Alfy , t ( 8 ) = 3 . 12 , p < 0 . 01 ( Ctrl: n = 4 , Alfy KO: n = 6; two litters ) .","Bars denote mean ± SEM .","ns , not significant; * , p<0 . 01 .","( D ) Proliferation , indicated by Ki67-positivity indicates the normal proliferation in newborn Alfy GT mice .","Boxed regions highlight the sub-ventricular zone ( VZ ) and are shown at higher magnification in images below .","Stereology was performed to quantify the number of Ki67 positive cells in the sub-VZ ( Gundersen Coeffecient of Error = 0 . 15 , Ctrl ( wildtype littermate , n = 3 ) = 187 , 221 cells , S . D . = 19 , 634 compared to Alfy GT ( n = 3 ) = 179 , 074 cells , S . D . = 28 , 917 , t ( 4 ) = 0 . 40 , p = 0 . 71 . ) ( E ) Immunohistochemistry ( brown ) against cleaved caspase-3 reveals no significant difference in cell death at embryonic age E15 . 5 in the absence of Alfy .","The enlarged images of the inset for the images are provided immediately below .","No cell loss was detected in cortex at this age .","As a positive control , the naturally occurring cell death observed in the trigeminal ganglia at this age is included ( leftmost panels ) .","No difference was observed across genotype ( n = 2 WT , 2 Alfy GT Het; 3 Alfy GT ) .","Tissue sections are counterstained with Nissl , which is shown in purple . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 01310 . 7554/eLife . 14810 . 014Figure 4—figure supplement 2 . Focal cortical dysplasias are observed in Alfy GT brains .","( A ) No gross difference in Doublecortin ( DCX , green ) staining can be observed in E15 . 5 Alfy GT brains ( n = 3 ) compared with littermate controls ( Ctrl , n = 3 ) .","( B ) Specification of deep layer , Tbr1-positive cortical projection neurons are observed in Alfy GT mice when compared to littermate controls ( Ctrl ) .","Three mice per genotype were stained Tbr1 ( Top , red ) and counterstained with Hoeschst 33342 ( bottom , blue ) , and all mice demonstrated this pattern .","( C–E )","Focal cortical dysplasia ( FCD ) in Alfy null brains .","H&E staining reveals the presence of FCD in coronal ( C ) and sagittal ( D ) sections of Alfy GT brains .","Boxed areas are shown at higher magnification below .","FCDs are labeled with arrows .","( E ) FCDs can be detected by Nissl staining as early as E15 in Alfy GT mice ( 6/6 ) but not in control embryos ( 0/6 ) .","Arrowheads point to multiple FCDs located within the same hemisphere of an Alfy GT embryo . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 014 Two cell populations that help axons navigate the developing forebrain during the genesis of the corpus callosum are the glial wedge ( gw ) and indusium griseum ( ig ) glia ( Shu and Richards , 2001; Shu et al . , 2003 ) .","In light of the presence of Alfy in astroglial cells , as well as the midline crossing defects , we hypothesized that migration abnormalities could contribute to the connectivity defects in Alfy GT and KO mice .","Thus , we next examined the callosal guidepost cells , a key population implicated in providing intermediate guidance cues to developing cortical axons .","Immunostaining against GFAP on P0 mouse brains revealed abnormalities in Alfy GT brains ( Figure 4D , E ) .","Unlike control brain , the ig cells were undetectable and the gw appeared disorganized and loosely packed .","The midline zipper glia ( mz ) , cells proposed to promote fusion of the two telencephalic hemispheres ( Silver , 1993 ) , were present but distributed abnormally ( Figure 4D ) , whereas cells in the fimbria and dentate gyrus of the hippocampus appear largely normal ( Figure 4E ) .","Taken together , the disorganization of the ig , gw and mz is consistent with the agenesis of the corpus callosum observed in the absence of Alfy .","These findings suggest that cells that act as important guideposts for decussating callosal axons ( Shu and Richards , 2001 ) fail to migrate properly in the absence of Alfy , contributing to the connectivity defects observed .","In addition to the localization of the midline glial cells , we also determined if intrinsic alterations in the proliferation or differentiation of cortical projection neurons were also contributing to the disruptions in forebrain axonal connectivity .","Nissl staining revealed that cortical layering appeared normal , however there was a significant but modest thinning of the cortical plate/subplate at P0 ( Figure 4—figure supplement 1A ) .","Although this may be due to the diminished NF staining observed in Figure 3 , we next determined if cell proliferation was affected .","Pulse-labeling with bromo-6-deoxy-uridine ( BrdU ) at E15 . 5 revealed cell division in the developing cortex at the expected location in Alfy GT mice ( Figure 4—figure supplement 1B ) .","To determine if the amount of proliferation was impacted by the loss of Alfy , stereology was performed on BrdU labeled embryos at ages E13 . 5 and E15 . 5 ( Figure 4—figure supplement 1C ) .","No significant difference across genotypes was detected at E13 . 5 , but a modest yet significant reduction in the number of BrdU-positive cells was detected in Alfy KO embryos at E15 . 5 .","This difference appeared limited to the cortex , since staining against Ki67 within the subventricular zone at P0 revealed no significant difference between Alfy null mice and controls ( Figure 4—figure supplement 1D ) .","Further , despite this modest decrease in embryonic proliferation , there were no detectable differences across genotypes in the amount of cell death in E15 . 5 brains , as indicated by immunohistochemistry against cleaved caspase-3 ( Figure 4—figure supplement 1E ) .","We next sought to determine if the loss of Alfy disrupted proliferation in the embryonic cerebral cortex .","Although qualitative immunofluorescence against expression markers for immature migrating neurons ( DCX ) and for early born layer VI neurons ( Tbr1 ) also were not significantly altered in the absence of Alfy , histological staining revealed focal cortical malformations known as focal cortical dysplasias ( FCD ) present throughout the cerebral cortex of Alfy KO mice , as described previously in a Wdfy3 hypomorph ( Figure 4—figure supplement 2C–E ) ( Orosco et al . , 2014 ) .","These structures were observed as early as E15 . 5 .","Thus , the loss of Alfy leads to subtle defects in proliferation , but no changes in cell death or specification in the developing cortical projection neurons .","Although columnar organization of discrete regions of the cerebral cortex can be disrupted by the absence of Alfy during embryonic development , it is unlikely that these defects can account for the midline crossing defects observed in Alfy null mice .","In addition to the absence of interhemispheric commissures , another striking feature revealed by NF staining in Alfy GT and KO brains was the evident disorganization and apparent loss of other CNS axon tracts .","One particularly prominent defect was found in the organization of the internal capsule , which carries the corticospinal tract , and corticothalamic and thalamocortical projections; the internal capsule of Alfy GT brains was disorganized and followed an unusual trajectory through the striatum ( Figure 3 and Figure 3—figure supplement 1 ) .","To gain a temporal perspective on the development of the internal capsule , we examined its development during E15 . 5 ( Figure 5A ) and E17 . 5 ( Figure 5B ) .","NF staining revealed the expected fasciculated axonal projections of the developing internal capsule en route to and from the cerebral cortex in controls .","In contrast , the thalamocortical projections in Alfy GT mice formed ventrally displaced bundles , with fewer projections observed traveling through the ganglionic eminence .","Similarly , Alfy GT embryonic brain sections labeled with the axonal marker NCAML1 also highlighted abnormal bundle-like structures in the ventral diencephalic-telencephalic border ( Figure 5—figure supplement 1 ) 10 . 7554/eLife . 14810 . 015Figure 5 . Axonal tracts develop abnormally in the Alfy mutant brain and spinal cord .","( A , B )","Coronal sections of diencephalon at ( A ) E15 . 5 ( n = 3/genotype ) and ( B ) E17 . 5 ( n = 3/genotype ) stained for neurofilament ( NF , red ) and counter-stained with the nuclear stain Hoechst 33342 ( blue ) .","The developing internal capsule is highlighted with a yellow box ( top ) and higher magnification , confocal images of NF staining are shown with ( middle ) and without ( bottom ) Hoechst 33342 .","In control brains ( Ctrl ) , axons that comprise the internal capsule form a fan-shaped projection of bundled axons within the ganglionic eminence .","The abnormal , ventrally displaced , knot-like bundles of axons found in Alfy GT are marked with white arrows .","Neural cell adhesion molecular L1 ( NCAML1 ) staining reveals similar defects ( Figure 5—figure supplement 1 ) .","( C , D )","TH staining reveals abnormal projections in DAergic cell populations .","( C ) Coronal sections .","A9 and A10 DAergic populations are present in Alfy GT brains appear immature ( top ) .","The hypothalamic A13 DAergic cell population and medium forebrain bundle ( mfb ) are present in the diencephalon in Alfy GT brains; however aberrant projections into the hypothalamus and abnormal midline-crossing events are observed ( arrows , bottom ) .","n = 3/genotype .","( D ) Sagittal sections counterstained with Nissl .","In Alfy GT brains , the mfb has ectopic ventral projections into the hypothalamus ( arrows , bottom ) .","Boxed regions are shown at higher magnification below .","n = 3/genotype .","( E ) NF staining of E13 . 5 cervical spinal cord .","Coronal sections are shown .","Higher magnification images of the dorsal spinal cord are shown below .","The abnormal NF patterning observed in Alfy GT embryos is highlighted with white arrows .","n = 3/genotype . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 01510 . 7554/eLife . 14810 . 016Figure 5—figure supplement 1 . NCAML1 staining confirms axonal defects of the internal capsule in the developing Alfy GT brain . Immunofluorescence against NCAML1 ( red ) in E15 . 5 brains counterstained with Hoechst 33342 ( blue ) ( top ) .","Boxed areas are shown at higher magnification with and without the counterstain ( bottom ) .","Arrow indicates abnormal bundle-like structures formed by the projections in the Alfy GT brain .","n = 4 littermate controls , 4 Alfy GT . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 016 We next examined the ventral midbrain ( vMB ) dopaminergic ( DAergic ) neurons and their projections to the striatum ( Figure 5C , D ) .","Tyrosine hydroxylase ( TH ) staining revealed that the projections and morphology of vMB DAergic neurons were clearly abnormal in the P0 Alfy KO brains .","Notably , the A9 and A10 cell populations , which represent the ventral tegmental area ( A9 ) and substantia nigra ( A10 ) , revealed that the morphology of the regions had an immature appearance relative to control littermates at the same age ( Figure 5C ) .","Moreover , rather than follow their typical trajectory through the medium forebrain bundle to striatal targets in the forebrain , in Alfy KO brain , ectopic TH fibers were observed projecting ventrolaterally into the hypothalamus , near the location of the supraoptic decussation ( Figure 5C , D ) .","Axonal abnormalities were not limited to the developing brain but were also detected in the developing spinal cord of mice lacking Alfy .","At E13 . 5 , disorganized projections into gray matter and the loss of axon bundles were evident in the cervical , thoracic and lumbar levels of the spinal cord in the absence of Alfy , but not in control littermates ( Figure 5E ) .","In summary , these data indicate that Alfy is essential for establishing axonal tracts throughout the CNS , from forebrain commissures to the spinal cord .","Using two independent mouse models , we find that the loss of Alfy leads to erroneous pathfinding throughout the CNS .","Several of these findings are suggestive that the anatomical phenotype observed in Alfy mutant mice may in part be due to defects in axon guidance .","For example , an abnormal dopaminergic commissure at the level of the diencephalon traveling through the hypothalamus from the MFB has been previously reported in the Slit1/Slit2 double KO ( Bagri et al . , 2002 ) , the Deleted in colorectal cancer ( Dcc ) KO ( Xu et al . , 2010 ) , and the NK2 Homeobox 1 ( Nkx2 . 1 ) KO ( Kawano et al . , 2003 ) mice .","Moreover , diminished guidance cue responsiveness could also account for the failure of the midline astroglia to migrate properly .","We therefore hypothesized that Alfy may be involved in the ability of neural cells to respond to guidance cues .","Prior to determining if Alfy is involved in axon guidance , we first determined where Alfy is expressed .","To do so , we reintroduced full length Alfy into Alfy null primary dissociated cultures to visualize its localization ( Figure 6A , Figure 6—figure supplement 1 ) .","Full length Alfy constructs with an N-terminus tag were transfected into DIV7 dissociated primary cortical cultures .","Alfy localized throughout the neuron , including within the axon .","Neighboring astroglia also showed a diffuse localization of Alfy ( Figure 6—figure supplement 1B ) .","Next , we performed subcellular fractionation of adult cortical lysates ( Figure 6B , C , Figure 6—figure supplement","2 ) ( Hallett et al . , 2008 ) .","Fractionation revealed that Alfy enriched in the light membrane fraction ( P3 ) along with synaptosomal membrane protein synaptophysin and a classic marker for autophagic vesicles , the membrane bound form of microtubule associated protein light chain 3 ( LC3-II ) ( Kabeya et al . , 2000 ) ( Figure 6B , C ) .","Additionally , Alfy was enriched in the crude synaptic vesicle fraction ( LP1 ) .","Taken together , these results suggest that Alfy may be involved in regulating the trafficking , sorting or signaling events that are required for brain wiring . 10 . 7554/eLife . 14810 . 017Figure 6 . Alfy localizes to axons and enriches to membrane fractions .","( A ) Immunofluorescence images showing MAP2-positive neurons expressing Alfy .","Merged color image demonstrates co-localization of mCherry-Alfy within a MAP2-positive neuron .","Alfy is found within the soma and co-localizes with MAP2 positive projections .","Transfections were replicated three times across three independent cultures .","Colocalization to βIII Tubulin projections is shown in Figure 6—figure supplement 1 .","( B , C )","Fractionation of adult cortical lysates reveals that Alfy enriches into membrane fractions .","( B ) Equal amounts of protein per fraction were analyzed by immunoblotting .","Alfy was present in membrane fractions that also enriched with LC3-II and synaptophysin ( P3 , LP1; boxes .","( C ) The fold enrichment measured relative to the total homogenate fraction ( H ) .","Bars represent mean enrichment ( n = 3 ) ± SEM .","A schematic depiction of the fractionation can be found in Figure 6—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 01710 . 7554/eLife . 14810 . 018Figure 6—figure supplement 1 . Alfy is expressed in the soma and axons of neurons .","( A , B )","Alfy control mixed primary cortical cultures were transfected with plasmids containing full-length or truncated tagged-Alfy cDNA .","( A ) Representative confocal images are shown of Alfy-tagged constructs detected with antibodies directed against the fluorophore tag and co-stained MAP2 or BIII tubulin .","Full-length and C-terminal fragments of Alfy are detected throughout MAP2- and BIII tubulin-positive projections ( arrows ) .","( B ) Representative confocal images are shown of Alfy-tagged constructs detected with antibodies directed against the fluorophore tag ( left ) and co-stained with GFAP .","Alfy are expressed throughout astroglia , although large vesicles and the nucleus are devoid of staining .","Studies were replicated across three independent cultures .","( C , D )","To determine the sub-cellular distribution of Alfy , Alfy KO or Ctrl ( A , B ) mixed primary cortical cultures were transfected with plasmids containing full-length tagged-Alfy .","To confirm transfection , COS-7 cells were transfected in parallel and immunoblotted .","Vinculin is shown as a loading control .","( C ) Full-length Alfy-tagged proteins migrate at the expected size and are detected with anti-mCherry and anti-Alfy antibodies .","Over-exposed images are necessary to show endogenous Alfy , since it is at a much lower abundance in non-neuronal cells ( circle ) .","( D ) Full length and truncated constructs of Alfy .","Studies were replicated across three cultures . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 01810 . 7554/eLife . 14810 . 019Figure 6—figure supplement 2 . Alfy enriches in membrane fractions from brain . A schematic depiction of the modified synaptosome preparation used for Figure 6A , based on a previously published protocol ( Hallett et al . , 2008 ) .","Abbreviations: H , homogenate; P1 , nuclei and large debris; S1 , soluble cytoplasmic; P2 , crude synaptosomal membranes; S2 , soluble cytoplasmic; P3 , light membrane; S3 , soluble cytoplasmic; LP1 , enriched synaptosomal membranes; LS1 , synaptic vesicles and synaptic proteins . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 019 To test this hypothesis , we next explored whether Alfy is required for the outgrowth of axons .","Dissociated cultures from P0 cortices were plated and examined for morphological differences at day in vitro 7 ( DIV7 ) ( Figure 7A , B; Figure 7—figure supplement 1A–C ) .","Alfy KO neurons displayed healthy growth in vitro and Scholl analysis confirmed that they were morphologically similar to control ( Figure 7B ) .","Staining also revealed that the neuronal cytoskeletal markers Tau-1 , βIII Tubulin and microtubule associated protein 2 ( MAP2 ) were expressed comparably across genotypes ( data not shown ) .","Taken together , these data indicate Alfy is not required for processes controlling non-directed neuronal outgrowth in a cell autonomous manner . 10 . 7554/eLife . 14810 . 020Figure 7 . Alfy is required for the ability of axons to respond to Netrin-1 . ( A , B ) Primary cortical neurons are morphologically similar across genotype .","( A ) Individual , GFP-transfected neurons were imaged by confocal microscopy and traced in ImageJ .","Representative maximum projection images of two heterozygous ( Het , n=5 brains ) and two Alfy KO neurons ( n = 5 brains ) are shown .","Neuronal cytoskeletal markers are expressed comparably across genotype ( Figure 7—figure supplement 1A , B ) .","( B ) Scholl analysis of neurons at DIV7 as represented in A . One-way ANOVA revealed no effect of genotype on the number of crossings ( n = 50 neurons/genotype; F ( 1 , 97 ) = 0 . 66 , p = 0 . 41 ) .","Analysis of branching ( Figure 7—figure supplement 1C ) similarly showed no effect of genotype .","( C , D )","Alfy KO cortical explants have attenuated responsiveness to Netrin-1 .","( C ) Representative images of Alfy Het or Alfy KO cortical explants in the presence of control ( Ctrl ) or Netrin-1 as described ( Figure 7—figure supplement","2 ) and stained with βIII Tubulin .","( D ) Quantification of the average length of outgrowth after 72 hr in culture on data collected from 54 explants ( Alfy Het treated with Ctrl: n = 15; Alfy Het treated with Netrin-1: n = 12; Alfy KO treated with Ctrl = 12 , Alfy KO treated with Netrin-1: n = 15 ) , from two litters of mice .","A two-way ANOVA revealed a significant effect of genotype ( F ( 1 , 50 ) = 7 . 69 , p < 0 . 01 ) and the presence of Netrin-1 ( F ( 1 , 50 ) = 7 . 71 , p < 0 . 01 ) on outgrowth , as well as a significant interaction between genotype and Netrin-1 exposure ( F ( 1 , 50 ) = 12 . 29 , p < 0 . 001 ) .","Fisher PLSD post hoc analysis revealed that exposure to Netrin-1 resulted in a significant increase in outgrowth in Alfy Hets ( p < 0 . 001 ) , but not in Alfy KO ( p = 0 . 56 ) .","Under control conditions , there is no difference in the amount of outgrowth between the genotypes ( p = 0 . 56 ) .","Netrin-1 attractive guidance was also assessed by determining the Guidance Ratio , which is achieved by measuring the amount of outgrowth from the side of the explant closest to the cue-expressing cell block ( proximal ) over the amount of growth on the side of the explant furthest from the cell block ( distal ) .","Under the conditions of our assay , directional growth in response to Netrin-1 was not observed in the control explants ( t ( 25 ) = 1 . 98 , p = 0 . 059 ) or KO explants ( t ( 22 ) = 1 . 77 , p = 0 . 091 ) , suggesting that under the conditions of our assay HEK293T cells secrete Netrin-1 above the threshold for selectively promoting attractive guidance .","Similar results were achieved after 48 hr in culture .","Bars represent mean ± SEM .","* , p < 0 . 001; ns , not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 02010 . 7554/eLife . 14810 . 021Figure 7—figure supplement 1 . Primary cortical neurons are similar across genotype .","( A , B )","The loss of Alfy does not affect the growth and differentiation of dissociated neurons in culture .","Day in vitro 7 ( DIV7 ) neurons from Ctrl and Alfy KO mice express cytoskeletal markers ( A ) Tau-1 ( red ) or ( B ) MAP2 ( red ) and βIII Tubulin ( βIII , green ) positive processes .","Merged images are shown in the top panel , whereas individual channels for each fluorophore are shown in grayscale images below .","Representative images shown from three independent cultures generated from Alfy Het or Alfy KO brains .","( C ) The loss of Alfy had no effect on neurite outgrowth in dissociated cortical neurons .","A one-way ANOVA reveals no significant difference across genotype ( F ( 1 , 97 ) = 0 . 19 , p = 0 . 66 ) .","Data represented as mean ± St . Dev .","Cortical cultures were prepared from five independent cultures with 10 neurons/culture/genotype imaged , resulting in 50 Alfy Het and 50 Alfy KO neurons analyzed . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 02110 . 7554/eLife . 14810 . 022Figure 7—figure supplement 2 . Responsiveness to guidance cues is attenuated in Alfy KO axons .","( A , B )","Cortical explant culture to examine axon outgrowth in response to Netrin-1 as described in Figure 7C , D .","( A ) Diagram of an E14 . 5 embryo with developing cerebral cortex highlighted in red .","Cortical explants were co-cultured with agarose-embedded HEK293T cells that were mock transfected ( Control ) or transfected with Netrin-1-Myc ( Netrin-1 ) .","Transiently transfected HEK293T cells robustly secrete Netrin-1-MYC into the culture media as revealed by immunoblotting for the MYC tag .","Probing for the cytoskeletal protein Vinculin demonstrates that the culture media fraction is predominately cell-free .","( B ) Low-power images , stitched together to show the entire amount of outgrowth after 72 hrs in culture , from cortical explants stained with βIII Tubulin ( red ) in the presence of control or Netrin-1 expressing HEK293T cells ( location of HEK293T cells denoted by white arrow ) .","( C ) The loss of Alfy does not affect guidance cue receptor levels in the telencephalon .","Lysates prepared from E14 . 5 telencephalon were probed on a Western blot with antibodies directed against Alfy , Robo1 , DCC .","The amount of DCC and Robo1 in Alfy KO telencephalon was equivalent to the abundance of the receptors detected in Alfy control samples ( DCC: t ( 5 ) = 0 . 45 , p = 0 . 67; Robo1: t ( 4 ) = 0 . 13 , p = 0 . 90 ) .","-DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 022 We next examined if Alfy is essential for neurons to respond appropriately to trophic agents , such as the bi-directional guidance cue Netrin-1 ( reviewed in [Leyva-Diaz and Lopez-Bendito , 2013] ) .","Explants from control and Alfy KO mice were co-cultured with mock transfected or Netrin-1-Myc transfected HEK293T cells embedded in agarose ( Figure 7—figure supplement 2A ) .","After 48 hr , the distance of growth outward from cortical explants was measured ( Figure 7C , D , Figure 7—figure supplement 2B ) .","In the presence of Netrin-1 , control explants demonstrate significantly increased growth whereas Alfy KO explants did not respond , despite maintaining their ability to extend their processes under non-stimulated conditions ( Figure 7D ) .","Under the conditions used , directional responsiveness could not be ascertained ( Figure 7D ) , likely due to the abundance of Netrin-1 expression due to the use of HEK293T cells ( Shirasaki et al . , 1996 ) .","These data indicate that Alfy is required for cortical neurons to respond to the trophic effects of Netrin-1 .","The loss of Alfy did not affect the total levels of the guidance cue receptor DCC ( Figure 7—figure supplement 2C ) .","Therefore , our data suggest that Alfy function could involve the regulation of intracellular membrane-based signaling events in response to changes in the extracellular milieu of the developing brain .","We and others previously found using stable cell lines that Alfy was an adaptor protein for the degradation of aggregated proteins by selective macroautophagy ( Clausen et al . , 2010; Filimonenko et al . , 2010; Lystad et al . , 2014 ) .","A key step in response to a guidance cue signaling is the recycling and degradation of large signaling protein complexes .","Moreover , upon responding to cues , the resultant neuronal remodeling is also accompanied by the local sequestration and turnover of various cellular components , including cytoskeletal proteins .","In light of the degradative capacity of macroautophagy , it is possible that this degradative pathway may be involved , and Alfy acts to sequester these cargoes .","As an adaptor protein , the absence of Alfy should not impact macroautophagic degradation overall .","Consistent with previous findings , non-selective macroautophagy remains intact in tissues and cells derived from Alfy null mice ( Filimonenko et al . , 2010 ) ( Figure 8A–D ) .","Mice lacking Alfy clearly mounted an autophagic response to starvation as indicated by the increased levels of LC3-II in lysates collected from the perinatal liver ( Figure 8A ) , which is consistent with the lack of feeding observed in the pups ( Figure 2E ) .","The presence of LC3 conversion is consistent with the formation of APs , which we find forms in response to starvation in the presence or absence of Alfy ( Figure 8—figure supplement 1A ) .","Although neurons can properly mount an autophagic response ( Figure 8D ) , no abnormal accumulation of LC3-II was observed in the brain ( Figure 8B ) , suggesting that autophagosome maturation also is unaffected .","Consistent with this finding , macroautophagic degradation of long lived proteins was also independent of Alfy expression ( Figure 8C ) . 10 . 7554/eLife . 14810 . 023Figure 8 . Alfy is an adaptor protein for selective macroautophagy ( A–D ) Non-selective macroautophagy proceeds normally in the absence of Alfy .","( A ) Liver lysates from perinatal Alfy GT pups reveal greater LC3 conversion to LC3-II , reflecting the starvation response due to their inability to feed properly ( Figure 2E ) .","Lysates from normally fed WT and heterozygous littermates mice predominantly contain LC3-I .","The bottom graphs quantify LC3 conversion as the ratio of LC3-II/LC3-I .","Bars represent mean ± St . Dev .","ANOVA reveals a significant effect of genotype on LC3 conversion ( n = 7 WT , 6 Alfy GT Het , 6 Alfy GT; F ( 2 , 16 ) = 11 . 07; p < 0 . 001 ) .","Fisher PLSD post hoc analyses indicate that LC3 conversion of Alfy GT mice differed significantly from mice with at least one copy of Alfy .","( B ) In the brain , there is no abnormal accumulation of LC3-II in the Alfy GT mice , suggesting that autophagosome maturation proceeds normally .","A single-factor ANOVA reveals no significant difference between the three genotypes ( n = 5/genotype; F ( 2 , 12 ) = 5 . 52; p = 0 . 90 ) .","( C ) Macroautophagic protein degradation in response to starvation is normal in the absence of Alfy .","Long lived protein degradation assay .","( LC3 puncta formation is shown in Figure 8—figure supplement 1A ) .","A two-factor ANOVA revealed a significant main effect for treatment ( n = 4/genotype; F ( 2 , 27 ) = 24 . 93 , p < 0 . 001 ) , but no significant effect of genotype on percent ( % ) Proteolysis ( n = 4/genotype; F ( 2 , 27 ) = 1 . 82 , p = 0 . 18 ) .","Fisher PLSD post hoc analysis revealed % Proteolysis was significantly increased in all genotypes during starvation compared with complete media ( p < 0 . 001 ) and the addition of 5 mM 3MA significantly attenuated the starvation-induced proteolysis ( p < 0 . 001 ) .","( D ) In the absence of Alfy , LC3 conversion is normal in primary neurons in response to trophic factor withdrawal ( n = 4/genotype ) .","To accumulate LC3-II , cells were starved in the presence of 20 µM leupeptin .","( E ) The loss of Alfy impedes the selective macroautophagic turnover of its cargo , ALIS .","Although ubiquitinated structures are readily found when all forms of macroautophagy is impeded ( Figure 8—figure supplement 1B ) , the turnover of only specific cargoes is affected by the loss of Alfy .","Primary Alfy GT versus Alfy heterozygous ( HET ) MEFs after 72 hr of Veh or 5 μM AraC .","Ubiquitin-positive ALIS bodies form in response to mitotic inhibition , as revealed by FK2 immunofluorescence ( green ) .","In the absence of Alfy , these structures accumulate more rapidly ( white arrows ) .","n = 3 .","Similar Alfy dependence can be observed with the aggregation prone expanded polyQ proteins ( Figure 8—figure supplement 1C , D ) .","For all graphs , bars represent mean ± SEM .","ns , not significant; * , p < 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 02310 . 7554/eLife . 14810 . 024Figure 8—figure supplement 1 . Alfy is an adaptor for selective macroautophagy .","( A ) In the presence of complete media , LC3 immunoreactivity ( green ) produces a diffuse staining pattern throughout the cytoplasm .","When MEF cultures are starved by incubation in HBSS , LC3 reactivity becomes localized to distinct puncta regardless of genotype , suggesting the formation of LC3-positive autophagosomes ( n = 4 ) .","( B ) The inhibition of macroautophagy leads to ALIS body accumulation in the absence of stress .","Immortalized Atg5 KO MEFs ( Kuma et al . , 2004 ) demonstrate the accumulation of ubiquitin positive aggregates ( white arrows ) under basal conditions as revealed by FK2 antibody ( green ) ( n = 3 ) .","( C , D )","The selective macroautophagy cargo , expanded polyglutamine inclusion , accumulates more rapidly in the absence of Alfy .","Transient transfection of 17aahtt103Q-eGFP in Ctrl and Alfy GT MEFs .","Aggregation over time is quantified in ( D ) .","The percentage of transfected cells with aggregates is higher in the Alfy GT MEFs compared with Ctrl .","Repeated measures ANOVA revealed a significant difference of genotype in the% transfected cells with aggregate genotype ( n = 3/genotype; F ( 1 , 8 ) = 154 . 643 , p = 0 . 002 ) , and a significant interaction between time and genotype ( n = 3/genotype; F ( 2 , 8 ) = 55 . 332 , p < 0 . 0001 ) .","Post hoc analyses ( Fisher PLSD ) revealed that in Ctrl MEFs , ‘% transfected cells with agg’ do not change between 24 and 48 hr ( p = 0 . 4144 ) , but decrease after 72 hr ( p = 0 . 005 ) .","In contrast , in Alfy GT MEFs , ‘% transfected cells with agg’ continuously increase over time ( comparison between 24 and 48 hr , p = 0 . 0047; comparison between 48 and 72 hr , p = 0 . 0105 ) .","Scale bar = 25 μm .","Data represent mean ± St . Dev . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 024 Since we confirmed that nonselective macroautophagy was unaffected , we next determine if the loss of Alfy impeded the turnover of its substrates .","One naturally occurring Alfy substrate are known as aggresome-like induced structures ( ALIS ) ( Clausen et al . , 2010 ) .","Cytosolic ubiquitinated ALIS were formed in MEFs upon mitotic inhibition ( Figure 8E ) .","In contrast to control MEFs , these structures accumulated more rapidly in the absence of Alfy , suggesting that in its absence , the turnover of ALIS are impeded .","Moreover , unlike in MEFs that lack all forms of macroautophagy ( Figure 8—figure supplement 1B ) ( Kuma et al . , 2004 ) , Alfy GT MEFs do not demonstrate basal protein accumulation , suggesting that Alfy is not required for the turnover of all ubiquitinated substrates .","Nonetheless , over-expression of a canonical aggregation-prone polyQ protein , a 17 amino acid fragment of the mutant huntingtin protein with 103 glutamines and an eGFP tag ( 17aahtt ( 103Q ) eGFP ) ( Kazantsev et al . , 1999 ) , accumulated more rapidly by the absence of Alfy ( Figure 8—figure supplement 1C , D ) .","Thus , in the absence of Alfy , the elimination of ubiquitinated proteins by selective macroautophagy , but not basal macroautophagy , is impaired .","Taken together with the rest of our findings , we hypothesize that Alfy-mediated sequestration of cargoes may help selectively eliminate cargoes at or around the membrane , thereby affecting the ability of axons to properly respond to guidance cues ."],["In this study , our genetic models revealed that Alfy influences how growing axons interact with their environment to form stereotyped axonal connectivity in the central nervous system ( CNS ) .","The homozygous loss of Wdfy3 disrupts the formation of many axonal connections , causing agenesis of all three forebrain commissural tracts and leading to profound changes in brain wiring throughout the CNS , including the optic chiasm , internal capsule , cerebral peduncle , medial forebrain bundle and spinal cord tracts .","At the cellular level , the loss of Alfy led to defects in the development of midline glial population , impacted embryonic cortical neuron proliferation and impeded the ability of axons to respond to Netrin-1 .","Furthermore , Alfy KO mice present a phenotype unlike mice lacking core autophagy genes such as Atg5 , suggesting the perinatal lethality is not due to a loss of general autophagic degradation , as Alfy mutant tissue and cells display the classic response to starvation .","We hypothesize that Alfy functions as a selective macroautophagy adaptor protein and that Alfy-dependent regulation of intracellular vesicle trafficking and signaling pathways upstream of macroautophagy can have a significant influence over brain development .","The widespread disruption of axonal wiring identified in the absence of Alfy supports a functional role for this protein in the sequestration and trafficking of substrates into the autophagic pathway .","In the adult brain , Alfy facilitates the sorting of ubiquitinated cargo into APs , and one possibility is that during development Alfy has an analogous function .","For instance , APs are present in the growth cone and axon tips ( Hollenbeck , 1993; Maday and Holzbaur , 2014; Maday et al . , 2012 ) .","We propose that Alfy could regulate the ability of growth cones to respond to cues by sequestering and sorting proteins into degradative vesicles .","Such regulation could be critical for responding to cues in the environment through changes in cell shape and motility .","It has been suggested previously that the ubiquitination of guidance receptor complexes could regulate the responsiveness of axons to guidance cues ( O'Donnell et al . , 2009 ) .","The trafficking and responsiveness of the axonal guidance receptors Robo1 and UNC5 has been shown to be regulated by the E3 ubiquitin ligases rpm-1 in C . elegans and USP33 in murine commissural axons , respectively ( Li et al . , 2008; Yuasa-Kawada et al . , 2009 ) .","Furthermore , deletion of the rpm-1 homologue , Phr1 , in mice , causes axonal wiring defects reminiscent of Alfy mutants ( Bloom et al . , 2007 ) .","One intriguing possibility is that Alfy might sequester cargo ubiquitinated by E3 ligases such as Phr1 or USP33 .","Alternatively , the association of Alfy with the GABAA receptor-associated protein ( GABARAP ) ( Lystad et al . , 2014 ) , implies Alfy could be involved in the selective sorting of plasma membrane receptors .","Based on this model , we predict that in the absence of Alfy , the efficient degradation of Alfy-associated cargo would be slowed , or the cargo would be trafficked into alternative pathways , disrupting the temporal regulation of signaling events within the growth cone .","Similarly , the disruption of the midline glial structures may also reflect the disruption of signaling events that promote the differentiation and migration of these cell populations .","Examination of substrates labeled with a Lys63 polyUb chain , which are also associated with APs ( Shaid et al . , 2013 ) , may identify other Alfy-interacting substrates .","Wdfy3 and its homologue bchs have been implicated in trafficking events associated with lysosomes , yet genetic disruptions yield distinct phenotypes in their respective model systems .","Both homologues have been shown to co-localize with markers for degradative vesicles , including APs in mammals and endolysosomes in Drosophila ( Lim and Kraut , 2009; Lystad et al . , 2014; Simonsen et al . , 2004 ) yet , despite the conservation of domain structure , bchs loss of function ( LoF ) mutants have no reported developmental neuroanatomical defects ( Khodosh et al . , 2006 ) .","Overexpression of bchs , however can impact synaptic and axonal morphology at the neuromuscular junction and the retina , revealing that bchs dosage plays a role in CNS development and function ( Khodosh et al . , 2006; Kraut et al . , 2001 ) .","The difference in phenotype between bchs LoF flies and Alfy mutant mice might be explained by the emergence of novel biological functions due to the evolutionary expansion of the BEACH family of genes from six in invertebrates to nine in vertebrates ( Cullinane et al . , 2013 ) .","Evolutionary divergence also has been described for the BEACH protein , Neurobeachin ( Nbea ) , which has evolved distinct biochemical properties from its Drosophila homologue rugose ( Volders et al . , 2012 ) .","We propose Alfy , which shares 48% protein sequence identity with bchs , may have similarly diverged , acquiring new functions that are essential for vertebrate development .","Whereas proteasome-mediated degradation is thought to be the primary catabolic pathway regulating neurodevelopmental signaling ( Yi and Ehlers , 2007 ) , recent studies indicate that macroautophagy may also play a critical role in morphogenesis .","For instance , the loss of Ambra1 , a regulator of the autophagy protein Beclin1 , causes neural tube closure defects in mice ( Fimia et al . , 2007 ) .","In humans , recessive mutations in TECPR2 and EPG5 , genes implicated in AP maturation ( Stadel et al . , 2015; Tian et al . , 2010 ) , are responsible , respectively , for spastic paraplegia 49 with brain malformations , including hypogenesis of the corpus callosum [OMIM 615031] and Vici syndrome with brain malformations , including agenesis of the corpus callosum [OMIM 242840] ( Cullup et al . , 2013; Oz-Levi et al . , 2012 ) .","Why loss of these autophagy-related proteins causes corpus callosum defects remains unclear .","Although little is known about the importance of core autophagy genes such as Atg5 , Atg7 and Atg14 in neurodevelopment , no obvious defects in axonal connectivity have been reported ( Hara et al . , 2006; Komatsu et al . , 2006; Liang et al . , 2010 ) .","One exception is that the conditional elimination of Atg7 in POMC neurons leads to a defect in postnatal axonal outgrowth ( Coupe et al . , 2012 ) , and recently , a partial LoF mutation in a core autophagy gene , Atg5 , have been reported to lead to cerebellar hypoplasia and developmental delay ( Kim et al . , 2016 ) , but connectivity of the callosum appears unaffected .","Given that degradation by autophagy is slower than proteasome-mediated turnover , these data suggest that the sorting and sequestration of cargo , rather than elimination of cargo , is critical for the timing of cellular processes regulated by macroautophagy .","Alternatively , as a scaffold for ubiquitinated structures for degradation by macroautophagy , the loss of Alfy might have indirect effects on proteasome activity .","For example , Alfy is recruited to aggregated structures that form upon proteasome-inhibition ( Clausen et al . , 2010; Simonsen et al . , 2004 ) ; when Alfy is absent , the persistence of structures that might otherwise be eliminated may impact the efficiency of proteasome-mediated degradation .","We have previously reported that the deletion of Alfy does not measurably affect the enzymatic activity of the proteasome ( Filimonenko et al . , 2010 ) and does not lead to the accumulation of ubiquitinated structures in Alfy null MEFs ( Figure 8 ) .","Nonetheless , it is still possible that localized , discrete disruptions of proteasome activity or modifications in activity enough to disrupt timing might be present , leading to the profound phenotype we observe here .","Genetic screening has revealed a possible role for the human Wdfy3 homolog ( WDFY3 ) as a genetic risk factor for intellectual and developmental disabilities .","Most recently , a missense mutation in WDFY3 has been linked to autosomal dominant primary microcephaly ( Kadir et al . , 2016 ) .","Furthermore , in autism spectrum disorder ( ASD ) , rare de novo nonsense mutations within WDFY3 have been identified across two independent studies ( De Rubeis et al . , 2014; Iossifov et al . , 2012 ) .","WDFY3 resides on chromosome 4q21 . 23 and this region of chromosome 4 was suggested to harbor a genetic predisposition to ASD ( Chen et al . , 2006 ) and to schizophrenia ( Faraone et al . , 2006; Paunio et al . , 2004 ) .","In addition , the dosage of WDFY3 is affected in a subset of individuals with a rare chromosome disorder known as 4q21 deletion syndrome [OMIM 613509] , characterized by intellectual disabilities ( ID ) , language impairment and congenital birth defects ( Bonnet et al . , 2010; Cooper et al . , 2011 ) .","Taken together , these data imply WDFY3 is a rare genetic risk factor for childhood neurological disorders .","Future studies should aim to determine whether WDFY3 dosage is critical for human brain development and how mutations could potentially disrupt the biological function of Alfy .","A network analysis study on genomic data found that CNVs in genes associated with autophagy were enriched in patients with ASD ( Poultney et al . , 2013 ) .","While it remains to be determined whether computational modeling can be used to accurately predict the cellular pathways disrupted in ASD , in light of our findings , we would propose that further study is warranted .","In conclusion , mice lacking Alfy survive embryogenesis , but the loss of Alfy function produces widespread CNS defects in axonal tracts , including loss of the major forebrain commissures .","The results of our study provide new insight into the genetic control of brain wiring and open the door to many new questions surrounding the physiological role of Alfy and selective autophagy in CNS development ."],["The following antibodies were used for western blot , immunocytochemistry , immunohistochemistry , in situ hybridization and immunoprecipitation experiments: Anti-Alfy ( rabbit polyclonal anti-COOH Alfy; generously provided by Dr . Anne Simonsen and used for western blots and immunoprecipitation ) , anti-Alfy-N1 ( rabbit polyclonal anti-NH3 Alfy; generously provided by Dr . Masaaki Komatsu and used for western blots ) , anti-BrdU clone BU-33 ( B2531 , Sigma-Aldrich [St . Louis , MO] ) , anti-Caspase 3 active/cleaved form ( AB3623 , EMD Millipore [Germany] ) , anti-Class βIII-Tubulin ( PRB-435P , Covance [Princeton , NJ] ) , anti-Human DCC ( 554222 , BD Biosciences [San Jose , CA] ) , anti-Digoxigenin-AP , Fab fragments ( Roche [Switzerland] ) , anti-Doublecortin ( ab18723 , Abcam [Cambridge , MA] ) , anti-GFAP , clone GA5 ( MAB3402 , EMD Millipore ) , anti-LC3 ( for immunofluorescence , rabbit polyclonal; generously provided by Ron Kopito ) , anti-LC3B ( for western blots , ab48394 , Abcam ) , anti-Ki67 clone B56 ( 550609 , BD Biosciences ) , anti-MAP-2 ( ab11267 , Abcam ) , anti-NCAML1 , clone 324 ( MAB5272 , EMD Millipore ) , anti-Neurofilament clone2H3 ( developed by TM Jessell and J Dodd and was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa ) , anti-Robo1 ( H-200 , Santa Cruz Biotechnology [Santa Cruz , CA] ) , anti-synaptophysin ( 101 002 , Synaptic Systems [Germany] ) , anti-Tau-1 , clone PC1C6 ( MAB3420 , EMD Millipore ) , anti-Tbr1 ( AB2261 , EMD Millipore ) , and anti-Ubiquitin , clone FK2 ( Enzo Life Sciences [Farmingdale , NY] ) .","The following reagents were generously provided: Atg5 knockout and wildtype MEFs ( Noboru Mizushima , [Itakura and Mizushima , 2010] ) and MYC-tagged Netrin-1 ( pGNET1-myc ) ( Marc Tessier-Lavigne [Serafini et al . , 1994] ) .","All animals and procedures complied with the Guide for Care and Use of Laboratory Animals and were approved by the IACUC committee at Columbia University .","Mice were maintained in a 12h/12h light/dark cycle in a temperature and humidity controlled environment , with ad libitum access to food and water .","Alfy GT mice ( 129/SvEv x C57BL/6; Wdfy3Gt ( OSTGST_5258_D3 ) Lex ) were generated at the Texas Institute for Genomic Medicine ( TIGM [College Station , TX] ) .","A complete description of the gene trap vector can be found in ( Zambrowicz et al . , 2003 ) .","The Alfy GT mice used in this study were derived from mating heterozygous mice .","Wdfy3 floxed mice were generated on a 129/SvEv x C57BL/6 background under contract with the University of Connecticut .","The floxed Wdfy3 allele has loxP sites flanking exon V that is predicted to produce a 66 amino acid peptide following Cre-mediated recombination , instead of the 3508 amino acid full-length Alfy protein .","The Wdfy3 deletion allele ( ∆ ) is created when male mice carrying Wdfy3 floxed alleles are crossed with Bl6/J female carrier of HprtCre/+ , a Cre-deleter stain with 100% Cre-mediated recombination in oocytes ( Tang et al . , 2002 ) .","All experiments included control and experimental littermates and represent data across multiple litters .","Genomic DNA was extracted and the alleles were identified using the following primers: Common Alfy forward: 5’-CTTGTTACACTTGTCCCACAGC-3’ , Alfy WT reverse 5’– TTAGACTTCTAAGCCCACGAGTACC-3’ , and Alfy GT reverse: 5’-ATAAACCCTCTTGCAGTTGCATC-3’ .","Genotyping for the Alfy WT , loxP and ∆ alleles was performed using following the primers in a multiplex PCR reaction: LoxP-F 5’-gaaagcaagctcgtttacgg-3’ , Frt-R 5’-aggttaccagccacaaccag -3’ , and Frt-F 5’-acttgggaagagggaagctc-3’ .","RNA from whole embryo was isolated using Trizol reagent ( Life Technologies [Carlsbad , CA] ) according to the manufacture’s recommendation .","This RNA was used in a reverse transcription reaction containing random 9mers ( NEB Biosciences ) and reverse transcriptase ( Superscript , Qiagen [Germany] ) according to manufacturer’s instructions .","Negative controls ( minus reverse transcriptase ) were run for each sample .","The resulting cDNA was used to determine the abundance of Wdfy3 mRNA relative to GFAP , βIII Tubulin or Actin .","The following primers were used: βIII Tubulin: Tuj1-1F: 5’-ctacgacatctgcttccgca -3’ , Tuj1-1R: 5’-gaagggaggtggtgactcca-3’; GFAP: GFAP-F: 5’-gagctcaatgaccgctttgc -3’ , GFAP-R: 5’-tccttggctcgaagctggt -3’ .","Primary dissociated cortical cultures were prepared from postnatal day 0 mouse pups ( P0 ) .","The cortical lobes were dissected in ice cold DMEM/F12 ( Life Technologies ) containing 10% heat- inactivated FBS and antibiotics .","Cortical tissue were trypsinized for 30 min at 37°C , then triturated in fresh 10% FBS DMEM/F12 through a fire-polished glass pipette and filtered through a 40 micron nylon cell strainer ( BD Biosciences ) .","For immunocytochemistry , cells were plated at a density of 1 . 0 x 105 cells/well onto coverslips coated with 20 μg/mL polyD-lysine ( Sigma Aldrich ) and 5 µg/mL mouse laminin ( Life Technologies ) .","The medium was changed 2 hr after plating to NB media ( Neurobasal media [Life Technologies] ) containing 0 . 5 mM L-glutamine , 1X B27 supplement ( Life Technologies ) , 2% heat inactivated FBS ( Life Technologies ) and 1X antibiotic ( Life Technologies ) .","Embryos were collected from deeply anesthetized pregnant dams at E14 . 5 .","Uterine horns were placed into ice cold Hank’s buffered saline solution ( HBSS ) .","Heart , liver and head were removed for genomic DNA extraction , the remaining tissue was trypsinized in equal volume of HBSS and 0 . 25% trypsin for 15 min . at 37°C .","Samples were triturated with a fire-polished glass pipette , then diluted into MEFs complete media: DMEM ( Life Technologies ) containing 10% FBS ( Life Technologies ) , 1X L-glutamine ( Life Technologies ) , 0 . 1 mM beta-mercaptoethanol ( Life Technologies ) , 1X Pen/strep ( Life Technologies ) .","Cells were filtered through a 100 micron sieve and plated .","In addition to genotype confirmation , RNA was extracted from the cultures to measure transcript levels and MEF cultures were confirmed to be mycoplasma free ( Takara ) .","To starve the MEF cultures , the complete media was aspirated off the cultures , followed by three PBS washes and cultures were placed in HBSS supplemented with 10 mM HEPES for four hours .","The subcellular fractionation of the brain tissue protocol was performed as described ( Hallett et al . , 2008 ) .","Briefly , adult control mice were euthanized and cerebral cortex was rapidly dissected and frozen on dry ice .","An aliquot of each fraction was saved for analysis .","Brain tissue was homogenized in TEVP buffer ( 10 mM Tris , 5 mM NaF , 1 mM Na3VO4 , 1 mM EDTA , 1 mM EGTA , pH . 7 . 4 ) containing 320 mM sucrose .","Homogenates ( H ) were fractionated sequentially as shown in Figure 6—supplement figure 2 at 800 xg , resulting in the first supernatant ( S1 ) and pellet ( P1 ) fractions , and 9200 xg to generate the second supernatant ( S2 ) and crude synaptosomal membranes ( P2 ) .","P2 was resuspended in TEVP buffer containing 35 . 6 mM sucrose for 30 min on ice to release vesicles and organelles .","P2 was then spun at 25 , 000 xg to segregate the supernatant ( LS1 ) from the membrane fraction ( LP1 ) .","Lastly , S2 was centrifuged for 2 hr at 165 , 000 xg to generate the supernatant fraction ( S3 ) and light membrane fraction ( P3 ) .","Protein quantification was performed on all fractions and equal amounts of protein were loaded onto SDS-PAGE .","Alfy MEFs as well as Atg5 knockout and wildtype MEFs were maintained in MEF complete media .","Starvation was achieved using a starvation medium of HBSS + 10 mM HEPES for 4 hr .","To slow lysosome-mediated degradation , cells were treated with 20 μM leupeptin .","Mitotic inhibition was achieved by treating cells with 10 μM arabinofuranosylcytidine ( AraC ) for 72 hr .","Transfections of cells were achieved with Lipofectamine 2000 ( Life Technologies ) in serum-free media .","Cells were transfected with constructs encoded by 17aahtt103Q tagged with eGFP as previously described ( Filimonenko et al . , 2010 ) .","Cells were fixed 24 , 48 and 72 hr later and imaged by epifluorescence microscopy .","Long lived protein degradation assay was performed as previously described ( Filimonenko et al . , 2010 ) .","Whole cell lysates and tissue lysates from freshly dissected brain and liver were generated using a modified RIPA buffer ( 50 mM Tris-HCl , pH 7 . 4 , 150 mM NaCl , 10 mM EDTA , 0 . 1% Triton-X 100 ) containing protease and protein phosphatase inhibitors ( Halt Inhibitor Cocktail , Roche ) .","Ten strokes in a dounce homogenizer were used to extract protein from tissues and samples were cleared by centrifugation at 15 , 000 xg for 60 min at 4°C unless otherwise indicated .","Protein concentration was determined using the DC Protein Assay ( Bio-rad Laboratories [Hercules , CA] ) and equal amounts of protein were prepared and loaded onto 4–12% Bis-Tris or 3–8% Tris-Acetate NuPAGE gels and blotted to PVDF membranes as described by the manufacture’s recommendations ( Life Technologies ) .","PVDF membranes were blocked in 5% BSA in TBS containing 1% Tween-20 ( TBST ) .","Antibodies were diluted in 1% BSA in TBST and incubated overnight at 4°C .","The appropriate HRP-conjugated secondary antibodies ( ThermoFisher Scientific ) were diluted in 1% BSA TBST and a chemiluminescent reaction ( West Dura SuperSignal , ThermoFisher Scientific ) was detected by the Versadoc Imaging System ( Bio-rad ) .","Fresh frozen tissue was sectioned at 15 μm onto Fisherband Superfrost plus slides and stored at −80°C until use .","On day one , slides were warmed at 37°C for 30 min , fixed in 4% paraformaldehyde for 10 min and washed in DECP-treated phosphate buffered saline ( PBS ) .","Slides were acetylated with 0 . 3 M acetic anhydride in 0 . 1 M triethanolamine for 10 min followed by three PBS washes .","Slides were prehybridized in formamide diluted 2X Prehyb buffer ( 5 M NaCl , 1 M Tris , pH 7 . 4 , 6% Ficoll , 6% polyvinylpyrrolidone , 6% Bovine serum albumin , 50 mg total Yeast RNA , 5 mg Yeast tRNA , 250 mM EDTA and 50 mg SS DNA ) for 1 hr at room temperature .","Wdfy3 riboprobes were prepared from a pCR II plasmid ( Life Technologies ) containing a PCR fragment of the 5’ UTR region of mouse Wdfy3 from 320–540 nt ( ref NM_172882 . 3 ) .","In vitro transcription was performed as described by the manufacturer ( Promega Corporation [Madison , WI] ) using the T7 polymerase for the antisense probe on template linearized with KpnI and Sp6 polymerase for the sense probe on template linearized with NotI .","Riboprobes were cleaned using the RNeasy MinElute Cleanup Kit ( Qiagen ) according the manufacturer’s protocol .","Riboprobes were heated to 80°C for 5 min , quenched on ice and added to 2X Hyb buffer ( 5 M NaCl , 1 M Tris , 6% Ficoll , 6% polyvinylpyrrolidone , 6% Bovine serum albumin , 50 mg total Yeast RNA , 5 mg Yeast tRNA , 250 mM EDTA , 50 mg SS DNA and 20% dextran sulphate ) diluted 1:1 with formamide .","Hyb solution was added directly to slides and they were incubated overnight at 68°C in a humidified chamber .","On day two , slides were washed in 5X SSC at 68°C for 10 min and coverslips were removed .","Three more washes 0 . 2X SSC were carried out at 68°C , followed by a 0 . 2X SSC wash at room temperature .","Slides were incubated in B1 buffer ( 0 . 1M Tris , pH 7 . 5 , 0 . 15M NaCl ) for 5 min then blocked in B1 containing 10% heat-inactivated sheep serum for 1 hr in a humidified chamber .","Anti-DIG antibody diluted 1:5000 in B1 buffer containing 1% heat-inactivated sheep serum and slides were incubated overnight at 4°C in a humidified chamber .","On day two , slides were washed in B1 buffer three times and equilibrated in B3 buffer ( 0 . 1 M Tris , pH 9 . 5 , 0 . 1 M NaCl , 50 mM MgCl2 ) for 5 min at room temperature .","The staining reaction was carried out using NBT/BCIP ( Promega ) diluted in B3 buffer in the dark at room temperature overnight in a humidified chamber and the staining reaction was stopped by washing the slides three times in TE ( 10 mM Tris , pH 8 . 0; 1 mM EDTA ) .","On DIV3 , cultures were transfected with 1 µg pEGFP-N3 ( Clontech ) using Lipofectamine 2000 ( Life Technologies ) in Optimem for 4 hr .","The next day , the media was changed to NB media + mitotic inhibitors: 10 µM AraC ( Sigma-Aldrich ) , 10 µM Uridine ( Sigma-Aldrich ) , and 10 µM 5-Fluoro-2’-deoxyuridine ( FDU , Sigma-Aldrich ) to prevent the excessive growth of glial cells .","On DIV7 , cells were washed with TBS , fixed for 15 min with 4% paraformaldehyde , permeabilized in 0 . 1% Triton-X 100 in TBS , blocked in 5% BSA in TBS and stained with primary antibodies diluted in 1% BSA in TBS overnight at 4°C .","Alexa Fluor conjugated secondary antibodies ( Life Technologies ) were diluted in 1% BSA TBS , incubated with coverslips for 1 hr at room temperature , counterstained with Hoechst 33 , 342 ( Life Technologies ) and mounted with prolong gold antifade ( Life Technologies ) onto glass slides .","A Leica SP5 confocal microscope was used to image cortical cultures .","GFP-transfected cells with the characteristic neuronal morphology , including a small triangular shaped soma were analyzed to compare morphology between Alfy null and Alfy control cultures .","Z-stacks were taken every micron using a 20X objective and 3X digital zoom .","For each culture , approximately10 individual neurons were imaged totaling 50 control and 49 knockout cortical neurons .","Confocal z-stacks were traced and analyzed using the simple neurite tracer segmentation plug-in for the FIJI version of ImageJ ( Longair et al . , 2011 ) .","Time-pregnant dams were sacrificed in accordance with humane protocols established by the Columbia University IACUC .","All dissections were carried out in ice-cold PBS .","The embryonic cerebral cortex was dissected out and finely cut into pieces , then 28 gauge needles were used to transfer and position explant pieces inside a drop ( 5 µL ) of Matrigel ( BD Biosciences ) on a collagen-coated glass bottom petri dish ( MatTek ) .","Once explants were placed , 350 µm diameter punches of solidified agarose cell blocks containing either untransfected or Netrin1-MYC transfected HEK293T cells were placed alongside cortical explant pieces .","Explants were grown for 48 hr , and then fixed with 4% PFA for 15 min .","Explants were imaged and measured using phase contrast microscopy using a 10X objective on a Nikon TiE microscope and NIS Elements software ( Nikon ) .","Across two experiments , outgrowth in microns towards the HEK293T cellblock was recorded for the three longest processes in a single plane .","The measurements were averaged for each explant and statistical analysis was performed using two-way ANOVA followed by the Fisher LSD post hoc test .","Following analysis of outgrowth , explants were further processed for immunocytochemistry as described above .","To prepare HEK293T agarose cell blocks , HEK293T cells were seeded at 600 , 000 cells per six well , then 16 hr later transiently transfected with pGNET1-myc using Lipofectamine 2000 .","The following day , untransfected HEK293T or HEK293T cells transfected with Netrin1-MYC were trypsinized , pelleted and resuspended in 900 µL of warm ( ~50°C ) 1% agarose dissolved in DMEM .","Punches of agarose cell blocks containing HEK293T cells were prepared with a 0 . 35 mm diameter Harris Uni-Core tissue punch .","To visualize retinal ganglion cell ( RGC ) axon projections through the optic chiasm , unilateral whole eye anterograde labeling with DiI ( Molecular Probes ) was performed on fixed tissue as described previously ( Kuwajima et al . , 2012 ) .","Samples were incubated in PBS + 0 . 02% sodium azide for 14 days at 37°C .","Ipsilateral versus contralateral projections were quantified by measuring the pixel intensity of ipsilateral and contralateral optic tracts in a 500 x 500 μm area lateral to the optic chiasm midline with the MetaMorph image analysis software .","An ipsilateral index was calculated by dividing the intensity of the ipsilateral projection by the sum of the contralateral and ipsilateral pixel intensities as described previously ( Erskine et al . , 2011; Kuwajima et al . , 2012 ) .","The ipsilateral index in Alfy null mice was normalized to the littermate WT ipsilateral index .","The number of Ki67 positive cells labeled with DAB within the subventricular zone of the ganglionic eminence was determined by stereology using StereoInvestigator software ( MicrobrightField ) by an experimenter blind to genotype .","For each animal , measurements were made at 60X bilaterally in five matched , adjacent sections spaced 200 μm apart .","There were three animals per genotype .","Timed pregnant dams were weighed and received an intraperitoneal injection with 50 mg/kg of BrdU at E15 . 5 .","One hour following the injection , the dams were deeply anesthetized and the uterine horns were dissected out .","Embryo heads were drop fixed in 4% PFA for 48 hr and then cryoprotected in 30% sucrose , embedded in OCT and sectioned at 25 µm .","Sections were processed using an antibody against BrdU as described in the immunohistochemistry section .","Statistical analyses were performed using Statview 5 . 0 ( SAS Institute ) .","Normally distributed data were subject to student t-test , or for multiple comparisons , analysis of variance ( ANOVA ) followed by the appropriate post hoc comparisons as indicated in the figure legends .","Complete F-statistics and calculated p-values are also available in the figure legends .","Power analyses for quantitative studies were performed to achieve a power of 0 . 8 with a confidence of 0 . 95 using G*Power 3 . 1 ( Faul et al . , 2007 , 2009 ) .","Effect sizes were based upon pilot studies &/or previously published or unpublished materials .","n-values are indicative of biological replicates and no data were excluded from analyses ."]],"string":"[\n [\n \"The Autophagy linked FYVE domain protein ( Alfy ) [gene name , WD40 repeat and FYVE domain protein 3 ( Wdfy3 ) ] is a member of the Beige and Chediak-Higashi ( BEACH ) domain containing proteins , a family of proteins implicated in vesicle trafficking and membrane dynamics ( Isakson et al . , 2013; Simonsen et al . , 2004 ) .\",\n \"As its name implies , Alfy has been implicated in the degradative pathway macroautophagy , by acting as a molecular scaffold between select cargo and core members of the mammalian autophagic machinery such as Atg5 , p62 and Atg8 homologs ( Clausen et al . , 2010; Filimonenko et al . , 2010; Lystad et al . , 2014; Simonsen et al . , 2004 ) .\",\n \"In addition , its functional FYVE zinc finger domain at the COOH-terminus permits partial co-localization to phosphatidylinositol-3-monophosphate ( PtdIns3P ) , especially at autophagosome membranes ( Simonsen et al . , 2004 ) .\",\n \"Wdfy3 is evolutionarily conserved and the most extensively studied homolog is Blue Cheese ( bchs ) in D . melanogaster ( Finley et al . , 2003 ) .\",\n \"In the developing and adult fly central nervous system ( CNS ) , Bchs is abundantly expressed , with preferential accumulation in axon terminals and at the growth cone ( Finley et al . , 2003; Khodosh et al . , 2006 ) .\",\n \"Adult bchs null flies have a shortened life span and show signs of adult onset neurodegeneration , including the accumulation of ubiquitinated aggregates ( Filimonenko et al . , 2010; Finley et al . , 2003; Khodosh et al . , 2006 ) .\",\n \"Loss-of-function ( LoF ) mutations in bchs disrupt the axonal transport of endolysosomal vesicles ( Lim and Kraut , 2009 ) , however no defects in axon guidance have been reported in bchs null larva ( Khodosh et al . , 2006 ) .\",\n \"Recently it has been reported that in vertebrates , genetically diminished levels of Alfy disrupts neurogenesis leading to altered forebrain morphology ( Orosco et al . , 2014 ) .\",\n \"Furthermore , genetic screening has revealed a possible role for the human homolog WDFY3 as a genetic risk factor for intellectual and developmental disabilities ( IDD ) , microcephaly and neuropsychiatric disorders ( Bonnet et al . , 2010; Iossifov et al . , 2012; Kadir et al . , 2016 ) .\",\n \"These findings raise the possibility that Alfy could have an important function in mammalian CNS development .\",\n \"Here , we present two new mouse models that eliminate Alfy expression and identify an essential role for Alfy during murine development .\",\n \"Constitutive elimination of Alfy leads to perinatal lethality , in conjunction with developmental brain wiring defects throughout the CNS , involving forebrain commissures , internal capsule , optic chiasm , spinal cord and longitudinal tracts such as the medial forebrain bundle .\",\n \"In the ventral midbrain , dopaminergic cell populations retain an immature morphology and their axons aberrantly project into the hypothalamic region , forming an ectopic commissure near the optic chiasm .\",\n \"Consistent with a failure of axon guidance mechanisms , localization of glial guidepost cells for callosal axons were disrupted , and sensitivity of Alfy knockout axons to the trophic effect of Netrin-1 was significantly diminished .\",\n \"Moreover , Alfy is enriched in membrane fractions , suggesting that it may play a key role in membrane trafficking events to establish neural connectivity in the mammalian brain .\"\n ],\n [\n \"To characterize the role of Alfy in mouse , we initially determined when and where Alfy/Wdfy3 is expressed .\",\n \"Multiplex , semi-quantitative RT-PCR revealed that Wdfy3 mRNA could be detected as early as embryonic day ( E ) 11 in CNS tissue , and remains detectable throughout gestation ( Figure 1A ) .\",\n \"Similar analysis in adult tissue revealed that the Wdfy3 transcript is ubiquitously expressed , and that the highest concentration of Alfy was observed in the brain ( Figure 1—figure supplement 1 ) , confirming previous results ( Simonsen et al . , 2004 ) .\",\n \"Wdfy3 transcript is detected throughout the both the perinatal and adult brain , as determined by in situ hybridization ( ISH ) ( Figure 1B and not shown ) .\",\n \"Immunoblotting revealed that expression of the protein was present uniformly throughout the brain ( Figure 1C ) .\",\n \"Using both primary neuronal and purified astroglial cultures , endogenous Alfy expression was detected in both cell types ( Figure 1—figure supplement 2 ) , supporting recent transcriptome analysis of the mouse cortex ( Zhang et al . , 2014 ) .\",\n \"Therefore , we conclude that Alfy is a CNS-enriched protein that is present in various neuronal and non-neuronal cell types in the developing and adult brain . 10 . 7554/eLife . 14810 . 003Figure 1 . Alfy is highly expressed throughout the developing and adult mouse CNS .\",\n \"( A ) ( Top ) RT-PCR demonstrates Alfy/Wdfy3 can be detected as early as embryonic day 11 ( E11 ) and remains abundant throughout gestation ( E19 ) .\",\n \"( A ) Multiplex PCR for the 5’ region of the gene encoding Alfy , Wdfy3 with Class III β-tubulin ( βIII , top ) and Glial fibrillary acid protein ( Gfap , bottom ) .\",\n \"( Btm )\",\n \"Quantification of transcript levels relative to βIII ( black ) and Gfap ( gray ) , n = 4 , bars represent mean ± SEM .\",\n \"( B ) In situ hybridization of sections from P0 mice reveals strong expression of Wdfy3 throughout the brain .\",\n \"Low magnification images stitched together to reveal a complete sagittal brain section representative of Wdfy3 mRNA distribution in a wildtype mouse .\",\n \"An abundance of mRNA is observed throughout the newborn CNS ( n = 4 ) , whereas no mRNA staining above background is observed in Alfy KO mice ( data not shown ) .\",\n \"The ISH reflects how Alfy is most highly expressed in brain ( Figure 1—figure supplement 1 ) and this expression is both neuronal and glial ( Figure 1—figure supplement 2 ) .\",\n \"Abbreviations: cerebellum ( cb ) , cerebral cortex ( ctx ) , hippocampus ( hp ) , hypothalamus ( ht ) , midbrain ( mb ) , olfactory bulb ( ob ) , striatum ( st ) , superior colliculus ( sc ) and thalamus ( th ) .\",\n \"( C ) Immunblotting reveals that Alfy expression is maintained throughout the adult brain .\",\n \"Lysates generated from different regions of the adult brain , including the brain stem ( bs ) were probed with an Alfy antibody raised against its N-terminus .\",\n \"γ-tubulin is shown as a loading control ( n = 4 ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 00310 . 7554/eLife . 14810 . 004Figure 1—figure supplement 1 . Alfy/Wdfy3 expression is highest in the brain .\",\n \"( A ) Semi-quantitative RT-PCR for Wdfy3 mRNA in different organs from adult mice .\",\n \"Relative levels of Wdfy3 mRNA ( corrected for β-Actin ) across the different organs is quantified below ( n = 5 ) .\",\n \"Bars represent mean ± SEM .\",\n \"Wdfy3 RNA is most highly expressed in brain , although the transcript can be detected in all tissues examined .\",\n \"( B ) Immunoblotting for Alfy from tissue samples collected from adult mice .\",\n \"Relative protein levels were normalized using the cytoskeletal protein vinculin as shown below ( n = 6 ) .\",\n \"Bars represent mean ± SEM .\",\n \"In confirmation with previous results , Alfy protein is most abundant in brain . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 00410 . 7554/eLife . 14810 . 005Figure 1—figure supplement 2 . Alfy is expressed in neurons and astroglia .\",\n \"( A ) Primary cortical cultures were grown for seven days with or without mitotic inhibitors .\",\n \"Mitotic inhibitors were added to cultures after plating to prevent glial proliferation and generate cultures that are enriched for neurons .\",\n \"After seven days , lysates were prepared from the cultures and western blots containing 5 µg of total protein were probed with antibodies against Alfy , the glial marker GFAP , and the neuronal specific tubulin , BIII .\",\n \"As expected , mitotically inhibited cultures had a minimal GFAP expression but contained abundant BIII Tubulin .\",\n \"Alfy was detected in mitotically inhibited cultures , implying neurons endogenously express Alfy .\",\n \"The experiment was replicated twice , using two litters of mice ( Control n = 3; KO n = 3 ) .\",\n \"( B ) Alfy was detected in lysates prepared from primary astroglia cultures that were passaged and allowed to differentiate for fourteen days .\",\n \"Western blots containing 3 µg of total protein were probed with antibodies against Alfy and GFAP .\",\n \"Results from two independent cultures are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 005 To investigate the consequence of the genetic deletion of Wdfy3 in mice , we generated and characterized two different Alfy deficient mouse lines: One using gene trap ( GT ) -mediated disruption and a second using a conditional strategy ( Figure 2 , Figure 2—figure supplement 1 ) .\",\n \"Whereas several GT lines disrupting the Wdfy3 locus were found , one that contained a GT insertion within the first intron was predicted to completely abolish production of the full length transcript .\",\n \"To confirm , mice heterozygous for this mutation ( Alfy GT Het ) were interbred .\",\n \"Homozygous mice ( Alfy GT ) were born at close to the expected Mendelian ratios ( WT: 19% , n = 20; Alfy GT Het: 52% , n = 54; Alfy GT: 28% , n= 29 ) .\",\n \"Primer pairs spanning the transcript indicated that the full length Wdfy3 transcript is not produced in Alfy GT mice ( Figure 2—figure supplement 1B ) , and antibodies directed against the NH3- or COOH-terminus of Alfy confirmed that full length Alfy protein was not detectable in brain lysates ( not shown and Figure 2B ) .\",\n \"This demonstrates that the GT insertion successfully mutagenized the Wdfy3 locus , leading to the loss of Alfy expression . 10 . 7554/eLife . 14810 . 006Figure 2 . Two lines of knockout ( KO ) mice reveal Alfy is essential for postnatal survival .\",\n \"( A , B )\",\n \"Alfy GT mice .\",\n \"( A ) The GT cassette introduces a splice acceptor site ( SA ) and causes the premature termination of transcription through the introduction of a poly-adenylation ( pA ) tail .\",\n \"The translation start site for Wdfy3 is located in exon IV .\",\n \"RT-PCR indicates that the gene trap insertion leads to a loss of Wdfy3 transcript ( Figure 2—figure supplement 1A , B ) .\",\n \"( B ) Western blot of brain lysates probed with an antibody against the COOH-terminus of Alfy and βIII ( n = 5 ) .\",\n \"Abbreviations: long terminal repeats ( LTR ) , neomycin ( NEO ) , Phosphoglycerate kinase-1 promoter and Bruton tyrosine kinase splice donor site ( PGK-BTK-SD ) .\",\n \"( C , D )\",\n \"Alfy KO mice .\",\n \"( C ) A conditional ( flox ) Wdfy3 allele is created by insertion of two loxP sites ( red triangles ) to flank exon 5 , leading to its excision upon exposure to Cre and the creation of the smaller knockout ( KO ) ‘Δ’ allele .\",\n \"The two forward and one reverse primers ( arrows ) used for genotyping are noted ( also see Figure 2—figure supplement 1C ) .\",\n \"( D ) Immunoblotting detects Alfy in brain lysates from heterozygous ( Alfy Het , n = 6 ) but not in Alfy KO mice ( n = 7 ) .\",\n \"( E–G )\",\n \"Characterization of newborn Alfy GT mice .\",\n \"( E ) Newborn Alfy GT and littermatecontrol ( Ctrl ) mice .\",\n \"Arrows highlight that control pups have stomachs full of milk whereas their GT littermates do not .\",\n \"( F ) Alfy GT pups are consistently smaller than control .\",\n \"After genotyping , the weights of the animals were percentile ranked and binned into four groups .\",\n \"Approximately 50% of Alfy GT mice ( 8/15 ) had birth weights in the lowest 25th percentile , whereas heterozygous and wildtype littermates made up 92% ( 12/13 ) of mice with birth weights in the 75th percentile or greater .\",\n \"Data were collected from 10 litters of mice , n = 55 .\",\n \"Similar differences were observed between the Alfy KO pups and their heterozygous littermates ( data not shown ) .\",\n \"( G ) Alfy GT pups lack a righting reflex .\",\n \"The amount of time it took each pup to perform the task was averaged over three trials and recorded as the latency to right .\",\n \"Alfy GT failed to rotate from back to their bellies within 60 sec .\",\n \"A single factor ANOVA revealed genotype had a significant effect on pup behavior ( n = 6 WT , 9 Alfy GT Het , 7 Alfy GT; F ( 2 , 17 ) = 20 . 90 , p < 0 . 001 ) .\",\n \"Fisher’s PLSD test indicated a significant difference between Alfy GT and WT ( p < 0 . 001 ) or heterozygous ( p < 0 . 001 ) littermates .\",\n \"Bars represent mean ± SEM .\",\n \"Similar differences were observed between Alfy KO pups and their heterozygous littermates ( Figure 2—figure supplement 1D ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 00610 . 7554/eLife . 14810 . 007Figure 2—figure supplement 1 . Creation and characterization of Alfy GT and Alfy KO mice .\",\n \"( A , B )\",\n \"Creation of Alfy GT mice .\",\n \"( A ) PCR based genotyping of wildtype ( WT ) , Alfy GT heterozygotes ( Alfy GT Het ) and Alfy GT mice .\",\n \"( B ) Semi-quantitative RT-PCR of Alfy GT mice .\",\n \"Both the 5’ and 3’ portions of the Wdfy3 mRNA transcript are undetectable in Alfy GT mice by RT-PCR .\",\n \"β-actin is shown as control .\",\n \"n = 2 WT , 3 Alfy GT Het , 3 Alfy GT ) ( C ) PCR genotyping of Alfy KO mice .\",\n \"PCR based genotyping of the homozygous and heterozygous conditional Wdfy3 allele ( Wdfy3loxP/+ and Wdfy3loxP/loxP , respectively ) and WT , Alfy KO heterozygous ( Alfy KO Het ) and Alfy KO mice .\",\n \"( D ) Mice are born with an inherent ability to right themselves from a supine to prone position .\",\n \"To determine if the loss of Alfy affected this behavior , marked and warmed pups were placed on their backs on a flat surface and given 60 s to rotate to a prone position .\",\n \"The amount of time it took each pup to perform the task was averaged over three trials and recorded as the latency to right .\",\n \"Alfy KO mice were not able to rotate from their backs to their bellies within 60 s , similar to Alfy GT mice ( Figure 2 ) .\",\n \"A single factor ANOVA revealed genotype had a significant effect on pup behavior ( n = 4/genotype; F ( 1 , 6 ) = 43 . 14 , p < 0 . 001 ) .\",\n \"Bars represent mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 007 The second Alfy-deficient mouse line was produced using a conditional strategy ( Figure 2C ) .\",\n \"Mice carrying a conditional Wdfy3 allele were crossed to HprtCre/+ females to generate a constitutive null '∆' allele ( Figure 2C , Figure 2—figure supplement 2C ) ( Tang et al . , 2002 ) .\",\n \"Similar to Alfy heterozygous for the GT insertion , mice heterozygous for the '∆' allele ( Alfy Het ) were healthy and fertile .\",\n \"Breeding of Wdfy3+/∆::HprtCre/+females with Wdfy3loxP/loxP::Hprt+/Y males generated mice heterozygous ( Alfy Het ) and homozygous ( Alfy KO ) for the null allele close to the expected Mendelian ratios ( Alfy Het: 56% , n = 40; Alfy KO: 44% , n = 32 ) .\",\n \"Alfy protein also was not detected in Alfy KO mice ( Figure 2D ) .\",\n \"Although neonatal Alfy mutant pups appeared grossly normal , were reactive , and showed no sign of cyanosis , they all died several hours after birth .\",\n \"Unlike their littermates , Alfy mutant pups failed to thrive and consistently showed an absence of milk in their stomachs ( Figure 2E ) .\",\n \"Despite being properly cleaned , live Alfy mutant pups were frequently the smallest pups within the litter ( Figure 2F ) and were selectively buried under nesting material , indicating that the dams were rejecting them due to diminished or uncoordinated movement ( Turgeon and Meloche , 2009 ) .\",\n \"To test this hypothesis , we performed a righting reflex test ( Figure 2G , Figure 2—figure supplement 1D , Video 1 ) .\",\n \"Littermates with at least one copy of Alfy were able to right themselves within the allotted test time .\",\n \"In contrast , mice lacking Alfy consistently failed at this test , and demonstrated uncoordinated behavior . 10 . 7554/eLife . 14810 . 008Video 1 . Mice lacking Alfy have an abnormal righting reflex . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 008 Histological examination of the newborn ( P0 ) brain by Nissl staining revealed that brains from Alfy GT and Alfy KO both demonstrated striking abnormalities in the forebrain , midbrain and hindbrain , including visibly smaller brains and gross enlargement of the lateral ventricles ( Figure 3A , B and Figure 3—figure supplement 1A ) .\",\n \"Concurrently , there was an apparent loss and disorganization of interhemispheric axonal tracts throughout the brain .\",\n \"Brains of Alfy GT Het or Alfy Het mice were indistinguishable from wildtype brains ( Figure 3A , B ) . 10 . 7554/eLife . 14810 . 009Figure 3 . Commissures fail to cross the midline appropriately in Alfy GT and KO brains .\",\n \"( A , B )\",\n \"Nissl stained forebrain from newborn ( P0 ) control or Alfy mutant ( Alfy GT or Alfy KO ) mice .\",\n \"Coronal sections are shown .\",\n \"Alfy mutants have many abnormal features , including enlarged ventricles ( * ) , and apparent white matter abnormalities , including absence of a corpus callosum ( cc ) at the midline ( black arrow ) , undetectable anterior commissure ( ac , white arrow ) , and dysmorphic internal capsule ( rectangle ) .\",\n \"More caudal sections can be found in Figure 3—figure supplement 1A .\",\n \"n = 6 WT , 3 Alfy GT Het , 7 Alfy GT; n = 3 Alfy Het , 3 Alfy KO .\",\n \"( C , D )\",\n \"Immunostaining highlights axonal abnormalities in Alfy mutant mice .\",\n \"Coronal sections are shown .\",\n \"The three major forebrain commissures fail to cross the midline in Alfy mutant brains .\",\n \"( C ) The cc , hippocampal commissure ( hc ) and ( D ) ac are absent in Alfy mutants .\",\n \"‘*’ denotes midline and lack of axonal connectivity .\",\n \"Boxed regions are shown enlarged below .\",\n \"n = 5/genotype .\",\n \"The habenular and posterior commissures can be found in Figure 3—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 00910 . 7554/eLife . 14810 . 010Figure 3—figure supplement 1 . Alfy mutant mice have aberrant and disorganized axon projections .\",\n \"( A ) H&E staining of neonatal brains reveal altered brain morphology in Alfy GT mice .\",\n \"Coronal sections are shown .\",\n \"In the absence of Alfy , the cerebral cortex ( ctx ) , thalamus ( th ) and hippocampus ( hp ) are dysmorphic .\",\n \"Additional abnormalities in the Alfy null mice include a reduced size of the thalamus .\",\n \"Alfy heterozygotes are indistinguishable from WT littermates ( data not shown ) .\",\n \"Wildtype mice are shown as control ( Ctrl ) .\",\n \"n = 3/genotype .\",\n \"Scale bar = 250 μm .\",\n \"( B ) NF staining reveals aberrant and disorganized axonal projections of Alfy mutant brains .\",\n \"Coronal sections are shown .\",\n \"Alfy GT mice show reduced staining in the ctx and abnormal NF staining in the internal capsule ( ic ) and tracts within the hp .\",\n \"The fasciculus retroflexus ( fr , square box ) , mammalothalamic tract ( mt , circle ) and cerebral peduncle ( arrow ) are evident in the Ctrl but present as aberrant , disorganized axonal tracts in Alfy GT ( dashed box ) .\",\n \"The external capsule ( arrowhead ) is present in Ctrl and Alfy null brains .\",\n \"n = 5/genotype .\",\n \"Additional abbreviations: hypothalamus ( ht ) , periaqueductal gray ( pag ) , striatum ( st ) , and superior colliculus ( sc ) .\",\n \"DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 01010 . 7554/eLife . 14810 . 011Figure 3—figure supplement 2 . Aberrant and disorganized projections of the habenular and posterior commisural axons .\",\n \"( A ) Low-power magnification of Ctrl and Alfy GT coronal sections are presented in the top panels .\",\n \"The arrow points to the habenular commissure ( hac ) , which is presented at higher magnification below .\",\n \"( B ) The posterior commissure ( pc ) .\",\n \"Both appeared morphologically abnormal in Alfy GT mice , although both crossed the midline .\",\n \"n = 3/genotype .\",\n \"Scale bar as indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 011 To examine the axonal defects further , P0 Alfy mutant brains were stained for neurofilament ( NF ) ( Figure 3C , D and Figure 3—figure supplement 2 ) .\",\n \"When compared to control , NF labeling of Alfy null brains was greatly reduced , suggesting fewer axonal tracts .\",\n \"A notable abnormality was the failure of the three major forebrain commissures to cross the midline .\",\n \"Axons of the corpus callosum , hippocampal commissure ( Figure 3C ) , and anterior commissure ( Figure 3D ) failed to decussate , leading to a separation of the two hemispheres .\",\n \"Two smaller , caudal commissures , the habenular and posterior commissures ( Figure 3—figure supplement 2 ) , were morphologically abnormal and reduced in size however a portion of their tracts crossed the midline .\",\n \"We next examined the retinal axon decussation at the optic chiasm by anterograde-labeling with DiI ( Figure 4A–C ) .\",\n \"Ipsilateral and contralateral projections were quantified by calculating the ipsilateral index ( Kuwajima et al . , 2012 ) .\",\n \"At P0 , the ipsilateral projection in Alfy GT mice ( 1 . 40 ± 0 . 06 ) was 40% larger than that in wildtype ( 1 . 0 ± 0 . 08 ) or heterozygous mice ( 1 . 1 ± 0 . 06 ) ( Figure 4C ) .\",\n \"This significant difference suggests that in Alfy mutants , contralateral axons less readily cross the optic chiasm midline .\",\n \"These data reinforce the findings that Alfy is required to properly establish commissures throughout the developing mouse brain . 10 . 7554/eLife . 14810 . 012Figure 4 . Midline crossing defects extend to the optic chiasm in Alfy GT and KO brains , possibly due to disrupted localization of guidepost glial cells .\",\n \"( A–C )\",\n \"Retinal decussation defects are observed in Alfy mutant mice .\",\n \"( A ) Whole mounts of P0 optic chiasm are unilaterally labeled with DiI at the optic disc .\",\n \"The ipsilateral projection denoted in the white dashed box , which is shown enlarged below .\",\n \"WT and GT heterozygous littermates were indistinguishable .\",\n \"A heterozygous mouse is shown as a control ( Ctrl ) .\",\n \"( B ) Schematic representation of how the ipsilateral index is calculated .\",\n \"Pixel intensities of contralateral and ipsilateral optic tracts are measured within 500 x 500 µm2 .\",\n \"( C ) The ipsilateral index of Alfy mutant mice is greater than both WT and heterozygous ( GT Het ) littermate controls , indicating the abnormally enlarged ipsilateral projection .\",\n \"Bars represent mean ± SEM , and statistical analysis was determined using one-way ANOVA followed by the Tukey’s post hoc test .\",\n \"Respective n-values per genotype are noted in the bars .\",\n \"*p < 0 . 05 .\",\n \"( D , E )\",\n \"Immunostaining for GFAP in P0 forebrain reveals mislocalization of guidepost glial cells .\",\n \"Coronal sections are shown .\",\n \"( D ) The glial wedge ( gw ) , the indusium griseum ( ig ) and midline zipper ( mz ) glia populations of glial cells were in the expected locations in WT littermate brains ( Ctrl ) .\",\n \"In Alfy GT brains , the ig was not detectable ( expected location denoted by '*' ) , and the gw and mz cells were disorganized .\",\n \"n = 3/genotype .\",\n \"( E ) Glial populations within the fimbria ( fi ) and dentate gyrus ( dg ) of the hippocampus were apparent in both genotypes .\",\n \"Boxed areas are shown at higher magnification below .\",\n \"n = 3/genotype .\",\n \"The examination of other potential intrinsic alterations that may contribute to forebrain axonal connectivity can be found in Figure 4—figure supplements 1 and 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 01210 . 7554/eLife . 14810 . 013Figure 4—figure supplement 1 . The loss of Alfy leads to modest changes in proliferation but not cell death in the neocortex .\",\n \"( A ) Loss of Alfy leads to a moderate but significant decrease in cortical plate thickness .\",\n \"Measurements of P0 cortex reveal that the cortical plate/subplate ( CP/SP ) of Alfy GT brains ( n = 3 ) are modestly but significantly thinner than control ( Ctrl , n = 3 ) brains , ( t ( 4 ) = 5 . 30 , p < 0 . 01 ) .\",\n \"The marginal zone ( MZ ) and intermediate zone ( IZ ) do not reach significance , ( t ( 4 ) = 1 . 67 , p = 0 . 17 and t ( 4 ) = 2 . 38 , p = 0 . 076 , respectively ) .\",\n \"Bars denote mean ± St . Dev; * , p < 0 . 01 .\",\n \"( B , C )\",\n \"Loss of Alfy leads to a subtle defect in cell proliferation at E15 . 5 but not at E13 . 5 .\",\n \"A single injection of BrdU was administered to time-pregnant dams at E13 . 5 or E15 . 5 and embryos were collected 1 hr post-injection .\",\n \"( B ) A clear band of BrdU-positive cells ( red ) was detected in the subventricular zone and deep cortical layer in all three littermate genotypes ( WT: n = 2 , heterozygous: n = 3 , and Alfy GT n = 3; two litters ) .\",\n \"Hoechst 33342 nuclear DNA stain is in blue .\",\n \"( C ) Colorimetric BrdU was used to quantify the number of proliferative cells in the developing cortical plate .\",\n \"At E13 . 5 , Quantification revealed no significant difference in the number of BrdU positive ( BrdU+ ) cells between Alfy KO and heterozygous littermate control ( Ctrl ) t ( 8 ) = 1 . 59 , p = 0 . 15 ( Ctrl: n = 5 , Alfy KO: n = 5; two litters ) .\",\n \"At E15 . 5 , there was a modest , but significant reduction in the absence of Alfy , t ( 8 ) = 3 . 12 , p < 0 . 01 ( Ctrl: n = 4 , Alfy KO: n = 6; two litters ) .\",\n \"Bars denote mean ± SEM .\",\n \"ns , not significant; * , p<0 . 01 .\",\n \"( D ) Proliferation , indicated by Ki67-positivity indicates the normal proliferation in newborn Alfy GT mice .\",\n \"Boxed regions highlight the sub-ventricular zone ( VZ ) and are shown at higher magnification in images below .\",\n \"Stereology was performed to quantify the number of Ki67 positive cells in the sub-VZ ( Gundersen Coeffecient of Error = 0 . 15 , Ctrl ( wildtype littermate , n = 3 ) = 187 , 221 cells , S . D . = 19 , 634 compared to Alfy GT ( n = 3 ) = 179 , 074 cells , S . D . = 28 , 917 , t ( 4 ) = 0 . 40 , p = 0 . 71 . ) ( E ) Immunohistochemistry ( brown ) against cleaved caspase-3 reveals no significant difference in cell death at embryonic age E15 . 5 in the absence of Alfy .\",\n \"The enlarged images of the inset for the images are provided immediately below .\",\n \"No cell loss was detected in cortex at this age .\",\n \"As a positive control , the naturally occurring cell death observed in the trigeminal ganglia at this age is included ( leftmost panels ) .\",\n \"No difference was observed across genotype ( n = 2 WT , 2 Alfy GT Het; 3 Alfy GT ) .\",\n \"Tissue sections are counterstained with Nissl , which is shown in purple . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 01310 . 7554/eLife . 14810 . 014Figure 4—figure supplement 2 . Focal cortical dysplasias are observed in Alfy GT brains .\",\n \"( A ) No gross difference in Doublecortin ( DCX , green ) staining can be observed in E15 . 5 Alfy GT brains ( n = 3 ) compared with littermate controls ( Ctrl , n = 3 ) .\",\n \"( B ) Specification of deep layer , Tbr1-positive cortical projection neurons are observed in Alfy GT mice when compared to littermate controls ( Ctrl ) .\",\n \"Three mice per genotype were stained Tbr1 ( Top , red ) and counterstained with Hoeschst 33342 ( bottom , blue ) , and all mice demonstrated this pattern .\",\n \"( C–E )\",\n \"Focal cortical dysplasia ( FCD ) in Alfy null brains .\",\n \"H&E staining reveals the presence of FCD in coronal ( C ) and sagittal ( D ) sections of Alfy GT brains .\",\n \"Boxed areas are shown at higher magnification below .\",\n \"FCDs are labeled with arrows .\",\n \"( E ) FCDs can be detected by Nissl staining as early as E15 in Alfy GT mice ( 6/6 ) but not in control embryos ( 0/6 ) .\",\n \"Arrowheads point to multiple FCDs located within the same hemisphere of an Alfy GT embryo . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 014 Two cell populations that help axons navigate the developing forebrain during the genesis of the corpus callosum are the glial wedge ( gw ) and indusium griseum ( ig ) glia ( Shu and Richards , 2001; Shu et al . , 2003 ) .\",\n \"In light of the presence of Alfy in astroglial cells , as well as the midline crossing defects , we hypothesized that migration abnormalities could contribute to the connectivity defects in Alfy GT and KO mice .\",\n \"Thus , we next examined the callosal guidepost cells , a key population implicated in providing intermediate guidance cues to developing cortical axons .\",\n \"Immunostaining against GFAP on P0 mouse brains revealed abnormalities in Alfy GT brains ( Figure 4D , E ) .\",\n \"Unlike control brain , the ig cells were undetectable and the gw appeared disorganized and loosely packed .\",\n \"The midline zipper glia ( mz ) , cells proposed to promote fusion of the two telencephalic hemispheres ( Silver , 1993 ) , were present but distributed abnormally ( Figure 4D ) , whereas cells in the fimbria and dentate gyrus of the hippocampus appear largely normal ( Figure 4E ) .\",\n \"Taken together , the disorganization of the ig , gw and mz is consistent with the agenesis of the corpus callosum observed in the absence of Alfy .\",\n \"These findings suggest that cells that act as important guideposts for decussating callosal axons ( Shu and Richards , 2001 ) fail to migrate properly in the absence of Alfy , contributing to the connectivity defects observed .\",\n \"In addition to the localization of the midline glial cells , we also determined if intrinsic alterations in the proliferation or differentiation of cortical projection neurons were also contributing to the disruptions in forebrain axonal connectivity .\",\n \"Nissl staining revealed that cortical layering appeared normal , however there was a significant but modest thinning of the cortical plate/subplate at P0 ( Figure 4—figure supplement 1A ) .\",\n \"Although this may be due to the diminished NF staining observed in Figure 3 , we next determined if cell proliferation was affected .\",\n \"Pulse-labeling with bromo-6-deoxy-uridine ( BrdU ) at E15 . 5 revealed cell division in the developing cortex at the expected location in Alfy GT mice ( Figure 4—figure supplement 1B ) .\",\n \"To determine if the amount of proliferation was impacted by the loss of Alfy , stereology was performed on BrdU labeled embryos at ages E13 . 5 and E15 . 5 ( Figure 4—figure supplement 1C ) .\",\n \"No significant difference across genotypes was detected at E13 . 5 , but a modest yet significant reduction in the number of BrdU-positive cells was detected in Alfy KO embryos at E15 . 5 .\",\n \"This difference appeared limited to the cortex , since staining against Ki67 within the subventricular zone at P0 revealed no significant difference between Alfy null mice and controls ( Figure 4—figure supplement 1D ) .\",\n \"Further , despite this modest decrease in embryonic proliferation , there were no detectable differences across genotypes in the amount of cell death in E15 . 5 brains , as indicated by immunohistochemistry against cleaved caspase-3 ( Figure 4—figure supplement 1E ) .\",\n \"We next sought to determine if the loss of Alfy disrupted proliferation in the embryonic cerebral cortex .\",\n \"Although qualitative immunofluorescence against expression markers for immature migrating neurons ( DCX ) and for early born layer VI neurons ( Tbr1 ) also were not significantly altered in the absence of Alfy , histological staining revealed focal cortical malformations known as focal cortical dysplasias ( FCD ) present throughout the cerebral cortex of Alfy KO mice , as described previously in a Wdfy3 hypomorph ( Figure 4—figure supplement 2C–E ) ( Orosco et al . , 2014 ) .\",\n \"These structures were observed as early as E15 . 5 .\",\n \"Thus , the loss of Alfy leads to subtle defects in proliferation , but no changes in cell death or specification in the developing cortical projection neurons .\",\n \"Although columnar organization of discrete regions of the cerebral cortex can be disrupted by the absence of Alfy during embryonic development , it is unlikely that these defects can account for the midline crossing defects observed in Alfy null mice .\",\n \"In addition to the absence of interhemispheric commissures , another striking feature revealed by NF staining in Alfy GT and KO brains was the evident disorganization and apparent loss of other CNS axon tracts .\",\n \"One particularly prominent defect was found in the organization of the internal capsule , which carries the corticospinal tract , and corticothalamic and thalamocortical projections; the internal capsule of Alfy GT brains was disorganized and followed an unusual trajectory through the striatum ( Figure 3 and Figure 3—figure supplement 1 ) .\",\n \"To gain a temporal perspective on the development of the internal capsule , we examined its development during E15 . 5 ( Figure 5A ) and E17 . 5 ( Figure 5B ) .\",\n \"NF staining revealed the expected fasciculated axonal projections of the developing internal capsule en route to and from the cerebral cortex in controls .\",\n \"In contrast , the thalamocortical projections in Alfy GT mice formed ventrally displaced bundles , with fewer projections observed traveling through the ganglionic eminence .\",\n \"Similarly , Alfy GT embryonic brain sections labeled with the axonal marker NCAML1 also highlighted abnormal bundle-like structures in the ventral diencephalic-telencephalic border ( Figure 5—figure supplement 1 ) 10 . 7554/eLife . 14810 . 015Figure 5 . Axonal tracts develop abnormally in the Alfy mutant brain and spinal cord .\",\n \"( A , B )\",\n \"Coronal sections of diencephalon at ( A ) E15 . 5 ( n = 3/genotype ) and ( B ) E17 . 5 ( n = 3/genotype ) stained for neurofilament ( NF , red ) and counter-stained with the nuclear stain Hoechst 33342 ( blue ) .\",\n \"The developing internal capsule is highlighted with a yellow box ( top ) and higher magnification , confocal images of NF staining are shown with ( middle ) and without ( bottom ) Hoechst 33342 .\",\n \"In control brains ( Ctrl ) , axons that comprise the internal capsule form a fan-shaped projection of bundled axons within the ganglionic eminence .\",\n \"The abnormal , ventrally displaced , knot-like bundles of axons found in Alfy GT are marked with white arrows .\",\n \"Neural cell adhesion molecular L1 ( NCAML1 ) staining reveals similar defects ( Figure 5—figure supplement 1 ) .\",\n \"( C , D )\",\n \"TH staining reveals abnormal projections in DAergic cell populations .\",\n \"( C ) Coronal sections .\",\n \"A9 and A10 DAergic populations are present in Alfy GT brains appear immature ( top ) .\",\n \"The hypothalamic A13 DAergic cell population and medium forebrain bundle ( mfb ) are present in the diencephalon in Alfy GT brains; however aberrant projections into the hypothalamus and abnormal midline-crossing events are observed ( arrows , bottom ) .\",\n \"n = 3/genotype .\",\n \"( D ) Sagittal sections counterstained with Nissl .\",\n \"In Alfy GT brains , the mfb has ectopic ventral projections into the hypothalamus ( arrows , bottom ) .\",\n \"Boxed regions are shown at higher magnification below .\",\n \"n = 3/genotype .\",\n \"( E ) NF staining of E13 . 5 cervical spinal cord .\",\n \"Coronal sections are shown .\",\n \"Higher magnification images of the dorsal spinal cord are shown below .\",\n \"The abnormal NF patterning observed in Alfy GT embryos is highlighted with white arrows .\",\n \"n = 3/genotype . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 01510 . 7554/eLife . 14810 . 016Figure 5—figure supplement 1 . NCAML1 staining confirms axonal defects of the internal capsule in the developing Alfy GT brain . Immunofluorescence against NCAML1 ( red ) in E15 . 5 brains counterstained with Hoechst 33342 ( blue ) ( top ) .\",\n \"Boxed areas are shown at higher magnification with and without the counterstain ( bottom ) .\",\n \"Arrow indicates abnormal bundle-like structures formed by the projections in the Alfy GT brain .\",\n \"n = 4 littermate controls , 4 Alfy GT . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 016 We next examined the ventral midbrain ( vMB ) dopaminergic ( DAergic ) neurons and their projections to the striatum ( Figure 5C , D ) .\",\n \"Tyrosine hydroxylase ( TH ) staining revealed that the projections and morphology of vMB DAergic neurons were clearly abnormal in the P0 Alfy KO brains .\",\n \"Notably , the A9 and A10 cell populations , which represent the ventral tegmental area ( A9 ) and substantia nigra ( A10 ) , revealed that the morphology of the regions had an immature appearance relative to control littermates at the same age ( Figure 5C ) .\",\n \"Moreover , rather than follow their typical trajectory through the medium forebrain bundle to striatal targets in the forebrain , in Alfy KO brain , ectopic TH fibers were observed projecting ventrolaterally into the hypothalamus , near the location of the supraoptic decussation ( Figure 5C , D ) .\",\n \"Axonal abnormalities were not limited to the developing brain but were also detected in the developing spinal cord of mice lacking Alfy .\",\n \"At E13 . 5 , disorganized projections into gray matter and the loss of axon bundles were evident in the cervical , thoracic and lumbar levels of the spinal cord in the absence of Alfy , but not in control littermates ( Figure 5E ) .\",\n \"In summary , these data indicate that Alfy is essential for establishing axonal tracts throughout the CNS , from forebrain commissures to the spinal cord .\",\n \"Using two independent mouse models , we find that the loss of Alfy leads to erroneous pathfinding throughout the CNS .\",\n \"Several of these findings are suggestive that the anatomical phenotype observed in Alfy mutant mice may in part be due to defects in axon guidance .\",\n \"For example , an abnormal dopaminergic commissure at the level of the diencephalon traveling through the hypothalamus from the MFB has been previously reported in the Slit1/Slit2 double KO ( Bagri et al . , 2002 ) , the Deleted in colorectal cancer ( Dcc ) KO ( Xu et al . , 2010 ) , and the NK2 Homeobox 1 ( Nkx2 . 1 ) KO ( Kawano et al . , 2003 ) mice .\",\n \"Moreover , diminished guidance cue responsiveness could also account for the failure of the midline astroglia to migrate properly .\",\n \"We therefore hypothesized that Alfy may be involved in the ability of neural cells to respond to guidance cues .\",\n \"Prior to determining if Alfy is involved in axon guidance , we first determined where Alfy is expressed .\",\n \"To do so , we reintroduced full length Alfy into Alfy null primary dissociated cultures to visualize its localization ( Figure 6A , Figure 6—figure supplement 1 ) .\",\n \"Full length Alfy constructs with an N-terminus tag were transfected into DIV7 dissociated primary cortical cultures .\",\n \"Alfy localized throughout the neuron , including within the axon .\",\n \"Neighboring astroglia also showed a diffuse localization of Alfy ( Figure 6—figure supplement 1B ) .\",\n \"Next , we performed subcellular fractionation of adult cortical lysates ( Figure 6B , C , Figure 6—figure supplement\",\n \"2 ) ( Hallett et al . , 2008 ) .\",\n \"Fractionation revealed that Alfy enriched in the light membrane fraction ( P3 ) along with synaptosomal membrane protein synaptophysin and a classic marker for autophagic vesicles , the membrane bound form of microtubule associated protein light chain 3 ( LC3-II ) ( Kabeya et al . , 2000 ) ( Figure 6B , C ) .\",\n \"Additionally , Alfy was enriched in the crude synaptic vesicle fraction ( LP1 ) .\",\n \"Taken together , these results suggest that Alfy may be involved in regulating the trafficking , sorting or signaling events that are required for brain wiring . 10 . 7554/eLife . 14810 . 017Figure 6 . Alfy localizes to axons and enriches to membrane fractions .\",\n \"( A ) Immunofluorescence images showing MAP2-positive neurons expressing Alfy .\",\n \"Merged color image demonstrates co-localization of mCherry-Alfy within a MAP2-positive neuron .\",\n \"Alfy is found within the soma and co-localizes with MAP2 positive projections .\",\n \"Transfections were replicated three times across three independent cultures .\",\n \"Colocalization to βIII Tubulin projections is shown in Figure 6—figure supplement 1 .\",\n \"( B , C )\",\n \"Fractionation of adult cortical lysates reveals that Alfy enriches into membrane fractions .\",\n \"( B ) Equal amounts of protein per fraction were analyzed by immunoblotting .\",\n \"Alfy was present in membrane fractions that also enriched with LC3-II and synaptophysin ( P3 , LP1; boxes .\",\n \"( C ) The fold enrichment measured relative to the total homogenate fraction ( H ) .\",\n \"Bars represent mean enrichment ( n = 3 ) ± SEM .\",\n \"A schematic depiction of the fractionation can be found in Figure 6—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 01710 . 7554/eLife . 14810 . 018Figure 6—figure supplement 1 . Alfy is expressed in the soma and axons of neurons .\",\n \"( A , B )\",\n \"Alfy control mixed primary cortical cultures were transfected with plasmids containing full-length or truncated tagged-Alfy cDNA .\",\n \"( A ) Representative confocal images are shown of Alfy-tagged constructs detected with antibodies directed against the fluorophore tag and co-stained MAP2 or BIII tubulin .\",\n \"Full-length and C-terminal fragments of Alfy are detected throughout MAP2- and BIII tubulin-positive projections ( arrows ) .\",\n \"( B ) Representative confocal images are shown of Alfy-tagged constructs detected with antibodies directed against the fluorophore tag ( left ) and co-stained with GFAP .\",\n \"Alfy are expressed throughout astroglia , although large vesicles and the nucleus are devoid of staining .\",\n \"Studies were replicated across three independent cultures .\",\n \"( C , D )\",\n \"To determine the sub-cellular distribution of Alfy , Alfy KO or Ctrl ( A , B ) mixed primary cortical cultures were transfected with plasmids containing full-length tagged-Alfy .\",\n \"To confirm transfection , COS-7 cells were transfected in parallel and immunoblotted .\",\n \"Vinculin is shown as a loading control .\",\n \"( C ) Full-length Alfy-tagged proteins migrate at the expected size and are detected with anti-mCherry and anti-Alfy antibodies .\",\n \"Over-exposed images are necessary to show endogenous Alfy , since it is at a much lower abundance in non-neuronal cells ( circle ) .\",\n \"( D ) Full length and truncated constructs of Alfy .\",\n \"Studies were replicated across three cultures . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 01810 . 7554/eLife . 14810 . 019Figure 6—figure supplement 2 . Alfy enriches in membrane fractions from brain . A schematic depiction of the modified synaptosome preparation used for Figure 6A , based on a previously published protocol ( Hallett et al . , 2008 ) .\",\n \"Abbreviations: H , homogenate; P1 , nuclei and large debris; S1 , soluble cytoplasmic; P2 , crude synaptosomal membranes; S2 , soluble cytoplasmic; P3 , light membrane; S3 , soluble cytoplasmic; LP1 , enriched synaptosomal membranes; LS1 , synaptic vesicles and synaptic proteins . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 019 To test this hypothesis , we next explored whether Alfy is required for the outgrowth of axons .\",\n \"Dissociated cultures from P0 cortices were plated and examined for morphological differences at day in vitro 7 ( DIV7 ) ( Figure 7A , B; Figure 7—figure supplement 1A–C ) .\",\n \"Alfy KO neurons displayed healthy growth in vitro and Scholl analysis confirmed that they were morphologically similar to control ( Figure 7B ) .\",\n \"Staining also revealed that the neuronal cytoskeletal markers Tau-1 , βIII Tubulin and microtubule associated protein 2 ( MAP2 ) were expressed comparably across genotypes ( data not shown ) .\",\n \"Taken together , these data indicate Alfy is not required for processes controlling non-directed neuronal outgrowth in a cell autonomous manner . 10 . 7554/eLife . 14810 . 020Figure 7 . Alfy is required for the ability of axons to respond to Netrin-1 . ( A , B ) Primary cortical neurons are morphologically similar across genotype .\",\n \"( A ) Individual , GFP-transfected neurons were imaged by confocal microscopy and traced in ImageJ .\",\n \"Representative maximum projection images of two heterozygous ( Het , n=5 brains ) and two Alfy KO neurons ( n = 5 brains ) are shown .\",\n \"Neuronal cytoskeletal markers are expressed comparably across genotype ( Figure 7—figure supplement 1A , B ) .\",\n \"( B ) Scholl analysis of neurons at DIV7 as represented in A . One-way ANOVA revealed no effect of genotype on the number of crossings ( n = 50 neurons/genotype; F ( 1 , 97 ) = 0 . 66 , p = 0 . 41 ) .\",\n \"Analysis of branching ( Figure 7—figure supplement 1C ) similarly showed no effect of genotype .\",\n \"( C , D )\",\n \"Alfy KO cortical explants have attenuated responsiveness to Netrin-1 .\",\n \"( C ) Representative images of Alfy Het or Alfy KO cortical explants in the presence of control ( Ctrl ) or Netrin-1 as described ( Figure 7—figure supplement\",\n \"2 ) and stained with βIII Tubulin .\",\n \"( D ) Quantification of the average length of outgrowth after 72 hr in culture on data collected from 54 explants ( Alfy Het treated with Ctrl: n = 15; Alfy Het treated with Netrin-1: n = 12; Alfy KO treated with Ctrl = 12 , Alfy KO treated with Netrin-1: n = 15 ) , from two litters of mice .\",\n \"A two-way ANOVA revealed a significant effect of genotype ( F ( 1 , 50 ) = 7 . 69 , p < 0 . 01 ) and the presence of Netrin-1 ( F ( 1 , 50 ) = 7 . 71 , p < 0 . 01 ) on outgrowth , as well as a significant interaction between genotype and Netrin-1 exposure ( F ( 1 , 50 ) = 12 . 29 , p < 0 . 001 ) .\",\n \"Fisher PLSD post hoc analysis revealed that exposure to Netrin-1 resulted in a significant increase in outgrowth in Alfy Hets ( p < 0 . 001 ) , but not in Alfy KO ( p = 0 . 56 ) .\",\n \"Under control conditions , there is no difference in the amount of outgrowth between the genotypes ( p = 0 . 56 ) .\",\n \"Netrin-1 attractive guidance was also assessed by determining the Guidance Ratio , which is achieved by measuring the amount of outgrowth from the side of the explant closest to the cue-expressing cell block ( proximal ) over the amount of growth on the side of the explant furthest from the cell block ( distal ) .\",\n \"Under the conditions of our assay , directional growth in response to Netrin-1 was not observed in the control explants ( t ( 25 ) = 1 . 98 , p = 0 . 059 ) or KO explants ( t ( 22 ) = 1 . 77 , p = 0 . 091 ) , suggesting that under the conditions of our assay HEK293T cells secrete Netrin-1 above the threshold for selectively promoting attractive guidance .\",\n \"Similar results were achieved after 48 hr in culture .\",\n \"Bars represent mean ± SEM .\",\n \"* , p < 0 . 001; ns , not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 02010 . 7554/eLife . 14810 . 021Figure 7—figure supplement 1 . Primary cortical neurons are similar across genotype .\",\n \"( A , B )\",\n \"The loss of Alfy does not affect the growth and differentiation of dissociated neurons in culture .\",\n \"Day in vitro 7 ( DIV7 ) neurons from Ctrl and Alfy KO mice express cytoskeletal markers ( A ) Tau-1 ( red ) or ( B ) MAP2 ( red ) and βIII Tubulin ( βIII , green ) positive processes .\",\n \"Merged images are shown in the top panel , whereas individual channels for each fluorophore are shown in grayscale images below .\",\n \"Representative images shown from three independent cultures generated from Alfy Het or Alfy KO brains .\",\n \"( C ) The loss of Alfy had no effect on neurite outgrowth in dissociated cortical neurons .\",\n \"A one-way ANOVA reveals no significant difference across genotype ( F ( 1 , 97 ) = 0 . 19 , p = 0 . 66 ) .\",\n \"Data represented as mean ± St . Dev .\",\n \"Cortical cultures were prepared from five independent cultures with 10 neurons/culture/genotype imaged , resulting in 50 Alfy Het and 50 Alfy KO neurons analyzed . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 02110 . 7554/eLife . 14810 . 022Figure 7—figure supplement 2 . Responsiveness to guidance cues is attenuated in Alfy KO axons .\",\n \"( A , B )\",\n \"Cortical explant culture to examine axon outgrowth in response to Netrin-1 as described in Figure 7C , D .\",\n \"( A ) Diagram of an E14 . 5 embryo with developing cerebral cortex highlighted in red .\",\n \"Cortical explants were co-cultured with agarose-embedded HEK293T cells that were mock transfected ( Control ) or transfected with Netrin-1-Myc ( Netrin-1 ) .\",\n \"Transiently transfected HEK293T cells robustly secrete Netrin-1-MYC into the culture media as revealed by immunoblotting for the MYC tag .\",\n \"Probing for the cytoskeletal protein Vinculin demonstrates that the culture media fraction is predominately cell-free .\",\n \"( B ) Low-power images , stitched together to show the entire amount of outgrowth after 72 hrs in culture , from cortical explants stained with βIII Tubulin ( red ) in the presence of control or Netrin-1 expressing HEK293T cells ( location of HEK293T cells denoted by white arrow ) .\",\n \"( C ) The loss of Alfy does not affect guidance cue receptor levels in the telencephalon .\",\n \"Lysates prepared from E14 . 5 telencephalon were probed on a Western blot with antibodies directed against Alfy , Robo1 , DCC .\",\n \"The amount of DCC and Robo1 in Alfy KO telencephalon was equivalent to the abundance of the receptors detected in Alfy control samples ( DCC: t ( 5 ) = 0 . 45 , p = 0 . 67; Robo1: t ( 4 ) = 0 . 13 , p = 0 . 90 ) .\",\n \"-DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 022 We next examined if Alfy is essential for neurons to respond appropriately to trophic agents , such as the bi-directional guidance cue Netrin-1 ( reviewed in [Leyva-Diaz and Lopez-Bendito , 2013] ) .\",\n \"Explants from control and Alfy KO mice were co-cultured with mock transfected or Netrin-1-Myc transfected HEK293T cells embedded in agarose ( Figure 7—figure supplement 2A ) .\",\n \"After 48 hr , the distance of growth outward from cortical explants was measured ( Figure 7C , D , Figure 7—figure supplement 2B ) .\",\n \"In the presence of Netrin-1 , control explants demonstrate significantly increased growth whereas Alfy KO explants did not respond , despite maintaining their ability to extend their processes under non-stimulated conditions ( Figure 7D ) .\",\n \"Under the conditions used , directional responsiveness could not be ascertained ( Figure 7D ) , likely due to the abundance of Netrin-1 expression due to the use of HEK293T cells ( Shirasaki et al . , 1996 ) .\",\n \"These data indicate that Alfy is required for cortical neurons to respond to the trophic effects of Netrin-1 .\",\n \"The loss of Alfy did not affect the total levels of the guidance cue receptor DCC ( Figure 7—figure supplement 2C ) .\",\n \"Therefore , our data suggest that Alfy function could involve the regulation of intracellular membrane-based signaling events in response to changes in the extracellular milieu of the developing brain .\",\n \"We and others previously found using stable cell lines that Alfy was an adaptor protein for the degradation of aggregated proteins by selective macroautophagy ( Clausen et al . , 2010; Filimonenko et al . , 2010; Lystad et al . , 2014 ) .\",\n \"A key step in response to a guidance cue signaling is the recycling and degradation of large signaling protein complexes .\",\n \"Moreover , upon responding to cues , the resultant neuronal remodeling is also accompanied by the local sequestration and turnover of various cellular components , including cytoskeletal proteins .\",\n \"In light of the degradative capacity of macroautophagy , it is possible that this degradative pathway may be involved , and Alfy acts to sequester these cargoes .\",\n \"As an adaptor protein , the absence of Alfy should not impact macroautophagic degradation overall .\",\n \"Consistent with previous findings , non-selective macroautophagy remains intact in tissues and cells derived from Alfy null mice ( Filimonenko et al . , 2010 ) ( Figure 8A–D ) .\",\n \"Mice lacking Alfy clearly mounted an autophagic response to starvation as indicated by the increased levels of LC3-II in lysates collected from the perinatal liver ( Figure 8A ) , which is consistent with the lack of feeding observed in the pups ( Figure 2E ) .\",\n \"The presence of LC3 conversion is consistent with the formation of APs , which we find forms in response to starvation in the presence or absence of Alfy ( Figure 8—figure supplement 1A ) .\",\n \"Although neurons can properly mount an autophagic response ( Figure 8D ) , no abnormal accumulation of LC3-II was observed in the brain ( Figure 8B ) , suggesting that autophagosome maturation also is unaffected .\",\n \"Consistent with this finding , macroautophagic degradation of long lived proteins was also independent of Alfy expression ( Figure 8C ) . 10 . 7554/eLife . 14810 . 023Figure 8 . Alfy is an adaptor protein for selective macroautophagy ( A–D ) Non-selective macroautophagy proceeds normally in the absence of Alfy .\",\n \"( A ) Liver lysates from perinatal Alfy GT pups reveal greater LC3 conversion to LC3-II , reflecting the starvation response due to their inability to feed properly ( Figure 2E ) .\",\n \"Lysates from normally fed WT and heterozygous littermates mice predominantly contain LC3-I .\",\n \"The bottom graphs quantify LC3 conversion as the ratio of LC3-II/LC3-I .\",\n \"Bars represent mean ± St . Dev .\",\n \"ANOVA reveals a significant effect of genotype on LC3 conversion ( n = 7 WT , 6 Alfy GT Het , 6 Alfy GT; F ( 2 , 16 ) = 11 . 07; p < 0 . 001 ) .\",\n \"Fisher PLSD post hoc analyses indicate that LC3 conversion of Alfy GT mice differed significantly from mice with at least one copy of Alfy .\",\n \"( B ) In the brain , there is no abnormal accumulation of LC3-II in the Alfy GT mice , suggesting that autophagosome maturation proceeds normally .\",\n \"A single-factor ANOVA reveals no significant difference between the three genotypes ( n = 5/genotype; F ( 2 , 12 ) = 5 . 52; p = 0 . 90 ) .\",\n \"( C ) Macroautophagic protein degradation in response to starvation is normal in the absence of Alfy .\",\n \"Long lived protein degradation assay .\",\n \"( LC3 puncta formation is shown in Figure 8—figure supplement 1A ) .\",\n \"A two-factor ANOVA revealed a significant main effect for treatment ( n = 4/genotype; F ( 2 , 27 ) = 24 . 93 , p < 0 . 001 ) , but no significant effect of genotype on percent ( % ) Proteolysis ( n = 4/genotype; F ( 2 , 27 ) = 1 . 82 , p = 0 . 18 ) .\",\n \"Fisher PLSD post hoc analysis revealed % Proteolysis was significantly increased in all genotypes during starvation compared with complete media ( p < 0 . 001 ) and the addition of 5 mM 3MA significantly attenuated the starvation-induced proteolysis ( p < 0 . 001 ) .\",\n \"( D ) In the absence of Alfy , LC3 conversion is normal in primary neurons in response to trophic factor withdrawal ( n = 4/genotype ) .\",\n \"To accumulate LC3-II , cells were starved in the presence of 20 µM leupeptin .\",\n \"( E ) The loss of Alfy impedes the selective macroautophagic turnover of its cargo , ALIS .\",\n \"Although ubiquitinated structures are readily found when all forms of macroautophagy is impeded ( Figure 8—figure supplement 1B ) , the turnover of only specific cargoes is affected by the loss of Alfy .\",\n \"Primary Alfy GT versus Alfy heterozygous ( HET ) MEFs after 72 hr of Veh or 5 μM AraC .\",\n \"Ubiquitin-positive ALIS bodies form in response to mitotic inhibition , as revealed by FK2 immunofluorescence ( green ) .\",\n \"In the absence of Alfy , these structures accumulate more rapidly ( white arrows ) .\",\n \"n = 3 .\",\n \"Similar Alfy dependence can be observed with the aggregation prone expanded polyQ proteins ( Figure 8—figure supplement 1C , D ) .\",\n \"For all graphs , bars represent mean ± SEM .\",\n \"ns , not significant; * , p < 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 02310 . 7554/eLife . 14810 . 024Figure 8—figure supplement 1 . Alfy is an adaptor for selective macroautophagy .\",\n \"( A ) In the presence of complete media , LC3 immunoreactivity ( green ) produces a diffuse staining pattern throughout the cytoplasm .\",\n \"When MEF cultures are starved by incubation in HBSS , LC3 reactivity becomes localized to distinct puncta regardless of genotype , suggesting the formation of LC3-positive autophagosomes ( n = 4 ) .\",\n \"( B ) The inhibition of macroautophagy leads to ALIS body accumulation in the absence of stress .\",\n \"Immortalized Atg5 KO MEFs ( Kuma et al . , 2004 ) demonstrate the accumulation of ubiquitin positive aggregates ( white arrows ) under basal conditions as revealed by FK2 antibody ( green ) ( n = 3 ) .\",\n \"( C , D )\",\n \"The selective macroautophagy cargo , expanded polyglutamine inclusion , accumulates more rapidly in the absence of Alfy .\",\n \"Transient transfection of 17aahtt103Q-eGFP in Ctrl and Alfy GT MEFs .\",\n \"Aggregation over time is quantified in ( D ) .\",\n \"The percentage of transfected cells with aggregates is higher in the Alfy GT MEFs compared with Ctrl .\",\n \"Repeated measures ANOVA revealed a significant difference of genotype in the% transfected cells with aggregate genotype ( n = 3/genotype; F ( 1 , 8 ) = 154 . 643 , p = 0 . 002 ) , and a significant interaction between time and genotype ( n = 3/genotype; F ( 2 , 8 ) = 55 . 332 , p < 0 . 0001 ) .\",\n \"Post hoc analyses ( Fisher PLSD ) revealed that in Ctrl MEFs , ‘% transfected cells with agg’ do not change between 24 and 48 hr ( p = 0 . 4144 ) , but decrease after 72 hr ( p = 0 . 005 ) .\",\n \"In contrast , in Alfy GT MEFs , ‘% transfected cells with agg’ continuously increase over time ( comparison between 24 and 48 hr , p = 0 . 0047; comparison between 48 and 72 hr , p = 0 . 0105 ) .\",\n \"Scale bar = 25 μm .\",\n \"Data represent mean ± St . Dev . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 024 Since we confirmed that nonselective macroautophagy was unaffected , we next determine if the loss of Alfy impeded the turnover of its substrates .\",\n \"One naturally occurring Alfy substrate are known as aggresome-like induced structures ( ALIS ) ( Clausen et al . , 2010 ) .\",\n \"Cytosolic ubiquitinated ALIS were formed in MEFs upon mitotic inhibition ( Figure 8E ) .\",\n \"In contrast to control MEFs , these structures accumulated more rapidly in the absence of Alfy , suggesting that in its absence , the turnover of ALIS are impeded .\",\n \"Moreover , unlike in MEFs that lack all forms of macroautophagy ( Figure 8—figure supplement 1B ) ( Kuma et al . , 2004 ) , Alfy GT MEFs do not demonstrate basal protein accumulation , suggesting that Alfy is not required for the turnover of all ubiquitinated substrates .\",\n \"Nonetheless , over-expression of a canonical aggregation-prone polyQ protein , a 17 amino acid fragment of the mutant huntingtin protein with 103 glutamines and an eGFP tag ( 17aahtt ( 103Q ) eGFP ) ( Kazantsev et al . , 1999 ) , accumulated more rapidly by the absence of Alfy ( Figure 8—figure supplement 1C , D ) .\",\n \"Thus , in the absence of Alfy , the elimination of ubiquitinated proteins by selective macroautophagy , but not basal macroautophagy , is impaired .\",\n \"Taken together with the rest of our findings , we hypothesize that Alfy-mediated sequestration of cargoes may help selectively eliminate cargoes at or around the membrane , thereby affecting the ability of axons to properly respond to guidance cues .\"\n ],\n [\n \"In this study , our genetic models revealed that Alfy influences how growing axons interact with their environment to form stereotyped axonal connectivity in the central nervous system ( CNS ) .\",\n \"The homozygous loss of Wdfy3 disrupts the formation of many axonal connections , causing agenesis of all three forebrain commissural tracts and leading to profound changes in brain wiring throughout the CNS , including the optic chiasm , internal capsule , cerebral peduncle , medial forebrain bundle and spinal cord tracts .\",\n \"At the cellular level , the loss of Alfy led to defects in the development of midline glial population , impacted embryonic cortical neuron proliferation and impeded the ability of axons to respond to Netrin-1 .\",\n \"Furthermore , Alfy KO mice present a phenotype unlike mice lacking core autophagy genes such as Atg5 , suggesting the perinatal lethality is not due to a loss of general autophagic degradation , as Alfy mutant tissue and cells display the classic response to starvation .\",\n \"We hypothesize that Alfy functions as a selective macroautophagy adaptor protein and that Alfy-dependent regulation of intracellular vesicle trafficking and signaling pathways upstream of macroautophagy can have a significant influence over brain development .\",\n \"The widespread disruption of axonal wiring identified in the absence of Alfy supports a functional role for this protein in the sequestration and trafficking of substrates into the autophagic pathway .\",\n \"In the adult brain , Alfy facilitates the sorting of ubiquitinated cargo into APs , and one possibility is that during development Alfy has an analogous function .\",\n \"For instance , APs are present in the growth cone and axon tips ( Hollenbeck , 1993; Maday and Holzbaur , 2014; Maday et al . , 2012 ) .\",\n \"We propose that Alfy could regulate the ability of growth cones to respond to cues by sequestering and sorting proteins into degradative vesicles .\",\n \"Such regulation could be critical for responding to cues in the environment through changes in cell shape and motility .\",\n \"It has been suggested previously that the ubiquitination of guidance receptor complexes could regulate the responsiveness of axons to guidance cues ( O'Donnell et al . , 2009 ) .\",\n \"The trafficking and responsiveness of the axonal guidance receptors Robo1 and UNC5 has been shown to be regulated by the E3 ubiquitin ligases rpm-1 in C . elegans and USP33 in murine commissural axons , respectively ( Li et al . , 2008; Yuasa-Kawada et al . , 2009 ) .\",\n \"Furthermore , deletion of the rpm-1 homologue , Phr1 , in mice , causes axonal wiring defects reminiscent of Alfy mutants ( Bloom et al . , 2007 ) .\",\n \"One intriguing possibility is that Alfy might sequester cargo ubiquitinated by E3 ligases such as Phr1 or USP33 .\",\n \"Alternatively , the association of Alfy with the GABAA receptor-associated protein ( GABARAP ) ( Lystad et al . , 2014 ) , implies Alfy could be involved in the selective sorting of plasma membrane receptors .\",\n \"Based on this model , we predict that in the absence of Alfy , the efficient degradation of Alfy-associated cargo would be slowed , or the cargo would be trafficked into alternative pathways , disrupting the temporal regulation of signaling events within the growth cone .\",\n \"Similarly , the disruption of the midline glial structures may also reflect the disruption of signaling events that promote the differentiation and migration of these cell populations .\",\n \"Examination of substrates labeled with a Lys63 polyUb chain , which are also associated with APs ( Shaid et al . , 2013 ) , may identify other Alfy-interacting substrates .\",\n \"Wdfy3 and its homologue bchs have been implicated in trafficking events associated with lysosomes , yet genetic disruptions yield distinct phenotypes in their respective model systems .\",\n \"Both homologues have been shown to co-localize with markers for degradative vesicles , including APs in mammals and endolysosomes in Drosophila ( Lim and Kraut , 2009; Lystad et al . , 2014; Simonsen et al . , 2004 ) yet , despite the conservation of domain structure , bchs loss of function ( LoF ) mutants have no reported developmental neuroanatomical defects ( Khodosh et al . , 2006 ) .\",\n \"Overexpression of bchs , however can impact synaptic and axonal morphology at the neuromuscular junction and the retina , revealing that bchs dosage plays a role in CNS development and function ( Khodosh et al . , 2006; Kraut et al . , 2001 ) .\",\n \"The difference in phenotype between bchs LoF flies and Alfy mutant mice might be explained by the emergence of novel biological functions due to the evolutionary expansion of the BEACH family of genes from six in invertebrates to nine in vertebrates ( Cullinane et al . , 2013 ) .\",\n \"Evolutionary divergence also has been described for the BEACH protein , Neurobeachin ( Nbea ) , which has evolved distinct biochemical properties from its Drosophila homologue rugose ( Volders et al . , 2012 ) .\",\n \"We propose Alfy , which shares 48% protein sequence identity with bchs , may have similarly diverged , acquiring new functions that are essential for vertebrate development .\",\n \"Whereas proteasome-mediated degradation is thought to be the primary catabolic pathway regulating neurodevelopmental signaling ( Yi and Ehlers , 2007 ) , recent studies indicate that macroautophagy may also play a critical role in morphogenesis .\",\n \"For instance , the loss of Ambra1 , a regulator of the autophagy protein Beclin1 , causes neural tube closure defects in mice ( Fimia et al . , 2007 ) .\",\n \"In humans , recessive mutations in TECPR2 and EPG5 , genes implicated in AP maturation ( Stadel et al . , 2015; Tian et al . , 2010 ) , are responsible , respectively , for spastic paraplegia 49 with brain malformations , including hypogenesis of the corpus callosum [OMIM 615031] and Vici syndrome with brain malformations , including agenesis of the corpus callosum [OMIM 242840] ( Cullup et al . , 2013; Oz-Levi et al . , 2012 ) .\",\n \"Why loss of these autophagy-related proteins causes corpus callosum defects remains unclear .\",\n \"Although little is known about the importance of core autophagy genes such as Atg5 , Atg7 and Atg14 in neurodevelopment , no obvious defects in axonal connectivity have been reported ( Hara et al . , 2006; Komatsu et al . , 2006; Liang et al . , 2010 ) .\",\n \"One exception is that the conditional elimination of Atg7 in POMC neurons leads to a defect in postnatal axonal outgrowth ( Coupe et al . , 2012 ) , and recently , a partial LoF mutation in a core autophagy gene , Atg5 , have been reported to lead to cerebellar hypoplasia and developmental delay ( Kim et al . , 2016 ) , but connectivity of the callosum appears unaffected .\",\n \"Given that degradation by autophagy is slower than proteasome-mediated turnover , these data suggest that the sorting and sequestration of cargo , rather than elimination of cargo , is critical for the timing of cellular processes regulated by macroautophagy .\",\n \"Alternatively , as a scaffold for ubiquitinated structures for degradation by macroautophagy , the loss of Alfy might have indirect effects on proteasome activity .\",\n \"For example , Alfy is recruited to aggregated structures that form upon proteasome-inhibition ( Clausen et al . , 2010; Simonsen et al . , 2004 ) ; when Alfy is absent , the persistence of structures that might otherwise be eliminated may impact the efficiency of proteasome-mediated degradation .\",\n \"We have previously reported that the deletion of Alfy does not measurably affect the enzymatic activity of the proteasome ( Filimonenko et al . , 2010 ) and does not lead to the accumulation of ubiquitinated structures in Alfy null MEFs ( Figure 8 ) .\",\n \"Nonetheless , it is still possible that localized , discrete disruptions of proteasome activity or modifications in activity enough to disrupt timing might be present , leading to the profound phenotype we observe here .\",\n \"Genetic screening has revealed a possible role for the human Wdfy3 homolog ( WDFY3 ) as a genetic risk factor for intellectual and developmental disabilities .\",\n \"Most recently , a missense mutation in WDFY3 has been linked to autosomal dominant primary microcephaly ( Kadir et al . , 2016 ) .\",\n \"Furthermore , in autism spectrum disorder ( ASD ) , rare de novo nonsense mutations within WDFY3 have been identified across two independent studies ( De Rubeis et al . , 2014; Iossifov et al . , 2012 ) .\",\n \"WDFY3 resides on chromosome 4q21 . 23 and this region of chromosome 4 was suggested to harbor a genetic predisposition to ASD ( Chen et al . , 2006 ) and to schizophrenia ( Faraone et al . , 2006; Paunio et al . , 2004 ) .\",\n \"In addition , the dosage of WDFY3 is affected in a subset of individuals with a rare chromosome disorder known as 4q21 deletion syndrome [OMIM 613509] , characterized by intellectual disabilities ( ID ) , language impairment and congenital birth defects ( Bonnet et al . , 2010; Cooper et al . , 2011 ) .\",\n \"Taken together , these data imply WDFY3 is a rare genetic risk factor for childhood neurological disorders .\",\n \"Future studies should aim to determine whether WDFY3 dosage is critical for human brain development and how mutations could potentially disrupt the biological function of Alfy .\",\n \"A network analysis study on genomic data found that CNVs in genes associated with autophagy were enriched in patients with ASD ( Poultney et al . , 2013 ) .\",\n \"While it remains to be determined whether computational modeling can be used to accurately predict the cellular pathways disrupted in ASD , in light of our findings , we would propose that further study is warranted .\",\n \"In conclusion , mice lacking Alfy survive embryogenesis , but the loss of Alfy function produces widespread CNS defects in axonal tracts , including loss of the major forebrain commissures .\",\n \"The results of our study provide new insight into the genetic control of brain wiring and open the door to many new questions surrounding the physiological role of Alfy and selective autophagy in CNS development .\"\n ],\n [\n \"The following antibodies were used for western blot , immunocytochemistry , immunohistochemistry , in situ hybridization and immunoprecipitation experiments: Anti-Alfy ( rabbit polyclonal anti-COOH Alfy; generously provided by Dr . Anne Simonsen and used for western blots and immunoprecipitation ) , anti-Alfy-N1 ( rabbit polyclonal anti-NH3 Alfy; generously provided by Dr . Masaaki Komatsu and used for western blots ) , anti-BrdU clone BU-33 ( B2531 , Sigma-Aldrich [St . Louis , MO] ) , anti-Caspase 3 active/cleaved form ( AB3623 , EMD Millipore [Germany] ) , anti-Class βIII-Tubulin ( PRB-435P , Covance [Princeton , NJ] ) , anti-Human DCC ( 554222 , BD Biosciences [San Jose , CA] ) , anti-Digoxigenin-AP , Fab fragments ( Roche [Switzerland] ) , anti-Doublecortin ( ab18723 , Abcam [Cambridge , MA] ) , anti-GFAP , clone GA5 ( MAB3402 , EMD Millipore ) , anti-LC3 ( for immunofluorescence , rabbit polyclonal; generously provided by Ron Kopito ) , anti-LC3B ( for western blots , ab48394 , Abcam ) , anti-Ki67 clone B56 ( 550609 , BD Biosciences ) , anti-MAP-2 ( ab11267 , Abcam ) , anti-NCAML1 , clone 324 ( MAB5272 , EMD Millipore ) , anti-Neurofilament clone2H3 ( developed by TM Jessell and J Dodd and was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa ) , anti-Robo1 ( H-200 , Santa Cruz Biotechnology [Santa Cruz , CA] ) , anti-synaptophysin ( 101 002 , Synaptic Systems [Germany] ) , anti-Tau-1 , clone PC1C6 ( MAB3420 , EMD Millipore ) , anti-Tbr1 ( AB2261 , EMD Millipore ) , and anti-Ubiquitin , clone FK2 ( Enzo Life Sciences [Farmingdale , NY] ) .\",\n \"The following reagents were generously provided: Atg5 knockout and wildtype MEFs ( Noboru Mizushima , [Itakura and Mizushima , 2010] ) and MYC-tagged Netrin-1 ( pGNET1-myc ) ( Marc Tessier-Lavigne [Serafini et al . , 1994] ) .\",\n \"All animals and procedures complied with the Guide for Care and Use of Laboratory Animals and were approved by the IACUC committee at Columbia University .\",\n \"Mice were maintained in a 12h/12h light/dark cycle in a temperature and humidity controlled environment , with ad libitum access to food and water .\",\n \"Alfy GT mice ( 129/SvEv x C57BL/6; Wdfy3Gt ( OSTGST_5258_D3 ) Lex ) were generated at the Texas Institute for Genomic Medicine ( TIGM [College Station , TX] ) .\",\n \"A complete description of the gene trap vector can be found in ( Zambrowicz et al . , 2003 ) .\",\n \"The Alfy GT mice used in this study were derived from mating heterozygous mice .\",\n \"Wdfy3 floxed mice were generated on a 129/SvEv x C57BL/6 background under contract with the University of Connecticut .\",\n \"The floxed Wdfy3 allele has loxP sites flanking exon V that is predicted to produce a 66 amino acid peptide following Cre-mediated recombination , instead of the 3508 amino acid full-length Alfy protein .\",\n \"The Wdfy3 deletion allele ( ∆ ) is created when male mice carrying Wdfy3 floxed alleles are crossed with Bl6/J female carrier of HprtCre/+ , a Cre-deleter stain with 100% Cre-mediated recombination in oocytes ( Tang et al . , 2002 ) .\",\n \"All experiments included control and experimental littermates and represent data across multiple litters .\",\n \"Genomic DNA was extracted and the alleles were identified using the following primers: Common Alfy forward: 5’-CTTGTTACACTTGTCCCACAGC-3’ , Alfy WT reverse 5’– TTAGACTTCTAAGCCCACGAGTACC-3’ , and Alfy GT reverse: 5’-ATAAACCCTCTTGCAGTTGCATC-3’ .\",\n \"Genotyping for the Alfy WT , loxP and ∆ alleles was performed using following the primers in a multiplex PCR reaction: LoxP-F 5’-gaaagcaagctcgtttacgg-3’ , Frt-R 5’-aggttaccagccacaaccag -3’ , and Frt-F 5’-acttgggaagagggaagctc-3’ .\",\n \"RNA from whole embryo was isolated using Trizol reagent ( Life Technologies [Carlsbad , CA] ) according to the manufacture’s recommendation .\",\n \"This RNA was used in a reverse transcription reaction containing random 9mers ( NEB Biosciences ) and reverse transcriptase ( Superscript , Qiagen [Germany] ) according to manufacturer’s instructions .\",\n \"Negative controls ( minus reverse transcriptase ) were run for each sample .\",\n \"The resulting cDNA was used to determine the abundance of Wdfy3 mRNA relative to GFAP , βIII Tubulin or Actin .\",\n \"The following primers were used: βIII Tubulin: Tuj1-1F: 5’-ctacgacatctgcttccgca -3’ , Tuj1-1R: 5’-gaagggaggtggtgactcca-3’; GFAP: GFAP-F: 5’-gagctcaatgaccgctttgc -3’ , GFAP-R: 5’-tccttggctcgaagctggt -3’ .\",\n \"Primary dissociated cortical cultures were prepared from postnatal day 0 mouse pups ( P0 ) .\",\n \"The cortical lobes were dissected in ice cold DMEM/F12 ( Life Technologies ) containing 10% heat- inactivated FBS and antibiotics .\",\n \"Cortical tissue were trypsinized for 30 min at 37°C , then triturated in fresh 10% FBS DMEM/F12 through a fire-polished glass pipette and filtered through a 40 micron nylon cell strainer ( BD Biosciences ) .\",\n \"For immunocytochemistry , cells were plated at a density of 1 . 0 x 105 cells/well onto coverslips coated with 20 μg/mL polyD-lysine ( Sigma Aldrich ) and 5 µg/mL mouse laminin ( Life Technologies ) .\",\n \"The medium was changed 2 hr after plating to NB media ( Neurobasal media [Life Technologies] ) containing 0 . 5 mM L-glutamine , 1X B27 supplement ( Life Technologies ) , 2% heat inactivated FBS ( Life Technologies ) and 1X antibiotic ( Life Technologies ) .\",\n \"Embryos were collected from deeply anesthetized pregnant dams at E14 . 5 .\",\n \"Uterine horns were placed into ice cold Hank’s buffered saline solution ( HBSS ) .\",\n \"Heart , liver and head were removed for genomic DNA extraction , the remaining tissue was trypsinized in equal volume of HBSS and 0 . 25% trypsin for 15 min . at 37°C .\",\n \"Samples were triturated with a fire-polished glass pipette , then diluted into MEFs complete media: DMEM ( Life Technologies ) containing 10% FBS ( Life Technologies ) , 1X L-glutamine ( Life Technologies ) , 0 . 1 mM beta-mercaptoethanol ( Life Technologies ) , 1X Pen/strep ( Life Technologies ) .\",\n \"Cells were filtered through a 100 micron sieve and plated .\",\n \"In addition to genotype confirmation , RNA was extracted from the cultures to measure transcript levels and MEF cultures were confirmed to be mycoplasma free ( Takara ) .\",\n \"To starve the MEF cultures , the complete media was aspirated off the cultures , followed by three PBS washes and cultures were placed in HBSS supplemented with 10 mM HEPES for four hours .\",\n \"The subcellular fractionation of the brain tissue protocol was performed as described ( Hallett et al . , 2008 ) .\",\n \"Briefly , adult control mice were euthanized and cerebral cortex was rapidly dissected and frozen on dry ice .\",\n \"An aliquot of each fraction was saved for analysis .\",\n \"Brain tissue was homogenized in TEVP buffer ( 10 mM Tris , 5 mM NaF , 1 mM Na3VO4 , 1 mM EDTA , 1 mM EGTA , pH . 7 . 4 ) containing 320 mM sucrose .\",\n \"Homogenates ( H ) were fractionated sequentially as shown in Figure 6—supplement figure 2 at 800 xg , resulting in the first supernatant ( S1 ) and pellet ( P1 ) fractions , and 9200 xg to generate the second supernatant ( S2 ) and crude synaptosomal membranes ( P2 ) .\",\n \"P2 was resuspended in TEVP buffer containing 35 . 6 mM sucrose for 30 min on ice to release vesicles and organelles .\",\n \"P2 was then spun at 25 , 000 xg to segregate the supernatant ( LS1 ) from the membrane fraction ( LP1 ) .\",\n \"Lastly , S2 was centrifuged for 2 hr at 165 , 000 xg to generate the supernatant fraction ( S3 ) and light membrane fraction ( P3 ) .\",\n \"Protein quantification was performed on all fractions and equal amounts of protein were loaded onto SDS-PAGE .\",\n \"Alfy MEFs as well as Atg5 knockout and wildtype MEFs were maintained in MEF complete media .\",\n \"Starvation was achieved using a starvation medium of HBSS + 10 mM HEPES for 4 hr .\",\n \"To slow lysosome-mediated degradation , cells were treated with 20 μM leupeptin .\",\n \"Mitotic inhibition was achieved by treating cells with 10 μM arabinofuranosylcytidine ( AraC ) for 72 hr .\",\n \"Transfections of cells were achieved with Lipofectamine 2000 ( Life Technologies ) in serum-free media .\",\n \"Cells were transfected with constructs encoded by 17aahtt103Q tagged with eGFP as previously described ( Filimonenko et al . , 2010 ) .\",\n \"Cells were fixed 24 , 48 and 72 hr later and imaged by epifluorescence microscopy .\",\n \"Long lived protein degradation assay was performed as previously described ( Filimonenko et al . , 2010 ) .\",\n \"Whole cell lysates and tissue lysates from freshly dissected brain and liver were generated using a modified RIPA buffer ( 50 mM Tris-HCl , pH 7 . 4 , 150 mM NaCl , 10 mM EDTA , 0 . 1% Triton-X 100 ) containing protease and protein phosphatase inhibitors ( Halt Inhibitor Cocktail , Roche ) .\",\n \"Ten strokes in a dounce homogenizer were used to extract protein from tissues and samples were cleared by centrifugation at 15 , 000 xg for 60 min at 4°C unless otherwise indicated .\",\n \"Protein concentration was determined using the DC Protein Assay ( Bio-rad Laboratories [Hercules , CA] ) and equal amounts of protein were prepared and loaded onto 4–12% Bis-Tris or 3–8% Tris-Acetate NuPAGE gels and blotted to PVDF membranes as described by the manufacture’s recommendations ( Life Technologies ) .\",\n \"PVDF membranes were blocked in 5% BSA in TBS containing 1% Tween-20 ( TBST ) .\",\n \"Antibodies were diluted in 1% BSA in TBST and incubated overnight at 4°C .\",\n \"The appropriate HRP-conjugated secondary antibodies ( ThermoFisher Scientific ) were diluted in 1% BSA TBST and a chemiluminescent reaction ( West Dura SuperSignal , ThermoFisher Scientific ) was detected by the Versadoc Imaging System ( Bio-rad ) .\",\n \"Fresh frozen tissue was sectioned at 15 μm onto Fisherband Superfrost plus slides and stored at −80°C until use .\",\n \"On day one , slides were warmed at 37°C for 30 min , fixed in 4% paraformaldehyde for 10 min and washed in DECP-treated phosphate buffered saline ( PBS ) .\",\n \"Slides were acetylated with 0 . 3 M acetic anhydride in 0 . 1 M triethanolamine for 10 min followed by three PBS washes .\",\n \"Slides were prehybridized in formamide diluted 2X Prehyb buffer ( 5 M NaCl , 1 M Tris , pH 7 . 4 , 6% Ficoll , 6% polyvinylpyrrolidone , 6% Bovine serum albumin , 50 mg total Yeast RNA , 5 mg Yeast tRNA , 250 mM EDTA and 50 mg SS DNA ) for 1 hr at room temperature .\",\n \"Wdfy3 riboprobes were prepared from a pCR II plasmid ( Life Technologies ) containing a PCR fragment of the 5’ UTR region of mouse Wdfy3 from 320–540 nt ( ref NM_172882 . 3 ) .\",\n \"In vitro transcription was performed as described by the manufacturer ( Promega Corporation [Madison , WI] ) using the T7 polymerase for the antisense probe on template linearized with KpnI and Sp6 polymerase for the sense probe on template linearized with NotI .\",\n \"Riboprobes were cleaned using the RNeasy MinElute Cleanup Kit ( Qiagen ) according the manufacturer’s protocol .\",\n \"Riboprobes were heated to 80°C for 5 min , quenched on ice and added to 2X Hyb buffer ( 5 M NaCl , 1 M Tris , 6% Ficoll , 6% polyvinylpyrrolidone , 6% Bovine serum albumin , 50 mg total Yeast RNA , 5 mg Yeast tRNA , 250 mM EDTA , 50 mg SS DNA and 20% dextran sulphate ) diluted 1:1 with formamide .\",\n \"Hyb solution was added directly to slides and they were incubated overnight at 68°C in a humidified chamber .\",\n \"On day two , slides were washed in 5X SSC at 68°C for 10 min and coverslips were removed .\",\n \"Three more washes 0 . 2X SSC were carried out at 68°C , followed by a 0 . 2X SSC wash at room temperature .\",\n \"Slides were incubated in B1 buffer ( 0 . 1M Tris , pH 7 . 5 , 0 . 15M NaCl ) for 5 min then blocked in B1 containing 10% heat-inactivated sheep serum for 1 hr in a humidified chamber .\",\n \"Anti-DIG antibody diluted 1:5000 in B1 buffer containing 1% heat-inactivated sheep serum and slides were incubated overnight at 4°C in a humidified chamber .\",\n \"On day two , slides were washed in B1 buffer three times and equilibrated in B3 buffer ( 0 . 1 M Tris , pH 9 . 5 , 0 . 1 M NaCl , 50 mM MgCl2 ) for 5 min at room temperature .\",\n \"The staining reaction was carried out using NBT/BCIP ( Promega ) diluted in B3 buffer in the dark at room temperature overnight in a humidified chamber and the staining reaction was stopped by washing the slides three times in TE ( 10 mM Tris , pH 8 . 0; 1 mM EDTA ) .\",\n \"On DIV3 , cultures were transfected with 1 µg pEGFP-N3 ( Clontech ) using Lipofectamine 2000 ( Life Technologies ) in Optimem for 4 hr .\",\n \"The next day , the media was changed to NB media + mitotic inhibitors: 10 µM AraC ( Sigma-Aldrich ) , 10 µM Uridine ( Sigma-Aldrich ) , and 10 µM 5-Fluoro-2’-deoxyuridine ( FDU , Sigma-Aldrich ) to prevent the excessive growth of glial cells .\",\n \"On DIV7 , cells were washed with TBS , fixed for 15 min with 4% paraformaldehyde , permeabilized in 0 . 1% Triton-X 100 in TBS , blocked in 5% BSA in TBS and stained with primary antibodies diluted in 1% BSA in TBS overnight at 4°C .\",\n \"Alexa Fluor conjugated secondary antibodies ( Life Technologies ) were diluted in 1% BSA TBS , incubated with coverslips for 1 hr at room temperature , counterstained with Hoechst 33 , 342 ( Life Technologies ) and mounted with prolong gold antifade ( Life Technologies ) onto glass slides .\",\n \"A Leica SP5 confocal microscope was used to image cortical cultures .\",\n \"GFP-transfected cells with the characteristic neuronal morphology , including a small triangular shaped soma were analyzed to compare morphology between Alfy null and Alfy control cultures .\",\n \"Z-stacks were taken every micron using a 20X objective and 3X digital zoom .\",\n \"For each culture , approximately10 individual neurons were imaged totaling 50 control and 49 knockout cortical neurons .\",\n \"Confocal z-stacks were traced and analyzed using the simple neurite tracer segmentation plug-in for the FIJI version of ImageJ ( Longair et al . , 2011 ) .\",\n \"Time-pregnant dams were sacrificed in accordance with humane protocols established by the Columbia University IACUC .\",\n \"All dissections were carried out in ice-cold PBS .\",\n \"The embryonic cerebral cortex was dissected out and finely cut into pieces , then 28 gauge needles were used to transfer and position explant pieces inside a drop ( 5 µL ) of Matrigel ( BD Biosciences ) on a collagen-coated glass bottom petri dish ( MatTek ) .\",\n \"Once explants were placed , 350 µm diameter punches of solidified agarose cell blocks containing either untransfected or Netrin1-MYC transfected HEK293T cells were placed alongside cortical explant pieces .\",\n \"Explants were grown for 48 hr , and then fixed with 4% PFA for 15 min .\",\n \"Explants were imaged and measured using phase contrast microscopy using a 10X objective on a Nikon TiE microscope and NIS Elements software ( Nikon ) .\",\n \"Across two experiments , outgrowth in microns towards the HEK293T cellblock was recorded for the three longest processes in a single plane .\",\n \"The measurements were averaged for each explant and statistical analysis was performed using two-way ANOVA followed by the Fisher LSD post hoc test .\",\n \"Following analysis of outgrowth , explants were further processed for immunocytochemistry as described above .\",\n \"To prepare HEK293T agarose cell blocks , HEK293T cells were seeded at 600 , 000 cells per six well , then 16 hr later transiently transfected with pGNET1-myc using Lipofectamine 2000 .\",\n \"The following day , untransfected HEK293T or HEK293T cells transfected with Netrin1-MYC were trypsinized , pelleted and resuspended in 900 µL of warm ( ~50°C ) 1% agarose dissolved in DMEM .\",\n \"Punches of agarose cell blocks containing HEK293T cells were prepared with a 0 . 35 mm diameter Harris Uni-Core tissue punch .\",\n \"To visualize retinal ganglion cell ( RGC ) axon projections through the optic chiasm , unilateral whole eye anterograde labeling with DiI ( Molecular Probes ) was performed on fixed tissue as described previously ( Kuwajima et al . , 2012 ) .\",\n \"Samples were incubated in PBS + 0 . 02% sodium azide for 14 days at 37°C .\",\n \"Ipsilateral versus contralateral projections were quantified by measuring the pixel intensity of ipsilateral and contralateral optic tracts in a 500 x 500 μm area lateral to the optic chiasm midline with the MetaMorph image analysis software .\",\n \"An ipsilateral index was calculated by dividing the intensity of the ipsilateral projection by the sum of the contralateral and ipsilateral pixel intensities as described previously ( Erskine et al . , 2011; Kuwajima et al . , 2012 ) .\",\n \"The ipsilateral index in Alfy null mice was normalized to the littermate WT ipsilateral index .\",\n \"The number of Ki67 positive cells labeled with DAB within the subventricular zone of the ganglionic eminence was determined by stereology using StereoInvestigator software ( MicrobrightField ) by an experimenter blind to genotype .\",\n \"For each animal , measurements were made at 60X bilaterally in five matched , adjacent sections spaced 200 μm apart .\",\n \"There were three animals per genotype .\",\n \"Timed pregnant dams were weighed and received an intraperitoneal injection with 50 mg/kg of BrdU at E15 . 5 .\",\n \"One hour following the injection , the dams were deeply anesthetized and the uterine horns were dissected out .\",\n \"Embryo heads were drop fixed in 4% PFA for 48 hr and then cryoprotected in 30% sucrose , embedded in OCT and sectioned at 25 µm .\",\n \"Sections were processed using an antibody against BrdU as described in the immunohistochemistry section .\",\n \"Statistical analyses were performed using Statview 5 . 0 ( SAS Institute ) .\",\n \"Normally distributed data were subject to student t-test , or for multiple comparisons , analysis of variance ( ANOVA ) followed by the appropriate post hoc comparisons as indicated in the figure legends .\",\n \"Complete F-statistics and calculated p-values are also available in the figure legends .\",\n \"Power analyses for quantitative studies were performed to achieve a power of 0 . 8 with a confidence of 0 . 95 using G*Power 3 . 1 ( Faul et al . , 2007 , 2009 ) .\",\n \"Effect sizes were based upon pilot studies &/or previously published or unpublished materials .\",\n \"n-values are indicative of biological replicates and no data were excluded from analyses .\"\n ]\n]"},"abstract":{"kind":"list like","value":["The regulation of protein degradation is essential for maintaining the appropriate environment to coordinate complex cell signaling events and to promote cellular remodeling .","The Autophagy linked FYVE protein ( Alfy ) , previously identified as a molecular scaffold between the ubiquitinated cargo and the autophagic machinery , is highly expressed in the developing central nervous system , indicating that this pathway may have yet unexplored roles in neurodevelopment .","To examine this possibility , we used mouse genetics to eliminate Alfy expression .","We report that this evolutionarily conserved protein is required for the formation of axonal tracts throughout the brain and spinal cord , including the formation of the major forebrain commissures .","Consistent with a phenotype reflecting a failure in axon guidance , the loss of Alfy in mice disrupts localization of glial guidepost cells , and attenuates axon outgrowth in response to Netrin-1 .","These findings further support the growing indication that macroautophagy plays a key role in the developing CNS ."],"string":"[\n \"The regulation of protein degradation is essential for maintaining the appropriate environment to coordinate complex cell signaling events and to promote cellular remodeling .\",\n \"The Autophagy linked FYVE protein ( Alfy ) , previously identified as a molecular scaffold between the ubiquitinated cargo and the autophagic machinery , is highly expressed in the developing central nervous system , indicating that this pathway may have yet unexplored roles in neurodevelopment .\",\n \"To examine this possibility , we used mouse genetics to eliminate Alfy expression .\",\n \"We report that this evolutionarily conserved protein is required for the formation of axonal tracts throughout the brain and spinal cord , including the formation of the major forebrain commissures .\",\n \"Consistent with a phenotype reflecting a failure in axon guidance , the loss of Alfy in mice disrupts localization of glial guidepost cells , and attenuates axon outgrowth in response to Netrin-1 .\",\n \"These findings further support the growing indication that macroautophagy plays a key role in the developing CNS .\"\n]"},"summary":{"kind":"list like","value":["Unlike many other cells in the body , neurons typically survive throughout the life of a mammal .","This long life suggests that they may be more vulnerable to damage from cellular debris .","Previous research has found that a protein called Alfy , which is abundant in the brain , is involved in cleaning up debris , such as those involved in neurodegenerative diseases , by a pathway known as autophagy .","Alfy guides the formation of spherical compartments called autophagosomes , which deliver the debris to another compartment known as the lysosome to permit degradation .","In developing embryos , neurons need to migrate to the right location within the central nervous system and extend projections called axons to communicate with other cells .","However , it was not clear whether this process requires cell materials to be selectively sent to lysosomes , and whether this involves the Alfy protein .","Dragich et al . addressed this question by studying mouse embryos that lack Alfy .","The brains of these mice developed abnormally and were missing the corpus callosum ( the dense band of fibers that normally connects the two halves of the brain ) .","Without Alfy , the growing axons could not navigate their way to the right places to connect with other neurons .","Furthermore , some neurons migrated to the wrong places in the developing brain , which resulted in the abnormal formation of cell-clusters .","The findings of Dragich et al . suggest that autophagy also plays an important role in normal brain development .","Future studies are now needed to work out exactly how Alfy controls neuron migration and the growth of axons .","The human gene WDFY3 is nearly identical to the gene that encodes the Alfy protein , and has been implicated in neurodevelopmental disorders such as autism and microencephaly .","Studying Alfy therefore may help us to understand human conditions that affect the developing or aging brain ."],"string":"[\n \"Unlike many other cells in the body , neurons typically survive throughout the life of a mammal .\",\n \"This long life suggests that they may be more vulnerable to damage from cellular debris .\",\n \"Previous research has found that a protein called Alfy , which is abundant in the brain , is involved in cleaning up debris , such as those involved in neurodegenerative diseases , by a pathway known as autophagy .\",\n \"Alfy guides the formation of spherical compartments called autophagosomes , which deliver the debris to another compartment known as the lysosome to permit degradation .\",\n \"In developing embryos , neurons need to migrate to the right location within the central nervous system and extend projections called axons to communicate with other cells .\",\n \"However , it was not clear whether this process requires cell materials to be selectively sent to lysosomes , and whether this involves the Alfy protein .\",\n \"Dragich et al . addressed this question by studying mouse embryos that lack Alfy .\",\n \"The brains of these mice developed abnormally and were missing the corpus callosum ( the dense band of fibers that normally connects the two halves of the brain ) .\",\n \"Without Alfy , the growing axons could not navigate their way to the right places to connect with other neurons .\",\n \"Furthermore , some neurons migrated to the wrong places in the developing brain , which resulted in the abnormal formation of cell-clusters .\",\n \"The findings of Dragich et al . suggest that autophagy also plays an important role in normal brain development .\",\n \"Future studies are now needed to work out exactly how Alfy controls neuron migration and the growth of axons .\",\n \"The human gene WDFY3 is nearly identical to the gene that encodes the Alfy protein , and has been implicated in neurodevelopmental disorders such as autism and microencephaly .\",\n \"Studying Alfy therefore may help us to understand human conditions that affect the developing or aging brain .\"\n]"},"year":{"kind":"string","value":"2016"}}},{"rowIdx":4298,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["short report","cancer biology"],"string":"[\n \"short report\",\n \"cancer biology\"\n]"},"title":{"kind":"string","value":"Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells"},"id":{"kind":"string","value":"elife-30224-v2"},"sections":{"kind":"list like","value":[["Amongst the most significant therapeutic breakthroughs in cancer has been the discovery of drug-sensitising genomic alterations .","Drugs such as the tyrosine kinase inhibitors ( TKIs ) developed against the BCR-ABL fusion product in chronic myeloid leukaemia ( CML ) and the receptor products of HER2 mutations in breast cancer have transformed the prognosis of these cancers ( Druker et al . , 2006 ) .","Malignant mesothelioma ( MM ) currently has no biomarker-driven therapies in routine clinical use .","The mainstay of medical therapy for all patients remains systemic cytotoxic chemotherapy that offers only limited survival benefit in unselected populations; as such the disease remains invariably fatal ( Vogelzang et al . , 2003 ) .","A plethora of genomic studies in MM has identified recurrent mutations in several genes considered to be tumour drivers .","CDKN2A , NF2 , BAP1 and TP53 are the most frequently mutated ( Guo et al . , 2015; Bueno et al . , 2016 ) and there has been increased focus on these genes and their associated signaling pathways as potential therapeutic targets ( LaFave et al . , 2015 ) .","We aimed to determine if the mutational status of these tumour driver genes could predict response to a range of existing anti-cancer compounds with a view to identifying genomic biomarkers for responsive subsets of MM .","We have previously reported on the ability of such unbiased high-throughput chemical screens in cancer cell lines to identify drug-sensitising mutations in other cancer types ( Garnett et al . , 2012 ) .","To this end , we conducted a high-throughput chemical screen of molecularly characterised MM cell lines seeking associations between MM driver gene mutations and compound response .","This strategy led to the discovery of a subset of MM cell lines defined by loss-of-function ( LOF ) mutations in BRCA associated protein-1 ( BAP1 ) that demonstrated heightened sensitivity to the death receptor agonist recombinant tumour necrosis factor ( TNF ) -related apoptosis-inducing ligand ( rTRAIL ) .","We validated this finding using in vitro , in vivo and ex vivo models supporting the use of BAP1 as a genomic biomarker to identify TRAIL-sensitive MM tumours and a novel stratified approach to treat MM . rTRAIL and other death receptor agonists selectively induce apoptosis in cancer cells and have long held promise as anti-cancer agents owing to their broad clinical utility and minimal off-target effects ( Wiley et al . , 1995; Pitti et al . , 1996; Ashkenazi et al . , 1999 ) .","Despite this , successful preclinical studies have not translated to clinical efficacy in trials of unselected populations ( Herbst et al . , 2010; Wainberg et al . , 2013; Soria et al . , 2010; Lemke et al . , 2014a ) ; there have been no trials to date in MM .","However , within these trials some patients showed signs of therapeutic benefit and differential sensitivity within cell lines is well known .","Retrospective biomarker identification has led to the stratified use of other anti-cancer therapies that initially failed in unselected trials such as activating EGFR mutations and EGFR TKIs ( Lynch et al . , 2004 ) .","We propose that BAP1 could potentially act as such a biomarker for the death receptor agonists .","BAP1 is a nuclear deubiquitinase and forms multi-protein complexes that regulate the transcription of genes involved in key cellular functions including cell cycle regulation and DNA repair ( Ismail et al . , 2014; Machida et al . , 2009 ) .","We investigated which BAP1 protein-binding partners , and thus which regulatory complexes , mediate TRAIL sensitivity identifying the BAP1-ASXL1 complex , the Polycomb repressive deubiquitinase ( PR-DUB ) , as key .","We further found that loss of BAP1 function modulates mRNA and protein expression of components of the extrinsic apoptotic pathway ."],["A 6 day viability screen using 94 drugs including small molecule inhibitors and cytotoxic chemotherapeutics ( Supplementary file","1 ) was performed on 15 MM cell lines ( Supplementary file","2 ) that had been characterised using whole-exome sequencing , copy number analysis and gene expression arrays .","We generated 1425 single agent activity data profiles across the 15 cell lines ( Figure 1A and Supplementary file 3 ) .","To detect novel markers of drug sensitivity , we sought statistical associations between drug response and the mutational status of the cell lines based on five genes identified as candidate drivers of tumourigenesis in MM ( Guo et al . , 2015 ) ( Figure 1—figure supplement 1 ) .","There were 24 significant associations ( false discovery rate ( FDR ) < 0 . 2 ) between single agent response and the presence of a genomic alteration .","The most statistically significant sensitising association seen was between BAP1 LOF mutations ( mt BAP1 ) and treatment with recombinant TRAIL ( rTRAIL; FDR = 0 . 18 , effect size −0 . 48 ) ( Figure 1B , C and Supplementary file 4 ) .","No significant effect on cell viability was observed in BAP1 wild-type ( wt BAP1 ) lines when treated with rTRAIL .","We subsequently confirmed this association in a larger panel of MM cell lines ( Figure 1D and Supplementary file 5 ) .","Strikingly , 6 of the 8 cell lines ( 75% ) harbouring a BAP1 LOF mutation were sensitive or partially sensitive to a dose range of rTRAIL while 7 of the 9 cell lines ( 78% ) harbouring wild-type BAP1 were resistant .","BAP1 LOF mutations correlated with a loss of BAP1 protein expression in the majority of cell lines ( Figure 1E ) .","No sensitising association with BAP1 was observed for pemetrexed or cisplatin , which are current first line agents for the treatment of MM ( Figure 1—figure supplement 2A and B ) .","A marginal trend towards increased sensitivity in BAP1 mutant MM lines in response to treatment with the agonistic FAS receptor antibody CH11 and a TNF-α/IAP inhibitor combination was observed .","However , this was not as pronounced as that observed with rTRAIL or the multivalent death receptor five superagonist MEDI3039 ( Figure 1—figure supplement 2C , D and E ) .","Thus , while the significant sensitising association observed in the screen appears most specific to death receptor agonists , the trend observed with other TNF superfamily agonists indicates the BAP1-rTRAIL association to be mediated by an underlying mechanism common to this family such as the cytoplasmic extrinsic apoptotic machinery .","To determine if knockdown of BAP1 in wild-type MM cells led to TRAIL sensitivity , we silenced BAP1 expression in four wt BAP1 MM cell lines using a lentiviral shRNA construct .","Knockdown of BAP1 resulted in increased cell death following rTRAIL treatment compared with empty vector ( EV ) control shRNA and the parental cell line in all four MM cell lines ( Figure 2A and Figure 2—figure supplement 1B and C ) .","Loss of BAP1 expression has also been identified in several other tumour types including uveal melanoma ( 47% ) ( Harbour et al . , 2010 ) , clear cell renal carcinoma ( CCRC ) ( 14% ) ( Peña-Llopis et al . , 2012 ) and cholangiocarcinoma ( 7% ) ( Fujimoto et al . , 2015 ) .","Notably , knockdown of BAP1 in two CCRC lines resulted in increased sensitivity to rTRAIL in addition to the MDAMB-231 breast cancer line ( Figure 2B and Figure 2—figure supplements 2 and 3 ) .","We also analysed a panel of 1001 cancer cell lines submitted for whole exome and copy number analysis as part of the COSMIC cell lines project ( Forbes et al . , 2015 ) and identified nine additional non-mesothelioma cell lines harbouring truncating mutations in BAP1 ( http://cancer . sanger . ac . uk/cancergenome/projects/cell_lines/ ) .","These include CCRC , bladder and breast cancer lines .","Treatment of cancer cell lines harbouring nonsense mutations in BAP1 with rTRAIL resulted in markedly reduced cell viability compared with cancer cell lines harbouring missense mutations ( Figure 2—figure supplement 4 ) .","BAP1 is a nuclear deubiquitinase that forms multi-protein complexes with transcription factors to regulate gene transcription ( Jensen et al . , 1998; Ventii et al . , 2008 ) .","To elucidate the mechanism by which BAP1 modulates sensitivity to TRAIL we generated expression vectors containing wild-type or mutant forms of BAP1 , each with an inactive functional site or protein-binding domain .","These included C91A ( mutation in the deubiquitination catalytic site ) ( Jensen et al . , 1998; Ventii et al . , 2008 ) , ΔHBM ( deletion of the HCF-1-binding site ) ( Misaghi et al . , 2009 ) , T493A ( mutation in the FOXK2-binding site ) ( Ji et al . , 2014 ) , ΔASXL ( deletion of the ASXL1/2 protein-binding site ) ( Daou et al . , 2015 ) and ΔCTD ( deletion of the C-terminal domain containing the nuclear localisation signal ) ( Ventii et al . , 2008 ) .","H226 MM cells , which harbour a homozygous deletion of BAP1 and demonstrate complete loss of BAP1 expression , were transduced with a GFP ( vector control ) , a wild-type BAP1 expression vector or one of these five mutant BAP1 expression vectors .","rTRAIL sensitivity of the parental BAP1-null H226 MM line was significantly diminished following expression of wild-type BAP1 and each of the mutant constructs except those with an inactive deubiquitinating or ASXL protein-binding site ( Figure 2C ) , implicating the function of these sites in BAP1-induced TRAIL resistance .","These effects were replicated using MEDI3039 ( Figure 2D ) .","Transduction of two further BAP1-mutant rTRAIL-sensitive cell lines , H28 and H2804 , with wild-type BAP1 also induced resistance to rTRAIL while sensitivity was maintained with transduction of the deubiquitinase mutant ( Figure 2—figure supplement 5 ) .","The BAP1 deubiquitinase and ASXL-binding sites are key to the function of the PR-DUB , an epigenetic transcriptional regulatory complex composed of BAP1 and ASXL1 .","Deubiquitination of the main substrate of the PR-DUB , H2AK119Ub , alters chromatin architecture to modulate gene transcription ( Scheuermann et al . , 2010 ) .","This led us to hypothesise that PR-DUB , rather than exclusively BAP1 , function might underlie rTRAIL sensitivity .","Consistent with this shRNA silencing of ASXL1 , but not ASXL2 , induced sensitivity to MEDI3039 and rTRAIL in the BAP1/ASXL1/ASXL2-wild-type MM line MPP-89 ( Figure 2E and Figure 2—figure supplement 6 ) .","Furthermore , H2AK119Ub expression was unaltered in the rTRAIL-sensitive H226 cells transduced with mutant constructs that disrupt PR-DUB activity , while the rTRAIL-resistant H226 cells transduced with a wild-type BAP1 construct exhibited lower H2AK119Ub levels ( Figure 2—figure supplement 7 ) .","Thus , as the PR-DUB complex is implicated in transcriptional regulation , differential modulation of specific transcriptional programmes by BAP1 may determine rTRAIL sensitivity .","We therefore compared differential gene expression data from BAP1-null H226 cells transduced with the C91A BAP1 mutant or with wild-type BAP1 and carried out a signalling pathway impact analysis ( SPIA ) ( ( Figure 2—figure supplements 8 and 9 [SPIA_H226 C91A mutant vs WT] ) ( http://www . genome . jp/dbget-bin/www_bget ? path:map04210 ) .","Among those pathways significantly altered when comparing wild-type versus C91A BAP1 ( FDR < 0 . 2 ) was that of apoptosis .","In particular , there was altered mRNA expression of components of the extrinsic death pathway ( Figure 2F and Supplementary file 6 ) .","This manifested as an imbalance in levels of pro- and anti-apoptotic mRNA expression with , for example , significantly decreased levels of the anti-apoptotic cIAP1/2 ( p=2 . 32E-10 ) and increased levels of the pro-apoptotic death receptor 5 ( p=7 . 79E-10 ) in the rTRAIL sensitive C91A BAP1-transduced cells relative to the rTRAIL resistant BAP1-wild-type transduced cells .","Immunoblot analysis confirmed reduced protein expression of cIAP1/2 and c-FLIP in both C91A and ΔASXL BAP1-transduced cells relative to BAP1-wild-type transduced cells ( Figure 2G ) .","Flow cytometry analysis confirmed reduced DR4 and DR5 expression in C91A BAP1 transduced relative to BAP1-wild-type-transduced cells .","Knockdown of BAP1 in the BAP1 wild-type H2818 line resulted in a significant increase in DR4 expression only ( Figure 2H ) .","To support the clinical relevance of our finding we extended our assays to two further models derived from primary tumour tissue .","25 human early passage MM lines from the UK Mesobank ( Rintoul et al . , 2016 ) were assessed for BAP1 expression by immunohistochemistry , a technique known to correlate strongly with BAP1 LOF mutations in the absence of strong nuclear staining ( Nasu et al . , 2015 ) .","When treated with rTRAIL , those without strong nuclear staining were significantly more sensitive than those with strong nuclear staining ( p=0 . 0067 ) .","Of the 12 lines that did not express nuclear BAP1 9 were sensitive , 2 partially sensitive and only one resistant to rTRAIL ( Table 1 , Figure 3A and Figure 3—figure supplement 1 ) .","Remarkably , rTRAIL treatment of tumour explants derived from three patients with MM also revealed increased levels of apoptosis ( as measured by poly ( ADP-ribose ) polymerase ( PARP ) cleavage ) in explants with low BAP1 expression compared with those with high BAP1 expression ( Figure 3B and C , Figure 3—figure supplement 2 ) .","To test the in vivo efficacy of TRAIL in inducing apoptosis in BAP1-mutant MM cells , we transduced the H226 BAP1-wild-type and the H226 C91A BAP1-mutant cell lines with luciferase and injected equal numbers of wild-type and mutant cells into the opposite flanks of mice ( Figure 3—figure supplement 3A ) .","On day 14 after injection the mice were divided into two groups and injected intraperitoneally with rTRAIL or vehicle for 6 days per week until day 40 .","At sacrifice rTRAIL-treated BAP1-mutant tumours weighed significantly less than rTRAIL-treated BAP1-wild-type tumours ( p=0 . 020 ) and vehicle-treated BAP1-mutant tumours ( p=0 . 019 ) ( Figure 3D and Figure 3—figure supplement 3B ) .","BAP1-wild-type tumours showed no response to rTRAIL compared with vehicle .","The growth rate of rTRAIL-treated BAP1-mutant tumours was also significantly suppressed compared with rTRAIL-treated BAP1-wild-type and vehicle-treated tumours ( p<0 . 05 ) as assessed by longitudinal bioluminescence intensity ( Figure 3E and F ) ."],["Malignant mesothelioma remains a devastating disease with limited systemic treatment options ( Vogelzang et al . , 2003 ) .","Biomarker-driven therapies have significantly improved the prognosis for subsets of patients within other cancer types however this strategy has yet to impact MM .","Our data support the use of loss of function of BAP1 as a genomic stratification tool to identify rTRAIL-sensitive MM tumours , an approach that may extend to other cancer subtypes .","We propose the underlying mechanism involves the transcriptional regulation of expression of components of the apoptotic pathway by the PR-DUB .","Our finding has potentially significant and immediately actionable clinical implications for both MM treatment and for the death receptor agonist field .","BAP1 has emerged as a key driver of tumorigenesis in MM ( Bueno et al . , 2016 ) .","As such , there has been increased focus on this nuclear deubiquitinase and its associated pathways ( LaFave et al . , 2015 ) .","While next-generation sequencing reveals MM BAP1 mutation rates in the order of 20–30% ( Guo et al . , 2015; Bueno et al . , 2016; Bott et al . , 2011 ) , immunohistochemical analysis has identified loss of BAP1 function in up to 67% of MM tumours ( Nasu et al . , 2015 ) opening our biomarker-driven approach to a significant proportion of MM patients .","BAP1 immunohistochemistry accurately identifies loss of BAP1 function as a consequence of genetic and non-genetic mechanisms ( Nasu et al . , 2015 ) and is already in clinical use as a diagnostic tool; hence the clinical tools for our proposed approach are validated and ready .","Our data indicate the BAP1-TRAIL association extends beyond MM to other tumours with loss of BAP1 function .","Chromosomal deletions and somatic inactivating mutations have been identified at high frequency in uveal melanoma ( Harbour et al . , 2010 ) , clear cell renal carcinoma ( Peña-Llopis et al . , 2012 ) and cholangiocarcinoma ( Fujimoto et al . , 2015 ) , increasing the potential clinical impact of our discovery .","Although loss of BAP1 function is seen at far lower rates in breast carcinoma ( 1% ) ( Stephens et al . , 2012 ) and non-small cell lung carcinoma ( 1% ) ( Owen et al . , 2017 ) , the high incidence of these cancers translates to a large cohort of patients .","Focus on death receptor agonists as anti-cancer agents has generated two decades of preclinical studies and the development of numerous clinically tested compounds , all of which have demonstrated limited therapeutic efficacy at phase I/II trials ( Herbst et al . , 2010; von Pawel et al . , 2014; Paz-Ares et al . , 2013; Forero-Torres et al . , 2013 ) .","Strategies to overcome this have included the development of increasingly potent death receptor agonists and combination therapies to address resistance factors within the apoptosis pathway ( Holland , 2013; Lemke et al . , 2014b ) .","As differential sensitivity has been observed in trials , it has been accepted that identification of a biomarker predicting the therapeutic outcome is of paramount importance ( Ashkenazi , 2015; von Karstedt et al . , 2017 ) .","There have been previous attempts to identify predictive biomarkers largely focused on molecular expression panels ( Passante et al . , 2013 ) .","Ours is the first unbiased approach to address how the genetic make-up of tumours predicts response to rTRAIL treatment .","The identification of BAP1 as a potential genomic biomarker has the potential to reignite the death receptor agonist field of research into which significant investment has already been made .","The value of retrospective analysis of clinical trials based on the genomic landscape has clearly been demonstrated in the past ( Lynch et al . , 2004 ) and we wait with interest whether this will be performed on archived tumour tissue , in the context of BAP1 status , from previous trials .","Notably there have been no trials of any death receptor agonists in MM or indeed any cancer with a high proportion of BAP1 mutations .","We suspect a significantly higher proportion of responders would have been identified in such trials .","Our findings also have implications for death receptor agonists as a therapy for BAP1-wild-type tumours as delineation of the underlying mechanism would offer a novel avenue by which to sensitise these tumours .","Our mechanistic data implicate transcriptional regulation by the PR-DUB as key to the capacity of BAP1 to modulate death receptor agonist sensitivity .","BAP1 is a master genetic regulator and is known to influence the transcription of thousands of genes as supported by our and others’ gene expression data ( Dey et al . , 2012 ) .","While we highlight the extrinsic apoptotic pathway and proteins as being significantly altered by BAP1 status , identifying a single factor to explain BAP1-induced TRAIL resistance is extremely challenging .","Of more direct clinical significance is our finding that loss of function of either component of the PR-DUB , BAP1 or ASXL1 , results in an increase in death receptor agonist sensitivity .","ASXL1 mutations have an important role in the pathogenesis of myeloid neoplasms primarily consisting of nonsense , missense and frameshift mutations resulting in a truncated ASXL1 protein that retains the BAP1-binding domain ( Boultwood et al . , 2010 ) .","It has yet to be clarified if this truncated protein possesses dominant-negative or gain-of-function properties in the context of PR-DUB activity ( Balasubramani et al . , 2015 ) .","In the case of the former , ASXL1 could potentially predict death receptor agonist sensitivity in myeloid neoplasms .","Further research is needed in these malignancies to determine this .","Confirmation of the clinical value of BAP1 as a targeting biomarker for death receptor agonists in early phase clinical trials of mesothelioma is the first priority .","The clinical tools for this approach are already validated and established facilitating the translation of our discovery into a desperately needed new therapy for this fatal thoracic cancer ."],["All cell lines were sourced from the Wellcome Trust Sanger Institute except the H226 line that was a kind gift from Dr Peter Szlosarek , Barts Cancer Institute .","All cell lines were authenticated by genotyping using Short Tandem Repeat ( STR ) and Sequenom profiling of a panel of 92 single nucleotide polymorphisms for each cell line to ensure non-synonymous cell lines were not used .","As a cell line classified as mesothelioma , H513 ( on the list of commonly misidentified cell lines ) was included in the drug screen of 15 mesothelioma cell lines conducted .","Use of this cell line however was not carried forward to further experiments in the paper .","The 25 early passage MM cultures were purchased from MesobanK ( Rintoul et al . , 2016 ) .","All cell lines and cultures were tested for mycoplasma contamination and confirmed to be negative .","Cell lines were cultured in RPMI-1640 or Dulbecco's modified Eagle's medium and nutrient mix 12 medium ( DMEM:F12 ) supplemented with 10% fetal bovine serum ( FBS ) , penicillin/streptavidin and sodium pyruvate .","Early passage human mesothelioma cultures were cultured in RPMI-1640 medium supplemented with 5% FBS , 25 mM HEPES , penicillin/streptavidin and sodium pyruvate .","293 T cells were cultured in Dulbecco's modified Eagle's medium ( DMEM ) supplemented with 10% fetal bovine serum ( FBS ) and 2 mM L-glutamine .","All cells were maintained in a humidified environment at 37°C and 5% CO2 .","Cells were lysed in radioimmunoprecipitation assay ( RIPA ) buffer ( Sigma-Aldrich , St . Louis , MO ) with protease inhibitors ( Complete-mini; Roche , Switzerland ) on ice to extract protein .","20 μg of protein samples were separated by SDS–PAGE and transferred onto nitrocellulose membranes .","Membranes were incubated with specific primary antibodies , washed , incubated with secondary antibodies and visualised using an ImageQuant LAS 4000 imaging system ( GE Healthcare , Little Chalfont , NY ) .","Antibodies used include BAP1 ( Santa Cruz Biotechnology , Santa Cruz , CA ) Cat# sc-28383 , RRID:AB_626723 ) , caspase 8 ( Cell Signaling Technology , Danvers , MA ) Cat# 9746 , RRID:AB_2275120 ) , c-FLIP ( Enzo Life Sciences , Farmingdale , NY ) Cat# ALX-804-961-0100 RRID:AB_2713915 ) , cIAP1 ( Cell Signaling Technology Cat# 7065S , RRID:AB_10890862 ) , cIAP2 ( Cell Signaling Technology Cat# 3130S , RRID:AB_10693298 ) , FADD ( Cell Signaling Technology Cat# 2782 , RRID:AB_2100484 ) , XIAP ( Cell Signaling Technology Cat# 2045 , RRID:AB_2214866 ) , survivin ( Cell Signaling Technology Cat# 2803 , RRID:AB_490807 ) , α-tubulin ( Cell Signaling #2125 ) , H2AK119Ub ( Cell Signaling Technology Cat# 8240P , RRID:AB_10891618 ) , H2A ( Cell Signaling Technology Cat# 12349 , RRID:AB_2687875 ) , anti-mouse HRP ( Cell Signaling Technology Cat# 7076 , RRID:AB_330924 ) and anti-rabbit HRP ( Cell Signaling Technology Cat# 7074 , RRID:AB_2099233 ) .","To detect the ubiquitination status of the histones , the cells were lysed with TBS buffer containing 1% SDS , protease and phosphatase inhibitors .","The cell extract was denatured by heating up at 95°C for 10 min and centrifuged at 13000 rpm for 10 min .","The supernatant was collected and immunoblotted as described above .","Cells were seeded in 96-well plates in 100 μl media per well at a density of 40 , 000 cells/ml 1 day prior to treatment with soluble recombinant TRAIL ( rTRAIL; Peprotech , UK ) or MEDI3039 ( Medimmune , UK ) .","XTT ( Applichem , UK; A8088 ) or MTT ( M-2128 , Sigma-Aldrich ) reagent was added on day 3 .","The absorbance was measured with a spectrophotometer at a wavelength of 490 nm or 560 nm for XTT or MTT respectively .","Relative cell viability was calculated as a fraction of viable cells relative to untreated cells .","Full-length BAP1 cDNA was amplified by PCR from pCMV6-AC BAP1 plasmid ( Origene ( Rockville , MD; SC117256 ) and cloned into the lentiviral plasmid pCCL-CMV-flT vector previously described ( Yuan et al . , 2015 ) in place of flT via BamHI and SalI sites , creating the BAP1 vector designated pCCL-CMV-BAP1 .","Vectors expressing mutant BAP1 constructs were generated by site-directed mutagenesis ( New England Biolabs ) of the pCCL-CMV-BAP1 vector .","The primers used are listed below .","All mutations were confirmed by sequencing .","BAP1-F CGTGGATCCGCCACCATGAATAAGGGCTGGCTGGA BAP1-R GTCGGTCGACTCACTGGCGCTTGGCCTTGTA C91A-F ATACCCAACTCTGCTGCAACTCATGCCTTGCTG C91A-R CAGCTGGTGGGCAAAGAACATGTTATTCACAATATCATC HBM-F CGCTGCTGCCAAGTCCCCCATGCAGGAGGA HBM-R GCAGCGTCTAGAAAGGCCGGCAGCCGCT CTD-F CGTGGATCCGCCACCATGAATAAGGGCTGGCTGGA CTD-R GTCGTTCGAATCAGTCAGGCTTCCGCTGCTTGTGG T493A-F GCAGACACGGCCTCTGAGATCGGCAGTGCT T493A-R ACTCTCATTGCTGGGGGTGGGTGA ASXL-F AACTACGATGAGTTCATCTGCACCT ASXL-R CTGGTCATCAATCTTGAACTTCTTCCTC The ZS-green luciferase plasmid , pHIV-Luc-ZsGreen ( a gift from Bryan Welm , Addgene plasmid #39196 ) was used for generating ZS-Green luciferase-expressing lentivirus to transduce the H226 cells used in animal experiments .","Short hairpin RNAs ( shRNAs ) were expressed as part of a mir30-based GIPZ lentiviral vector ( Dharmacon , Lafayette , CO ) .","The clones used in this study include BAP1 ( V2LHS_41473 ) , ASXL1 ( V2LHS_78829 ) , ASXL2 ( V3LHS_313940 ) and the empty GIPZ control vector .","Lentiviral vectors were produced by co-transfection of 293 T cells with construct plasmids together with the packaging plasmids pCMV-dR8 . 74 and pMD2 . G ( kind gifts from Dr Adrian Thrasher , UCL , Addgene plasmid #22036 and #12259 ) in the presence of a DNA transfection reagent jetPEI ( Source Bioscience UK Ltd ) .","Lentiviruses were concentrated by ultracentrifugation at 17 , 000 rpm ( SW28 rotor , Optima LE80K Ultracentrifuge , Beckman Coulter , Brea , CA ) for 2 hr at 4°C .","To determine the titres of prepared lentiviruses 293 T cells were transduced with serial dilutions of viruses in the presence of 8 μg/ml Polybrene ( Sigma-Aldrich ) and BAP1 expression was assessed by flow cytometry .","shRNA- and luciferase-expressing vectors were assessed by analysis of GFP expression .","Cell lines were transduced in the presence of 8 μg/ml Polybrene at a range of MOIs and transduction efficacy was assessed by flow cytometry for BAP1 expression .","We pre-processed and normalised raw CEL files from Affymetrix Human Genome U219 array plate hybridisations with the Multi-Array Average ( RMA ) method ( Irizarry et al . , 2003 ) .","We discarded transcripts with low sample variance and consolidated duplicated genes by averaging their expression values across duplicates .","The resulting data were subsequently normalised ( μ = 0 , σ = 1 ) sample-wise and gene-median centred .","Gene expression was averaged across three biological replicates of H226 transduced cells with either a C91A mutant or a wild-type BAP1 construct .","SPIA pathway analysis as described in Tarca et al ( Tarca et al . , 2009 ) was performed on those genes with an adjusted p<0 . 05 and a fold change of >1 .","All flow cytometry analysis was performed on a LSR Fortessa analyser ( Becton Dickinson , Franklin Lakes , NJ ) .","For analysis of BAP1 expression cells were stained with primary antibody to BAP1 ( Santa Cruz Biotechnology Cat# sc-28383 , RRID:AB_626723; 1:50 ) and then with an AlexaFluor 488-conjugated anti-mouse antibody ( Thermo Fisher Scientific Cat# A-21202 , RRID:AB_141607; 1:200 ) .","For analysis of apoptosis and cell death all floating and adherent cells were harvested and stained with an Annexin V AlexaFluor 647-conjugated antibody ( Thermo Fisher Scientific Cat# A23204 , RRID:AB_2341149 ) and 4’ , 6-diamidino-2-phenylindole ( DAPI; Sigma-Aldrich , 200 μg/ml ) .","For analysis of DR4 and DR5 expression on cell surface cells were stained with PE-conjugated antibody ( DR4 - BioLegend , San Diego , CA ) Cat# 307205 , RRID:AB_314669 , DR5 - BioLegend Cat# 307405 , RRID:AB_314677 , Isotype control - Biolegend #400112; 1:100 ) .","FlowJo software was used to analyse all data .","H226 cells were seeded at 2 . 5 × 103 cells per well into 96-well Greiner micro-clear imaging plates in DMEM 10% FBS .","After 48 hr , cells were fixed in 4% PFA for 10 min at room temperature and permeabilised in 0 . 3% NP-40 in PBS for 10 min .","Cells were blocked in 1% BSA in 0 . 1% PBS tween for 1 hr at room temperature .","Ubiquityl-histone H2A ( Lys119 ) primary antibody ( Cell Signaling , #8240 ) was incubated overnight at 4°C , before incubating for 1 hr at room temperature with Alexafluor 488-conjugated anti-rabbit secondary antibody .","Nuclei were stained with Hoechst 33342 ( Thermo Fisher Scientific Cat# 62249 ) .","Images were acquired ( n = 3 ) with a BioTek Cytation3 Multimode reader .","Using a 10x objective 4 fields of view were acquired per well ( n = 3 ) and the level of nuclear ubiquityl-histone H2A intensity was determined within the primary nuclear mask and normalised to total cell number .","BAP1 immunohistochemistry of human early passage cell lines was conducted on sections of cell pellets mounted on slides .","Automated staining on a Leica Bond III staining platform was used .","Slides were incubated with BAP1 primary antibody ( Santa Cruz Biotechnology Cat# sc-28383 , RRID:AB_626723; 1:150 ) for 15 min at room temperature .","Epitope retrieval was completed using HIER using Leica Bond ER2 ( high pH ) for 30 min and a Leica Bond Polymer Refine with DAB chromogen detections system used .","Appropriate ethical approval was obtained from the NHS Health Research Authority National Research Ethics Service to carry out this work ( reference 14/LO/1527 ) .","Informed consent to conduct research on samples collected and to publish results was obtained from patients .","The diagnosis of mesothelioma was confirmed histologically for all patients prior to consent and surgery .","Patients underwent pleurectomy , following which primary pleural tissue was sectioned into fragments measuring approximately 2 mm3 .","These tissue explants were cultured in 50% neurobasal and 50% DMEM:F12 , supplemented with B27 ( 2% ) , EGF ( 20 ng/ml ) and FGF ( 10 ng/ml ) .","After 24 hr the explants were treated with rTRAIL ( vehicle , 50 ng/ml , 100 ng/ml or 200 ng/ml ) for a further 24 hr , following which explants were either fixed for PARP immunohistochemistry .","The explants were fixed in 10% neutral-buffered formalin ( NBF ) for 24 hr and then transferred into 70% ethanol followed by paraffin embedding .","Subsequently , 5 μm sections were used for immunohistochemistry , as previously described ( Busacca et al . , 2016 ) .","All animal studies were approved by the University College London Biological Services Ethical Review Committee and licensed under the UK Home Office regulations and the Evidence for the Operation of Animals ( Scientific Procedures ) Act 1986 ( Home Office , London , UK ) .","Mice were purchased from Charles River , kept in individually ventilated cages under specific pathogen-free conditions and had access to sterile irradiated food and autoclaved water ad libitum .","12 8 week old NOD . CB17-Prkdcscid/NcrCrl ( NOD SCID ) mice ( Charles River , UK; RRID:IMSR_CRL:394 ) were injected with 1 × 106 H226 cells transduced with a plasmid containing wild-type BAP1 and luciferase on the right flank and with a plasmid containing a catalytically inactive BAP1-mutant ( C91A ) and luciferase on the left flank in a 1:1 mixture of Matrigel ( Corning , Corning , NY ) and medium .","Tumour size was assessed by bioluminescence in vivo imaging system ( IVIS , PerkinElmer , Waltham , MA ) 15 min following intraperitoneal injection of 0 . 2 ml ( 2 mg ) luciferin .","Tumours were allowed to establish for 2 weeks prior to baseline assessment of size at day 13 .","Mice were then divided into two groups each of which received either 600 μg TRAIL or vehicle 6 days a week from day 14 until day","40 . Bioluminescence was measured on days 0 , 13 , 19 , 26 and","41 . Mice were sacrificed on day 42 and tumours harvested for measurement .","TRAIL used in the mouse experiment was made in Henning Walczak’s laboratory as per the established protocol ( Ganten et al . , 2006 ) .","Statistical analysis was performed using GraphPad Prism ( GraphPad Software , CA , USA ) .","t-test was used to analyse differences between two groups whilst the analysis of variance ( ANOVA ) test with a Tukey post-hoc analysis was used to analyse differences between three groups .","For multiple groups measured over multiple time points repeated measures ANOVA was used .","All in vitro tests were performed in triplicate and all data are represented as mean values ± standard error of mean unless otherwise stated ."]],"string":"[\n [\n \"Amongst the most significant therapeutic breakthroughs in cancer has been the discovery of drug-sensitising genomic alterations .\",\n \"Drugs such as the tyrosine kinase inhibitors ( TKIs ) developed against the BCR-ABL fusion product in chronic myeloid leukaemia ( CML ) and the receptor products of HER2 mutations in breast cancer have transformed the prognosis of these cancers ( Druker et al . , 2006 ) .\",\n \"Malignant mesothelioma ( MM ) currently has no biomarker-driven therapies in routine clinical use .\",\n \"The mainstay of medical therapy for all patients remains systemic cytotoxic chemotherapy that offers only limited survival benefit in unselected populations; as such the disease remains invariably fatal ( Vogelzang et al . , 2003 ) .\",\n \"A plethora of genomic studies in MM has identified recurrent mutations in several genes considered to be tumour drivers .\",\n \"CDKN2A , NF2 , BAP1 and TP53 are the most frequently mutated ( Guo et al . , 2015; Bueno et al . , 2016 ) and there has been increased focus on these genes and their associated signaling pathways as potential therapeutic targets ( LaFave et al . , 2015 ) .\",\n \"We aimed to determine if the mutational status of these tumour driver genes could predict response to a range of existing anti-cancer compounds with a view to identifying genomic biomarkers for responsive subsets of MM .\",\n \"We have previously reported on the ability of such unbiased high-throughput chemical screens in cancer cell lines to identify drug-sensitising mutations in other cancer types ( Garnett et al . , 2012 ) .\",\n \"To this end , we conducted a high-throughput chemical screen of molecularly characterised MM cell lines seeking associations between MM driver gene mutations and compound response .\",\n \"This strategy led to the discovery of a subset of MM cell lines defined by loss-of-function ( LOF ) mutations in BRCA associated protein-1 ( BAP1 ) that demonstrated heightened sensitivity to the death receptor agonist recombinant tumour necrosis factor ( TNF ) -related apoptosis-inducing ligand ( rTRAIL ) .\",\n \"We validated this finding using in vitro , in vivo and ex vivo models supporting the use of BAP1 as a genomic biomarker to identify TRAIL-sensitive MM tumours and a novel stratified approach to treat MM . rTRAIL and other death receptor agonists selectively induce apoptosis in cancer cells and have long held promise as anti-cancer agents owing to their broad clinical utility and minimal off-target effects ( Wiley et al . , 1995; Pitti et al . , 1996; Ashkenazi et al . , 1999 ) .\",\n \"Despite this , successful preclinical studies have not translated to clinical efficacy in trials of unselected populations ( Herbst et al . , 2010; Wainberg et al . , 2013; Soria et al . , 2010; Lemke et al . , 2014a ) ; there have been no trials to date in MM .\",\n \"However , within these trials some patients showed signs of therapeutic benefit and differential sensitivity within cell lines is well known .\",\n \"Retrospective biomarker identification has led to the stratified use of other anti-cancer therapies that initially failed in unselected trials such as activating EGFR mutations and EGFR TKIs ( Lynch et al . , 2004 ) .\",\n \"We propose that BAP1 could potentially act as such a biomarker for the death receptor agonists .\",\n \"BAP1 is a nuclear deubiquitinase and forms multi-protein complexes that regulate the transcription of genes involved in key cellular functions including cell cycle regulation and DNA repair ( Ismail et al . , 2014; Machida et al . , 2009 ) .\",\n \"We investigated which BAP1 protein-binding partners , and thus which regulatory complexes , mediate TRAIL sensitivity identifying the BAP1-ASXL1 complex , the Polycomb repressive deubiquitinase ( PR-DUB ) , as key .\",\n \"We further found that loss of BAP1 function modulates mRNA and protein expression of components of the extrinsic apoptotic pathway .\"\n ],\n [\n \"A 6 day viability screen using 94 drugs including small molecule inhibitors and cytotoxic chemotherapeutics ( Supplementary file\",\n \"1 ) was performed on 15 MM cell lines ( Supplementary file\",\n \"2 ) that had been characterised using whole-exome sequencing , copy number analysis and gene expression arrays .\",\n \"We generated 1425 single agent activity data profiles across the 15 cell lines ( Figure 1A and Supplementary file 3 ) .\",\n \"To detect novel markers of drug sensitivity , we sought statistical associations between drug response and the mutational status of the cell lines based on five genes identified as candidate drivers of tumourigenesis in MM ( Guo et al . , 2015 ) ( Figure 1—figure supplement 1 ) .\",\n \"There were 24 significant associations ( false discovery rate ( FDR ) < 0 . 2 ) between single agent response and the presence of a genomic alteration .\",\n \"The most statistically significant sensitising association seen was between BAP1 LOF mutations ( mt BAP1 ) and treatment with recombinant TRAIL ( rTRAIL; FDR = 0 . 18 , effect size −0 . 48 ) ( Figure 1B , C and Supplementary file 4 ) .\",\n \"No significant effect on cell viability was observed in BAP1 wild-type ( wt BAP1 ) lines when treated with rTRAIL .\",\n \"We subsequently confirmed this association in a larger panel of MM cell lines ( Figure 1D and Supplementary file 5 ) .\",\n \"Strikingly , 6 of the 8 cell lines ( 75% ) harbouring a BAP1 LOF mutation were sensitive or partially sensitive to a dose range of rTRAIL while 7 of the 9 cell lines ( 78% ) harbouring wild-type BAP1 were resistant .\",\n \"BAP1 LOF mutations correlated with a loss of BAP1 protein expression in the majority of cell lines ( Figure 1E ) .\",\n \"No sensitising association with BAP1 was observed for pemetrexed or cisplatin , which are current first line agents for the treatment of MM ( Figure 1—figure supplement 2A and B ) .\",\n \"A marginal trend towards increased sensitivity in BAP1 mutant MM lines in response to treatment with the agonistic FAS receptor antibody CH11 and a TNF-α/IAP inhibitor combination was observed .\",\n \"However , this was not as pronounced as that observed with rTRAIL or the multivalent death receptor five superagonist MEDI3039 ( Figure 1—figure supplement 2C , D and E ) .\",\n \"Thus , while the significant sensitising association observed in the screen appears most specific to death receptor agonists , the trend observed with other TNF superfamily agonists indicates the BAP1-rTRAIL association to be mediated by an underlying mechanism common to this family such as the cytoplasmic extrinsic apoptotic machinery .\",\n \"To determine if knockdown of BAP1 in wild-type MM cells led to TRAIL sensitivity , we silenced BAP1 expression in four wt BAP1 MM cell lines using a lentiviral shRNA construct .\",\n \"Knockdown of BAP1 resulted in increased cell death following rTRAIL treatment compared with empty vector ( EV ) control shRNA and the parental cell line in all four MM cell lines ( Figure 2A and Figure 2—figure supplement 1B and C ) .\",\n \"Loss of BAP1 expression has also been identified in several other tumour types including uveal melanoma ( 47% ) ( Harbour et al . , 2010 ) , clear cell renal carcinoma ( CCRC ) ( 14% ) ( Peña-Llopis et al . , 2012 ) and cholangiocarcinoma ( 7% ) ( Fujimoto et al . , 2015 ) .\",\n \"Notably , knockdown of BAP1 in two CCRC lines resulted in increased sensitivity to rTRAIL in addition to the MDAMB-231 breast cancer line ( Figure 2B and Figure 2—figure supplements 2 and 3 ) .\",\n \"We also analysed a panel of 1001 cancer cell lines submitted for whole exome and copy number analysis as part of the COSMIC cell lines project ( Forbes et al . , 2015 ) and identified nine additional non-mesothelioma cell lines harbouring truncating mutations in BAP1 ( http://cancer . sanger . ac . uk/cancergenome/projects/cell_lines/ ) .\",\n \"These include CCRC , bladder and breast cancer lines .\",\n \"Treatment of cancer cell lines harbouring nonsense mutations in BAP1 with rTRAIL resulted in markedly reduced cell viability compared with cancer cell lines harbouring missense mutations ( Figure 2—figure supplement 4 ) .\",\n \"BAP1 is a nuclear deubiquitinase that forms multi-protein complexes with transcription factors to regulate gene transcription ( Jensen et al . , 1998; Ventii et al . , 2008 ) .\",\n \"To elucidate the mechanism by which BAP1 modulates sensitivity to TRAIL we generated expression vectors containing wild-type or mutant forms of BAP1 , each with an inactive functional site or protein-binding domain .\",\n \"These included C91A ( mutation in the deubiquitination catalytic site ) ( Jensen et al . , 1998; Ventii et al . , 2008 ) , ΔHBM ( deletion of the HCF-1-binding site ) ( Misaghi et al . , 2009 ) , T493A ( mutation in the FOXK2-binding site ) ( Ji et al . , 2014 ) , ΔASXL ( deletion of the ASXL1/2 protein-binding site ) ( Daou et al . , 2015 ) and ΔCTD ( deletion of the C-terminal domain containing the nuclear localisation signal ) ( Ventii et al . , 2008 ) .\",\n \"H226 MM cells , which harbour a homozygous deletion of BAP1 and demonstrate complete loss of BAP1 expression , were transduced with a GFP ( vector control ) , a wild-type BAP1 expression vector or one of these five mutant BAP1 expression vectors .\",\n \"rTRAIL sensitivity of the parental BAP1-null H226 MM line was significantly diminished following expression of wild-type BAP1 and each of the mutant constructs except those with an inactive deubiquitinating or ASXL protein-binding site ( Figure 2C ) , implicating the function of these sites in BAP1-induced TRAIL resistance .\",\n \"These effects were replicated using MEDI3039 ( Figure 2D ) .\",\n \"Transduction of two further BAP1-mutant rTRAIL-sensitive cell lines , H28 and H2804 , with wild-type BAP1 also induced resistance to rTRAIL while sensitivity was maintained with transduction of the deubiquitinase mutant ( Figure 2—figure supplement 5 ) .\",\n \"The BAP1 deubiquitinase and ASXL-binding sites are key to the function of the PR-DUB , an epigenetic transcriptional regulatory complex composed of BAP1 and ASXL1 .\",\n \"Deubiquitination of the main substrate of the PR-DUB , H2AK119Ub , alters chromatin architecture to modulate gene transcription ( Scheuermann et al . , 2010 ) .\",\n \"This led us to hypothesise that PR-DUB , rather than exclusively BAP1 , function might underlie rTRAIL sensitivity .\",\n \"Consistent with this shRNA silencing of ASXL1 , but not ASXL2 , induced sensitivity to MEDI3039 and rTRAIL in the BAP1/ASXL1/ASXL2-wild-type MM line MPP-89 ( Figure 2E and Figure 2—figure supplement 6 ) .\",\n \"Furthermore , H2AK119Ub expression was unaltered in the rTRAIL-sensitive H226 cells transduced with mutant constructs that disrupt PR-DUB activity , while the rTRAIL-resistant H226 cells transduced with a wild-type BAP1 construct exhibited lower H2AK119Ub levels ( Figure 2—figure supplement 7 ) .\",\n \"Thus , as the PR-DUB complex is implicated in transcriptional regulation , differential modulation of specific transcriptional programmes by BAP1 may determine rTRAIL sensitivity .\",\n \"We therefore compared differential gene expression data from BAP1-null H226 cells transduced with the C91A BAP1 mutant or with wild-type BAP1 and carried out a signalling pathway impact analysis ( SPIA ) ( ( Figure 2—figure supplements 8 and 9 [SPIA_H226 C91A mutant vs WT] ) ( http://www . genome . jp/dbget-bin/www_bget ? path:map04210 ) .\",\n \"Among those pathways significantly altered when comparing wild-type versus C91A BAP1 ( FDR < 0 . 2 ) was that of apoptosis .\",\n \"In particular , there was altered mRNA expression of components of the extrinsic death pathway ( Figure 2F and Supplementary file 6 ) .\",\n \"This manifested as an imbalance in levels of pro- and anti-apoptotic mRNA expression with , for example , significantly decreased levels of the anti-apoptotic cIAP1/2 ( p=2 . 32E-10 ) and increased levels of the pro-apoptotic death receptor 5 ( p=7 . 79E-10 ) in the rTRAIL sensitive C91A BAP1-transduced cells relative to the rTRAIL resistant BAP1-wild-type transduced cells .\",\n \"Immunoblot analysis confirmed reduced protein expression of cIAP1/2 and c-FLIP in both C91A and ΔASXL BAP1-transduced cells relative to BAP1-wild-type transduced cells ( Figure 2G ) .\",\n \"Flow cytometry analysis confirmed reduced DR4 and DR5 expression in C91A BAP1 transduced relative to BAP1-wild-type-transduced cells .\",\n \"Knockdown of BAP1 in the BAP1 wild-type H2818 line resulted in a significant increase in DR4 expression only ( Figure 2H ) .\",\n \"To support the clinical relevance of our finding we extended our assays to two further models derived from primary tumour tissue .\",\n \"25 human early passage MM lines from the UK Mesobank ( Rintoul et al . , 2016 ) were assessed for BAP1 expression by immunohistochemistry , a technique known to correlate strongly with BAP1 LOF mutations in the absence of strong nuclear staining ( Nasu et al . , 2015 ) .\",\n \"When treated with rTRAIL , those without strong nuclear staining were significantly more sensitive than those with strong nuclear staining ( p=0 . 0067 ) .\",\n \"Of the 12 lines that did not express nuclear BAP1 9 were sensitive , 2 partially sensitive and only one resistant to rTRAIL ( Table 1 , Figure 3A and Figure 3—figure supplement 1 ) .\",\n \"Remarkably , rTRAIL treatment of tumour explants derived from three patients with MM also revealed increased levels of apoptosis ( as measured by poly ( ADP-ribose ) polymerase ( PARP ) cleavage ) in explants with low BAP1 expression compared with those with high BAP1 expression ( Figure 3B and C , Figure 3—figure supplement 2 ) .\",\n \"To test the in vivo efficacy of TRAIL in inducing apoptosis in BAP1-mutant MM cells , we transduced the H226 BAP1-wild-type and the H226 C91A BAP1-mutant cell lines with luciferase and injected equal numbers of wild-type and mutant cells into the opposite flanks of mice ( Figure 3—figure supplement 3A ) .\",\n \"On day 14 after injection the mice were divided into two groups and injected intraperitoneally with rTRAIL or vehicle for 6 days per week until day 40 .\",\n \"At sacrifice rTRAIL-treated BAP1-mutant tumours weighed significantly less than rTRAIL-treated BAP1-wild-type tumours ( p=0 . 020 ) and vehicle-treated BAP1-mutant tumours ( p=0 . 019 ) ( Figure 3D and Figure 3—figure supplement 3B ) .\",\n \"BAP1-wild-type tumours showed no response to rTRAIL compared with vehicle .\",\n \"The growth rate of rTRAIL-treated BAP1-mutant tumours was also significantly suppressed compared with rTRAIL-treated BAP1-wild-type and vehicle-treated tumours ( p<0 . 05 ) as assessed by longitudinal bioluminescence intensity ( Figure 3E and F ) .\"\n ],\n [\n \"Malignant mesothelioma remains a devastating disease with limited systemic treatment options ( Vogelzang et al . , 2003 ) .\",\n \"Biomarker-driven therapies have significantly improved the prognosis for subsets of patients within other cancer types however this strategy has yet to impact MM .\",\n \"Our data support the use of loss of function of BAP1 as a genomic stratification tool to identify rTRAIL-sensitive MM tumours , an approach that may extend to other cancer subtypes .\",\n \"We propose the underlying mechanism involves the transcriptional regulation of expression of components of the apoptotic pathway by the PR-DUB .\",\n \"Our finding has potentially significant and immediately actionable clinical implications for both MM treatment and for the death receptor agonist field .\",\n \"BAP1 has emerged as a key driver of tumorigenesis in MM ( Bueno et al . , 2016 ) .\",\n \"As such , there has been increased focus on this nuclear deubiquitinase and its associated pathways ( LaFave et al . , 2015 ) .\",\n \"While next-generation sequencing reveals MM BAP1 mutation rates in the order of 20–30% ( Guo et al . , 2015; Bueno et al . , 2016; Bott et al . , 2011 ) , immunohistochemical analysis has identified loss of BAP1 function in up to 67% of MM tumours ( Nasu et al . , 2015 ) opening our biomarker-driven approach to a significant proportion of MM patients .\",\n \"BAP1 immunohistochemistry accurately identifies loss of BAP1 function as a consequence of genetic and non-genetic mechanisms ( Nasu et al . , 2015 ) and is already in clinical use as a diagnostic tool; hence the clinical tools for our proposed approach are validated and ready .\",\n \"Our data indicate the BAP1-TRAIL association extends beyond MM to other tumours with loss of BAP1 function .\",\n \"Chromosomal deletions and somatic inactivating mutations have been identified at high frequency in uveal melanoma ( Harbour et al . , 2010 ) , clear cell renal carcinoma ( Peña-Llopis et al . , 2012 ) and cholangiocarcinoma ( Fujimoto et al . , 2015 ) , increasing the potential clinical impact of our discovery .\",\n \"Although loss of BAP1 function is seen at far lower rates in breast carcinoma ( 1% ) ( Stephens et al . , 2012 ) and non-small cell lung carcinoma ( 1% ) ( Owen et al . , 2017 ) , the high incidence of these cancers translates to a large cohort of patients .\",\n \"Focus on death receptor agonists as anti-cancer agents has generated two decades of preclinical studies and the development of numerous clinically tested compounds , all of which have demonstrated limited therapeutic efficacy at phase I/II trials ( Herbst et al . , 2010; von Pawel et al . , 2014; Paz-Ares et al . , 2013; Forero-Torres et al . , 2013 ) .\",\n \"Strategies to overcome this have included the development of increasingly potent death receptor agonists and combination therapies to address resistance factors within the apoptosis pathway ( Holland , 2013; Lemke et al . , 2014b ) .\",\n \"As differential sensitivity has been observed in trials , it has been accepted that identification of a biomarker predicting the therapeutic outcome is of paramount importance ( Ashkenazi , 2015; von Karstedt et al . , 2017 ) .\",\n \"There have been previous attempts to identify predictive biomarkers largely focused on molecular expression panels ( Passante et al . , 2013 ) .\",\n \"Ours is the first unbiased approach to address how the genetic make-up of tumours predicts response to rTRAIL treatment .\",\n \"The identification of BAP1 as a potential genomic biomarker has the potential to reignite the death receptor agonist field of research into which significant investment has already been made .\",\n \"The value of retrospective analysis of clinical trials based on the genomic landscape has clearly been demonstrated in the past ( Lynch et al . , 2004 ) and we wait with interest whether this will be performed on archived tumour tissue , in the context of BAP1 status , from previous trials .\",\n \"Notably there have been no trials of any death receptor agonists in MM or indeed any cancer with a high proportion of BAP1 mutations .\",\n \"We suspect a significantly higher proportion of responders would have been identified in such trials .\",\n \"Our findings also have implications for death receptor agonists as a therapy for BAP1-wild-type tumours as delineation of the underlying mechanism would offer a novel avenue by which to sensitise these tumours .\",\n \"Our mechanistic data implicate transcriptional regulation by the PR-DUB as key to the capacity of BAP1 to modulate death receptor agonist sensitivity .\",\n \"BAP1 is a master genetic regulator and is known to influence the transcription of thousands of genes as supported by our and others’ gene expression data ( Dey et al . , 2012 ) .\",\n \"While we highlight the extrinsic apoptotic pathway and proteins as being significantly altered by BAP1 status , identifying a single factor to explain BAP1-induced TRAIL resistance is extremely challenging .\",\n \"Of more direct clinical significance is our finding that loss of function of either component of the PR-DUB , BAP1 or ASXL1 , results in an increase in death receptor agonist sensitivity .\",\n \"ASXL1 mutations have an important role in the pathogenesis of myeloid neoplasms primarily consisting of nonsense , missense and frameshift mutations resulting in a truncated ASXL1 protein that retains the BAP1-binding domain ( Boultwood et al . , 2010 ) .\",\n \"It has yet to be clarified if this truncated protein possesses dominant-negative or gain-of-function properties in the context of PR-DUB activity ( Balasubramani et al . , 2015 ) .\",\n \"In the case of the former , ASXL1 could potentially predict death receptor agonist sensitivity in myeloid neoplasms .\",\n \"Further research is needed in these malignancies to determine this .\",\n \"Confirmation of the clinical value of BAP1 as a targeting biomarker for death receptor agonists in early phase clinical trials of mesothelioma is the first priority .\",\n \"The clinical tools for this approach are already validated and established facilitating the translation of our discovery into a desperately needed new therapy for this fatal thoracic cancer .\"\n ],\n [\n \"All cell lines were sourced from the Wellcome Trust Sanger Institute except the H226 line that was a kind gift from Dr Peter Szlosarek , Barts Cancer Institute .\",\n \"All cell lines were authenticated by genotyping using Short Tandem Repeat ( STR ) and Sequenom profiling of a panel of 92 single nucleotide polymorphisms for each cell line to ensure non-synonymous cell lines were not used .\",\n \"As a cell line classified as mesothelioma , H513 ( on the list of commonly misidentified cell lines ) was included in the drug screen of 15 mesothelioma cell lines conducted .\",\n \"Use of this cell line however was not carried forward to further experiments in the paper .\",\n \"The 25 early passage MM cultures were purchased from MesobanK ( Rintoul et al . , 2016 ) .\",\n \"All cell lines and cultures were tested for mycoplasma contamination and confirmed to be negative .\",\n \"Cell lines were cultured in RPMI-1640 or Dulbecco's modified Eagle's medium and nutrient mix 12 medium ( DMEM:F12 ) supplemented with 10% fetal bovine serum ( FBS ) , penicillin/streptavidin and sodium pyruvate .\",\n \"Early passage human mesothelioma cultures were cultured in RPMI-1640 medium supplemented with 5% FBS , 25 mM HEPES , penicillin/streptavidin and sodium pyruvate .\",\n \"293 T cells were cultured in Dulbecco's modified Eagle's medium ( DMEM ) supplemented with 10% fetal bovine serum ( FBS ) and 2 mM L-glutamine .\",\n \"All cells were maintained in a humidified environment at 37°C and 5% CO2 .\",\n \"Cells were lysed in radioimmunoprecipitation assay ( RIPA ) buffer ( Sigma-Aldrich , St . Louis , MO ) with protease inhibitors ( Complete-mini; Roche , Switzerland ) on ice to extract protein .\",\n \"20 μg of protein samples were separated by SDS–PAGE and transferred onto nitrocellulose membranes .\",\n \"Membranes were incubated with specific primary antibodies , washed , incubated with secondary antibodies and visualised using an ImageQuant LAS 4000 imaging system ( GE Healthcare , Little Chalfont , NY ) .\",\n \"Antibodies used include BAP1 ( Santa Cruz Biotechnology , Santa Cruz , CA ) Cat# sc-28383 , RRID:AB_626723 ) , caspase 8 ( Cell Signaling Technology , Danvers , MA ) Cat# 9746 , RRID:AB_2275120 ) , c-FLIP ( Enzo Life Sciences , Farmingdale , NY ) Cat# ALX-804-961-0100 RRID:AB_2713915 ) , cIAP1 ( Cell Signaling Technology Cat# 7065S , RRID:AB_10890862 ) , cIAP2 ( Cell Signaling Technology Cat# 3130S , RRID:AB_10693298 ) , FADD ( Cell Signaling Technology Cat# 2782 , RRID:AB_2100484 ) , XIAP ( Cell Signaling Technology Cat# 2045 , RRID:AB_2214866 ) , survivin ( Cell Signaling Technology Cat# 2803 , RRID:AB_490807 ) , α-tubulin ( Cell Signaling #2125 ) , H2AK119Ub ( Cell Signaling Technology Cat# 8240P , RRID:AB_10891618 ) , H2A ( Cell Signaling Technology Cat# 12349 , RRID:AB_2687875 ) , anti-mouse HRP ( Cell Signaling Technology Cat# 7076 , RRID:AB_330924 ) and anti-rabbit HRP ( Cell Signaling Technology Cat# 7074 , RRID:AB_2099233 ) .\",\n \"To detect the ubiquitination status of the histones , the cells were lysed with TBS buffer containing 1% SDS , protease and phosphatase inhibitors .\",\n \"The cell extract was denatured by heating up at 95°C for 10 min and centrifuged at 13000 rpm for 10 min .\",\n \"The supernatant was collected and immunoblotted as described above .\",\n \"Cells were seeded in 96-well plates in 100 μl media per well at a density of 40 , 000 cells/ml 1 day prior to treatment with soluble recombinant TRAIL ( rTRAIL; Peprotech , UK ) or MEDI3039 ( Medimmune , UK ) .\",\n \"XTT ( Applichem , UK; A8088 ) or MTT ( M-2128 , Sigma-Aldrich ) reagent was added on day 3 .\",\n \"The absorbance was measured with a spectrophotometer at a wavelength of 490 nm or 560 nm for XTT or MTT respectively .\",\n \"Relative cell viability was calculated as a fraction of viable cells relative to untreated cells .\",\n \"Full-length BAP1 cDNA was amplified by PCR from pCMV6-AC BAP1 plasmid ( Origene ( Rockville , MD; SC117256 ) and cloned into the lentiviral plasmid pCCL-CMV-flT vector previously described ( Yuan et al . , 2015 ) in place of flT via BamHI and SalI sites , creating the BAP1 vector designated pCCL-CMV-BAP1 .\",\n \"Vectors expressing mutant BAP1 constructs were generated by site-directed mutagenesis ( New England Biolabs ) of the pCCL-CMV-BAP1 vector .\",\n \"The primers used are listed below .\",\n \"All mutations were confirmed by sequencing .\",\n \"BAP1-F CGTGGATCCGCCACCATGAATAAGGGCTGGCTGGA BAP1-R GTCGGTCGACTCACTGGCGCTTGGCCTTGTA C91A-F ATACCCAACTCTGCTGCAACTCATGCCTTGCTG C91A-R CAGCTGGTGGGCAAAGAACATGTTATTCACAATATCATC HBM-F CGCTGCTGCCAAGTCCCCCATGCAGGAGGA HBM-R GCAGCGTCTAGAAAGGCCGGCAGCCGCT CTD-F CGTGGATCCGCCACCATGAATAAGGGCTGGCTGGA CTD-R GTCGTTCGAATCAGTCAGGCTTCCGCTGCTTGTGG T493A-F GCAGACACGGCCTCTGAGATCGGCAGTGCT T493A-R ACTCTCATTGCTGGGGGTGGGTGA ASXL-F AACTACGATGAGTTCATCTGCACCT ASXL-R CTGGTCATCAATCTTGAACTTCTTCCTC The ZS-green luciferase plasmid , pHIV-Luc-ZsGreen ( a gift from Bryan Welm , Addgene plasmid #39196 ) was used for generating ZS-Green luciferase-expressing lentivirus to transduce the H226 cells used in animal experiments .\",\n \"Short hairpin RNAs ( shRNAs ) were expressed as part of a mir30-based GIPZ lentiviral vector ( Dharmacon , Lafayette , CO ) .\",\n \"The clones used in this study include BAP1 ( V2LHS_41473 ) , ASXL1 ( V2LHS_78829 ) , ASXL2 ( V3LHS_313940 ) and the empty GIPZ control vector .\",\n \"Lentiviral vectors were produced by co-transfection of 293 T cells with construct plasmids together with the packaging plasmids pCMV-dR8 . 74 and pMD2 . G ( kind gifts from Dr Adrian Thrasher , UCL , Addgene plasmid #22036 and #12259 ) in the presence of a DNA transfection reagent jetPEI ( Source Bioscience UK Ltd ) .\",\n \"Lentiviruses were concentrated by ultracentrifugation at 17 , 000 rpm ( SW28 rotor , Optima LE80K Ultracentrifuge , Beckman Coulter , Brea , CA ) for 2 hr at 4°C .\",\n \"To determine the titres of prepared lentiviruses 293 T cells were transduced with serial dilutions of viruses in the presence of 8 μg/ml Polybrene ( Sigma-Aldrich ) and BAP1 expression was assessed by flow cytometry .\",\n \"shRNA- and luciferase-expressing vectors were assessed by analysis of GFP expression .\",\n \"Cell lines were transduced in the presence of 8 μg/ml Polybrene at a range of MOIs and transduction efficacy was assessed by flow cytometry for BAP1 expression .\",\n \"We pre-processed and normalised raw CEL files from Affymetrix Human Genome U219 array plate hybridisations with the Multi-Array Average ( RMA ) method ( Irizarry et al . , 2003 ) .\",\n \"We discarded transcripts with low sample variance and consolidated duplicated genes by averaging their expression values across duplicates .\",\n \"The resulting data were subsequently normalised ( μ = 0 , σ = 1 ) sample-wise and gene-median centred .\",\n \"Gene expression was averaged across three biological replicates of H226 transduced cells with either a C91A mutant or a wild-type BAP1 construct .\",\n \"SPIA pathway analysis as described in Tarca et al ( Tarca et al . , 2009 ) was performed on those genes with an adjusted p<0 . 05 and a fold change of >1 .\",\n \"All flow cytometry analysis was performed on a LSR Fortessa analyser ( Becton Dickinson , Franklin Lakes , NJ ) .\",\n \"For analysis of BAP1 expression cells were stained with primary antibody to BAP1 ( Santa Cruz Biotechnology Cat# sc-28383 , RRID:AB_626723; 1:50 ) and then with an AlexaFluor 488-conjugated anti-mouse antibody ( Thermo Fisher Scientific Cat# A-21202 , RRID:AB_141607; 1:200 ) .\",\n \"For analysis of apoptosis and cell death all floating and adherent cells were harvested and stained with an Annexin V AlexaFluor 647-conjugated antibody ( Thermo Fisher Scientific Cat# A23204 , RRID:AB_2341149 ) and 4’ , 6-diamidino-2-phenylindole ( DAPI; Sigma-Aldrich , 200 μg/ml ) .\",\n \"For analysis of DR4 and DR5 expression on cell surface cells were stained with PE-conjugated antibody ( DR4 - BioLegend , San Diego , CA ) Cat# 307205 , RRID:AB_314669 , DR5 - BioLegend Cat# 307405 , RRID:AB_314677 , Isotype control - Biolegend #400112; 1:100 ) .\",\n \"FlowJo software was used to analyse all data .\",\n \"H226 cells were seeded at 2 . 5 × 103 cells per well into 96-well Greiner micro-clear imaging plates in DMEM 10% FBS .\",\n \"After 48 hr , cells were fixed in 4% PFA for 10 min at room temperature and permeabilised in 0 . 3% NP-40 in PBS for 10 min .\",\n \"Cells were blocked in 1% BSA in 0 . 1% PBS tween for 1 hr at room temperature .\",\n \"Ubiquityl-histone H2A ( Lys119 ) primary antibody ( Cell Signaling , #8240 ) was incubated overnight at 4°C , before incubating for 1 hr at room temperature with Alexafluor 488-conjugated anti-rabbit secondary antibody .\",\n \"Nuclei were stained with Hoechst 33342 ( Thermo Fisher Scientific Cat# 62249 ) .\",\n \"Images were acquired ( n = 3 ) with a BioTek Cytation3 Multimode reader .\",\n \"Using a 10x objective 4 fields of view were acquired per well ( n = 3 ) and the level of nuclear ubiquityl-histone H2A intensity was determined within the primary nuclear mask and normalised to total cell number .\",\n \"BAP1 immunohistochemistry of human early passage cell lines was conducted on sections of cell pellets mounted on slides .\",\n \"Automated staining on a Leica Bond III staining platform was used .\",\n \"Slides were incubated with BAP1 primary antibody ( Santa Cruz Biotechnology Cat# sc-28383 , RRID:AB_626723; 1:150 ) for 15 min at room temperature .\",\n \"Epitope retrieval was completed using HIER using Leica Bond ER2 ( high pH ) for 30 min and a Leica Bond Polymer Refine with DAB chromogen detections system used .\",\n \"Appropriate ethical approval was obtained from the NHS Health Research Authority National Research Ethics Service to carry out this work ( reference 14/LO/1527 ) .\",\n \"Informed consent to conduct research on samples collected and to publish results was obtained from patients .\",\n \"The diagnosis of mesothelioma was confirmed histologically for all patients prior to consent and surgery .\",\n \"Patients underwent pleurectomy , following which primary pleural tissue was sectioned into fragments measuring approximately 2 mm3 .\",\n \"These tissue explants were cultured in 50% neurobasal and 50% DMEM:F12 , supplemented with B27 ( 2% ) , EGF ( 20 ng/ml ) and FGF ( 10 ng/ml ) .\",\n \"After 24 hr the explants were treated with rTRAIL ( vehicle , 50 ng/ml , 100 ng/ml or 200 ng/ml ) for a further 24 hr , following which explants were either fixed for PARP immunohistochemistry .\",\n \"The explants were fixed in 10% neutral-buffered formalin ( NBF ) for 24 hr and then transferred into 70% ethanol followed by paraffin embedding .\",\n \"Subsequently , 5 μm sections were used for immunohistochemistry , as previously described ( Busacca et al . , 2016 ) .\",\n \"All animal studies were approved by the University College London Biological Services Ethical Review Committee and licensed under the UK Home Office regulations and the Evidence for the Operation of Animals ( Scientific Procedures ) Act 1986 ( Home Office , London , UK ) .\",\n \"Mice were purchased from Charles River , kept in individually ventilated cages under specific pathogen-free conditions and had access to sterile irradiated food and autoclaved water ad libitum .\",\n \"12 8 week old NOD . CB17-Prkdcscid/NcrCrl ( NOD SCID ) mice ( Charles River , UK; RRID:IMSR_CRL:394 ) were injected with 1 × 106 H226 cells transduced with a plasmid containing wild-type BAP1 and luciferase on the right flank and with a plasmid containing a catalytically inactive BAP1-mutant ( C91A ) and luciferase on the left flank in a 1:1 mixture of Matrigel ( Corning , Corning , NY ) and medium .\",\n \"Tumour size was assessed by bioluminescence in vivo imaging system ( IVIS , PerkinElmer , Waltham , MA ) 15 min following intraperitoneal injection of 0 . 2 ml ( 2 mg ) luciferin .\",\n \"Tumours were allowed to establish for 2 weeks prior to baseline assessment of size at day 13 .\",\n \"Mice were then divided into two groups each of which received either 600 μg TRAIL or vehicle 6 days a week from day 14 until day\",\n \"40 . Bioluminescence was measured on days 0 , 13 , 19 , 26 and\",\n \"41 . Mice were sacrificed on day 42 and tumours harvested for measurement .\",\n \"TRAIL used in the mouse experiment was made in Henning Walczak’s laboratory as per the established protocol ( Ganten et al . , 2006 ) .\",\n \"Statistical analysis was performed using GraphPad Prism ( GraphPad Software , CA , USA ) .\",\n \"t-test was used to analyse differences between two groups whilst the analysis of variance ( ANOVA ) test with a Tukey post-hoc analysis was used to analyse differences between three groups .\",\n \"For multiple groups measured over multiple time points repeated measures ANOVA was used .\",\n \"All in vitro tests were performed in triplicate and all data are represented as mean values ± standard error of mean unless otherwise stated .\"\n ]\n]"},"abstract":{"kind":"list like","value":["Malignant mesothelioma ( MM ) is poorly responsive to systemic cytotoxic chemotherapy and invariably fatal .","Here we describe a screen of 94 drugs in 15 exome-sequenced MM lines and the discovery of a subset defined by loss of function of the nuclear deubiquitinase BRCA associated protein-1 ( BAP1 ) that demonstrate heightened sensitivity to TRAIL ( tumour necrosis factor-related apoptosis-inducing ligand ) .","This association is observed across human early passage MM cultures , mouse xenografts and human tumour explants .","We demonstrate that BAP1 deubiquitinase activity and its association with ASXL1 to form the Polycomb repressive deubiquitinase complex ( PR-DUB ) impacts TRAIL sensitivity implicating transcriptional modulation as an underlying mechanism .","Death receptor agonists are well-tolerated anti-cancer agents demonstrating limited therapeutic benefit in trials without a targeting biomarker .","We identify BAP1 loss-of-function mutations , which are frequent in MM , as a potential genomic stratification tool for TRAIL sensitivity with immediate and actionable therapeutic implications ."],"string":"[\n \"Malignant mesothelioma ( MM ) is poorly responsive to systemic cytotoxic chemotherapy and invariably fatal .\",\n \"Here we describe a screen of 94 drugs in 15 exome-sequenced MM lines and the discovery of a subset defined by loss of function of the nuclear deubiquitinase BRCA associated protein-1 ( BAP1 ) that demonstrate heightened sensitivity to TRAIL ( tumour necrosis factor-related apoptosis-inducing ligand ) .\",\n \"This association is observed across human early passage MM cultures , mouse xenografts and human tumour explants .\",\n \"We demonstrate that BAP1 deubiquitinase activity and its association with ASXL1 to form the Polycomb repressive deubiquitinase complex ( PR-DUB ) impacts TRAIL sensitivity implicating transcriptional modulation as an underlying mechanism .\",\n \"Death receptor agonists are well-tolerated anti-cancer agents demonstrating limited therapeutic benefit in trials without a targeting biomarker .\",\n \"We identify BAP1 loss-of-function mutations , which are frequent in MM , as a potential genomic stratification tool for TRAIL sensitivity with immediate and actionable therapeutic implications .\"\n]"},"summary":{"kind":"list like","value":["Two patients with the same disease who receive the same treatment may respond in different ways .","This variation often arises from differences in each patient’s genetic code .","Genes encode proteins , and proteins are the targets of most medical drugs and thus determine the patient’s response to treatment .","A major advance in the 21st century is that doctors recognise that patients can respond differently to the same treatment and now try to predict which patients will respond best to which drug – an approach known as personalised medicine .","Cancer treatment has been at the forefront of personalised medicine because mutations in different genes underlie each different cancer .","By analysing which mutations are present in a cancer , doctors can thus predict which drug ( or combination of drugs ) will be most effective .","This approach has been used successfully in several cancers , including breast and lung cancer , leading to fewer patients being exposed to ineffective treatments and their associated side effects and costs .","Mesothelioma is a cancer of the lining of the lung that is associated with exposure to the mineral asbestos .","Current treatment options for mesothelioma are unfortunately limited and not very effective .","No personalised treatments are currently in use and new treatment approaches are desperately needed .","Kolluri , Alifrangis , Kumar , Ishii et al . set out to determine if any of the mutations commonly seen in mesothelioma affected how the cancer would respond to 94 anticancer drugs that are either in use or in development .","In the laboratory , mesothelioma cells that have mutations in the gene that codes for a protein known as BRCA associated protein-1 ( or BAP1 for short ) were killed much more effectively by a drug known as TNF-related apoptosis-inducing ligand ( TRAIL ) .","The same link was seen in experiments with tumours of mesothelioma cells that had been transplanted into mice , and for fragments of mesothelioma tumours taken from patients .","When Kolluri et al . studied why these tumours might be killed more effectively with TRAIL , they found that mutations in the gene for BAP1 result in a change in the levels of proteins that transmit the signal from the receptors targeted by the TRAIL drug .","These findings may one day result in a new approach to treating patients with mesothelioma .","But first , the next step would be to conduct a clinical trial of TRAIL in patients with mesothelioma and assess if those with tumours that have mutations in the gene for BAP1 do indeed respond better .","If this proves to be the case , this would result in a new personalised treatment option for patients that suffer from this disease ."],"string":"[\n \"Two patients with the same disease who receive the same treatment may respond in different ways .\",\n \"This variation often arises from differences in each patient’s genetic code .\",\n \"Genes encode proteins , and proteins are the targets of most medical drugs and thus determine the patient’s response to treatment .\",\n \"A major advance in the 21st century is that doctors recognise that patients can respond differently to the same treatment and now try to predict which patients will respond best to which drug – an approach known as personalised medicine .\",\n \"Cancer treatment has been at the forefront of personalised medicine because mutations in different genes underlie each different cancer .\",\n \"By analysing which mutations are present in a cancer , doctors can thus predict which drug ( or combination of drugs ) will be most effective .\",\n \"This approach has been used successfully in several cancers , including breast and lung cancer , leading to fewer patients being exposed to ineffective treatments and their associated side effects and costs .\",\n \"Mesothelioma is a cancer of the lining of the lung that is associated with exposure to the mineral asbestos .\",\n \"Current treatment options for mesothelioma are unfortunately limited and not very effective .\",\n \"No personalised treatments are currently in use and new treatment approaches are desperately needed .\",\n \"Kolluri , Alifrangis , Kumar , Ishii et al . set out to determine if any of the mutations commonly seen in mesothelioma affected how the cancer would respond to 94 anticancer drugs that are either in use or in development .\",\n \"In the laboratory , mesothelioma cells that have mutations in the gene that codes for a protein known as BRCA associated protein-1 ( or BAP1 for short ) were killed much more effectively by a drug known as TNF-related apoptosis-inducing ligand ( TRAIL ) .\",\n \"The same link was seen in experiments with tumours of mesothelioma cells that had been transplanted into mice , and for fragments of mesothelioma tumours taken from patients .\",\n \"When Kolluri et al . studied why these tumours might be killed more effectively with TRAIL , they found that mutations in the gene for BAP1 result in a change in the levels of proteins that transmit the signal from the receptors targeted by the TRAIL drug .\",\n \"These findings may one day result in a new approach to treating patients with mesothelioma .\",\n \"But first , the next step would be to conduct a clinical trial of TRAIL in patients with mesothelioma and assess if those with tumours that have mutations in the gene for BAP1 do indeed respond better .\",\n \"If this proves to be the case , this would result in a new personalised treatment option for patients that suffer from this disease .\"\n]"},"year":{"kind":"string","value":"2018"}}},{"rowIdx":4299,"cells":{"headings":{"kind":"list like","value":["Introduction","Results","Discussion","Materials and methods"],"string":"[\n \"Introduction\",\n \"Results\",\n \"Discussion\",\n \"Materials and methods\"\n]"},"keywords":{"kind":"list like","value":["developmental biology","neuroscience"],"string":"[\n \"developmental biology\",\n \"neuroscience\"\n]"},"title":{"kind":"string","value":"Imp/IGF2BP levels modulate individual neural stem cell growth and division through myc mRNA stability"},"id":{"kind":"string","value":"elife-51529-v2"},"sections":{"kind":"list like","value":[["The many cells of the brain are produced through the highly regulated repeated divisions of a small number of neural stem cells ( NSCs ) .","NSCs grow and divide rapidly in order to supply the cells of the developing brain , but must be restrained to prevent tumour formation .","Individual NSCs produce characteristic lineages of progeny cells ( Kriegstein and Alvarez-Buylla , 2009; Merkle et al . , 2007 ) , which vary in number suggesting differences in division and growth rates during development .","However , the mechanisms differentially regulating the growth and division of individual NSCs are currently unknown .","Many of the processes and factors regulating neurogenesis are conserved between mammals and insects , making Drosophila an excellent model system to study NSC regulation ( Homem and Knoblich , 2012 ) .","During Drosophila neurogenesis , NSCs , also known as neuroblasts ( NBs ) , divide asymmetrically , budding-off a small progeny cell , the ganglion mother cell ( GMC ) , which divides into neurons that progress through differentiation .","During larval neurogenesis , the NB divides on average once every 80 min ( Homem et al . , 2013 ) and regrows between divisions to replace its lost volume , maintaining the proliferative potential of the cell ( Homem and Knoblich , 2012 ) .","However , average measurements of growth and division mask considerable heterogeneity between the behaviour of individual NBs in the brain over developmental time .","Individual NBs produce unique lineages of neurons ( Pereanu and Hartenstein , 2006 ) , with characteristically different clone sizes ( Yu et al . , 2013 ) .","Individual NBs also have differing division frequencies ( Hailstone et al . , 2019 ) and terminate division at different times ( NB decommissioning ) ( Yang et al . , 2017a ) .","This individual control ensures that the appropriate number of each neuron type is produced in the correct location during the construction of the brain .","Systemic signals such as insulin and ecdysone signalling drive NB growth and division , with a particularly strong influence at the transitions between developmental stages ( Chell and Brand , 2010; Géminard et al . , 2009; Homem et al . , 2014; Ren et al . , 2017; Rulifson et al . , 2002; Sousa-Nunes et al . , 2011; Syed et al . , 2017 ) .","However , the reproducible heterogeneity between individual NBs implies the existence of an unknown local or cell-intrinsic signal , acting in addition to the systemic signals to determine the proliferation of each NB .","The temporal regulation of NB proliferation and progeny fate has been well studied in the embryo and larva , and many key factors have been identified ( Doe , 2017; Li et al . , 2013; Miyares and Lee , 2019; Rossi et al . , 2017 ) .","The developmental progression of larval NBs is characterised by the levels of two conserved RNA-binding proteins ( RBPs ) , IGF2 mRNA-binding protein ( Imp/IGF2BP2 ) and Syncrip ( Syp/hnRNPQ ) ( Liu et al . , 2015 ) .","Imp and Syp negatively regulate each other and are expressed in opposing temporal gradients through larval brain development ( Liu et al . , 2015 ) : Imp level in the NB declines through larval development while Syp level correspondingly increases .","Imp and Syp play numerous key roles in larval neurogenesis .","The levels of Imp and Syp are known to determine the different types of neuron produced by the NBs over time , through post-transcriptional regulation of the transcription factor ( TF ) chinmo ( Liu et al . , 2015; Ren et al . , 2017 ) .","The loss of Syp results in an enlarged central brain , in part due to an increase in NB proliferation rate ( Hailstone et al . , 2019 ) .","In pupal NBs , declining Imp expression allows NB shrinkage and Syp promotes NB termination ( Yang et al . , 2017a ) .","Temporal regulation of the Imp/Syp gradients depends on the upstream temporal patterning system ( Narbonne-Reveau et al . , 2016; Ren et al . , 2017; Syed et al . , 2017 ) .","The timing and rates of change of these RBP levels differ substantially between classes of NB , and to a lesser degree between NBs of the same class ( Liu et al . , 2015; Syed et al . , 2017; Yang et al . , 2017a ) .","However , it is unknown if the intrinsic levels of Imp and Syp in each NB play a role in controlling the growth and division rates of individual NBs during their main proliferative window in the larva .","Imp and Syp are RBPs and can modify the protein complement of a cell via post-transcriptional modulation of mRNA localisation , stability and translation rates ( Boylan et al . , 2008; Geng and Macdonald , 2006; Hobor et al . , 2018; McDermott et al . , 2012; McDermott et al . , 2014; Medioni et al . , 2014; Munro et al . , 2006 ) .","Cell growth and proliferation are classically thought to be regulated at the level of transcription by pro-proliferative TFs .","Various signalling pathways converge to promote cell growth and proliferation through transcriptional upregulation of the conserved TF and proto-oncogene , Myc ( Dang , 2012; Delanoue et al . , 2010; Levens , 2010; Teleman et al . , 2008 ) .","Myc interacts with a binding partner , Max , to exert widespread transcriptional effects , binding upwards of 2000 genes in Drosophila ( Orian et al . , 2003 ) .","In Drosophila , Myc is best known for its role in promoting cell growth through increased ribosome biogenesis ( Grewal et al . , 2005 ) , and also accelerates progression through the G1 phase of the cell cycle in the developing wing , though this does not affect overall cell cycle length ( Johnston et al . , 1999 ) .","It is unclear whether the transcriptional activation of pro-proliferative TFs , such as Myc and its downstream targets , is overlaid by post-transcriptional regulatory mechanisms executed by RBPs , such as Imp and Syp , which could increase the precision and flexibility of the system .","Here , we examine the role of the Imp/Syp temporal gradient in regulating NB size and division during larval neurogenesis .","We show that the upregulation of Imp increases NB division and size , while Syp influences these processes indirectly via its negative regulation of Imp .","We use a genome-wide approach to determine the mRNA targets bound by Imp in the brain and identify myc mRNA among the top 15 targets of Imp .","Single molecule fluorescent in situ hybridisation ( smFISH ) shows that myc mRNA is stabilised by Imp , leading to increased Myc protein levels , NB growth and proliferation .","We compare NB types with different Imp levels and find that low Imp levels result in unstable myc mRNA , which restrains NB growth and division .","Finally , at an earlier time point , when Imp expression is heterogeneous between individual NBs , we find that higher Imp correlates with increased myc mRNA half-life .","We propose a model in which Imp post-transcriptionally regulates myc mRNA stability to fine-tune individual NB size and division rate in their appropriate developmental context ."],["To investigate the roles of the opposing Imp and Syp gradients in the NB , we used RNAi knockdown to manipulate the level of these RBPs ( Figure 1—figure supplement 1 ) .","We studied the type I NBs , the most numerous NB type in the brain , which are also very convenient to analyse , as they have a simple division hierarchy with each asymmetric division producing a GMC that divides only once more to produce two neurons or glia ( Bello et al . , 2008; Boone and Doe , 2008; Bowman et al . , 2008 ) .","In the wandering L3 stage ( wL3 ) brains all type I NBs express high levels of Syp and low of Imp ( Figure 1—figure supplement 1A ) .","We depleted Syp or Imp from the NBs with Syp knockdown and Imp knockdown RNAi constructs using the GAL4-UAS system , driven by insc-GAL4 ( Betschinger et al . , 2006 ) .","In NBs Imp and Syp negatively regulate each other and therefore the Syp knockdown results in Imp upregulation ( Figure 1—figure supplement 1B ) ( Liu et al . , 2015 ) .","We distinguished between direct effects of Syp depletion and indirect effects due to upregulated Imp expression by analysing Imp Syp double knockdown mutants ( Figure 1—figure supplement 1C ) ( Yang et al . , 2017a ) .","We also examined Imp overexpression brains , but the UAS overexpression construct only produces a very limited upregulation of Imp in the type I NB at the wL3 stage ( Figure 1—figure supplement 1D ) , as previously observed ( Liu et al . , 2015; Yang et al . , 2017a ) .","Therefore we primarily use the Syp knockdown to upregulate Imp .","We first examined the roles that Imp and Syp play in influencing type I NB size .","Our results show that higher Imp promotes larger size of type I NBs at wL3 , and Syp acts indirectly through its negative regulation of Imp .","Imp-depleted NBs are almost half the size of wild type NBs and NBs that overexpress Imp are 1 . 4-fold larger in midpoint area ( Figure 1A , A’ , Materials and methods ) .","Syp-depleted NBs are 1 . 5-fold larger than wild type .","We tested whether this effect is direct or indirect by studying the size of NBs in the Imp Syp double knockdown .","Our results show that Imp depletion suppresses the increase in NB size observed in Syp knockdown mutants , which indicates that Syp only plays an indirect role in type I NB size , through its repression of Imp .","NB size is affected by both cell growth and division rate so we then tested whether NB division rate is also sensitive to Imp levels .","We incubated ex vivo explanted brains in 5-ethynyl-2’-deoxyuridine ( EdU ) -containing media for four hours to label the progeny cells produced during this time ( see Materials and methods ) .","The number of labelled progeny was decreased by more than half in the Imp RNAi brains compared to wild type ( Figure 1B , B’ ) , which suggests that the decreased NB size in the Imp knockdown is not due to an increased division rate .","The number of progeny was increased 1 . 4-fold in the Imp overexpressing brains and increased 1 . 6-fold in the Syp RNAi brains , in which Imp is strongly upregulated , compared to wild type .","This phenotype is consistent with the increased proliferation rate previously observed in Syp knockdown brains with ex vivo culture and live imaging ( Hailstone et al . , 2019 ) .","However , the increased proliferation was lost in the Imp Syp double knockdown brains .","These results , together with our previous findings that Imp overexpression prevents NB shrinkage in the pupa and extends NB lifespan ( Yang et al . , 2017a ) , suggest that low levels of Imp in the late larval NBs restrains NB growth and division , ensuring the brain growth is limited appropriately during its development .","Imp is an RBP , so is likely to exert its function in the NB through regulation of the RNA metabolism of its key target mRNA transcripts .","In an effort to identify strong candidate targets , we identified the transcripts bound by Imp in the brain .","To achieve this aim we performed Imp RNA immunoprecipitation and sequencing ( RIPseq ) in larval brain lysates ( see Materials and methods ) .","We identified 318 mRNA targets that were significantly enriched in the Imp pulldown compared to input brain RNAseq ( using the thresholds DESeq2 . padj < 0 . 01 and DESeq2 . log2FoldChange > 2 ) ( Figure 2—figure supplement 1A , B , Supplementary file 1 ) .","The list of targets includes known Imp targets such as chickadee ( target rank: 37 ) ( Medioni et al . , 2014 ) , as well as mRNAs that have previously been shown to be regulated by Imp .","Imp binds syp mRNA ( target rank: 103 ) , which indicates a post-transcriptional mechanism for the previously observed negative regulation of Syp by Imp ( Liu et al . , 2015 ) .","Another Imp target is chinmo ( target rank: 55 ) , which is known to be post-transcriptionally regulated by Imp to determine the progeny fate of NBs in the mushroom body ( MB ) , the centre for memory and learning .","Chinmo is also regulated by Imp in type II NBs ( Liu et al . , 2015; Ren et al . , 2017; Syed et al . , 2017 ) and during NB self renewal ( Dillard et al . , 2018; Narbonne-Reveau et al . , 2016 ) .","Imp binds a number of long non-coding RNAs , including CR43283/cherub ( target rank: 5 ) .","cherub is also a binding target of Syp and facilitates Syp asymmetric segregation during type II NB division ( Landskron et al . , 2018 ) .","The large number of Imp targets identified by RIPseq indicates that Imp has a broad range of roles in the developing brain .","Imp has been shown to regulate mRNA localisation , stability , and translation ( Degrauwe et al . , 2016 ) .","Our results suggest that examining the Imp targets will provide further insight into the role of Imp in neurogenesis and the critical importance of post-transcriptional regulation .","To identify the key candidate mRNA targets responsible for the Imp NB size and division phenotypes , we examined the gene ontology ( GO ) annotations of the top 40 Imp targets ( Figure 2A ) .","We searched for genes annotated to play a role in cell growth , cell size , cell cycle and neural development , as well as regulatory genes with RNA-binding or DNA-binding function ( Figure 2B , Supplementary file 1 ) .","We identified myc ( target rank: 13 ) as the top candidate that could explain the Imp phenotype , based on these GO categories .","As discussed in the introduction , myc is a master transcription factor regulator of growth and division in diverse model systems .","In Drosophila it is primarily known as a driver of cell growth ( Grewal et al . , 2005 ) , and is a determinant of self renewal in the type II NB ( Betschinger et al . , 2006 ) .","We also identified a second member of the Myc transcriptional network , mnt , as an mRNA target bound by Imp ( target rank: 36 ) .","Mnt competes with Myc for binding to Max , and promotes opposed transcriptional effects ( Loo et al . , 2005; Orian et al . , 2003 ) .","We first focussed on myc , and later investigated mnt .","myc is the 13th most enriched target of Imp and is a very promising candidate as a direct mediator of the Imp phenotype in NBs .","To further examine the interaction between Imp and myc mRNA , we reanalysed a previously published dataset of Imp iCLIP ( individual nucleotide resolution cross-linking and immunoprecipitation ) performed in S2 cells ( Hansen et al . , 2015 ) .","The iCLIP data shows that Imp directly binds the myc transcript ( Figure 2—figure supplement 1C ) , which supports our identification of myc mRNA as an Imp target in the brain .","The iCLIP experiment identifies Imp binding sites primarily in the myc untranslated regions ( UTRs ) and binding signal is enriched in the extended 3’ UTR of the longer mRNA isoform .","In our brain Imp RIPseq dataset , we also see reads throughout the extended 3’ UTR , suggesting that Imp binds to the long myc mRNA isoform ( Figure 2—figure supplement 1D ) .","Notably , the full myc 3’ UTR extension is expressed in the brain ( Figure 2—figure supplement 1E ) but it is truncated early in the S2 cells ( Figure 2—figure supplement 1F ) , so the fully extended transcript in the brain may contain additional Imp binding sites .","The results in S2 cells support our identification of myc mRNA as a target of Imp in the brain , highlighting the hypothesis that Imp is a key regulator of myc in the NB .","To test the hypothesis that Myc protein levels are regulated by Imp , we used antibody staining in wild type and knockdown type I NB lineages .","We found that Imp is required to maintain correct Myc levels in the NB .","We observed Myc protein expression in type I NBs , but not in the surrounding GMCs or neurons ( Figure 3A ) .","Myc protein level was increased more than 2-fold in the Syp RNAi NBs compared to wild type ( Figure 3B , quantitated in 3C ) , while this effect was lost in the double Imp Syp depleted NBs .","Directly overexpressing Imp resulted in a small increase in Myc protein level ( 1 . 2-fold increase on wild type level ) ( Figure 3C ) .","The effect of Imp overexpression on Myc protein level is smaller than that in Syp knockdown NBs as the overexpression construct produces a smaller upregulation of Imp ( Figure 1—figure supplement 1 ) .","Imp knockdown produced a small decrease in Myc protein level ( Figure 3C ) , as expected because Imp levels are already very low in wild type type I NBs .","These data indicate that Imp upregulation increases Myc protein level in the NB , while Syp’s effect on Myc is indirect , as it requires Imp .","We next examined the effect of Imp and Syp on Mnt , the antagonist of Myc , also identified as an Imp target .","Using antibody staining , we found that Mnt protein is expressed in the type I NB , as well as in the progeny cells of the lineage ( Figure 3—figure supplement 1A ) .","However , knockdowns of Imp and Syp have no effect on the levels of Mnt protein .","Therefore , we conclude that Mnt is not likely to be a key target responsible for the NB growth and division phenotype of Imp .","We then asked whether the upregulation of Myc by Imp could be responsible for the phenotype of increased type I NB growth and division .","We overexpressed the Myc open reading frame ( ORF ) in type I NBs ( Figure 3—figure supplement 1B , Materials and methods ) and found a significant 1 . 3-fold increase in NB size ( Figure 3D ) .","Myc knockdown produced a small and not significant decrease in NB size .","We used a Myc Syp double knockdown to confirm that upregulated Myc is responsible for the increased size of Syp knockdown NBs ( in which Imp is upregulated ) .","We found that the increased NB size in the Syp knockdown is lost in the Myc Syp double knockdown brains ( Myc_Syp RNAi NBs are 0 . 7x the size of wild type ) , supporting the hypothesis that Imp regulates NB size through upregulation of Myc .","We tested the effect of Myc overexpression on type I NB division rate , and observed an increased division rate in the Myc OE compared to wild type ( Myc OE: 4 . 04 EdU-labelled progeny per NB , Figure 3E ) .","The observed increase in division rate is a surprising result as previous work in the wing disc showed that Myc overexpression increased cell size without affecting division rate ( Johnston et al . , 1999 ) , highlighting that Myc could regulate cell size and division rate in distinct ways in different tissue contexts .","In the NB , we find that increased Myc protein levels can explain the increased size and division rate that occur in response to overexpressing Imp .","However , Imp levels are very low in wL3 wild type type I NBs ( Figure 1—figure supplement 1 ) , which may limit Myc protein expression and restrain NB growth and division .","In order to further characterise the regulation of myc mRNA by Imp , we visualised myc mRNA transcripts using smFISH in type I NBs ( Yang et al . , 2017b ) .","The two annotated RNA isoforms of myc are identical except that the longer isoform includes a 3’ UTR extension of 5 . 7 kb ( Figure 4A ) ( FlyBase , Thurmond et al . , 2019 ) .","This additional UTR sequence potentially includes substantial regulatory sequence , including multiple binding sites for Imp according to iCLIP in S2 cells ( Hansen et al . , 2015 ) ( Figure 2—figure supplement 1C ) , which could allow differential regulation of the two isoforms .","smFISH probes against the myc intron and common exon show myc transcription and mature myc transcripts in the type I NB ( Figure 4A , B , Figure 4—figure supplement 1A , Supplementary file 2 ) .","Co-staining with the common exon probe and a long-UTR-specific probe , showed that all cytoplasmic transcripts in the type I NB are positive for both probes ( Figure 4A , C ) .","This result shows that the extended UTR isoform of myc ( myclong ) is the predominant isoform expressed in the NB .","Therefore , we used probes specifically against the myclong isoform for the following quantitative experiments .","Imp binds to myc mRNA and could upregulate Myc protein either through increasing myc mRNA levels or increasing Myc translation .","To distinguish between these possibilities , we stained brains with myclong-specific smFISH probes and quantitated the RNA expression in individual NBs within the mixed-cell tissue ( Figure 4—figure supplement 1B , C , Materials and methods , Mueller et al . , 2013 ) .","We measured the effects of Imp knockdown , Imp upregulation using the Syp knockdown , and suppression in the Imp Syp double knockdown .","Due to the minimal upregulation of Imp with the Imp overexpression construct ( Figure 1—figure supplement 1 ) and correspondingly small upregulation of Myc protein ( Figure 3C ) , we did not quantitate the myc mRNA expression in the Imp overexpression brains ( Figure 4—figure supplement 1B ) .","The number of myclong transcripts per NB is significantly reduced in the Imp knockdown , and is significantly increased in the Syp knockdown ( Figure 4D , E ) .","The transcript number is similar to wild type levels in the Imp Syp double knockdown , showing that Imp , rather than Syp , is the primary regulator of the number of myclong transcripts observed in the NB .","We interpret our results as showing that the increase in myc transcript number observed when Imp is upregulated causes the observed increase in Myc protein level .","In contrast , Imp is unlikely to upregulate Myc protein levels primarily through an increase in myc translation efficiency , although the data does not exclude the possibility that this mechanism makes a minor contribution to Myc protein upregulation .","The number of mature transcripts is affected by both transcription rate and mRNA stability .","In order to distinguish between a role for Imp in regulating myc transcription rate or myc transcript stability , we used smFISH measurements to estimate the transcription rate and mRNA half-life of myclong in each NB ( Bahar Halpern and Itzkovitz , 2016 ) .","We used the average intensity of a single transcript to calculate the number of nascent transcripts at the transcription foci , which indicates the relative transcription rate ( Mueller et al . , 2013 , Materials and methods ) .","We found that while the number of nascent transcripts is not significantly changed in the Imp knockdown or the Syp knockdown , it is significantly reduced in the Imp Syp double knockdown ( Figure 4F ) .","We used this measurement to estimate the transcription rate and showed that myclong transcription is unchanged in the single knockdowns , but is significantly reduced in the Imp Syp double knockdown ( Figure 4G , Materials and methods , [Bahar Halpern and Itzkovitz , 2016] ) .","This change in myc transcription in Imp Syp double knockdown NBs is unexpected , and may be an indirect effect through other transcription factors that Imp and Syp regulate , or a feedback loop of Myc autoregulation .","To determine the post-transcriptional role of Imp in regulating myc transcript level we calculated the myc mRNA half-life , allowing direct comparison between genotypes despite differing transcription rates ( Materials and methods , [Bahar Halpern and Itzkovitz , 2016] ) .","We found that the half-life of myclong is not significantly changed in the Imp knockdown , but is significantly increased in the Syp knockdown , in which Imp is upregulated ( wild type = 18 . 6 mins , Syp RNAi = 43 . 2 mins ) ( Figure 4H ) .","This increase in myclong mRNA half-life is suppressed in Imp Syp double knockdown NBs , in which there is no significant difference compared to wild type .","It is not surprising that the Imp knockdown has no effect on myc mRNA half-life when compared to wild type NBs , because Imp levels are very low in wild type type I NBs at the wL3 stage .","We find that Imp’s main direct role is to promote myclong mRNA stability and this results in upregulation of Myc protein , which promotes NB growth and division .","To characterise the regulation of Myc in other cells in the type I NB lineage , we used smFISH to observe myc transcription and cytoplasmic transcripts in the whole lineage ( Figure 4B , C , Figure 4—figure supplement 1 ) .","We found that while myc is transcribed and transcripts are present in all cells in the lineage , Myc protein is limited to the NB only ( Figure 3A ) , suggesting that myc transcripts are translationally repressed in the progeny GMCs and neurons .","The repression of Myc protein expression in the progeny cells was unaffected by manipulation of Imp and Syp levels , driven by insc-GAL4 ( Figure 3B ) , suggesting that these two RBPs are not responsible for translational regulation of myc .","While in the type II NB lineage , Brat is thought to translationally repress myc in progeny cells ( Betschinger et al . , 2006 ) , it is not known to act in the type I lineage .","We conclude that Myc is regulated in the NB lineages by mRNA stability through Imp and by translation , perhaps through a different RBP .","The gradient of Imp level decline with developmental age is different between different NB types ( Liu et al . , 2015; Syed et al . , 2017; Yang et al . , 2017a ) .","Therefore , we used smFISH to explore whether myc mRNA is also differentially stable in distinct NB types .","Imp level declines more slowly in MB NBs compared to the rest of the type I NBs in the central brain and higher Imp expression remains in the MB NBs at wL3 ( Liu et al . , 2015; Yang et al . , 2017a ) .","In each NB , we used smFISH to measure myclong transcription , myclong mRNA half-life and myclong transcript number as well as NB size and Imp protein level ( Figure 5A ) .","We identified MB NBs by their elevated Imp expression ( Figure 5A , B ) .","We found that MB NBs are 1 . 5-fold larger than type I NBs ( Figure 5C ) .","The myc mRNA half-life is 2 . 5-fold higher in the MB NBs ( type I NBs = 18 . 79 mins , MB NBs , 51 . 34 mins ) ( Figure 5D , Materials and methods ) , while myc transcription rate is slightly reduced in the MB NBs compared to the type I NBs ( Figure 5E ) .","Plotting these variables together shows clear differences between the type I NBs and MB NBs .","While type I NBs show low Imp , unstable myc mRNA and small NB size , the MB NBs have higher Imp , more stable myc mRNA and larger NB size ( Figure 5F ) .","These results support our earlier finding that higher Imp promotes myc mRNA stability and NB growth and indicates that Imp is a key regulator of differences between different classes of NBs .","We also measured Myc protein levels and NB division rates in MB NBs and type I NBs , although these could not be multiplexed into the same images as the smFISH measurements .","We found that Myc protein level is 1 . 4-fold higher in MB NBs compared to type I NBs ( Figure 5G ) .","Finally , we measured NB division rate by incubation with EdU , which showed that MB NBs have a faster division rate than type I NBs ( Figure 5H ) .","Collectively , these results suggest that the higher level of Imp maintained into the late L3 stage in the MB NBs increases myc mRNA stability , causing increased Myc protein levels and increased NB growth and division relative to type I NBs at the same stage .","Imp levels decline in NBs as larval development progresses ( Liu et al . , 2015 ) so we next asked what role Imp plays in myc regulation in earlier larval neurogenesis .","We studied brains at 72 hr after larval hatching ( ALH ) when the Imp protein level in the NB is higher than at the later wL3 stage and there is substantial heterogeneity in Imp expression level between the individual NBs ( Figure 6A ) .","We first compared the average populations of 72 hr ALH NBs to wL3 NBs .","Imp protein levels were measured from endogenous GFP-tagged Imp and found to be significantly increased in the 72 hr ALH NBs compared to wL3 , as expected ( Figure 6B ) .","We then measured NB size and found that NBs are significantly larger at 72 hr ALH ( Figure 6C ) .","smFISH quantitation of myclong transcription and half-life at 72 hr ALH showed that myclong half-life is increased at 72 hr ALH ( Figure 6D ) , but there was no significant difference in myclong transcription rate ( Figure 6E ) .","To validate the role of Imp in early larval neurogenesis , we measured NB size in Imp-depleted early NBs .","NBs were much smaller in the Imp knockdown than in Imp::GFP ( wild type ) brains at 72 hr ALH ( Figure 6F ) .","This data supports the model that the decline in Imp levels during larval development reduces myc mRNA stability , restraining NB growth and division at the end of the larval stage .","Pooled averages hide the substantial variation in between individual NBs at 72 hr ALH so we asked whether the Imp level in each NB determines myclong half-life .","We used a correlation matrix to examine the relationships between the variables measured in each individual NB at 72 hr ALH ( Figure 6G , Figure 6—figure supplement 1 ) and found that Imp level correlates with myclong half-life ( r = 0 . 344 , p<0 . 01 ) in individual NBs .","We also found a significant correlation between myclong transcript number and NB size ( r = 0 . 281 , p<0 . 05 ) , which supports the hypothesis that Myc is a significant regulator of NB size at this stage .","However , we found no significant correlation between Imp levels and myclong transcript numbers or NB size .","The myc transcript number is controlled on multiple levels through both transcriptional and post-transcriptional mechanisms , and transcriptional activation of myc is a downstream consequence of many signalling pathways in the brain .","Imp regulates myc mRNA stability to modify the final number of transcripts in each cell and as Imp levels decline through development myc mRNA stability also decreases .","These results support the hypothesis that intrinsic Imp levels provide a mechanism to fine-tune the amount of Myc protein produced in each NB , allowing NB growth and division to be determined in each NB independently throughout its lifespan ."],["Myc is known to promote stem cell character and must be switched off in progeny cells to allow correct differentiation ( Betschinger et al . , 2006; Gallant , 2013 ) .","We found that Myc overexpression increases both type I NB size and division rate , which is a very interesting result since Myc is best known to drive cell growth through activation of ribosome biogenesis ( Grewal et al . , 2005 ) .","Myc also promotes a shortened G1 phase in the wing disc , but this does not increase division rate as the G2 phase is proportionately lengthened ( Johnston et al . , 1999 ) .","In the NB , the increased division rate we observe with Myc overexpression could be the result of a direct effect of Myc driving cell cycle progression , which would be mechanistically different from the cells of the wing disc .","Alternatively , division rate may be increased indirectly as a result of the larger cell size .","Further experiments will be required to uncover the precise mechanism of Myc action in the NB .","Our discovery of Imp-dependent modulation of Myc levels adds another dimension of regulation allowing cell-intrinsic modulation of NB growth and division tailored to individual NBs .","It has been shown that Brat , an RBP , translationally represses Myc in type II NB progeny cells ( intermediate neural progenitors ) to prevent formation of ectopic NBs ( Bello et al . , 2008; Betschinger et al . , 2006; Boone and Doe , 2008; Bowman et al . , 2008 ) .","Together these findings emphasise the importance of the complex network of RBPs that play crucial post-transcriptional roles to control growth and division in individual NBs and their progeny in brain development .","Our work also suggests a new potential mechanism by which NB growth and division is restrained toward the end of the stem cell lifespan , in preparation for the terminal division in the pupa .","The intrinsic regulation of myc mRNA stability by Imp could explain why NBs are insensitive to the general growth signalling pathways at their late stages ( Homem et al . , 2014 ) .","Homem et al . , show that activation or inhibition of signalling through insulin-like peptides or their effector FOXO , has no effect on NB shrinkage or termination .","Our results demonstrate that in the late larval NBs , there is insufficient Imp to stabilise myc mRNA , so that upregulation of myc transcription would still lead to low levels of Myc protein .","MB NBs are the longest lived NBs in the larval brain and their growth and division only finally slows at about 72 hr after pupal formation ( Siegrist et al . , 2010 ) , 24 hr after the termination of the other type I NBs ( Yang et al . , 2017a ) .","It was previously shown that NB decommissioning is initiated through a metabolic response to ecdysone signalling , via Mediator ( Homem et al . , 2014 ) .","Elevated Imp level inhibits Mediator in the MB NBs to extend their lifespan by preventing NB shrinkage ( Yang et al . , 2017a ) .","However , Yang et al . ( 2017a ) , found that inhibition of the Mediator complex only partially explained the lack of cell shrinkage in the long-lived MB NBs , suggesting that other targets of Imp also play a role in MB NBs .","Imp stabilisation of myc mRNA might additionally promote NB growth to contribute to extending the MB NB proliferative lifespan .","In contrast , Imp levels decline faster in the other type I NBs , which would restrain their growth and division in preparation for their earlier decommissioning .","We also examined the role of Imp earlier in larval development , at 72 hr ALH when Imp levels are higher and heterogeneous between individual NBs .","Type I NBs at 72 hr ALH have higher myc mRNA stability and increased cell size compared to type I NBs at wL3 .","Our measurements of multiple variables in single cells allowed us to examine the function of Imp expression heterogeneity between individual NBs .","We found that Imp levels correlate with myc mRNA stability in individual NBs at 72 hr ALH , providing a cell intrinsic mechanism to modulate NB growth and division .","However , Imp levels do not correlate with NB size , unlike at the later wL3 stage .","In the early larva , Imp and Myc levels are rapidly changing so a snapshot measurement of NB size may not be a suitable proxy for cell growth at each time point .","Resolving this issue will require more sophisticated methods for long-term imaging of live whole brains that allow direct measurement of the growth and division rates of each NB at the same time as the Imp and Myc levels .","We have identified a mechanism of cell-intrinsic regulation of individual NB division and growth , which we suggest plays a key role in ensuring the correct number of progeny is produced in each lineage to build the correct sub-regions and circuits in the brain .","This intrinsic regulatory mechanism must be integrated with extrinsic growth signals in the brain to determine the growth and division of each stem cell throughout development .","Systemic insulin and ecdysone signalling are known to promote the timing of developmental switches in NBs , at the exit from quiescence after larval hatching and the decommissioning of the NB in the pupa .","In the final stages of larval development , brain growth is also driven locally to protect it from nutrient restriction , in a process called brain sparing , by which Jelly-Belly expressed by the glial niche bypasses the insulin signalling pathway ( Cheng et al . , 2011 ) .","It is plausible that this local extrinsic regulation might also be specific to individual NBs , for example through controlled expression level of Jelly-Belly in each glial niche .","Future experiments will determine the interplay between the intrinsic regulation of myc stability by Imp that we have shown here , and other extrinsic systemic and local regulators of NB growth and division .","c-myc , the mammalian homologue of Drosophila myc , is best known for its role in cancers , and so its regulation has been studied extensively ( reviewed in Conacci-Sorrell et al . , 2014; Farrell and Sears , 2014 ) .","It is therefore interesting to consider to what extent the mechanism we have uncovered is conserved between c-myc and Drosophila myc .","The mammalian homologue of Imp , IGF2BP1 , binds to c-myc mRNA and regulates its stability .","However , IGF2BP binds to c-myc mRNA in the coding sequence , whereas Imp binds to myc UTRs in Drosophila .","IGF2BP1 is known to stabilise c-myc transcripts by blocking translation-coupled decay ( Bernstein et al . , 1992; Doyle et al . , 1998; Lemm and Ross , 2002; Weidensdorfer et al . , 2009 ) , but in Drosophila , Imp’s exact mechanism of stabilisation is not yet known .","Nevertheless , the similarity of the two cases suggests that Imp regulation of myc stability might play a conserved role , coordinating stem cell growth and division with developmental progression .","The activity of stem cells in every context must be precisely restrained to prevent uncontrolled proliferation , and produce the correct numbers of each cell type to build the organ .","We have discovered an important new regulatory mechanism , that Imp acts through myc mRNA stability to modulate cell growth and division appropriately in each stem cell and each stage of development .","During development , lengthening of the G1 phase to extend the cell cycle length of NSCs is correlated with a switch from expansion to differentiation in the mouse ventricular zone ( Takahashi et al . , 1995 ) .","It has been proposed that Myc is a critical link between cell cycle length and pluripotency ( Singh and Dalton , 2009 ) .","In parallel , Imp expression levels have been shown to occur in declining temporal gradients in diverse stem cells including the Drosophila testis ( Toledano et al . , 2012 ) and , in vertebrates , mouse foetal NSCs ( Nishino et al . , 2013 ) .","These diverse studies support our proposal of a new general principal that Imp temporal gradients limit stem cell proliferative potential towards the end of their developmental lifespan , by reducing myc mRNA stability and leading to low Myc protein level .","Future experiments in a wide range of other organs and systems will now be required to test our model , and to examine the extent of Imp expression heterogeneity in other stem cell systems ."],["Drosophila melanogaster fly stocks were kept at 18°C , but transferred to 25°C for crosses and experimental use .","OregonR was the wild type strain .","Flies were raised on standard cornmeal-agar medium .","smFISH probes were designed using the Stellaris Probe Designer version 4 . 2 .","The sequences against which the probes were designed are shown in Supplementary file 2 .","Stellaris DNA probes were gently resuspended in 95 μl fresh TE buffer and 5 ul RNAse inhibitor ( RNAsin Plus RNase Inhibitor , Promega ) , and frozen at −80°C in 10 μl aliquots .","Dissected brains from male larvae were rinsed once with 0 . 3% PBSTX and then fixed in 4% PFA ( in 0 . 3% PSTX ) for 25 min ( for wL3 ) or 15 min ( for 72 hr ALH ) at RT .","Samples were rinsed briefly and then washed 3 × 15 min in 0 . 3% PBSTX at RT .","Samples were washed for 5 min in Wash Buffer ( 10% deionised formamide ( stored at −80°C ) and 2x SSC in DEPC water ) and then incubated with 250 nM Stellaris DNA probes in Hybridisation Buffer ( 10% deionised formamide , 2x SSC and 5% dextran sulphate in DEPC water ) overnight at 37°C on a rocker .","Samples were rinsed briefly 3x in Wash Buffer , and then washed 3 × 15 min in Wash Buffer at 37°C .","For nuclear staining DAPI ( 4’ , 6-diamidino-2-phenylindole ) was included at 1:500 in the second wash .","Brains were mounted in VECTASHIELD anti-fade mounting medium ( Vector Labs ) .","Slides were either imaged immediately or stored at −20°C .","DAPI was used to stain nuclei , and was added at 1:500 in one of the final wash steps before mounting .","Phalloidin was used to label F-actin and was added in one of the final wash steps and incubated for 1 hr at 37°C .","Fluorescein 488 phalloidin was used at 5 μl per 100 μl , 647 Phalloidin was used at 2 . 5 μl per 100 μl .","Brains were dissected in Schneider’s medium and then transferred to Brain Culture Medium ( 80% Schneider’s medium , 20% fetal bovine serum ( Gibco ThermoFisher ) , 0 . 1 mg/ml insulin ( Sigma ) ) with 25 μM EdU for 4 hr .","Brains were then washed with Schneider’s medium and fixed for 25 min in 4% PFA in 0 . 3% PBSTX at RT .","The samples were rinsed and then washed 3 × 15 min in 0 . 3% PBSTX at RT before blocking for 1 hr at RT in Blocking Buffer .","Samples were incubated with anti-Dpn antibody in Blocking Buffer overnight at 4°C .","The following day , samples were washed in Blocking Buffer and then incubated with Alexa Fluor secondary antibody ( Thermofisher ) at 1:200 in Blocking Buffer and samples were incubated for 1 hr at RT in the dark .","Samples were washed 3 × 15 min in 0 . 3% PBSTX at RT and then fixed in 1% PFA in 0 . 3% PBSTX at RT for 15 min .","Samples were washed and then incubated in Blocking Buffer for 1 hr .","The Click-iT reaction was carried out with the Click-iT EdU Alexa Fluor 488 Imaging Kit ( Invitrogen ) following manufacturer’s instructions for 30 min at RT .","Samples were washed in 0 . 3% PBST with 5 mM EDTA , once including DAPI , and then mounted in VECTASHIELD anti-fade mounting medium ( Vector Labs ) .","Samples were imaged on the same day .","An inverted Olympus FV3000 Laser Scanning Microscope was used for fixed imaging of larval brains .","Images were acquired using 60x/1 . 30 NA Si UApoN objective .","For smFISH quantitation images , pixel size was 74 nm in x and y , and 200 nm in z .","The presented RNA sequencing data has been deposited with Gene Expression Omnibus ( GEO ) , with accession number GSE140704 .","Further details of the analysis and code are available in Source code 1 ."]],"string":"[\n [\n \"The many cells of the brain are produced through the highly regulated repeated divisions of a small number of neural stem cells ( NSCs ) .\",\n \"NSCs grow and divide rapidly in order to supply the cells of the developing brain , but must be restrained to prevent tumour formation .\",\n \"Individual NSCs produce characteristic lineages of progeny cells ( Kriegstein and Alvarez-Buylla , 2009; Merkle et al . , 2007 ) , which vary in number suggesting differences in division and growth rates during development .\",\n \"However , the mechanisms differentially regulating the growth and division of individual NSCs are currently unknown .\",\n \"Many of the processes and factors regulating neurogenesis are conserved between mammals and insects , making Drosophila an excellent model system to study NSC regulation ( Homem and Knoblich , 2012 ) .\",\n \"During Drosophila neurogenesis , NSCs , also known as neuroblasts ( NBs ) , divide asymmetrically , budding-off a small progeny cell , the ganglion mother cell ( GMC ) , which divides into neurons that progress through differentiation .\",\n \"During larval neurogenesis , the NB divides on average once every 80 min ( Homem et al . , 2013 ) and regrows between divisions to replace its lost volume , maintaining the proliferative potential of the cell ( Homem and Knoblich , 2012 ) .\",\n \"However , average measurements of growth and division mask considerable heterogeneity between the behaviour of individual NBs in the brain over developmental time .\",\n \"Individual NBs produce unique lineages of neurons ( Pereanu and Hartenstein , 2006 ) , with characteristically different clone sizes ( Yu et al . , 2013 ) .\",\n \"Individual NBs also have differing division frequencies ( Hailstone et al . , 2019 ) and terminate division at different times ( NB decommissioning ) ( Yang et al . , 2017a ) .\",\n \"This individual control ensures that the appropriate number of each neuron type is produced in the correct location during the construction of the brain .\",\n \"Systemic signals such as insulin and ecdysone signalling drive NB growth and division , with a particularly strong influence at the transitions between developmental stages ( Chell and Brand , 2010; Géminard et al . , 2009; Homem et al . , 2014; Ren et al . , 2017; Rulifson et al . , 2002; Sousa-Nunes et al . , 2011; Syed et al . , 2017 ) .\",\n \"However , the reproducible heterogeneity between individual NBs implies the existence of an unknown local or cell-intrinsic signal , acting in addition to the systemic signals to determine the proliferation of each NB .\",\n \"The temporal regulation of NB proliferation and progeny fate has been well studied in the embryo and larva , and many key factors have been identified ( Doe , 2017; Li et al . , 2013; Miyares and Lee , 2019; Rossi et al . , 2017 ) .\",\n \"The developmental progression of larval NBs is characterised by the levels of two conserved RNA-binding proteins ( RBPs ) , IGF2 mRNA-binding protein ( Imp/IGF2BP2 ) and Syncrip ( Syp/hnRNPQ ) ( Liu et al . , 2015 ) .\",\n \"Imp and Syp negatively regulate each other and are expressed in opposing temporal gradients through larval brain development ( Liu et al . , 2015 ) : Imp level in the NB declines through larval development while Syp level correspondingly increases .\",\n \"Imp and Syp play numerous key roles in larval neurogenesis .\",\n \"The levels of Imp and Syp are known to determine the different types of neuron produced by the NBs over time , through post-transcriptional regulation of the transcription factor ( TF ) chinmo ( Liu et al . , 2015; Ren et al . , 2017 ) .\",\n \"The loss of Syp results in an enlarged central brain , in part due to an increase in NB proliferation rate ( Hailstone et al . , 2019 ) .\",\n \"In pupal NBs , declining Imp expression allows NB shrinkage and Syp promotes NB termination ( Yang et al . , 2017a ) .\",\n \"Temporal regulation of the Imp/Syp gradients depends on the upstream temporal patterning system ( Narbonne-Reveau et al . , 2016; Ren et al . , 2017; Syed et al . , 2017 ) .\",\n \"The timing and rates of change of these RBP levels differ substantially between classes of NB , and to a lesser degree between NBs of the same class ( Liu et al . , 2015; Syed et al . , 2017; Yang et al . , 2017a ) .\",\n \"However , it is unknown if the intrinsic levels of Imp and Syp in each NB play a role in controlling the growth and division rates of individual NBs during their main proliferative window in the larva .\",\n \"Imp and Syp are RBPs and can modify the protein complement of a cell via post-transcriptional modulation of mRNA localisation , stability and translation rates ( Boylan et al . , 2008; Geng and Macdonald , 2006; Hobor et al . , 2018; McDermott et al . , 2012; McDermott et al . , 2014; Medioni et al . , 2014; Munro et al . , 2006 ) .\",\n \"Cell growth and proliferation are classically thought to be regulated at the level of transcription by pro-proliferative TFs .\",\n \"Various signalling pathways converge to promote cell growth and proliferation through transcriptional upregulation of the conserved TF and proto-oncogene , Myc ( Dang , 2012; Delanoue et al . , 2010; Levens , 2010; Teleman et al . , 2008 ) .\",\n \"Myc interacts with a binding partner , Max , to exert widespread transcriptional effects , binding upwards of 2000 genes in Drosophila ( Orian et al . , 2003 ) .\",\n \"In Drosophila , Myc is best known for its role in promoting cell growth through increased ribosome biogenesis ( Grewal et al . , 2005 ) , and also accelerates progression through the G1 phase of the cell cycle in the developing wing , though this does not affect overall cell cycle length ( Johnston et al . , 1999 ) .\",\n \"It is unclear whether the transcriptional activation of pro-proliferative TFs , such as Myc and its downstream targets , is overlaid by post-transcriptional regulatory mechanisms executed by RBPs , such as Imp and Syp , which could increase the precision and flexibility of the system .\",\n \"Here , we examine the role of the Imp/Syp temporal gradient in regulating NB size and division during larval neurogenesis .\",\n \"We show that the upregulation of Imp increases NB division and size , while Syp influences these processes indirectly via its negative regulation of Imp .\",\n \"We use a genome-wide approach to determine the mRNA targets bound by Imp in the brain and identify myc mRNA among the top 15 targets of Imp .\",\n \"Single molecule fluorescent in situ hybridisation ( smFISH ) shows that myc mRNA is stabilised by Imp , leading to increased Myc protein levels , NB growth and proliferation .\",\n \"We compare NB types with different Imp levels and find that low Imp levels result in unstable myc mRNA , which restrains NB growth and division .\",\n \"Finally , at an earlier time point , when Imp expression is heterogeneous between individual NBs , we find that higher Imp correlates with increased myc mRNA half-life .\",\n \"We propose a model in which Imp post-transcriptionally regulates myc mRNA stability to fine-tune individual NB size and division rate in their appropriate developmental context .\"\n ],\n [\n \"To investigate the roles of the opposing Imp and Syp gradients in the NB , we used RNAi knockdown to manipulate the level of these RBPs ( Figure 1—figure supplement 1 ) .\",\n \"We studied the type I NBs , the most numerous NB type in the brain , which are also very convenient to analyse , as they have a simple division hierarchy with each asymmetric division producing a GMC that divides only once more to produce two neurons or glia ( Bello et al . , 2008; Boone and Doe , 2008; Bowman et al . , 2008 ) .\",\n \"In the wandering L3 stage ( wL3 ) brains all type I NBs express high levels of Syp and low of Imp ( Figure 1—figure supplement 1A ) .\",\n \"We depleted Syp or Imp from the NBs with Syp knockdown and Imp knockdown RNAi constructs using the GAL4-UAS system , driven by insc-GAL4 ( Betschinger et al . , 2006 ) .\",\n \"In NBs Imp and Syp negatively regulate each other and therefore the Syp knockdown results in Imp upregulation ( Figure 1—figure supplement 1B ) ( Liu et al . , 2015 ) .\",\n \"We distinguished between direct effects of Syp depletion and indirect effects due to upregulated Imp expression by analysing Imp Syp double knockdown mutants ( Figure 1—figure supplement 1C ) ( Yang et al . , 2017a ) .\",\n \"We also examined Imp overexpression brains , but the UAS overexpression construct only produces a very limited upregulation of Imp in the type I NB at the wL3 stage ( Figure 1—figure supplement 1D ) , as previously observed ( Liu et al . , 2015; Yang et al . , 2017a ) .\",\n \"Therefore we primarily use the Syp knockdown to upregulate Imp .\",\n \"We first examined the roles that Imp and Syp play in influencing type I NB size .\",\n \"Our results show that higher Imp promotes larger size of type I NBs at wL3 , and Syp acts indirectly through its negative regulation of Imp .\",\n \"Imp-depleted NBs are almost half the size of wild type NBs and NBs that overexpress Imp are 1 . 4-fold larger in midpoint area ( Figure 1A , A’ , Materials and methods ) .\",\n \"Syp-depleted NBs are 1 . 5-fold larger than wild type .\",\n \"We tested whether this effect is direct or indirect by studying the size of NBs in the Imp Syp double knockdown .\",\n \"Our results show that Imp depletion suppresses the increase in NB size observed in Syp knockdown mutants , which indicates that Syp only plays an indirect role in type I NB size , through its repression of Imp .\",\n \"NB size is affected by both cell growth and division rate so we then tested whether NB division rate is also sensitive to Imp levels .\",\n \"We incubated ex vivo explanted brains in 5-ethynyl-2’-deoxyuridine ( EdU ) -containing media for four hours to label the progeny cells produced during this time ( see Materials and methods ) .\",\n \"The number of labelled progeny was decreased by more than half in the Imp RNAi brains compared to wild type ( Figure 1B , B’ ) , which suggests that the decreased NB size in the Imp knockdown is not due to an increased division rate .\",\n \"The number of progeny was increased 1 . 4-fold in the Imp overexpressing brains and increased 1 . 6-fold in the Syp RNAi brains , in which Imp is strongly upregulated , compared to wild type .\",\n \"This phenotype is consistent with the increased proliferation rate previously observed in Syp knockdown brains with ex vivo culture and live imaging ( Hailstone et al . , 2019 ) .\",\n \"However , the increased proliferation was lost in the Imp Syp double knockdown brains .\",\n \"These results , together with our previous findings that Imp overexpression prevents NB shrinkage in the pupa and extends NB lifespan ( Yang et al . , 2017a ) , suggest that low levels of Imp in the late larval NBs restrains NB growth and division , ensuring the brain growth is limited appropriately during its development .\",\n \"Imp is an RBP , so is likely to exert its function in the NB through regulation of the RNA metabolism of its key target mRNA transcripts .\",\n \"In an effort to identify strong candidate targets , we identified the transcripts bound by Imp in the brain .\",\n \"To achieve this aim we performed Imp RNA immunoprecipitation and sequencing ( RIPseq ) in larval brain lysates ( see Materials and methods ) .\",\n \"We identified 318 mRNA targets that were significantly enriched in the Imp pulldown compared to input brain RNAseq ( using the thresholds DESeq2 . padj < 0 . 01 and DESeq2 . log2FoldChange > 2 ) ( Figure 2—figure supplement 1A , B , Supplementary file 1 ) .\",\n \"The list of targets includes known Imp targets such as chickadee ( target rank: 37 ) ( Medioni et al . , 2014 ) , as well as mRNAs that have previously been shown to be regulated by Imp .\",\n \"Imp binds syp mRNA ( target rank: 103 ) , which indicates a post-transcriptional mechanism for the previously observed negative regulation of Syp by Imp ( Liu et al . , 2015 ) .\",\n \"Another Imp target is chinmo ( target rank: 55 ) , which is known to be post-transcriptionally regulated by Imp to determine the progeny fate of NBs in the mushroom body ( MB ) , the centre for memory and learning .\",\n \"Chinmo is also regulated by Imp in type II NBs ( Liu et al . , 2015; Ren et al . , 2017; Syed et al . , 2017 ) and during NB self renewal ( Dillard et al . , 2018; Narbonne-Reveau et al . , 2016 ) .\",\n \"Imp binds a number of long non-coding RNAs , including CR43283/cherub ( target rank: 5 ) .\",\n \"cherub is also a binding target of Syp and facilitates Syp asymmetric segregation during type II NB division ( Landskron et al . , 2018 ) .\",\n \"The large number of Imp targets identified by RIPseq indicates that Imp has a broad range of roles in the developing brain .\",\n \"Imp has been shown to regulate mRNA localisation , stability , and translation ( Degrauwe et al . , 2016 ) .\",\n \"Our results suggest that examining the Imp targets will provide further insight into the role of Imp in neurogenesis and the critical importance of post-transcriptional regulation .\",\n \"To identify the key candidate mRNA targets responsible for the Imp NB size and division phenotypes , we examined the gene ontology ( GO ) annotations of the top 40 Imp targets ( Figure 2A ) .\",\n \"We searched for genes annotated to play a role in cell growth , cell size , cell cycle and neural development , as well as regulatory genes with RNA-binding or DNA-binding function ( Figure 2B , Supplementary file 1 ) .\",\n \"We identified myc ( target rank: 13 ) as the top candidate that could explain the Imp phenotype , based on these GO categories .\",\n \"As discussed in the introduction , myc is a master transcription factor regulator of growth and division in diverse model systems .\",\n \"In Drosophila it is primarily known as a driver of cell growth ( Grewal et al . , 2005 ) , and is a determinant of self renewal in the type II NB ( Betschinger et al . , 2006 ) .\",\n \"We also identified a second member of the Myc transcriptional network , mnt , as an mRNA target bound by Imp ( target rank: 36 ) .\",\n \"Mnt competes with Myc for binding to Max , and promotes opposed transcriptional effects ( Loo et al . , 2005; Orian et al . , 2003 ) .\",\n \"We first focussed on myc , and later investigated mnt .\",\n \"myc is the 13th most enriched target of Imp and is a very promising candidate as a direct mediator of the Imp phenotype in NBs .\",\n \"To further examine the interaction between Imp and myc mRNA , we reanalysed a previously published dataset of Imp iCLIP ( individual nucleotide resolution cross-linking and immunoprecipitation ) performed in S2 cells ( Hansen et al . , 2015 ) .\",\n \"The iCLIP data shows that Imp directly binds the myc transcript ( Figure 2—figure supplement 1C ) , which supports our identification of myc mRNA as an Imp target in the brain .\",\n \"The iCLIP experiment identifies Imp binding sites primarily in the myc untranslated regions ( UTRs ) and binding signal is enriched in the extended 3’ UTR of the longer mRNA isoform .\",\n \"In our brain Imp RIPseq dataset , we also see reads throughout the extended 3’ UTR , suggesting that Imp binds to the long myc mRNA isoform ( Figure 2—figure supplement 1D ) .\",\n \"Notably , the full myc 3’ UTR extension is expressed in the brain ( Figure 2—figure supplement 1E ) but it is truncated early in the S2 cells ( Figure 2—figure supplement 1F ) , so the fully extended transcript in the brain may contain additional Imp binding sites .\",\n \"The results in S2 cells support our identification of myc mRNA as a target of Imp in the brain , highlighting the hypothesis that Imp is a key regulator of myc in the NB .\",\n \"To test the hypothesis that Myc protein levels are regulated by Imp , we used antibody staining in wild type and knockdown type I NB lineages .\",\n \"We found that Imp is required to maintain correct Myc levels in the NB .\",\n \"We observed Myc protein expression in type I NBs , but not in the surrounding GMCs or neurons ( Figure 3A ) .\",\n \"Myc protein level was increased more than 2-fold in the Syp RNAi NBs compared to wild type ( Figure 3B , quantitated in 3C ) , while this effect was lost in the double Imp Syp depleted NBs .\",\n \"Directly overexpressing Imp resulted in a small increase in Myc protein level ( 1 . 2-fold increase on wild type level ) ( Figure 3C ) .\",\n \"The effect of Imp overexpression on Myc protein level is smaller than that in Syp knockdown NBs as the overexpression construct produces a smaller upregulation of Imp ( Figure 1—figure supplement 1 ) .\",\n \"Imp knockdown produced a small decrease in Myc protein level ( Figure 3C ) , as expected because Imp levels are already very low in wild type type I NBs .\",\n \"These data indicate that Imp upregulation increases Myc protein level in the NB , while Syp’s effect on Myc is indirect , as it requires Imp .\",\n \"We next examined the effect of Imp and Syp on Mnt , the antagonist of Myc , also identified as an Imp target .\",\n \"Using antibody staining , we found that Mnt protein is expressed in the type I NB , as well as in the progeny cells of the lineage ( Figure 3—figure supplement 1A ) .\",\n \"However , knockdowns of Imp and Syp have no effect on the levels of Mnt protein .\",\n \"Therefore , we conclude that Mnt is not likely to be a key target responsible for the NB growth and division phenotype of Imp .\",\n \"We then asked whether the upregulation of Myc by Imp could be responsible for the phenotype of increased type I NB growth and division .\",\n \"We overexpressed the Myc open reading frame ( ORF ) in type I NBs ( Figure 3—figure supplement 1B , Materials and methods ) and found a significant 1 . 3-fold increase in NB size ( Figure 3D ) .\",\n \"Myc knockdown produced a small and not significant decrease in NB size .\",\n \"We used a Myc Syp double knockdown to confirm that upregulated Myc is responsible for the increased size of Syp knockdown NBs ( in which Imp is upregulated ) .\",\n \"We found that the increased NB size in the Syp knockdown is lost in the Myc Syp double knockdown brains ( Myc_Syp RNAi NBs are 0 . 7x the size of wild type ) , supporting the hypothesis that Imp regulates NB size through upregulation of Myc .\",\n \"We tested the effect of Myc overexpression on type I NB division rate , and observed an increased division rate in the Myc OE compared to wild type ( Myc OE: 4 . 04 EdU-labelled progeny per NB , Figure 3E ) .\",\n \"The observed increase in division rate is a surprising result as previous work in the wing disc showed that Myc overexpression increased cell size without affecting division rate ( Johnston et al . , 1999 ) , highlighting that Myc could regulate cell size and division rate in distinct ways in different tissue contexts .\",\n \"In the NB , we find that increased Myc protein levels can explain the increased size and division rate that occur in response to overexpressing Imp .\",\n \"However , Imp levels are very low in wL3 wild type type I NBs ( Figure 1—figure supplement 1 ) , which may limit Myc protein expression and restrain NB growth and division .\",\n \"In order to further characterise the regulation of myc mRNA by Imp , we visualised myc mRNA transcripts using smFISH in type I NBs ( Yang et al . , 2017b ) .\",\n \"The two annotated RNA isoforms of myc are identical except that the longer isoform includes a 3’ UTR extension of 5 . 7 kb ( Figure 4A ) ( FlyBase , Thurmond et al . , 2019 ) .\",\n \"This additional UTR sequence potentially includes substantial regulatory sequence , including multiple binding sites for Imp according to iCLIP in S2 cells ( Hansen et al . , 2015 ) ( Figure 2—figure supplement 1C ) , which could allow differential regulation of the two isoforms .\",\n \"smFISH probes against the myc intron and common exon show myc transcription and mature myc transcripts in the type I NB ( Figure 4A , B , Figure 4—figure supplement 1A , Supplementary file 2 ) .\",\n \"Co-staining with the common exon probe and a long-UTR-specific probe , showed that all cytoplasmic transcripts in the type I NB are positive for both probes ( Figure 4A , C ) .\",\n \"This result shows that the extended UTR isoform of myc ( myclong ) is the predominant isoform expressed in the NB .\",\n \"Therefore , we used probes specifically against the myclong isoform for the following quantitative experiments .\",\n \"Imp binds to myc mRNA and could upregulate Myc protein either through increasing myc mRNA levels or increasing Myc translation .\",\n \"To distinguish between these possibilities , we stained brains with myclong-specific smFISH probes and quantitated the RNA expression in individual NBs within the mixed-cell tissue ( Figure 4—figure supplement 1B , C , Materials and methods , Mueller et al . , 2013 ) .\",\n \"We measured the effects of Imp knockdown , Imp upregulation using the Syp knockdown , and suppression in the Imp Syp double knockdown .\",\n \"Due to the minimal upregulation of Imp with the Imp overexpression construct ( Figure 1—figure supplement 1 ) and correspondingly small upregulation of Myc protein ( Figure 3C ) , we did not quantitate the myc mRNA expression in the Imp overexpression brains ( Figure 4—figure supplement 1B ) .\",\n \"The number of myclong transcripts per NB is significantly reduced in the Imp knockdown , and is significantly increased in the Syp knockdown ( Figure 4D , E ) .\",\n \"The transcript number is similar to wild type levels in the Imp Syp double knockdown , showing that Imp , rather than Syp , is the primary regulator of the number of myclong transcripts observed in the NB .\",\n \"We interpret our results as showing that the increase in myc transcript number observed when Imp is upregulated causes the observed increase in Myc protein level .\",\n \"In contrast , Imp is unlikely to upregulate Myc protein levels primarily through an increase in myc translation efficiency , although the data does not exclude the possibility that this mechanism makes a minor contribution to Myc protein upregulation .\",\n \"The number of mature transcripts is affected by both transcription rate and mRNA stability .\",\n \"In order to distinguish between a role for Imp in regulating myc transcription rate or myc transcript stability , we used smFISH measurements to estimate the transcription rate and mRNA half-life of myclong in each NB ( Bahar Halpern and Itzkovitz , 2016 ) .\",\n \"We used the average intensity of a single transcript to calculate the number of nascent transcripts at the transcription foci , which indicates the relative transcription rate ( Mueller et al . , 2013 , Materials and methods ) .\",\n \"We found that while the number of nascent transcripts is not significantly changed in the Imp knockdown or the Syp knockdown , it is significantly reduced in the Imp Syp double knockdown ( Figure 4F ) .\",\n \"We used this measurement to estimate the transcription rate and showed that myclong transcription is unchanged in the single knockdowns , but is significantly reduced in the Imp Syp double knockdown ( Figure 4G , Materials and methods , [Bahar Halpern and Itzkovitz , 2016] ) .\",\n \"This change in myc transcription in Imp Syp double knockdown NBs is unexpected , and may be an indirect effect through other transcription factors that Imp and Syp regulate , or a feedback loop of Myc autoregulation .\",\n \"To determine the post-transcriptional role of Imp in regulating myc transcript level we calculated the myc mRNA half-life , allowing direct comparison between genotypes despite differing transcription rates ( Materials and methods , [Bahar Halpern and Itzkovitz , 2016] ) .\",\n \"We found that the half-life of myclong is not significantly changed in the Imp knockdown , but is significantly increased in the Syp knockdown , in which Imp is upregulated ( wild type = 18 . 6 mins , Syp RNAi = 43 . 2 mins ) ( Figure 4H ) .\",\n \"This increase in myclong mRNA half-life is suppressed in Imp Syp double knockdown NBs , in which there is no significant difference compared to wild type .\",\n \"It is not surprising that the Imp knockdown has no effect on myc mRNA half-life when compared to wild type NBs , because Imp levels are very low in wild type type I NBs at the wL3 stage .\",\n \"We find that Imp’s main direct role is to promote myclong mRNA stability and this results in upregulation of Myc protein , which promotes NB growth and division .\",\n \"To characterise the regulation of Myc in other cells in the type I NB lineage , we used smFISH to observe myc transcription and cytoplasmic transcripts in the whole lineage ( Figure 4B , C , Figure 4—figure supplement 1 ) .\",\n \"We found that while myc is transcribed and transcripts are present in all cells in the lineage , Myc protein is limited to the NB only ( Figure 3A ) , suggesting that myc transcripts are translationally repressed in the progeny GMCs and neurons .\",\n \"The repression of Myc protein expression in the progeny cells was unaffected by manipulation of Imp and Syp levels , driven by insc-GAL4 ( Figure 3B ) , suggesting that these two RBPs are not responsible for translational regulation of myc .\",\n \"While in the type II NB lineage , Brat is thought to translationally repress myc in progeny cells ( Betschinger et al . , 2006 ) , it is not known to act in the type I lineage .\",\n \"We conclude that Myc is regulated in the NB lineages by mRNA stability through Imp and by translation , perhaps through a different RBP .\",\n \"The gradient of Imp level decline with developmental age is different between different NB types ( Liu et al . , 2015; Syed et al . , 2017; Yang et al . , 2017a ) .\",\n \"Therefore , we used smFISH to explore whether myc mRNA is also differentially stable in distinct NB types .\",\n \"Imp level declines more slowly in MB NBs compared to the rest of the type I NBs in the central brain and higher Imp expression remains in the MB NBs at wL3 ( Liu et al . , 2015; Yang et al . , 2017a ) .\",\n \"In each NB , we used smFISH to measure myclong transcription , myclong mRNA half-life and myclong transcript number as well as NB size and Imp protein level ( Figure 5A ) .\",\n \"We identified MB NBs by their elevated Imp expression ( Figure 5A , B ) .\",\n \"We found that MB NBs are 1 . 5-fold larger than type I NBs ( Figure 5C ) .\",\n \"The myc mRNA half-life is 2 . 5-fold higher in the MB NBs ( type I NBs = 18 . 79 mins , MB NBs , 51 . 34 mins ) ( Figure 5D , Materials and methods ) , while myc transcription rate is slightly reduced in the MB NBs compared to the type I NBs ( Figure 5E ) .\",\n \"Plotting these variables together shows clear differences between the type I NBs and MB NBs .\",\n \"While type I NBs show low Imp , unstable myc mRNA and small NB size , the MB NBs have higher Imp , more stable myc mRNA and larger NB size ( Figure 5F ) .\",\n \"These results support our earlier finding that higher Imp promotes myc mRNA stability and NB growth and indicates that Imp is a key regulator of differences between different classes of NBs .\",\n \"We also measured Myc protein levels and NB division rates in MB NBs and type I NBs , although these could not be multiplexed into the same images as the smFISH measurements .\",\n \"We found that Myc protein level is 1 . 4-fold higher in MB NBs compared to type I NBs ( Figure 5G ) .\",\n \"Finally , we measured NB division rate by incubation with EdU , which showed that MB NBs have a faster division rate than type I NBs ( Figure 5H ) .\",\n \"Collectively , these results suggest that the higher level of Imp maintained into the late L3 stage in the MB NBs increases myc mRNA stability , causing increased Myc protein levels and increased NB growth and division relative to type I NBs at the same stage .\",\n \"Imp levels decline in NBs as larval development progresses ( Liu et al . , 2015 ) so we next asked what role Imp plays in myc regulation in earlier larval neurogenesis .\",\n \"We studied brains at 72 hr after larval hatching ( ALH ) when the Imp protein level in the NB is higher than at the later wL3 stage and there is substantial heterogeneity in Imp expression level between the individual NBs ( Figure 6A ) .\",\n \"We first compared the average populations of 72 hr ALH NBs to wL3 NBs .\",\n \"Imp protein levels were measured from endogenous GFP-tagged Imp and found to be significantly increased in the 72 hr ALH NBs compared to wL3 , as expected ( Figure 6B ) .\",\n \"We then measured NB size and found that NBs are significantly larger at 72 hr ALH ( Figure 6C ) .\",\n \"smFISH quantitation of myclong transcription and half-life at 72 hr ALH showed that myclong half-life is increased at 72 hr ALH ( Figure 6D ) , but there was no significant difference in myclong transcription rate ( Figure 6E ) .\",\n \"To validate the role of Imp in early larval neurogenesis , we measured NB size in Imp-depleted early NBs .\",\n \"NBs were much smaller in the Imp knockdown than in Imp::GFP ( wild type ) brains at 72 hr ALH ( Figure 6F ) .\",\n \"This data supports the model that the decline in Imp levels during larval development reduces myc mRNA stability , restraining NB growth and division at the end of the larval stage .\",\n \"Pooled averages hide the substantial variation in between individual NBs at 72 hr ALH so we asked whether the Imp level in each NB determines myclong half-life .\",\n \"We used a correlation matrix to examine the relationships between the variables measured in each individual NB at 72 hr ALH ( Figure 6G , Figure 6—figure supplement 1 ) and found that Imp level correlates with myclong half-life ( r = 0 . 344 , p<0 . 01 ) in individual NBs .\",\n \"We also found a significant correlation between myclong transcript number and NB size ( r = 0 . 281 , p<0 . 05 ) , which supports the hypothesis that Myc is a significant regulator of NB size at this stage .\",\n \"However , we found no significant correlation between Imp levels and myclong transcript numbers or NB size .\",\n \"The myc transcript number is controlled on multiple levels through both transcriptional and post-transcriptional mechanisms , and transcriptional activation of myc is a downstream consequence of many signalling pathways in the brain .\",\n \"Imp regulates myc mRNA stability to modify the final number of transcripts in each cell and as Imp levels decline through development myc mRNA stability also decreases .\",\n \"These results support the hypothesis that intrinsic Imp levels provide a mechanism to fine-tune the amount of Myc protein produced in each NB , allowing NB growth and division to be determined in each NB independently throughout its lifespan .\"\n ],\n [\n \"Myc is known to promote stem cell character and must be switched off in progeny cells to allow correct differentiation ( Betschinger et al . , 2006; Gallant , 2013 ) .\",\n \"We found that Myc overexpression increases both type I NB size and division rate , which is a very interesting result since Myc is best known to drive cell growth through activation of ribosome biogenesis ( Grewal et al . , 2005 ) .\",\n \"Myc also promotes a shortened G1 phase in the wing disc , but this does not increase division rate as the G2 phase is proportionately lengthened ( Johnston et al . , 1999 ) .\",\n \"In the NB , the increased division rate we observe with Myc overexpression could be the result of a direct effect of Myc driving cell cycle progression , which would be mechanistically different from the cells of the wing disc .\",\n \"Alternatively , division rate may be increased indirectly as a result of the larger cell size .\",\n \"Further experiments will be required to uncover the precise mechanism of Myc action in the NB .\",\n \"Our discovery of Imp-dependent modulation of Myc levels adds another dimension of regulation allowing cell-intrinsic modulation of NB growth and division tailored to individual NBs .\",\n \"It has been shown that Brat , an RBP , translationally represses Myc in type II NB progeny cells ( intermediate neural progenitors ) to prevent formation of ectopic NBs ( Bello et al . , 2008; Betschinger et al . , 2006; Boone and Doe , 2008; Bowman et al . , 2008 ) .\",\n \"Together these findings emphasise the importance of the complex network of RBPs that play crucial post-transcriptional roles to control growth and division in individual NBs and their progeny in brain development .\",\n \"Our work also suggests a new potential mechanism by which NB growth and division is restrained toward the end of the stem cell lifespan , in preparation for the terminal division in the pupa .\",\n \"The intrinsic regulation of myc mRNA stability by Imp could explain why NBs are insensitive to the general growth signalling pathways at their late stages ( Homem et al . , 2014 ) .\",\n \"Homem et al . , show that activation or inhibition of signalling through insulin-like peptides or their effector FOXO , has no effect on NB shrinkage or termination .\",\n \"Our results demonstrate that in the late larval NBs , there is insufficient Imp to stabilise myc mRNA , so that upregulation of myc transcription would still lead to low levels of Myc protein .\",\n \"MB NBs are the longest lived NBs in the larval brain and their growth and division only finally slows at about 72 hr after pupal formation ( Siegrist et al . , 2010 ) , 24 hr after the termination of the other type I NBs ( Yang et al . , 2017a ) .\",\n \"It was previously shown that NB decommissioning is initiated through a metabolic response to ecdysone signalling , via Mediator ( Homem et al . , 2014 ) .\",\n \"Elevated Imp level inhibits Mediator in the MB NBs to extend their lifespan by preventing NB shrinkage ( Yang et al . , 2017a ) .\",\n \"However , Yang et al . ( 2017a ) , found that inhibition of the Mediator complex only partially explained the lack of cell shrinkage in the long-lived MB NBs , suggesting that other targets of Imp also play a role in MB NBs .\",\n \"Imp stabilisation of myc mRNA might additionally promote NB growth to contribute to extending the MB NB proliferative lifespan .\",\n \"In contrast , Imp levels decline faster in the other type I NBs , which would restrain their growth and division in preparation for their earlier decommissioning .\",\n \"We also examined the role of Imp earlier in larval development , at 72 hr ALH when Imp levels are higher and heterogeneous between individual NBs .\",\n \"Type I NBs at 72 hr ALH have higher myc mRNA stability and increased cell size compared to type I NBs at wL3 .\",\n \"Our measurements of multiple variables in single cells allowed us to examine the function of Imp expression heterogeneity between individual NBs .\",\n \"We found that Imp levels correlate with myc mRNA stability in individual NBs at 72 hr ALH , providing a cell intrinsic mechanism to modulate NB growth and division .\",\n \"However , Imp levels do not correlate with NB size , unlike at the later wL3 stage .\",\n \"In the early larva , Imp and Myc levels are rapidly changing so a snapshot measurement of NB size may not be a suitable proxy for cell growth at each time point .\",\n \"Resolving this issue will require more sophisticated methods for long-term imaging of live whole brains that allow direct measurement of the growth and division rates of each NB at the same time as the Imp and Myc levels .\",\n \"We have identified a mechanism of cell-intrinsic regulation of individual NB division and growth , which we suggest plays a key role in ensuring the correct number of progeny is produced in each lineage to build the correct sub-regions and circuits in the brain .\",\n \"This intrinsic regulatory mechanism must be integrated with extrinsic growth signals in the brain to determine the growth and division of each stem cell throughout development .\",\n \"Systemic insulin and ecdysone signalling are known to promote the timing of developmental switches in NBs , at the exit from quiescence after larval hatching and the decommissioning of the NB in the pupa .\",\n \"In the final stages of larval development , brain growth is also driven locally to protect it from nutrient restriction , in a process called brain sparing , by which Jelly-Belly expressed by the glial niche bypasses the insulin signalling pathway ( Cheng et al . , 2011 ) .\",\n \"It is plausible that this local extrinsic regulation might also be specific to individual NBs , for example through controlled expression level of Jelly-Belly in each glial niche .\",\n \"Future experiments will determine the interplay between the intrinsic regulation of myc stability by Imp that we have shown here , and other extrinsic systemic and local regulators of NB growth and division .\",\n \"c-myc , the mammalian homologue of Drosophila myc , is best known for its role in cancers , and so its regulation has been studied extensively ( reviewed in Conacci-Sorrell et al . , 2014; Farrell and Sears , 2014 ) .\",\n \"It is therefore interesting to consider to what extent the mechanism we have uncovered is conserved between c-myc and Drosophila myc .\",\n \"The mammalian homologue of Imp , IGF2BP1 , binds to c-myc mRNA and regulates its stability .\",\n \"However , IGF2BP binds to c-myc mRNA in the coding sequence , whereas Imp binds to myc UTRs in Drosophila .\",\n \"IGF2BP1 is known to stabilise c-myc transcripts by blocking translation-coupled decay ( Bernstein et al . , 1992; Doyle et al . , 1998; Lemm and Ross , 2002; Weidensdorfer et al . , 2009 ) , but in Drosophila , Imp’s exact mechanism of stabilisation is not yet known .\",\n \"Nevertheless , the similarity of the two cases suggests that Imp regulation of myc stability might play a conserved role , coordinating stem cell growth and division with developmental progression .\",\n \"The activity of stem cells in every context must be precisely restrained to prevent uncontrolled proliferation , and produce the correct numbers of each cell type to build the organ .\",\n \"We have discovered an important new regulatory mechanism , that Imp acts through myc mRNA stability to modulate cell growth and division appropriately in each stem cell and each stage of development .\",\n \"During development , lengthening of the G1 phase to extend the cell cycle length of NSCs is correlated with a switch from expansion to differentiation in the mouse ventricular zone ( Takahashi et al . , 1995 ) .\",\n \"It has been proposed that Myc is a critical link between cell cycle length and pluripotency ( Singh and Dalton , 2009 ) .\",\n \"In parallel , Imp expression levels have been shown to occur in declining temporal gradients in diverse stem cells including the Drosophila testis ( Toledano et al . , 2012 ) and , in vertebrates , mouse foetal NSCs ( Nishino et al . , 2013 ) .\",\n \"These diverse studies support our proposal of a new general principal that Imp temporal gradients limit stem cell proliferative potential towards the end of their developmental lifespan , by reducing myc mRNA stability and leading to low Myc protein level .\",\n \"Future experiments in a wide range of other organs and systems will now be required to test our model , and to examine the extent of Imp expression heterogeneity in other stem cell systems .\"\n ],\n [\n \"Drosophila melanogaster fly stocks were kept at 18°C , but transferred to 25°C for crosses and experimental use .\",\n \"OregonR was the wild type strain .\",\n \"Flies were raised on standard cornmeal-agar medium .\",\n \"smFISH probes were designed using the Stellaris Probe Designer version 4 . 2 .\",\n \"The sequences against which the probes were designed are shown in Supplementary file 2 .\",\n \"Stellaris DNA probes were gently resuspended in 95 μl fresh TE buffer and 5 ul RNAse inhibitor ( RNAsin Plus RNase Inhibitor , Promega ) , and frozen at −80°C in 10 μl aliquots .\",\n \"Dissected brains from male larvae were rinsed once with 0 . 3% PBSTX and then fixed in 4% PFA ( in 0 . 3% PSTX ) for 25 min ( for wL3 ) or 15 min ( for 72 hr ALH ) at RT .\",\n \"Samples were rinsed briefly and then washed 3 × 15 min in 0 . 3% PBSTX at RT .\",\n \"Samples were washed for 5 min in Wash Buffer ( 10% deionised formamide ( stored at −80°C ) and 2x SSC in DEPC water ) and then incubated with 250 nM Stellaris DNA probes in Hybridisation Buffer ( 10% deionised formamide , 2x SSC and 5% dextran sulphate in DEPC water ) overnight at 37°C on a rocker .\",\n \"Samples were rinsed briefly 3x in Wash Buffer , and then washed 3 × 15 min in Wash Buffer at 37°C .\",\n \"For nuclear staining DAPI ( 4’ , 6-diamidino-2-phenylindole ) was included at 1:500 in the second wash .\",\n \"Brains were mounted in VECTASHIELD anti-fade mounting medium ( Vector Labs ) .\",\n \"Slides were either imaged immediately or stored at −20°C .\",\n \"DAPI was used to stain nuclei , and was added at 1:500 in one of the final wash steps before mounting .\",\n \"Phalloidin was used to label F-actin and was added in one of the final wash steps and incubated for 1 hr at 37°C .\",\n \"Fluorescein 488 phalloidin was used at 5 μl per 100 μl , 647 Phalloidin was used at 2 . 5 μl per 100 μl .\",\n \"Brains were dissected in Schneider’s medium and then transferred to Brain Culture Medium ( 80% Schneider’s medium , 20% fetal bovine serum ( Gibco ThermoFisher ) , 0 . 1 mg/ml insulin ( Sigma ) ) with 25 μM EdU for 4 hr .\",\n \"Brains were then washed with Schneider’s medium and fixed for 25 min in 4% PFA in 0 . 3% PBSTX at RT .\",\n \"The samples were rinsed and then washed 3 × 15 min in 0 . 3% PBSTX at RT before blocking for 1 hr at RT in Blocking Buffer .\",\n \"Samples were incubated with anti-Dpn antibody in Blocking Buffer overnight at 4°C .\",\n \"The following day , samples were washed in Blocking Buffer and then incubated with Alexa Fluor secondary antibody ( Thermofisher ) at 1:200 in Blocking Buffer and samples were incubated for 1 hr at RT in the dark .\",\n \"Samples were washed 3 × 15 min in 0 . 3% PBSTX at RT and then fixed in 1% PFA in 0 . 3% PBSTX at RT for 15 min .\",\n \"Samples were washed and then incubated in Blocking Buffer for 1 hr .\",\n \"The Click-iT reaction was carried out with the Click-iT EdU Alexa Fluor 488 Imaging Kit ( Invitrogen ) following manufacturer’s instructions for 30 min at RT .\",\n \"Samples were washed in 0 . 3% PBST with 5 mM EDTA , once including DAPI , and then mounted in VECTASHIELD anti-fade mounting medium ( Vector Labs ) .\",\n \"Samples were imaged on the same day .\",\n \"An inverted Olympus FV3000 Laser Scanning Microscope was used for fixed imaging of larval brains .\",\n \"Images were acquired using 60x/1 . 30 NA Si UApoN objective .\",\n \"For smFISH quantitation images , pixel size was 74 nm in x and y , and 200 nm in z .\",\n \"The presented RNA sequencing data has been deposited with Gene Expression Omnibus ( GEO ) , with accession number GSE140704 .\",\n \"Further details of the analysis and code are available in Source code 1 .\"\n ]\n]"},"abstract":{"kind":"list like","value":["The numerous neurons and glia that form the brain originate from tightly controlled growth and division of neural stem cells , regulated systemically by important known stem cell-extrinsic signals .","However , the cell-intrinsic mechanisms that control the distinctive proliferation rates of individual neural stem cells are unknown .","Here , we show that the size and division rates of Drosophila neural stem cells ( neuroblasts ) are controlled by the highly conserved RNA binding protein Imp ( IGF2BP ) , via one of its top binding targets in the brain , myc mRNA .","We show that Imp stabilises myc mRNA leading to increased Myc protein levels , larger neuroblasts , and faster division rates .","Declining Imp levels throughout development limit myc mRNA stability to restrain neuroblast growth and division , and heterogeneous Imp expression correlates with myc mRNA stability between individual neuroblasts in the brain .","We propose that Imp-dependent regulation of myc mRNA stability fine-tunes individual neural stem cell proliferation rates ."],"string":"[\n \"The numerous neurons and glia that form the brain originate from tightly controlled growth and division of neural stem cells , regulated systemically by important known stem cell-extrinsic signals .\",\n \"However , the cell-intrinsic mechanisms that control the distinctive proliferation rates of individual neural stem cells are unknown .\",\n \"Here , we show that the size and division rates of Drosophila neural stem cells ( neuroblasts ) are controlled by the highly conserved RNA binding protein Imp ( IGF2BP ) , via one of its top binding targets in the brain , myc mRNA .\",\n \"We show that Imp stabilises myc mRNA leading to increased Myc protein levels , larger neuroblasts , and faster division rates .\",\n \"Declining Imp levels throughout development limit myc mRNA stability to restrain neuroblast growth and division , and heterogeneous Imp expression correlates with myc mRNA stability between individual neuroblasts in the brain .\",\n \"We propose that Imp-dependent regulation of myc mRNA stability fine-tunes individual neural stem cell proliferation rates .\"\n]"},"summary":{"kind":"list like","value":["The brain is a highly complex organ made up of huge numbers of different cell types that connect up to form a precise network .","All these different cell types are generated from the repeated division of a relatively small pool of cells called neural stem cells .","The division of these cells needs to be carefully regulated so that the correct number and type of nerve cells are produced at the right time and place .","But it remains unclear how the division rate of individual neural stem cells is controlled during development .","Controlling these divisions requires the activity of countless genes to be tightly regulated over space and time .","When a gene is active , it is copied via a process called transcription into a single-stranded molecule known as messenger RNA ( or mRNA for short ) .","This molecule provides the instructions needed to build the protein encoded within the gene .","Proteins are the functional building blocks of all cells .","The conventional way of controlling protein levels is to vary the number of mRNA molecules made by transcription .","Now , Samuels et al . reveal a second mechanism of determining protein levels in the brain , through regulating the stability of mRNA after it is transcribed .","Samuels et al . discovered that a key regulatory protein called Imp controls the growth and division of individual neural stem cells in the brains of developing fruit flies .","The experiments showed that Imp binds to mRNA molecules that contain the code for a protein called Myc , which is known to drive cell growth and division in many different cell types .","Both human Imp and Myc have been implicated in cancer .","Using a technique that images single molecules of mRNA , Samuels et al . showed that the Imp protein in stem cells stabilises the mRNA molecule coding for Myc .","This means that when more Imp is present , more Myc protein gets produced .","Thus , the level of Imp in each individual neural stem cell fine-tunes the rate at which the cell grows and divides: the higher the level of Imp , the larger the stem cell and the faster it divides .","These findings underscore how important post-transcriptional processes are for regulating gene activity in the developing brain .","The methods used in this study to study mRNA molecules in single cells also provide new insights that could not be derived from the average measurements of many cells .","Similar methods could also be applied to other developmental systems in the future ."],"string":"[\n \"The brain is a highly complex organ made up of huge numbers of different cell types that connect up to form a precise network .\",\n \"All these different cell types are generated from the repeated division of a relatively small pool of cells called neural stem cells .\",\n \"The division of these cells needs to be carefully regulated so that the correct number and type of nerve cells are produced at the right time and place .\",\n \"But it remains unclear how the division rate of individual neural stem cells is controlled during development .\",\n \"Controlling these divisions requires the activity of countless genes to be tightly regulated over space and time .\",\n \"When a gene is active , it is copied via a process called transcription into a single-stranded molecule known as messenger RNA ( or mRNA for short ) .\",\n \"This molecule provides the instructions needed to build the protein encoded within the gene .\",\n \"Proteins are the functional building blocks of all cells .\",\n \"The conventional way of controlling protein levels is to vary the number of mRNA molecules made by transcription .\",\n \"Now , Samuels et al . reveal a second mechanism of determining protein levels in the brain , through regulating the stability of mRNA after it is transcribed .\",\n \"Samuels et al . discovered that a key regulatory protein called Imp controls the growth and division of individual neural stem cells in the brains of developing fruit flies .\",\n \"The experiments showed that Imp binds to mRNA molecules that contain the code for a protein called Myc , which is known to drive cell growth and division in many different cell types .\",\n \"Both human Imp and Myc have been implicated in cancer .\",\n \"Using a technique that images single molecules of mRNA , Samuels et al . showed that the Imp protein in stem cells stabilises the mRNA molecule coding for Myc .\",\n \"This means that when more Imp is present , more Myc protein gets produced .\",\n \"Thus , the level of Imp in each individual neural stem cell fine-tunes the rate at which the cell grows and divides: the higher the level of Imp , the larger the stem cell and the faster it divides .\",\n \"These findings underscore how important post-transcriptional processes are for regulating gene activity in the developing brain .\",\n \"The methods used in this study to study mRNA molecules in single cells also provide new insights that could not be derived from the average measurements of many cells .\",\n \"Similar methods could also be applied to other developmental systems in the future .\"\n]"},"year":{"kind":"string","value":"2020"}}}],"truncated":false,"partial":false},"paginationData":{"pageIndex":42,"numItemsPerPage":100,"numTotalItems":4346,"offset":4200,"length":100}},"jwt":"eyJhbGciOiJFZERTQSJ9.eyJyZWFkIjp0cnVlLCJwZXJtaXNzaW9ucyI6eyJyZXBvLmNvbnRlbnQucmVhZCI6dHJ1ZX0sImlhdCI6MTc1MTk3NDEyNCwic3ViIjoiL2RhdGFzZXRzL3NhbmR5MzcvZWxpZmUiLCJleHAiOjE3NTE5Nzc3MjQsImlzcyI6Imh0dHBzOi8vaHVnZ2luZ2ZhY2UuY28ifQ.tQHcxodnBX9P1PD1H3gVWrg00X-5g0tCtzvVp4a2TY9EYYRVXQAAS8uK-F4DFcF0c6M2FbQZhM3sVhgGQhEJBQ","displayUrls":true},"discussionsStats":{"closed":0,"open":1,"total":1},"fullWidth":true,"hasGatedAccess":true,"hasFullAccess":true,"isEmbedded":false,"savedQueries":{"community":[],"user":[]}}">
headings
sequence | keywords
sequence | title
stringlengths 30
189
| id
stringlengths 14
14
| sections
sequence | abstract
sequence | summary
sequence | year
stringclasses 11
values |
|---|---|---|---|---|---|---|---|
[
"Introduction",
"Results and discussion",
"Materials and methods"
] | [
"developmental biology"
] | Multipotent versus differentiated cell fate selection in the developing Drosophila airways | elife-09646-v2 | [
[
"Multipotent stem cells are essential for both growth and generation of cell diversities in developing organs .",
"They also serve as reservoir to replace damaged or aged cells during physiological or pathological tissue homeostasis .",
"The discovery of a small number of transcription factors that can induce pluripotent cells ( Takahashi and Yamanaka , 2006 ) has fueled major research efforts to reveal the mechanisms biasing the choice between pluripotent/multipotent and more differentiated cell fates in vivo ( O'Brien and Bilder , 2013 ) .",
"Using the Drosophila airways ( tracheal system ) ( Manning and Krasnow , 1993; Samakovlis et al . , 1996b ) ( Figure 1A ) , we studied how the initial selection of different potencies within an organ becomes first predisposed and then regionally confined . 10 . 7554/eLife . 09646 . 003Figure 1 . The proximo-distal cell fate organization of the Drosophila airways .",
"( A ) A sketch of the embryonic Drosophila airways at stage 10/11 and late stage 12 .",
"( B ) A representation of the ectodermal expression the secreted signaling molecules wg/WNT , hh ( upper panel ) , and dpp ( lower panel ) in relation to the airway primordia at stage 10 .",
"( C–J )",
"Expression of the P/D-fate markers at different stages of the airway development , which is summarized schematically in C , where the regional diversification of the Drosophila airway according to the proximo-distal axis is also shown in different colors .",
"The P-fate region: spiracular branch ( SB ) .",
"The D-fate region: transverse connectives ( TC ) and six primary branches ( dorsal branch/DB , dorsal trunk anterior/DTa , dorsal trunk posterior/DTp , visceral branch/VB , lateral trunk anterior/LTa , and ganglionic branch/GB/lateral trunk posterior/LTp ) .",
"Expression of the D-fate markers , btl ( D , E ) , mab2A12 ( F , J ) and the P-fate markers , P0144-lacZ ( E , F , I , J ) and upd ( G-I ) , relative to each other or trh-lacZ ( D , G , H ) .",
"In this and other figures , arrowheads mark one of the 10 metameres .",
"In E-F , enlarged picture of Tr5 is also shown , where the P-fate cells are bracketed .",
"btl expression at stage 11 ( D ) is concentrated in the central parts of the primordia .",
"At stage 13 ( E ) , btl expression and P0144-lacZ expression ( arrowheads ) rarely overlap .",
"At stage 16 , mab2A12 ( anti-Gasp antibody ) strongly labels the lumens of the D-cells while P0144-lacZ is strongly detected in the P-cells ( F , arrowheads ) .",
"At stage 11 ( G ) , upd transcript is detected at the peripheral area of each primordium ( arrowheads ) .",
"At stage 13 ( H , I ) , upd is expressed in the P-cells that express P0144-lacZ ( arrowheads ) .",
"Note that compared to the control , where upd and P0144-lacZ are not expressed in Tr10 ( arrows in F , H ) , the P-fate ( P0144-lacZ ) is established in Tr10 of AbdB mutants ( asterisks in J ) .",
"Scale bar is 2 μm in the enlarged panels of E-F .",
"Scale bar in the remaining panels is 50 μm .",
"( K ) summarizes typical gene expression patterns of various marker genes in the airway primordium and in the mature airways .",
"See text for details . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 00310 . 7554/eLife . 09646 . 004Figure 1—figure supplement 1 . The centro-peripheral organization of the airway primordia . Expression of rho ( A-E ) , pnt ( F-O ) , h ( P-Q ) , or salm ( R-T ) in the airways before or after invagination .",
"( A–E ) rho expression in the trachea .",
"At stage 10 ( A ) , the dorso-central part of each primordium expresses rho ( arrowheads ) .",
"By stage 11 ( B , C ) , when the tracheal cells invaginate , rho expression expands to cover more cells of the central/distal areas ( arrowheads ) .",
"At late stage 11 ( E , F ) , rho expression in the dorsal part of the distal trachea becomes weak ( D , arrowheads ) , while the ventral part of the distal trachea still retains strong rho expression ( E , arrowheads ) .",
"( F–O ) pnt expression in the trachea .",
"At early stage 11 ( F ) , pnt is expressed in the dorso-central part of each primordium ( arrowheads ) , which expands to the whole cental/distal areas ( G-U ) .",
"The proximal area expresses less pnt ( H , arrowheads ) than the distal area ( I , arrowheads ) as also evident in a section from a ventral view ( J , arrowheads ) .",
"At stage 11 , compared to the wild type ( K ) , pnt expression in the primordia is barely detectable in rho mutants ( L , arrowheads ) .",
"In the wild type ( M ) , pnt-lacZ is expressed in the distal trachea , with its expression upregulated in a pan-tip pattern due to the Bnl/dFGF-dependent activation of Btl/dFGFR ( arrowheads ) .",
"In btl mutants ( N , O ) where this upregulation is absent , pnt-lacZ is expressed in the D-cells ( arrowheads in Tr7 ) but is absent in the P-cells ( arrows in Tr7 ) .",
"( P , Q ) h expression in the trachea .",
"At stage 10/11 ( P ) , h is expressed in the dorso-central part of each primordium ( arrowheads ) .",
"At stage 13 ( Q ) , expression of h-lacZ is detected in DB , DT , and VB ( arroheads in Tr5 ) , whereas TC and SB does not express it ( arrows in Tr5 ) .",
"Note that the earlier pair-rule pattern of h-lacZ expression is also evident ( asterisks ) .",
"( R–T ) salm expression in the trachea .",
"At stage 10/11 ( R ) , salm is expressed in the dorsal part of each primordium ( arrowheads ) .",
"At stage 13 ( S , T ) , salm-gal4 driven UAS-nGFP labels DB , DT ( arrowheads in Tr7 ) , and part of TC and SB ( arrows in Tr7 ) .",
"( U , V )",
"Requirements of trh for the P/D fate selection .",
"In trh mutants , expression of the P-fate markers upd ( U , stage 13 ) and P0144-lacZ ( V , stage 14/15 ) and the D-fate marker mab2A12 ( V ) is abolished ( asterisks ) .",
"Scale bar: 50 μm .",
"DB , dorsal branch; DT , dorsal trunk; SB , spiracular branch; TC , transverse connectives; VB , visceral branch . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 004 By mid-embryogenesis , Drosophila embryos are metamerically divided along the anterior-posterior ( AP ) axis ( Ingham , 1988; Sanson , 2001 ) .",
"Each unit ( segment or parasegment ) is also subdivided along the dorso-ventral ( DV ) embryo axis ( Anderson et al . , 1985b; Wharton et al . , 1993 ) .",
"Generation of the airway primordia is spatially restricted along these AP or DV axis , by the local concentrations of repressing and activating signals ( Bier et al . , 1989; Isaac and Andrew , 1996; Perrimon et al . , 1991; Wilk et al . , 1996 ) .",
"For example , Wingless ( Wg ) expressed in stripes along the AP axis and Decapentaplegic ( Dpp ) expressed in the dorsal ectoderm ( Figure 1B ) repress the airway primordia specification ( Isaac and Andrew , 1996; Wilk et al . , 1996 ) .",
"As a result , 10 metameric groups of cells expressing a master gene , Trachealess ( Trh ) are specified on each side of the lateral ectoderm ( Figure 1A–B ) .",
"Invagination of each primordial cluster transforms the two-dimensional ( 2D ) shape of each cluster to 3D tubes ( Figure 1C ) ( Campos-Ortega and Hartenstein , 1985; Turner and Mahowald , 1977 ) .",
"Within the invaginated primitive sacs/tubes , the proximal cells form a narrow cord , the spiracular branch/SB ( P-fate ) ( Figure 1C , K ) and stay multipotent to later proliferate and to generate many parts of the pupal/adult airways ( Manning and Krasnow , 1993; Pitsouli and Perrimon , 2010; Weaver and Krasnow , 2008 ) .",
"Within the remaining distal cells ( D-fate ) , the more distal parts achieve a series of morphogenetic events , extending six primary branches , fusing with branches from neighboring metameres and supplying oxygen directly to the target cells ( Manning and Krasnow , 1993; Samakovlis et al . , 1996a; Samakovlis et al . , 1996b ) , while the more proximal parts ( transverse connectives/TC ) connect SB with the primary branches ( Figure 1C , K ) .",
"The primary branches constitute D-fate cells located distally from TC , and their morphological diversification depends on the activation of various signaling pathways .",
"For example , rhomboid ( rho ) is expressed in each primordium ( Bier et al . , 1990 ) and activates dEGFR ( also called torpedo or faint little ball ) ( Price et al . , 1989; Schejter and Shilo , 1989 ) by generating the secreted active ligand Spitz ( s-Spi ) ( Gabay et al . , 1997; Golembo et al . , 1996; Schweitzer et al . , 1995b; Urban et al . , 2001; Wappner et al . , 1997 ) .",
"Activated dEGFR instructs the cytoskeletal changes that coordinate invagination ( Brodu and Casanova , 2006; Kondo and Hayashi , 2013; Llimargas and Casanova , 1999; Nishimura et al . , 2007 ) .",
"Branchless ( Bnl ) /dFGF is expressed in patches of surrounding tissues to guide primary branching ( Sutherland et al . , 1996 ) by activating Breathless ( Btl ) /dFGFR on the airway cells ( Klambt et al . , 1992; Lee et al . , 1996 ) .",
"Apart from receptor tyrosine kinase ( RTK ) signaling , Decapentaplegic ( Dpp ) /BMP , Wingless ( Wg ) /WNT , and Hedgehog ( Hh ) signals combinatorially establish different primary branch identities ( Affolter et al . , 1994; Chen et al . , 1998; Chihara and Hayashi , 2000; Glazer and Shilo , 2001; Llimargas , 2000; Llimargas and Lawrence , 2001; Matsuda et al . , 2015; Vincent et al . , 1997 ) .",
"Although many studies focused on the differentiation and subsequent morphogenesis of the D-fate cells ( Beitel and Krasnow , 2000; Cabernard et al . , 2004; Ghabrial et al . , 2011; Hosono et al . , 2015 ) , generation of the P-fate has been underexplored .",
"Our current dissection of P/D fate establishment in the embryo , together with metamere-specific control programs of differentiation/de-differentiation in the larval airways ( Djabrayan et al . , 2014; Guha et al . , 2008; Sato et al . , 2008; Weaver and Krasnow , 2008 ) may broaden our understanding of the strategies for stem cell selection and maintenance in vivo ."
],
[
"Every cell in the main airways derives from 20 primordial cell clusters expressing the master regulator TF Trh ( Figure 1A–C , K ) ( Isaac and Andrew , 1996; Perrimon et al . , 1991; Wilk et al . , 1996 ) .",
"However , the expression of several other genes is enriched either distally or proximally in the airways .",
"rho is expressed preferentially in the central and distal regions of the airway primordia before and during invagination ( Figure 1—figure supplement 1A–E ) .",
"btl/dFGFR is also expressed preferentially in the distal part ( Figure 1D–E ) , and its expression is upregulated by Bnl/dFGF in the most distal leading cells of the primary branch tips ( Figure 1E ) ( Ohshiro et al . , 2002; Ohshiro and Saigo , 1997 ) .",
"mAb2A12 detecting the putative chitin-biding protein Gasp ( Tiklova et al . , 2013 ) preferentially labels the distal airways during stages 14-17 ( Figure 1F ) ( Samakovlis et al . , 1996a ) .",
"On the other hand , unpaired ( upd ) , a ligand activating the JAK/STAT signaling is expressed only in the proximal SB cells from stage 12 on ( Figure 1G–I ) ( Harrison et al . , 1998 ) .",
"From stage 13 onwards , the SB cells also show stronger expression of the P0144 enhancer trap marker ( Figure 1E , F , I ) ( http://flyview . uni-muenster . de ) .",
"Compared to the anterior metameres , the 10th tracheal primordium ( Tr10 ) does not establish the P0144-postive P-fate due to the high level of a Hox protein Abdominal-B ( AbdB ) in this part of the embryo ( Figure 1F , J ) , ( Celniker et al . , 1989; Matsuda et al . , 2015 ) .",
"In trh mutants , both the D-fate marker ( Chung et al . , 2011; Jin et al . , 2001; Ohshiro and Saigo , 1997; Zelzer and Shilo , 2000 ) and the P-fate marker expressions ( Figure 1—figure supplement 1U , V ) are lost .",
"To define the origins of the proximo-distal organization of the airway tree , we labeled cell groups during the 2D primordial stage and recorded their fates in the 3D tree .",
"pointed ( pnt ) is a general transcriptional target and mediator of RTK signaling ( Brunner et al . , 1994; Klambt , 1993; O'Neill et al . , 1994 ) .",
"rho induces pnt expression in the central parts of the airway primordia ( Figure 1—figure supplement 1F–O ) and btl/dFGFR upregulates pnt expression in the tips of the primary branches ( Figure 1—figure supplement 1M–O ) ( Samakovlis et al . , 1996a ) .",
"Markers of the central parts of the airway primordia ( pnt-lacZ and btl-CD8-GFP ) become preferentially expressed in the distal part of the airway tree ( Figure 1K , Figure 1—figure supplement 1N–O ) .",
"Similarly , hairy ( h ) expression in the airway primordia ( Carroll et al . , 1988; Hooper et al . , 1989; Zhan et al . , 2010 ) is confined to the dorso-central part ( Figure 1—figure supplement 1P ) and its reporter ( h-lacZ ) becomes active in most of the DB , VB , and DT cells but not in TC , SB nor in ventral branches ( Figure 1K , Figure 1—figure supplement 1Q ) ( Samakovlis et al . , 1996a ) .",
"In contrast , salm labels the dorsal part of the airway primordia ( Figure 1—figure supplement 1R ) , ( Kuhnlein and Schuh , 1996 ) and salm-gal4 driven UAS-GFP marks DB and DT , and parts of TC and SB in the airways ( Figure 1K , Figure 1—figure supplement 1S–T ) .",
"These results suggest that the dorso-peripheral sector of each primordium , which is positive for salm but lacking h , composes half of the proximal SB cells in the airway tree .",
"The other half may originate from the ventro-peripheral sector of the primordia .",
"Consistently , around the completion of invagination , the P-fate marker upd is enriched in a horseshoe-like peripheral ring ( Figure 1G ) ( Harrison et al . , 1998 ) , which eventually marks the SB cells at stage 12 ( Figure 1H , I , K ) .",
"Collectively , the gene expression analysis suggests a fate map of the airway primordia , where the centro-peripheral axis inversely correlates both with the ordered pattern of tracheal cell invagination ( Brodu and Casanova , 2006; Nishimura et al . , 2007 ) and the PD axis of airway branching ( Figure 1C , K ) .",
"As activation of RTKs propels several morphogenetic programs in the D-cells , we first assessed their effects on the discrimination of the P- and the D-fate .",
"In btl/dFGFR mutants , where active branch extension does not occur , apoptotic staining in the distal cells increases from stage 12 ( Figure 2—figure supplement 1A , B ) , suggesting that btl/dFGFR signaling directly or indirectly suppresses apoptosis of the distal airways .",
"In dEGFR btl/dFGFR double mutants , massive cell death staining accompanies loss of most tracheal cells compared to either single mutant ( Figure 2—figure supplement 1B–D ) .",
"Suppression of apoptosis by removing pro-apoptotic genes using Df ( H99 ) ( Figure 2—figure supplement 1E ) ( White et al . , 1994 ) or by transgenic expression of the apoptosis inhibitor , DIAP ( Figure 2—figure supplement 1F ) ( Hay et al . , 1995 ) partially restores the survival of tracheal cells in dEGFR btl/dFGFR double mutants ( Figure 2—figure supplement 1E–J ) .",
"Thus , dEGFR and Btl/dFGFR cooperate to suppress apoptosis in the airway cells .",
"To analyze the effects of RTK signaling in the P/D fate choice independently of cell survival , we introduced Df ( H99 ) into some of the genotypes used in the analysis described below .",
"In bnl/dFGF mutants , the feedback upregulation of the D-fate marker btl/dFGFR is lost in the tips of the primary branches ( Figure 2A , B ) ( Ohshiro et al . , 2002 ) .",
"However , the distal part of the invaginated stump retains the mab2A12 signal ( Lee et al . , 1996 ) and the basal btl/dFGFR expression ( Figure 2B ) .",
"Similarly , expression of the P-fate markers ( upd and P0144-lacZ ) in btl/dFGFR mutants remains largely comparable to the wild type ( Figure 2E–F , I–J , M ) .",
"These results suggest that Btl/dFGFR signaling does not play a major role in the P-D discrimination .",
"In rho mutants , however , the refinement of upd expression to the primordia periphery is retarded ( Figure 2—figure supplement 1K , L ) , and the expression domains of upd and P0144-lacZ become expanded ( Figure 2G , K , M ) .",
"Correspondingly , the domain of btl-expressing cells is decreased ( Figure 2C ) suggesting that the rho-mediated dEGFR signaling drives the D-fate selection at the expense of the P-fate .",
"We investigated the possible redundant functions of the two RTKs in D-fate definition .",
"In rho bnl/dFGF double mutants , btl expression is further reduced or even lost in some segments ( Figure 2D ) .",
"Correspondingly , in rho btl/dFGFR double mutants , another D-fate marker mab2A12/Gasp becomes reduced or undetectable in some segments , accompanied by a concomitant increase of the proximal P0144-lacZ expression domain ( Figure 2L , N ) .",
"Consistent with the phenotypes of rho btl/dFGFR mutants , P0144-lacZ expression is also increased in pnt mutants , ( Figure 2M , Figure 2—figure supplement 1P ) .",
"Taken together , we conclude that dEGFR signaling in cooperation with btl/dFGFR signaling promotes the D-fate selection at the expense of the P-fate partly through pnt ( Figure 2O ) .",
"The residual weak expression of the D-fate markers in rho btl or dEGFR btl/dFGFR double mutants ( Figure 2L , Figure 2—figure supplement 1M–O ) suggests additional inputs in the D fate selection , independent of RTK signaling downstream of dEGFR and btl/dFGFR ( Figure 2O ) . 10 . 7554/eLife . 09646 . 005Figure 2 . RTK activation drives the D-fate selection . Expression of the D-fate ( btl in A-D and mab2A12 in I-L ) and the P-fate ( upd in E-H and P0144-lacZ in I-L ) markers upon loss of dEGFR and/or btl/dFGFR signaling .",
"( A–H )",
"Stage 12/13 .",
"( I–L )",
"Stage 16 .",
"Compared to the control ( A , E , I ) , in bnl mutants ( B ) , btl mutants ( F ) or Df ( H99 ) btl mutants ( J ) , expression of the D-fate markers btl ( A ) and mab2A12 ( J ) and the P-fate markers upd ( F ) and P0144-lacZ ( J ) appears similar .",
"Note , however , that upregulation of btl in a pan-tip pattern is abolished in bnl mutants ( B ) ( Ohshiro et al . , 2002 ) .",
"In rho mutants ( C , G ) or Df ( H99 ) rho mutants ( K ) , the expression area of the P-fate markers upd ( G ) and P0144-lacZ ( K , arrowheads ) is increased , leading to the decreased area of the D-fate marker expression ( C , K ) .",
"In rho bnl double mutants ( D ) , rho btl double mutants ( H ) or Df ( H99 ) rho btl triple mutants ( L ) , the P-fate area is further expanded with concomitant reduction of the D-fate markers .",
"Note that in D , L expression of the D-fate markers becomes reduced very much in a few segments ( asterisk ) .",
"Scale bar is 2 μm in the enlarged panels of I-L where the P-fate cells are bracketed .",
"Scale bar in the remaining panels is 50 μm .",
"( M , N )",
"Scatter plots of the number of P0144-lacZ positive P-fate cells in Tr5 and Tr6 of the indicated genotypes at stage 16 .",
"All p-values were calculated by Student’s t-test .",
"Source files are supplied in Figure 2—source data 1 , 2 .",
"( O ) A scheme of P/D-fate selection by RTK signaling components .",
"RTK , receptor tyrosine kinase . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 00510 . 7554/eLife . 09646 . 006Figure 2—source data 1 . Source data for Figure 2M . The number of P0144-lacZ-positive P-fate cells in Tr5 and Tr6 of the indicated genotypes at stage 16 . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 00610 . 7554/eLife . 09646 . 007Figure 2—source data 2 . Source data for Figure 2N . The number of P0144-lacZ-positive P-fate cells in Tr5 and Tr6 of the indicated genotypes at stage 16 . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 00710 . 7554/eLife . 09646 . 008Figure 2—figure supplement 1 . Multiple roles of dEGFR and btl/dFGFR signaling .",
"( A–D )",
"RTKs promote cell survival of the tracheal cells .",
"Single metameres in stage 13 embryos are marked with arrowheads .",
"Compared to the control , many tracheal cells become positive for TUNEL signals in btl mutants ( B ) .",
"Upon loss of both RTKs , dEGFR and btl/dFGFR ( C ) , more cells become positive for TUNEL signals , compared to either single mutants ( B , D ) .",
"( E–J )",
"Branching phenotypes and rescue of dEGFR btl/dFGFR double mutants .",
"Single metameres in stage 15 embryos are marked with arrowheads .",
"In dEGFR btl/dFGFR double mutants ( E ) , there is no branching and almost no mab2A12 signals .",
"By suppressing apoptosis with Df ( H99 ) ( F ) or DIAP overexpression ( G ) , as well as by expression of RasV12 ( H ) , dEGFR ( I ) , or btl/dFGFR ( J ) mab2A12 signals are significantly restored .",
"( K–P )",
"Roles of RTK signaling components in the P/D fate selection .",
"Compared to the control ( K ) , in rho mutants ( L ) , repression of the P fate marker upd in the D cells is retarded at stage 11/12 ( arrowheads ) .",
"In Df ( H99 ) dEGFR double mutants ( M ) , there are metameres with significantly less P0144-lacZ-positive cells than in Df ( H99 ) rho double mutants ( arrowheads ) .",
"Df ( H99 ) dEGFR btl/dFGFR triple mutants ( N ) additionally lose mab2A signals in some metameres ( arrowheads ) .",
"Ras85D maternal and zygotic mutants harboring Df ( H99 ) ( O ) have similar phenotypes as Df ( H99 ) dEGFR btl/dFGFR triple mutants ( arrowheads ) .",
"In Df ( H99 ) pnt double mutants ( P ) , P0144-lacZ-positive cells are increased in number as in Df ( H99 ) rho double mutants ( arrowheads ) .",
"Scale bar: 50 μm .",
"RTKs , receptor tyrosine kinases . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 008 The expression of two key components of RTK signaling , rho in the central part of the airway primordia and btl in the extending distal airways , partly depends on the POU-domain transcription factor vvl ( Anderson et al . , 1996; Llimargas and Casanova , 1997 ) .",
"We confirmed that in vvl mutants , expression of the D-fate markers rho and btl is reduced ( Figure 3A , B , E , F ) .",
"Moreover , mAb2A12 staining is almost diminished ( Figure 3N , O ) in the residual distal branches , which is restored by UAS-vvl driven with btl-gal4 ( Figure 3—figure supplement 1T ) .",
"By contrast , the expression of the P-fate marker upd is expanded to the distal branches at stage 12 ( Figure 3J–K ) and the P0144-lacZ domain is significantly increased at stage 15 in vvl mutants ( Figure 2N , 3O ) .",
"This indicates that vvl promotes the D-fate at the expense of the P-fate , and a part of this function may be achieved through vvl-dependent expression of rho and btl .",
"In wild type , vvl expression is enriched in the distal trachea , indicating that vvl itself is a D-fate marker ( Figure 3—figure supplement 1A–C ) . 10 . 7554/eLife . 09646 . 009Figure 3 . Hh and Vvl drive the D-fate selection . Expression of the D-fate markers ( rho at stage 11 in A–D , btl at stages 11-13 in E–I and mab2A12 at stages 15/16 in N–Q ) and the P-fate markers ( upd at stages 12/13 in J–M , P0144-lacZ in N-Q ) upon loss of vvl and/or hh .",
"Note that the genotypes in ( N-Q ) additionally carry the Df ( H99 ) mutation .",
"Compared to the control ( column 1 ) , upon loss of vvl ( column 2 ) , the expression area of the D-fate markers rho ( B ) , btl ( F ) , and mab2A12 ( Q ) is decreased while expression of the P-fate markers upd ( K ) and P0144-lacZ ( Q ) is expanded .",
"Note that mab2A12 signals are hardly detectable in O . hh mutants ( column 3 ) show the same trend as vvl mutants .",
"Expression of btl ( G ) and mab2A12 ( P ) is variably lost in Tr3/4 and upd expression ( P ) expands to the whole distal area .",
"In hh vvl double mutants ( column 4 ) , expression of the D-fate markers ( D , I ) is lost , while expression of the P-fate markers ( M , Q ) persist in the whole trachea .",
"Note that weak btl expression is detectable in the primordia ( H ) but is lost soon after ( I ) .",
"Scale bar: 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 00910 . 7554/eLife . 09646 . 010Figure 3—figure supplement 1 . Hh , Vvl , and RTKs collaborate to drive the D-fate selection .",
"( A–D ) vvl expression .",
"vvl is expressed in the epidermis and in the central/distal part of each primordium/metamere ( A ) .",
"At stage 13 ( B ) , tracheal vvl expression is predominant in the D-cells that are negative for P0144-lacZ .",
"Compared to the wild type ( C ) , vvl expression is variably decreased in hh mutants ( D ) .",
"( E–I )",
"Comparison of double or triple mutants of vvl and RTKs .",
"Loss of vvl and dEGFR signaling: vvl rho double mutants ( E ) or Df ( H99 ) vvl rho triple mutants ( J ) .",
"Loss of vvl and btl/dFGFR signaling: vvl bnl double mutants ( F ) or Df ( H99 ) vvl btl triple mutants ( K ) .",
"Loss of vvl and RTK signaling: vvl rho bnl triple mutants ( G , H ) and Df ( H99 ) vvl rho btl quadruple mutants ( L ) .",
"Compared to loss of vvl and single RTK signaling components , upon loss of vvl and 2 RTK signaling factors , more cells lose expression of the D fate marker btl ( E-H ) .",
"Note that in vvl rho bnl triple mutants ( G , H ) , btl expression initiates in the primordia ( G ) but is not maintained ( H ) .",
"Instead , the tracheal cells express the P-fate marker upd ( I ) .",
"A similar trend is seen for another pair of the P/D cell fate markers , mab2A12 and P0144-lacZ ( J-L ) .",
"( M–S )",
"Comparison of double and triple loss of hh and dEGFR signaling .",
"Loss of hh and dEGFR signaling: hh rho double mutants ( M ) or Df ( H99 ) hh rho triple mutants ( Q ) .",
"Loss of hh and btl/dFGFR signaling: hh bnl double mutants ( N ) or Df ( H99 ) hh btl triple mutants ( R ) .",
"Loss of hh and 2 RTK signaling components: hh rho bnl triple mutants ( O ) and Df ( H99 ) vvl btl rho quadruple mutants ( S ) .",
"Compared to loss of hh and a single RTK signaling factor , upon loss of hh and 2 RTK signaling components , more cells lose expression of the D fate markers btl ( O ) and mab2A12 ( R ) .",
"Concomitantly , many of the tracheal cells become positive for the P-fate marker upd ( P ) and P0144-lacZ ( S ) .",
"( T ) Loss of mab2A12 signal in vvl mutants is partially restored by btl-gal4 mediated overexpression of vvl .",
"Scale bar: 50 μm .",
"RTK , receptor tyrosine kinase . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 010 The residual expression of the RTK signaling components in vvl mutant may function as D-factors in the absence of vvl .",
"To evaluate the relative contributions of vvl and RTKs in the P/D fate selection , we examined double or triple mutants of vvl and components of the two RTK signaling pathways .",
"Compared to single or double mutants ( Figure 3—figure supplement 1E , F ) , in vvl rho bnl triple mutant embryos , the expression of the D-fate marker btl is initiated at stage11 but is not maintained by stage 12 ( Figure 3—figure supplement 1G , H ) .",
"Consistently , the mab2A12 signal is undetectable in vvl rho btl mutants ( Figure 3J–L ) .",
"In contrast , expression of the P-fate markers upd and P0144-lacZ appears to be expressed in all tracheal cells ( Figure 2N , Figure 3—figure supplement 1I , L ) .",
"These results suggest that the two RTK pathways act both downstream of and in parallel to vvl to drive the D-fate selection at the expense of the P-fate .",
"Hh is expressed just anteriorly to the airway primordia ( Glazer and Shilo , 2001 ) and is proposed to enhance the primordial vvl expression ( Llimargas and Casanova , 1997 ) .",
"We confirmed that in hh mutants , vvl expression is variably decreased or lost ( Figure 3—figure supplement 1D ) .",
"Consistently , expression of the D-fate markers rho and btl is also variably decreased or lost in hh mutants ( Figure 3C , G , in 6 out of 26 embryos for btl expression ) .",
"In contrast , expression of the P-fate marker upd is expanded and sometimes occupies the whole trachea in some segments of hh mutants ( Figure 3L ) .",
"A similar effect is observed on the expression of a different set of the P/D fate markers , mab2A12/Gasp and P0144-lacZ ( Figure 3P ) .",
"These results suggest that Hh signals from the anterior border of the airway primordia to orient the P/D fate selection toward the D-fate direction .",
"Similar to the cooperative activities of rho , btl , and vvl , hh could synergize with vvl to drive the D-fate .",
"Indeed , in the absence of both hh and vvl , although expression of the D-fate markers rho and btl is variably detected in the primordia ( Figure 3D , H ) , btl expression is completely absent later ( Figure 3I ) .",
"Correspondingly , the expression of P-fate markers expands to all tracheal cells ( Figure 3M , Q ) .",
"A similar trend was observed in mutants for hh and RTK signaling components ( Figure 3—figure supplement 1M–S ) .",
"Taken together , we conclude that Hh and Vvl cooperatively drive the D fate selection , partly through the activation of dEGFR and btl/dFGFR signaling in the central part of the primordium and in the distal branches .",
"The identification of several signaling pathways converging in the initiation and establishment of the D-fate in the airways prompted us to interrogate what promotes the P-fate .",
"We revealed the GATA-family TF Grn ( Brown and Castelli-Gair Hombria , 2000; Garces and Thor , 2006; Lin et al . , 1995 ) as the P-fate promoting factor .",
"grn is preferentially expressed in the peripheral parts of the airway primordia from stage 11 on ( Figure 4—figure supplement 1A ) .",
"At stage 13 , the P-fate cells of the SB and the overlying lateral ectoderm are positive for grn ( Figure 4—figure supplement 1B–C ) .",
"grn expression is repressed in the D-cells by the D-fate determinants , hh , vvl , and RTKs ( Figure 4—figure supplement 1D–G ) , while grn expression in the lateral ectoderm is largely intact in the absence of tracheal cells in upd or trh mutants ( Figure 4—figure supplement 1H–I ) .",
"Grn overexpression results in the upregulation of the P-fate marker P0144-lacZ in the more distal TC branches ( Figure 4A–C ) .",
"Conversely , in grn mutants , expression of the P-fate markers upd and P0144-lacZ is lost ( Figure 4D–F , Figure 4—figure supplement 1J ) , resulting in mAb2A12-positive tubes directly connecting to the epidermis .",
"Counting the Trh-positive cells in Tr5 and in TC5/SB5 reveals that around 10 cells are selectively lost from the TC/VB region ( Figure 4G ) .",
"As halving of the airway cell number in CycA mutants ( Beitel and Krasnow , 2000 ) does not abolish the P-fate ( Figure 4H , Figure 4—figure supplement 1K ) , we suggest that reduction of cell number does not account for the loss of the P-fate in grn mutants .",
"Also , arm-gal4-mediated overexpression of Trh in grn mutants does not restore the P-fate ( Figure 4H , Figure 4—figure supplement 1L ) .",
"These results suggest that grn functions in two ways , namely to establish Trh expression in the P-region and to induce the P-fate marker expression ( Figure 4K ) .",
"Accordingly , we designate grn a master regulator of the P-fate . 10 . 7554/eLife . 09646 . 011Figure 4 . Grn promotes the P fate . Compared to the control ( A ) , grn overexpression with arm-gal4 ( B ) or two copies of btl-gal4 and trh-gal4 ( C ) slightly expands the area of P0144-lacZ positive cells to more distal TC areas , especially in Tr2-5 ( B-C , arrows ) .",
"In grn mutants , expression of the P-fate markers upd ( D ) or P0144-lacZ ( E ) disappears while arm-gal4-mediated Grn overexpression restores P0144-lacZ expression in the proximal cells ( F ) .",
"( G ) Scatter plots of the Trh-positive cell numbers in the whole Tr5 or in the SB/TC subregion of the indicated genotypes at stage 14 ( mean ± SEM , N = 5; all p-values were calculated by Student’s t-test ) .",
"The cell number is decreased by around 10 cells in grn mutants in Tr5 and in the SB/TC subregion .",
"A source file is supplied in Figure 4—source data",
"1 . ( H ) Scatter plots of the number of metameres with any number of P0144-lacZ-positive cells of the indicated genotypes at stages15/16 ( NS: not significant by Student’s t-test ) .",
"Note that grn mutants occasionally possess a few P0144-lacZ cells in anterior metameres .",
"A source file is supplied in Figure 4—source data",
"2 . ( I ) summarizes the two functions of grn in P-fate promotion .",
"Scale bar: 50 μm .",
"SB , spiracular branch; SEM , standard error of the mean , TC , transverse connectives . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 01110 . 7554/eLife . 09646 . 012Figure 4—source data 1 . Source data for Figure 4G . The Trh-positive cell numbers in the whole Tr5 or in the SB/TC subregion of the indicated genotypes at stage 14 . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 01210 . 7554/eLife . 09646 . 013Figure 4—source data 2 . Source data for Figure 4H . The number of metameres with any number of P0144-lacZ-positive cells of the indicated genotypes at stages 15/16 . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 01310 . 7554/eLife . 09646 . 014Figure 4—figure supplement 1 . Regulation and function of grn expression .",
"( A–C ) grn expression in wild type .",
"At stage 11 ( A ) , grn is preferentially expressed in the periphery of the tracheal primordia ( arrowheads ) .",
"At stage 13 ( B , C ) , grn is detected in the P-cells ( B ) positive for P0144-lacZ ( C ) ( arrowheads ) as well as in the lateral ectoderm .",
"( D–I ) grn expression in mutants .",
"grn expression expands to the D-cells in hh vvl double mutants ( D , stage 12 ) or vvl rho btl triple mutants ( E , stage 12/13 ) .",
"Loss of two RTK signaling components ( F , stage 12 , arrowheads in Tr7 ) or the downstream mediator Ras85D ( G , stage 12 ) has weaker effects compared to hh vvl double mutants and the distal area variably becomes positive for grn ( arrowheads ) .",
"Note that the Df ( H99 ) mutation is introduced in ( F , G ) to suppress apoptosis .",
"In Df ( upd ) ( H , stage 13 ) or trh mutants ( I , stage 13 ) , grn expression in the lateral ectoderm is largely intact , with some defects in Df ( upd ) mutants .",
"Expression of the P-fate marker upd is abolished in grn mutants ( J , stage 13 ) .",
"In CycA mutants ( K , stage 15 ) , the number of P0144-lacZ-positive cells becomes reduced but not abolished , while arm-gal4-mediated overexpression of trh does not restore P0144-lacZ expression in grn mutants ( L , stage 15 ) .",
"Scale bar: 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 014 Having identified several regulators of the D- and P-fates , we examined their epistatic relationships .",
"Compared to the control ( Figure 5A , Figure 5—figure supplement 1E , J ) , in grn hh double- ( Figure 5C , Figure 5—figure supplement 1G , L ) or grn rho btl triple mutants ( Figure 5—figure supplement 1I , N ) , the P-fate marker expression is not robustly restored .",
"In grn vvl double mutants , the trh expression area appears to be very much reduced ( Figure 5F ) , probably because the P-fate cells depend on grn for trh expression .",
"However , in this reduced area of the distal region , we detected weak restoration of the P-fate markers upd and P0144-lacZ ( Figure 5B , Figure 5—figure supplement 1F , K ) .",
"These results may suggest the presence of another factor in the distal area that promotes the P-fate when grn and vvl are inactivated .",
"Alternatively , the default airway cell fate is the proximal and multipotent cell fate ( the P-fate ) and an important aspect of the grn functions in P-fate selection/promotion maybe to antagonize the D-factor vvl .",
"In the absence of both the D- and P-factors ( Figure 5H , Figure 5—figure supplement 2A–B ) , trh expression initiates at stage 10 but becomes drastically reduced by stage 12/13 ( Figure 5E–H ) .",
"In these mutants ( Figure 5D , Figure 5—figure supplement 1D , H , M , Figure 5—figure supplement 2A–B ) , the expression of both proximal and distal markers is almost completely eliminated ( Figure 5A–D . Figure 5—figure supplement 1A–N ) .",
"We conclude that the regulators of the D- and P-fate cooperatively promote and maintain the airway cell identity defined by trh expression .",
"Below , we first describe roles of the embryo axis determinants on P/D-fate selection and then proceed to dissect the function of D-factors by gain-of-function approaches . 10 . 7554/eLife . 09646 . 015Figure 5 . Genetic interactions of grn , hh , and vvlExpression of the P-fate marker upd ( A–D , stages 12-13 ) or the pan-tracheal marker trh ( E–H , stages 12-13 ) are shown .",
"Loss of upd expression in grn mutants ( A ) is partially reversed by vvl mutation ( B , arrowheads ) but not by hh mutation ( C ) .",
"In the absence of all , grn , hh , and vvl , upd expression is virtually lost ( D ) .",
"This accompanies severe reduction of trh expression ( H , arrowheads ) , compared to either grn ( E ) , grn vvl ( F ) , or grn hh double mutants ( G ) .",
"Note the trh-lacZ expression in H , reflecting initial trh expression at the primordia stage .",
"Also , expression of trh and upd in Tr1 is often detected at stage 12 .",
"Asterisks in G marks loss of trh expression in Tr3 .",
"Scale bar: 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 01510 . 7554/eLife . 09646 . 016Figure 5—figure supplement 1 . Genetic analysis of grn , hh , vvl , and RTK components . Expression of the D-fate marker ( btl in A–D or mab2A12 in E–I ) or the P-fate marker ( P0144-lacZ in E–N ) is shown .",
"Compared to grn ( A ) , grn vvl ( B ) , or grn hh ( C ) mutants , in grn hh vvl triple mutants , btl expression is largely lost as in hh vvl double mutants .",
"Loss of P0144-lacZ expression in grn mutants ( E , J ) is partially restored in grn vvl double mutants ( F , K ) but not significantly in grn hh double mutants ( G , L ) or in grn rho btl triple mutants ( I , N ) .",
"Restoration of the P-fate in grn vvl double mutants is suppressed in grn vvl hh triple mutants ( H , M ) .",
"Note the presence of Df ( H99 ) mutation in J–N .",
"Arrowheads in E-N mark the proximal location of the tracheal cells .",
"Scale bar: 50 μm .",
"RTK , receptor tyrosine kinase . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 01610 . 7554/eLife . 09646 . 017Figure 5—figure supplement 2 . Phenotypes caused by simultaneous loss of the D- and P-factors . Expression of pan-tracheal marker ( trh ) and the P-fate marker ( P0144-lacZ ) .",
"Either in Df ( H99 ) grn hh vvl quadruple ( A ) or Df ( H99 ) grn vvl rho btl quintuple mutants ( B ) , the P-fate is not established and Trh expression becomes significantly reduced by stage 13/14 .",
"Arrows point persistent Trh expression in part of the posterior spiracle , which originates from a primordium specified in connection to Tr10 ( Hu and Castelli-Gair , 1999 ) .",
"Scale bar: 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 017 A key aspect of the P/D-fate selection in the Drosophila airways is the separation of the trh positive airway primordia into a D-fate circle ( vvl , rho and btl positive ) surrounded by a concentric P-fate ring ( grn and upd positive ) .",
"To investigate the origins of this rough setup , we examined the effects of Wg/WNT and Dpp/BMP ( Figure 1B ) , two major axis determinants along the AP or DV axis , respectively ( DiNardo et al . , 1994; Wharton et al . , 1993 ) .",
"In wg/WNT mutants , expression of the D-fate markers vvl ( de Celis et al . , 1995 ) , mab2A12 ( Llimargas , 2000 ) , and btl expands along the AP axis to cover most , if not all lateral parts of the embryos as a single band ( Figure 6A–B , I , Figure 6—figure supplement 1B ) .",
"On the other hand , expression of the P-fate markers grn , upd , and P0144-lacZ also expands along the AP axis , but this time , approximately forms two narrow stripes , dorsal and ventral , sandwiching the D-fate ( Figure 6C , I , Figure 6—figure supplement 1A–B ) .",
"These results suggest that Wg/WNT confines both the D- and P-fates along the AP axis ( Figure 6I ) . 10 . 7554/eLife . 09646 . 018Figure 6 . Regulation of the P/D fate selection by Wg/WNT and Dpp/BMP . Expression regions of the D-fate markers ( vvl in A , D , btl in B , E or Gasp in H ) or the P-fate markers ( upd in C , F or P0144-lacZ in H ) are shown for wg mutants ( A–C ) , dpp hypomorph ( dpphr92 ) ( D–F ) or dpphr92 wg double mutants ( H ) .",
"( D-F ) are dorsal views where both left ( L ) and right ( R ) sides are seen .",
"In wg mutants , expression domains of the D-fate markers expand along the AP axis ( A , B ) , and form a single lateral band ( bracket ) with occasional interruptions before the initiation of primordia invagination ( A , stage 10 ) .",
"After invagination ( B , C , stage 12 ) , some D-fate cells remain exposed at the embryo surface to form a lateral band ( B ) .",
"The P-fate markers form a dorsal and a lateral band of expression at the ectoderm ( C , arrowheads ) .",
"In dpphr92 mutants ( D-F ) , the airway primordia expand to the dorsal midline ( D , stage 10 ) .",
"Concomitantly , both the D-fate ( D , E ) and the P-fate ( F ) expand to the dorsal midline .",
"Note that the P-fate encircles the D-fate ( arrowheads ) and that the anterior cells often do not express the P-fate marker upd in ( F , stage 11/12 ) , probably due to the distalization by Hh that is expressed in the anterior border of each primordium .",
"In dpphr92 Df ( wg ) double mutants ( G , H ) , the whole dorsal area of the trunk/abdominal body parts become Trh positive at stage 11 ( G ) .",
"At stage 14/15 ( H ) , the P-fate ( arrowheads ) is specified abutting the D-fate only at the ventral edge .",
"Note that in the posterior part , the P-fate is not established as it is not established in the wild type .",
"Scale bar: 50 μm .",
"( I ) Interpretation of data in each genotype regarding specification of the airway field ( left parts ) and P/D-fate selection ( right parts ) .",
"Expression domains of dpp and wg are colored pink and orange , respectively .",
"In wg mutants , the airway field ( light blue ) expands along the AP axis ( arrows ) and the P-fate is established approximately as a dorsal and a ventral stripes ( light green ) .",
"In dpp hypomorphs ( dpphr92 ) , the airway field expands dorsally to the dorsal midline and the P-fate is established in the periphery except for the anterior and dorsal margin .",
"In double mutants of wg and dpp hypomorph , the airway field expands both dorsally and along the AP axis , and the P-fate is only established in a ventral stripe . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 01810 . 7554/eLife . 09646 . 019Figure 6—figure supplement 1 . Role of wg in P/D fate selection . Expression of the P-fate marker grn ( A ) or P0144-lacZ ( B ) or the D-fate marker mab2A12 ( B ) in wg/WNT mutants .",
"Note that at stage 14/15 ( B ) , the P-fate cells ( arrowheads ) that reside at the embryo surface surround the D-fate cell fraction that still remain at the surface ( bracket ) .",
"Scale bar: 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 01910 . 7554/eLife . 09646 . 020Figure 6—figure supplement 2 . Roles of dpp in P/D fate selection .",
"( A–X )",
"Expression of the D fate markers ( vvl in A , F , btl in B , C , G ) or the P-fate markers ( upd in D , H or grn in E , I , J ) in dpp null mutants ( dppH46 ) ( A-E ) , dppH46/dpphr92 heterozygotes ( F-I ) or dpphr92 homozygotes .",
"In dppH46 mutants , expression of both the D-fate ( A-C , arrowheads ) and the P-fate ( D , E ) is not established .",
"Note that the weak early expression of btl ( B , arrowheads ) is not maintained ( C ) .",
"In dppH46/dpphr92 mutants , both the D fate ( F , G ) and the P fate ( H , I ) are established only around the dorsal midline ( arrowheads ) .",
"In dpphr92 mutants , grn expression surrounding the airway cells expands to the dorsal midline ( J , arrowheads ) .",
"Scale bar: 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 02010 . 7554/eLife . 09646 . 021Figure 6—figure supplement 3 . Roles of D-factors in h expression . Expression of h-lacZ ( A–D , stage 13 ) or h transcript ( E–H , stage 10/11 ) is shown .",
"h-lacZ marks the dorso-distal area of the trachea in btl mutants ( A , arrowheads in Tr5 ) as in the wild type .",
"This distal expression is significantly reduced ( arrowheads ) in hh ( B ) , rho ( C ) or pnt mutants ( D ) .",
"Compared to the control ( E ) , in wg mutants ( F ) h expression slightly expands along the AP axis with many gaps remaining .",
"Upon overactivation of hh in wg mutants by hh overexpression ( G ) or ptc mutation ( H ) , h expression expands along the AP axis to form a continuous band .",
"Scale bar: 50 μm .",
"AP , anterior-posterior . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 021 The dorsal and ventral stripes of the proximal markers surrounding the expanded airways in wg/WNT mutants suggest that there are cues driving the P/D fate selection along the DV axis .",
"Dpp/BMP presents a top candidate for such a signal ( Isaac and Andrew , 1996; Wilk et al . , 1996 ) .",
"In the absence of dpp , trh expression expands to the dorsal midline ( Figure 6—figure supplement 2A , D ) ( Isaac and Andrew , 1996 ) and the cells initiate btl expression ( Figure 6—figure supplement 2B ) .",
"However , neither the D-fate nor the P-fate is established ( Figure 6—figure supplement 2A–E ) , suggesting that Dpp also functions as a positive regulator of early airway development , in addition to its repressive effect on the earliest trh expression .",
"In dpp hypomorphs ( Figure 6D–F , Figure 6—figure supplement 2F–J ) , trh expression is expanded to the dorsal midline and P/D specification is confined near the dorsal midline , depending on the allele severities or the residual Dpp/BMP activity in the hypomorphic conditions .",
"In the dorsally expanded primordia of dpphr92 homozygotes , expression of the D-factors vvl and btl is detected as stripes straddling the dorsal midline ( Figure 6D–E ) , and P-fate marker expression typically encircles the entire airway primordium except for its anterior border ( Figure 6F , I , Figure 6—figure supplement 2J ) .",
"The loss of the P-fate markers at the anterior border in dpphr92 homozygotes could be due to the activity of the D-fate inducer Hh , which is expressed just anterior to the airway primordia ( Figure 1B ) ( Glazer and Shilo , 2001 ) .",
"Consistent with the Hh-dependent anterior bias of the D-fate selection , the expression of the dorsal-distal marker h-lacZ , is lost in hh mutant ( Figure 6—figure supplement 3A–B ) .",
"Similarly , expression of h-lacZ is lost in rho and pnt mutants but not in btl mutants ( Figure 6—figure supplement 3A , C , D ) .",
"On the other hand , overactivation of Hh signaling in wg/WNT mutants expands h expression along the AP axis ( Figure 6—figure supplement 1N–Q ) .",
"Collectively , this analysis suggests that the graded Dpp/BMP activity controls the P/D fate selection at the dorsal border of the airway primordia .",
"Wg/WNT signals impinge on the P/D-fate selection along the AP axis , while Hh modulates P/D differentiation from the anterior border .",
"Consistent with the predominant roles of Dpp/BMP and Wg/WNT in airway primordia specification , in Df ( wg ) dpphr92 double mutants , the whole dorsal area of the trunk/abdominal parts of the embryo takes the airway cell fate ( Figure 6G , I ) .",
"In this situation , the dorsal part of the airway primordia takes the D-fate and only the ventral periphery takes the P fate ( Figure 6H , I ) , supporting our model that Dpp/BMP and Wg/WNT are key regulators of the P/D fate selection .",
"Several other cues including inducers of the airway fate ( Brown et al . , 2001 ) are expected to contribute to the P/D fate selection .",
"At the ventral border for example , apart from the dorso-ventral gradient of Dpp/BMP , the ventro-dorsal gradient of dEGFR signaling ( Gabay et al . , 1997; Golembo et al . , 1996; Raz and Shilo , 1993 ) and another cue originating from the ventro-lateral gradient of the TF Dorsal ( Anderson et al . , 1985a; Roth et al . , 1991; Zhao and Skeath , 2002 ) may orient the P/D fate selection .",
"The exact mechanisms by which Wg/WNT , Dpp/BMP , and these other cues impinge on P/D fate selection await future research and the development of appropriate cell-specific gene inactivation methods .",
"In order to investigate how the P- and D-fates are balanced during airway cell differentiation , we analyzed the effects of overactivation of the D-fate determinants , namely RTK signaling , hh signaling and vvl .",
"argos ( aos ) is a secreted negative feedback regulator of dEGFR signaling ( Schweitzer et al . , 1995a ) while anterior open ( aop ) encodes an ETS TF that antagonizes RTK signaling ( Rebay and Rubin , 1995 ) .",
"Upon activation of RTK signaling , aos is induced , while Aop is excluded from the nucleus and becomes degraded .",
"In the airway primordia , aos expression gradually expands from the center to the periphery ( Figure 7—figure supplement 1A–E ) .",
"At early stage 12 , both aos and Aop are preferentially detected in the proximal airways ( Figure 7—figure supplement 1E–F ) .",
"In aop mutants , the number of cells expressing P-fate markers is very much reduced ( Figure 7A , Figure 7—figure supplement 2A ) .",
"Moreover , compared to either of the single mutants ( Figure 7B , Figure 7—figure supplement 2A ) , in aop aos double mutants , expression of the P-fate marker P0144-lacZ is virtually abolished ( Figure 7C , Figure 7—figure supplement 2A ) .",
"Similar phenotypes are observed upon tracheal overexpression of RasV12 ( Figure 7D ) or s-spi ( Figure 7E ) with btl-gal4 or trh-gal4 in an aos mutant background .",
"In double mutants of aos and Gap1 , a GTPase-activating protein for Ras85D ( Gaul et al . , 1992 ) , expression of the P-fate markers upd and grn is variably reduced or abolished ( Figure 7G , H ) .",
"The expression of another P fate marker P0144-lacZ is also lost ( Figure 7F , Figure 7—figure supplement 2A–B ) .",
"Concomitantly , the distal mAb2A12/Gasp marker staining appears on the embryo outer surface ( Figure 7E–F ) .",
"These results suggest that aos , Gap1 , and aop are parts of a negative regulatory mechanism balancing the D-fate inducing activity of RTK signaling to assure P-fate selection . 10 . 7554/eLife . 09646 . 022Figure 7 . Overactivation of the D-fate determinants abrogates the P-fate specification .",
"( A–I )",
"Effects of RTK signaling overactivation .",
"Proximal areas are marked by arrowheads in Tr7 .",
"In aop mutants ( A ) , expression of the P-fate marker P0144-lacZ is variably decreased or lost .",
"Compared to either single mutant ( A , B ) , in aop aos double mutants ( C ) , P0144-lacZ expression is virtually abolished .",
"Note that the epidermal signals in C ( brackets ) are from other lineages .",
"aos mutation , combined with RasV12 overexpression by btl-gal4 ( D ) , s-spi overexpression by trh-gal4 ( E ) or Gap1 mutation ( F ) abolish P0144-lacZ expression .",
"Note that in E and F , after enhancement of mab2A12/Gasp signals , the epidermal surface staining is evident ( asterisks ) .",
"In aos Gap1 double mutants , expression of the other P-fate markers ( upd in G and grn in H ) is variably reduced or lost , while grn overexpression ( I ) partially restores P0144-lacZ expression to the proximal areas of aos Gap1 double mutants ( arrows ) .",
"( J–P )",
"Effects of overactivation of hh or vvl .",
"Overactivation of hh signaling by ptc mutation ( J , M ) or hh overexpression ( K , L ) , either in the wild-type background ( J , K ) or wg mutant background ( M , L ) reduces the P0144-lacZ-positive cells .",
"salm-gal4 mediated overexpression cirep increases the area of P0144-lacZ-positive P-fate cells in the dorsal stripe ( N ) .",
"The dorsal and the ventral stripes are marked with D and V in ( M , N ) .",
"vvl overexpression reduces or abolishes the expression region of the P-fate markers upd ( O ) or P0144-lacZ ( P ) .",
"Scale bar: 50 μm .",
"RTK , receptor tyrosine kinase . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 02210 . 7554/eLife . 09646 . 023Figure 7—source data 1 . Source data for Figure 7—figure supplement 2A . The number of metameres with any number of P0144-lacZ-positive cells of the indicated genotypes at stages 15/16 . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 02310 . 7554/eLife . 09646 . 024Figure 7—source data 2 . Source data for Figure 7—figure supplement 2J . The number of metameres with any number of P0144-lacZ-positive cells of the indicated genotypes at stages 15/16 . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 02410 . 7554/eLife . 09646 . 025Figure 7—figure supplement 1 . Expression of aos and Aop . Expression of aos ( A–E ) or Aop ( F ) during stages 10-11 .",
"aos expression in the primordia ( arrowheads ) expands from the central part of the primordia ( A ) to the peripheral areas ( B ) to cover all the tracheal cells ( C ) .",
"By late stage 11 , aos expression in the distal area decreases ( D arrows in Tr3 ) , while expressions of both aos and Aop are detected in the proximal area ( E , F , arrowheads ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 02510 . 7554/eLife . 09646 . 026Figure 7—figure supplement 2 . Requirement of the overactivated RTK signaling in the abrogation of the P-fate selection . Expression of the D-fate marker mab2A12 and the P-fate marker P0144-lacZ is shown .",
"Scatter plots of the number of metameres with any number of P0144-lacZ-positive cells of the genotypes at stages 15/16 relating to aos Gap1 mutants or overexpression of RasV12 , s-spi or vvl are summarized in ( A , J ) .",
"All p-values were calculated by Student’s t-test .",
"*p < 5 × 10−2 , ***p < 1 × 10−3 , ****p < 1 × 10-4 , NS: Not significant .",
"Source file is supplied in Figure 7—source data 1 , 2 .",
"In Gap1 mutants ( B ) , P/D fate selection is comparable to aos mutants .",
"Loss of P0144-lacZ expression in aos Gap1 double mutants is variably suppressed by mutations of vvl ( C ) , hh ( D ) , rho ( E ) , pnt ( F ) , or Ras85D ( G ) but not by mutations of sim ( H ) or btl ( I ) .",
"Loss of P0144-lacZ expression upon overexpression of s-spi/TGF-alpha ( K ) is variably suppressed by mutations of vvl ( L ) , pnt ( O ) or Ras85D ( P ) , but not by hh mutation ( M ) nor rho btl double mutations ( N ) .",
"Overexpression of Bnl/dFGF does not abolish P0144-lacZ expression ( Q ) while simultaneous overexpression of an active form of btl/dFGFR with its mediator , downstream of FGFR ( dof ) variably diminished P0144-lacZ expression ( R ) .",
"Note that overactivation of RTK signaling in hh mutant background ( D , M ) results in expansion of the field expressing airway markers .",
"Scale bar: 50 μm .",
"RTK , receptor tyrosine kinase . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 02610 . 7554/eLife . 09646 . 027Figure 7—figure supplement 3 . Effects of vvl or s-spi overexpression in wg/WNT mutant embryos . Compared to single wg/WNT mutants ( A , arrowheads ) , the P0144-lacZ-positive P-fate is not established upon arm-gal4-mediated overexpression of vvl ( B , asterisks ) or s-spi ( C , asterisks ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 027 The loss of P0144-lacZ expression in aos Gap1 double mutants is partially suppressed by overexpression of the P fate determinant Grn ( Figure 7I , Figure 7—figure supplement 2A ) .",
"Reversion of the aos Gap1 mutant phenotypes is also detected by introducing vvl , hh , rho , Ras85D ( Simon et al . , 1991 ) or pnt mutations but not by btl or by single-minded ( sim ) mutations ( Figure 7—figure supplement 2A–I ) .",
"sim governs the ventral midline cell fate , which is one source of dEGFR signaling before stage 10 ( Golembo et al . , 1996; Mayer and Nusslein-Volhard , 1988 ) .",
"Thus , these results illustrate the essential roles of vvl , hh , and RTK signaling components as D-fate determinants and suggest that neither the CNS midline nor btl/dFGFR are involved in the RTK overactivation observed in the aos Gap1 double mutants .",
"Similarly , arm-gal4 mediated , ubiquitous overexpression of secreted active spitz ( s-spi ) results in loss of the P-fate marker P0144-lacZ ( Figure 7—figure supplement 2J–K ) .",
"This defect is also suppressed by mutations of vvl , pnt , or Ras85D but not by btl , rho , or hh mutations ( Figure 7—figure supplement 2J–P ) .",
"This suggests that vvl has an additional essential role in dEGFR signaling other than facilitating active dEGFR ligand production .",
"In contrast to s-spi , bnl overexpression does not abolish P0144-lacZ expression ( Figure 7—figure supplement 1Q ) .",
"However , simultaneous overexpression of an activated form of btl/dFGFR and its downstream mediator downstream of FGFR ( dof ) ( Imam et al . , 1999; Michelson et al . , 1998; Vincent et al . , 1998 ) reduced P0144-lacZ expression ( Figure 7—figure supplement 1R ) suggesting that dEGFR and btl/dFGFR share the downstream signaling pathway for P/D fate selection .",
"Overactivation of hh signaling , either by mutation of the inhibitory receptor ptc ( Chen and Struhl , 1996; Ingham et al . , 1991 ) or by arm-gal4-mediated hh overexpression , reduces the number of cells expressing P-fate markers ( Figure 7J–K ) .",
"Although overactivation of Hh signaling is expected to increase the expression of Wg/WNT ( DiNardo et al . , 1994 ) , a negative regulator of trh expression ( Wilk et al . , 1996 ) , reduction of the P-fate cell number upon hh overactivation still occurs in wg/WNT mutant backgrounds ( Figure 7M–N ) .",
"Moreover , driving a repressor form of Cubitus interruptus ( Cirep ) , a mediator TF of Hh signaling ( Hepker et al . , 1997; Methot and Basler , 2001 ) at the dorsal ectoderm with salm-gal4 in ptc wg mutants locally increases the P-fate cell number in the dorsal side ( Figure 7N ) .",
"These results imply that overactivation of hh signaling autonomously represses the P-fate selection independent of its effect on wg/WNT .",
"Overexpression of vvl with arm-gal4 , like s-spi overexpression reduces or abolishes the expression of the P-fate markers , upd or P0144-lacZ ( Figure 7O–P ) , and this occurs independently of the presence of wg/WNT ( Figure 7—figure supplement 3A–C ) .",
"Collectively , the analysis indicates that expansion of the D-fate-inducing activities of RTKs , Hh or Vvl is deleterious to the P-fate selection and that the negative regulators of RTK signaling ( aos , aop or Gap1 ) and hh signaling ( ptc ) ensure the selection or maintenance of the P-fate .",
"The hitherto analysis indicates that aos sensitizes the circuit of P/D-fate selection .",
"We noticed that crossing of a weaker driving strain of UAS-RasV12 with arm-gal4 causes loss of P0144-lacZ expression only in the aos mutant background while the stronger UAS-RasV12strain is sufficient to eliminate P0144-lacZ expression on its own ( Figure 8—figure supplement 1A–D ) .",
"Using the weaker UAS-RasV12 strain , we demonstrate that within the D-fate group , the cells of the TC and the remaining primary branches have differential competence ( Figure 8—figure supplement 1E ) .",
"In wild-type embryos , Kni and Knrl are induced in a subset of the distal primary branches ( DB , LT and GB ) in response to Dpp/BMP ( Chen et al . , 1998; Vincent et al . , 1997 ) .",
"arm-gal4-mediated overexpression of Dpp/BMP or the active form of its co-receptor tkvQD ( Nellen et al . , 1996 ) is sufficient to induce ectopic Kni in additional branches ( Figure 8A , B ) ( Vincent et al . , 1997 ) .",
"However , Kni levels in the TC cells is weaker compared to the more distal cells , suggesting that TC cells are less competent in inducing the D-fate marker Kni in response to Dpp/BMP signaling .",
"When both UAS-dpp and the weaker line of UAS-RasV12 are simultaneously driven by arm-gal4 , kni expression becomes homogenously induced in both the TC cells and the more distal cells ( Figure 8C ) .",
"This suggests that graded activity of the D-fate inducers , like RTKs may act to generate three different cell states along the PD axis of the airways .",
"The most distal part ( the primary branches ) is established in response to the highest activity , the intermediate domain ( TC ) requires weaker activity , whereas the level of the D-factors needs to be low in the most proximal part ( Figure 8—figure supplement 1E ) .",
"Slight expansion of the P-fate only in the TC region upon Grn overexpression ( Figure 4A–C ) may also reflect this differential competence of TC and the remaining primary branches . 10 . 7554/eLife . 09646 . 028Figure 8 . TC cells may define an intermediate fate between the more distal primary branches and the SB . Expression of Kni is shown together with the D-fate marker btl-CD8GFP and the P-fate marker P0144-lacZ upon overexpression of dpp/BMP and/or RasV12 .",
"Arrowheads and arrows mark the P-cells and TC in Tr7 , respectively .",
"Compared to the control , dpp/BMP overexpression induces Kni expression in all the D-cells .",
"But the Kni expression level is weaker in TC .",
"The differential Kni expression levels in the primary branches and TC ( B ) become equalized by additional overexpression of RasV12 ( C ) .",
"Upon RasV12 overexpression alone ( D ) , Kni expression becomes detected along the TC .",
"Note that in all cases , P0144-lacZ-positive P-cells do not express Kni .",
"Scale bar: 50 μm .",
"SB , spiracular branch; TC , transverse connectives . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 02810 . 7554/eLife . 09646 . 029Figure 8—source data 1 . Source data for Figure 8—figure supplement 1D . The number of metameres displaying P0144-lacZ-positive cells in the indicated genotypes at stages 15/16DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 02910 . 7554/eLife . 09646 . 030Figure 8—figure supplement 1 . Characterization of the effects of the weaker and the stronger UAS-RasV12 lines . Driving the stronger UAS-RasV12 line/RasV12 ( s ) with arm-gal4 ( A ) causes loss of P0144-lacZ expression ( arrowheads in Tr7 ) , while the weaker UAS-RasV12 line/RasV12 ( w ) ( C ) needs aos mutation ( B ) to cause loss of P0144-lacZ expression ( arrowheads in Tr7 ) .",
"Scale bar: 50 μm .",
"( D ) Scatter plots of the number of metameres displaying P0144-lacZ-positive cells in the indicated genotypes at stages 15/16 .",
"All p-values were calculated by Student’s t-test .",
"**p < 2 × 10−3 , ****p < 1 × 10−4 .",
"A source file is supplied in Figure 8—source data",
"1 . ( E ) illustrates the separation of the airway tree into three different developmental competences , SB ( P-fate ) , TC and the remaining primary branches ( both D-fates ) .",
"SB , spiracular branch; TC , transverse connectives . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 030 The early development of the airway primordia includes two essential aspects: first , the selection and maintenance of the airway field , and second , the selection of the P/D-fates , both of which originate from the AP and DV axis determinants of the embryo ( Figure 9 ) .",
"More specifically , we identified a positive role of Dpp/BMP and the P/D fate factors in the establishment of the airway field .",
"In the absence of Dpp/BMP or both P/D fate regulators , the airway field is lost .",
"The latter phenotype could be explained in the following way in terms of P/D fate selection .",
"Without the D-factors , the airway field is expected to uniformly take the P-fate that is promoted by grn .",
"In this situation , grn becomes indispensable for maintenance of trh expression in the main airways .",
"However , Wg/WNT , Hh , Dpp/BMP and other cues generate a centrally enriched expression field of the D-fate determinants vvl , rho , and btl , which then cooperate to repress the P fate , leaving the grn-trh regulation operative only in the P-region .",
"Restoration of the P-fate in grn vvl double mutants suggests that the proximal multipotent fate is the default state for airway cells or that there is another factor that promotes the P-fate in the absence of grn and vvl . 10 . 7554/eLife . 09646 . 031Figure 9 . Genetic circuits explaining the early development of the Drosophila airways .",
"( A ) Establishment of the airway field is regulated by Dpp/BMP and Wg/WNT .",
"In each metameric unit of the Drosophila embryo ( parasegment , PS ) ( Sanson , 2001 ) , an airway primordium is specified just posterior to the hh expression domain ( a posterior compartment of a segment , P ) .",
"This is combinatorially controlled along the DV and AP axis .",
"Dpp/BMP expressed in the dorsal region functions as both a repressor ( Isaac and Andrew , 1996; Wilk et al . , 1996 ) and an activator of the airway field ( this study ) while Wg/WNT functions as a repressor along the AP axis .",
"( B ) Initiation of the P/D-fate selection by Dpp/BMP , Wg/WNT , and Hh .",
"Each airway primordium is roughly subdivided into two regions , anterior-central ( D-fate , dark green ) and the peripheral ( P-fate , light green ) .",
"The patterning cues along the AP and DV embryo axis roughly set up this radial patterning .",
"Along the DV axis , Dpp/BMP expressed in the dorsal region functions to discriminate the P/D-fates , at least at the dorsal edge .",
"Dpp/BMP may also discriminate the P/D-fates at the ventral edge .",
"Alternatively , ventral cues dependent on the Dorsal TF gradient may discriminate the P/D-fates at the ventral edge .",
"Along the AP axis , Wg/WNT expressed in transverse stripes may discriminate the P/D-fates at each edge .",
"Hh from the anterior border stimulates the D-fate .",
"( C ) Establishing the P/D-fates by the P/D-factors .",
"The ordered Invagination of the primordia converts the centro-peripheral patterning to the proximo-distal organization along the airway tree .",
"By completion of invagination , D-factors establish the D-fate at the expense of the P-fate .",
"Within the D-fate , there are two groups of cells having different responsiveness to overexpression of Dpp/BMP or Grn , e . g . , TC ( green ) and the primary branches ( dark green ) .",
"See text for details .",
"AP , anterior-posterior; DV , dorso-ventral . DOI: http://dx . doi . org/10 . 7554/eLife . 09646 . 031 Intriguingly , the Drosophila legs and the trachea are supposed to have evolved form common ancestor appendages ( Franch-Marro et al . , 2006 ) .",
"The proximo-distal patterning mechanisms of these two organs are similar in that RTK activation distalizes the field ( Campbell , 2002; Galindo et al . , 2002 ) while initiation of the distal identity is conferred by the same set of signaling molecules ( Dpp/BMP , Wg/WNT , and Hh ) from the outside ( for the airways ) or the inside ( for the legs ) ( Estella et al . , 2012 ) .",
"The proximal location of multipotent cells in the Drosophila airway tree resembles the conspicuous proximal localization of multipotent tracheal basal cells in the mouse lung ( Rock et al . , 2009; Rock et al . , 2010 ) Given the prominent roles of FGFR signaling in branching morphogenesis in both flies ( Sutherland et al . , 1996 ) and mice ( Min et al . , 1998; Sekine et al . , 1999 ) , the identification of a genetic mechanism for P- and D-fate selection in flies may aid future studies aiming to identify the mechanisms that spatially confine multipotent cell selection in the embryonic vertebrate lung ."
],
[
"Flies kept over balancer chromosomes ( Lindsley et al . , 1992 ) were grown in standard medium .",
"We obtained the appropriate genotypes by standard genetic crosses .",
"For overexpression of genes , we used the Gal4/UAS system ( Brand and Perrimon , 1993 ) .",
"Germline clones of Ras85Dc40b mutants were made by FLP-DFS technique ( Chou and Perrimon , 1996; Hou et al . , 1995 ) .",
"Mutant embryos were identified by the expression of twi-lacZ , ftz-lacZ , hb-lacZ , Ubx-lacZ , or GMR-dfd-GFP constructs inserted on balancer chromosomes .",
"We identified mutants harboring dpp mutations by selecting embryos with previously reported phenotypes in the embryo DV patterning .",
"For collection of large numbers of virgins , we used a Y chromosome harboring hs-hid construct developed by R . Lehmann and M . Van Doren ( Starz-Gaiano et al . , 2001 ) .",
"See Flybase ( St Pierre et al . , 2014 ) for details of strains described below .",
"Mutant strains; AbdBM1 ( a gift from I . Lohmann ) ( Lohmann et al . , 2002 ) , btl∆oh10 and btl∆oh24-1 ( Ohshiro and Saigo , 1997 ) , Df ( os ) 1A ( a gift from D . Harrison ) ( Harrison et al . , 1998 ) , Gap1B2 ( a gift from N . Perrimon ) ( Hou et al . , 1995 ) , grn7L12 ( a gift from J . Hombria ) ( Brown and Castelli-Gair Hombria , 2000 ) , hh13C ( Hosono et al . , 2003 ) , Ras85D∆c40b ( a gift from N . Perrimon and C . A . Berg ) ( Hou et al . , 1995; Schnorr and Berg , 1996 ) , rho∆38 ( a gift from D . Andrew ) ( Bradley and Andrew , 2001 ) , rho7M ( a gift from J . Skeath ) ( Skeath , 1998 ) , topf24 ( a gift from K . Moses ) ( Kumar et al . , 1998 ) utH599 = vvlH599 ( a gift from A . Salzberg ) ( Inbal et al . , 2003 ) .",
"arm4 was obtained from National Institute of Genetics ( NIG ) , Mishima , Japan .",
"aop1 , aos∆7 , bnlP1 , Df ( 3R ) Dl-BX12 as Df ( bnl ) , dpphr92 , dppH46 , pnt∆88 , Df ( H99 ) , hhAC , pnt2 , ptc9 , Df ( 2R ) Exel7098 as Df ( ptc ) , sim2 , topf2 , trh2 , Df ( 3L ) Exel6109 as Df ( vvl ) , wgCX4 , Df ( 2L ) Exel6017 as Df ( wg ) were obtained from Bloomington stock center ( BDSC ) , Indiana .",
"Enhancer trap strains; 1-eve-1 as trh-lacZ ( a gift from N . Perrimon ) ( Perrimon et al . , 1991 ) and P0144-lacZ ( a gift from W . Janning , Flyview ) , pnt-lacZ ( a gift from M . Krasnow ) ( Samakovlis et al . , 1996a ) .",
"h-lacZ was obtained from BDSC .",
"Enhancer reporter strains; btl-CD8-GFP ( a gift from M . Sato ) ( Sato and Kornberg , 2002 ) , kni- ( dpp ) -lacZ ( Chen et al . , 1998 ) , and salm-TSE-lacZ ( Kuhnlein and Schuh , 1996 ) ( gifts from R . Schuh ) .",
"Gal4 and UAS strains; btl-gal4 and trh66-gal4 on sencond and third chromosomes ( gifts from S . Hayashi ) ( Kondo and Hayashi , 2013; Shiga et al . , 1996 ) , salm-gal4 ( a gift from M . Llimargas ) ( Llimargas , 2000 ) , UAS-bnl ( a gift from M . Krasnow ) ( Sutherland et al . , 1996 ) , UAS-btl/dFGFR ( R . Matsuda and K . Saigo ) , UAS-ci75 ( a gift from S . Ishii ) ( Dai et al . , 2003 ) , UAS-cirep ( a gift from A . Moore ) ( Karim and Moore , 2011 ) , UAS-DIAP ( a gift from E . Kuranaga and M . Miura ) ( Kuranaga et al . , 2002 ) , UAS-dof ( a gift from M . Affolter ) ( Vincent et al . , 1998 ) , UAS-dpp ( a gift from K . Basler ) ( Ruberte et al . , 1995 ) , UAS-grn ( a gift from J . Hombria ) ( Brown and Castelli-Gair Hombria , 2000 ) , UAS-hh ( Hosono et al . , 2003 ) , UAS-torso4021-btl ( a gift from E . Hafen ) ( Feldmann et al . , 1999 ) , UAS-RasV12 ( gifts from G . M . Rubin ) ( Karim and Rubin , 1998 ) , UAS-sspi ( a gift from B . Shilo ) ( Schweitzer et al . , 1995b ) , UAS-vvl and UAS-vvl vvlH599 ( gifts from A . Salzberg ) ( Inbal et al . , 2003 ) .",
"arm-gal4 , UAS-dEGFR and UAS-nGFP were obtained from BDSC .",
"Eggs were collected on apple/grape juice plates at 25°C .",
"Embryos were bleached and fixed as previously described ( Patel , 1994 ) for 15–30 min with a 1:1 mixture of heptane and a fix solution ( 3 . 7% formaldehyde , 0 . 1 M Hepes pH6 . 9 , 2 mM MgSO4 ) .",
"Embryos were dechorionated with methanol and incubated in 0 . 1% PBT supplemented with 0 . 5% BSA .",
"Staging of embryos was done as previously described ( Campos-Ortega and Hartenstein , 1997 ) .",
"For immunostaining , the following primary antibodies were used .",
"Guinea-pig anti-Gasp ( 1:1000 ) ( Tiklova et al . , 2013 ) , Guinea-pig anti-Kni ( 1:300 ) , ( developed by J . Reinitz and distributed by Y . Hiromi , East Asian Segmentation Antibody Center , Mishima , Japan ) ( Kosman et al . , 1998 ) , rabbit anti-Trh ( 1:50 ) .",
"Mouse anti-Abd-B ( 1:10 , donated by S . Celniker ) ( Celniker et al . , 1989 ) , mouse anti-Aop ( 1:10 , donated by I . Rebay and G . M . Rubin ) ( Rebay and Rubin , 1995 ) , mouse Dcad ( 1:10 , donated by T . Uemura ) ( Oda et al . , 1994 ) , mouse mab2A12 ( anti-Gasp ) ( 1:5 , donated by M . Krasnow , N . Patel and C . Goodman ) ( Samakovlis et al . , 1996b; Tiklova et al . , 2013 ) were obtained from Developmental Studies Hybridoma Bank ( DSHB ) , Iowa .",
"Commercially available antibodies were anti-LacZ ( E . coli . β-Galactosidase ) antibodies made in goat ( 1:500 , Biogenesis , UK ) or rabbit ( 1:1000 , Cappel , Netherlands ) and anti-GFP antibodies made in rabbit ( 1:500 , JL-8 Clontech , Mountain View , CA ) or mouse ( 1:1000 , GFP20 Sigma , St . Louis , MO ) .",
"Donkey or goat biotin- or fluorescently labeled secondary antibodies made against the host species of primary antibodies were purchased from Jackson Laboratories , Sacramento , CA .",
"Streptavidin coupled with AMCA , FITC , or Cy5 were used when necessary .",
"For mab2A12 detection TSA amplification ( PerkinElmer , Waltham , MA ) was used .",
"For detection of apoptosis , a TUNEL kit from Roche ( Switzerland ) was used .",
"Double fluorescent labeling with RNA probes and antibodies was carried out as described ( Goto and Hayashi , 1997 ) .",
"The following cDNA clones were used to make hybridization probes; btl ( Ohshiro and Saigo , 1997 ) , h ( a gift from D . Ish-Horowicz ) ( Hooper et al . , 1989 ) , grn ( a gift from J . Hombria ) ( Brown and Castelli-Gair Hombria , 2000 ) , pnt ( a gift from C . Klambt ) ( Klambt , 1993 ) , salm ( a gift from R . Schuh ) ( Kuhnlein and Schuh , 1996 ) , upd ( a gift from D . Harrison ) ( Harrison et al . , 1998 ) , vvl ( a gift from J . Casanova ) ( Llimargas and Casanova , 1997 ) .",
"DNA clones of aos , rho and trh were obtained from Drosophila Genomics Resource Center ( DGRC ) , Indiana , USA .",
"Confocal images were taken by Bio-Rad ( Hercules , CA ) MRC1024 , Olympus ( Japan ) Fluoview 1000 or Zeiss ( Germany ) LSM780 .",
"Images of controls and mutants taken by the same confocal microscopes were used for comparison .",
"Images were processed by ImageJ and figures were prepared with Adobe Photoshop and Illustrator ."
]
] | [
"Developmental potentials of cells are tightly controlled at multiple levels .",
"The embryonic Drosophila airway tree is roughly subdivided into two types of cells with distinct developmental potentials: a proximally located group of multipotent adult precursor cells ( P-fate ) and a distally located population of more differentiated cells ( D-fate ) .",
"We show that the GATA-family transcription factor ( TF ) Grain promotes the P-fate and the POU-homeobox TF Ventral veinless ( Vvl/Drifter/U-turned ) stimulates the D-fate .",
"Hedgehog and receptor tyrosine kinase ( RTK ) signaling cooperate with Vvl to drive the D-fate at the expense of the P-fate while negative regulators of either of these signaling pathways ensure P-fate specification .",
"Local concentrations of Decapentaplegic/BMP , Wingless/Wnt , and Hedgehog signals differentially regulate the expression of D-factors and P-factors to transform an equipotent primordial field into a concentric pattern of radially different morphogenetic potentials , which gradually gives rise to the distal-proximal organization of distinct cell types in the mature airway ."
] | [
"Many organs are composed of tubes of different sizes , shapes and patterns that transport vital substances from one site to another .",
"In the fruit fly species Drosophila melanogaster , oxygen is transported by a tubular network , which divides into finer tubes that allow the oxygen to reach every part of the body .",
"Different parts of the fruit fly’s airways develop from different groups of tracheal precursor cells .",
"P-fate cells form the most 'proximal' tubes ( which are found next to the outer layer of the fly ) .",
"These cells are 'multipotent' stem cells , and have the ability to specialize into many different types of cells during metamorphosis .",
"The more 'distal' branches that emerge from the proximal tubes develop from D-fate cells .",
"These are cells that generally acquire a narrower range of cell identities .",
"By performing a genetic analysis of fruit fly embryos , Matsuda et al . have now identified several proteins and signaling molecules that control whether tracheal precursor cells become D-fate or P-fate cells .",
"For example , several signaling pathways work with a protein called Ventral veinless to cause D-fate cells to develop instead of P-fate cells .",
"However , molecules that prevent signaling occurring via these pathways help P-fate cells to form .",
"Different amounts of the molecules that either promote or hinder these signaling processes are present in different parts of the fly embryo; this helps the airways of the fly to develop in the correct pattern .",
"This work provides a comprehensive view of how cell types with different developmental potentials are positioned in a complex tubular network .",
"This sets a basis for future studies addressing how the respiratory organs – and indeed the entire organism – are sustained ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"cell biology"
] | Radially patterned cell behaviours during tube budding from an epithelium | elife-35717-v4 | [
[
"During early embryonic development , simple tissue structures are converted into complex organs through highly orchestrated morphogenetic movements .",
"We use the formation of a simple tubular epithelial structure from a flat epithelial sheet as a model system to dissect the processes and forces that drive this change .",
"Many important organ systems in both vertebrates and invertebrates are tubular in structure , such as lung , kidney , vasculature , digestive system and many glands .",
"The formation of the salivary glands from an epithelial placode in the Drosophila embryo constitutes such a simple model of tubulogenesis ( Girdler and Röper , 2014; Sidor and Röper , 2016 ) .",
"Each of the two placodes on the ventral side of the embryo ( Figure 1A ) consists of about 100 epithelial cells , and cells in the dorso-posterior corner of the placode begin the process of tube formation through constriction of their apical surfaces , leading to the formation of an invagination pit through which all cells eventually internalise ( Girdler and Röper , 2014; Sidor and Röper , 2016 ) .",
"Apical constriction , a cell behaviour of epithelial cells that can transform columnar or cuboidal cells into wedge-shaped cells and can thereby induce and assist tissue bending , has emerged as a key morphogenetic module utilised in many different events ranging from mesoderm invagination in flies , Xenopus and zebrafish to lens formation in the mouse eye ( Lee and Harland , 2007; Martin and Goldstein , 2014; Martin et al . , 2009; Plageman et al . , 2011 ) .",
"Apical constriction relies on the apical accumulation of actomyosin , that when tied to junctional complexes can exert pulling forces on the cell cortex and thereby reduce apical cell radius ( Blanchard et al . , 2010; Mason et al . , 2013 ) .",
"Two pools of apical actomyosin have been identified: junctional actomyosin , closely associated with apical adherens junctions , as well as apical-medial actomyosin , a highly dynamic pool underlying the free apical domain ( Levayer and Lecuit , 2012; Röper , 2015 ) .",
"Another prominent cell behaviour during morphogenesis in all animals is cell intercalation , the directed exchange of neighbours , that is for instance the driving force behind events such as convergence and extension of tissues during gastrulation .",
"Also during cell intercalation apical actomyosin activity is crucial to processes such as junction shrinkage and junction extension that underlie this cell behaviour ( Bertet et al . , 2004; Collinet et al . , 2015; Rauzi et al . , 2010; Zallen and Wieschaus , 2004 ) .",
"Importantly , all cell behaviours during morphogenesis require close coordination between neighbouring cells .",
"This is achieved on the one hand through tight coupling of cells at adherens junctions , but also through coordination of actomyosin behaviour within groups of cells , often leading to seemingly supracellular actomyosin structures in the form of interlinked meshworks and cables ( Blankenship et al . , 2006; Röper , 2012 , 2013 ) .",
"The coordination between cells at the level of adherens junctions as well as actomyosin organisation and dynamics allows a further important aspect of morphogenesis to be implemented: the force propagation across cells and tissues .",
"There is mounting evidence from different processes in Drosophila that force generated in one tissue can have profound effects on morphogenetic behaviour and cytoskeletal organisation in another tissue .",
"For instance , during germband extension in the fly embryo , the pulling force exerted by the invagination of the posterior midgut leads to both anisotropic cell shape changes in the germband cells ( Lye et al . , 2015 ) and also assists the junction extension during neighbour exchanges ( Collinet et al . , 2015 ) .",
"During mesoderm invagination in the fly embryo , anisotropic tension due to the elongated geometry of the embryo leads to a clear anisotropic polarisation and activity of apical actomyosin within the mesodermal cells ( Chanet et al . , 2017 ) .",
"We have previously shown that in the salivary gland placode during early tube formation when the cells just start to invaginate , the placodal cells contain prominent junctional and apical-medial actomyosin networks ( Booth et al . , 2014 ) .",
"The highly dynamic and pulsatile apical-medial pool is important for apical constriction of the placodal cells , and constriction starts in the position of the future pit and cells near the pit continue to constrict before they invaginate ( Booth et al . , 2014 ) .",
"The GPCR-ligand Fog is important for apical constriction and myosin activation in different contexts in the fly ( Kerridge et al . , 2016; Manning et al . , 2013 ) , and fog expression is also clearly upregulated in the salivary gland placode downstream of two transcription factors , Fkh and Hkb ( Chung et al . , 2017; Myat and Andrew , 2000b ) .",
"Fkh is a key factor expressed in the placode directly downstream of the homeotic factor Sex combs reduced ( Scr ) that itself is necessary and sufficient to induce gland fate ( Myat and Andrew , 2000a; Panzer et al . , 1992 ) .",
"fkh mutants fail to invaginate cells from the placode , with only a central depression within the placode forming over time ( Myat and Andrew , 2000a ) .",
"Here , we use morphometric methods , in particular strain rate analysis ( Blanchard et al . , 2009 ) , to quantify the changes occurring during early tube formation in the salivary gland placode .",
"Many morphogenetic processes are aligned with the major embryonic axes of anterio-posterior and dorso-ventral .",
"An excellent example is germband extension in the fly embryo , where polarised placement of force-generating actomyosin networks is downstream of the early anterio-posterior patterning cascade ( Blankenship et al . , 2006; Simões et al . , 2010 ) .",
"In the case of the salivary gland placode , the primordium of the secretory cells that invaginate first is roughly circular , with an off-center focus due to the invagination point being located in the dorsal-posterior corner , prompting us to assess changes within a radial coordinate framework .",
"In addition to previously characterised apical constriction ( Booth et al . , 2014; Chung et al . , 2017 ) , we demonstrate that circumferentially oriented directional intercalation of placodal cells plays a major contribution to ordered invagination at early stages .",
"In addition , we compare quantitative planar strain rate analysis at different apical-basal depths of the tissue , and link cells between the planes to calculate rates of change of local geometry in depth , as a proxy for a full 3D analysis .",
"We uncover that cell geometries and behaviours in 3D are also radially patterned: near the pit of invagination , where apical-medial myosin II is strong ( Booth et al . , 2014 ) , cells are isotropically constricting apically leading to apical cell wedging , and with distance from the pit cells progressively tilt towards the pit .",
"Cells also interleave apically towards each other in a circumferential direction , which can lead to different neighbour connectivity along their basal-to-apical length , equivalent to a T1 transition in depth .",
"This strongly suggests apically driven active intercalation behaviours .",
"We further show that several measures of ‘geometrical stress’ have signatures indicating that circumferential intercalation in the cells away from the pit is active .",
"In addition , across the placode junctional myosin II is enriched in circumferential junctions , suggesting polarised initiation of cell intercalation through active junction shrinkage .",
"This is followed by polarised resolution of exchanges towards the pit , thereby contributing to tissue invagination .",
"forkhead ( fkh ) mutants , that fail to form an invagination , still show cell intercalations within the placode at a high rate , further supporting the active nature of the intercalations .",
"Thus , tube budding depends on a radial organisation of 3D cell behaviours , that are themselves patterned by the radially patterned and polarised activity of apical myosin II pools , with apical-medial myosin dominating near the invagination point and polarised junctional myosin dominating further away from the pit .",
"The continued initiation of cell intercalation but lack of polarised resolution in the fkh mutant , where the invagination is lost , could suggest that a tissue-intrinsic mechanical interplay also contributes to successful tube budding ."
],
[
"Upon specification of the placode of cells that will form the embryonic salivary gland at the end of embryonic stage 10 , the first apparent change within the apical domain of placodal cells is apical constriction at the point that will form the first point of invagination or pit ( Figure 1A , B; [Booth et al . , 2014; Myat and Andrew , 2000b] ) .",
"Apical constriction at this point in fact preceded actual tissue bending ( Figure 1B ) .",
"We have previously shown that apical constriction is clustered around the pit and is important for proper tissue invagination and tube formation ( Booth et al . , 2014 ) .",
"In order to discover if any further cell behaviours in addition to apical constriction contribute to tissue bending and tube invagination in the early placode , we employed quantitative morphometric tools to investigate the process in comparable wild-type time-lapse movies using strain rate analysis ( Blanchard et al . , 2009 ) .",
"We imaged embryos expressing a lateral plasma membrane label in all epidermal cells as well as a marker that allows identification of placodal cells ( Figure 1C and Video 1; for genotypes see Table 1 ) .",
"Time-lapse movies were segmented and cells tracked using previously developed computational tools that allow for the curvature of the tissue to be taken into account ( Figure 1C–F ) ( Blanchard et al . , 2009; Booth et al . , 2014 ) .",
"The cells of the salivary gland placode that will later form the secretory part of the gland are organised into a roughly circular patch of tissue prior to invagination and maintain this shape during the process ( Figure 1C ) .",
"Within this circular patch , the invagination pit is located at the dorsal-posterior edge rather than within the centre of the placode .",
"We therefore employed a radial coordinate system , with the forming pit as its origin , in which to analyse and express any changes ( Figure 1E ) .",
"We locally projected 2D strain ( deformation ) rates and other oriented measures onto these radial ( ‘rad’ ) and circumferential ( ‘circ’ ) axes ( Figure 1F ) .",
"We focused our analysis on the early stages of tissue bending and invagination , defining as t = 0 the time point before the first curvature change at the point of invagination at the tissue level could be observed .",
"Dynamic analysis of the changes in apical area of placodal cells , in the time interval of 18 min prior to and 18 min after the first tissue bending , revealed distinct zones of apical cell behaviour .",
"Grouping the placodal cells into ~ 2 cell-wide stripes concentric to the pit for apical area change analysis ( Figure 1G; Figure 1—figure supplement 1 and Video 2 ) revealed a split into cells whose apical area over this time interval did not change or changed only slightly ( Figure 1G’ , red , yellow and green lines ) and cells whose apical area progressively decreased ( Figure 1G’ , blue and purple lines ) .",
"The clear split into two zones of differing cell behaviours , defined by radial distance from the invagination point at zero minutes ( when the pit starts to invaginate ) , prompted us to analyse these regions independently in our strain rate analyses ( Figure 1H ) .",
"Methods have been developed previously to calculate strain ( deformation ) rates for small patches of tissue , and to further decompose these into the additive contributions of the rates of cell shape change and of the continuous process of cell rearrangement ( intercalation ) ( Blanchard , 2017; Blanchard et al . , 2009 ) ( Figure 2A , B ) .",
"The levels of both contributions vary dramatically between different morphogenetic processes ( Blanchard et al . , 2010; Bosveld et al . , 2012; Butler et al . , 2009; Etournay et al . , 2015; Guirao et al . , 2015 ) .",
"Cell divisions have ceased in the placode around the time of specification , and there is also no cell death , therefore none of these processes contributed to the overall tissue deformation in this case .",
"Upon segmentation of cell outlines , the rate of tissue deformation was calculated from the relative movement of cell centroids in small spatio-temporal domains of a central cell surrounded by its immediate neighbours and over three movie frames ( ~6 min ) .",
"Independently , for the same domains , rates of cell shape change were calculated mapping best-fitted ellipses to actual cell shapes over time .",
"The rate of cell intercalation could then be deduced as the difference between these two measures ( Figure 2A , B and Video 3; see Materials and methods for a detailed description ) .",
"The three types of strain rate measure were then projected onto our radial coordinate system .",
"Strain rate analysis of changes within the apical domain of the epithelial cells in the early salivary gland placode clearly confirmed the existence of two distinct zones of cell behaviour .",
"Spatial plots summarising ~ 36 mins of data centred around the first tissue bending event ( combined from the analysis of 9 wild-type embryos; Figure 2—figure supplement 1A ) revealed strong isotropic tissue contraction near the invagination pit ( Figure 2C’ and Figure 2—figure supplement 1B , green ) that was mainly contributed by apical cell constriction ( Figure 2C’’ and Figure 2—figure supplement 1B’ ) .",
"At a distance from the pit , tissue elongation dominated in the radial direction ( Figure 2C’ , magenta ) and was contributed mostly by cell intercalation ( Figure 2C’’ , C’’’ ) .",
"The split in behaviour and the strong contribution of cell intercalation to the early invagination and tube formation was even more apparent from time resolved strain rate analyses .",
"At the whole tissue level , apical cell constriction began more than 10 min before any curvature change at the tissue level ( Figure 2—figure supplement 1C , C’ , green curve ) , and this change was most pronounced in the cells near the invagination pit ( Figure 2D , D’ versus E , E’ , green curve ) .",
"In addition , cell intercalation also commenced about 10 min prior to tissue bending ( Figure 2—figure supplement 1C , C’ , orange curve ) , but in this case , the stronger contribution came from cells far from the pit ( Figure 2E , E’ versus D , D’ , orange curve ) .",
"Although constriction was isotropic near the pit , with equally large magnitudes contributing both radially and circumferentially ( Figure 2D , D’ , green curves ) , intercalation was clearly polarised towards the invagination pit , with expansion radially in the orientation of the pit ( Figure 2D , E and Figure 2—figure supplement 1C , ‘rad’ , orange curves ) , and contraction circumferentially ( Figure 2D’ , E’ and Figure 2—figure supplement 1C' , ‘circ’ , orange curves; see also Figure 2—figure supplement 1F ) .",
"Thus , in addition to apical constriction , directional cell intercalation constitutes a major second cell behaviour occurring during tissue bending and invagination of a tube .",
"Furthermore , the amount of both cell behaviours occurring was radially patterned across the placode .",
"However , it was not clear whether both these behaviours were entirely being driven by active apical mechanisms , or whether further basal events contributed , so we next investigated the 3D behaviours of placode cells .",
"The apical constriction as observed in the placodal cells near the pit is indicative of a redistribution of cell volume more basally , which could result in a combination of cell wedging and/or cell elongation in depth .",
"The 3D-cell behaviour of acquiring a wedge-like shape is of particular interest in the placode because it is capable of tissue bending .",
"Apical constriction ( coupled with corresponding basal expansion if cell volume is maintained , as in the case of the salivary gland placode [data not shown] ) is known to deform previously columnar or cuboidal epithelial cells into wedge-shaped cells ( Martin and Goldstein , 2014; Wen et al . , 2017a ) .",
"This is also true in the salivary gland placode , where cross-sections in xz of fixed samples and time-lapse movies confirm the change from columnar to wedged morphology ( Figure 1B , B' ) ( Girdler and Röper , 2014; Myat and Andrew , 2000b ) .",
"Most analyses of morphogenetic processes are conducted with a focus on events within the apical domain given the prevalent apical accumulation of actomyosin and junctional components ( Figure 3A , A’ ) ( Bosveld et al . , 2012; Butler et al . , 2009; Martin et al . , 2009; Rauzi et al . , 2010; Simões et al . , 2010 ) .",
"However , this apico-centric view neglects most of the volume of the cells .",
"For instance , in the case of the salivary gland placode , cells extend up to 15 µm in depth ( Figure 3B ) .",
"We thus decided to analyse cell and tissue behaviour during early stages of tube formation from the placode in a 3D context .",
"Automated cell segmentation and tracking of 3D behaviours is still unreliable in the case of tissues with a high amount of curvature , such as in the salivary gland placode once invagination begins .",
"To circumvent this issue , we used strain rate analysis at different depth as a proxy for a full 3D analysis ( Figure 3A’ , C ) .",
"After accounting for the overall curvature of the tissue , we segmented and tracked placodal cells not only within the apical region ( as shown in Figures 1 and 2; Figure 3A’ , C pink ) , but also at a more basal level , ~8 µm below the apical domain ( Figure 3A’ , C blue ) , and repeated the strain rate analysis at this depth ( Figure 3D–E’’ and Figure 3—figure supplement 1 and Figure 3—figure supplement 2 ) .",
"We used the same radial coordinate system with the pit as the origin for both layers , so we were able to compare cell behaviours between depths .",
"Segmentation and analysis at the most-basal level of placodal cells was not reliably possible due low signal-to-noise ratio of all analysed membrane reporters at this depth ( data not shown ) .",
"Overall , the spatial pattern of change at mid-basal level was similar to changes within the apical domain during 33 min centred around the start of tissue bending ( Figure 3—figure supplement 1B , C ) .",
"Isotropic tissue contraction at mid-basal depth was clustered around the position of the invagination pit ( Figure 3—figure supplement 1B , C , green ) , and this was due to cell constriction at this level ( Figure 3—figure supplement 1B’ , C’ ) , whereas the tissue expansion at a distance from the pit in the radial direction ( Figure 3—figure supplement 1C ) was due primarily to cell intercalation ( Figure 3—figure supplement 1C’’ ) .",
"Similarly to events at the apical level , this radial expansion towards the pit was accompanied by a circumferential contraction , again due primarily to cell intercalation in the region away from the pit ( Figure 3—figure supplement 1B , B’’ ) .",
"Comparing apical and basal strain rates at the cell and tissue level with respect to their radial and circumferential contributions revealed an interesting picture .",
"In temporally resolved plots , isotropic cell constriction dominated apically in cells near the pit ( Figure 3D , pink ) , but with a slower rate of constriction at the mid-basal depth ( Figure 3D , blue ) .",
"This was confirmed by cross-section images that show that , near the pit and once tissue-bending had commenced , the basal surface of the cells was displaced even further basally than in the rest of the placode , and cells were expanded at this level , leading to an overall wedge-shape ( Figure 1B’ ) .",
"In cells away from the pit , similar to the apical region , cell shapes did not change much ( Figure 3E ) .",
"In contrast to cell shape changes that diverged at depth at least in the cells near the pit , intercalation behaviour appeared to be highly coordinated between apical and basal levels with near identical contributions at both particularly in cells far from the pit ( Figure 3D’ , E’ and Figure 3—figure supplement 1D’’ ) .",
"In cells near to the pit , the faster rate of apical constriction implies that cell wedging is occurring , but we have not confirmed this in the same dataset by measuring the relative sizes of apical and basal cell diameters .",
"Similarly , although the rates of apical and basal intercalation are remarkably similar , this does not rule out that the actual arrangement of cells at one level is ‘tipped’ ahead of the other , through interleaving in depth ( akin to a T1 transition along the apical-basal axis , see z-sections in Figure 4C and Figure 4—figure supplement 1Dd'’ ) .",
"For example , if cell rearrangement is being driven by an active apical mechanism ( see below and Figure 6 ) , we predict that apical cell contours would be intercalating ahead of basal cell contours , even while their rates remain the same .",
"Both wedging ( Figure 4B ) and interleaving ( Figure 4C ) have implications for the tilt , or lean , of cells relative to epithelial surface normals ( Figure 4D and Figure 4—figure supplement 1A–C ) , with a gradient of tilt expected for constant wedging or interleaving in a flat epithelium .",
"We therefore set out to quantify 3D wedging , interleaving and cell tilt with new methods .",
"We used the placodes ( n = 5 ) for which we have tracked cells at both apical and mid-basal levels .",
"First , we developed a semi-automated method to accurately match cell identities correctly between depths ( Figure 4A , Video 4 , and see Materials and methods ) .",
"We then borrowed ideas from recent methods developed to account for epithelial curvature in terms of the additive contributions of cell wedging and interleaving in depth ( Deacon , 2012 ) .",
"In the early developing salivary gland placode , average tissue curvature is very slight , so we simplified the above methods for flat epithelia .",
"We applied exactly the same methods that we have used in Figures 2 and 3 to calculate strain rates for small cell domains , but rather than quantifying rates of deformation over time , now we quantify rates of deformation in depth ( see z-level illustrations in Figure 4B , C , D and Figure 4—figure supplement 1D ) .",
"The cell shape strain rate becomes a wedging strain in depth , in units of proportional shape change per micron in z ( Figure 4B , B’ and Figure 4—figure supplement 1Dc , c' ) , the intercalation strain rate becomes the interleaving strain in depth , in the same units ( Figure 4C , C’ and Figure 4—figure supplement 1Dd , d' ) , and translation velocity becomes the cell tilt ( Figure 4D , D’ and Figure 4—figure supplement 1A , Da , a’; see Materials and methods for details ) .",
"Once again , we projected the z-strain rates and tilt onto our radial coordinate system .",
"Cells across the placode started out at −18 min before pit invagination unwedged and mostly untilted in radial and circumferential orientations ( Figure 4B’ , D’ ) .",
"Cells near the pit became progressively wedge-shaped over the next 30 min , with smaller apices ( Figure 4B’ , orange lines; Figure 4—figure supplement 1C ) .",
"Cell wedging was reasonably isotropic , but with circumferential wedging always stronger than radial .",
"Away from the pit , progressive wedging was less rapid , again with a strong circumferential contribution but nearly no radial contribution ( Figure 4B’ , purple lines ) .",
"That cells were less wedged radially might be because this is the orientation in which cells move into the pit , releasing radial pressure due to apical constriction near the pit .",
"The wedging anisotropy is also compatible with active circumferential contraction .",
"Indeed , circumferential interleaving was always more negative than radial interleaving , often significantly so ( Figure 4C’ , solid vs dashed lines ) .",
"Thus , interleaving contributes a circumferential tissue contraction apically , with a concomitant radial expansion .",
"This pattern is thus also compatible with an apical circumferential contraction mechanism , possibly driving cell rearrangements .",
"Cell tilt , a measure of the divergence of a cell’s in-line from the surface normal ( Figure 4D and Figure 4—figure supplement 1A , B , D ) , increased continuously in the radial direction towards the pit over the period of our analysis ( Figure 4D’ , solid lines ) .",
"A stronger tilt was observed for the cells further from the pit ( Figure 4D’ , purple solid line ) , which is expected from the radial wedging seen near the pit ( Figure 4B’ , solid orange line ) .",
"Hence , the relatively isotropic rates of apical constriction near the pit and the very similar rates of intercalation apically versus basally were in fact grounded in anisotropic wedging near the pit and in an interleaving difference between apical and basal .",
"3D tissue information such as wedging , interleaving and tilt are therefore essential to fully understand planar cell behaviours such as cell shape change and intercalation .",
"Overall , our combined analysis so far suggests that isotropic apical constriction near the pit combines with an apically led circumferential contraction mechanism .",
"We now investigate the possible origins of the latter .",
"Our strain rate analysis has revealed that the intercalation strain rate , representing the continuous process of slippage of cells past each other , was a major contributor to early tube formation and was highly coordinated between apical and basal domains .",
"3D domain interleaving further revealed that cell rearrangement convergence is more advanced apically in the circumferential orientation .",
"Both these findings are measured from small groups of cells across the placode but are agnostic about neighbour exchange events or more complicated multi-neighbour exchanges .",
"Neighbour exchanges or cell intercalations are usually thought to occur through one of two mechanisms: groups of four cells can exchange contacts through the formation of a transient four-cell vertex structure , in a typical T1 exchange ( Figure 5A ) , whereas groups of more than four cells can form an intermediary structure termed a rosette , followed by resolution of the rosette to create new neighbour contacts ( Figure 5B ) .",
"During convergence and extension of tissues in both vertebrates and invertebrates , the formation and resolution of these intermediate structures tend to be oriented along embryonic axis , with the resolution occurring perpendicular to the formation ( Blankenship et al . , 2006; Lienkamp et al . , 2012 ) .",
"We therefore decided to also analyse intercalation in the placode by following individual events to identify the underlying mechanistic basis .",
"From our database of apical cell tracks and their connectivity , we identified all T1 transitions , classifying the time point when pairs of cells become new neighbours as ‘neighbour gains’ .",
"We further sub-classified neighbour gains as being either radially or circumferentially oriented , depending on which orientation was closest to the centroid-centroid line of two cells involved in a neighbour gain ( Figure 5C ) .",
"T1s occurred at a constant rate over our study period , and we observed T1s in both orientations , revealing that neighbour connectivity was quite dynamic ( Figure 5C’ , D ) .",
"Nevertheless , over two-thirds of neighbour gains were oriented circumferentially , a bias that correlates with the intercalation strain rate contraction circumferentially ( see for example Figure 2E’ ) .",
"This bias was also evident when visualising interface losses and gains over time for an individual placode , with losses preferentially occurring for circumferential interfaces and gains for radial interfaces ( Figure 5D and Figure 5—figure supplement 1 ) .",
"We defined the number of productive neighbour gains as the difference between circumferential and radial gains , since equal numbers of both would cancel each other out .",
"In order to control for any variability between placodes or between the number of cells tracked per placode , we expressed the number of productive gains as a proportion of the number of cell-cell contacts that were available to perform a circumferential T1 per time step ( see Materials and methods ) .",
"The proportion of productive circumferential gains was approximately constant , which lead to a steady net gain over time ( Figure 5C’’ ) .",
"Furthermore , the cumulative strain attributable to discrete T1s calculated here ( e0 . 09 = 1 . 09 ) is in good agreement with the cumulative strain over the same time window that is attributed to the continuous process of intercalation ( 1 . 1 in Figure 2E ) .",
"In addition to typical T1 exchanges , multicellular rosette structures could easily be identified amongst the placodal cells ( Figure 5E–F ) .",
"Rosette formation began prior to the first sign of tissue-bending , but the number of rosettes per placode increased afterwards ( Figure 5E , E’ ) .",
"Rosettes were usually formed of five to seven cells , with most involving only five cells ( Figure 5E’ , F ) .",
"By contrast , rosettes observed during Drosophila germband extension can be formed of up to 12 cells ( Blankenship et al . , 2006 ) .",
"The strain rate analysis already indicated that , overall , intercalation events should be polarised to produce a contraction in the circumferential orientation with the corresponding expansion polarised towards the pit ( Figure 2C–E’ ) .",
"Analysis of rosette formation and resolution in our time-lapse datasets of embryos expressing a membrane marker demonstrated that groups of cells contracted in a circumferential orientation to form a rosette , the resolution of which then moved individual cells towards the invaginating pit ( Figure 5F and Video 5 ) , thereby leading to the expansion observed in the strain rate analysis .",
"Non-muscle myosin II is the major driver of cell shape changes in many different contexts ( Levayer and Lecuit , 2012; Röper , 2013 , 2015 ) , and it has been shown to play an important role in T1 transitions and in rosette formation driving the convergence and extension events during germband extension in the Drosophila embryo ( Blankenship et al . , 2006; Fernandez-Gonzalez et al . , 2009; Rauzi et al . , 2010 ) .",
"We imaged embryos expressing palmitoylated RFP ( Ubi-TagRFP-CAAX ) as a membrane label and a GFP-tagged version of non-muscle myosin II regulatory light chain ( called spaghetti squash , sqh , in Drosophila ) under control of its own promoter in the null mutant background ( sqhAX[3]; sqhGFP42 ) to assess myosin II distribution and intensity as a proxy for myosin activity ( Video 6 ) .",
"In all individual rosette formation-resolution examples analysed ( n = 29 ) , junctional myosin II appeared particularly enriched in the form of short cable-like structures at the central contact sites of the rosette ( Figure 5G , H ) .",
"These cables initially spanned several cell diameters and shortened concomitant with the cells being drawn into a central vertex ( Figure 5I ) .",
"The orientation of the short myosin cables correlated with the direction of rosette-formation , but in contrast to germband extension was not always oriented parallel to the dorsal-ventral axis , but rather following the circumferential coordinates of the placode .",
"We now investigated whether these circumferential intercalations were actively driven within the apical domain to promote invagination .",
"Even though the quasi-3D analysis detailed above strongly indicates that the intercalation of cells far from the pit initiates from the apical domain , the process of initiation itself could be either actively driven or a passive response .",
"Intercalation far from the pit would be active if it arose as an intrinsic property of this part of the tissue and actively drove circumferential contraction of the tissue ( Figure 6A; red curved arrows ) .",
"The capacity of these cells for such active behaviour would likely be achieved through genetic patterning .",
"Intercalation would be passive if the active apical constriction near the pit drove a passive ‘funnelling’ of far cells towards the pit , the radial pull leading to passive polarised intercalations ( Figure 6A’; blue arrows ) .",
"In order to distinguish between an active mechanism and a passive mechanism , we made four predictions ( Figure 6B–E’ ) , comparing metrics of junction length , angles at vertices , cell elongation and junctional myosin II accumulation .",
"For each metric , we compared circumferentially oriented junctions with radial junctions .",
"We also distinguished whether junctions were or were not shrinking junctions , about to be involved in a T1 event ( see Materials and methods ) .",
"We compared T1 data with similarly oriented non-T1 data to ask if there was a more active signature to shrinking junctions .",
"We also compared radial versus circumferential non-T1 data to ask whether junctions as a whole in one orientation had a more active signature .",
"For the first two predictions ( Figure 6B–C’ ) , we compared real interface lengths and vertex angles with interface lengths and angles predicted by a Voronoi tessellation ( see Materials and methods ) .",
"We considered that a Voronoi tessellation generated from cell centroid seeds represents a mechanically neutral configuration for the cell-cell junctions and angles , and controlled for variation in local geometry around focal junctions .",
"The direction in which real junction lengths or angles deviated from neutral Voronoi geometries was indicative of an active or a passive mechanism .",
"The deviation of real junction length from predicted Voronoi junction length ( Figure 6B , B’ ) has previously been used as a geometric proxy for junction stress in the germband ( Tetley et al . , 2016 ) .",
"In the salivary gland placode , junction lengths were shorter than Voronoi predicted lengths for shortening T1 junctions compared to non-T1 junctions , most strongly for circumferentially oriented junctions ( Figure 6F and Figure 6—figure supplement 1B ) .",
"Circumferential non-T1 interfaces also deviated from neutral geometries by being shorter on average than expected , with radial junctions longer ( Figure 6F ) .",
"This suggests that circumferential junctions , and circumferential T1s in particular , were contracted , possibly by an intrinsic contractile mechanism , rather than the cells being pulled away from each other radially by the pit .",
"We predicted that active circumferential junction contraction would impose more acute angles at their associated vertices ( Figure 6C , [Rauzi et al . , 2008] ) , whereas a radial pull would lead to more obtuse angles ( Figure 6C’ ) .",
"On average , angles linked to circumferential junctions were indeed more acute , relative to neutral Voronoi geometry , than those linked to radial junctions ( Figure 6G and Figure 6—figure supplement 1C ) .",
"Similarly , cells were on average more elongated circumferentially ( Figure 6H and Figure 6—figure supplement 1D ) as predicted by actively contracting circumferential junctions and incompatible with a radial pull .",
"These three geometrical measures for cells far from the pit , where intercalation dominates , are therefore consistent with an active intercalation mechanism , leading to circumferential neighbour gains and thus circumferential convergence .",
"We also predicted that active circumferential junction contraction would be driven by myosin II accumulation at circumferential junctions ( Figure 6E ) .",
"Recent studies have indicated that in some tissues , as response to mechanical pulling , myosin II accumulates and becomes polarised at junctions parallel to the pulling force ( Duda et al . , 2018; Fernandez-Gonzalez et al . , 2009 ) .",
"Hence if intercalation occurred as a passive response , we would expect either no myosin II polarisation or mechanically induced localisation to radially oriented junctions ( Figure 6E’ ) .",
"The localisation and increase in myosin II observed at the central junctions during rosette formation/resolution in the placode , as shown above ( Figure 5G–I ) , indicated that some junctional myosin II was circumferentially enriched .",
"We now set out to analyse junctional myosin II distribution systematically across the whole placode .",
"When apical junctional myosin intensity was quantified in fixed samples of sqhGFP embryos across the early placode , a clear enrichment of myosin II was apparent in circumferentially compared to radially oriented junctions ( Figure 7A , B ) .",
"Such tissue-wide circumferential versus radial polarisation of myosin clearly supported an active mechanism of cell intercalation , with circumferential myosin II likely assisting both rosette and T1 vertex formation during the polarised cell intercalation events .",
"To compare and correlate junctional myosin II dynamics with the above strain rate analysis , we analysed myosin II polarisation dynamically across the whole tissue ( Figure 7C , C’ ) .",
"Quantitative tools that allow junctional polarisation of myosin and other players to be quantified have previously been established ( Figure 7D , D’’; [Tetley et al . , 2016] ) .",
"Myosin II polarisation at circumferential junctions could either occur through enrichment at two opposite junctions or sides , termed bi-polarity ( Figure 7D’ and Video 7 ) , or through enrichment at a single junction or side within a cell , termed unipolarity ( Figure 7D’’ and Video 7 ) .",
"Starting about 10 min prior to tissue bending , bi-polar enrichment of myosin II at circumferential junctions was significantly stronger than at radial junctions when measured across the whole placode ( Figure 7E ) .",
"Similarly , starting ~ 5 min later , unipolar enrichment of myosin II at circumferential junctions dominated over radial enrichment when measured across the whole placode ( Figure 7F ) .",
"This clear and increasing circumferential junctional polarisation of myosin II together with the geometrical signatures discussed above strongly supported an active mechanism of T1 vertex and rosette formation ( Figure 6A ) .",
"Less clear is whether the resolution of rosettes and vertices is equally actively driven .",
"Instead , this part of the intercalation events could respond to cell-extrinsic cues , such as the pulling of the invaginating pit , akin to the role of the posterior midgut invagination during germ band extension ( Collinet et al . , 2015; Lye et al . , 2015 ) or pulling forces generated through micro-aspiration re-orienting intercalations in embryonic mouse tissues ( Wen et al . , 2017b ) .",
"In cell intercalation events during germband extension , myosin polarisation is complementary to enrichment of Par3/Bazooka ( Baz ) as well as Armadillo/β-catenin , with both enrichments controlled by the upstream patterning and positioning of transmembrane Toll receptors and Rho-kinase ( Rok ) ( Blankenship et al . , 2006; Paré et al . , 2014; Simões et al . , 2010 ) .",
"We therefore analysed whether such complementarity was also present in the early salivary gland placode .",
"Antibody labelling of Baz often showed a complementarity in membrane enrichment to myosin II ( Figure 7G–I ) , most pronounced where myosin II was organised into circumferential mini cables during intercalation events ( Figure 7G , H ) .",
"Baz was enriched at radially oriented junctions where myosin II was low , and vice versa at circumferential junctions , though in contrast to germband extension , the Baz polarisation did not extend uniformly across the tissue and was overall less strong than the myosin II polarisation ( Figure 7I ) .",
"Thus , in order to adapt to a circular tissue geometry and to the need for ordered invagination through a focal point during the process of tube budding , a conserved molecular pattern of myosin-Baz complementarity is apparently imprinted onto the salivary gland placode in a radial coordinate pattern , rather than the prevailing A-P/D-V pattern of the earlier embryo during germband extension ( Figure 7J , K ) .",
"In order to address how the radial pattern of behaviours and molecular factors across the placode is established , we analysed mutants in a key factor of salivary gland tube invagination , the transcription factor Fork head ( Fkh ) .",
"Fkh is expressed just upon specification of the placodal cells in a dynamic pattern spreading across the whole placode ( Figure 8—figure supplement 1A–C ) , directly downstream of the homeotic factor Scr ( Zhou et al . , 2001 ) .",
"In fkh mutants , invagination of the placode fails , and towards the end of embryogenesis salivary gland-fated cells undergo apoptosis as Fkh appears to prevent activation of pro-apoptotic factors ( Jürgens and Weigel , 1988; Myat and Andrew , 2000a ) .",
"Previous studies have concluded that Fkh promotes cell shape changes important for invagination , in particular apical constriction ( Chung et al . , 2017; Myat and Andrew , 2000a ) .",
"Fkh is not the only transcription factor important for correct changes during invagination , but works in parallel to for example Huckebein ( Hkb ) , the lack of which also confers significant problems with apical cell shape changes and invagination ( Myat and Andrew , 2000b , 2002 ) .",
"We combined a fkh null mutant ( fkh[6] ) with markers allowing membrane labeling and cell segmentation ( Ubi-TagRFP-CAAX ) as well as myosin II quantification ( sqhGFP; see Video 8 ) .",
"When fkh mutant placodes were compared to wild-type ones at late stage 11 ( beyond the time window analysed here ) then fkh mutant placodes showed no sign of a dorsal-posterior invagination point ( Figure 8A–B’; Figure 8—figure supplement 1D–E’’ ) .",
"In fact , many placodes beyond late stage 11 showed a centrally located shallow depression ( Figure 8A , A’ , yellow dotted lines ) .",
"Strain rate analysis of five segmented movies of fkh[6] mutant placodes , spanning the equivalent time period to the wild-type movies analysed above ( Figure 8—figure supplement 2C ) , showed that there was no constriction at the tissue level near the pit ( Figure 8C–E and Figure 8—figure supplement 2D , D’ ) .",
"In fact , if anything , there was a slight tissue expansion ( Figure 8E ) caused by a slight expansion at the cell level ( Figure 8E’ ) , with zero intercalation ( Figure 8E’’ ) .",
"Away from the pit fkh[6] mutant placodes expanded slightly ( Figure 8F and Figure 8—figure supplement 2E , E’ ) , again mostly due to cell shape changes ( Figure 8F’ , F’’ ) .",
"Is this strong reduction of cell behaviours due to a complete ‘freezing’ of the placode in the fkh[6] mutants ?",
"We analysed whether junctional myosin II was still polarised in the fkh[6] mutant placodes .",
"In fixed and live samples , fkh[6] mutant placodes still showed the circumferential actomyosin cable surrounding the placode ( Figure 8—figure supplement 1E’’ , compare to D’’; [Röper , 2012] ) .",
"When myosin II unipolarity and bi-polarity were quantified from five segmented and tracked movies , at the tissue-level there was a strong and significant reduction in myosin II polarisation , with no difference between radial and circumferential orientations in either measure and myosin II bi-polarity indistinguishable from zero ( Figure 9A , B ) .",
"Nonetheless , cells were not static but in fact neighbour exchanges were present , both in form of T1 exchanges and rosettes ( Figure 9C–F ) .",
"The quantitative analysis of neighbour gains from segmented and tracked movies in the fkh[6] mutant revealed that gains accumulated both circumferentially as well as radially , at a rate comparable to that observed in the control ( Figure 9E ) .",
"However , in contrast to the control , where circumferential gains significantly outweighed radial neighbour gains leading to a positive net rate of productive circumferential gains ( Figure 9F , green line ) , in the fkh[6] mutant circumferential and radial gains occurred in equal amounts ( Figure 9E ) leading to a near zero net gain ( Figure 9F , pink line ) .",
"Interestingly , when focusing on individual events such as rosettes , despite a loss of tissue-wide myosin II polarisation , myosin II was still enriched at the central constricting junctions ( Figure 9Cd’ ) , and in fixed embryos smaller regions of myosin II-Baz complementarity could be identified ( Figure 8—figure supplement 1F–G ) .",
"Therefore , although at the tissue level fkh[6] mutant placodes appear near static , the close analysis of individual events revealed a highly dynamic but unpolarised intercalation behaviour across the mutant placodes .",
"Without an actively invaginating pit and focussed apical constriction taking place in the fkh[6] mutant , the unpolarised intercalation events are not being resolved radially because of the absence of the pull from the pit .",
"This finding therefore also suggests an active intercalation mechanism , where the remaining local increases in junctional myosin II still support formation of T1 vertices and rosettes , but without any overall directionality to their resolution ."
],
[
"Morphogenesis sculpts many differently shaped tissues and structures during embryogenesis .",
"A core set of molecular factors that are the actual morphogenetic effectors , such as actomyosin allowing contractility or cell-cell adhesion components allowing coordination and mechanical propagation of cell behaviours across tissues , are used iteratively in different tissues and at different times .",
"By contrast , the activity of upstream activating gene regulatory networks leading to tissue identity , but also initial tissue geometry and mechanical constraints , are highly tissue-specific .",
"During tube formation of the salivary glands in the fly embryo , we observe a clear tissue-level radial organisation of cell behaviours , with the off-centre located invaginating pit as the organising focal point ( Figure 10A ) .",
"When analysed in 2D within the apical domain of the placodal epithelial cells , apical constriction dominates at the future pit of invagination and directional cell intercalation dominates further away from the pit .",
"This intercalation achieves a circumferential convergence and radial extension of the tissue towards the invagination point ( Figure 10B ) .",
"Interestingly , these cell behaviours ( cell shape change and cell intercalation ) have previously been shown to drive other morphogenetic processes ( Butler et al . , 2009; Collinet et al . , 2015; Lee and Harland , 2007; Lye et al . , 2015; Martin and Goldstein , 2014; Martin et al . , 2009; Plageman et al . , 2011; Rauzi et al . , 2010 ) , but in our system they are utilised within a radial coordinate system , with the morphogenetic outcome being the formation of a narrow tube of epithelial cells from a round and flat placode primordium .",
"In order to understand complex organ formation , it is important to understand cause and effect during the process .",
"Cell behaviours can either be actively driven through for instance patterned actomyosin activity or they can be a mechanical response to events , or a combination of the two .",
"An excellent example for active behaviours intersecting and being influenced by nearby events is the extension of the germband during Drosophila embryogenesis .",
"In this tissue , an active mechanism of polarised intercalation combines with an extrinsic pulling force from the invaginating posterior midgut that helps the directional resolution of T1s ( Butler et al . , 2009; Collinet et al . , 2015; Lye et al . , 2015 ) .",
"During tube formation of the salivary gland placode , a similar intersection of active and passive mechanisms could be taking place: actively initiated intercalations combine with an active apical constriction at the pit that polarises the resolution of the exchanges ( Figure 10C , D ) .",
"The ongoing but unproductive intercalations in the fkh mutant , that lack directional formation and resolution , support the notion that the pulling of the constricting and invaginating pit reinforces the directionality of resolution in the wild type .",
"The setup of active constriction and active intercalation in adjacent regions combined with some aspect of mechanical coupling between the two would thus be similar to the germband and posterior midgut , although with the placode being radially organised and the germband axially patterned .",
"Thus , our work suggests the existence of iteratively used morphogenetic mechanisms that are highly adaptable to a particular tissue geometry and size .",
"Compared to germband extension ( Tetley et al . , 2016 ) , the signatures of active apical intercalation behaviour analysed here are reduced in magnitude .",
"In particular , interfaces contract much more strongly away from the neutral Voronoi geometry in the dorso-ventral axis in the germband , compared to the circumferential axis in the placode .",
"Furthermore , individual interfaces approaching a T1 are not significantly different from similarly oriented interfaces in the placode , whereas the strongly increasing fluorescence density of myosin associated with junction shortening in the germband distinguishes the geometry of these interfaces from non-T1 interfaces .",
"This could be due to the overall much smaller size of the tissue , with changes accumulating at a tissue level that restrict further drastic deformation prior to a T1 .",
"This aspect can be addressed in future studies comparing even more active intercalation events in other tissues or through modelling approaches .",
"It will also be crucial to determine what underlies the patterning of accumulation of the different myosin pools across the placode that likely allows the mechanical coupling of behaviours ( Figure 10B ) .",
"The quasi-3D analysis comparing apical to basal sections also revealed a radial organisation of cell behaviours , specifically cell wedging , tilting and interleaving ( Figure 10C , D ) .",
"The constriction near the pit leads to strong cell wedging , with less wedging at a distance from the pit .",
"Also in 3D , cells are tilting towards the point of invagination , with more tilt at a distance .",
"In addition , interleaving of cells ( the continuous change in arrangement of cells along their basal to apical length that can be but is not necessarily associated with discrete changes in neighbour connectivity in depth ) , occurs across the placode and is compatible with active apical circumferential convergence and radial extension of the tissue overall .",
"Thus , our work revealed novel patterns of cell behaviours that could only be uncovered by considering the 3D context of a developing tissue .",
"Non-equilibrium 3D cell geometries in a flat epithelium , such as those caused by the wedging , interleaving and tilting analysed here , evolve during early placode morphogenesis in revealing patterns ( Figure 10C , D ) .",
"The appearance and progressive increase of these out-of-equilibrium geometries in the placode precede the start of 3D pit invagination by more than 10 min .",
"This could suggest that during this phase a pre-pattern of tension could build up across the placode that would make the pit invagination more efficient , once initiated .",
"Some of the cell behaviours we observe and the resulting complex 3D shapes might then be the integrated results of the balance of forces in the changing mechanical context of the placode .",
"This will be possible to test experimentally by interference with certain behaviours or through ectopic induction of others , and can also be tested in silico in the future .",
"Our strain rate analysis in depth has revealed many interesting features of epithelial geometry and behaviour .",
"What is still quite unclear during epithelial morphogenesis is whether morphogenetic behaviours are always initiated apically and propagated basally , or whether there is in fact an active contribution through events initiated at lateral or basal sides .",
"A few recent reports suggest that not all is apically initiated ( Monier et al . , 2015; Sun et al . , 2017 ) , but whether this is a general principle or highly tissue-specific is unclear .",
"It is also unclear how any cell behaviour , whether initiated apically or basally , is communicated and propagated across the length of the cell .",
"In many morphogenetic processes including the tube budding from the salivary gland placode , actomyosin and other morphogenetic effectors are concentrated within the apical junctional domain .",
"Our data at depth reveal that there is close coordination of intercalation rates between apical and mid-basal levels , and that the apically led interleaving and progressive wedging strongly support an active apical mechanism that is followed further basally .",
"In summary , our work uncovers a dynamic interplay of highly patterned cell behaviours within a radially organised tissue .",
"Future research will show whether such radial patterning of myosin and cell behaviours is conserved across other tube-forming tissue primordia ."
],
[
"The following transgenic fly lines were used: sqhAX3; sqh::sqhGFP42 ( Royou et al . , 2004 ) and fkhGal4 ( Henderson and Andrew , 2000; Zhou et al . , 2001 ) [kind gift of Debbie Andrew]; Scribble-GFP ( DGRC Kyoto ) , UAS-palmYFP ( generated from membrane Brainbow; [Hampel et al . , 2011] ) , y1 w* cv1 sqhAX3; P{w+mC = sqh-GFP . RLC}C-42 M{w+mC = Ubi-TagRFP-T-CAAX}ZH-22A ( Kyoto DGRC Number 109822 , referred to as sqhAX3;sqhGFP; UbiRFP ) ; P{w + mC = sqh-GFP . RLC}C-42 M{w + mC = Ubi-TagRFP-T-CAAX}ZH-22A; fkh[6]/TM3 Sb Twi-Gal4::UAS-GFP ( fkh[6] allele from Bloomington ) .",
"See Table 1 for details of genotypes used for individual figure panels .",
"Embryos were collected on apple juice-agar plates and processed for immunofluorescence using standard procedures .",
"Briefly , embryos were dechorionated in 50% bleach , fixed in 4% formaldehyde , and stained with primary and secondary antibodies in PBT ( PBS plus 0 . 5% bovine serum albumin and 0 . 3% Triton X-100 ) .",
"anti-Crumbs and anti-E-Cadherin antibodies were obtained from the Developmental Studies Hybridoma Bank at the University of Iowa; anti-Baz was a gift from Andreas Wodarz ( Wodarz et al . , 1999 ) ; anti-Fkh was a gift from Herbert Jäckle ( Weigel et al . , 1989 ) .",
"Secondary antibodies used were Alexa Fluor 488/Fluor 549/Fluor 649 coupled ( Molecular Probes ) and Cy3 and Cy5 coupled ( Jackson ImmunoResearch Laboratories ) .",
"Samples were embedded in Vectashield ( Vectorlabs ) .",
"Images of fixed samples were acquired on an Olympus FluoView 1200 or a Zeiss 780 Confocal Laser scanning system as z-stacks to cover the whole apical surface of cells in the placode .",
"Z-stack projections were assembled in ImageJ or Imaris ( Bitplane ) , 3D rendering was performed in Imaris .",
"For live time-lapse experiments embryos from [Scribble-GFP , UAS-palmYFP fkhGal4] , [sqhAX3; sqhGFP , UbiRFP] or [sqhGFP , UbiRFP; fkh[6]] were dechorionated in 50% bleach and extensively rinsed in water .",
"Embryos were manually aligned and attached to heptane-glue coated coverslips and mounted on custom-made metal slides; embryos were covered using halocarbon oil 27 ( Sigma ) and viability after imaging after 24 h was controlled prior to further data analysis .",
"Time-lapse sequences were imaged under a 40x/1 . 3NA oil objective on an inverted Zeiss 780 Laser scanning system , acquiring z-stacks every 0 . 8–2 . 6 min with a typical voxel xyz size of 0 . 22 × 0 . 22 × 1 μm .",
"Z-stack projections to generate movies in Supplementary Material were assembled in ImageJ or Imaris .",
"The absence of fluorescent Twi-Gal4::UAS-GFP was used to identify homozygous fkh[6] mutant embryos .",
"During the early stages of salivary gland placode morphogenesis analysed here , fkh[6] mutants showed no reduction in cell number or initiation of apoptosis ( data not shown ) .",
"The membrane channel images from time-lapse experiments were denoised using nd-safir software ( Boulanger et al . , 2010 ) .",
"Cell tracking was performed using custom software written in IDL ( code provided in ( Blanchard et al . , 2009 ) or by email from G . B . B . ) .",
"First , the curved surface of the embryonic epithelium was located by draping a ‘blanket’ down onto all image volumes over time , where the pixel-detailed blanket was caught by , and remained on top of binarised cortical fluorescence signal .",
"Different quasi-2D image layers were then extracted from image volumes at specified depths from the surface blanket .",
"We took image layers at 1–3 and 7–8 μm for the apical and mid-basal depths , respectively .",
"Image layers were local projections of 1–3 z-depths , with median , top-hat or high/low frequency filters applied as necessary to optimise subsequent cell tracking .",
"Cells in image layers at these two depths were segmented using an adaptive watershedding algorithm as they were simultaneously linked in time .",
"Manual correction of segmented cell outlines was performed for all fixed and time-lapse data .",
"The segmentation of all the movies used in this study was manually corrected to ensure at least 90% tracking coverage of the placode at all times .",
"Tracked cells were subjected to various quality filters ( lineage length , area , aspect ratio , relative velocity ) so that incorrectly tracked cells were eliminated prior to further analysis .",
"The number of embryos analysed and number of cells can be found in Tables 1 and 2 ( also see Figure 2—figure supplement 1 ( WT apical ) , Figure 3—figure supplement 1 ( WT basal ) and Figure 8—figure supplement 1 ( f , K , h ) ) .",
"WT movies were aligned in time using as t = 0 min the frame just before the first sign of invagination of cell apices at the future tube pit was evident .",
"fkh[6] mutants were aligned using as a reference of embryo development the level of invagination of the tracheal pits that are not affected in the fkh[6] mutant as well as other morphological markers such as appearance and depth of segmental grooves in the embryo .",
"Cells belonging to the salivary placode ( without the future duct cells that comprise the two most ventral rows of cells in the primordium ) were then manually outlined at t = 0 mins using the surrounding myosin II cable as a guide and ramified forwards and backwards in time .",
"Only cells of the salivary placode were included in subsequent analyses .",
"At t = 0 min , the centre of the future tube pit was specified manually as the origin of a radial coordinate system , with radial distance ( in µm ) increasing away from the pit ( e . g . Figure 1G ) .",
"Circumferential angle was set to zero towards Posterior , proceeding anti-clockwise for the placode on the left-hand side of the embryo , and clockwise for the placode on the right so that data collected from different sides could be overlaid .",
"The radial coordinate system was ‘mobile’ , in the sense that its origin tracked the centre of the pit , forwards and backwards in time , as the placode translated within the field of view due to embryo movement or to on-going morphogenesis .",
"Detailed spatial patterns of the rates of deformation across the placode and over time quantify the outcome of active stresses , viscoelastic material properties and frictions both from within and outside the placode .",
"We quantified strain ( deformation ) rates over small spatio-temporal domains composed of a focal cell and one corona of immediate neighbours over a ~5 min interval ( [Blanchard et al . , 2009] and reviewed in [Blanchard , 2017] ) .",
"On such 2D domains , strain rates are captured elliptically , as the strain rate in the orientation of greatest absolute strain rate , with a second strain rate perpendicular to this ( Figure 2B ) .",
"For the early morphogenesis of the salivary gland placode , in which there is no cell division or gain/loss of cells from the epithelium , three types of strain rate can be calculated .",
"First , total tissue strain rates are calculated for all local domains using the relative movements of cell centroids , extracted from automated cell tracking .",
"This captures the net effect of cell shape changes and cell rearrangements within the tissue , but these can also be separated out .",
"Second , domain cell shape strain rates are calculated by approximating each cell with its best-fit ellipse and then finding the best mapping of a cell’s elliptical shape to its shape in the subsequent time point , and averaging over the cells of the domain .",
"Third , intercalation strain rates that capture the continuous process of cells in a domain sliding past each other in a particular orientation , is calculated as the difference between the total tissue strain rates and the cell shape strain rates of cells .",
"Strain rates were calculated using custom software written in IDL ( code provided in ( Blanchard et al . , 2009 ) or by email from G . B . B . ) .",
"The three types of elliptical strain rate were projected onto our radial coordinate system ( see Figure 1F ) , so that we could analyse radial and circumferential contributions .",
"Strain rates in units of proportional size change per minute can easily be averaged across space or accumulated over time .",
"We present instantaneous strain rates over time for spatial subsets of cells in the placode , and cumulative strain ratios for the same regions over time .",
"These plots were made from exported data using MATLAB R2014b .",
"We calculated strain rates in layers at two depths for WT placodes .",
"Because we applied a radial coordinate system originating in the centre of the future tube pit to both depths , and used the same reference t = 0 min , we were able to compare strain rates at the same spatio-temporal locations on the placode between the depths .",
"We also wanted to characterise the 3D geometries of cells within small domains , using the cell shapes and arrangements in the two depths we had tracked .",
"To do this , we needed to correctly match cells between apical and mid-basal depths .",
"We manually seeded 3–5 apico-basal cell matches per placode , then used an automated method to fill out the remaining cell matches across the placode and over time .",
"We did this by sequentially looking at all unmatched apical cells that were next to one or more matched cell .",
"The location of the unmatched basal centroid was predicted by adding the vector between matched and unmatched apical cell centroids to the matched basal centroid .",
"The nearest actual basal centroid to this predicted basal centroid location was chosen as the match , on condition that the prediction accuracy was within 0 . 25 of the apical centroid distance .",
"Information from multiple matched neighbours was used , if available , improving the basal centroid prediction .",
"Progressively matching cells out from known matched cells filled out placodes for all embryos .",
"We visually checked apical-basal matches for each embryo in movies of an overlay of apical and basal cell shapes with apico-basal centroid connections drawn ( Video 3 ) .",
"Having matched cells between apical and mid-basal layers , we have access to information about approximate 3D cell shapes .",
"For single cells , we can measure how wedged the cell shape is in any orientation and how tilted is the apical centroid to basal centroid ‘in-line’ relative to the surface normal ( Figure 4A ) .",
"In particular , we are interested in the amount of wedging and tilt in our radial and circumferential placode orientations ( Figure 4A ) .",
"However , an important aspect is missing which is that cells can be arranged differently apically versus basally , independently of any cell wedging .",
"That is they can be to a greater or less extent interleaved .",
"Interleaving in depth is completely analogous to cell intercalation in time ( Figure 4—figure supplement 1D ) .",
"The degree of interleaving is a multi-cell phenomenon , so we return to our small domains of a cell and its immediate neighbours .",
"Within these small domains we want to quantify the amount of cell wedging , interleaving and cell tilt .",
"To correctly separate these quantities , we borrow from methods developed to separate out the additive contributions of wedging , interleaving and tilt that account for epithelial curvature ( Deacon , 2012 ) .",
"Deacon shows that for small domains of epithelial cells , curvature across the domain is the sum of their wedging and interleaving , while cell tilt has no direct implication for curvature .",
"During our study period , salivary placodes have minimal curvature ( average curvature is less than 0 . 05 µm−1 in both AP and DV axes ) .",
"We therefore simplify the problem to flat ( uncurved ) domains , uncurving any local curvature so that apical and basal cell outlines are flat and parallel .",
"Usefully , we can then treat small domains in exactly the same way as we have done above to calculate strain rates above , but instead of quantifying the rate of deformation over time , here we calculate rate of deformation in depth , or ‘z-strain rates’ ( Figure 4—figure supplement 1D ) .",
"The cell shape strain rate is equivalent to the wedging z-strain rate ( Figure 4B ) and the intercalation strain rate is the interleaving z-strain rate ( Figure 4D ) .",
"Both of these add up to a total or tissue strain rate and a total or tissue z-strain rate ( Figure 4—figure supplement 1E ) , respectively .",
"Note that the total z-strain rate of a domain can be the result exclusively of wedging or of interleaving ( as in Figure 4B , D; as with temporal strain rates , Figure 2A ) , but some combination of the two is more likely .",
"Domain translation and rotation for temporal domains become domain tilt ( Figure 4F ) and twist in the 3D domain geometries , respectively .",
"Domain twist is very weak in spatial or temporal averages of the placode ( data not shown ) .",
"The units of wedging and interleaving are proportional size change per µm in z , and tilt is simply a rate ( xy µm/z µm ) .",
"We use the convention that change is from basal to apical , so a bottle-shape has negative cell wedging .",
"The number of embryos and cells analysed for 3D cell geometries can be found in Tables 1 and",
"2 . We used changes in neighbour connectivity in our tracked cell data to identify neighbour exchange events ( T1 processes ) .",
"Neighbour exchange events were defined by the identity of the pair of cells that lost connectivity in t and the pair that gained connectivity at t + 1 . The orientation of gain we defined as the orientation of the centroid-centroid line of the gaining pair at t + 1 . We further classified gains as either radially or circumferentially oriented , depending on which the gain axis was most closely aligned to locally .",
"We did not distinguish between solitary T1s and T1s involved in rosette-like structures .",
"From visual inspection , we knew that some T1s were subsequently reversed , so we characterised not only the total number of gains in each orientation but also the net gain in the circumferential axis , by subtracting the number of radial gains .",
"Furthermore , when comparing embryos and genotypes , we controlled for differences in numbers of tracked cells by expressing the net circumferential gain per time step as a proportion of half of the total number of tracked cell-cell interfaces in that time step .",
"We accumulated numbers of gains , net gains , and proportional rate of gain over time for WT ( Figure 5C ) and fkh ( Figure 9E , F ) embryos .",
"Two sample Kolmogorov-Smirnov test was used to determine significance at p<0 . 05 for data in Figure 9F .",
"In the region of the placode away from the pit , we considered that active circumferential intercalation driven by junctional shortening would produce a different signature in the geometries of cells from a pull from the actively constricting cells at the pit ( as depicted in Figure 4A , A’ ) .",
"We took an approach that avoided using simplistic raw measures of junction lengths and angles as these do not take into account differences in the geometry of cells immediately surrounding a junction .",
"First , we considered that junctions approaching a T1-transition that are being actively shortened by myosin II motors would be shorter than expected according to some neutral geometry .",
"We used a Voronoi tessellation based on actual cell centroids as our reference neutral geometry , allowing us to compare actual junction lengths with predicted neutral lengths ( as used in [Tetley et al . , 2016] ) .",
"The Voronoi tessellation provides cell junctions as the set of points equidistant between neighbouring cell centroids ( see magenta dashed lines in Figure 6B , B’ ) .",
"We calculated the deviation from Voronoi in µm , with a negative value indicating that junctions were likely to be actively shortened .",
"A positive value would indicate a passively elongated junction .",
"In practice , placode cells ( and germ-band cells , see Figure 5D–F in [Tetley et al . , 2016] ) are less ordered than a Voronoi tessellation , with the result that cell-cell interfaces are on average longer than tessellated interfaces .",
"The baseline average value is therefore positive and not zero ( see dashed line in Figure 6F ) .",
"In principle , it would be better to generate neutral geometries using a mechanical model but that would require a further set of untested assumptions about the rheology of the placode tissue .",
"We chose the Voronoi tessellation because it offers a very simple way of generating a plausible neutral geometry .",
"Second , we measured the vertex angles in the cells at opposite ends of focal junctions ( see Figure 6C , C’ ) .",
"Raw angles would be expected to reduce from 120° at three-way junctions to 90° at a four-way T1-transition .",
"However , again to control for variation in local cell geometry , we compared angles relative to those predicted by a Voronoi tessellation constructed from cell centroid seeds .",
"Our measure is therefore the angular deviation from the Voronoi tessellation , in degrees , with a negative value indicating a narrower angle , which we would interpret as being drawn out by an actively contracting junction .",
"Our angular deviation measure is measured as the average for the two vertices at either end of a focal junction .",
"Third , we calculated the elongation of the pair of cells at either end of a focal junction ( Figure 6D , D’ ) .",
"Again , we considered that an actively shrinking focal junction will tend to elongate these cells , whereas a contractile pit would elongate cells in the radial direction towards the pit .",
"We classified junctions as being in the run up to a T1 ( T1 data ) or not involved in T1 ( baseline data ) , and according to their ( radial or circumferential ) orientation and asked two types of question .",
"We compared circumferential baseline to radial baseline data , asking if there was an overall active signature bias in the tissue .",
"We also compared T1 data to baseline data for similarly oriented junctions , asking whether junctions involved in a T1 have a specific effect on local geometries .",
"For statistical tests , we again employed the mixed-effects models ( see Statistics section below ) .",
"In order to avoid using temporally correlated data in the comparisons we selected one time point for each data set we compared .",
"For baseline data , we could choose any developmental time point because the baseline data for all three measures did not vary with developmental time ( Figure 6—figure supplement 1B–D ) .",
"We therefore chose the time point −2–0 min from the start of pit invagination , which maximised the number of placodes ( see Figure 2—figure supplement 1A ) and hence N cells .",
"For T1 data we chose −3 min from neighbour exchange , near the middle of the time window before exchange where junction length proceeds consistently to extinction ( Figure 6—figure supplement 1A ) .",
"Embryos of the genotype sqhAX3; sqh::sqhGFP42 ( Royou et al . , 2004 ) were labelled with either with anti-Bazooka and anti-DE-Cadherin or anti-DE-Cadherin and phalloidin to highlight cell membranes .",
"For Figure 7B , myosin II signal intensity of junctions oriented either circumferentially or radially from five placodes was quantified using Image J . For Figure 7I fluoresce intensity of myosin II and Bazooka of junctions such as the ones indicated by arrows in Figure 7H’ , that were oriented either circumferentially or radially from 10 placodes was quantified using Image J . 3-pixel wide lines were manually drawn along each junction .",
"Intensity values were normalised to average fluorescence outside the placodes .",
"We used a paired t-test for comparison of intensities within in the same junction , unpaired t-tests for comparison of circumferential myosin II vs radial myosin II and circumferential Baz vs radial Baz .",
"Whereas previously we quantified apicomedial Myosin II ( Booth et al . , 2014 ) , here we focused on junctional Myosin II .",
"We extracted a quasi-2D layer image from the Myosin II channel at a depth that maximised the capture of the junctional Myosin .",
"We background-subtracted the Myosin images and quantified the average intensity of Myosin along each cell-cell interface within the placode .",
"To calculate the average intensity , we set the width of cell-cell interfaces as the cell edge pixels plus two pixels in a perpendicular direction either side .",
"This captured the variable width of Myosin signal at interfaces .",
"We further summarised the uni- and bipolarity of Myosin for each cell .",
"Methods to calculate Myosin II unipolarity and bipolarity are described in detail in Tetley et al . ( 2016 ) .",
"Briefly , the average interface fluorescence intensity around each cell perimeter as a function of angle is treated as a periodic signal and decomposed using Fourier analysis .",
"The amplitude component of period two corresponds to the strength of Myosin II bipolarity ( equivalent to planar cell polarity ) ( Figure 7D’ ) .",
"Similarly , the amplitude of period one is attributed to myosin unipolarity ( junctional enrichment in a particular interface ) ( Figure 7D” ) .",
"The extracted phase of periods 1 and 2 ( bi/uni-polarity ) represent the orientation of cell polarity .",
"We projected both polarities onto our radial coordinate system .",
"Both polarity amplitudes are expressed as a proportion of the mean cell perimeter fluorescence .",
"Cells at the border of the placode neighbouring the supra-cellular actomyosin cable were excluded from the analysis .",
"The number of embryos and number of cells analysed can be found in Tables 1 and",
"2 . Statistical tests to determine significance of data shown are indicated in the figure legends .",
"Significance in time-lapse movies was calculated for bins of 4 . 5 min using a mixed-effects model implemented in R ( ‘lmer4’ package as in [Butler et al . , 2009; Lye et al . , 2015] ) with a significance threshold of p<0 . 05 .",
"A mixed-effect model has fixed effects and random effects .",
"The former are variables associated with an entire population and are expected to have an effect on the dependent variable .",
"Random effects are factors which are associated with individual experimental units drawn at random from a population and introduce variation that is desirable to account for ( Pinheiro and Bates , 2000 ) .",
"Here , to test for differences in instantaneous strain rates we used as fixed effect the genotypes ( wild type and forkhead ) while the variation between embryos ( from the same genotype ) was considered a random effect .",
"The advantage of using a linear mixed-effect model is that this framework allows testing for differences on certain variables while accounting for sources of variation that are present in the full data set .",
"In the main figures we generally present cumulative strains , which are generated from instantaneous strain rate data shown in Figure supplements .",
"The cumulative strains are calculated only from instantaneous data averages and so do not carry any of the distributions that were used to calculate instantaneous means and associated confidence intervals .",
"We therefore perform all statistics on the instantaneous data , using the mixed-effects model above .",
"For mixed-effects models , one cannot portray a single overall confidence interval .",
"Instead , we have a choice to show one of within- or between-genotype confidence intervals , and we have chosen the former , as has been done previously ( for example in [Butler et al . , 2009; Gorfinkiel et al . , 2009; Lye et al . , 2015; Tetley et al . , 2016] ) .",
"Therefore , error bars in time-lapse plots show an indicative confidence interval of the mean , calculated as the mean of within-embryo variances .",
"The between-embryo variation is not depicted , even though both are accounted for in the mixed effects tests ."
]
] | [
"The budding of tubular organs from flat epithelial sheets is a vital morphogenetic process .",
"Cell behaviours that drive such processes are only starting to be unraveled .",
"Using live-imaging and novel morphometric methods , we show that in addition to apical constriction , radially oriented directional intercalation of cells plays a major contribution to early stages of invagination of the salivary gland tube in the Drosophila embryo .",
"Extending analyses in 3D , we find that near the pit of invagination , isotropic apical constriction leads to strong cell-wedging .",
"Further from the pit cells interleave circumferentially , suggesting apically driven behaviours .",
"Supporting this , junctional myosin is enriched in , and neighbour exchanges are biased towards the circumferential orientation .",
"In a mutant failing pit specification , neither are biased due to an inactive pit .",
"Thus , tube budding involves radially patterned pools of apical myosin , medial as well as junctional , and radially patterned 3D-cell behaviours , with a close mechanical interplay between invagination and intercalation ."
] | [
"Tubes form many of the organs in the animal body , from lungs to kidneys to intestines; but how are these structures created during development ?",
"For example , the tube that composes the salivary gland of the fruit fly emerges from a flat patch of cells .",
"First , a dimple develops in the cell layer and moves inwards to create the tube pit .",
"Then , like water down a plughole , the rest of the cells flow towards this point and fold downwards into the tube .",
"However , it is still unclear exactly which mechanisms drive this process .",
"Here , Sanchez-Corrales et al . combine microscopy and computational approaches to follow how cells behave in a fruit fly embryo as they build the future salivary gland .",
"The results confirmed that the tube starts forming because of ‘apical constriction’: cells , which normally look like prisms , squeeze into a wedge shape .",
"This helps the tissue to bend and create the tube pit .",
"Other mechanisms contribute to the extension of the tube by turning the flat surface of cells into a curved one .",
"In particular , further away from the dimple , cells become tilted towards the cylinder as they move into it .",
"Another process reshuffles how these cells are connected to each other – a mechanism known as neighbour exchange – which leads to an overall movement towards the dimple .",
"As the tube develops , this creates an increasing number of smaller rings of cells around the pit , which helps the cells to form the walls of the cylinder .",
"Many developmental processes are similar across organs and even species , and a next step could be to explore whether the mechanisms described by Sanchez-Corrales et al . are also present outside of the fruit fly’s salivary glands .",
"If so , this could shed light on what happens when tubes fail to form correctly in an embryo , and on how we could create these structures in the laboratory ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"computational and systems biology"
] | A simple biophysical model emulates budding yeast chromosome condensation | elife-05565-v1 | [
[
"One of the most recognizable phenomena of dividing cells is the compaction of chromatin that occurs when cells enter mitosis .",
"In mitosis , centimeters of DNA are compacted into micrometer-sized , rod-shaped chromosomes .",
"This allows genetic material to be packed small enough to be faithfully segregated to opposite cell halves , and compact enough to withstand the forces generated during this process .",
"Over the past two decades , accumulating lines of evidence have indicated that the chromosomal condensin complex is a principal mediator of chromosome condensation .",
"Condensin promotes interactions between its chromosomal binding sites ( Haeusler et al . , 2008 ) , its depletion or genetic mutation in organisms from yeast to vertebrates leads to defective chromosome condensation , reduced mechanical chromosome stability , and consequent chromosome segregation errors ( Hirano and Mitchison , 1994; Saka et al . , 1994; Strunnikov et al . , 1995; Hagstrom et al . , 2002; Hudson et al . , 2003; Hirota et al . , 2004; Oliveira et al . , 2005; Thadani et al . , 2012 ) .",
"Condensin is a member of the structural maintenance of chromosomes ( SMC ) family of large ring-shaped multisubunit protein complexes .",
"These are thought to bind to DNA by topological embrace ( Nasmyth and Haering , 2005; Cuylen et al . , 2011; Murayama and Uhlmann , 2014 ) .",
"How condensin promotes chromosome condensation has remained unclear .",
"Two main ideas about a possible mechanism of condensin function have been put forward .",
"In the more traditional view , condensin forms higher order assemblies within chromosomes , thought of as part of a chromosome scaffold , to which loops of DNA are attached .",
"This view is supported by cytological observations of condensin localization and early biochemical analyses , but also by recent simulations of chromatin interactions within human chromosomes ( Maeshima and Laemmli , 2003; Swedlow and Hirano , 2003; Naumova et al . , 2013; Maeshima et al . , 2014 ) .",
"In a contrasting model , condensin has been proposed to act by providing DNA interactions between its chromosomal binding sites in a more stochastic manner , without the need to engage into higher order assemblies .",
"This idea is supported by measurements of the biophysical properties of chromosomes and high resolution electron tomographic imaging of chromosomes in their close to native state ( Poirier and Marko , 2002; König et al . , 2007; Thadani et al . , 2012; Maeshima et al . , 2014 ) .",
"However , technical limitations mean that it remains a hitherto insurmountable challenge to directly visualize the path that a DNA strand takes inside a chromosome and how and where condensin acts .",
"In this study , we use an ab initio coarse-grained Brownian dynamics simulation of a budding yeast chromosome to explore chromatin behavior during chromosome condensation .",
"We make no assumptions about chromatin behavior other than known physical properties of a nucleosome chain ( Robert , 1995; Grassia and Hinch , 1996; Luger et al . , 1997 ) .",
"We then introduce condensin to provide intrachromosomal interactions .",
"These interactions are modeled to be",
"( i ) stochastic pairwise interactions between two chromosomal binding sites ( Type I model ) , or",
"( ii ) stochastic but able to engage in higher order assemblies where more than two condensin binding sites meet ( Type II model ) .",
"We compare the predictions from these simulations with experimental chromatin proximity data obtained by 4C analysis on budding yeast chromosome 5 and with other measured biophysical chromosome properties .",
"This analysis shows that stochastic pairwise interactions of a chromatin chain , mediated by condensin , provide a close fit to observed chromosome behavior in budding yeast ."
],
[
"We constructed a computational model to simulate emergent behavior of a large chromosome fragment , consisting of a string of 2000 nucleosomes , representing approximately 300 kb in genomic distance .",
"This is longer than the two smallest budding yeast chromosomes , and similar for example , to the length of the long arm of budding yeast chromosome 5 .",
"We applied a ‘bead-spring’ model in which the chromatin chain is represented as a series of nucleosome beads , joined by DNA linkers whose dynamic behavior is approximated as springs ( Figure 1A ) .",
"The nucleosome string thus behaves as a chain , where the DNA linkers regulate the movement of joined nucleosomes according to Hooke's law .",
"The movement of each nucleosome bead follows a Brownian dynamic trajectory , approximating the solution state in the nucleus .",
"Nucleosomes exclude each other in space though , during our simulations , the linker DNA can cross itself with a small probability , which , under in vivo conditions would be achieved by the enzyme topoisomerase II .",
"A weak force corrects the angle at which DNA emerges from the nucleosome surface ( Luger et al . , 1997; Bednar et al . , 1998; Engelhardt , 2007 ) ( see ‘Materials and methods’ for details of the model ) . 10 . 7554/eLife . 05565 . 003Figure 1 . A computational chromosome model .",
"( A ) Schematic of the forces enacted during simulation .",
"Inter-joined grey beads represent nucleosomes , condensin binding sites are highlighted in red .",
"Fentropic ( blue arrows ) move each bead in a Brownian dynamic trajectory , constrained by Ftension ( red arrows ) , a spring force that connects nucleosome beads , Frepulsion ( green arrows ) that avoids overlaps between beads , Fattraction ( purple arrows ) , a weak force that corrects the angle at which DNA linkers emanate from the nucleosomes and Fcondensin that maintains the vicinity of two condensin binding sites , if they meet .",
"( B ) Condensin localization along a 300 kb region on the right arm of budding yeast chromosome 5 , showing condensin binding sites ( red vertical lines ) at approximately 10 kb intervals .",
"( C ) View of a relaxed starting conformation of the simulated 300 kb nucleosome chain .",
"( D ) Illustration of Type I and Type II interactions , where pairs of condensin binding sites interact , or where one binding site interacts with up to two others , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 05565 . 00310 . 7554/eLife . 05565 . 004Figure 1—figure supplement 1 . Nucleosome displacement over time in our computational chromosome model . The histograms show the distribution of the nucleosome bead displacement within 30 ms timeframes , collated from 1000 randomly chosen observations from 10 independent simulations of both the Type I and Type II models in an interphase state . DOI: http://dx . doi . org/10 . 7554/eLife . 05565 . 00410 . 7554/eLife . 05565 . 005Figure 1—figure supplement 2 . α angle distribution of DNA entry and exit from nucleosomes in simulated chromosomes . The α angle distribution of all nucleosomes was recorded from 1000 snapshots , collected from 10 independent simulations of both the Type I and Type II models . DOI: http://dx . doi . org/10 . 7554/eLife . 05565 . 005 To analyze the impact of a chromatin crosslinker on chromosome behavior , we introduced condensin binding sites along the chromatin chain .",
"This was guided by the experimentally visualized condensin distribution along budding yeast chromosomes using chromatin immunoprecipitation ( Wang et al . , 2005; D'Ambrosio et al . , 2008 ) , which revealed condensin-enriched peaks with an average distance of approximately 10 kb ( Figure 1B ) .",
"We therefore assigned a condensin binding site approximately every 10 kb along the chromatin chain .",
"Figure 1C shows a relaxed conformation of the resultant chromatin chain at the beginning of our simulations .",
"If , on their stochastic trajectories , two condensin binding sites come within 40 nm of each other , an interaction is established between them .",
"While we are as yet naïve about how condensin bridges two binding sites , this assumption lies at the heart of the majority of models for condensin action .",
"The attraction radius of 40 nm is based on condensin's molecular architecture ( Anderson et al . , 2002 ) , the ability of chromosomes to compact was insensitive to its exact value .",
"Once established , the interactions dissolve with a model-specific dissociation probability , equivalent to an off-rate of the dynamically chromosome-bound condensin complex ( Gerlich et al . , 2006 ) .",
"We compared two distinct modes of how condensin might act .",
"In our Type I model , each condensin binding site can interact with exactly one other site at a time , thus leading to stochastic pairwise interactions between chromatin segments ( Figure 1D ) .",
"In the Type II model , each condensin binding site can interact with up to two others , thereby allowing the formation of higher order condensin binding site assemblies .",
"This model provides a means to interrogate chromosome behavior based on first principles .",
"We first compared chromosome dimensions in our model to those observed in vivo .",
"To do so , we used a budding yeast strain in which two loci on the chromosome 5 long arm , at 144 kb distance from each other , were fluorescently marked in distinct colors using the TetO/TetR-YFP and LacO/LacI-CFP systems , respectively ( Rohner et al . , 2008 ) .",
"This allowed us to measure their in vivo 3D distance with great precision , yielding interphase distances consistent with previous measurements using loci marked with the same fluorophore ( Guacci et al . , 1994; Bystricky et al . , 2004; D'Ambrosio et al . , 2008 ) .",
"In G1-arrested cells , the mean distance between the loci was 670 nm ( Figure 2A ) , which was similar to the distance of similarly spaced loci in the relaxed starting configuration of our model ( Figure 2B ) .",
"This striking correspondence suggests that interphase chromatin in vivo adopts a configuration of similar dimensions as compared to the dimensions of an unconstrained nucleosome fiber . 10 . 7554/eLife . 05565 . 006Figure 2 . Chromosome dimensions during experimental and computational condensation .",
"( A ) Scheme showing the location of the two loci whose distance was recorded in vivo and during each simulation .",
"Example micrographs of wild type cells in interphase and mitosis are shown , together with a graph depicting the median , upper , and lower quartiles , with whiskers at 2 . 5 and 97 . 5% , outliers also plotted , for both wild type strains in interphase and mitosis , as well as for a strain in mitosis in which condensin has been depleted from the nucleus using the brn1-aa allele ( Haruki et al . , 2008; O'Reilly et al . , 2012; Charbin et al . , 2014 ) .",
"Statistical significance of the differences was assessed using a Wilcoxon–Mann–Whitney test .",
"( B ) Example of an interphase conformation ( Type I model , condensin interaction dissociation rate 10−3 ) of a simulated chromosome , the two marker loci are highlighted , as well as mitotic conformations ( dissociation rate 10−4 ) generated by the Type I and Type II models .",
"( C ) Traces of marker distance over time after the dissociation rates were set to the indicated values at t = 0 .",
"Shown are the mean and the standard error of 30 simulations .",
"The linear compaction ratios are noted for the indicated comparisons . DOI: http://dx . doi . org/10 . 7554/eLife . 05565 . 00610 . 7554/eLife . 05565 . 007Figure 2—figure supplement 1 . Traces of marker distances over time in the Type II model at dissociation probability 5 × 10−4 . Four representative cases of marker distance traces from simulations of the Type II model using dissociation probability 5 × 10−4 .",
"Distance between the two markers was ∼144 kb in the middle section of the chromatin chain , as in Figure 2 .",
"The dissociation rate was changed from 1 × 10−3 to 5 × 10−4 at time 0 .",
"Examples of simulations that remain in a stable equilibrium , either extended or compact , or that transition in either direction between the two states , are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 05565 . 007 To see how condensin action impinges on chromosome behavior in our computational model , we allowed either Type I or Type II interactions between condensin binding sites and followed the marker distance over time .",
"At a high dissociation probability ( 10−3 , i . e . , a probability of 10−3 per simulation step that an existing interaction is lost ) , chromosome dimensions remained largely unchanged .",
"In case of Type I interactions , the marker distance fluctuated around 600–700 nm ( Figure 2C ) .",
"Allowing Type II interactions resulted in a slightly more compacted chromosome and marker distances between 500—600 nm .",
"Taken together , chromatin interactions with a high off-rate are compatible with interphase chromosome dimensions .",
"In mitotically arrested cells , the mean in vivo distance between the two loci decreased to 497 nm , corresponding to an approximately 25% length compaction , equivalent to a just over twofold volume compaction , which depended on the condensin complex ( Figure 2A ) .",
"The compaction ratio was slightly less than reported in previous studies ( Guacci et al . , 1994; Strunnikov et al . , 1995; Vas et al . , 2007; D'Ambrosio et al . , 2008 ) .",
"A likely reason is our use of two colors to distinguish the two loci .",
"This allows us to assign a discrete distance even to close foci in mitosis that would have been considered to be at zero distance due to the resolution limit in single color observations .",
"We note that a twofold volume compaction during budding yeast chromosome condensation is similar to what is observed for example , when comparing human interphase and mitotic chromosomes ( Mora-Bermúdez et al . , 2006 ) .",
"When we reduced condensin's dissociation probability in our simulations , akin to the stabilization of condensin binding to chromosomes that has been observed as human cells enter mitosis ( Gerlich et al . , 2006 ) , chromosomes began to compact .",
"A 10-fold reduction of dissociation probability in the Type I model resulted in a 25% length compaction , comparable to what we observed in vivo ( Figure 2B , C and Video 1 ) .",
"Further reduction of the dissociation probability led to further gradual compaction .",
"In the Type II model , a 10-fold reduction of the dissociation probability caused a length compaction of over 40% , more than what is observed in vivo ( Figure 2B , C and Video 2 ) .",
"A smaller reduction of the dissociation probability by only twofold , to 5 × 10 −4 , led to a bistable behavior of the chromosome , its compaction fluctuating between open and closed equilibrium states of similar dimensions as those obtained at 1 × 10−4 and 1 × 10−3 , respectively ( Figure 2—figure supplement 1 ) .",
"These observations suggest that the half-life of condensin-mediated interactions along a chromatin fiber has the potential to determine the chromosome condensation status .",
"Type I interactions allow compaction to be tuned within a physiological range .",
"In contrast , Type II interactions result in a more stepwise compaction , the degree of which exceeds what we observed in vivo . 10 . 7554/eLife . 05565 . 008Video 1 . Condensation of a single chromatin chain in the Type I model . The video shows two representative stages of simulated chromosome condensation: ( 1 ) the initial extended chromosomal structure ( 5 s are shown ) , followed by the 8th min , when the chromosome has reached a compacted steady-state ( 20 s are shown ) .",
"Nucleosomes are shown as grey spheres and condensin binding sites are in red .",
"If more than two condensin binding sites come within 40 nm of each other , they are highlighted by a yellow sphere to indicate ‘rosette’ formation .",
"Chromatin loops that connect condensin binding sites within rosettes are tinted cyan .",
"A balanced co-existence of rosette and web-like structures in the compacted mitotic stage becomes apparent . DOI: http://dx . doi . org/10 . 7554/eLife . 05565 . 00810 . 7554/eLife . 05565 . 009Video 2 . Condensation of a single chromatin chain in the Type II model . As Video 1 , but the simulation followed the Type II model .",
"Now the rosette-like topologies become dominant and the overall structure is densely packed . DOI: http://dx . doi . org/10 . 7554/eLife . 05565 . 009 To further evaluate our chromosome model , we compared simulated with experimental intrachromosomal contact frequency maps .",
"We utilized the 4C ( circularized chromosome conformation capture ) ( Dekker et al . , 2013 ) technique combined with high throughput sequencing to generate high resolution interaction maps of 4 loci along the budding yeast chromosome 5 long arm ( Figure 3A ) .",
"This revealed an interaction pattern in interphase that was largely contained within approximately 100 kb around the view point ( Figure 3B ) .",
"Two condensin binding sites showed increased local interactions , compared to two viewpoints that were relatively depleted of condensin .",
"Intrachromosomal interactions markedly increased in mitotic cells , both in the vicinity of the view point as well as longer range interactions beyond 100 kb .",
"This increase depended on the condensin complex and was largely reduced when condensin was depleted from nuclei using the brn1-aa allele ( Charbin et al . , 2014 ) .",
"Together this suggests that condensin binding sites are hubs of intrachromosomal interactions , and that these are augmented in mitosis . 10 . 7554/eLife . 05565 . 010Figure 3 . Experimental and computational intrachromosomal interaction frequency maps .",
"( A ) Close-up of the chromosomal viewpoints selected for 4C analysis .",
"Condensin localization along part of the chromosome 5 right arm is shown together with genomic HindIII recognition sites and the four 4C view points that do ( 1 and",
"4 ) or do not ( 2 and",
"3 ) contain a condensin binding site .",
"( B ) Experimental 4C interaction maps of the four regions , in both interphase and mitosis .",
"Shown is also a 4C map of region 4 in mitosis after condensin has been depleted from the nucleus using the brn1-aa allele .",
"The y-axis shows sequencing read counts normalized to the total number of mapped reads in each sample .",
"The percentage of interactions that extend farther than 100 kb from the viewpoint is indicated .",
"( C ) Averaged computational intrachromosomal interaction maps of 6 viewpoints within 50 kb from the chromosome ends , on or between condensin binding sites , generated using both the Type I and Type II model and sampled over 1000 time points and 30 simulations in interphase and mitosis ( condensin interaction dissociation rates 10−3 and 10−4 , respectively ) .",
"The y-axis shows interaction frequencies of the viewpoints normalized to all interactions .",
"( D ) Percentage of interactions that extend beyond 100 kb from the viewpoint under the indicated conditions .",
"The mean of the four experimental fragments , or of the simulated distributions , is shown together with the standard deviation .",
"*p < 0 . 0001 , Wilcoxon–Mann–Whitney test . DOI: http://dx . doi . org/10 . 7554/eLife . 05565 . 010 If condensin promotes interactions between its binding sites , these might become detectable as interaction peaks in our 4C interaction maps .",
"However , such peaks were not clearly discernible ( Figure 3B ) .",
"A possible explanation for this is that condensin-enriched sites are relatively broad features and their approximately 10 kb spacing is close to the resolution limit of the 4C technique , given by the sizes of the HindIII restriction fragments used in our analysis that are in a similar size range .",
"Alternatively , we cannot exclude that condensin engages in interactions not only between its binding sites , but also with chromatin features between binding sites , for example , histones ( Tada et al . , 2011 ) .",
"For ease of analysis , in our present study , the computational analysis focuses on interactions between condensin binding sites .",
"The resultant simulated intrachromosomal contact frequency map derived from our Type I chromatin interactions showed striking qualitative resemblance to the experimental interaction maps .",
"The similarities extended to",
"( i ) the distribution of interactions and their reach to approximately 100 kb ,",
"( ii ) a greater number of interactions that emanate from condensin binding sites ,",
"( iii ) an increase of interactions in mitosis ( Figure 3C ) .",
"The Type II model produced a contact frequency distribution of a broader shape , extending farther from the view point than observed , especially in mitosis .",
"As a quantitative measure to compare the experimental and computational interaction maps , we recorded the percentage of intrachromosomal interactions that extend beyond 100 kb .",
"While overall intrachromosomal contacts increased in mitosis , the fraction of interactions beyond 100 kb was in a similar range between interphase and mitosis ( Figure 3D ) .",
"The same was observed in simulations using the Type I model , while the Type II model predicts the appearance of significantly more mitosis-specific long-range interactions than observed .",
"We next studied the implications of the Type I and Type II models on the pattern and dynamics of intrachromosomal interactions .",
"Pairwise interactions between condensin binding sites in the Type I model gave rise to a loose web-like architecture spanning much of the chromosome volume in interphase ( Figure 4A ) .",
"Interactions within the web were dynamic and frequently interchanging .",
"In mitosis , rosette-shaped foci formed that contained more than two binding sites within condensin's interaction radius of 40 nm .",
"These foci were maintained for short periods of time by alternating pairwise binding site interactions , before they dissolved again ( Figure 4B and Video 1 ) . 10 . 7554/eLife . 05565 . 011Figure 4 . Web and rosette characteristics of the intrachromosomal interaction pattern .",
"( A ) 3D distance maps of the condensin binding sites , a snapshot of an interphase simulation is shown .",
"Each position along the x axis represents a condensin binding site , the color-coded distance between each is shown above .",
"The corresponding snapshot of the chromosome is partitioned into web ( grey ) and rosette ( blue ) compartments .",
"Yellow spheres highlight the core of the rosette structures where more than two condensin binding sites are in proximity .",
"( B ) as ( A ) , but snapshots are shown from simulations in mitosis .",
"A summary of the percentage , life-span , and size of rosette structures within the chromosome , averaged over 3000 time intervals and 30 simulations is given in the table . DOI: http://dx . doi . org/10 . 7554/eLife . 05565 . 011 An interphase Type II chromosome was only in part characterized by a web-like architecture .",
"Instead , over one third of its length was organized in rosettes in which more than 2 condensin binding sites interacted ( Figure 4A ) .",
"In mitosis , most of the chromosome consisted of extended rosette structures that persisted for longer and involved a greater number of condensin binding sites as compared to the Type I model ( Figure 4B and Video 2 ) .",
"Many chromosomal activities , for example , gene regulatory interactions or recombination events , are thought to involve rapid genome scanning for correct contacts which we expect is facilitated by a dynamic chromosome organization .",
"In addition to the mean distance between marker loci , used above to benchmark chromosome dimensions , we also compared the distance distributions between cells in a population to those from the computer simulations .",
"The kurtosis ( K ) is a dimensionless quantity that describes the difference of a distribution from normal , and is a distinctive feature of polymer models ( Gennes , 1979; Balanda and MacGillivray , 1988; Barbieri et al . , 2012 ) .",
"Figure 5A plots K values for a range of chromosomal distances , observed at 100 timepoints in 30 repeats of our chromosome simulations .",
"They range between 2 . 2 and 2 . 8 , with the Type II model showing slightly smaller values compared to Type I , indicative of distance distributions that have a broader peak than a normal distribution ( K = 3 ) .",
"We compared this to the K of the experimental distance distribution ( Figure 2A ) and to a published dataset of in vivo distance measurements in budding yeast ( Bystricky et al . , 2004 ) .",
"The observed K values agreed well with those seen in our simulations , they lay somewhat closer to the values seen in the Type I as compared to the Type II model .",
"Thus , the K value of the simulated distance distributions agrees with those observed in vivo . 10 . 7554/eLife . 05565 . 012Figure 5 . Polymer characteristics of simulated and native budding yeast chromatin .",
"( A ) Kurtosis values calculated from the simulations and experimental measurements in interphase .",
"Experimental data were from Figure 2 and from published measurements ( Bystricky et al . , 2004 ) .",
"( B ) The persistence length Lp of chromatin in the Type I and Type II model as a function of genomic distance .",
"100 chromosome conformations of each model were exhaustively sampled with the orientation correlation function , the means and standard deviations of Lp are plotted .",
"The values in the table are from the 100 kb cut-off , a range similar to that used in the experimental measurements ( Bystricky et al . , 2004; Dekker , 2008 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 05565 . 012 A defining characteristic of polymer models is the bending rigidity ( or persistence length , Lp ) .",
"We estimated Lp of the simulated structures from both Type I and II models using an orientational correlation function ( see ‘Materials and methods’ ) .",
"In both models , Lp increases with greater genomic distances , as expected from loop polymers ( Strobl , 1997 ) .",
"At smaller genomic distances , up to ∼150 kb , the two models show comparable persistence lengths , with values in agreement with experimental Lp estimates in budding yeast of 58–134 nm , based on physical distance and 3C interaction frequency measurements in this distance range ( Dekker , 2008 ) ( Figure 5B ) .",
"At the smallest distances in our simulations the persistence length approaches that of the free chromatin chain without loops , again showing values in line with an experimental Lp estimate for the local persistence length of budding yeast chromatin of approximately 30 nm ( Hajjoul et al . , 2013 ) .",
"At distances greater than 150 kb , the persistence lengths of Type I and II chromosomes becomes significantly distinct .",
"To our knowledge , there is currently no experimental estimate for Lp at these distances , which may be in part due to the difficulty of constraining Lp values by fitting experimental data to analytical functions at these distances .",
"We therefore do not know which model better describes the rigidity of yeast chromosomes at greater genomic distances .",
"The 16 budding yeast chromosomes lie in close contact to each other in the nucleus ( Duan et al . , 2010 ) .",
"If chromosome condensation indeed occurs by stochastic pairwise interactions between condensin binding sites , how does condensin discriminate intrachromosomal interactions that condense a chromosome from interchromosomal interactions that lead to unproductive chromosome crosslinks ?",
"To explore this , we simulated chromosome condensation of two 300 kb long chromatin chains lying adjacent to each other ( Figure 6 and Videos 3 , 4 ) .",
"As expected , we observed condensin-mediated interactions both within and between chromosomes .",
"In the Type I model , intrachromosomal interactions became dominant over time and the two chromosomes formed individual entities , while maintaining occasional dynamic contact between their surfaces .",
"Starting from the same chromosome positions , the Type II model also displayed a tendency for chromosomes to individualize .",
"However , the more stable nature of rosette-like interaction hubs that formed both within and between chromosomes often maintained the two chromosomes in an entangled state and prevented their complete separation .",
"These results show that condensin-mediated interactions individualize chromosomes , as condensin is more likely to encounter binding sites along the same nucleosome string compared to binding sites on two independently moving chains .",
"Dynamic pairwise interactions between condensin binding sites are better able to prevent persistent cross-linking of chromosomes than rosette-like interaction hubs . 10 . 7554/eLife . 05565 . 013Figure 6 . Chromosome individualization during condensation . Snapshots are shown of chromosomes and their 3D distance maps , after 5 min of simulated chromosome condensation of two adjacent chromosomes using the Type I and Type II models .",
"The average number of interchromosome contacts over the 10 min condensation timecourse are indicated .",
"Statistical significance of the difference was assessed using a Wilcoxon–Mann–Whitney test . DOI: http://dx . doi . org/10 . 7554/eLife . 05565 . 01310 . 7554/eLife . 05565 . 014Video 3 . Condensation of two nearby chromatin chains in the Type I model . 25 seconds of two chromatin chains compacting next to each other using the Type I model are shown , illustrating chain separation during condensation ( chain 1 , nucleosomes in light blue , condensin binding sties in red; chain 2 , nucleosomes in yellow , condensin binding sites in green ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 05565 . 01410 . 7554/eLife . 05565 . 015Video 4 . Condensation of two nearby chromatin chains in the Type II model . As Video 3 , but using the Type II model .",
"The two chromatin chains fail to separate during condensation .",
"The full-length , high resolution versions of all the videos can be found with the digital object identifier doi:10 . 5061/dryad . 78622 at http://datadryad . org . DOI: http://dx . doi . org/10 . 7554/eLife . 05565 . 015"
],
[
"In this study , we explore chromosome architecture by building a simple computational model of a budding yeast chromosome , consisting of coarse-grained representations of the two essential elements for condensation , nucleosomes , and a chromatin cross-linking protein , modeled in our simulations to follow the distribution and behavior of condensin .",
"We compare the emergent model behavior to a panel of existing and new in vivo measurements .",
"Previous models have studied the behavior of short chromatin pieces , including details about nucleosome structure ( Woodcock et al . , 1993; Grigoryev et al . , 2009; Schlick and Perisić , 2009; Diesinger and Heermann , 2010 ) .",
"Other models were aimed at describing whole genome organization in the yeast nucleus ( Duan et al . , 2010; Tjong et al . , 2012; Tokuda et al . , 2012; Wong et al . , 2012 ) , or chromosome behavior at a larger scale but lower resolution ( Barbieri et al . , 2012; Brackley et al . , 2013; Naumova et al . , 2013; Giorgetti et al . , 2014 ) .",
"A defining feature of our coarse-grained nucleosome polymer model is that it allows higher order chromosome structure to arise from first principles , the physics of a solvated polymer chain driven by non-specific entropic forces that generate Brownian motion .",
"The model uses a simulated 10 nm nucleosome chain , to which we add two different scenarios of how the chromosomal condensin complex might act .",
"The emergent chromosome architecture is strikingly in line with numerous features that we and others have measured in vivo , offering quasi-molecular insight into what the inside of a chromosome might look like .",
"A first surprise came from the realization that the dimensions of a relaxed computational nucleosome chain are similar to those determined experimentally for interphase budding yeast chromatin .",
"The reason for this surprise is that a nucleosome chain is often portrayed as beads on a straight line .",
"However , a straight line presents a highly ordered state and entropic forces work to fold this into a much more irregular configuration .",
"The angle at which DNA emanates from a nucleosome further promotes generation of a more rugged path of the nucleosome chain .",
"These results suggest that interphase chromatin exists in a relatively relaxed state inside a budding yeast nucleus and that there may be no need to invoke major forces , scaffolds or other organizing principles to explain its packing .",
"The modeled chromatin packing density , when scaled up to the entirety of the budding yeast genome , allows its comfortable fit within the budding yeast nucleus ( see ‘Materials and methods’ ) .",
"We explored the consequences of condensin-mediated interactions along the chromatin chain .",
"Our 4C results suggest that condensin binding sites are hubs of intrachromosomal interactions , and that these interactions are promoted by the condensin complex .",
"We do not yet know how condensin promotes intrachromosomal interactions .",
"One condensin ring might sequentially topologically entrap DNA at two of its binding sites , or two condensin complexes at their respective binding sites might interact with each other .",
"In addition , condensin might engage in interactions with chromatin or nucleosomes at places distinct from its primary binding sites , and such additional contacts might display similar or different characteristics from topological DNA interactions .",
"While the molecular biology underlying condensin's binding mechanism is important to explore , our simulations are oblivious to the molecular details that underlie the interactions .",
"Instead , we found that the half-life and topology of interactions that each condensin binding site can engage in profoundly affects chromosome behavior .",
"In our Type I model , interactions are pairwise , while the Type II model allows each condensin binding site to interact with up to two others .",
"The latter mode allows the seeding and propagation of higher order assemblies of condensin binding sites , in a way that is often portrayed in models of condensin action .",
"Having compared the two models in our simulations , we conclude that Type I interactions fare better at generating a wide spectrum of chromosomal features that match in vivo observations , including genomic to physical distance distributions and intrachromosomal interaction maps in both interphase and mitosis .",
"The Type I interactions also perform better in individualizing neighboring chromosomes during their mitotic condensation .",
"It has been suggested , on theoretical grounds , that a ‘weak chromatin glue’ is required to allow chromosome individualization during condensation ( Marko and Siggia , 1997 ) .",
"In our Type I model , this weakness is achieved by ongoing dynamic reorganization of the chromatin web , compared to the more stable rosette structures formed by Type II interactions .",
"From a physical point of view , a dynamic web structure allows the small advantage of intrachromosomal interactions , arising from the physical continuity of the chromatin chain , to separate chromosomes over time .",
"In other words , it provides a safeguard to reduce the degree of entanglement , should it occur .",
"We cannot exclude that both Type I and II interaction modes occur simultaneously on chromosomes .",
"Indeed , rosette-like interaction hubs transiently form during simulations based on Type I interactions .",
"It remains possible that modifications to condensin , for example due to different levels of phosphorylation , could alter the balance between the Type I and II mechanisms .",
"In any event , our simulations suggest that maintaining a dynamic aspect of chromatin interactions confers advantages during the chromosome condensation process .",
"Is chromosome architecture in budding yeast , and potentially other organisms , sufficiently described by a self organizing chromatin chain , constrained by condensin-mediated interactions ?",
"On the one hand , a web-like chromosome architecture is consistent with biophysical and microscopic analyses also of higher eukaryotic chromosomes ( Poirier and Marko , 2002; König et al . , 2007; Nishino et al . , 2012; Thadani et al . , 2012 ) .",
"On the other hand , our model is doubtless an oversimplification .",
"Condensin is only one of at least three SMC family complexes in eukaryotes , all of which are likely to act as chromatin crosslinkers by promoting DNA interactions .",
"In addition , many organisms contain more than one type of condensin complex , whose chromosomal distributions appear to be distinct .",
"Condensin might also interact with histones and histones in turn engage with each other , while both types of contact are regulated by posttranslational modifications .",
"Linker histones , in organisms that encode them , alter the flexibility of the nucleosome chain .",
"We expect that a plethora of inputs have the potential to modulate or add parameters in our model .",
"What our study shows is that simple assumptions based on first principles go a long way towards explaining chromosome behavior .",
"It will be fascinating to extend similar simulations to much longer nucleosome chains .",
"Will topologically associating domains , that are seen in large chromosomes ( Dixon et al . , 2012; Mizuguchi et al . , 2014 ) , emerge from subtle inhomogeneities in the condensin distribution ?",
"Which additional principles will have to be incorporated into the model to make an iconic metazoan X-shaped mitotic chromosome gain shape ?"
],
[
"The detection of intrachromosomal interactions along budding yeast chromosomes was based on previously published protocols ( Singh et al . , 2009; Splinter et al . , 2012 ) .",
"Cells were grown to mid log phase and arrested in G1 ( interphase ) by α-factor or a-factor treatment ( O'Reilly et al . , 2012 ) , or in metaphase by nocodazole treatment .",
"Rapamycin treatment to deplete nuclear condensin using the anchor-away technique ( Haruki et al . , 2008 ) was performed as described ( Lopez-Serra et al . , 2013; Charbin et al . , 2014 ) .",
"Uniform arrest was confirmed by cell morphology and FACS analysis of DNA content .",
"400 ml of culture were crosslinked at room temperature with 1% formaldehyde for 15 min followed by quenching with 125 mM glycine for 5 min .",
"Cells were washed twice in ice cold TBS and resuspended in 1 ml FA buffer ( 50 mM HEPES/KOH pH7 . 9 , 140 mM NaCl , 1 mM EDTA , 1% Triton X-100 , 0 . 1% sodium deoxycholate , protease inhibitors ) .",
"Cells were broken in a multi bead shocker ( Yasui Kikai Corporation , Japan ) using acid-washed glass beads .",
"Chromatin was pelleted by centrifugation .",
"Samples were taken at each step to assess the chromatin state by agarose gel electrophoresis and by quantitative real time PCR .",
"The chromatin pellet was resuspended in 500 μl NEBuffer 2 ( New England Biolabs , Ipswich , MA ) .",
"SDS was added to a final concentration of 0 . 1% and extraction was allowed for 10 min at 65°C followed by quenching of the SDS with 1% Triton X-100 .",
"0 . 1 mg/ml RNase A was added and incubated for 2 hr at room temperature .",
"Now , 2000 units HindIII ( New England Biolabs ) were added and the digest kept at 37°C with frequent mixing .",
"After 2 hr the same amount of enzyme was added again and incubation continued over night .",
"In the morning , the enzyme was inactivated by incubation at 65°C for 20 min .",
"For proximity ligation , the crosslinked chromatin was diluted 20-fold ( to 10 ml ) with DNA ligase buffer and incubated with 1600 units of T4 DNA ligase ( New England Biolabs ) at 16°C for 2 hr .",
"The crosslinks were now reversed by addition of 100 μl proteinase K ( 20 mg/ml ) and 1% SDS and incubation at 65°C over night .",
"DNA was purified by Phenol/Chloroform extraction and precipitated by addition of 200 mM NaCl and 70% ( final ) of ice cold ethanol .",
"The resulting 3C library was dissolved in 500 μl 10 mM Tris/HCl pH 7 . 9 .",
"440 μl of the above 3C library were adjusted to NEBuffer DpnII and digested with 100 units DpnII ( New England Biolabs ) for 4 hr at 37°C , followed by heat inactivation .",
"Dilution , a second round of proximity ligation and DNA purification were repeated as above .",
"The resulting final 4C library was dissolved in 500 μl 10 mM Tris pH 7 . 9 .",
"1 μl of the 4C library was used as template for PCR amplification using oligonucleotide primers adjacent to pairs of HindIII and DpnII sites on the long arm of budding yeast chromosome 5 .",
"Aliquots of the PCR reactions were analyzed by agarose gel electrophoresis , the remainder was applied to QIAquick PCR Purification Kit ( Qiagen , Netherlands ) for DNA purification .",
"In preparation for sequencing , the DNA samples were end repaired , poly-A tailed and Single End Adapters ( Illumina , San Diego , CA ) were ligated .",
"The manufacturers ‘ChIP-Seq’ protocol was adjusted for our samples .",
"We used Agencourt AMPure XP beads ( Beckman Coulter , Brea , CA ) at a 0 . 8× ratio to remove adapter dimers after ligation and replaced the Illumina Phusion enzyme with the Kapa HiFi HotStart ready mix ( Kapa Biosystems , Wilmington , MA ) .",
"For post library PCR , we used AMPure XP beads at a 1× ratio and optimized pH and salt concentration to maintain size distribution of the library .",
"To improve visualization , size selection was performed after post library PCR on a 2% agarose gel .",
"The region covering 125–1000 bp was excised and gel residue was removed using the QIAquick Gel Extraction Kit ( Qiagen ) .",
"After final quality control on a BioAnalyser 2100 using a DNA 1000 chip ( Agilent , Santa Clara , CA ) , the 4C libraries were used for flowcell cluster formation on a cluster station and 36 bp single end sequencing was performed on a Genome Analyzer IIx .",
"Sequence data were scanned for the respective primer/restriction site pairs associated with the region of interest .",
"Any sequences not matching perfectly these criteria were discarded .",
"The remaining sequences were trimmed to remove the primer and then mapped to the Saccharomyces cerevisiae genome using PatMaN ( Prüfer et al . , 2008 ) .",
"This was performed in two rounds .",
"Firstly , aligning with no mismatches , removing the matching sequences from the input file and then aligning again with 1 mismatch .",
"The two sets of PatMaN results were then merged for each genomic location and then counts were produced for each position .",
"Images of live budding yeast cells containing two differentially fluorescently marked loci at 144 kb distance from each other on the long arm of chromosome 5 ( Rohner et al . , 2008 ) were acquired using a DeltaVision Olympus IX70 inverted microscope with a 100× ( NA = 1 . 40 ) PlanApo objective .",
"100 pairs of two independent biological repeats , each , were scored .",
"Distances between the brightest centers of the two fluorescent foci were measured in 3 dimensional reconstructions of the cells using Softworx ( DeltaVision , GE Healthcare , Pittsburgh , PA ) .",
"For each simulation , a virtual chromosome of ∼300 kb is constructed .",
"Along this length , 2000 nucleosomes are represented as spheres of 10 nm diameter , with condensin binding sites placed at ∼10 kb intervals .",
"The distance between the centers of each consecutive nucleosome bead is set to 15 nm , with the interconnection between them modeled as a spring .",
"The conformation of a self-avoiding chromosome structure used in each Brownian dynamics simulation is obtained in two steps .",
"Firstly , a 3D Hilbert curve filling the space of a 150 nm length cube is generated .",
"Secondly , using Brownian dynamics this compacted chain is allowed to expand to a cylinder of diameter 200 nm and of unlimited length .",
"After this , spatial constraints are removed to obtain a chromatin chain topology of a relaxed interphase state .",
"Brownian dynamics simulation is run until a steady state of the chromosome compaction ratio is achieved ( as shown in Figure 2C where the compaction of chromosome stabilized from ∼5 min onwards ) .",
"The Type I and II models are conceptually similar to a dynamic loop model ( Zhang and Heermann , 2011 ) , in a way that genomically distant sections of the chromatin fibre can cross-link for a fixed amount of time , as facilitated by condensin molecules , when they come into physical proximity of each other .",
"An important feature of our Type I and II model is the probabilistic nature of the cross-linking mechanism , which allows the organization of the chromatin fibre to be dynamic , rather than being a fixed structure .",
"Another important feature of the Type I and II models is the application of physical forces at the nucleosome level .",
"The trajectory of the fiber backbone is regulated by forces , as described below , based on first principle physics .",
"The balance of all forces is calibrated based on experimental measurements of nucleosome movements and the angles between neighboring nucleosome linkers .",
"Without arbitrary parameters to pre-define the conformation of the chromosome chain ( i . e . , no conformation parameters ) , the models allow a direct examination of how chromosome conformations emerge from different modes of condensin interactions ( i . e . , stochastic pairwise interactions and higher order condensing assemblies for the Type I and II models , respectively ) .",
"We calculated the Pearson's definition of K , defined as the fourth central moment divided by the square of the variance , using the following function: ( 11 ) K=μ4σ4 , where μ4 is the fourth moment about the mean and σ is the standard deviation .",
"We applied an orientational correlation function , Kor , to estimate the persistence length , Lp , at different genomic distances ( Strobl , 1997 ) .",
"Kor gauges the rigidity of a polymer by the orientational correlation of pairs of locations .",
"Let u ( L ) be a tangent unit vector describing the direction of the chain at contour length L . The correlation between two points separated by contour length L′ is: ( 12 ) u ( L ) ⋅u ( L+L′ ) .",
"The Kor value is the average of correlations , considering all pairs of tangent vectors separated by contour distance L′ , extracted from an ensemble of equilibrated structures: ( 13 ) Kor=〈u ( L ) ⋅u ( L+L′ ) 〉 .",
"Eventually the Lp value is estimated as the integral width of the Kor function: ( 14 ) LP=∫0∞Kor ( L′ ) d ( L′ ) .",
"We note that this persistence length determination applies to a loop-based chromatin model ( Zhang and Heermann , 2011 ) .",
"Loops constrain free movement of the chromatin fibre and , unlike in a worm-like model , Lp increases with greater genomic distances .",
"We estimated the volume of the 300 kb chromosome chain in our simulations through gridded spaces .",
"Firstly , we defined a space big enough to enclose the chromosome then we partitioned the space into joined 3D grids .",
"We counted the number of grid cells occupied by at least one nucleosome .",
"The volume of the chromosome was the sum of the volumes of the occupied grid cells .",
"The accuracy of this approach depends on the grid size used to partition the space .",
"Small grids underestimate the chromosome volume by omitting internal spaces that are surrounded by the chromosome and that are hence not reachable by other chromosomes .",
"Large grids tend to overestimate the volume by including an excess of external surrounding space that could be available to neighboring chromosomes .",
"Here , we use a grid of 50 nm cubes , corresponding to the observed persistence length at small distances in our simulated structures .",
"As the chromosome volume is determined by the curvature of the chromatin chain , closely related to its persistence length , this is aimed to reduce the effect of both over- or underestimating chromosome volume .",
"Using this approach , the estimated volumes of Type I and Type II chromosomes in interphase were 5 . 0 ± 0 . 17 × 10−2 μm3 and 4 . 8 ± 0 . 3 × 10−2 μm3 , respectively , based on the 60 sampled structures .",
"By scaling both numbers by a factor of 40 we project the volume of our 300 kb chromosome to the volume of the 12 Mb budding yeast genome .",
"This yields approximately 2 ± 0 . 07 μm3 for chromosomes that behave according to the Type I model .",
"This accounts for half the volume of the yeast nucleus , assuming a diameter of 2 μm and corresponding volume of approximately 4 μm3 ( Gasser , 2002 ) .",
"This suggests that the budding yeast nucleus comfortably accommodates the yeast genome based on the size of our simulated chromosome structures ."
]
] | [
"Mitotic chromosomes were one of the first cell biological structures to be described , yet their molecular architecture remains poorly understood .",
"We have devised a simple biophysical model of a 300 kb-long nucleosome chain , the size of a budding yeast chromosome , constrained by interactions between binding sites of the chromosomal condensin complex , a key component of interphase and mitotic chromosomes .",
"Comparisons of computational and experimental ( 4C ) interaction maps , and other biophysical features , allow us to predict a mode of condensin action .",
"Stochastic condensin-mediated pairwise interactions along the nucleosome chain generate native-like chromosome features and recapitulate chromosome compaction and individualization during mitotic condensation .",
"Higher order interactions between condensin binding sites explain the data less well .",
"Our results suggest that basic assumptions about chromatin behavior go a long way to explain chromosome architecture and are able to generate a molecular model of what the inside of a chromosome is likely to look like ."
] | [
"The genetic material of living things is made up of long strands of DNA .",
"Human cells contain about two meters of DNA split between 46 chromosomes .",
"These chromosomes carry all the instructions to build a human body .",
"To fit all of this information inside each human cell , the DNA is wrapped around hundreds of thousands of proteins such that the chromosomes each resemble a string of beads .",
"Most of the time the chromosomes in a cell are only loosely arranged .",
"But , when a cell prepares to divide into two new cells , its chromosomes become more compacted .",
"This allows the DNA to withstand the physical forces involved when the copies of the chromosomes are pulled into the two daughter cells , and it makes it easier for the cell to handle its genetic material .",
"If a chromosome breaks during cell division , it can result in diseases such as cancer .",
"Several proteins—collectively called condensins—work to compact ( or condense ) the chromosomes .",
"These proteins are found in a wide range of species , but it remains poorly understood how they cause chromosomes to become more compact .",
"Due to the technical limitations of current imaging methods , it has not been possible to directly visualize the path of the DNA strand within a compacted chromosome .",
"However , Cheng et al . have now overcome this limitation by combining experimental analyses and computational simulations .",
"Cheng et al . used computer modeling to simulate a piece of chromosome that was about the same size as a chromosome from a single-celled microorganism called budding yeast .",
"This model could accurately recreate the behavior of chromosomes as observed in non-dividing cells—and revealed that these chromosomes are in a relaxed state .",
"Cheng et al . then modeled what happens when condensins are introduced .",
"As expected , the chromosomes became more compacted and the model's behavior was then validated using further experiments .",
"This predicted that condensin complexes , bound to regions along the chromosome's length , interact to form pairs that continually separate and form new pairs with other condensins; and that these ‘dynamic pairwise’ interactions compact the chromosome .",
"The current model describes a relatively small chromosome and , in the future , extending the model to larger chromosomes could shed insight ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | Metaphase chromosome structure is dynamically maintained by condensin I-directed DNA (de)catenation | elife-26120-v2 | [
[
"Mitotic chromosome assembly , although poorly understood at the molecular level ( Piskadlo and Oliveira , 2016 ) , fulfils three major tasks essential for faithful chromosome segregation: First , it ensures chromosome compaction making cell division feasible within the cell space .",
"Secondly , it provides chromosomes with the right mechanical properties ( e . g . bendiness and rigidity ) to facilitate their drastic movements during mitosis .",
"Lastly , it ensures the resolution of the topological constrains that exist between the two sister DNA molecules , as well as between neighbouring chromosomes ( chromosome individualization ) , a key requisite for efficient chromosome partitioning .",
"At the heart of these structural changes are the condensin complexes .",
"Condensin complexes , one of the most abundant non-histone complexes on mitotic chromosomes ( Cuylen and Haering , 2011; Hirano et al . , 1997; Ono et al . , 2003 ) , are composed of two structural maintenance of chromosome ( SMC ) proteins ( SMC2 and SMC4 ) bridged by a kleisin subunit ( Barren/Cap-H for condensin I and Barren2/Cap-H2 for condensin II ) ( Cuylen and Haering , 2011; Hirano et al . , 1997; Ono et al . , 2003 ) .",
"Despite extensive research over the last years , how condensins contribute to chromosome morphology is not completely understood .",
"Biochemical and phenotypic analysis of condensin depletion suggest several possible activities for these complexes , including the resolution of DNA entanglements ( Gerlich et al . , 2006; Hagstrom et al . , 2002; Hirano , 2006; Hudson et al . , 2003; Oliveira et al . , 2005; Ribeiro et al . , 2009; Steffensen et al . , 2001 ) and structural integrity by conferring chromosome rigidity ( Gerlich et al . , 2006; Houlard et al . , 2015; Oliveira et al . , 2005; Ribeiro et al . , 2009 ) .",
"Whether or not these complexes also promote chromatin compaction remains controversial ( Hagstrom et al . , 2002; Hirano , 2006; Hirano et al . , 1997; Hudson et al . , 2003; Kimura and Hirano , 1997; Lavoie et al . , 2002; Oliveira et al . , 2005; Steffensen et al . , 2001 ) .",
"The multiple phenotypes observed on mitotic chromosomes upon depletion of condensin complexes raise the possibility that these complexes may have distinct roles at different times of mitosis .",
"Additionally , several lines of evidence support that these complexes also influence interphase chromosome structure ( Cobbe et al . , 2006; Hartl et al . , 2008 ) .",
"Thus , it is difficult , if not impossible to interpret the results when condensins are depleted prior to mitotic entry using conventional depletion approaches .",
"To circumvent this limitation , we adopt a ‘reverse and acute’ approach to dissect the role of condensin I in the maintenance of chromosome organization .",
"We find that inactivation of one condensin I specifically during metaphase leads to over-compaction at the majority of chromosomal regions .",
"We further demonstrate that upon condensin I cleavage previously separated sister DNA molecules undergo topoisomerase II-dependent re-intertwining and complete failure in chromosome segregation ."
],
[
"To study the role of condensin complexes in the maintenance of chromosome structure , specifically during metaphase , we developed a system to enable analysis of chromosomal structural changes upon rapid and temporally controlled inactivation of condensin in Drosophila melanogaster .",
"Our analysis focused on condensin I complex as prior studies reveal a minor role for condensin II in mitotic chromosome organization in Drosophila ( Hartl et al . , 2008; Herzog et al . , 2013; Savvidou et al . , 2005 ) .",
"We developed a fast inactivation system to disrupt condensin I in the living fly ( Figure 1 and Figure 1—figure supplement 1 ) , following a similar strategy previously used for the structurally related complex cohesin ( Oliveira et al . , 2010; Pauli et al . , 2008; Uhlmann et al . , 2000 ) .",
"This system is based on the use of an exogenous protease ( Tobacco Etch Virus , TEV ) to cleave an engineered protein of interest that contains TEV-cleavage sites and allows specific , rapid and efficient protein inactivation in a tissue- and/or time-dependent manner ( Figure 1 , Figure 1—figure supplement 1 and data not shown ) .",
"To produce flies carrying solely TEV-sensitive condensin I complexes , we produced four versions of the kleisin subunit Barren that contain three consecutive TEV-cleavage sites at four different positions: aa175 , aa389 , aa437 , aa600 ( Figure 1—figure supplement 1 ) .",
"All versions are fully functional as they were able to complement the lethality associated with the Barren null allele BarrL305 ( Bhat et al . , 1996 ) ( Figure 1—figure supplement 1b and data not shown ) .",
"In vitro cleavage experiments reveal that all modified proteins are efficiently cleaved by TEV protease ( Figure 1B and Figure 1—figure supplement 1 ) .",
"The construct Barren3xTEV175-myc was chosen for future analysis based on the healthiness of the rescued strains ( referred as BarrenTEV hereafter ) . 10 . 7554/eLife . 26120 . 003Figure 1 . TEV-mediated cleavage of Barren disrupts condensin I function within a few minutes .",
"( a ) Schematic representation of condensin complex indicating the position of the 3xTEV cleavage sites in the kleisin subunit Barren ( aa175 ) .",
"( b ) In vitro cleavage of BarrenTEV-myc .",
"Extracts were prepared from ovaries of flies expressing solely TEV-cleavable Barren and incubated with TEV protease for the indicated time points ( periods of time ) .",
"The presence of full-length and cleaved Barren was monitored by western blot using myc antibodies .",
"Tubulin was used as loading control .",
"( c ) Early embryos ( 0–30 min old ) expressing HisH2AvD-mRFP1 ( red ) were injected with mRNA coding for BarrenTEV-EGFP ( green ) .",
"Embryos were aged for 1 hr-1hr 30m to allow for protein expression .",
"Embryos were injected with 12 mg/ml UbcH10C114S protein to arrest in metaphase and subsequently with TEV-protease; images depict the same region before and after TEV injection; times ( minutes:seconds ) are relative to the time of injection; scale bar is 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 00310 . 7554/eLife . 26120 . 004Figure 1—figure supplement 1 . A TEV-cleavable system to destroy condensin I .",
"( a ) graphic representation of the four positions used to introduce three consecutive TEV-protease consensus sites within the linker region of Drosophila Barren .",
"Conservation with other species is shown and conserved regions are colour-coded .",
"( b ) Western blot analysis of the strain carrying solely Barren175TEV-myc , compared to wild-type strains , confirming the absence of endogenous protein .",
"Extracts were prepared from ovaries and probed using the indicated antibodies .",
"( c–f )",
"Western blot analysis of in vitro cleavage of different versions of myc-tagged BarrenTEV or Rad21TEV .",
"Extracts were prepared from ovaries of flies expressing TEV-cleavable Barren/Rad21 and incubated with TEV protease for the indicated time points .",
"The presence of full-length and cleaved Barren was monitored by western blot using myc antibodies .",
"Tubulin was used as loading control . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 004 TEV protease-mediated inactivation of condensin complexes has been previously successfully applied in yeast and mouse oocytes ( Cuylen et al . , 2011; Houlard et al . , 2015 ) .",
"However , in both cases the inactivation of condensin complexes took place within over an hour after TEV protease induction .",
"Direct injection of TEV-protease into syncytial embryos , in contrast , allowed cleavage and the removal of chromosome-associated BarrenTEV within 8–15 min ( Figure 1b , c ) , enabling the analysis of the immediate consequences upon disruption of this complex .",
"To confirm that TEV-protease was able to inactivate condensin I efficiently within a few minutes , by cleavage of BarrenTEV , we injected TEV protease in embryos derived from females surviving solely on BarrenTEV ( ectopic expression of BarrenTEV in a BarrL305 null background ) .",
"Injection of TEV-protease in early interphase embryos leads to complete failure of the subsequent mitosis ( which takes place within ~15 min in these embryos ) .",
"Although chromosomes were able to condense upon nuclear envelope breakdown , centromeres , monitored by the Cenp-A ortholog Cid-EGFP , display significant stretching upon microtubule attachment ( Figure 2 , Video 1 and Video 2 ) .",
"Moreover , resolution of sister chromatids is completely impaired , as chromatids appeared as a fused chromatin mass or display very thick bridges during the attempted anaphase ( Figure 2 , Video 1 and Video 2 ) .",
"These results are in accordance with previous findings for condensin I depletion ( Gerlich et al . , 2006; Hagstrom et al . , 2002; Hirano , 2006; Hudson et al . , 2003; Oliveira et al . , 2005; Ribeiro et al . , 2009; Steffensen et al . , 2001 ) , which ensures the developed system is efficient at promoting rapid condensin I inactivation . 10 . 7554/eLife . 26120 . 005Figure 2 . Condensin I inactivation prior to mitotic entry . Embryos surviving solely on BarrenTEV were injected with buffer",
"( a ) or 13 mg/ml TEV protease",
"( b ) ~10–15 min before mitosis; Embryos also express His2A–mRFP1 ( red ) and Cid-EGFP ( green ) ; scale bars , 10 μm .",
"Bottom rows show higher magnifications ( ~3x ) of a single nuclear division .",
"Times ( minutes:seconds ) are relative to the time of anaphase onset . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 00510 . 7554/eLife . 26120 . 006Video 1 . Mitosis in Drosophila embryos . Embryos were injected with buffer in early interphase and monitored throughout the subsequent mitosis .",
"Embryos express HisH2Av-mRFP1 ( red ) and Cid-EGFP ( green ) .",
"Times are relative to injection time .",
"Scale bar is 10 um . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 00610 . 7554/eLife . 26120 . 007Video 2 . Mitosis upon condensin I inactivation in Drosophila embryos . Embryos surviving solely on BarrenTEV were injected with TEV protease in early interphase and monitored in the subsequent mitosis .",
"Embryos express HisH2Av-mRFP1 ( red ) and Cid-EGFP ( green ) .",
"Times are relative to injection time .",
"Scale bar is 10 um . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 007 To test the role of condensin I in the maintenance of chromosome architecture , we allowed mitotic chromosomes to assemble without any perturbation on their structure and subsequently disrupted condensin I during the metaphase-arrest .",
"Embryos were arrested in metaphase , with a functional mitotic spindle , by the use of a catalytically dead dominant-negative form of the E2 ubiquitin ligase necessary for anaphase onset ( UbcH10C114S ) ( Oliveira et al . , 2010; Rape et al . , 2006 ) .",
"Arrested embryos were subsequently injected with TEV protease to destroy condensin I . Given the known role of condensin I in the rigidity of pericentromeric region of chromosomes ( Gerlich et al . , 2006; Oliveira et al . , 2005; Ribeiro et al . , 2009 ) , we first tested the effect of TEV protease injection at those chromosomal sites .",
"Whereas injection of buffer causes no change in the distance between centromeres , TEV protease injection in strains containing solely TEV-cleavable Barren results in rapid separation of centromeres , that appear to stretch towards the poles , leaving behind the majority of the chromatin mass ( Figure 3 , Video 3 and Video 4 ) .",
"These findings imply that condensin I is not only required for the establishment of a rigid structure at the pericentromeric domains prior to metaphase , but also for the maintenance of such organization . 10 . 7554/eLife . 26120 . 008Figure 3 . Condensin I inactivation in pre-assembled chromosomes leads to disruption of centromere structure and hyper-compaction of mitotic chromosomes .",
"( a ) Schematic representation of the experimental layout .",
"Embryos expressing solely BarrenTEV were injected with 12 mg/ml of a dominant-negative form of the human E2 ubiquitin-conjugating enzyme ( UbcH10C114S ) to induce a metaphase arrest .",
"Embryos were subsequently injected with buffer",
"( b ) , 13 mg/ml TEV protease",
"( c ) , 280 μM ICRF",
"( d ) or a mixture containing 13 mg/ml TEV protease and 280 μM ICRF",
"( e ) ; Images depict embryos before the second injection and 14 min after .",
"Embryos also express His2A–mRFP1 ( red ) and Cid-EGFP ( green ) ; scale bars , 10 μm .",
"Insets show higher magnifications ( 2 . 5x ) of a single metaphase .",
"Times ( minutes:seconds ) are relative to the time of the second injection .",
"( f ) Quantitative analysis of centromere positioning 10 min after the second injection , as above; graph shows average ± SEM of individual embryos ( n ≥ 7 embryos for each experimental condition ) ; for each embryo , a minimum of 8 metaphases was measured;",
"( g ) quantifications of mean voxel intensity , volume and surface area of the entire metaphase plate quantified in 3D , over time , and normalized to the time of the second injection .",
"Graphs represent the average ± SEM of individual embryos ( n ≥ 10 embryos for each experimental condition ) ; for each embryo , a minimum of 8 metaphases was quantified . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 008 10 . 7554/eLife . 26120 . 009Figure 3—source data 1 . Centromere displacement and chromosome compaction measurements upon condensin I and topoII inactivation . Individual measurements of centromeres displacement ( Figure 3f ) and relative Mean voxel intensity , relative volume and relative surface area ( Figure 3g ) .",
"Each data set is presented on a separate sheet . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 009 10 . 7554/eLife . 26120 . 010Figure 3—figure supplement 1 . - Chromosome condensation induced by TEV-protease depends on TEV cleavage sites present in BarrenTEV .",
"( a ) Representative images from embryos that do not contain TEV-cleavage sites in Barren .",
"Embryos were injected with UbcH10C114S to induce a metaphase arrest and subsequently injected with 13 mg/ml TEV protease .",
"Embryos also express HisH2Av-RFP1 ( red ) and Cid-EGFP ( green ) ; scale bar is 10 μm .",
"( b ) Quantifications of mean voxel intensity , volume and surface area of the entire metaphase plate quantified in 3D , over time , and normalized to the time of the second injection .",
"Graphs represent the average ± SEM of individual embryos ( n ≥ 10 embryos for each experimental condition ) ; for each embryo , a minimum of 8 metaphases was quantified . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 01010 . 7554/eLife . 26120 . 011Video 3 . Buffer injection in metaphase-arrested embryos . Embryos expressing solely BarrenTEV were injected with 12 mg/ml of a dominant-negative form of the human E2 ubiquitin-conjugating enzyme ( UbcH10C114S ) , to induce a metaphase arrest , and subsequently injected with buffer .",
"Embryos also express His2A–mRFP1 ( red ) and Cid-EGFP ( green ) ; scale bars , 10 μm .",
"Times ( minutes:seconds ) are relative to the time of buffer injection . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 01110 . 7554/eLife . 26120 . 012Video 4 . Condensin I inactivation in metaphase-arrested embryos . Embryos expressing solely BarrenTEV were injected with 12 mg/ml of a dominant-negative form of the human E2 ubiquitin-conjugating enzyme ( UbcH10C114S ) , to induce a metaphase arrest , and subsequently injected with 13 mg/ml TEV protease .",
"Embryos also express His2A–mRFP1 ( red ) and Cid-EGFP ( green ) ; scale bars , 10 μm .",
"Times ( minutes:seconds ) are relative to the time of TEV injection . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 012 Surprisingly , non-centromeric regions do not follow similar disorganization and in fact appeared to become more compacted .",
"We defined chromosome compaction by degree of chromatin density , inferred from the signal of fluorescently labelled histone H2Av-mRFP1 .",
"To quantify the changes in chromosome compaction upon condensin inactivation , we used quantitative imaging analysis to monitor the mean voxel intensity , volume and surface area of each metaphase plate , over time , in 3D ( Figure 3d ) .",
"We found that injection of TEV protease in strains surviving only on BarrenTEV leads to an overall over-compaction of the entire chromatin mass , as evidenced by an increase in the mean voxel intensity and a decrease in both the surface area and the volume of the metaphase plate ( Figure 3c , d ) .",
"Injection of the protease in strains that do not contain TEV-cleavage sites does not result in any evident change in chromosome compaction relative to controls ( Figure 3—figure supplement 1 ) , implying that chromosome over-compaction is specific of condensin I inactivation .",
"In contrast , inactivation of Topoisomerase II ( TopoII ) using a small molecule inhibitor ( ICRF-193 ) , leads to rapid de-compaction of chromosomes ( Figure 3d , g and Video 5 ) .",
"TopoII has been previously implicated in chromosome compaction although its role in the process remains controversial ( Piskadlo and Oliveira , 2016 ) .",
"Although we cannot exclude that chromosome decompaction may be exacerbated by the fact that ICRF-193 traps TopoII onto chromatin , our results support that TopoII may contribute to chromosome compaction in metaphase , consistent with previous observations ( Samejima et al . , 2012 ) , possibly by promoting self-entanglements within the same chromatid ( Kawamura et al . , 2010 ) .",
"Importantly , combined inactivation of both TopoII and condensin I results in chromosome decompaction similar to TopoII inhibition alone ( Figure 3e , g and Video 6 ) .",
"This finding implies that chromatin hyper-compaction observed upon loss of condensin I depends on TopoII activity . 10 . 7554/eLife . 26120 . 013Video 5 . Topoisomerase II inhibition in metaphase-arrested embryos . Embryos expressing solely BarrenTEV were injected with 12 mg/ml of a dominant-negative form of the human E2 ubiquitin-conjugating enzyme ( UbcH10C114S ) , to induce a metaphase arrest , and subsequently injected with 280 μM ICRF-193 .",
"Embryos also express His2A–mRFP1 ( red ) and Cid-EGFP ( green ) ; scale bars , 10 μm .",
"Times ( minutes:seconds ) are relative to the time of ICRF injection . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 01310 . 7554/eLife . 26120 . 014Video 6 . Concomitant inactivation of Topoisomerase II and Condenin I in metaphase-arrested embryos . Embryos expressing solely BarrenTEV were injected with 12 mg/ml of a dominant-negative form of the human E2 ubiquitin-conjugating enzyme ( UbcH10C114S ) , to induce a metaphase arrest , and subsequently injected with a mix of 280 μM ICRF-193 and 13 mg/ml TEV protease .",
"Embryos also express His2A–mRFP1 ( red ) and Cid-EGFP ( green ) ; scale bars , 10 μm .",
"Times ( minutes:seconds ) are relative to the time of the second injection . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 014 The unexpected finding that condensin I inactivation promotes further chromosome compaction , together with the observation that TopoII inhibition reverts this hyper-compaction phenotype , lead us to hypothesize that the observed increase in compaction stems from re-entanglements of DNA strands , which would consequently lead to an increase in chromatin density .",
"Enzymatically , TopoII can promote both the decatenation and the concatenation of DNA strands .",
"Efficient chromosome segregation requires that TopoII is strongly biased towards decatenation prior to anaphase onset but it is conceivable that TopoII can additionally drive the concatenation of native metaphase chromosomes , in vivo .",
"To test whether condensin I removal leads to re-catenation of previously separated sisters , we tested several predictions that arise from this hypothesis: First , the hyper-compaction observed in metaphase , if derived from sister-chromatid re-intertwining , should be dependent on the proximity between DNA molecules .",
"The physical separation of sister chromatids will increase the distance between these two molecules , placing them too far apart , and consequently decreasing the likelihood of their re-entanglement , as recently proposed ( Sen et al . , 2016 ) .",
"Secondly , re-intertwining in late metaphase should lead to severe segregation failures .",
"And lastly , DNA entanglements and the consequent segregation defects should depend on TopoII activity .",
"To evaluate the effect of sister chromatid proximity in chromosome condensation upon condensin inactivation we combined TEV-mediated cleavage of condensin I and cohesin by the use of strains carrying TEV-sensitive Rad21 ( cohesin ) and Barren ( condensin ) proteins .",
"We took advantage of the fact that Rad21TEV cleavage is more efficient than BarrenTEV ( Figure 1—figure supplement 1 ) , which allowed the analysis of changes in chromosome architecture upon condensin inactivation after artificial separation of sister chromatids .",
"Upon TEV protease injection , pole-ward chromosome segregation is initiated within 2 to 5 min and with similar kinetics in both strains ( Figure 4a ) . 10 . 7554/eLife . 26120 . 015Figure 4 . Condensin I inactivation in separated sister chromatids reduces their movement .",
"( a ) Representative images of the initial separation after TEV-mediated cleavage of Rad21TEV and Rad21TEV + BarrenTEV .",
"Graph plots the relative distribution of HisH2B-RFP at the maximal state of sister chromatid separation triggered by TEV-mediated cleavage of Rad21TEV , in strains that contain solely Rad21TEV or both Rad21TEV and BarrenTEV .",
"A 15 μm line was used to measure plot profiles along the segregation plane , measured 3–5 min after TEV protease injection .",
"Graphs plot the average ± SEM of individual embryos ( n ≥ 7 embryos for each experimental condition ) .",
"For each embryo , between 8 and 12 anaphases were analysed .",
"( b ) Example of chromosome movement analysis; left panel represents average of the binary images of three consecutive frames , used to estimate chromosome displacements: blue , non-overlapping pixels; green , two- out of three-frame overlap; grey , three-frame overlap .",
"Scale bar is 10 μm .",
"( c ) Frequency of overlapping pixels to estimate chromosome displacement ( as in",
"b ) , over time , after TEV protease injection . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 015 10 . 7554/eLife . 26120 . 016Figure 4—source data 1 . Measurements of segregation efficiency and chromosome movement upon cohesin/condensin inactivation . Individual measurements of segregation efficiency ( Figure 4a ) and chromosome displacement ( Figure 4c . ) Each data set is presented on a separate sheet . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 016 After the initial pole-ward chromatid movement , isolated chromatids shuffle between the poles , consistent with previous observations ( Oliveira et al . , 2010 ) .",
"To quantify the degree of movement , we used a displacement-quantification method that infers chromosome movement by the overlap between chromosome positions on consecutive frames ( Mirkovic et al . , 2015 ) .",
"Cohesin cleavage alone leads to strong shuffling of isolated single chromatids , as previously described .",
"However , concomitant inactivation of condensin and cohesin results in much slower chromatid movements , with chromatids accumulating in the middle of the segregation plane ( Figure 4b , c ) .",
"Condensin I is thus important for efficient movement of isolated chromatids .",
"This may be due abnormal centromere/kinetochore structure and/or to a possible role for condensin in the error-correction process , as recently proposed ( Peplowska et al . , 2014; Verzijlbergen et al . , 2014 ) .",
"The reduced chromosome movement observed upon condensin I inactivation leads to considerable differences in chromatid positioning in both experimental set-ups .",
"Thus , we restricted our chromosome morphology/compaction analysis to measurements of isolated single sisters , as quantifying the entire chromatin mass would include many confounding variables .",
"Measurements of isolated single chromatids were performed at 20 min after injections and normalized to the values at 5 min after protease injection ( once complete separation has occurred but no significant changes in chromosome structure was yet observed ) .",
"Chromatids considerably change their shape , becoming thicker and shorter ( Figure 5b , c , Video 7 and Video 8 ) , as previously described ( Green et al . , 2012; Ono et al . , 2003 ) .",
"To directly measure the degree of compaction of these isolated sisters , we measured their mean voxel intensity .",
"This analysis revealed that despite the significant changes in chromatid organization , there is no overall change in the mean voxel intensity of single chromatids ( Figure 5d ) , indicating that shape changes are not accompanied by an overall increase in chromatin compaction .",
"We therefore conclude that condensin I inactivation does not promote further chromosome compaction if sister chromatids are physically apart , in contrast to the effect observed in metaphase-arrested chromosomes ( Figure 3 ) .",
"These results support that over-compaction observed in metaphase chromosomes may arise from sister chromatid re-intertwining , consistent with previous observations using yeast circular mini-chromosomes ( Sen et al . , 2016 ) .",
"It is conceivable that condensin I inactivation also promotes abnormal re-entanglement in cis ( between distal regions of the same chromatid ) .",
"The shape changes observed upon condensin inactivation from isolated sisters ( shorter and thicker chromatids ) could indeed be explained by an excess of concatenation within the same DNA molecule , leading to the shortening of the longitudinal axis .",
"However , our compaction measurements indicate that such changes , if occur , do not lead to detectable increase in chromatin density . 10 . 7554/eLife . 26120 . 017Figure 5 . Chromosome over-compaction depends on sister-chromatid proximity .",
"( a ) Stills from metaphase-arrested embryos after injection of TEV protease in strains surviving solely on Rad21TEV ( cohesin cleavage ) or Rad21TEV+BarrenTEV ( cohesin and condensin cleavage ) ; embryos also express HisH2B-RFP; scale bars , 5 μm .",
"Insets show higher magnifications ( 3x ) of single chromatids 20 min after TEV injection .",
"Times ( minutes:seconds ) are relative to the time of TEV injection .",
"( b–c )",
"Relative frequency of sister chromatid length",
"( b ) and width",
"( c ) at 20 min after TEV injections ( n ≥ 120 single chromatids from seven independent embryos for each experimental condition ) .",
"( d ) Mean voxel intensity of isolated single chromatids 20 min after TEV injections , normalized to mean voxel intensity 5 min past injection .",
"( n ≥ 10 embryos for each experimental condition ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 017 10 . 7554/eLife . 26120 . 018Figure 5—source data 1 . Measurements of isolated chromatids upon cohesin/condensin inactivation . Individual measurements of chromosome thickness , length and mean voxel intensity upon TEV-mediated cleavage of Read21TEV and Rad21TEV+BarrenTEV .",
"Each data set is presented on a separate sheet .",
"File includes descriptive statistics . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 018 10 . 7554/eLife . 26120 . 019Video 7 . Artificial induction of sister chromatid separation in metaphase-arrested embryos . Embryos expressing solely Rad21TEV and wild-type Barren were injected with 12 mg/ml of a dominant-negative form of the human E2 ubiquitin-conjugating enzyme ( UbcH10C114S ) , to induce a metaphase arrest , and subsequently injected with 13 mg/ml TEV protease .",
"Embryos also express His2B–RFP; scale bars , 10 μm .",
"Times ( minutes:seconds ) are relative to the time of the second injection . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 01910 . 7554/eLife . 26120 . 020Video 8 . Effect of condensin I inactivation on isolated sister chromatids . Embryos expressing uniquely TEV-sensitive Rad21 and Barren were injected with 12 mg/ml of a dominant-negative form of the human E2 ubiquitin-conjugating enzyme ( UbcH10C114S ) , to induce a metaphase arrest , and subsequently injected with 13 mg/ml TEV protease .",
"Embryos also express His2B–RFP; scale bars , 10 μm .",
"Times ( minutes:seconds ) are relative to the time of the second injection . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 020 Next , we sought to evaluate chromosome segregation defects , which serve as an indirect read-out for the amount of DNA catenations bridging DNA molecules .",
"To monitor segregation defects when condensin I or TopoII are inactivated specifically in metaphase , we developed conditions in which unperturbed chromosomes would be transiently arrested in metaphase by the dominant negative UbcH10C114S , for ~3–5 min , and subsequently injected with the respective perturbing factor in metaphase .",
"Embryos were subsequently injected with a wild-type version of UbcH10 14 min later , which causes anaphase onset and mitotic exit in about 4–8 min ( Figure 6a ) .",
"We monitored the segregation efficiency during anaphase by quantitative analysis of the profile of Histone H2AvD-mRFP ( to visualize chromatin separation ) and Cid-EGFP ( to infer centromere segregation ) along the segregation plane ( Figure 6 ) .",
"In this assay , injection of buffer causes virtually no defects in the segregation of sister chromatids ( Figure 6b , Figure 6—figure supplement 1 and Video 9 ) . 10 . 7554/eLife . 26120 . 021Figure 6 . Condensin I inactivation results in TopoII-dependent sister chromatids intertwines and segregation failure .",
"( a ) Schematic representation of the experimental set-up .",
"Embryos were arrested with 12 mg/ml UbcH10C114S and injected with buffer",
"( b ) , 280 μM ICRF-193",
"( c ) , 13 mg/ml TEV protease",
"( d ) or TEV+ICRF-193 , while in metaphase; Embryos were subsequently injected with 14 mg/ml of a wild-type version of UbcH10 to release them from the arrest .",
"Images depict representative images of the anaphase; Graphs plot the relative distribution of HisH2Av-mRFP1 and Cid-EGFP across the 15 μm segregation plane , measured 4–6 min after anaphase onset .",
"Graphs plot the average +_SEM of individual embryos ( n ≥ 10 embryos for each experimental condition ) .",
"For each embryo , at least eight anaphases were analysed .",
"( f ) Quantification of centromere distances during UbcH10wt-induced anaphase as in ( b–e ) .",
"Graphs plot the distances between segregating centromeres measured 6 min after anaphase onset ( n ≥ 10 embryos for each experimental condition; for each embryo , at least eight anaphases were analysed ) .",
"Statistical analysis was performed using the non-parametric Kruskal-Wallis test; ns p>0 . 05 , *p<0 . 05; ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 021 10 . 7554/eLife . 26120 . 022Figure 6—source data 1 . Measurements of segregation efficiency after metaphase-specific inactivation of condensin and/or Topoisomerase II . Anaphase profiles for HisH2Av-mRFP1 and Cid-EGFP measured 4–6 min after anaphase onset ( Figure 6b-2 ) .",
"Each measurement represents the average for independent embryos ( resulting from at least eight anaphases measured ) .",
"Individual sheets include either the same measurement for the four experimental conditions or both Cid-EGFP and HisH2Av-mRFP1 for the same experiment , as indicated .",
"Centromere separation measurements ( Figure 6f ) also include descriptive statistics . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 022 10 . 7554/eLife . 26120 . 023Figure 6—figure supplement 1 . – Comparative analysis of segregation efficiency for condensin and/TopoII inhibition before mitosis ( light colour ) and during metaphase arrest/release ( dark colour ) ; Graphs plot the relative distribution of HisH2Av-mRFP1 ( red ) and Cid-EGFP ( green ) across a 20 μm segregation plane , measured 4–6 min after anaphase onset . Graphs plot the average +_SEM of individual embryos ( n ≥ 8 embryos for each experimental condition ) .",
"For each embryo , at least eight anaphases were analysed . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 02310 . 7554/eLife . 26120 . 024Video 9 . Induced anaphase in control embryos . Embryos expressing solely BarrenTEV were injected with 12 mg/ml of a dominant-negative form of the human E2 ubiquitin-conjugating enzyme ( UbcH10C114S ) , to induce a metaphase arrest , and subsequently injected with buffer .",
"After 14 min embryos were injected a wild-type version of UbcH10 to induce anaphase .",
"Embryos also express His2A–mRFP1 ( red ) and Cid-EGFP ( green ) ; scale bars , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 024 Inactivation of TopoII under these conditions leads mostly to the appearance of fine chromatid bridges ( Figure 6c and Video 10 ) .",
"These residual bridges are insufficient to delay centromere separation ( 11 , 01 ± 2 , 03 μm upon ICRF-193 treatment compared to 10 , 72 ± 1 , 69 μm in buffer-injected embryos; Figure 6f ) .",
"The extent of chromatin bridges observed upon metaphase-specific inactivation of TopoII is considerably lower when compared to experiments where this enzyme is inhibited prior to mitotic entry ( Figure 6—figure supplement 1 ) .",
"These findings imply that in metaphase-arrested chromosomes the amount of unresolved catenations is residual .",
"In contrast , inactivation of condensin I during metaphase leads to complete impairment of the segregation process , as revealed by the high frequency of ‘fused’ chromatin masses , with the chromosomes remaining in the centre of the segregation plane , and a significant decrease in the distance between segregating centromeres ( 6 , 08 ± 0 , 92 μm ) ( Figure 6d , f and Video 11 ) .",
"The degree of segregation defects observed in these metaphase-inactivation experiments , is even higher than the defects observed upon condensin inactivation prior to mitotic entry ( Figure 6—figure supplement 1 ) .",
"The severity of segregation impairment upon metaphase-specific condensin I inactivation indicates that in the absence of this complex previously resolved sister DNA molecules undergo re-catenation . 10 . 7554/eLife . 26120 . 025Video 10 . Induced anaphase after timely inhibition of topoisomerase II . Embryos expressing solely BarrenTEV were injected with 12 mg/ml of a dominant-negative form of the human E2 ubiquitin-conjugating enzyme ( UbcH10C114S ) , to induce a metaphase arrest , and subsequently injected with 280 μM ICRF-193 .",
"After 14 min embryos were injected a wild-type version of UbcH10 to induce anaphase .",
"Embryos also express His2A–mRFP1 ( red ) and Cid-EGFP ( green ) ; scale bars , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 02510 . 7554/eLife . 26120 . 026Video 11 . Induced anaphase after timely inhibition of Condensin I . Embryos expressing solely BarrenTEV were injected with 12 mg/ml of a dominant-negative form of the human E2 ubiquitin-conjugating enzyme ( UbcH10C114S ) , to induce a metaphase arrest , and subsequently injected with 13 mg/ml TEV protease .",
"After 14 min embryos were injected a wild-type version of UbcH10 to induce anaphase .",
"Embryos also express His2A–mRFP1 ( red ) and Cid-EGFP ( green ) ; scale bars , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 026 To directly test this hypothesis , we accessed whether or not de novo chromatin intertwines take place upon condensin inactivation , as the formation of these new links should depend on TopoII activity .",
"If TopoII-dependent re-catenation occurs upon condensin I inactivation , one would expect that the combination of TopoII and condensin I inactivation should reduce the amount of chromatin trapped in the middle of the segregation plane .",
"On the contrary , if segregation defects result from pre-existing catenations , combined inhibition of condensin I and TopoII should increase , or at least maintain , the density of chromosome bridges and segregation impairment .",
"To address this issue , we used the same experimental layout as above but induced concomitant inactivation of condensin I and TopoII during the metaphase arrest .",
"These experiments reveal a partial rescue of the retained chromatin mass , inferred by HisH2Av-mRFP1 profile ( Figure 6e and Video 12 ) .",
"Chromosome positioning may not be linearly correlated with the amount of linkages bridging the two sister chromatids and thus the reduction on chromosome intertwines may be even more pronounced than inferred by histone profiles .",
"In accordance with this notion , the efficiency of chromosome separation inferred by the position of centromeres returns to levels indistinguishable from wild-type ( Figure 6e , f and Video 12 ) .",
"Thus , concomitant inactivation of condensin I and TopoII significantly reverts the defects associated with condensin I removal .",
"We therefore conclude that the segregation defects observed upon metaphase-specific condensin I inactivation are caused by de novo TopoII-dependent re-intertwining of previously separated sister chromatids . 10 . 7554/eLife . 26120 . 027Video 12 . Induced anaphase after timely inhibition of Condensin I and topoisomerase II . Embryos expressing solely BarrenTEV were injected with 12 mg/ml of a dominant-negative form of the human E2 ubiquitin-conjugating enzyme ( UbcH10C114S ) , to induce a metaphase arrest , and subsequently injected with a mix of 280 μM ICRF-193 and 13 mg/ml TEV protease .",
"After 14 min embryos were injected a wild-type version of UbcH10 to induce anaphase .",
"Embryos also express His2A–mRFP1 ( red ) and Cid-EGFP ( green ) ; scale bars , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 027"
],
[
"The role of condensin complexes in chromosome compaction has been extensively debated .",
"Here we provide evidence that temporally controlled inactivation of condensin I , specifically during metaphase , causes an increase in the overall levels of chromosome compaction in non-centromeric regions .",
"These findings strongly argue that condensin I is required to maintain chromosomal architecture but not to sustain their compacted state .",
"Studies using similar inactivation techniques in mouse oocytes have proposed that condensins confirm longitudinal rigidity , as chromosomes disassemble upon condensin inactivation ( particularly condensin II ) ( Houlard et al . , 2015 ) .",
"At first sight , these findings may be perceived as in sharp contrast to our current observations .",
"It should nevertheless be noted that meiotic chromosomes are under very different force-balance than their mitotic counterparts .",
"In particular , spindle attachment on meiotic bivalents imposes stretching along the longitudinal axis of chromosomes , similarly to what we report here for the pericentromeric region in mitotic chromosomes .",
"Our results now demonstrate that when chromosomes are not subjected to significant additional forces , condensin I inactivation results in an overall chromatin over-compaction rather than chromosome de-condensation .",
"This force-dependent phenotype may explain several inconsistencies in prior analysis on condensins depletion that as sample preparation , chromosome state , presence/absence of microtubules , or even the cell division type ( mitosis vs meiosis ) may play a major role in chromosome morphology .",
"In contrast to condensin I inactivation , TopoII inhibition leads to rapid chromosome decompaction .",
"These finding are consistent with the idea that metaphase chromosome structure is organized as a chromatin network resultant from self-entanglements of DNA strands , as initially proposed by biophysical studies on isolated chromosomes ( Kawamura et al . , 2010 ) .",
"Restricting/favouring chromosome entanglements may thus dictate the state of chromosome compaction .",
"Condensin has been previously proposed to interplay with TopoII , both for chromosome compaction and sister chromatid resolution .",
"The exact details for this interaction , however , remain elusive .",
"Both condensins and TopoII inactivation impair sister chromatid resolution ( Bhat et al . , 1996; Clarke et al . , 1993; Gerlich et al . , 2006; Hagstrom et al . , 2002; Hirano , 2006; Hudson et al . , 2003; Oliveira et al . , 2005; Ribeiro et al . , 2009; Steffensen et al . , 2001; Uemura et al . , 1987 ) , suggesting these two molecules have cooperative roles on chromosome resolution .",
"In contrast , cytological analyses suggest that condensin and TopoII have opposite roles in shaping mitotic chromatin ( Samejima et al . , 2012 ) , raising further doubts on their functional interaction .",
"It has long been hypothesized that condensin may impose directionality on TopoII reactions ( Baxter et al . , 2011; Charbin et al . , 2014; Coelho et al . , 2003; Leonard et al . , 2015 ) , as this enzyme is able to both decatenate and catenate DNA strands .",
"But this model has been very difficult to formally prove .",
"Studies in yeast using artificial circular mini-chromosomes , in which the levels of catenation can be directly measured , support that full decatenation by TopoII requires condensin activity ( Baxter and Aragón , 2012; Baxter et al . , 2011; Charbin et al . , 2014 ) .",
"Whether the same is true in large and linear native chromosomes remained to be addressed , particularly as circular chromosomes are under different topological constrains when compared to linear ones .",
"The experimental approach used in our study allowed the manipulation of native chromosomes , in their natural environment , providing evidence that upon removal of condensin I , previously separated sister chromatids re-intertwine in a TopoII-dependent manner .",
"These findings are in agreement with a recent study that revealed that the resolution of sister chromatids from circular minichromosomes can be reverted by increased expression of TopoII ( Sen et al . , 2016 ) .",
"All together , these results support that condensin I is not directly necessary for TopoII catalytic activity , but rather to impose directionality on TopoII reactions , favouring resolution of the sister DNA molecules rather than their intertwine .",
"Upon condensin I removal , creation of new links between previously separated DNA strands leads to their increased proximity , which may underlie the observed increase in chromosome compaction .",
"Importantly , our studies reveal that TopoII is able to promote erroneous re-entanglements of sister chromatids throughout mitosis , an activity that needs to be constantly opposed by condensin I .",
"How condensin I is able to confer such directionality remains to be addressed .",
"Condensins are enriched at the chromosome axis where they have been proposed to promote interactions within the same chromatid ( Ono et al . , 2003; Steffensen et al . , 2001 ) .",
"Condensin I was shown to display significant turn-over on mitotic chromosomes ( Gerlich et al . , 2006; Oliveira et al . , 2007 ) highlighting that its mode of action relies in dynamic reactions rather than statically holding chromatin loops .",
"Bringing strands of DNA from the same chromatid in close proximity could alone favour sister chromatid decatenation by limiting the probability contacts between sister DNA molecules .",
"Models that predict that DNA loops can extrude away from condensin have been hypothesized ( Goloborodko et al . , 2016; Nasmyth , 2001 ) and are better at explaining the directionally issue , as they provide a mechanism that inherently explains how condensins distinguish intra- versus inter-chromosomal looping .",
"Random intrachromatid linkages are also possible ( Cheng et al . , 2015; Cuylen et al . , 2011 ) , although in this case additional mechanisms may ensure that connections in cis are favoured over linkages between sister- ( and nearby ) chromatids .",
"Condensin I- mediated supercoiling of the DNA molecule has also been proposed to change DNA structure to favour DNA decatenation activity ( Baxter and Aragón , 2012; Baxter et al . , 2011; Sen et al . , 2016 ) , although it is yet to be determined whether the supercoiling activity of this complex can account for all the phenotypes associated with condensin loss .",
"Our analysis further reveals that maintenance of chromosome architecture , particularly sister chromatid resolution , is not a unidirectional process but instead a much more dynamic reaction than previously anticipated .",
"It is conceivable that the highly compacted chromatin state present in metaphase chromosomes could , on its own , shift TopoII reaction towards sister chromatid re-entanglement given the increased proximity between DNA strands .",
"Condensin I would thus counteract an inherent tendency of chromosomes to re-intertwine , a reaction necessary throughout metaphase .",
"Additionally , it is possible that a dynamic balance of chromosome entanglements allows remodelling of chromosome architecture , providing chromosomes with plasticity to counteract the cytoplasmic drag faced during dynamic movements .",
"Energy released during these reactions could potentially be used to further facilitate chromosome movement .",
"Mitotic chromosomes should thus be visualized as highly dynamic structures during mitosis , whose re-shaping may be fundamental for the fidelity of their own segregation ."
],
[
"To destroy condensin by TEV protease-mediated cleavage , strains carrying solely TEV-sensitive Barren versions were produced .",
"A construct carrying a ~4 . 7 kb Barren genomic region was used as a starting point ( kindly provided by Beat Suter , Institute of Cell Biology , University of Bern ) .",
"This region contains the regulatory sequences and was previously shown to restore Barren function ( Masrouha et al . , 2003 ) .",
"This construct was engineered to add a 10xMyc sequence at the C-terminus of Barren .",
"Three consecutive TEV recognition sites were placed at different positions ( corresponding to a . a . 175 , a . a . 389 , a . a . 437 and a . a 600 ) .",
"Cloning details are available upon request .",
"Each variant of genomic Barren with different TEV sites was cloned into pCaSpeR4 vector used for fly transformation .",
"Transgenic flies were produced by P-element integration ( BestGene Inc , Chino Hills , CA ) .",
"Transgenes were placed in a BarrL305 background , a Barren null allele ( Bhat et al . , 1996 ) , over a deficiency for the corresponding genomic region ( Df ( 2L ) Exel7077 , stock #7850 from Bloomington stock center ) .",
"To destroy cohesin by TEV-protease we used strains carrying Rad21TEV , previously described ( Oliveira et al . , 2010; Pauli et al . , 2008 ) .",
"Fly strains also expressed His2AvD–mRFP1 or polyubiquitin His2B–RFP , to monitor DNA and EGFP–Cid to monitor centromeres ( Schuh et al . , 2007 ) .",
"A list with detailed genotypes can be found in Table 1 . 10 . 7554/eLife . 26120 . 028Table 1 . List of fly strains used in this studyDOI: http://dx . doi . org/10 . 7554/eLife . 26120 . 028CHR#*GenotypeReference1418BarrL305/CyOBhat et al . ( 1996 ) ( RRID:BDSC_4402 ) 1421Df ( 2L ) Exel7077/CyOBlommington #7850 ( RRID:BDSC_7850 ) 1513w;; Barr ( 175 - 3TEV ) -myc10 III . 5This study1509w; Barr ( 175 - 3TEV ) -myc10 II . 1;This study1522w;; Barr ( 389 - 3TEV ) -myc10 III . 2This study1514w;; Barr ( 437 - 3TEV ) -myc10 III . 1This study1520w;; Barr ( 600 - 3TEV ) -myc10 III . 3This study1525w;; Barr ( wt ) -myc10 III . 1This study1560w; BarrL305/ Df ( 2L ) Exel7077; Barr ( 175 - 3TEV ) -myc10 III . 5This study820w;; HisH2AvD-mRFP1 III . 1 , CGC ( CID-EGFP ) III . 1Schuh et al . ( 2007 ) 1564Df ( 2L ) Exel7077 / CyO; HisH2AvD-mRFP1 III . 1 , CGC ( CID-EGFP ) III . 1This studyw; BarrL305/ Df ( 2L ) Exel7077; Barr ( 175 - 3TEV ) -myc10 III . 5/ HisH2AvD-mRFP1 III . 1 , CGC ( CID-EGFP ) III . 1629w;; Rad21ex15 , polyubiq-H2B-RFP , tubpr-Rad21 ( 550-3TEV ) -myc10Oliveira et al . ( 2010 ) 1646w; BarrL305 , Barr ( 175 - 3TEV ) -myc10 II . 1; +/+This study1648w; BarrL305 , Barr ( 175 - 3TEV ) -myc10 II . 1; Rad21ex15 , polyubiq-H2B-RFP , tubpr-Rad21 ( 550-3TEV ) -myc10This study*Reference number in our internal lab fly database Microinjection experiments were performed as previously described ( Oliveira et al . , 2010 ) .",
"1–1 . 5 hr old embryos ( or 0–30 min for mRNA injections ) were collected and processed according to standard protocols , and embryos were injected at the posterior pole ( up to three sequential injections ) using a Burleigh Thorlabs Micromanipulator , a Femtojet microinjection system ( Eppendorf , Germany ) , and pre-pulled Femtotip I needles ( Eppendorf ) .",
"Embryos were injected with buffer , drugs or proteins purified from E . coli at the following concentrations: Buffer ( 20 mM Tris-HCl at pH 8 . 0 , 1 mM EDTA , 50 mM NaCl and 2 mM DTT ) , 13 mg/ml TEV protease in TEV buffer , 12 mg/ml UbcH10C114S , 14 mg/ml UbcH10wt and/or 280 μM ICRF-193 ( Sigma-Aldrich , St Louis , MO ) .",
"Purified TEV protease was described previously ( Haering et al . , 2008 ) .",
"Purification of UbcH10wt and UbcH10C114S was performed from BL21 cells as previously described ( Oliveira et al . , 2010 ) , with minor modifications , as follows .",
"Bacterial cells were grown for 16 hr at 37°C , 225 rpm .",
"This pre-culture was used to inoculate fresh LB media and cells were allowed to grow until 0 . 8/1 ODs .",
"Cultures were then induced with 1 mM IPTG and after 4 hr of induction at 37°C , 225 rpm , cells were harvested .",
"Pellets were ressuspended in Lysis Buffer ( 20 mM Tris-HCL pH7 . 5 , 0 . 5M NaCl , 5 mM Imidazole with protease inhibitors ) and sonicated 5x on ice in 30 s cycles ( power 5- Sonicator XL2020 , Misonix , Farmingdale , NY ) .",
"The soluble fraction of the extracts was then incubated in TALON Metal Affinity Resin ( Takara Bio Inc . , Japan ) , according to manufacturer’s instructions .",
"After several washes with Lysis Buffer , the resin coated with the protein was packed into a Poly-Prep Chromatography Column ( Biorad , Hercules , CA ) .",
"Proteins were eluted in the same buffer with 300 mM imidazole .",
"For buffer exchange , purified UbcH10wt and UbcH10C114S proteins were dialyzed overnight , at 4°C , in a Slide-a-Lyzer 7 KDa Dialysis cassettes ( Thermo Scientific , Waltham , MA ) .",
"Final storage buffer was 20 mM Tris-HCL pH7 . 5 , 0 . 3M NaCl ) .",
"The purified proteins were concentrated in a Vivaspin 6 Centrifugal Concentrator MWCO 10 . 000 KDa ( GE Healthcare , Issaquah , WA ) .",
"Barren175TEV-EGFP was cloned into a pRNA plasmid and mRNA was synthesized by in vitro transcription with the mMessage mMachine T3 kit ( Ambion , Austin , TX ) , followed by purification with RNeasy kit ( Qiagen , Germany ) , and elution in RNase-free water .",
"To probe for the efficiency of BarrenTEV removal ( Figure 1C ) , 0–30 min old embryos surviving only on BarrenTEV-Myc were injected with BarrenTEV-EGFP mRNA in pure water at ~2 . 2 μg/μl .",
"Embryos were left to develop at 22°C for 1 , 5–2 hr , to allow for protein translation , before the subsequent injections .",
"Ovaries were dissected from females and homogenized in PBS .",
"Extracts were sonicated for 2 min in a water-bath ( power 5- Sonicator XL2020 , Misonix ) .",
"After centrifugation for 10 min at 15 . 000 rpm at 4°C , the supernatant was removed and adjusted to a final concentration of 2 μg/μl .",
"For cleavage experiments , 80 μl of extract were incubated with 2 μg of TEV protease .",
"At the indicated time points , 10 μl of the reaction were diluted with sample buffer , boiled and stored at −20°C .",
"Samples were loaded on a 10% SDS-gel for electrophoresis and transferred onto a membrane ( Immun-Blot PVDF , Biorad ) .",
"Western-blot analysis was performed according to standard protocols using the following antibodies: anti myc-tag ( 1:200 , Santa Cruz Biotechnology , Dallas , TX , Cat# sc-47694 RRID:AB_627266 ) , anti-α-tubulin ( 1:50 . 000 , DM1A , Sigma-Aldrich Cat# T9026 RRID:AB_477593 ) and anti-Barren ( 1:3000 , kindly provided by Hugo Bellen , ( Bhat et al . , 1996 ) , RRID:AB_2567044 ) .",
"Aligned embryos on coverslips were covered with Series 700 halocarbon oil ( Sigma-Aldrich ) .",
"Imaging of embryos after mRNA injection ( Figure 1c ) was performed with a spinning disc Revolution XD microscope ( Andor , UK ) at 22°C .",
"Stacks of around 20 frames 1 μm were taken at indicated times using a 100 × 1 . 4 oil immersion objective ( Nikon , Japan ) and iXon +512 EMCCD camera ( Andor ) .",
"Time-lapse microscopy was performed with an inverted wide-field DeltaVision microscope ( Applied Precision Inc . , Issaquah , WA ) at 18–20°C in a temperature-controlled room .",
"One stack of ~20 frames ( 0 . 8 μm apart ) was acquired every 1 or 2 min using a 100 × 1 . 4 oil immersion objective ( Olympus , Japan ) and an EMCCD camera ( Roper Cascade 1024 , Roper Technologies , Inc . , Sarasota , FL ) .",
"Widefield images were restored by deconvolution with the Huygens v15 . 10/16 . 10 deconvolution software using a calculated point-spread function ( RRID:SCR_014237 , Scientific Volume Imaging , The Netherlands ) .",
"Movies were assembled using FIJI software ( RRID:SCR_002285 ) ( Schindelin et al . , 2012 ) and selected stills were processed with Photoshop CS6 ( Adobe Systems Incorporated , San Jose , CA ) .",
"For the quantification of chromosome condensation presented in Figure 3g and Figure 3—figure supplement 1 , deconvolved images were analyzed using Imaris v6 . 1 software ( RRID:SCR_007370 , Bitplane , Switzerland ) .",
"The same metaphase was tracked over time and average values for mean voxel intensity , volume and surface area were normalized to the first frame after injection .",
"For the fluorescence profiles presented in Figures 3f and 6b–e , a wide 15 μm-long line was placed manually along the segregation plane and measured using the ‘Plot Profile’ function on FIJI software .",
"For each data set , values were normalized to the maximum .",
"Measurements of single chromatids width and length were performed on projected images ( maximum intensity projection ) , using FIJI software and single chromatids mean voxel intensity measurements were performed using Imaris software .",
"Quantification of chromosome movement ( Figure 4 ) was performed as previously described ( Mirkovic et al . , 2015 ) .",
"Briefly , HisH2B-RFP was imaged at 1 min intervals .",
"Images were segmented to select the chromosomal regions , based on an automatic threshold ( set in the first frame after TEV injection ) , to create binary images .",
"For each movie , a walking average of 3 frames was produced ( using kymograph plug-in , written by J . Rietdorf and A . Seitz , EMBL , Heidelberg , Germany ) creating a merged image in which the intensity is proportional to the overlap between consecutive frames .",
"Intensity profiles were used to estimate the percentage of non-overlapping , 2- frame overlap and 3-frame overlap pixels .",
"Graphic representation was performed using Prism seven software ( RRID:SCR_002798 , GraphPad , La Jolla , CA ) .",
"To compare the average of the centromere distances between each experimental condition ( Figure 6f ) , at least 10 independent embryos were analyzed .",
"Statistical analysis was performed using Prism seven software ( RRID:SCR_002798 ) .",
"Given that some datasets did not pass the normality test ( D'Agostino and Pearson normality test ) , multiple comparisons were performed using the non-parametric Krustal-Wallis test ."
]
] | [
"Mitotic chromosome assembly remains a big mystery in biology .",
"Condensin complexes are pivotal for chromosome architecture yet how they shape mitotic chromatin remains unknown .",
"Using acute inactivation approaches and live-cell imaging in Drosophila embryos , we dissect the role of condensin I in the maintenance of mitotic chromosome structure with unprecedented temporal resolution .",
"Removal of condensin I from pre-established chromosomes results in rapid disassembly of centromeric regions while most chromatin mass undergoes hyper-compaction .",
"This is accompanied by drastic changes in the degree of sister chromatid intertwines .",
"While wild-type metaphase chromosomes display residual levels of catenations , upon timely removal of condensin I , chromosomes present high levels of de novo Topoisomerase II ( TopoII ) -dependent re-entanglements , and complete failure in chromosome segregation .",
"TopoII is thus capable of re-intertwining previously separated DNA molecules and condensin I continuously required to counteract this erroneous activity .",
"We propose that maintenance of chromosome resolution is a highly dynamic bidirectional process ."
] | [
"Living cells can contain huge amounts of genetic information encoded in long strands of DNA .",
"In total several metres of DNA are packed into a small space inside each human cell and these strands can easily become entangled and knotted .",
"When a cell divides to produce new cells the DNA is duplicated and the two copies must be reliably separated , meaning all the knots must be undone .",
"If the DNA strands are not properly separated it can cause extensive damage to genes when the cell tries to divide .",
"Enzymes called topoisomerases work to undo the tangles in DNA allowing it to be divided into two cells .",
"A large protein complex called “condensin I” plays also an important part in organising DNA , and it has also been implicated in helping to resolve knots in the DNA .",
"However , it was not known how condensin I contributes to the successful separation of DNA into new cells , or when in the course of a cell dividing the knots finally get untangled .",
"Cell division is similar in humans and the fruit fly Drosophila melanogaster , and so the fly is often used as a simple way to study this process in the laboratory .",
"Now , Piskadlo et al . have examined the role of condensin I in dividing fruit fly cells by using recently developed techniques that rapidly shut down key molecular machineries while cells divide .",
"The results show that condensin I and an enzyme called Topoisomerase II work together to separate entangled DNA .",
"Topisomerase II can both entangle and disentangle DNA strands and it is condensin I that guides this process to ensure that ultimately all the knots are removed .",
"These findings show that successful cell division requires constant attention from condensin I to make sure Topoisomerase II aids cell division , rather than making the DNA more tangled .",
"Overall this requires more active and constant work to disentangle DNA than expected , and further work is now needed to explain why .",
"Understanding how cells avoid DNA damage during division clarifies why errors in this process cause diseases .",
"For example , changes to condensin I are common in certain cancers and can also lead to disrupted brain development ( e . g . microcephaly ) ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"ecology",
"microbiology and infectious disease"
] | A novel ATP dependent dimethylsulfoniopropionate lyase in bacteria that releases dimethyl sulfide and acryloyl-CoA | elife-64045-v2 | [
[
"The organosulfur molecule dimethylsulfoniopropionate ( DMSP ) is produced in massive amounts by many marine phytoplankton , macroalgae , angiosperms , bacteria , and animals ( Curson et al . , 2018; Stefels , 2000; Otte et al . , 2004; Curson et al . , 2017; Raina et al . , 2013 ) .",
"DMSP can function as an antioxidant , osmoprotectant , predator deterrent , cryoprotectant , protectant against hydrostatic pressure , chemoattractant and may enhance the production of quorum-sensing molecules ( Sunda et al . , 2002; Cosquer et al . , 1999; Wolfe et al . , 1997; Karsten et al . , 1996; Zheng et al . , 2020; Seymour et al . , 2010; Johnson et al . , 2016 ) .",
"DMSP also has important roles in global sulfur and nutrient cycling ( Kiene et al . , 2000; Charlson et al . , 1987 ) .",
"Environmental DMSP can be taken up and catabolised as a carbon and/or sulfur source by diverse microbes , particularly bacteria ( Curson et al . , 2011b ) .",
"DMSP catabolism can release volatile dimethyl sulfide ( DMS ) and/or methanethiol ( MeSH ) ( Reisch et al . , 2011a ) .",
"DMS is a potent foraging cue for diverse organisms ( Nevitt , 2011 ) and the primary biological source of sulfur transferred from oceans to the atmosphere ( Andreae , 1990 ) , which may participate in the formation of cloud condensation nuclei , and influence the global climate ( Vallina and Simó , 2007 ) .",
"Bacteria can metabolize DMSP via three known pathways , the demethylation pathway ( Howard et al . , 2006 ) , the recently reported oxidation pathway ( Thume et al . , 2018 ) , and the lysis pathway ( Curson et al . , 2011b; Figure 1 ) .",
"The nomenclature of these pathways is based on the reaction type of the enzyme catalyzing the first step of DMSP catabolism .",
"In the demethylation pathway , DMSP demethylase DmdA first demethylates DMSP to produce methylmercaptopropionate ( MMPA ) ( Howard et al . , 2006; Reisch et al . , 2008 ) , which can be further catabolized to MeSH and acetaldehyde ( Figure 1; Reisch et al . , 2011b; Bullock et al . , 2017; Shao et al . , 2019 ) .",
"In the oxidation pathway , DMSP is oxidized to dimethylsulfoxonium propionate ( DMSOP ) , which is further metabolized to dimethylsulfoxide ( DMSO ) and acrylate; however , enzymes involved in this pathway are unknown ( Thume et al . , 2018; Figure 1 ) .",
"In the lysis pathway , diverse lyases cleave DMSP to produce DMS and acrylate or 3-hydroxypropionate-CoA ( 3-HP-CoA ) , which are further metabolized by ancillary enzymes ( Curson et al . , 2011b; Johnston et al . , 2016; Figure 1 ) .",
"There is large biodiversity in DMSP lysis , with eight different known DMSP lyases that encompass four distinct protein families ( DddD a CoA-transferase; DddP a metallopeptidase; cupin containing DddL , DddQ , DddW , DddK , and DddY; and Alma1 an aspartate racemase ) functioning in diverse marine bacteria , algae , and fungi ( Figure 1; Curson et al . , 2011b; Johnston et al . , 2016 ) .",
"With the exception of DddD , which catalyzes an acetyl-CoA-dependent CoA transfer reaction , all other DMSP lyases directly cleave DMSP ( Bullock et al . , 2017; Todd et al . , 2007; Alcolombri et al . , 2014; Lei et al . , 2018; Li et al . , 2014 ) .",
"Recently , several bacterial isolates were reported to produce DMS from DMSP but lack known DMSP lyases in their genomes ( Liu et al . , 2018; Zhang et al . , 2019 ) , suggesting the presence of novel enzyme ( s ) for DMSP degradation in nature .",
"A common feature of previously characterized DMSP metabolic pathways is that the metabolites ( i . e . MMPA , acrylate ) need to be ligated with CoA for further catabolism ( Figure 1; Curson et al . , 2011b; Reisch et al . , 2011b ) .",
"Currently , there is no known pathway whereby DMSP is ligated with free CoA , and it is tempting to speculate that there may be such a novel DMSP metabolic pathway .",
"In this study , we screened DMSP-catabolizing bacteria from Antarctic samples , and obtained a strain Psychrobacter sp .",
"D2 that grew on DMSP and produced DMS .",
"Genetic and biochemical work showed that Psychrobacter sp .",
"D2 possesses a novel DMSP lyase termed DddX for DMSP catabolism ( Figure 1 ) .",
"DddX is an ATP-dependent DMSP lyase which catalyzes a two-step reaction: the ligation of DMSP and CoA , and the cleavage of DMSP-CoA to produce DMS and acryloyl-CoA .",
"We further solved the crystal structure of DddX and elucidated the molecular mechanism for its catalysis based on structural and biochemical analyses .",
"DddX is found in both Gram-negative and Gram-positive bacteria .",
"Our results provide novel insights into the microbial metabolism of DMSP by this novel enzyme ."
],
[
"Using DMSP ( 5 mM ) as the sole carbon source , DMSP-catabolizing bacteria were isolated from five Antarctic samples including alga , sediments , and seawaters ( Figure 2—figure supplement 1 , Supplementary file 1a ) .",
"In total , 175 bacterial strains were obtained ( Figure 2—figure supplement 1B ) .",
"Among these bacterial strains , Psychrobacter sp .",
"D2 , a marine gammaproteobacterium , grew well in the medium containing DMSP as the sole carbon source , but not acrylate ( Figure 2A ) .",
"Moreover , gas chromatography ( GC ) analysis showed that Psychrobacter sp .",
"D2 could catabolize DMSP and produce DMS ( 44 . 8 ± 1 . 8 nmol DMS min–1 mg protein–1 ) ( Figure 2B ) .",
"To identify the genes involved in DMSP degradation in Psychrobacter sp .",
"D2 , we sequenced its genome and searched homologs of known DMSP lyases .",
"However , no homologs of known DMSP lyases with amino acid sequence identity higher than 30% were found in its genome ( Supplementary file 1b ) , implying that this strain may possess a novel enzyme or a novel pathway for DMSP catabolism .",
"We then sequenced the transcriptomes of this strain when grown with and without DMSP as the sole carbon source .",
"Transcriptional data analyses showed that the transcripts of four genes ( 1696 , 1697 , 1 , 698 , and 1699 ) that compose a gene cluster were all highly upregulated ( Figure 2—figure supplement 2 ) when DMSP was supplied as the sole carbon source , which was further confirmed by RT-qPCR analysis ( Figure 2C ) .",
"These results suggest that this gene cluster may participate in DMSP catabolism within Psychrobacter sp .",
"D2 .",
"In the gene cluster , 1696 is annotated as a betaine-carnitine-choline transporter ( BCCT ) , sharing 32% amino acid identity with DddT , the predicted DMSP transporter in Marinomonas sp .",
"MWYL1 ( Sun et al . , 2012; Todd et al . , 2007 ) ; 1 , 697 is annotated as an acetate-CoA ligase , and shares 26% sequence identity with the acetyl-CoA synthetase ( ACS ) in Giardia lamblia ( Sánchez et al . , 2000 ) ; 1 , 698 is annotated as an aldehyde dehydrogenase , sharing 72% sequence identity with DddC in Marinomonas sp .",
"MWYL1 ( Todd et al . , 2007 ) ; and 1 , 699 is annotated as an alcohol dehydrogenase , sharing 65% sequence identity with DddB in Marinomonas sp .",
"MWYL1 ( Todd et al . , 2007 ) .",
"DddT , DddC , and DddB have been reported to be involved in DMSP import and catabolism ( Sun et al . , 2012; Todd et al . , 2007; Todd et al . , 2010 ) .",
"The pattern of the identified gene cluster 1696–1699 in Psychrobacter sp .",
"D2 is similar to the patterns of those DMSP-catabolizing clusters reported in Pseudomonas , Marinomonas , and Halomonas , in which dddT , dddB and dddC are clustered with the DMSP lyase gene dddD¸ but which is missing in 1696–1699 and is replaced by 1 , 697 ( Todd et al . , 2007; Todd et al . , 2010; Curson et al . , 2010; Figure 2D ) .",
"These data further support that the 1696–1699 gene cluster is involved in Psychrobacter sp .",
"D2 DMSP catabolism and 1697 encodes a DMSP lyase equivalent to DddD .",
"However , the sequence identity between 1 , 697 and DddD is less than 15% , suggesting that 1 , 697 is unlikely a DddD homolog .",
"With these data we predicted that 1 , 697 encodes a novel DMSP lyase in Psychrobacter sp .",
"D2 , which we term as DddX hereafter .",
"To identify the possible function of dddX in DMSP catabolism , we first deleted the majority of the dddX gene within the Psychrobacter sp .",
"D2 genome to generate a ΔdddX mutant strain ( Figure 2—figure supplement 3 ) .",
"The ΔdddX mutant was unable to grow on DMSP as the sole carbon source , but its ability to utilize DMSP was fully restored to wild type levels by cloned of dddX ( in pBBR1MCS-dddX ) ( Figure 3A ) , indicating that dddX is essential for strain D2 to utilize DMSP .",
"Furthermore , the ΔdddX mutant lost DMSP lyase activity , that is it no longer produced DMS when cultured in marine broth 2 , 216 medium with DMSP .",
"DMSP lyase activity was fully restored to wild type levels in the complemented strain ( ΔdddX/pBBR1MCS-dddX ) ( Figure 3B ) , indicating that dddX encodes a functional DMSP lyase enzyme degrading DMSP to DMS .",
"To verify the enzymatic activity of DddX on DMSP , we cloned the dddX gene , overexpressed it in Escherichia coli BL21 ( DE3 ) , and purified the recombinant DddX ( Figure 3—figure supplement 1 ) .",
"Sequence analysis suggests that DddX is an acetate-CoA ligase , which belongs to the acyl-CoA synthetase ( ACD ) superfamily and requires CoA and ATP as co-substrates for catalysis ( Musfeldt and Schonheit , 2002; Mai and Adams , 1996 ) .",
"Thus , we added CoA and ATP into the reaction system when measuring the enzymatic activity of the recombinant DddX on DMSP .",
"GC analysis showed that the recombinant DddX directly acted on DMSP and produce DMS ( Figure 3C ) .",
"HPLC analysis uncovered ADP and an unknown product as DMS co-products ( Figure 3D ) .",
"The chromatographic retention time of the unknown product was consistent with it being acryloyl-CoA ( Wang et al . , 2017; Cao et al . , 2017 ) .",
"Indeed , liquid chromatography-mass spectrometry ( LC-MS ) analysis found the molecular weight ( MW ) of the unknown product to be 822 . 1317 , exactly matching acryloyl-CoA ( Figure 3E ) .",
"These data demonstrate that DddX is a functional ATP-dependent DMSP lyase that can catalyze DMSP degradation to DMS and acryloyl-CoA .",
"The biochemical results above suggest that DddX catalyzes a two-step degradation of DMSP , a CoA ligation reaction and a cleavage reaction .",
"To perform this two-step reaction , there are two alternative pathways:",
"( i ) , DMSP is first cleaved to form DMS and acrylate , and subsequently CoA is ligated with acrylate ( Figure 3—figure supplement 2A ) .",
"In this case , the intermediate acrylate is produced .",
"( ii ) , CoA is primarily ligated with DMSP to form DMSP-CoA .",
"Then , DMSP-CoA is cleaved , producing DMS and acryloyl-CoA ( Figure 3—figure supplement 2B ) .",
"In this scenario , the intermediate DMSP-CoA is produced .",
"To determine the catalytic process of DddX , we monitored the occurrence of acrylate and/or DMSP-CoA in the reaction system via LC-MS .",
"While acrylate was not detectable in the reaction system , a small peak of DMSP-CoA emerged after a 2 min reaction ( Figure 3F ) , indicating that DMSP-CoA is primarily formed in the catalytic reaction of DddX , which is then cleaved to generate DMS and acryloyl-CoA .",
"Knowing the DddX enzyme activity , we examined its in vitro properties .",
"The DddX enzyme had an optimal temperature and pH of 40°C and 8 . 5 , respectively ( Figure 3—figure supplement 3A and B ) .",
"The apparent KM of DddX for ATP and CoA was 2 . 5 mM ( Figure 3—figure supplement 3C ) and 0 . 4 mM ( Figure 3—figure supplement 3D ) , respectively .",
"DddX had an apparent KM value of 0 . 4 mM for DMSP ( Figure 3—figure supplement 3E ) , which is lower than that of most other reported DMSP lyases and the DMSP demethylase DmdA ( Supplementary file 1c ) .",
"The kcat of DddX for DMSP was 0 . 7 s–1 , with an apparent kcat/KM of 1 . 6 × 103 M–1 s–1 .",
"The catalytic efficiency of DddX toward DMSP is higher than known DMSP lyases DddK , DddP , DddD , but lower than DddY and Alma1 ( Supplementary file 1c ) .",
"Despite DddX belongs to the ACD superfamily , the amino acid identity between DddX and known ACD enzymes is relatively low , with the highest being 26 % between DddX and the Giardia lamblia ACS ( Sánchez et al . , 2000 ) .",
"The kcat/KM value of DddX towards DMSP is lower than several reported ACS enzymes towards acetate ( Chan et al . , 2011; You et al . , 2017 ) .",
"Because ACS enzymes were reported to have promiscuous activity toward different short chain fatty acids , such as acetate and propionate ( Patel and Walt , 1987 ) , we tested the substrate specificity of DddX .",
"The recombinant DddX exhibited no activity towards acetate or propionate ( Figure 3—figure supplement 4 ) , and the presence of acetate or propionate had little effects on the enzymatic activity of DddX toward DMSP ( Figure 3—figure supplement 5 ) , indicating that DddX cannot utilize acetate or propionate as a substrate .",
"Furthermore , we tested the ability of the strain D2 to grow with acetate or propionate as the sole carbon source .",
"The wild-type strain D2 could use acetate or propionate as sole carbon source but deletion of dddX has little effect on the growth of strain D2 on these substrates ( Figure 3—figure supplement 6 ) , suggesting that dddX is unlikely to be involved in acetate and propionate catabolism .",
"Together , these results indicate that DddX does not function as an acetate-CoA ligase .",
"To elucidate the structural basis of DddX catalysis , we solved the crystal structure of DddX in complex with ATP by the single-wavelength anomalous dispersion method using a selenomethionine derivative ( Se-derivative ) ( Supplementary file 1d ) .",
"Although there are four DddX monomers arranged as a tetramer in an asymmetric unit ( Figure 4—figure supplement 1A ) , gel filtration analysis indicated that DddX maintains a dimer in solution ( Figure 4—figure supplement 1B ) .",
"Each DddX monomer contains a CoA-binding domain and an ATP-grasp domain ( Figure 4A ) , with one loop ( Gly280-Tyr300 ) of the CoA-binding domain inserting into the ATP-grasp domain .",
"ATP is bound in DddX mainly via hydrophilic interactions , including hydrogen bonds and salt bridges ( Figure 4B ) .",
"The overall structure of DddX is similar to that of NDP-forming acetyl-CoA synthetase ACD1 ( Weiße et al . , 2016; Figure 4—figure supplement 2 ) , with a root mean square deviation ( RMSD ) between these two structures of 4 . 6 Å over 581 Cα atoms .",
"ACD1 consists of separate α- and β-subunits ( Weiße et al . , 2016 ) , which corresponds to the CoA-binding domain and the ATP-grasp domain of DddX , respectively .",
"Both DddX and ACS belong to the ACD superfamily , which also contains the well-studied ATP citrate lyases ( ACLY ) ( Weiße et al . , 2016; Verschueren et al . , 2019; Hu et al . , 2017 ) .",
"The biochemistry of DddX catalysis is similar to that of ACLY , which converts citrate to acetyl-CoA and oxaloacetate with ATP and CoA as co-substrates ( Verschueren et al . , 2019; Hu et al . , 2017 ) .",
"The catalytic processes of enzymes in the ACD superfamily involve a conformational change of a ‘swinging loop’ or ‘phosphohistidine segment’ , in which a conserved histidine is phosphorylated ( Weiße et al . , 2016; Verschueren et al . , 2019; Hu et al . , 2017 ) .",
"Sequence alignment indicated that His292 of DddX is likely the conserved histidine residue to be phosphorylated , and Gly280-Tyr300 is likely the ‘swinging loop’ ( Figure 4—figure supplement 3 ) .",
"In the crystal structure of DddX , His292 from loop Gly280-Tyr300 directly forms a hydrogen bond with the γ-phosphate of ATP ( Figure 4B ) , suggesting a potential for phosphorylation , which is further supported by mutational analysis .",
"Mutation of His292 to alanine abolished the activity of DddX ( Figure 4C ) , indicating the key role of His292 during catalysis .",
"Circular-dichroism ( CD ) spectroscopy analysis showed that the secondary structure of His292Ala exhibits little deviation from that of wild-type ( WT ) DddX ( Figure 4—figure supplement 4 ) , indicating that the enzymatic activity loss was caused by amino acid replacement rather than by structural change .",
"Altogether , these data suggest that His292 is phosphorylated in the catalysis of DddX on DMSP .",
"Having solved the crystal structure of the DddX-ATP complex , we next sought to determine the crystal structures of DddX in complex with CoA and DMSP .",
"However , the diffractions of these crystals were poor and all attempts to solve the structures failed .",
"Thus , we docked DMSP and CoA into the structure of DddX .",
"In the docked structure , the CoA molecule is bound in the CoA-binding domain , while the DMSP molecule is bound in the interface between two DddX monomers ( Figure 4—figure supplement 5A ) .",
"Because our biochemical results demonstrated that DMSP-CoA is an intermediate of DddX catalysis ( Figure 3F ) , we further docked DMSP-CoA into DddX .",
"DMSP-CoA also locates between two DddX monomers ( Figure 4—figure supplement 5B ) , and two aromatic residues ( Trp391 and Phe435 ) form cation-π interactions with the sulfonium group of DMSP-CoA ( Figure 4—figure supplement 5C ) .",
"Mutations of these two residues significantly decreased the enzymatic activities of DddX ( Figure 4C ) , suggesting that these residues play important roles in DddX catalysis .",
"To cleave DMSP-CoA into DMS and acryloyl-CoA , a catalytic base is necessary to deprotonate DMSP-CoA .",
"Structure analysis showed that Tyr181 , Asp208 , and Glu432 are close to the DMSP moiety ( Figure 4D ) and may function as the general base .",
"Mutational analysis showed that the mutation of Glu432 to alanine abolished the enzymatic activity of DddX , while mutants Tyr181Ala and Asp208Ala still maintained ~40% activities ( Figure 4C ) , indicating that Glu432 is the most probable catalytic residue for the final cleavage of DMSP-CoA .",
"CD spectra of these mutants were indistinguishable from that of WT DddX ( Figure 4—figure supplement 4 ) , suggesting that the decrease in the enzymatic activities of the mutants were caused by residue replacement rather than structural alteration of the enzyme .",
"Based on structural and mutational analyses of DddX , and the reported molecular mechanisms of the ACD superfamily ( Weiße et al . , 2016; Verschueren et al . , 2019; Hu et al . , 2017 ) , we proposed the molecular mechanism of DddX catalysis on DMSP ( Figure 5 ) .",
"Firstly , His292 is phosphorylated by ATP , forming phosphohistidine ( Figure 5A ) , which will be brought to the CoA-binding domain through the conformational change of the swinging loop Gly280-Tyr300 .",
"Next , the phosphoryl group is most likely transferred to DMSP to generate DMSP-phosphate ( Figure 5B ) , which is subsequently attacked by CoA to form DMSP-CoA intermediate ( Figure 5C ) .",
"The last step is the cleavage of DMSP-CoA probably initiated by the base-catalyzed deprotonation of Glu432 ( Figure 5D ) .",
"Finally , acryloyl-CoA and DMS are generated ( Figure 5E ) and released from the catalytic pocket of DddX .",
"We next set out to determine the diversity and distribution of DddX in bacteria with sequenced genomes .",
"We searched the NCBI Reference Sequence Database using the DddX sequence of Psychrobacter sp .",
"D2 as the query .",
"The data presented in Figure 6 showed that DddX homologs are present in several diverse groups of bacteria , including Alphaproteobacteria , Gammaproteobacteria , and Firmicutes .",
"Multiple sequence alignment showed the presence of the key residues involved in phosphorylation ( H292 ) , co-ordination of the substrate ( e . g . W391 ) and catalysis ( D432 ) , suggesting that these DddX homologs are likely functional in bacterial DMSP catabolism .",
"To further validate that these DddX homologs are indeed functional DMSP degrading enzymes , we chemically synthesized representative dddX sequences from Alphaproteobacteria ( Pelagicola sp . LXJ1103 ) , Gammaproteobacteria ( Psychrobacter sp . P11G5; Marinobacterium jannaschii ) , and Firmicutes ( Sporosarcina sp . P33 ) .",
"These candidate DddX enzymes were purified and all were shown to degrade DMSP and produce acryloyl-CoA confirming their predicted activity ( Figure 6—figure supplement 1 ) .",
"We predict that bacteria containing DddX will have DMSP lyase activity , but this will depend on the expression of this enzyme in the host and substrate availability ."
],
[
"DMSP is widespread in nature and cleavage of DMSP produces DMS , an important mediator in the global sulfur cycle .",
"In this study , we report the identification of a novel ATP-dependent DMSP lyase DddX from marine bacteria .",
"DddX belongs to the ACD superfamily , and catalyzes the conversion of DMSP to DMS and acryloyl-CoA , with CoA and ATP as co-substrates .",
"DddX homologs are found in both Gram-positive and Gram-negative bacterial lineages .",
"This study offers new insights into how diverse bacteria cleave DMSP to generate the climatically important gas DMS ."
],
[
"Strains and plasmids used in this study are shown in Supplementary file 1f .",
"Isolates were cultured in the marine broth 2 , 216 medium or the basal medium ( Supplementary file 1g ) with 5 mM DMSP as the sole carbon source at 15°C–25°C .",
"Psychrobacter sp .",
"D2 was cultured in the marine broth 2 , 216 medium or the basal medium ( Supplementary file 1g ) supplied with different carbon sources ( sodium pyruvate , acrylate or DMSP at a final concentration of 5 mM ) at 15–25°C .",
"The E . coli strains DH5α and BL21 ( DE3 ) were grown in the Lysogeny Broth ( LB ) medium at 37°C .",
"Diaminopimelic acid ( 0 . 3 mM ) was added to culture the E . coli WM3064 strain ."
]
] | [
"Dimethylsulfoniopropionate ( DMSP ) is an abundant and ubiquitous organosulfur molecule in marine environments with important roles in global sulfur and nutrient cycling .",
"Diverse DMSP lyases in some algae , bacteria , and fungi cleave DMSP to yield gaseous dimethyl sulfide ( DMS ) , an infochemical with important roles in atmospheric chemistry .",
"Here , we identified a novel ATP-dependent DMSP lyase , DddX .",
"DddX belongs to the acyl-CoA synthetase superfamily and is distinct from the eight other known DMSP lyases .",
"DddX catalyses the conversion of DMSP to DMS via a two-step reaction: the ligation of DMSP with CoA to form the intermediate DMSP-CoA , which is then cleaved to DMS and acryloyl-CoA .",
"The novel catalytic mechanism was elucidated by structural and biochemical analyses .",
"DddX is found in several Alphaproteobacteria , Gammaproteobacteria , and Firmicutes , suggesting that this new DMSP lyase may play an overlooked role in DMSP/DMS cycles ."
] | [
"The global sulfur cycle is a collection of geological and biological processes that circulate sulfur-containing compounds through the oceans , rocks and atmosphere .",
"Sulfur itself is essential for life and important for plant growth , hence its widespread use in fertilizers .",
"Marine organisms such as bacteria , algae and phytoplankton produce one particular sulfur compound , called dimethylsulfoniopropionate , or DMSP , in massive amounts .",
"DMSP made in the oceans gets readily converted into a gas called dimethyl sulfide ( DMS ) , which is the largest natural source of sulfur entering the atmosphere .",
"In the air , DMS is converted to sulfate and other by-products that can act as cloud condensation nuclei , which , as the name suggests , are involved in cloud formation .",
"In this way , DMS can influence weather and climate , so it is often referred to as ‘climate-active’ gas .",
"At least eight enzymes are known to cleave DMSP into DMS gas with a few by-products .",
"These enzymes are found in algae , bacteria and fungi , and are referred to as lyases , for the way they breakdown their target compounds ( DMSP , in this case ) .",
"Recently , researchers have identified some bacteria that produce DMS from DMSP without using known DMSP lyases .",
"This suggests there are other , unidentified enzymes that act on DMSP in nature , and likely contribute to global sulfur cycling .",
"Li , Wang et al . set out to uncover new enzymes responsible for converting the DMSP that marine bacteria produce into gaseous DMS .",
"One new enzyme called DddX was identified and found to belong to a superfamily of enzymes quite separate to other known DMSP lyases .",
"Li , Wang et al . also showed how DddX drives the conversion of DMSP to DMS in a two-step reaction , and that the enzyme is found across several classes of bacteria .",
"Further experiments to characterise the protein structure of DddX also revealed the molecular mechanism for its catalytic action .",
"This study offers important insights into how marine bacteria generate the climatically important gas DMS from DMSP , leading to a better understanding of the global sulfur cycle .",
"It gives microbial ecologists a more comprehensive perspective of these environmental processes , and provides biochemists with data on a family of enzymes not previously known to act on sulfur-containing compounds ."
] | 2021 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology",
"immunology and inflammation"
] | Small molecule inhibition of Csk alters affinity recognition by T cells | elife-08088-v2 | [
[
"SFKs are ubiquitous regulators of basal and inducible receptor signaling .",
"The seven family members are expressed in various combinations in different cell types , have unique substrate specificity and are differentially regulated by phosphatases and localization ( Lowell , 2004 ) .",
"However , they share a common negative regulator Csk .",
"How Csk maintains basal and inducible receptor signaling is still unclear .",
"In T cells , the strength and duration of T cell antigen receptor ( TCR ) signaling in response to antigen stimulation dictates the magnitude and quality of primary and secondary immune responses ( Smith-Garvin et al . , 2009; Chakraborty and Weiss , 2014 ) .",
"The TCR activation threshold , agonist affinity discrimination and signal termination must therefore be tightly regulated .",
"This is achieved through the concerted action of multiple positive and negative regulators acting basally and during inducible signaling .",
"The SFK Lck regulates TCR signaling by phosphorylating tyrosines in the cytoplasmic segments of the TCR ζ and CD3 chains , basally and during antigen recognition , as well as by phosphorylating downstream kinases such as ZAP-70 and ITK ( Chan et al . , 1995; Heyeck et al . , 1997; Smith-Garvin et al . , 2009 ) .",
"The availability and recruitment of active Lck has been proposed to be the rate-limiting step in discriminating agonist affinity ( Stepanek et al . , 2014 ) .",
"Weak agonists with shorter half-lives of pMHC ( peptide-bound major histocompatibility complex ) -TCR interaction have less time to recruit active CD4 or CD8 co-receptor bound Lck .",
"Active Lck ( phosphorylated on Y394 ) is critical for downstream signaling and can be detected in the basal state , but its level does not change appreciably after TCR stimulation ( Nika et al . , 2010 ) .",
"Although the molecular regulation of Lck is understood , its localization and changes in activity during TCR signaling are still unclear ( Chakraborty and Weiss , 2014 ) .",
"Lck is tightly regulated by phosphorylation on two conserved tyrosines .",
"Trans-autophosphorylation of its kinase domain activation loop tyrosine , Y394 , increases its catalytic activity , whereas phosphorylation of its C-terminal tail inhibitory tyrosine , Y505 , promotes its closed , inactive conformation ( Palacios and Weiss , 2004; Chakraborty and Weiss , 2014 ) .",
"In T cells , the receptor-like protein tyrosine phosphatase CD45 regulates Lck activity positively and negatively by dephosphorylating its inhibitory tyrosine and activation loop tyrosine ( McNeill et al . , 2007; Zikherman et al . , 2010 ) .",
"However , the major negative regulator of Lck is Csk , which phosphorylates its inhibitory tail tyrosine ( Bergman et al . , 1992; Levinson et al . , 2008 ) .",
"Csk is a cytoplasmic protein , yet its predominant role is to phosphorylate the inhibitory tyrosine of SFKs that are localized to membranes ( Bergman et al . , 1992 ) .",
"Hence , multiple adaptors have been implicated in regulating its activity by influencing its localization .",
"Transmembrane PAG and Lime , as well as the cytosolic PH-domain containing Dok1/2 , may play roles in recruiting Csk to the cell membrane in a phosphorylation-dependent manner .",
"The association of Csk with PAG in biochemically defined lipid rafts has been observed to diminish in response to TCR stimulation , leading to its proposed involvement in regulating TCR signaling ( Torgersen et al . , 2001; Davidson et al . , 2003 ) .",
"Since Csk returns to these fractions upon signal termination , it has also been implicated in the down-regulation of signaling ( Torgersen et al . , 2001; Davidson et al . , 2003 ) .",
"However individual genetic deficiencies in PAG , LIME or Dok1/2 have had little effect on TCR signaling and on T cell function ( Brdickova et al . , 2003; Dobenecker et al . , 2005; Xu et al . , 2005; Yasuda et al . , 2007 ) , indicating that our understanding of Csk regulation during TCR signal initiation , propagation and termination remains incomplete .",
"The precise role of Csk in TCR signaling has also been difficult to study by genetic inactivation ( Imamoto and Soriano , 1993; Nada et al . , 1993; Schmedt and Tarakhovsky , 2001 ) .",
"Germline deletion of Csk leads to embryonic lethality ( Imamoto and Soriano , 1993; Nada et al . , 1993 ) .",
"Conditional deletion of Csk in the T lineage leads to TCR-MHC-independent , but Lck-dependent , passage through both thymic beta selection and positive selection checkpoints , both of which require perception of pre-TCR or TCR signaling , respectively ( Schmedt and Tarakhovsky , 2001 ) .",
"However , since Csk deficiency leads to aberrant T cell development and compensatory changes in the basal state , studying the role of Csk in ligand-induced signaling in unmanipulated primary T cells has been hindered .",
"We recently generated BAC transgenic mice that express a mutant of Csk ( CskAS ) in the absence of endogenous Csk ( Tan et al . , 2014 ) .",
"CskAS kinase activity can be specifically and rapidly inhibited by an analog of the general kinase inhibitor PP1 , 3-iodo-benzyl-PP1 ( 3-IB-PP1 ) .",
"Studies in thymocytes in this model system support the notion that the combined actions of CD45 and Csk establish the basal activity of SFK ( Tan et al . , 2014 ) .",
"However , it is not clear whether Csk plays a role in setting the TCR signaling threshold or in signal termination .",
"In this study , we utilized CD4+ and CD8+ T cells from CskAS mice to investigate how Csk modulates the threshold for TCR signal activation , affinity recognition and TCR signal termination .",
"Our data suggest that by controlling the amount of available active Lck , Csk regulates the TCR signaling threshold and affinity recognition .",
"Notably , even at a low level of Csk inhibition , T cell activation by weak agonists is greatly enhanced ."
],
[
"To determine whether Csk activity regulates the TCR activation threshold , we stimulated CskAS CD4+ T cells or wild type CD4+ T cells ( as a specificity control ) by titrating anti-CD3ε antibody in the absence or presence of a high dose of the CskAS inhibitor , 3-IB-PP1 , and examined proximal TCR signaling tyrosine phosphorylation events .",
"As previously shown , inhibition of CskAS alone induced strong activation of Lck , as well as Fyn , which was indicated by the hyperphosphorylation of their activation loop tyrosines and the reduced phosphorylation of their inhibitory tyrosines ( Tan et al . , 2014 ) .",
"In contrast , ligation of the TCR alone did not detectably alter Lck phosphorylation , consistent with an earlier report ( Nika et al . , 2010 ) .",
"The striking Lck activation after CskAS inhibition was associated with only relatively weak downstream phosphorylation of ZAP-70 , LAT and PLC-γ1 ( Figure 1A ) .",
"However , this downstream signaling was very strongly enhanced when the CskAS inhibitor treatment was combined with anti-CD3ε crosslinking , as evidenced by the marked increase in ZAP-70 , LAT and PLC-γ1 phosphorylation ( Figure 1A ) .",
"To determine whether this early enhanced proximal signaling was transmitted further downstream , we examined ERK1/2 phosphorylation ( p-ERK ) .",
"The magnitude of p-ERK was increased by CskAS inhibition in a 3-IB-PP1 dose-dependent manner , with the greatest enhancement seen when using a low dose of anti-CD3ε ( Figure 1B ) .",
"At a high dose of anti-CD3ε the increased p-ERK signal intensity , as indicated by the slight peak shift for 3-IB-PP1 treated cells , might reflect a more rapid or more complete peak response at the time point assayed .",
"Notably , CskAS inhibition alone induced only a small amount of protein phosphorylation downstream of ZAP-70 ( i . e . , LAT and ERK1/2 ) , likely due to spurious activation of a few cells ( Figure 1A , B ) .",
"Instead , CskAS inhibition synergized with TCR stimulation leading to downstream signaling .",
"Our data strongly suggest that Csk activity plays a crucial role in establishing the TCR activation threshold .",
"Specifically , a reduction in Csk activity lowered the threshold for signal activation and increased TCR sensitivity . 10 . 7554/eLife . 08088 . 003Figure 1 . Inhibiting Csk increases the magnitude of ligand-induced TCR signaling and reduces the threshold for TCR activation .",
"( A ) Purified CskAS ( AS ) or wildtype ( WT ) CD4+ T cells stimulated for 2 min with 1 μg/ml , 5 μg/ml or 10 μg/ml anti-CD3ε antibody in the presence of DMSO or 10 μM 3-IB-PP1 were analyzed by immunoblotting for the phosphorylation of the activation loop tyrosine of Lck and Fyn ( Src pY416 antibody ) and the inhibitory tyrosine of Lck ( Lck pY505 ) , phosphorylated ZAP-70 ( ZAP70 pY319 ) , LAT ( LAT pY132 ) and PLC-γ1 ( PLCγ1 pY783 ) , as well as total actin ( loading control ) .",
"Data are representative of at least three independent experiments .",
"( B ) Purified total CskAS ( AS ) or wildtype ( WT ) T cells stimulated with the indicated dose of anti-CD3ε antibody for 2 min in the presence of DMSO or 5 μM , 1 μM or 0 . 4 μM 3-IB-PP1 were analyzed for phosphorylated ERK ( p-ERK ) by phosphoflow .",
"Histograms were gated on CD4+ cells .",
"Data are representative of at least three independent experiments for AS cells and two independent experiments for WT cells . DOI: http://dx . doi . org/10 . 7554/eLife . 08088 . 00310 . 7554/eLife . 08088 . 004Figure 1—figure supplement 1 . Inhibiting Csk during TCR stimulation prolongs TCR signals .",
"( A ) Purified CskAS CD4+ T cells stimulated for 2 , 5 or 10 min with anti-CD3ε antibody in the presence of DMSO or 10 μM 3-IB-PP1 were analyzed by immunoblotting for the phosphorylated ZAP-70 , LAT and PLC-γ1 as well as total GAPDH ( loading control ) .",
"Data are representative of at least three independent experiments .",
"( B ) Purified total CskAS T cells stimulated with anti-CD3ε antibody for 2 , 5 or 10 min in the presence of DMSO or 5 μM , 1 μM or 0 . 4 μM 3-IB-PP1 were analyzed for phosphorylated ERK ( p-ERK ) by phosphoflow .",
"Histograms were gated on CD4+ cells .",
"Data are representative of at least three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 08088 . 004 It has been proposed that Csk plays an important role in TCR signal termination ( Torgersen et al . , 2001; Davidson et al . , 2003 ) .",
"To test this model we examined a time-course of CskAS CD4+ T cells stimulation with anti-CD3ε antibody .",
"CskAS inhibition prolonged phosphorylation of ZAP-70 , LAT and PLC-γ1 ( Figure 1—figure supplement 1A ) .",
"Moreover , there was still a clear digital response to anti-CD3 stimulation that was augmented by CskAS inhibition at the earliest time point ( Figure 1—figure supplement 1B ) .",
"There was evidence of 3-IB-PP1 dose-dependent delay in downregulation of p-ERK , with the down-regulation seeming more heterogeneous and , perhaps , asynchronous compared to the digital up-regulation of the response ( Figure 1—figure supplement 1B ) .",
"However , Csk inhibition did not prevent eventual signal attenuation over time , indicating that Csk has only a partial role in signal termination .",
"Other negative regulatory mechanisms must contribute to the termination of TCR signaling , albeit more slowly in the absence of Csk ( Naramura et al . , 2002; Rhee and Veillette , 2012 ) .",
"To examine more physiologically relevant regulatory mechanisms induced by peptide-MHC stimulation , we generated CskAS mice expressing the ovalbumin peptide/MHCII-reactive OTII TCR transgene ( Fu et al . , 2013; Salmond et al . , 2014 ) .",
"A tetramer was used as the stimulus to allow for detailed biochemical analyses that would not have been possible with the confounding contribution of proteins from antigen presenting cells ( APCs ) .",
"Stimulation of CskAS;OTII CD4+ T cells with an agonistic OVA pMHC tetramer during CskAS inhibition resulted in markedly enhanced phosphorylation of ZAP-70 , LAT and PLC-γ1 ( Figure 2A ) and led to increased and prolonged ERK1/2 phosphorylation ( Figure 2B ) .",
"Thus , our findings with the physiologic OVA pMHC TCR ligand parallel those using anti-CD3ε , confirming that Csk plays an important role in setting the TCR activation threshold and has a partial role in signal termination . 10 . 7554/eLife . 08088 . 005Figure 2 . Inhibiting Csk reduces the threshold for TCR activation and prolongs signaling induced by pMHC engagement .",
"( A ) Purified CskAS;OTII CD4+ T cells stimulated for 3 min with 5 μg/ml bead-bound control pMHC tetramer or 5 μg/ml , 2 . 5 μg/ml or 1 . 25 μg/ml bead-bound OVA pMHC tetramer in the presence of DMSO or 10 μM 3-IB-PP1 were analyzed by immunoblotting for the phosphorylated ZAP-70 , LAT and PLC-γ1 , as well as total actin ( loading control ) .",
"Data are representative of three independent experiments .",
"( B ) Purified CskAS;OTII CD4+ T cells stimulated with 5 μg/ml bead-bound control-pMHC tetramer or 2 . 5 μg/ml bead-bound OVA pMHC tetramer for 2 , 5 or 10 min in the presence of DMSO or 5 μM 3-IB-PP1 were analyzed for phosphorylated ERK ( p-ERK ) by phosphoflow .",
"Histograms were gated on CD4+ Vα2+ cells .",
"Data are representative of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 08088 . 00510 . 7554/eLife . 08088 . 006Figure 2—figure supplement 1 . Csk inhibition shifts the threshold for cellular proliferation in response to anti-CD3 or p-MHC tetramer stimulation .",
"( A ) Purified CD4+ T cells from CskAS mice were loaded with CFSE and stimulated with 3 μg/ml , 1 μg/ml or 0 . 3 μg/ml plate-bound anti-CD3ε and 1 μg/ml anti-CD28 for 72 hr in the presence or absence of 5 μM 3-IB-PP1 .",
"Data are representative of three independent experiments .",
"( B ) Purified CD4+ T cells from CskAS;OT-II mice were loaded with CFSE and stimulated with 0 . 3 μg/ml , 0 . 1 μg/ml or 0 . 04 μg/ml plate-bound OVA pMHC tetramer and 1 μg/ml anti-CD28 for 72 hr in the presence or absence of 5 μM 3-IB-PP1 .",
"( A , B )",
"Cells were then analyzed by flow cytometry for CFSE intensity .",
"Histograms were gated on CD4+ cells .",
"Data are representative of three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 08088 . 006 To assess whether the reduced TCR activation threshold for proximal signaling events following Csk inhibition had a functional impact on downstream T cell activation , we monitored cell proliferation of CskAS;OTII T cells stimulated with plate-bound anti-CD3ε ( Figure 2—figure supplement 1A ) or OVA pMHC tetramer ( Figure 2—figure supplement 1B ) in the presence of anti-CD28 costimulation .",
"Each stimulus was titrated to doses that yield minimal to maximum responses since antibody and tetramer have different molecular weights and physiological potency .",
"At low doses , both anti-CD3ε and OVA pMHC tetramer induced greater cellular proliferation when CskAS was inhibited , consistent with the increased magnitude of proximal TCR signaling .",
"Therefore , by controlling Csk activity , the threshold for TCR signaling and resultant responses can be modulated .",
"Next , we investigated how much perturbation of Csk and Lck is necessary to induce physiological changes in the T cell response .",
"Here we used CskAS-expressing CD8+ T cells or transgenic CD8+ T cells with the OTI TCR specific for an OVA peptide/MHC class I complex .",
"Titration of 3-IB-PP1 in CskAS CD8+ T cells revealed a maximum threefold to fourfold enhancement of Lck pY394 , which correlated with increased Lck activity ( Figure 3A ) ( Chakraborty and Weiss , 2014 ) .",
"Thus at resting state 25–33% of Lck was phosphorylated at Y394 , which agrees with some previous estimates ( Nika et al . , 2010 ) and is higher than others ( Ballek et al . , 2015 ) .",
"It should be noted that this is only an inference from the plateau with high drug doses .",
"This study is not designed to estimate the percentage of active Lck at the basal state , since some Lck may not be available for activation by Csk inhibition .",
"Low doses of 3-IB-PP1 ( 1 μM or less ) induced at most a 50% upregulation of pY394 , while doses of 5–10 μM induced a threefold to fourfold upregulation .",
"Surprisingly , phosphorylation of the inhibitory tail Y505 , the direct target of Csk , did not decrease as appreciably .",
"This observation suggests that Lck is actively phosphorylated by Csk , but a substantial proportion of pY505 is either inaccessible for dephosphorylation by CD45 or is dephosphorylated over a longer timescale .",
"This , however , does not explain how the small reduction in pY505 , particularly so in CD8+ T cells , results in a large increase of pY394 . 10 . 7554/eLife . 08088 . 007Figure 3 . Weak Csk inhibition and Lck activation potentiate agonist signaling .",
"( A ) CskAS CD8+ T cells were rested and stimulated with indicated doses of 3-IB-PP1 for 3 min , lysed , and immunoblotted for activating ( pY394 ) and inhibitory ( pY505 ) site Lck phosphorylation .",
"Below is quantification ( ± sem ) of three independent experiments immunoblot .",
"( B ) CskAS;OTI naive CD8+ T cells were rested , stimulated with bead-bound BSA or 10 μg/ml pMHC-OVA for 3 min , lysed and assayed by immunoblot .",
"Data are representative of at least two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 08088 . 00710 . 7554/eLife . 08088 . 008Figure 3—figure supplement 1 . Comparison of Csk inhibition and Lck activation in CD4+ vs CD8+ T cells .",
"( A ) CskAS CD4+ and CskAS CD8+ T cells were rested and stimulated with indicated doses of 3-IB-PP1 or DMSO for 2 min and then lysed .",
"Lysates were immunoblotted for activating ( pY394 ) and inhibitory ( pY505 ) site Lck phosphorylation , total Lck , and GAPDH ( loading control ) .",
"Data are representative of three independent experiments .",
"( B , C )",
"Quantification of Lck pY505 and Lck pY394 of three independent experiments ( as in A ) , with levels normalized to Lck levels per cell type .",
"Error bars are standard deviation . DOI: http://dx . doi . org/10 . 7554/eLife . 08088 . 008 We explored whether the effects of Csk inhibition has differential effects on CD4+ vs CD8+ T cells .",
"However , we found that CD4+ and CD8+ T cells have identical responses to Csk inhibition when normalized to their basal level of Lck phosphorylation ( Figure 3—figure supplement 1 ) .",
"Interestingly , CD8+ T cells express ∼15% more Lck ( Olszowy et al . , 1995 ) and have a higher ( ∼20% ) basal amount of inhibitory tail pY505 phosphorylation .",
"In both CD4+ and CD8+ T cells , pY394 increased much more markedly than the associated decrease in pY505 in response to low doses of the Csk inhibitor .",
"The molecular mechanism for this response is unknown .",
"One possibility to consider is that even a small pool of active Lck that is dephosphorylated at Y505 and becomes phosphorylated at Y394 enables a trans-autocatalytic mechanism which can become amplified within the larger pool of Lck molecules that are not phosphorylated on pY394 .",
"Interestingly , it has also been observed that stimulation with anti-TCR and anti-CD4 antibody leads to substantial increase in pY394 without much loss of pY505 ( Ballek et al . , 2015 ) .",
"Short-term CskAS;OTI CD8+ T cell stimulation with bead-bound OVA-MHC tetramer was enhanced even by low levels of Csk inhibition ( <1 μM ) ( Figure 3B ) , as demonstrated by the degree of PLC-γ1 and ERK1/2 phosphorylation .",
"At this inhibitor dose , Lck pY394 did not increase by more than 50% , suggesting that even subtle changes in Lck activity can potentiate proximal signaling .",
"Higher doses of the inhibitor potentiated Lck signaling even further , suggesting Csk and Lck activity can be titrated over a wide range .",
"Lck not only initiates TCR-dependent signaling but is also proposed to be the crucial gate-keeper in kinetic proof-reading models that attempt to explain the exquisite affinity discrimination of T cells of agonists with only slightly different half-lives ( Palmer and Naeher , 2009; Stepanek et al . , 2014 ) .",
"We hypothesized that Lck hyperactivation may differentially regulate strong and weak agonists .",
"To address this question , we utilized a panel of well-characterized altered OVA peptides ( Daniels et al . , 2006 ) .",
"When CskAS;OTI CD8+ T cells were stimulated by APCs preloaded with peptides of different agonist potency during strong CskAS inhibition ( 5 μM ) , we observed marked augmentation of CD69 expression in responses to weak ( T4 , Q4H7 , G4 ) agonists , contrasting with only slight enhancement of the responses to strong ( OVA , Q4R7 ) agonists ( Figure 4A ) .",
"Although the OVA response was not fully saturated at this early time point ( 4 hr ) , it was only slightly enhanced by strong Csk inhibition .",
"Upon stimulation with OVA , but not altered peptides , the TCR is quickly downregulated due to its phosphorylation and retention in intracellular vesicles ( Cai et al . , 1997 ) .",
"Csk inhibition alone induced some ligand-independent TCR downregulation , but additional downregulation was observed with all peptides , even the weakest G4 , which argues for altered signaling of low-affinity peptides at a very upstream step ( Figure 4—figure supplement 1 ) .",
"Importantly , the null agonist VSV-loaded APCs did not induce similar activation or TCR downregulation , demonstrating that Csk inhibition works strictly in synergy with a cognate TCR ligand .",
"Moreover , although the spleen-derived APCs should still present endogenous self-peptides , only loading of cognate peptides led to sustained T cell activation after Csk inhibition . 10 . 7554/eLife . 08088 . 009Figure 4 . Csk inhibition potentiates response to weak agonists .",
"( A ) CskAS;OTI naive CD8+ T cells were stimulated with MitoC-treated WT splenocytes , preloaded with indicated peptides , in presence of DMSO or 5 μM 3-IB-PP1 for 4 hr and CD69 upregulation was measured by flow cytometry .",
"( B ) CskAS;OTI naive CD8+ T cells were stimulated as in A with varying doses of 3-IB-PP1 with dose–response fit .",
"( C ) EC50 of 3-IB-PP1 ( nM ) for % CD69 positive cells for data in B . Data are representative of two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 08088 . 00910 . 7554/eLife . 08088 . 010Figure 4—figure supplement 1 . TCR Valpha2 downregulation in cells in Figure 4A . Only OVA triggers peptide-dependent TCR downregulation .",
"5 μM 3-IB-PP1 induces ligand-independent downregulation of TCR , as seen in VSV peptide .",
"In the presence of CskAS inhibition , all the other altered peptides induce further , dose-dependent loss of TCR . DOI: http://dx . doi . org/10 . 7554/eLife . 08088 . 010 We speculated that the striking enhancement of weak agonist recognition is due to the strong inhibition of Csk and activation of Lck ( threefold at 5 μM 3-IB-PP1 ) .",
"To test the extent of Csk inhibition necessary to boost weak agonist signaling , we titrated 3-IB-PP1 and assayed responses to weak agonists Q4H7 and G4 ( Figure 4B ) .",
"Surprisingly we observed dose-dependent enhancement of signaling at 100–1000 nM 3-IB-PP1 , where increased Lck pY394 was weakly detectable and at most 50% above the basal level .",
"Beyond this inhibitor dose , we observed a saturating effect , despite the lower overall plateau with lower peptide dose .",
"Thus , relatively weak Lck hyperactivation was sufficient to enhance the recognition of weak agonists .",
"The lower plateau suggests that there are Csk-independent mechanisms for sensing agonist dose .",
"Remarkably , the EC50 concentration of Csk inhibition for CD69 upregulation was still dependent on agonist quality and dose ( Figure 4C ) .",
"At a lower agonist dose , the weaker peptide G4 required double the amount of 3-IB-PP1 than Q4H7 .",
"Similarly , the EC50 depended on peptide dose .",
"These observations argue that Csk inhibition fine-tunes the response to agonist dose and affinity sensing in peripheral T cells ."
],
[
"Our results raise the question of how Csk activity controls SFK activity and specifically the TCR activation threshold .",
"Since a substantial amount of active Lck is present in the basal state and does not change with TCR ligation , it has been hypothesized that the pre-existing pool of active Lck is responsible for the highly sensitive and rapid initiation of TCR signaling in response to ligand engagement ( Nika et al . , 2010 ) .",
"Hence , a likely explanation for our observations is that by controlling the amount of active Lck present , Csk regulates the responsiveness of the T cell to receptor stimulus .",
"Our data supports the model that the size of the active pool of Lck is especially critical for affinity discrimination of different agonistic peptides .",
"Strong agonists have a long half-life of pMHC-TCR and have sufficient time to recruit active Lck and initiate downstream events , while weak agonists with short half-lives are most sensitive to the availability of active Lck before the pMHC-TCR complex falls apart .",
"The different amounts of Lck loaded on the CD4 and CD8 co-receptors can explain the different half-life range of positively and negatively selecting ligands for CD4 and CD8 cells ( Stepanek et al . , 2014 ) ; however , this is a genetically established parameter .",
"Our data supports the model that recruitment of active Lck is critical for affinity discrimination , but offers an alternative mechanism for its regulation .",
"By controlling the fraction of active Lck , Csk can also modulate the activation threshold and affinity discrimination of T cells .",
"Notably , since Csk localization and activity is dynamically controlled by phosphorylation-dependent recruitment to transmembrane and cytoplasmic adaptors , this could enable flexibility in affinity recognition in different settings .",
"For example , Csk has been found to be differentially localized in naive and antigen-experienced T cells ( Borger et al . , 2013 ) .",
"Our results add to the emerging understanding of how signaling by agonists of dissimilar affinity are differentially regulated .",
"Recently , the adaptor molecule Themis was characterized as critical for the suppression of weak agonists in thymocytes , while PTPN22 serves a similar role in in effector cells ( Fu et al . , 2013; Salmond et al . , 2014 ) .",
"Here , we show that Csk , via its regulation of Lck , regulates naive T cell priming to agonists of different potency .",
"The ability to acutely titrate the activity of Csk , and consequently Lck , allowed us to assess how graded increases of Lck activity affects TCR signaling without concerns about basal level adaptation and long-term compensation that occurs when using genetic manipulation of Csk amounts or function .",
"Surprisingly , even very small changes in Lck activity , caused by CskAS inhibition at 200 nM 3-IB-PP1 , led to relatively large pY394 changes in the setting of small changes in pY505 .",
"Since Y394 phosphorylation is the result of trans-autophosphorylation , it seems likely that a small reduction in pY505 induced by low levels of Csk inhibition , could lead to autoactivation of a large pool of Lck .",
"Such a larger pool of active Lck could then show strong synergy with the TCR stimulus and is sufficient to markedly enhance the response to very weak agonists .",
"The synergy we observed between TCR stimulus and Csk inhibition , strikingly , had its greatest impact on the activation of downstream signaling molecules .",
"This is most likely due to positive feedback/signal amplification along the pathway .",
"Eventually , the integration of stronger signaling over hours led to profound differences in the fraction of cells upregulating CD69 or proliferating .",
"On the other hand , this minimal change in Lck activity highlights how tightly it must be regulated in both the basal and inducible states by kinases and phosphatases and their respective adaptors .",
"It is possible that , although small changes in Lck activity greatly sensitize the TCR to weak agonists , such small changes do not as effectively engage negative feedback mechanisms .",
"Loss of Csk from the cell membrane , or biochemically defined lipid raft fractions has been proposed to lead to TCR signal initiation via activation of Lck ( Torgersen et al . , 2001; Davidson et al . , 2003 ) .",
"This model is contradicted by the lack of appreciable change in Lck phosphorylation upon TCR stimulation and by unaltered signaling in cells deficient in the lipid-raft-resident Csk adapter PAG ( Dobenecker et al . , 2005; Xu et al . , 2005 ) .",
"Our data demonstrate that acute activation of Lck , via weak or strong Csk inhibition , does not lead to full TCR signaling .",
"Strong Csk inhibition in mature T cells leads to small amounts of phosphorylation of downstream targets like ZAP-70 and PLC-γ1 , but not ERK activation or long-term proliferation in the absence of TCR engagement .",
"Thus , Lck activation alone is insufficient to trigger complete TCR signaling .",
"However , when combined with cognate TCR ligands , very small amounts of Csk inhibition and resultant larger amounts of Lck activation , synergize to enhance early biochemical events and downstream activation .",
"Therefore , it is possible that during physiological TCR stimulation , localization of only a small number of Csk molecules is altered and leads to the activation of a small fraction of Lck molecules that would be difficult to detect with current biochemical or imaging methods ( Paster et al . , 2009; Nika et al . , 2010 ) but might be sufficient to influence downstream signaling .",
"Csk has also been proposed to regulate TCR signal downregulation by returning to the plasma membrane after signal initiation .",
"It has been challenging to study this role using genetic ablation approaches since Csk deficiency affects TCR basal state signaling .",
"Using acute inhibition of Csk concurrently with TCR stimulation , we were able to follow TCR signal downregulation in the absence of Csk activity and observed that it is delayed but not abolished .",
"Our data suggest that other molecules are involved in signal termination more prominently than Csk .",
"New insights into how Csk is regulated will enable the development of strategies to manipulate Csk activity and hence the sensitivity of T cells to various activation stimuli .",
"Such manipulation of Csk activity by small molecule inhibitors could be useful therapeutically , for example in pathologic settings where suppression ( i . e . , autoimmunity ) or augmentation ( i . e . , vaccines or cancer ) of T cell responses to antigen is desirable ."
],
[
"Mice used for these studies were 6–12 weeks of age .",
"All mice were housed in a specific pathogen-free facility at UCSF according to the University Animal Care Committee and National Institutes of Health ( NIH ) guidelines .",
"3-IB-PP1 has been described ( Schoenborn et al . , 2011 ) .",
"CD4 PerCP-Cy5 . 5 ( 550954 ) , TCR Vα2 PE ( 553289 ) and Lck-pY505 ( BD Biosciences 612390 ) are from BD Biosciences ( San Jose , CA ) ; p44/42 MAPK pThr202/Tyr204 ( 4377 ) , Src 416 ( 2101 ) , ZAP-70-pY319 ( 2701 ) are from Cell Signaling ( Danvers , MA ) ; LAT-pY132 ( 44-224 ) , PLC-γ1-pY783 ( 44-696G ) and CFSE ( C34554 ) are from Life Technologies ( Life Technologies , Thermo Fisher Scientific , Waltham , MA ) ; actin ( Sigma Aldrich A2066; Sigma Aldrich , St . Louis , MO ) ; GAPDH ( Abcam ab8245; Abcam , Cambridge , MA ) ; Goat anti-armenian hamster IgG ( H+L ) ( 127-005-160 ) and donkey anti-rabbit IgG Ab conjugated to APC ( 711-136-152 ) are from Jackson Immunoresearch ( West Grove , PA ) ; Horseradish peroxidase ( HRP ) -conjugated goat antibody α-rabbit IgG ( H+L ) ( 4050-05 ) , α-mouse IgG ( H+L ) ( 1031-50 ) , Alexa647-conjugated α-mouse IgG ( H+L ) ( 1010-31 ) are from Southern Biotech ( Birmingham , AL ) ; I-A ( b ) tetramers loaded with human class II-associated invariant chain peptide 103–117 or chicken OVA peptide 328–337 are from NIH tetramer core facility .",
"Stained cells were analyzed on a BD Fortessa ( BD Biosciences; San Jose , CA ) .",
"Data analysis was performed using FlowJo software ( Treestar Incorporated; Ashland , OR ) and GraphPad Prism ( GraphPad Software , Inc . ; La Jolla , CA ) and ImageLab ( BioRad Inc . ; Hercules , CA ) .",
"Before stimulations , cells were serum-starved at 37°C for at least 20 min .",
"Stimulations were performed in serum-free RPMI at 37°C .",
"CD3ε crosslinking was induced by addition of anti-CD3ε followed by goat anti-armenian hamster IgG ( H+L ) to a final concentration of 50 μg/ml .",
"For bead-based stimulations , 4 . 5 μm styrene beads ( Polyscience; Niles , IL ) were coated overnight at 4°C or for 1 hr at room temperature with p-MHC tetramers or αCD3 ( 2c11 ) in PBS under continuous rotation at 4°C .",
"Biotinylated p-MHC for CD8 stimulation were precoated with 5 μg/ml streptavidin .",
"Beads were then saturated with 1% BSA in PBS under continuous rotation for 2 hr at room temperature , and washed with serum-free RPMI before use .",
"Ice-cold rested cells and beads were span together and signaling was initiated by transfer to 37°C .",
"Intracellular phospho-ERK was performed as previously described ( Schoenborn et al . , 2011 ) .",
"Immunoblotting was performed as previously described ( Tan et al . , 2014 ) .",
"Enriched populations of pan T cells were obtained by negative selection according to manufacturer's protocol ( STEMCELL Technologies , 19751 , 19851; STEMCELL Technologies; Vancouver , BC , Canada ) .",
"Enriched populations of CD4+ or CD8+ T cells were obtained by negative selection according to manufacturer's protocol ( STEMCELL Technologies 19852 ) or with an in-house procedure as follows: The following biotinylated antibodies were combined and dialyzed in 1× PBS with Slide-a-lyzer 10 , 000 Molecular Weight cutoff dialysis cassette ( Thermo catalog# 66810; Thermo Fisher Scientific , Waltham , MA ) , then filter sterilized: biotin anti-CD8a ( clone 53-6 . 7 ) or biotin anti-CD4 ( clone GK1 . 5 ) , biotin anti-CD11b ( clone M1/70 ) , biotin anti-CD11c ( clone N418 ) , biotin anti-CD19 ( clone 1D3 ) from were Tonbo Biosciences ( San Diego , CA ) ; biotin anti-CD24 ( clone M1/69 ) , biotin anti-CD45R ( B220 ) ( clone RA3-6B2 ) , biotin anti-CD49b ( clone DX5 ) , biotin anti-TER119 ( clone TER-119 ) were from Biolegend ( La Jolla , CA ) .",
"ACK-lysed splenocytes and lymphocytes were incubated at room temperature with the antibody cocktail for 10 min in PBS with 1% FBS , 2 mM EDTA and 5% normal rat serum at 108 cells/ml .",
"Cells were then washed once with PBS with 1% FBS , 2 mM EDTA , resuspended at 0 . 85 ml per 108 cells , and mixed with 0 . 15 ml anti-biotin MACSiBead per 108 cells ( Miltenyi Biotec; San Diego , CA ) .",
"After 5 min at room temperature , cell-bead mix was placed in separation magnet for 5 min and the unbound cells were collected .",
"Purified CD4+ or CD8+ T cells were resuspended in PBS , labeled for 4 min at room temperature with 2 μM CFSE , and quenched with 100% FBS .",
"Labeled CD4+ T cells were stimulated by plate-coated OVA-MHC or α-CD3 ( 2c11 ) for 72 hr .",
"At indicated timepoint , DAPI were analyzed for CFSE dilution .",
"Purified CD8+ OTI T cells were stimulated for indicated time by Mitomycin C-treated Calpha−/− or BoyJ splenocytes , preloaded for 1 hr with indicated peptide and washed of excess peptide .",
"At indicated time , cells were fixed and stained for CD69 , TCRValpha2 and CD45 . 1 , and analyzed by flow cytometry ."
]
] | [
"The C-terminal Src kinase ( Csk ) , the primary negative regulator of Src-family kinases ( SFK ) , plays a crucial role in controlling basal and inducible receptor signaling .",
"To investigate how Csk activity regulates T cell antigen receptor ( TCR ) signaling , we utilized a mouse expressing mutated Csk ( CskAS ) whose catalytic activity is specifically and rapidly inhibited by a small molecule .",
"Inhibition of CskAS during TCR stimulation led to stronger and more prolonged TCR signaling and to increased proliferation .",
"Inhibition of CskAS enhanced activation by weak but strictly cognate agonists .",
"Titration of Csk inhibition revealed that a very small increase in SFK activity was sufficient to potentiate T cell responses to weak agonists .",
"Csk plays an important role , not only in basal signaling , but also in setting the TCR signaling threshold and affinity recognition ."
] | [
"The immune system has ‘T’ cells that recognize when the body is infected with a virus or bacterium and mount an immune response that is targeted to that microbe .",
"They can also find and eliminate cancer cells .",
"The microbes and cancer cells produce molecules called antigens that are detected by proteins on the surface of the T cell called T cell receptors ( TCR ) .",
"Antigen recognition causes the TCRs to transmit signals to the inside of the T cell that trigger an immune response .",
"The degree to which the TCRs are active , and the length of time that they transmit signals regulates the size of the immune response .",
"Therefore , developing new drugs that manipulate the activity of TCRs could be useful to treat many diseases .",
"An enzyme called Csk inhibits the activities of a small family of proteins involved in a variety of different processes .",
"One protein that Csk targets is called Lck and is required for the activation of immune responses in T cells .",
"However , it is not clear whether Csk interacting with this protein stops the release of signals from TCRs , or whether it alters the level to which TCRs need to be activated before they transmit the signals .",
"Manz , Tan et al . studied T cells from mice that had a mutant form of Csk that is inhibited by a drug called 3-iodo-benzyl-PP1 ( 3-IB-PP1 ) , but otherwise works normally .",
"When these cells detected an antigen in the presence of the drug , its TCRs were more highly activated and transmitted signals for a longer period of time than cells not exposed to the drug .",
"The drug especially enhanced immune responses to very weak antigens , ones that might not activate T cells under normal circumstances .",
"Manz , Tan et al . 's findings confirm that Csk plays a negative role in the activation of T cells and suggest that Csk may be a useful target for drug therapies that aim to fine-tune immune responses .",
"The next challenge is to find a drug that can inhibit normal Csk enzymes and test this in mice and other animals ."
] | 2015 |
[
"Materials and methods"
] | [
"developmental biology",
"short report"
] | TAF7L modulates brown adipose tissue formation | elife-02811-v2 | [
[
"Full-length green fluorescent protein ( GFP ) or Taf7l cDNAs were inserted into p3XFLAG-CMV-10 vector to construct pCMV-FLAG-V5-GFP/TAF7L or into pCS2+ vector to construct pCS2+HA-TAF7L; full-length PRDM16 cDNA was inserted into p3XFLAG-CMV-10 vector to construct pCMV-FLAG-PRDM16 .",
"TAF4 ( 612054; BD , San Jose , CA ) , TBP ( 62126; abcam , Cambridge , MA ) , V5 ( R960-25; Invitrogen , Carlsbad , CA ) , HA ( 9110; abcam ) , FLAG ( F31655; Sigma , Saint Louis , MI ) MYHC ( 05-716; Millipore , Billerica , MA ) , FABP4 ( 66682; abcam ) , UCP1 ( 10983; abcam ) , TAF7L ( prepared in-house , Covance , Denver , PA ) , Pol II ( monoclonal 8WG16 , protein-A purified ) , PPARγ ( sc-7196 ) , ANTI-FLAG M2 Affinity Gel ( A2220; Sigma ) , Anti-V5-agarose affinity gel ( A7345; Sigma ) .",
"C3H10T1/2 and C2C12 cells were cultured in high glucose DMEM with 10% fetal bovine serum at 5% CO2 .",
"C3H10T1/2 cells stably expressing FLAG-V5-GFP/TAF7L were established by transfection of the pCMV-FLAG-V5-GFP/TAF7L plasmids followed by 2 weeks of 1 µg/µl neomycin selection .",
"For adipogenesis , C3H10T1/2 , control and FLAG-TAF7L-expressing C2C12 , and FLAG-V5-GFP/TAF7L-expressing C3H10T1/2 cells were grown in high glucose DMEM supplemented with 10% fetal bovine serum .",
"At confluence , cells were exposed to induction medium containing dexamethasone ( 1 μM ) ( 265005 , CalBiochem , San Diego , CA ) , isobutylmethylxanthine ( IBMX , 0 . 5 mM ) ( I-5879 , Sigma ) , Indomethacin ( 0 . 125 mM ) ( I-7378 , Sigma ) , and 10% FBS .",
"3 days later , cells were further cultured in high glucose DMEM-containing insulin ( 5 μg/ml ) ( 19278 , Sigma ) , T3 ( 0 . 1 μM ) ( T-2877 , Sigma ) , and rosiglitazone ( 5 μM ) ( 71740 , Cayman , Ann Arbor , MI ) until they were ready for harvest .",
"For Oil red O staining , pre- and post-differentiated C3H10T1/2 cells , shGFP and shTAF7L-treated C3H10T1/2 cells , C2C12 . CNTL and C2C12 . TAF7L cells were washed once in PBS and fixed with freshly prepared 4% formaldehyde in 1 × PBS for 30 min , followed by standard Oil red O staining method described previously ( Zhou et al . , 2013a ) .",
"C2C12 myogenesis procedure followed a previous study ( Zhou et al . , 2013a ) .",
"Total RNA from cultured cells or mouse tissues was isolated using QIAGEN RNeasy Plus mini columns according to the manufacturer's instructions ( Qiagen , Germany ) .",
"For RT-qPCR analysis , 1 μg total RNA was reverse transcribed using cDNA reverse transcription kit ( Invitrogen ) .",
"PCR reactions using the SYBR Green PCR Master Mix ( Applied Biosystems , Grand Island , NY ) were performed according to the manufacturer's instruction using an ABI 7300 real time PCR machine ( Applied Biosystems ) .",
"Relative expression of mRNA was determined after normalization to cyclophilin gene .",
"Student's t test was used to evaluate statistical significance ( Zhou et al . , 2013b ) .",
"Whole cell extracts were prepared from cells by homogenization in lysis buffer containing 50 mM Tris-Cl , pH 8 . 0 , 500 mM NaCl , and 0 . 1% Triton X-100 , 10% glycerol , and 1 mM EDTA , supplemented with protease inhibitor cocktail ( Roche , Indianapolis , IN ) and phenylmethylsulphonyl fluoride ( PMSF ) .",
"15 μg of whole-cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membrane .",
"For immunoblotting , membranes were blocked in 10% milk , 0 . 1% Tween-20 in TBS for 30 min , and then incubated with TAF7L , UCP1 , PPARγ , and POL II antibodies for 2 hr at room temperature; detailed Western blotting procedure was performed as previously described ( Zhou et al . , 2006 ) .",
"3 mg whole-cell extracts from FLAG-V5-GFP/TAF7L post-differentiated adipocytes were immunoprecipitated with 100 µl of ANTI-FLAG M2 Affinity Gel under the conditions of 0 . 3 M NaCl , 0 . 2% NP-40 at 4°C overnight .",
"After extensive washing with buffer containing 0 . 3 M NaCl and 0 . 1% NP-40 , proteins were eluted from the affinity gel with 100 µg/ml FLAG peptide in 0 . 1 M NaCl Tris buffer .",
"Elutions from both samples were subsequently immunoprecipitated with 40 µl Anti-V5-agarose affinity gel antibody with a similar procedure as above .",
"After extensive washes , proteins were eluted with 40 µl pH2 . 5 0 . 1 M glycine for three times and immediately neutralized with 12 µl of pH9 . 0 2 M Tris-Cl .",
"10 µl of eluted samples were subjected to 10% SDS-PAGE and followed by standard silver staining or by western blotting analysis with V5 , PPARγ , TAF4 , and TBP antibodies to detect tagged-proteins in the inputs and associated proteins as previously described ( Harms and Seale , 2013 ) .",
"The remaining samples were precipitated by 20% trichloroacetic acid , and the precipitates were sent to liquid chromatography-tandem mass spectrometry ( LC-MS/MS ) to detect peptides derived from proteins pulled-down by FLAG-V5-GFP/TAF7L .",
"500 μg whole-cell extracts from 293T cells transfected with HA-TAF7L and FLAG-PRDM16 were immunoprecipitated with FLAG or HA antibodies at 4°C for overnight under the conditions of 0 . 3 M NaCl and 0 . 2% NP-40 , 30 μl protein A/G beads were added and incubated for additional 2 hr at 4°C , after extensive washing with buffer containing 0 . 3 M NaCl and 0 . 1% NP-40 , remaining beads were subjected to 10% SDS-PAGE and followed by western blotting analysis with FLAG and HA antibodies to detect tagged-proteins in the inputs and IPs as described previously ( Zhou et al . , 2013a ) .",
"The derivation of Taf7l KO mice has been previously described ( Kajimura et al . , 2009 ) .",
"All animal experiments were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health .",
"All of the animals were handled according to animal use protocols ( #R007 ) approved by the Institutional Animal Care and Use Committee ( IACUC ) of the University of California , Berkeley .",
"Mice were maintained on a standard rodent chow diet with 12 hr light and dark cycles .",
"Taf7l KO mouse line was maintained on a C57BL/6J background .",
"Genotyping was performed by PCR as previously described ( Cheng et al . , 2007 ) .",
"For histological analysis on interscapular BAT of E18 . 5 embryos from WT and Taf7l KO mice , freshly-harvested mouse embryos were genotyped and the interscapular regions of embryos were transversally dissected and then fixed in 10% formaldehyde for 24 hr at 4°C; tissue was embedded in paraffin using the microwave method as described and then sectioned into 8–10 μm to mount on slides ( Zhou et al . , 2013b ) .",
"The following immunohistochemistry by haematoxylin and eosin ( H&E ) staining and FABP4 , UCP1 , and MYHC immunostaining were performed using the method described previously ( Zhou et al . , 2013a ) .",
"Fresh interscapular brown adipose tissues were removed from 3-week-old euthanized WT and Taf7l KO mice and finely minced , digested with 0 . 25% trypsin for 30 min at 37°C , and centrifuged for 5 min at 2 , 000×g to get rid of debris .",
"The pellet was resuspended in culture media before plated on gelatin coated plates .",
"Cells were cultured at 37°C in high glucose DMEM supplemented with 20% FBS .",
"Brown adipocyte differentiation and staining were followed the same procedure as C3H10T1/2 cells .",
"Total RNA was extracted from BAT , carefully excised to get rid of surrounding tissue based on the texture and color , of 6 WT and 6 Taf7l KO mice .",
"RNA was extracted separately for each mouse by RNeasy Plus Mini Kit ( Qiagen ) and then pooled for WT or Taf7l KO samples; 4 μg of each RNA pool was used to purify mRNA using oligo ( dT ) and subsequently converted into multiplexed mRNA-seq libraries using mRNA-Seq Trueseq Kit ( Illumina , San Diego , CA ) .",
"Samples were multiplexed and sequenced in one lane on an Illumina HiSeq 2000 sequencer ( QB3 Vincent J Coates Genomics Sequencing Library , University of California , Berkeley , CA ) .",
"50 bp single-end reads were used for both samples; each sample produced over 30 million reads .",
"Reads were mapped to the mouse transcriptome ( mm10 ) , using TopHat ( Langmead et al . , 2009; Trapnell et al . , 2009 ) , version v1 . 4 . 0 . , with default parameters .",
"We then applied cufflinks ( Trapnell et al . , 2010 ) , version v1 . 3 . 0 , using the default parameters except: --max-mle-iterations 1 , to estimate the digital expression levels at each transcript .",
"Gene ontology analysis was done using DAVID Bioinformatics Resources 6 . 7 .",
"3C analysis was performed as previously described on WT and Taf7l KO BAT ( Liu et al . , 2011 ) , which was carefully excised to get rid of all other possible tissue based on the brown color and the tissue texture .",
"2 mg of freshly dissected interscapular BAT from 6 WT or 6 Taf7l KO mice were minced , homogenized extensively to nearly single cells and washed , crosslinked with 1% formaldehyde for 15 min at 4°C and then quenched with 0 . 125 M glycine for 5 min .",
"Crosslinked BATs were lysed and the chromatin was digested with 8 U HaeIII ( NEB ) for the Cidea and Scd1 loci .",
"Digested fragments were cleaned and subsequently ligated with 60 units T4 DNA ligase ( Invitrogen ) for 4 hr at 16°C .",
"Following proteinase K digestion and decrosslinking at 65°C overnight , DNA fragments was recovered by phenol–chloroform extraction .",
"Control templates were generated using individual BAC clones covering Cidea or Scd1 locus ( Bacpac ) .",
"10 µg of BAC DNA was digested with 20 units HaeIII and then randomly ligated with 10 units T4 DNA ligase in 50 µl volume .",
"3C primers were designed upstream and downstream of the core promoter site ( P ) .",
"Primers annealing to distal enhancers ( D ) corresponding to TAF7L and PPARγ binding sites on either Cidea or Scd1 were used as anchor points .",
"3C analysis was done by qPCR using as a primer pair the anchor point primer and one annealing to region under investigation .",
"Each data point in WT and Taf7l KO BAT was normalized by the BAC control template and presented as interaction frequency .",
"Raw and mapped sequencing reads are available from the National Center for Biotechnology Information's GEO database ( http://www . ncbi . nlm . nih . gov/geo/ ) under accession number GSE55797 .",
"Primer sequences are listed in Supplementary file 1 ."
]
] | [
"Brown adipose tissue ( BAT ) plays an essential role in metabolic homeostasis by dissipating energy via thermogenesis through uncoupling protein 1 ( UCP1 ) .",
"Previously , we reported that the TATA-binding protein associated factor 7L ( TAF7L ) is an important regulator of white adipose tissue ( WAT ) differentiation .",
"In this study , we show that TAF7L also serves as a molecular switch between brown fat and muscle lineages in vivo and in vitro .",
"In adipose tissue , TAF7L-containing TFIID complexes associate with PPARγ to mediate DNA looping between distal enhancers and core promoter elements .",
"Our findings suggest that the presence of the tissue-specific TAF7L subunit in TFIID functions to promote long-range chromatin interactions during BAT lineage specification ."
] | [
"Mammals produce two distinct types of adipose tissue: white adipose tissue ( white fat ) is the more common type and is used to store energy; brown adipose tissue ( brown fat ) is mostly found in young animals and infants , and it plays an important role in dissipating energy as heat rather than storing it in fat for future use .",
"In adults , higher levels of brown fat are associated with lower levels of fat overall , so there is considerable interest in learning more about this form of fat to help address rising levels of obesity in the world .",
"Building on previous work in which they showed that a gene control protein called TAF7L has a central role in the development of the cells that make up white adipose tissue , Zhou et al . now show that this protein also helps to regulate the development of brown adipose tissue .",
"Mice lacking the gene for this protein developed embryos with 40% less brown fat than wild-type mice with the gene .",
"Moreover , these mice developed muscle-like cells in the regions that should have contained brown fat .",
"Gene expression analysis revealed that ‘knocking out’ the gene for TAF7L changed the expression of more than a thousand genes in these mice .",
"Zhou et al . suggest that TAF7L works as a ‘molecular switch’ that determines whether certain precursor cells ( called mesenchymal stem cells ) go on to become brown fat cells or muscle cells .",
"A future challenge will be to devise interventions to regulate the activity or levels of TAF7L as a potential means of modulating brown fat depots in animals and humans ."
] | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Predicting development of adolescent drinking behaviour from whole brain structure at 14 years of age | elife-44056-v1 | [
[
"Adolescence is a critically vulnerable time for the development of alcohol drinking habits that may lead to considerable consequences later in life including the development of alcohol addiction .",
"Importantly , the period of adolescence coincides with substantial behavioural changes together with structural and functional brain development .",
"Cortico-striatal regions play an important role in the regulation of behaviour and might therefore play a role in progress and maintenance of habits such as drinking ( Heinz , 2002 ) .",
"In particular , it has been proposed that drug addiction involves dysfunctions of brain circuitry related to the neurotransmitter dopamine that lead to alterations in both impulsive and compulsive behaviour ( Koob and Kreek , 2007 ) .",
"Since early exposure to drugs may alter brain development during adolescence this may set the stage for cognitive problems in adulthood , which translate into behavioral consequences throughout life ( Jacobus and Tapert , 2013 ) .",
"Hence , it is of particular importance to predict the acceleration of alcohol use as early as possible during adolescence in order to intervene timely .",
"Nees and colleagues reported that reward-related brain activation aided in the prediction of early-onset drinking in adolescents at age 14 years in a subset of the data used in the present paper ( Nees et al . , 2012 ) .",
"In a functional imaging study on youth of age 12–14 years , prior to initiation of alcohol use , it was found that teens classified as transitioning to heavy alcohol use by age 18 had less blood oxygen level dependent ( BOLD ) activation in frontal , temporal , and parietal cortices in a response inhibition task ( Norman et al . , 2011 ) .",
"These reports support the notion that brain differences present early during adolescence may leave certain youth vulnerable to addictive behaviors .",
"In an earlier publication on the same data set , we focussed on the prediction of changes in alcohol-related problems between age 14 and 16 years based on gyrification of the orbitofrontal cortex ( Kühn et al . , 2016 ) .",
"We argued that it is important to use behavioral difference scores when aiming at predicting prospective behavior rather than absolute measures at the prospective time point only .",
"In applying difference scores rather than absolute measures , all available information can be taken into account to evaluate potentially problematic behaviour .",
"Within the present study , we want to go beyond previous prediction attempts by including a trajectory of alcohol-related behavior over a longer period of time , from age 14 to 16 and 19 years of age .",
"Obtaining three measurement occasions enables the modelling of change on a latent level within a structural equation modelling ( SEM ) framework .",
"We set out to investigate brain structural predictors at age 14 of the latent change trajectory of the alcohol use score on a voxel-based whole-brain basis .",
"Methodologically this goes beyond previous studies with an SEM approach including brain data , since commonly only extracted regions of interests or global brain measures ( such as intracranial volume , white matter hyperintensities ) have been employed in an SEM context ( Ritchie et al . , 2015; Kievit et al . , 2014 ) .",
"The innovation of this paper’s approach lies in analysing the whole brain on voxel level .",
"In doing so , we are able to on the one hand extract possible predictors for change in alcohol consumption on a fine-grade level ( i . e . voxel ) , and on the other hand to exploratory analyse the whole brain in search of predictive regions .",
"This approach offers the major advantage of giving consideration to both , specific structures of the brain on a rather fine-graded level , and the broad , exploratory search for generally influential areas ."
],
[
"We started the analyses with estimating the two-part latent growth curve model on the clinical data only containing an intercept and a linear growth factor for both alcohol use vs . non-use as well as for the alcohol use score .",
"Table 1 shows the classification of the AUDIT-scores concerning severity of use ( Figure 1 ) .",
"In the discrete part of the model , a linear growth model demonstrated better fit to alcohol use vs . non-use than an intercept-only model , Δχ2 ( Koob and Kreek , 2007 ) ( 3 , N = 1814 ) =653 . 63 , p<0 . 001 .",
"Inclusion of a quadratic growth factor did not improve model fit , Δχ2 ( Koob and Kreek , 2007 ) ( 1 , N = 1814 ) =−0 . 74 , p=0 . 390 , therefore we refrained from a quadratic growth factor .",
"In the continuous part of the model , a linear growth model likewise demonstrated a better fit to alcohol use scores than an intercept-only model , Δχ2 ( Koob and Kreek , 2007 ) ( 3 , N = 1814 ) =860 . 58 , p<0 . 001 .",
"Inclusion of a quadratic growth factor did not improve model fit , Δχ2 ( Koob and Kreek , 2007 ) ( 1 , N = 1814 ) =−0 . 58 , p=0 . 450 .",
"Therefore , we accepted the model with intercept and a linear growth factor on the clinical data as our working model .",
"We then added the nuisance covariates age , sex , scanner and total brain volume to the model by predicting the slope of the continuous part of the model ( no matter whether the regression paths were significant or not ) , since it is common practice in neuroimaging studies to control for these nuisance variables .",
"The final model on the clinical data , not yet including the brain data ( since this varied for each voxel of the brain ) demonstrated an acceptable model fit , χ2 = 444 , df = 65 , RMSEA = 0 . 057 , CFI = 0 . 785 , SRMR = 0 . 065 .",
"Concerning the latent intercept and slope for both parts of the model , intercepts were significantly different from zero and variances of the intercepts for both parts of the model were significant , suggesting significant interindividual heterogeneity around the estimated mean level of alcohol drinking use vs . non-use and the alcohol use score at age 14 ( for estimates see Table 2 ) .",
"The covariance between the intercepts of the two parts of the model was 0 . 124 , p<0 . 001 , indicating that adolescents with a higher propensity to engage in alcohol drinking also engaged in it more frequently and vice versa .",
"Turning to the growth parameters , for the continuous part of the model the estimated mean of the slope was not significantly different from zero , indicating that , on average , no change in drinking habits emerged over time .",
"However , the variance was significantly different from zero , indicating interindividual differences in change of drinking behaviour between participants .",
"For the discrete part of the model both the mean and the variance of the slope were significantly different from zero , indicating change on average as well as interindividually .",
"The positive mean of the slope indicated an increasing propensity for drinking across time .",
"Intercept and slope covaried significantly ( −0 . 033 , p<0 . 001 ) within the discrete part of the model , however , numerically the correlation coefficient was so small that we do not think the association necessitates in-depth interpretation .",
"No significant correlation emerged between intercept and slope for the continuous part of the model , indicating that a relation between alcohol use score at age 14 and change in this behavior was not captured in our model .",
"Based on this two-part latent growth curve model on the clinical data , we entered the brain data and conducted a whole-brain analysis on grey matter probability maps at age 14 years predicting change in alcohol use score , that is the latent slope in the continuous part of the model , over time from each voxel in the brain ( Figure 2 ) .",
"Within the model an increase in alcohol use score is reflected as a positive and a decrease in alcohol use score as a negative latent slope mean .",
"We found a positive association within bilateral caudate nucleus ( around −14 , 24 , 7 and 17 , 24 , 6 ) and left cerebellum ( Lobule VIII/IX , around −16 , –53 , −56 ) , where higher grey matter volume predicts greater change in alcohol use scores ( p<0 . 001 , cluster >100 voxels , Figure 3 ) .",
"We repeated the same analysis on log-transformed data , to address the problem of skewness in the data with almost identical results .",
"The same analysis on white matter probability maps did not result in any significant clusters .",
"Neither did repeating the same analyses when investigating a regression path between white or grey matter voxels and the change in alcohol use vs . non-use .",
"Moreover , we conducted the same whole-brain analysis while predicting the latent intercept of the continuous part of the model , which reflects how high individuals score on AUDIT on average .",
"Here also no significant clusters emerged , neither for white nor grey matter maps ."
],
[
"The direction of the association , namely between increase in alcohol use scores and higher brain volume is remarkable , since it may reflect a disturbance of brain development during adolescence .",
"Caudate and cerebellum have been described as undergoing changes over the lifetime that resemble an inverted U-shape in gray matter volume that peaks during adolescence ( Durston et al . , 2001; Brenhouse and Andersen , 2011 ) .",
"However , on a longitudinal data set the changes of caudate over the course of adolescence were the smallest in comparison to other subcortical brain structures with caudate , putamen and nucleus accumbens peaking at earlier ages than amygdala and hippocampus ( Goddings et al . , 2014 ) .",
"In tendency females seem to show a peak around age 11 years and males seem to show a decrease in caudate volume over adolescence ( Goddings et al . , 2014; Brain Development Cooperative Group , 2012 ) .",
"The pattern of changes in the cerebellum seem less clear with one study reporting decreases across adolescence ( Ostby et al . , 2009 ) and another showing an inverse U-shape pattern with a peak around 15 years of age ( Brain Development Cooperative Group , 2012 ) .",
"However , the literature on age related changes in brain volume during adolescence does not help to solve the question whether our observed effects reflect a deceleration of pruning in bilateral caudate and cerebellum or an overproduction that surpasses the normal overproduction of synapses .",
"The present finding of a predictive value of bilateral caudate volume for the trajectory of alcohol use during adolescence fits nicely to previous studies in search of brain structural predictors of drinking .",
"One study on 40 adolescents using FreeSurfer showed that at baseline ( 12–17 years of age ) participants who transitioned into heavy drinking after 3 years showed smaller left cingulate , pars triangularis , and rostral anterior cingulate volume , and less right cerebellar white matter volumes ( Squeglia et al . , 2014 ) .",
"Other studies focussed on activation of the striatum , for example during an fMRI reward paradigm , reporting increases in brain activation in caudate nucleus at reward delivery in participants who had their first drink during puberty as compared to those who started after puberty ( Boecker-Schlier et al . , 2017 ) .",
"In another study focussing on brain function , 43 postpubertal participants ( aged 18–21 years ) were followed over a year and classified into moderate and heavy drinkers or transitioners who started drinking heavily .",
"In a cue-reactivity paradigm showing alcohol stimuli , transitioners showed higher activation in bilateral caudate nucleus , orbitofrontal cortex medial frontal cortex/anterior cingulate and left insula ( Dager et al . , 2014 ) .",
"However , the two latter results have not been corrected for grey matter volume , and it could well be that the stronger brain activation observed in caudate ( among others ) was actually driven by higher grey matter volume ( to correct for these structural effects dedicated toolboxes have been developed; Casanova et al . , 2007 ) .",
"Of note , it is very interesting that the cluster in caudate nucleus and cerebellum found to be predictive of the future changes in alcohol use scores were not related to the mean level of alcohol use scores in each individual , since we observe no significant regions in the analysis where the latent intercept of the continuous part of the model predicts the respective brain voxels .",
"This indicates that the grey matter volume in caudate nucleus and cerebellum has a value in predicting changes , but not the average alcohol use in general at the timepoint analysed .",
"On the basis of the present analyses , one might argue that preventive measures at this age should focus on the development of alcohol use rather than on drinking habits at the age of 14 , as the results suggest that specific characteristics of those brain regions prepare the ground for future alcohol consumption .",
"The fact that the brain-based prediction of the latent slope of the dichotomous part of the model showed no significant resulting clusters indicates that , using the presented methods , brain data is not indicative for the prognosis when individuals start drinking .",
"In this sense , the present results could be regarded as in line with a previous publication from the IMAGEN data set ( Whelan et al . , 2014 ) in which it was shown that brain information ( at age 14 years ) added only moderately in the prediction of binge drinking at age 16 years .",
"Moreover , rather unspecific brain measures have been added in the analyses , namely overall regional grey matter volume and the ratio of grey and white matter volume .",
"In a different data set ( Squeglia et al . , 2017 ) , likewise only diffuse regions added to the prediction of initiation of alcohol use during adolescence .",
"This later observation makes the prediction of the changes in alcohol use scores over time from very distinct brain regions the more remarkable .",
"While we learned from previous studies that brain characteristics might not be the key factor in explaining the start of alcohol consumption in adolescence , we learn from the present study that brain structural characteristics are of relevance when considering the development of alcohol use in adolescence .",
"Interestingly , a recent meta-analysis on brain imaging studies focussing on brain structural alterations across psychiatric disorders has revealed consistency in increases within bilateral striatum when comparing psychiatric patients to controls ( Goodkind et al . , 2015 ) .",
"From this meta-analysis on cross-sectional data , it is unclear whether these striatal increases in psychiatric patients were already present during adolescence or whether they occurred around disease onset or over the course of the disease .",
"But it is interesting that the direction of the effect and the localisation bear resemblance to the results of our study , although we focussed on trajectories of alcohol use which were , for most of the participants , far from the actual diagnosis of alcohol addiction .",
"To our knowledge up to now structural equation modeling on brain imaging data has been conducted solely based on data derived from regions of interest ( ROIs ) ( Kievit et al . , 2014; Kühn et al . , 2017; McArdle et al . , 2004; Raz et al . , 2005 ) .",
"However , this has the strong disadvantage that the obtained results are restricted to the regions selected for the analysis at hand .",
"The present study demonstrates the feasibility of running separate structural equation models for each and every voxel of the brain and therefore plot the voxel-wise results of structural equation models back into brain space .",
"This approach is not restricted to growth curve models but can be applied to all models in an SEM context .",
"It can for example also be applied to measurement models , in order to relate brain structure or function not only to a separate task performance measure but rather to the latent factor representing the shared variance of a set of different performance measures from the same cognitive domain .",
"This offers a new avenue of structural equation modeling on neuroimaging data in an unbiased , comprehensive way .",
"The present study revealed structural brain predictors ( at 14 years of age ) of the trajectory of alcohol use scores between the age of 14 and 19 years .",
"A two-part latent growth curve model was utilized to decompose the semicontinuous AUDIT outcome measure into a dichotomous use vs . non-use and a continuous alcohol use scale part .",
"We predicted the slope of use vs . non-use and of alcohol use scores by voxel-wise grey and white matter probability maps at baseline .",
"To obtain brain maps as a result , we plotted the statistics of the regression path between brain and slope back into brain space .",
"We observed that higher grey matter volume in bilateral caudate nucleus and in left cerebellum at age 14 years was associated with a stronger increase in alcohol use scores .",
"This finding fits well to previous studies pointing at an association between increases in striatum and psychiatric disease .",
"Potentially this is due to neurodevelopmental interindividual differences since adolescence is a period of brain structural in- and decreases .",
"Our finding may reflect a deceleration of pruning or an overproduction that surpasses the normal developmental overproduction of synapses .",
"Future research with repeated neuroimaging measurements is needed to solve this neurodevelopmental question ."
],
[
"We used data of 1794 healthy 14-year-old adolescents ( mean age = 14 . 4 , SD = 0 . 45 years; 54% males ) who were recruited within the scope of the IMAGEN project , a European multi-centre genetic-neuroimaging study in adolescence ( Schumann et al . , 2010 ) .",
"The selection of the participants was based on the fact that structural imaging data at age of 14 years was present .",
"At the time of analyses reported here ( age 14 years ) , retest data at age 16–17 years was present for 1439 participants ( mean age = 16 . 6 , SD = 0 . 64 years; 55% males ) and at age 19 years for 1284 participants ( mean age = 19 . 0 , SD = 0 . 77 years; 53% males ) .",
"Written informed consent was obtained from all participants as well as from their legal guardians .",
"The adolescents were recruited from secondary schools .",
"The study was approved by all local ethics committees separately ( in Germany this was accomplished by the medical ethics committee of the University of Heidelberg , reference number: 2007-024N-MA ) and approved by the head teachers of the respective schools .",
"Participants with a medical condition or neurological disorders were excluded .",
"All participating subjects were assessed by means of self-rating and two external ratings ( by their parents and a psychiatrist specialized in pediatrics ) based on ICD-10 as well as DSM-IV ( The Development and Well-Being Assessment Interview , DAWBA; Goodman et al . , 2000 ) .",
"We administered the Alcohol Use Disorder Identification Test ( AUDIT , Babor and Higgins-Biddle , 2001 ) at Baseline ( age 14 years ) , Follow-up 1 ( age 16–17 years ) and Follow-up 2 ( age 19 years ) to identify alcohol use .",
"We computed the total score by adding the scores of all 10 items .",
"Structural MRI was performed on 3 Tesla scanners from three manufacturers ( Siemens: five sites; Philips: two sites; and General Electric: two sites ) .",
"The details of the entire MR protocol are described elsewhere ( Schumann et al . , 2010 ) .",
"In this study , we used the T1-weighted images .",
"These high-resolution anatomical MRIs were obtained using a three-dimensional magnetization prepared gradient-echo ( MPRAGE ) sequence based on the ADNI protocol ( http://adni . loni . usc . edu/methods/documents/mri-protocols/; modified for the IMAGEN study to give a 1 . 1 × 1 . 1 × 1 . 1 mm3 voxel size ) .",
"For the present report , structural MR data of 2072 adolescents were available .",
"We excluded all participants where the image quality was suboptimal , most likely due to movement .",
"The visual quality control was carried out by 10 independent raters .",
"Structural data acquired at age 14 years was preprocessed by means of the VBM8 toolbox ( http://dbm . neuro . uni-jena . de/vbm . html ) and SPM8 ( http://www . fil . ion . ucl . ac . uk/spm ) with default parameters .",
"The VBM8 toolbox involves bias correction , tissue classification and affine registration .",
"The affine registered grey matter ( GM ) and white matter ( WM ) segmentations were used to build a customized DARTEL ( diffeomorphic anatomical registration through exponentiated lie algebra ) template .",
"Then warped GM and WM segments were created .",
"Modulation was applied in order to preserve the volume of a particular tissue within a voxel by multiplying voxel values in the segmented images by the Jacobian determinants derived from the spatial normalization step .",
"In effect , the analysis of modulated data tests for regional differences in the absolute amount ( volume ) of GM/WM .",
"Images were smoothed with a FWHM ( full-width at half maximum ) kernel of 8 mm .",
"Analyses were conducted within a structural equation modeling ( SEM ) framework using MPlus and R . We implemented a two-part latent growth curve model ( Muthen , 2001; Olsen and Schafer , 2001 ) since the AUDIT scores were zero inflated .",
"In a two-part latent growth curve model , zeros are valid values with its own meaning and not just proxies for missingness .",
"Information that is contained by zeros and the specific values of the non-zeros is qualitatively different and might even be differentially influenced by covariates ( cf . , Olsen and Schafer , 2001 ) .",
"In a two-part latent growth curve model , the presence or absence of a behavior and , if present , the manifestation of a specific behavior can be modeled simultaneously in one model .",
"In our study , the original distribution of the alcohol use variable ( AUDIT-score ) was decomposed into two parts ( see Figure 1 ) .",
"Then , each was modeled by separate , but correlated , growth functions ( see Figure 2 ) .",
"For the discrete part of the model scores of zero were separated from the rest of the distribution by creating a binary indicator variable that distinguished any positive alcohol use score ( =1 ) from nouse ( =0 ) ( lower part of Figure 2 ) .",
"For the continuous part of the model , the continuous indicator variables representing the AUDIT score , given that it was above zero , were used ( upper part of Figure 2 ) .",
"In this latter part of the model , substance non-use within each time point was treated as missing data , following standard assumptions of data missing at random ( MAR; Little and Rubin , 1987 ) .",
"In that way participants who did not drink alcohol throughout the study contributed little information to the growth parameter estimates , but all information to alcohol use was used to estimate the growth parameters .",
"We used Maximum Likelihood estimator for our analyses .",
"We controlled for the effects of age at baseline , sex , total brain volume ( TBV ) and site ( by dummy coding eight of all nine neuroimaging sites , for simplicity we represent this by only one manifest variable in Figure 2 ) onto the slope of the continuous part of the growth model .",
"As criteria for model fit we report Root Mean Square Error of Approximation ( RMSEA ) , Comparative Fit Index ( CFI ) , and Standardized Root Mean Square Residual ( SRMR ) .",
"Values of the CFI above 0 . 90 denote a well-fitting model , whereas for the RMSEA and the SRMR values less than 0 . 08 may be interpreted as acceptable model-fit .",
"Since our main interest was , which voxels of the brain from grey matter and white matter maps at age 14 years predict the continuous slope representing the increase of alcohol drinking over time , the final SEM models were conducted including a brain variable .",
"For this purpose , we ran separate two-part growth mixture models for each and every voxel of the brain and repeated this analyses for the grey and white matter maps separately .",
"This approach is exploratory as no specific areas of the brain are extracted ( as would be the case in the more traditional analyses of ROI ) .",
"While it bears the risk of inflated type-II-errors , it also enables the investigation of links between brain structures and behavior that have not been strictly established yet .",
"For each SEM that was generated with a specific voxel as covariate , we plotted the estimate/p-value of the regression path from brain to the slope back into the brain , to be able to plot the resulting image using R ( the scripts can be obtained from the first author , more flexible functions that may be helpful in conducting similar analyses can be found in the 'neuropointillist' R package , https://github . com/IBIC/neuropointillist , Madhyastha et al . , 2018 ) .",
"This approach is not limited to growth curve models but can easily be extended to all types of models that can be implemented in MPlus ) .",
"Finally , we thresholded the resulting maps with a threshold of p<0 . 001 and a cluster threshold of k > 100 .",
"We repeated the same analyses while predicting the discrete slope from voxels of the grey and white matter maps , although our main interest was on brain-wise predictors of the slope derived from the continuous part of the model ."
]
] | [
"Adolescence is a common time for initiation of alcohol use and development of alcohol use disorders .",
"The present study investigates neuroanatomical predictors for trajectories of future alcohol use based on a novel voxel-wise whole-brain structural equation modeling framework .",
"In 1814 healthy adolescents of the IMAGEN sample , the Alcohol Use Disorder Identification Test ( AUDIT ) was acquired at three measurement occasions across five years .",
"Based on a two-part latent growth curve model , we conducted whole-brain analyses on structural MRI data at age 14 , predicting change in alcohol use score over time .",
"Higher grey-matter volumes in the caudate nucleus and the left cerebellum at age 14 years were predictive of stronger increase in alcohol use score over 5 years .",
"The study is the first to demonstrate the feasibility of running separate voxel-wise structural equation models thereby opening new avenues for data analysis in brain imaging ."
] | [
"Puberty is a time of transformation .",
"Physical changes in the body occur alongside changes in personality and behaviour .",
"Compared to children , adolescents tend to be risk-takers and novelty-seekers .",
"They crave new sensations and experiences , as well as social interaction with their peers .",
"It is around puberty that many people try alcohol for the first time .",
"But it is not clear why people differ in their drinking habits , and why a small minority of young adults go on to become dependent on alcohol .",
"Part of the answer may lie in changes in the brain .",
"Differences in the size and structure of brain regions contribute to differences in behaviour between individuals .",
"During adolescence , the brain undergoes extensive re-modelling .",
"It forms new connections , while also pruning away connections that are unused .",
"Could differences in brain structure at puberty lead to differences in alcohol consumption in early adulthood ?",
"Kühn et al . scanned the brains of about 1 , 800 healthy adolescents at the age of 14 and then again at 19 ( within the context of the IMAGEN study ) .",
"At three time points , the teenagers also filled in questionnaires about their use of alcohol .",
"Two areas of the brain – the caudate nucleus and the left cerebellum – were larger at age 14 in teenagers who would increase their alcohol consumption by age 19 .",
"The larger the areas at age 14 , the bigger the increase in alcohol consumption over time .",
"Notably , there was no relationship between the size of either brain area at the age of 14 and how much alcohol the individuals drank at the same age .",
"These results may help us to understand why some young adults develop harmful drinking habits , whereas most do not .",
"The findings are part of a large and complex picture .",
"Other factors , such as social influences , also shape alcohol consumption .",
"However , the findings of Kühn et al . suggest that differences in brain structure may make some individuals more likely to increase how much alcohol they drink than others .",
"Understanding these biological differences could help researchers to develop measures to prevent addiction in young adults ."
] | 2019 |
[
"Introduction",
"Results",
"Discussion"
] | [
"stem cells and regenerative medicine",
"developmental biology"
] | Altered bone growth dynamics prefigure craniosynostosis in a zebrafish model of Saethre-Chotzen syndrome | elife-37024-v1 | [
[
"Craniofacial anomalies are among the most common congenital defects , encompassing cleft lip and palate , facial malformations , and abnormalities in the flat bones forming the top of the skull .",
"Craniosynostosis involves the premature fusion of the skull bones at sutures , fibrous structures that join the bones .",
"During normal development , cranial sutures hold bones of the skull in place while providing malleability required during childbirth .",
"Cranial sutures also contain stem cells that allow for continued skull bone growth as the brain and head structure expands ( Zhao et al . , 2015 ) .",
"While human skull bones eventually fuse later in life , precocious bone fusion in craniosynostosis correlates with region-specific defects in bone growth that often negatively impact growth of the underlying brain .",
"Genetic causes of craniosynostosis include mutations in genes that participate in developmental signaling pathways , including FGFRs 1 , 2 , and 3 ( Hajihosseini , 2008 ) , TGFBRs 1 and 2 ( Loeys et al . , 2005 ) , and the Notch ligand JAGGED1 ( Kamath et al . , 2002 ) .",
"A central unanswered question is the extent to which defects in these pathways result in a failure to maintain postnatal stem cells once the sutures have formed , or whether these pathways have roles in the specification and maintenance of bone progenitors during earlier phases of bone growth preceding suture formation ( Flaherty et al . , 2016 ) .",
"Most studies have focused on the maintenance of postnatal sutural stem cells , as these stem cells have only been recently marked with a variety of reporters in mice , for example based on Gli1 ( Zhao et al . , 2015 ) , Axin2 ( Maruyama et al . , 2016 ) , and Prrx1 ( Wilk et al . , 2017 ) .",
"Less is known , however , about how potential defects in the embryonic progenitors that grow the skull bones may prefigure suture loss , although a few reports describe altered bone formation during embryonic stages in craniosynostosis mutants ( Merrill et al . , 2006 ) .",
"The ex utero development and transparency of zebrafish provide a unique opportunity to track the progenitors that form the skull bones , and to better understand how defects in the dynamics of bone growth relate to a later ability to form and maintain sutures once the bones come together ( Quarto and Longaker , 2005; Laue et al . , 2011; Kague et al . , 2016; Topczewska et al . , 2016 ) .",
"A striking feature of many forms of craniosynostosis is that only particular sutures are affected .",
"For example , in Saethre-Chotzen syndrome , the second most common form of craniosynostosis , the coronal suture is selectively lost .",
"The majority of Saethre-Chotzen patients harbor heterozygous loss-of-function mutations in TWIST1 or TCF12 , which encode basic helix-loop-helix transcription factors ( el Ghouzzi et al . , 1997; Howard et al . , 1997; Sharma et al . , 2013 ) .",
"Similarly , mice lacking one copy of Twist1 , or compound heterozygous for Twist1 and Tcf12 , display loss of just the coronal suture ( Sharma et al . , 2013 ) .",
"Analysis of mouse models has led to an appreciation of proper cell migration ( Yoshida et al . , 2008; Ting et al . , 2009; Roybal et al . , 2010; Deckelbaum et al . , 2012 ) , segregation of osteogenic and non-osteogenic cells at the sutural boundary ( Merrill et al . , 2006; Ting et al . , 2009; Yen et al . , 2010 ) , and maintenance of postnatal stem cells ( Zhao et al . , 2015 ) in suture patency .",
"However , it remains unknown the extent to which defects in early progenitors versus postnatal stem cells account for suture loss .",
"As fish are amenable to repeated live imaging of calvarial bone growth and suture development outside the mother , this model provides an opportunity to correlate early defects in osteoprogenitors and bone growth with later suture loss in individual mutants , particularly in cases where the synostosis phenotype is variably penetrant .",
"However , a potential complication is that the coronal suture of zebrafish ( Kague et al . , 2012; Mongera et al . , 2013 ) , as well as those of some amphibians ( Piekarski et al . , 2014 ) and birds ( Matsuoka et al . , 2005 ) , forms at a mesoderm/mesoderm boundary , contrasting with the mammalian coronal suture that forms at a unique interface between the neural-crest-derived frontal bone and the mesoderm-derived parietal bone ( Ishii et al . , 2015 ) ( Figure 1A ) .",
"Mammalian sutures may therefore not be considered evolutionarily homologous to the sutures of these other vertebrates , at least from a strict embryological perspective .",
"Should the neural-crest/mesoderm boundary be a factor in coronal sensitivity , non-mammalian coronal sutures might not be susceptible to the same genetic perturbations that cause coronal-specific synostosis in mammals .",
"We report in this study the generation of a zebrafish model of Saethre-Chotzen syndrome that faithfully recapitulates the craniosynostosis phenotype seen in mice and humans with heterozygous mutations in TCF12 and TWIST1 .",
"The similarity in the genetic interaction between Twist1 and Tcf12 in mice , humans , and fish , despite differences in the cell lineages that give rise to the bones , suggests that the underlying processes of coronal suture development and craniosynostosis are deeply conserved .",
"We demonstrate that in tcf12; twist1b mutant fish , the frontal and parietal bones grow abnormally .",
"In mutants , skull bones initiate normally , yet early bone growth is accelerated across the skull .",
"However , subsequent bone growth selectively stalls at the future coronal suture that is destined to fuse .",
"Moreover , sequential live imaging of individual mutant fish shows that the degree of later bone stalling predicts which animals will lose the coronal suture .",
"We observe a similar misregulation of bone growth in Tcf12+/-; Twist1+/- mutant mice , with tissue-specific removal of Twist1 resulting in selective overgrowth of the frontal or parietal bones .",
"Further , Twist1 function must be perturbed within both neural crest- and mesoderm-derived bones , and not just the mesoderm-derived postnatal sutural mesenchyme , to prevent suture formation .",
"At least in mice , we find that these altered bone growth dynamics may be due to changes in osteoblast proliferation and eventual depletion of Gli1+ and Gremlin1+ putative bone progenitors prior to the fusion of the bones .",
"These findings demonstrate that Tcf12 and Twist1 have a conserved early function during skull bone growth to regulate the sustained production of osteoblasts , possibly through maintenance of osteoprogenitors , and that the coronal suture is particularly sensitive to defects in this process ."
],
[
"In order to investigate requirements for Tcf12 and Twist1 homologs in zebrafish suture formation , we designed TALE nucleases to generate mutant alleles for tcf12 and both zebrafish Twist1 homologs , twist1a and twist1b .",
"The tcf12el548 , twist1ael570 , and twist1bel571 alleles result in premature truncations before the helix-loop-helix domains required for DNA-binding and dimerization , thus likely abrogating all protein function ( Figure 1—figure supplement 1 ) .",
"Whereas individual homozygous mutants displayed no gross defects as embryos or adults and had patent sutures across the head ( Figure 1B and Figure 1—figure supplement 2 ) , 38% of tcf12; twist1b double mutant adults developed unilateral or bilateral coronal synostosis , as revealed by Alizarin Red staining of bone ( Figure 1B ) .",
"We confirmed loss of the coronal suture by micro-computed tomography scans and histology ( Figure 1C , D ) .",
"As in mice and humans , coronal suture loss correlated with reduced anterior-posterior growth of the frontal and parietal bones , and in cases where the suture was lost unilaterally we consistently observed reduced anterior-posterior growth on that side of the skull ( Figure 1C ) .",
"In contrast , we did not detect a requirement for twist1a in suture development; sutures formed normally in tcf12-/-; twist1a-/- mutants , and loss of twist1a alleles did not increase the severity or penetrance of suture defects in tcf12-/-; twist1b-/- fish ( Figure 1—figure supplement 3 , Supplementary file 1 ) .",
"Loss of tcf12 appears essential for synostosis , as rare adult viable twist1a-/-; twist1b-/- fish had normal sutures .",
"While we only detected fusions of the coronal suture in tcf12-/-; twist1b-/- fish , we did observe other abnormalities in skull bones in different mutant combinations , including ectopic sutures and small gaps between the parietal bones ( Figure 1—figure supplement 3 , Supplementary file 1 ) .",
"Our results demonstrate that , as in humans and mice with reduced TCF12 and/or TWIST1 dosage , mutations in the homologous genes in zebrafish primarily result in loss of the coronal suture , although other calvarial defects are also rarely observed .",
"Given the synergistic effect of tcf12 and twist1b loss on coronal suture formation , we examined whether tcf12 also interacts genetically with Twist1 genes in earlier craniofacial development .",
"Similar to previous reports of zebrafish with antisense morpholino reduction of twist1a and twist1b ( Das and Crump , 2012 ) , and mice with neural-crest-specific deletion of Twist1 ( Bildsoe et al . , 2009 ) , twist1a-/-; twist1b-/- zebrafish embryos displayed defects in the specification of skeletogenic ectomesenchyme from the neural crest .",
"In wild-type embryos , neural crest expression of sox10 is down-regulated by 20 hr post-fertilization ( hpf ) as ectomesenchyme neural crest cells populate the pharyngeal arches .",
"In twist1a-/-; twist1b-/- embryos , we observed persistent sox10 expression in arch ectomesenchyme and reductions in facial cartilage and bone at 5 days post-fertilization ( dpf ) ( Figure 2A , B ) .",
"Dorsal facial cartilages ( e . g . palatoquadrate and hyosymplectic ) were most affected ( Figure 2C , Figure 2—source data 1 ) , potentially reflecting the greater sensitivity of these elements to general neural crest defects ( Cox et al . , 2012 ) and/or post-migratory roles of Twist1 genes in branchial arch development ( Askary et al . , 2017 ) .",
"Interestingly , loss of tcf12 suppressed rather than enhanced the severity of facial skeletal defects in twist1a-/-; twist1b-/- larvae , with partial suppression seen with loss of just one tcf12 allele ( Figure 2B , D , Figure 2—source data 2 ) .",
"The suppression did not appear to be due to a rescue of ectomesenchyme specification , as similarly persistent sox10 was evident in twist1a-/-; twist1b-/- embryos with or without tcf12 loss ( Figure 2A ) .",
"We also observed that loss of at least one copy of tcf12 enhanced adult viability of twist1a-/-; twist1b-/- mutants ( Figure 2E , Figure 2—source data 3 ) .",
"These findings indicate temporally distinct genetic interactions between Twist1 and Tcf12 , with Tcf12 acting antagonistically to Twist1 during arch development and synergistically during later skull bone growth and suture formation .",
"Given the selective loss of the coronal suture in mutants , we examined whether tcf12 and twist1b genes might be selectively expressed in this suture .",
"However , at a stage when sutures have just formed ( 14 mm standard length ) , we observed expression of tcf12 and twist1b within the mesenchyme of not only the coronal but also the metopic and sagittal sutures ( Figure 3 ) .",
"Whereas twist1b expression was largely restricted to the suture mesenchyme , tcf12 expression was observed more broadly in the suture mesenchyme and cells surrounding the skull bones .",
"Expression of tcf12 and twist1b at multiple sutures in fish is consistent with similarly broad suture expression of Twist1 in mice ( Rice et al . , 2000 ) and argues against genetic sensitivity of the coronal suture being due to selective expression of tcf12 and twist1b at this suture .",
"We next investigated whether suture defects in tcf12; twist1b mutants might result from an earlier misregulation of skull bone growth , to which the coronal suture might be particularly sensitive .",
"In wild-type zebrafish , the anlage of the frontal and parietal bones can first be seen at 6 mm standard length , with the coronal suture forming at the interface of these bones in a lateral to medial progression ( Kague et al . , 2016 ) .",
"Live mineralization stains revealed that initiation of the frontal and parietal bones was unaffected in mutants , yet accelerated growth of these bones became detectable in mutants by 8 mm , and more so by 9 mm ( Figure 4A ) .",
"Staining with the mineralization dye Calcein Green revealed accelerated frontal bone fronts by 10 . 25 ± 0 . 25 mm in mutants , with no difference in the degree of increased bone between sides that developed synostosis and those that did not ( Figure 4B , C , Figure 4—source data 1 ) .",
"Although the overall area of the mutant parietal bone was comparable to that of wild types at 10 . 25 ± 0 . 25 mm , both the mutant frontal and parietal bones were aberrantly shaped .",
"In particular , both the frontal and parietal bones exhibited enhanced growth along an axis diagonal to the anterior-posterior and medial-lateral axes , which themselves exhibit little to no changes in directional growth ( Figure 4C , Figure 4—source data 2 ) .",
"Such enhanced diagonal growth would be expected to bring the frontal and parietal bones closer together at the forming coronal suture , particularly in the medial region most commonly affected in mutants ( Figure 4B ) .",
"Likewise , the lack of enhanced medial-lateral growth of the parietal bones correlates with the sagittal suture being unaffected in mutants .",
"Subsequent sequential staining with Alizarin Red unveiled a marked decrease in bone growth in mutants from 10 . 25 ± 0 . 25 to 14 ± 0 . 5 mm at the future coronal but not the metopic or sagittal sutures ( Figure 4D , Figure 4—source data 3 , Figure 4—source data 4 , Figure 4—source data 5 ) .",
"We further took advantage of the variable penetrance of suture defects to correlate the degree of bone stalling with later suture loss in individual tcf12; twist1b mutants .",
"Consistently , synostotic sides exhibited greater reductions in earlier bone growth compared to mutant sides that had patent sutures .",
"Thus , the degree to which the growth of the mutant parietal and frontal bones slows in the coronal zone predicts later loss of this suture .",
"Reciprocally , the absence of bone stalling at the future metopic and sagittal zones is consistent with these sutures being unaffected in mutants .",
"We next examined the cellular mechanisms underlying altered bone growth in mutants .",
"Analysis of an sp7:EGFP transgenic line , which labels Sp7+ osteoblasts , revealed accelerated frontal and parietal bone fronts along the diagonal axis in tcf12; twist1b mutants versus sibling controls at 10 mm ( Figure 5—figure supplement 1 ) , consistent with our analysis using mineralization dyes .",
"We then used BrdU incorporation in combination with anti-Sp7 antibody staining to assess the numbers of proliferative cells at the growing fronts of the frontal and parietal bones at an earlier 9 mm stage .",
"Analysis of Sp7+ osteoblasts revealed acceleration of frontal and parietal bone growth , with the leading edges of the bones appearing uneven ( Figure 5A; additional examples in Figure 5—figure supplement 2 ) .",
"After digitally extracting the bone fronts to avoid signals from the highly proliferative skin , we quantified the numbers of proliferative cells at the fronts .",
"Along both the mutant frontal and parietal bone fronts , we observed an increase in the percentage of Sp7+ osteoblasts undergoing proliferation , and a trend toward increased numbers of proliferative Sp7- cells just ahead of the bone fronts ( Figure 5B , C , Figure 5—source data 1 , Figure 5—source data 2 ) .",
"These results suggest that increased proliferation of early osteoblasts , and potentially also osteoprogenitors , contribute to the initial acceleration of bone growth across the mutant skull .",
"Given the similarity of coronal suture defects in tcf12-/-; twist1b-/- fish and Tcf12+/-; Twist1+/- mice , we investigated whether earlier alterations in calvarial bone growth might also prefigure suture loss in mice .",
"At embryonic day ( E ) 13 . 5 , when skull bone rudiments are first apparent , alkaline phosphatase staining revealed accelerated frontal and parietal bones that were in closer apposition in Tcf12+/-; Twist1+/- mutants versus wild-type sibling controls ( Figure 6A ) .",
"At birth , mutant frontal and parietal bones were more closely apposed than in controls and had abnormal shapes ( Figure 6B ) .",
"Next , we assessed proliferation rates and osteoblast density at the forming coronal suture ( E14 . 5 ) and sagittal suture ( E16 . 5 ) ( Figure 6C , outlined regions of bottom panels ) .",
"In mutants , we observed thicker bone fronts , as marked by Sp7+ cells ( Figure 6—figure supplement 1 , Figure 6-figure supplement 1-Source Data 1 ) , as well as a marked increase in the number of Sp7+ osteoblasts at the bones fronts predestined for the coronal and sagittal sutures ( Figure 6D , Figure 6—source data 1 ) .",
"We also observed an increase in the number of proliferative Sp7- cells immediately adjacent to osteoblasts at the bone fronts , with this increase more evident at the forming coronal versus sagittal suture ( Figure 6E , Figure 6—source data 2 ) .",
"In contrast to fish , we noted a decrease in the number of proliferative osteoblasts at the forming coronal suture , and no change at the forming sagittal suture ( Figure 6F , Figure 6—source data 3 ) .",
"These findings largely support a conserved role for Tcf12 and Twist1 in negatively regulating the number of proliferative cells at the growing bone fronts in fish and mice .",
"We next investigated whether the lack of continued bone growth at the mutant coronal fronts might reflect an exhaustion of osteoprogenitor cells .",
"In mouse , sutural stem cells express Prrx1 ( Wilk et al . , 2017 ) and Gli1 ( Zhao et al . , 2015 ) .",
"Grem1 also marks skeletal stem cells throughout the animal ( Worthley et al . , 2015 ) , yet a role in the skull and sutures has not been previously examined .",
"Using RNAscope in situ hybridization technology in zebrafish , we find that prrx1a is broadly expressed at the parietal and frontal bone fronts destined for the coronal and sagittal sutures , as well as the periosteum , at 10 mm , and in the sutures and periosteum at adult stages ( Figure 7A , B ) .",
"The expression of gli1 and grem1a appears more restricted to the growing bone fronts and suture mesenchyme , although we also see more general periosteal expression at earlier stages .",
"In tcf12; twist1b mutants , we still observe cells expressing gli1 , grem1a , and prrx1a at the forming coronal and sagittal sutures , as well as within the periosetum ( Figure 7A , B ) , with quantitation revealing similar levels of each gene on a per cell basis ( Figure 7—figure supplement 1A , Figure 7—figure supplement 1—source data 1 , Figure 7—figure supplement 1—source data 2 ) .",
"At the coronal suture , we observed that the mutant frontal and parietal bones were more closely apposed than in stage-matched wild-type siblings , which could possibly be a consequence of depleted progenitors at the bone fronts .",
"In adult mutants with fused coronal sutures , we failed to detect gli1+ , grem1a+ , or prrx1a+ cells in the coronal suture region , whereas prrx1a was still expressed in the periosteum ( Figure 7A ) .",
"Normal expression of all three markers was observed at the patent sagittal suture .",
"We also examined Fgf signaling in zebrafish mutants , as the expression of Fgfr2 has been reported to be altered in mouse Twist1 heterozygous animals ( Rice et al . , 2000; Connerney et al . , 2006 ) .",
"As with gli1 , grem1a , and prrx1a , we still observed cells expressing fgfr2 and the Fgf target gene dusp6 at the mutant coronal suture ( Figure 7—figure supplement 1B , Figure 7—figure supplement 1—source data 3 ) , arguing against the calvarial phenotypes being due to wholesale loss of Fgfr2 signaling at the bone fronts .",
"Given the difficulty in quantitating the numbers of osteoprogenitors at the forming coronal suture zone of mutant zebrafish , owing in part to the small sizes of these bone fronts , we also examined putative progenitors in mutant mice .",
"As in zebrafish , we observed cells expressing Gli1 and Grem1 protein at and around the fronts of the embryonic frontal and parietal bones .",
"In Tcf12+/-; Twist1+/- mice , we observed a marked reduction in the number of Gli1+ and Grem1+ putative progenitors at and around the developing coronal but not the sagittal bone fronts ( Figure 7C , D ) .",
"These findings highlight a conserved molecular signature of putative osteoprogenitors and sutural stem cells of zebrafish and mice and suggest , at least in mice , a selective exhaustion of osteoprogenitors at the developing coronal suture .",
"To investigate whether Twist1 functions tissue-intrinsically for proper skull bone growth , we took advantage of the unique germ-layer origins of the mammalian frontal and parietal bones to remove one copy of Twist1 in each tissue .",
"At postnatal day ( P ) 21 , we found that reduced dosage of Twist1 in the neural-crest-derived precursors of the frontal bone , in Wnt1-Cre; Twist1flox/+ mice , resulted in the overgrowth of the frontal bone relative to the parietal bone , which we quantified by measuring the ratio of the sagittal to metopic suture ( Figure 8 , Figure 8—source data 1 ) .",
"Reciprocally , removing one copy of Twist1 from the mesoderm-derived parietal bone , in Mesp1-Cre; Twist1flox/+ mice , resulted in its overgrowth relative to the frontal bone .",
"Reduced dosage of Twist1 in both the neural crest and mesoderm , in Wnt1-Cre; Mesp1-Cre; Twist1flox/+ mice , normalized the relative sizes of the frontal and parietal bones and resulted in loss of the coronal suture , a phenotype not seen upon deletion of Twist1 in neural crest or mesoderm alone .",
"We conclude that Twist1 negatively regulates bone growth in both the neural-crest- and mesoderm-derived portions of the skull , and that Twist1 must be mutated in not only the mesoderm-derived parietal bone and suture mesenchyme , but also the neural-crest-derived frontal bone , to impact coronal suture formation ."
],
[
"Discovery of a selective requirement for tcf12 and twist1b in coronal suture formation in zebrafish has allowed us to gain a better understanding of the developmental basis of suture loss in Saethre-Chotzen syndrome .",
"The similarity of coronal suture defects from humans to mice to zebrafish is striking , although each species displays unique dosage sensitivities to loss of TWIST1 and TCF12 .",
"In humans , haploinsufficiency of TWIST1 or TCF12 can lead to suture loss .",
"In mice , haploinsufficiency of Twist1 also results in coronal suture loss , yet haploinsufficiency of Tcf12 does not , despite enhancing the penetrance of suture defects in Twist1+/- mice ( Sharma et al . , 2013 ) .",
"In zebrafish , only loss of one of two Twist1 homologs ( twist1b ) along with loss of tcf12 results in suture loss .",
"It remains unclear why humans are more sensitive to Twist1 and Tcf12 dosage than mice and zebrafish , although a similar phenomenon has been observed with other synostosis genes ( e . g . JAG1 ) ( Teng et al . , 2017 ) .",
"Repeated live imaging of individual mutant fish revealed a strong correlation between the extent of altered bone growth and later suture loss .",
"Whereas the initiation of the frontal and parietal bones was largely unaffected in mutants , we observed increased proliferation and osteoblast production at the mutant bone fronts in both fish and mice .",
"There were some subtle differences between fish and mice , with larger increases in proliferative osteoblasts in mutant fish than mice , which might reflect species-specific or staging differences .",
"Nonetheless , mutants in both species displayed accelerated growth of both the frontal and parietal bones , which often resulted in abnormal shapes likely due to growth variations along individual bone fronts .",
"In particular , we found that increased diagonal growth in mutants brought the parietal and frontal bones together at the prospective medial regions of the coronal suture much earlier than in wild type animals , which correlates with the medial region of the coronal suture being most commonly fused in mutants .",
"This altered directional growth might be one reason why the coronal suture is preferentially affected in both zebrafish and mouse Twist1/Tcf12 mutants , despite the different origins of the coronal suture in these species .",
"Another prominent finding was a lack of continued bone growth at the future coronal but not other sutures in mutants , with the degree of bone stalling predicting synostosis in individuals .",
"We identified Gli1/gli1 and Grem1/grem1a as conserved markers of putative osteoprogenitors in both the growing bone fronts and mature sutures of mice and zebrafish , although both genes display broader expression at early stages of calvarial bone development , including in some periosteal cells .",
"Future lineage tracing experiments should help reveal the extent to which gli1 in fish and Grem1/grem1a in both species mark similar populations of embryonic osteoprogenitors and postnatal sutural stem cells .",
"A previous study had observed reduced expression of Gli1 in the sutures of adult Twist1+/- mice ( Zhao et al . , 2015 ) .",
"Here , we extend this finding to embryonic stages when the coronal suture is forming , and uncover the existence of Grem1+ cells in and around the bone fronts of the nascent coronal suture that become depleted in mutant mice .",
"Whether similar osteoprogenitors become exhausted at the developing coronal suture of zebrafish remains to be determined , as it was difficult to precisely quantify the numbers of these progenitors by RNA expression alone .",
"One possibility is that reduced Twist1 and Tcf12 function alters the balance between long-term sutural stem cells ( i . e . those marked by Gli1 ) and proliferative osteoblasts and their immediate progenitors .",
"In such a model , the early increase in osteoblast production would come at the expense of long-term progenitors , thus leading to a later failure of continued bone growth and a loss of the sutural stem cells that would normally separate the skull bones .",
"In addition to further verifying this model , an important next step will be to determine why the fronts of the parietal and frontal bones abutting the future coronal suture are most sensitive to progenitor exhaustion in mutants .",
"We did not observe preferential expression of tcf12 and twist1b at the coronal suture in fish , consistent with similarly broad sutural expression of Twist1 in mice ( Rice et al . , 2000 ) .",
"Instead , osteoprogenitors in the coronal zone could be fewer in number at initial stages and/or more sensitive to loss of Tcf12 and Twist1 , for example due to compensation by related genes at other sutures .",
"Further support for suture defects arising from much earlier changes in bone growth come from our conditional Twist1 deletion experiments in mouse .",
"Whereas the frontal bone arises from neural crest and the parietal bone from mesoderm , the mesenchyme within the postnatal coronal suture is largely mesoderm-derived ( Yen et al . , 2010 ) .",
"However , suture loss was only observed upon conditional deletion of one allele of Twist1 from both the embryonic mesoderm and neural crest .",
"This finding is inconsistent with Twist1 functioning solely in the mesoderm-derived postnatal suture mesenchyme for suture patency , instead suggesting that misregulated growth of both the frontal and parietal bones is required to later disrupt the coronal suture in Twist1 mutants .",
"Our study highlights a selective role for Tcf12 in the later growth of the skull bones and patency of the coronal suture .",
"In contrast to animals lacking Twist1 homologs , zebrafish lacking tcf12 do not display embryonic lethality or defects in ectomesenchyme and facial cartilage formation .",
"Instead , loss of tcf12 partially suppresses the facial cartilage defects and lethality of twist1a; twist1b mutants .",
"Suppression of embryonic defects could be due to loss of competition of Tcf12 with other Twist binding partners , such as Hand2 ( Firulli et al . , 2005 ) .",
"In this scenario , maternal Twist1a/b and/or other Twist family members ( e . g . twist2 and twist3 ) could compensate for lack of zygotic Twist1a/b .",
"Loss of tcf12 would then allow remaining Twist proteins to more effectively form homodimers or alternate heterodimers important for embryogenesis .",
"Whereas a previous report indicates that Tcf12 and Twist1 can form heterodimers ( Connerney et al . , 2006 ) , it is also possible that Tcf12 has Twist1-independent functions that antagonize Twist1 during embryogenesis .",
"Future efforts to directly visualize Tcf12-Twist1 complexes will help to resolve how Tcf12 promotes Twist1 function during skull bone growth while counteracting it during embryonic neural crest development .",
"Development of homologous structures often employs ancestrally conserved gene regulatory networks .",
"For example , a requirement for Pax6 genes in eye development in a wide range of animals has been used to argue for deep homology of eye structures ( Gehring and Ikeo , 1999 ) .",
"Here , we show remarkably specific loss of a single anatomical structure , the coronal suture , in zebrafish and mice lacking Tcf12 and Twist1 , despite this suture occurring at a mesoderm/mesoderm interface in zebrafish and a neural-crest/mesoderm interface in mice .",
"Hence , sensitivity of the coronal suture in Saethre-Chotzen syndrome is unlikely to be due to its location at a unique neural-crest/mesoderm interface .",
"In addition , there are several other sutures occurring at a neural-crest/mesoderm interface in mice and fish that are not affected by loss of Tcf12 and Twist1 .",
"There has been on-going debate , given these distinct tissue boundaries , as to whether coronal sutures are truly homologous across vertebrates ( Maddin et al . , 2016 ) .",
"Our data indicate that the conserved genetic dependence of the coronal suture in fish and mammals likely reflects a similar sensitivity to early bone growth changes , perhaps owing to similar developmental and anatomical constraints irrespective of the embryonic origins of the bones flanking this suture ."
]
] | [
"Cranial sutures separate the skull bones and house stem cells for bone growth and repair .",
"In Saethre-Chotzen syndrome , mutations in TCF12 or TWIST1 ablate a specific suture , the coronal .",
"This suture forms at a neural-crest/mesoderm interface in mammals and a mesoderm/mesoderm interface in zebrafish .",
"Despite this difference , we show that combinatorial loss of TCF12 and TWIST1 homologs in zebrafish also results in specific loss of the coronal suture .",
"Sequential bone staining reveals an initial , directional acceleration of bone production in the mutant skull , with subsequent localized stalling of bone growth prefiguring coronal suture loss .",
"Mouse genetics further reveal requirements for Twist1 and Tcf12 in both the frontal and parietal bones for suture patency , and to maintain putative progenitors in the coronal region .",
"These findings reveal conservation of coronal suture formation despite evolutionary shifts in embryonic origins , and suggest that the coronal suture might be especially susceptible to imbalances in progenitor maintenance and osteoblast differentiation ."
] | [
"Some of the most common birth defects involve improper development of the head and face .",
"One such birth defect is called craniosynostosis .",
"Normally , an infant’s skull bones are not fully fused together .",
"Instead , they are held together by soft tissue that allows the baby’s skull to more easily pass through the birth canal .",
"This tissue also houses specialized cells called stem cells that allow the brain and skull to grow with the child .",
"But in craniosynostosis these stem cells behave abnormally , which fuses the skull bones together and prevents the skull and brain from growing properly during childhood .",
"One form of craniosynostosis called Saethre-Chotzen syndrome is caused by mutations in one of two genes that ensure the proper separation of two bones in the roof of the skull .",
"Mice with mutations in the mouse versions of these genes develop the same problem and are used to study this condition .",
"Mouse studies have looked mostly at what happens after birth .",
"Studies looking at what happens in embryos with these mutations could help scientists learn more .",
"One way to do so would be to genetically engineer zebrafish with the equivalent mutations .",
"This is because zebrafish embryos are transparent and grow outside their mother’s body , making it easier for scientists to watch them develop .",
"Now , Teng et al . have grown zebrafish with mutations in the zebrafish versions of the genes that cause Saethre-Chotzen syndrome .",
"In the experiments , imaging tools were used to observe the live fish as they developed .",
"This showed that the stem cells in their skulls become abnormal much earlier than previous studies had suggested .",
"Teng et al . also showed that similar stem cells are responsible for growth of the skull in zebrafish and mice .",
"Babies with craniosynostosis often need multiple , risky surgeries to separate their skull bones and allow their brain and head to grow .",
"Unfortunately , these bones often fuse again because they have abnormal stem cells .",
"Teng et al . provide new information on what goes wrong in these stem cells .",
"Hopefully , this new information will help scientists to one day correct the defective stem cells in babies with craniosynostosis , thus reducing the number of surgeries needed to correct the problem ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"epidemiology and global health"
] | Ebola virus disease in the Democratic Republic of the Congo, 1976-2014 | elife-09015-v1 | [
[
"Ebola virus disease ( EVD ) outbreaks are rare and knowledge of the transmission and clinical features of this disease is sparse .",
"As of May 2015 , the devastating outbreak in West Africa has resulted in more than ten times the number of cases reported in all previous outbreaks and will ultimately provide improved insights into EVD .",
"Here , for the first time , all the databases from EVD outbreaks in the Democratic Republic of the Congo ( DRC ) have been cleaned and compiled into one anonymised individual-level dataset ( See Supplementary file 1 ) .",
"The data provided are an invaluable addition to the West Africa data and will allow a more complete picture of the disease .",
"The DRC is the country that has experienced the most outbreaks of EVD .",
"Since the virus' discovery in 1976 , there have been six major outbreaks ( Yambuku 1976 , Kikwit 1995 , Mweka 2007 , Mweka 2008/2009 , Isiro 2012 , and Boende 2014 ) and one minor outbreak ( Tandala 1977 ) reported in the DRC , four in the northern Equateur and Orientale provinces and three in the southern provinces of Bandundu and Kasai-Occidental ( Figure 1 ) .",
"Some of these have been described in the literature ( World Health Organization , 1978; Heymann et al . , 1980; Khan et al . , 1999; Muyembe-Tamfum et al . , 1999 , 2012; Maganga et al . , 2014 ) .",
"However , the individual-level data and corresponding lessons from these outbreaks have not been collated or made publicly available; by doing so , we aim to permit a more powerful statistical analysis and a fuller understanding of the disease .",
"The end of the most recent outbreak in the DRC was declared on the 21st of November 2014 .",
"This provides an unparalleled opportunity to assemble all the information gathered about EVD in the DRC through almost four decades , learn from the Congolese experience with this disease , and compare the features of EVD in DRC with the epidemic that has had such a devastating effect in West Africa . 10 . 7554/eLife . 09015 . 003Figure 1 . Map and historical timeline of the EVD outbreaks in the DRC .",
"( A ) Map of the Democratic Republic of the Congo ( DRC ) where the area of the circles are proportional to the number of cases ( probable and confirmed ) per outbreak .",
"( B ) The outbreaks ( in orange ) and relevant wars ( in purple ) are positioned in time . DOI: http://dx . doi . org/10 . 7554/eLife . 09015 . 003"
],
[
"The number of cases and case-fatality ratios ( CFRs ) varied greatly between outbreaks ( Table 2 ) .",
"It can also be observed that laboratory confirmation became more readily available over time .",
"Across all outbreaks , 57% of cases were female ( 95% CI = 53 . 9–60 . 1 ) .",
"In the second Mweka outbreak and in the Isiro outbreak , more than 70% of cases were females .",
"However , in the other outbreaks , the percentage of females was lower ( 53–59% ) .",
"When comparing the probable and confirmed cases by age with the overall DRC population ( Figure 2 ) , we observed a high concentration of cases in the 25–64 age category compared to the baseline population .",
"This might be because at this age individuals are more likely to be carers .",
"The occupation was only recorded during three outbreaks: Kikwit , Boende and Isiro .",
"During Kikwit , 23% ( 73/317 ) of cases were known healthcare workers ( HCWs ) and 0 . 6% ( 2/317 ) were possible HCWs .",
"During Boende , the occupation was recorded for 85% ( 58/68 ) of cases .",
"14% ( 8 ) were known HCWs and 3% ( 2 ) were possible HCWs .",
"During Isiro , occupation was reported for 94% ( 49/52 ) of cases .",
"27% ( 13 ) were HCW .",
"Although occupation was not recorded on an individual level , during Yambuku , 13 of the 17 Yambuku Hospital workers contracted EVD ( World Health Organization , 1978 ) . 10 . 7554/eLife . 09015 . 006Figure 2 . Incidence of cases by age and sex in the DRC outbreaks in comparison to the demographics of the national 1975–2010 population . DOI: http://dx . doi . org/10 . 7554/eLife . 09015 . 006 The epidemic curves were plotted for the six major outbreaks ( Figure 3 ) .",
"The date of infection was based on symptom onset when available ( 701/995 ) .",
"When it was not , hospitalisation dates were used ( 5/995 ) .",
"In cases where these were also absent ( 281/995 ) , the notification dates were used as proxy .",
"For Mweka 2007 , the date of infection was mostly based on the notification date ( 98% ) , whereas in the other outbreaks , infection dates refer to onset of symptoms almost exclusively ( >90% ) .",
"In time , case definitions became more specific .",
"With the exception of Kikwit , in which notification and the closure of healthcare facilities coincided closely in time , outbreaks seemed to peak before major interventions were initiated . 10 . 7554/eLife . 09015 . 007Figure 3 . Time course of the EVD outbreaks in DRC . Confirmed cases are plotted in red , probable cases in orange , suspected in light blue , cases that were either suspected or probable cases in dark blue , and cases for whom the definition was unknown in purple .",
"The dashed lines represent important events that occurred during the outbreaks ( in orange , the first records of the disease , in red , the first notifications , and in black , important interventions carried out ) .",
"For Yambuku , this was the closure of Yambuku Mission Hospital; for Kikwit , the closure of all hospitals , health centres , and laboratories in the area; for Mweka 2007 , the opening of two mobile laboratories; for Mweka 2008 , the opening of the first isolation centre; for Isiro , first the opening of the isolation centre and later the opening of the laboratory; and for Boende , the opening of the first isolation centre .",
"Notification dates were when the cases were first notified to the Direction de Lutte contre la Maladie ( DLM ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 09015 . 007 The proportion of probable and confirmed cases reporting EVD symptoms is shown in Figure 4 .",
"Overall , the most commonly reported symptom was fever , which was reported by 95% of cases ( 95% CI = 92 . 6–97 . 3% ) and at least 90% of cases in every outbreak .",
"Reports of vomiting were also similarly common across all major outbreaks , reported by 75% of cases ( 95% CI = 69 . 3–79 . 2 ) and between 57% and 76% of cases for all major outbreaks .",
"There was considerable variation in how frequently the remaining symptoms were reported for different outbreaks .",
"In particular , hemorrhagic symptoms were present in 61% ( 95% CI = 51–71 ) of cases during Kikwit but only 10% ( 95% CI = 5–18 ) during Mweka 2007 .",
"The Bundibugyo ebolavirus ( Isiro outbreak ) did not present a symptom profile that was particularly different from that seen for the Zaire ebolavirus ( all other outbreaks ) .",
"However , this was difficult to conclude given the large variation between outbreaks . 10 . 7554/eLife . 09015 . 008Figure 4 . Percentage of probable and confirmed cases with abdominal pain , diarrhoea , fever , haemorrhagic symptoms , headache , and vomiting . These were calculated by dividing the number of probable and confirmed cases with symptoms by the number of probable and confirmed cases with symptoms , no symptoms , and blanks for cases for who the presence or absence of at least one symptom was reported .",
"Note that the majority of cases in the Mweka 2007 outbreak were diagnosed a posteriori using recorded symptoms . DOI: http://dx . doi . org/10 . 7554/eLife . 09015 . 008 The mean CFR overall was 79% ( 95% CI = 76 . 4–81 . 6 ) , but there were significant differences between epidemics and within epidemics over time ( Figure 5 and Figure 5—figure supplement 1 ) .",
"The highest average CFR was seen during the first outbreak in Yambuku ( mean = 96% , 95% CI = 92 . 6–97 . 9 in our subset of 262/318 cases ) .",
"Kikwit , Mweka 2007 , and Boende had high average CFRs ranging from 74% to 78% .",
"During the Isiro and Mweka 2008 outbreaks , the CFR was lower , at 54 and 44% ( 95% CI = 39 . 5–67 . 8 and 26 . 4–62 . 3 ) , respectively . 10 . 7554/eLife . 09015 . 009Figure 5 . Evolving case-fatality ratios with time after the start of the outbreak . Monthly point estimates are presented with 95% binomial confidence intervals .",
"The dashed horizontal line indicates the average case-fatality ratio ( CFR ) during each outbreak .",
"The vertical dashed lines represent important events that occurred during the outbreaks ( in red , the first notifications , and in black , important interventions carried out ) .",
"For Yambuku , this was the closure of Yambuku Mission Hospital; for Kikwit , the closure of all hospitals , health centres , and laboratories; for Mweka 2007 , the opening of two mobile laboratories; for Mweka 2008 , the opening of the first isolation centre; for Isiro , first the opening of the isolation centre and later the opening of the laboratory; and for Boende , the opening of the first isolation centre .",
"Notification dates were when the cases were first notified to the DLM . DOI: http://dx . doi . org/10 . 7554/eLife . 09015 . 00910 . 7554/eLife . 09015 . 010Figure 5—figure supplement 1 . CFR by age groups for each outbreak . DOI: http://dx . doi . org/10 . 7554/eLife . 09015 . 01010 . 7554/eLife . 09015 . 011Figure 5—figure supplement 2 . Aggregated CFRs for all outbreaks by age group . DOI: http://dx . doi . org/10 . 7554/eLife . 09015 . 011 All EVD patients under 2 years of age died ( N = 29 , Figure 5—figure supplement 1 ) .",
"CFRs generally decreased during childhood and then increased again to plateau at around 70–80% in adulthood ( Figure 5—figure supplement 2 ) .",
"This pattern was less readily observed for the CFRs in the Yambuku outbreak , which remained high and similar for all ages .",
"In the regression model that included the delay between symptom onset and hospitalisation as a factor but excluded three outbreaks for missing data ( Table 3 ) , the baseline CFR in individuals over 15 years of age during the first month of an EVD outbreak who were admitted to hospital after 0 . 3 days ( the average time from symptom onset to admission to hospital ) during the Boende outbreak was 74% ( 95% CI = 17 . 8–99 . 3 ) .",
"The CFR was similar during the Isiro outbreak but was significantly higher during the Kikwit outbreak ( 94% ) .",
"The CFR in 0–5 year olds was 76% , and in 5–15 year olds , it was significantly lower at 36% .",
"The odds of dying declined on average by 31% ( 95% CI = 3 . 1–52 . 0% ) each month after the start of an outbreak and increased by 11% ( 95% CI = 1 . 8–20 . 7% ) per day that a symptomatic person is not hospitalised ( Table 3 ) . 10 . 7554/eLife . 09015 . 012Table 3 . Odds of dying from EVDDOI: http://dx . doi . org/10 . 7554/eLife . 09015 . 012OR2 . 50%97 . 50%Outbreak Isiro 20120 . 890 . 312 . 59Outbreak Kikwit 19955 . 441 . 4321 . 87Age [0 , 5 ) 1 . 120 . 236 . 63Age [5 , 15 ) 0 . 20 . 050 . 7Months since first case0 . 690 . 480 . 97Delay onset to hospitalisation1 . 111 . 021 . 21EVD , Ebola virus disease . Estimated through binomial regression with age group and year of outbreak as factorial covariates and the number of months since the start of the outbreak and the delay from symptom onset to hospitalisation as continuous covariates .",
"In the regression model that included all major outbreaks , the CFR for individuals over 15 years of age during the first month of the outbreak during the Boende outbreak was estimated at 79% ( 95% CI = 25 . 8–99 . 5 ) .",
"The Yambuku , Kikwit , and Mweka 2007 outbreaks had significantly higher CFRs ( 96% , 94% and 93% ) and the Mweka 2008 outbreak had a significantly lower CFR ( 48% ) .",
"0–5 year olds had significantly higher CFRs ( 90% ) than those over 15 years of age .",
"For the 5–15 year olds , the CFR was significantly lower ( 57% ) .",
"The odds of dying declined on average by 35% ( 95% CI = 22 . 6–45 . 9 ) each month after the start of each outbreak ( Table 4 ) . 10 . 7554/eLife . 09015 . 013Table 4 . Odds of dying from EVDDOI: http://dx . doi . org/10 . 7554/eLife . 09015 . 013CovariatesOR2 . 50%97 . 50%Outbreak Isiro 20120 . 670 . 291 . 55Outbreak Kikwit 19954 . 631 . 9610 . 94Outbreak Mweka 20073 . 831 . 619 . 18Outbreak Mweka 20080 . 250 . 10 . 63Outbreak Yambuku 19767 . 113 . 1316 . 75Age [0 , 5 ) 2 . 491 . 126 . 34Age [5 , 15 ) 0 . 360 . 210 . 63Months since first case0 . 650 . 540 . 77EVD , Ebola virus disease . Estimated through binomial regression with age group and year of outbreak as factorial covariates and the number of months since the start of the outbreak as continuous covariate .",
"Changes in the effective reproduction number , R , over the course of the outbreaks were plotted in Figure 6 .",
"In Yambuku , Mweka 2008 , and Boende 2014 , R dropped below one within 3–5 weeks after the initial case and the outbreak was rapidly brought under control .",
"In these settings , the spread of EVD during the first 2 weeks had been high ( R > 3 ) .",
"By contrast , in Kikwit 1995 , Mweka 2007 , and Isiro 2012 , where the initial transmission rate was lower , spread of EVD was sustained for more than 13 weeks .",
"Overall , we can see that R declines before the major interventions occurred , which could point to behavioural changes that occurred spontaneously in the populations . 10 . 7554/eLife . 09015 . 014Figure 6 . Evolving effective reproduction numbers with time after the start of the outbreak and adjusted weekly incidence . Weekly point estimates of the effective reproduction numbers are presented with 95% confidence intervals .",
"The dashed horizontal line indicates the threshold R = 1 .",
"The vertical dashed lines represent important events that occurred during the outbreaks ( in red , the first notifications , and in black , important interventions carried out ) .",
"For Yambuku , this was the closure of Yambuku Mission Hospital; for Kikwit , the closure of all hospitals , health centres , and laboratories; for Mweka 2007 , the opening of two mobile laboratories; for Mweka 2008 , the opening of the first isolation centre; for Isiro , first the opening of the isolation centre and later the opening of the laboratory; and for Boende , the opening of the first isolation centre .",
"The light grey bars represent the weekly incidence of Ebola virus disease ( EVD ) ( omitting suspected cases ) rescaled by dividing by seven . DOI: http://dx . doi . org/10 . 7554/eLife . 09015 . 014 The delay distributions from onset of symptoms to notification , from onset of symptoms to hospitalisation , from onset of symptoms to death , length of hospital stay , and from hospitalisation to death were plotted for each outbreak ( when available ) in Figure 7 .",
"The largest delays between symptom onset to notification and to hospitalisation were seen during the Kikwit outbreak ( 12 . 9 days and 5 . 0 days , respectively ) .",
"The largest delay between symptom onset and death and the longest duration of hospitalisation were seen during the Isiro outbreak ( 11 . 4 and 8 . 0 days , respectively ) .",
"However , this was only recorded for the Kikwit , Mweka 2008 , and Isiro outbreaks .",
"The longest delay between hospitalisation and death was observed during the Mweka 2008 outbreak ( 11 . 0 days ) ( Table 5 ) . 10 . 7554/eLife . 09015 . 015Figure 7 . Delay distributions for the EVD outbreaks in the DRC . The bars represent the observed frequency distributions of the delay from onset of symptoms to notification , onset of symptoms to hospitalisation , onset of symptoms to death , length of hospitalisation , and date of hospitalisation to death .",
"Delays were censored at 30 days .",
"The red line represents the respective fit of a gamma distribution . DOI: http://dx . doi . org/10 . 7554/eLife . 09015 . 01510 . 7554/eLife . 09015 . 016Table 5 . Mean values and standard deviations corresponding to the delay distributionsDOI: http://dx . doi . org/10 . 7554/eLife . 09015 . 016OutbreakDelayMeanSD1995 KikwitOnset to notification12 . 917 . 721995 KikwitOnset to hospitalisation5 . 023 . 911995 KikwitOnset to death9 . 474 . 441995 KikwitLength of hospital stay5 . 725 . 671995 KikwitHospitalisation to death4 . 53 . 692007 MwekaOnset to notification002008 MwekaOnset to notification10 . 048 . 472008 MwekaOnset to hospitalisation002008 MwekaOnset to death7 . 624 . 442008 MwekaLength of hospital stay1 . 52 . 122008 MwekaHospitalisation to death119 . 852012 IsiroOnset to notification8 . 838 . 292012 IsiroOnset to hospitalisation43 . 272012 IsiroOnset to death11 . 375 . 412012 IsiroLength of hospital stay86 . 562012 IsiroHospitalisation to death7 . 595 . 522014 BoendeOnset to notification6 . 235 . 082014 BoendeOnset to hospitalisation4 . 953 . 322014 BoendeOnset to death9 . 395 . 672014 BoendeHospitalisation to death4 . 864 . 23Delays distributions ( delay from onset of symptoms to notification , onset of symptoms to hospitalisation , onset of symptoms to death , length of hospitalisation and date of hospitalisation to death ) ."
],
[
"This article provides for the first time a description and a line list for all outbreaks that have occurred in the DRC .",
"This represents almost 40 years of surveillance data , seven outbreaks , and 996 suspected , probable , or confirmed cases .",
"It is an invaluable resource for studying the epidemiology and clinical features of EVD .",
"We highlight the importance of reducing the delay between symptom onset and hospitalisation , as the odds of dying increase by 11% per day that a patient is not hospitalised .",
"We also observe higher incidence in those between 25 and 64 years of age and a higher CFR in patients under 5 or over 15 years of age than in those between 5 and 15 years old .",
"These trends mirror those observed during the West African outbreak , where cumulative incidence was highest in those between 16 and 44 years of age and CFR progressively dropped from 89 . 5% in those under 1 year of age to 52 . 1% in those between 10 and 15 years , to rise again to 78 . 7% in those over 45 years old ( WHO Ebola Response Team et al . , 2015a ) .",
"These distinctions could inform the choice of target age groups for interventions such as vaccination .",
"Another important finding is that during outbreaks with an initially lower reproduction number , R , ( ≤3 ) national and international response was slower , outbreaks took longer to control , and ( with the exception of Yambuku , where the virus was first discovered ) were larger outbreaks than those with initially high R . This occurred during the current outbreak in West Africa , where the basic reproduction numbers for Guinea , Sierra Leone , and Liberia have been estimated at 1 . 51 , 2 . 53 , and 1 . 59 , respectively , and indicates the need for any future EVD to be met with rapid national and international response ( Althaus , 2014 ) .",
"Our estimates largely coincide with those recently reviewed in the literature ( Van Kerkhove et al . , 2015 ) .",
"The basic reproduction numbers reported for the Kikwit outbreak ( 3 . 00 ) is comprised in the range found by other studies ( 1 . 36–3 . 65 ) ( Chowell et al . , 2004; Ferrari et al . , 2005; Lekone and Finkenstädt , 2006; Legrand et al . , 2007; Forsberg White and Pagano , 2008; Ndanguza et al . , 2011 ) , and our estimate for the Yambuku outbreak ( 5 . 00 ) is similar to that reported by Camacho et al . ( 4 . 71 , range = 3 . 92–5 . 66 ) ( Camacho et al . , 2014 ) .",
"The mean delay of onset of symptoms to hospitalisation and to death estimated here for Kikwit ( 5 . 0 and 9 . 5 , respectively ) was also similar to that found by other authors ( 4–5 [Khan et al . , 1999; Rowe et al . , 1999] and 9 . 6–10 . 1 [Bwaka et al . , 1999; Khan et al . , 1999] ) .",
"Our estimated mean delay of onset of symptoms to death during the Boende outbreak ( 9 . 4 ) was slightly lower than that found by other authors ( 11 . 3 ) but included in their reported range ( 1–30 ) ( Maganga et al . , 2014 ) .",
"The delay between hospitalisation and death during the Kikwit outbreak found in the literature ( 4 . 6 ) coincided with our estimate ( 4 . 5 ) ( Khan et al . , 1999 ) .",
"In addition , our estimates of the overall CFR for Kikwit and Boende ( 78% and 74% , respectively ) coincided with other estimates reported in the literature ( 74–81% [Muyembe and Kipasa , 1995; Khan et al . , 1999; Ndambi et al . , 1999; Sadek et al . , 1999; Chowell et al . , 2004] and 74% [Maganga et al . , 2014] , respectively ) .",
"The remaining outbreak estimates have not been studied by other authors and are reported here for the first time .",
"Overall , CFRs and delays between symptom onset and hospitalisation , symptom onset and death , and hospitalisation and death reported in our study do not differ substantially with those reported for the current outbreak ( WHO Ebola Response Team et al . , 2015b ) .",
"The data presented were originally collected for the containment of the outbreaks rather than for providing the basis of an epidemiological study of the disease .",
"As such , variables are not recorded consistently across all outbreaks and there are missing data .",
"This dataset does not take into consideration undetected cases .",
"A surveillance study carried out in northwestern DRC between 1981 and 1985 , through clinical records and serological testing , detected 21 cases likely to be EVD , suggesting that sporadic cases do occur ( Jezek et al . , 1999 ) .",
"Another serosurvey carried out in Yambuku after the outbreak suggested that that 17% of the population in the village was infected asymptomatically ( Breman et al . , 1978 ) .",
"Under-reporting may differ between and during outbreaks and may impact the calculated estimates such as CFRs , which limits the validity of direct comparisons of values between outbreaks .",
"Other limitations include the different case definitions employed in different outbreaks and that the method used to calculate the effective reproduction numbers is susceptible to changes in reporting during the outbreak ( as most methods are ) .",
"However , it is robust if the extent of underreporting remains constant during each outbreak .",
"Moreover , it is robust to different reporting sensitivity between outbreaks .",
"The regular re-emergence of EVD in human hosts is likely to be connected to the presence of the virus in animal reservoirs , such as bats and monkeys ( Leroy et al . , 2009; Muyembe-Tamfum et al . , 2012 ) .",
"The presence of vast tropical rainforests covering entire regions of the DRC and the strong link existing between local economies and the forest makes a re-emergence of the virus in the country in the near future very likely ( Pigott et al . , 2014 ) .",
"Although the Mweka 2007 outbreak has been linked to the consumption of fruit bats that migrate to the area ( Leroy et al . , 2000 ) , the epidemiological link between index cases ( when known ) and animal reservoirs has not been found for any of these outbreaks .",
"All outbreaks except for the 2007 Mweka outbreak have involved hospital transmission during the early part of the outbreak , illustrating the amplifying effect that poor infection control can have on EVD epidemics .",
"A study of the 1976 outbreak has highlighted the importance of community infection to transmission ( Camacho et al . , 2014 ) .",
"Traditional burials are an important mechanism of transmission of EVD .",
"Funeral data can help inform mathematical models that explore the importance of this route of transmission and can help guide resource allocation .",
"This will be explored in subsequent analysis .",
"Mweka 2008 was the shortest and smallest outbreak with the lowest CFR .",
"This could be due to the short delay between the first notification and the opening of the isolation centre ( 10 days ) .",
"The low CFR during Isiro could be due to infection by a less virulent type of virus ( B . ebolavirus ) and is in line with what has been reported for this virus in other outbreaks ( Van Kerkhove et al . , 2015 ) .",
"In most outbreaks , major interventions arrived when the reproduction number , R , was less than one and the epidemic was already under control .",
"This suggests an important role of other factors , such as changes in contact behaviour , in shaping the changes of R . For example , there is evidence that an increase in the proportion of patients admitted to hospital was associated with a reduction in the size of EVD transmission chains in Guinea in 2014 ( Faye et al . , 2015 ) and the community acceptance of EVD control measures in West Africa improved dramatically over the course of the epidemic , which led to better infection control ( Dhillon and Kelly , 2015 ) .",
"The Boende outbreak began whilst the West African outbreak was gaining international importance .",
"This much smaller outbreak , with an initial R of five , which consisted of 68 cases , lasted only 10 weeks .",
"The more remote setting , a background antibody presence in the area and a greater preparedness to EVD ( that led to its notification 3 weeks after the first case and the opening of the first isolation centre a month later ) could have contributed to the avoidance of a larger outbreak ( Heymann et al . , 1980; Busico et al . , 1999; Maganga et al . , 2014 ) .",
"The high number of EVD cases between 25 and 64 years of age compared to the background demographics , the high CFR in children under five , the decrease in CFRs in those 5 to 15 , and the subsequent increase in CFR during adulthood are phenomena that warrant further investigation .",
"The variation in symptoms reported during different outbreaks is also a matter for further research ."
],
[
"Line list data and reports for each outbreak were retrieved from the Direction de Lutte contre la Maladie ( DLM ) ( Ministère de la Santé Publique ( Direction de la Lutte contre la Maladie ) , 2007 , 2009 , 2012; Ministère de la Santé Publique ( Comité National de Coordination ) , 2014 ) .",
"The DLM is the public body in charge of containing EVD outbreaks in the DRC .",
"These data were designed for outbreak containment rather than for epidemiological analysis; therefore , appropriate cleaning was undertaken .",
"The fields selected were age , sex , date of symptom onset , date of hospitalisation , date of hospital discharge , outcome , case definition , date of notification ( when the case was first reported to the DLM ) , date of death , occupation , fever , diarrhoea , abdominal pain , headache , vomiting , hiccups , and hemorrhagic symptoms .",
"Where this information was not available , it was left blank .",
"A unique ID was assigned to each patient in the dataset .",
"The Tandala outbreak ( 1977 ) included only one reported case; therefore , only the context and history of this outbreak was analysed .",
"We included 262 of the 318 cases reported in Yambuku ( those for which these data were available ) .",
"The aggregated line lists can be found in Supplementary file 1 .",
"According to the WHO EVD case definitions for outbreak settings; suspected cases are all individuals ( alive or dead ) who had a fever and had contact with a suspected , probable , or confirmed EVD case or a sick or dead animal; any individual with a fever and more than three additional EVD symptoms; or any person with unexplained bleeding or whose death is unexplained ( World Health Organization , 2014 ) .",
"Probable cases are suspected cases that have a clear epidemiological link with a confirmed case .",
"Confirmed cases are individuals who were tested positive via PCR .",
"In the DRC setting , the case definitions employed varied somewhat between outbreaks ( Appendix 1 , Section A ) .",
"Unless stated otherwise , where the case definitions distinguished susceptible cases from probable and confirmed cases , all estimates presented ( CFRs , symptom delays , and reproduction numbers ) were computed omitting suspected cases .",
"DRC national demographics between 1975 and 2010 were used as reported by the UN Department of Economic and Social Affairs ( United Nations ( Department of Economic and Social Affairs ) , 2013 ) .",
"For temporal comparison of patient reports , we used the date of infection .",
"When available , we used the date of symptom onset .",
"When these were unavailable , hospitalisation dates were used instead .",
"If these were also absent , the notification dates were used as proxy .",
"When calculating the proportion of confirmed and probable cases that presented with EVD symptoms , we assumed that patients for whom the presence or absence of at least one symptom was reported did not display any additional symptoms unless those were also reported .",
"The odds of dying from EVD were estimated through binomial regression with age group and year of outbreak as factorial covariates and the number of months since the start of the outbreak and the delay from symptom onset to hospitalisation as continuous covariates .",
"The age groups used were 0–5 years , 5–15 years , and >15 years .",
"The delay from symptom onset to hospitalisation was present for 63% of probable or confirmed cases .",
"These dates were not recorded for Yambuku and Mweka 2007 and only for four cases for Mweka 2008 .",
"For this reason , these outbreaks were excluded from this first analysis .",
"A second regression model was conducted that excluded the delays from symptom onset to hospitalisation as an explanatory variable , enabling the use of data from all major outbreaks and increasing statistical power .",
"The start of an outbreak was defined by the earliest onset of symptoms of any detected case .",
"The CIs were calculated using profiled log-likelihood .",
"We calculated the weekly effective R , the average number of individuals that were infected by a typical EVD case during the period of infectiousness , by reconstructing the transmission tree of each outbreak on the basis of date of infection for each case ( Wallinga and Teunis , 2004 ) .",
"To link a case to its most likely source , we assumed a serial interval of 15 . 3 days with a standard deviation of 9 . 3 days as reported during the current outbreak in West Africa ( Maganga et al . , 2014 ) .",
"Delays in care were only calculated for those outbreaks for which the necessary dates were recorded .",
"R-3 . 1 . 2 was used for the cleaning , analysis , and plotting of figures ( R Development Core Team , 2011 ) .",
"This study was approved by the LSHTM Research Ethics Committee ( approval number PR/1541/1541 ) .",
"The funders had no role in the design , collection , analysis , and interpretation of data , or in the writing of the manuscript .",
"The corresponding authors had full access to all the data and were responsible for the final decision to submit for publication ."
]
] | [
"The Democratic Republic of the Congo has experienced the most outbreaks of Ebola virus disease since the virus' discovery in 1976 .",
"This article provides for the first time a description and a line list for all outbreaks in this country , comprising 996 cases .",
"Compared to patients over 15 years old , the odds of dying were significantly lower in patients aged 5 to 15 and higher in children under five ( with 100% mortality in those under 2 years old ) .",
"The odds of dying increased by 11% per day that a patient was not hospitalised .",
"Outbreaks with an initially high reproduction number , R ( >3 ) , were rapidly brought under control , whilst outbreaks with a lower initial R caused longer and generally larger outbreaks .",
"These findings can inform the choice of target age groups for interventions and highlight the importance of both reducing the delay between symptom onset and hospitalisation and rapid national and international response ."
] | [
"Ebola virus disease commonly causes symptoms such as high fever , vomiting , and diarrhoea .",
"It may also cause muscle pain , headaches , and bleeding , and often leads to death .",
"There have been seven outbreaks of Ebola virus disease in the Democratic Republic of the Congo ( DRC ) since 1976 .",
"The DRC is the country that has had the most outbreaks of this disease in the world .",
"The most recent outbreak in the DRC was in 2014; this was separate from the outbreak that started in West Africa in the same year .",
"Rosello , Mossoko et al . have now compiled the data from all seven of the outbreaks in the DRC into a single dataset , which covers almost 1000 patients .",
"Analysing this data revealed that people between 25 and 64 years of age were most likely to be infected by the Ebola virus , possibly because most healthcare workers fall into this category .",
"Age also affected how likely a patient was to die , with those aged under 5 and over 15 more likely to die than those aged between 5 and 15 .",
"Delaying going to hospital once symptoms had started , even by one day , also increased the likelihood of death .",
"Rosello , Mossoko et al . also examined the Ebola virus effective reproduction number , which indicates how many people , on average , an infected person passes the virus on to .",
"Outbreaks that initially featured viruses with a reproduction number larger than three tended to be stemmed quickly .",
"However , when the reproduction number was lower , national and international organisations were slower to respond to the signs of the outbreak , leading to outbreaks that lasted longer .",
"Further research is needed to understand why the likelihood of death is different for different age groups and to investigate the effect of the different routes of transmission of the virus on interventions such as vaccination ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"structural biology and molecular biophysics",
"computational and systems biology"
] | CryoEM and computer simulations reveal a novel kinase conformational switch in bacterial chemotaxis signaling | elife-08419-v2 | [
[
"Bacterial chemotaxis is a ubiquitous , two-component signal transduction system that allows cells to extract information from environmental chemical gradients and place themselves within the nutrient-optimal portion of their habitat ( Wadhams and Armitage , 2004; Capra and Laub , 2012; Eisenbach , 2004 ) .",
"Though the topology and complexity of the protein networks employed in bacterial chemotaxis vary by species , each uses the histidine kinase CheA ( component",
"1 ) and response regulator CheY ( component",
"2 ) to set up an intracellular phosphorylation cascade that regulates the motile behavior of the cell ( Szurmant and Ordal , 2004 ) .",
"CheA , in particular , is a multi-domain protein , consisting of five separate and functionally distinct domains ( P1-P5 ) : P1-phosphoryl transfer domain , P2-substrate binding domain , P3-dimerization domain , P4-kinase domain and P5-regulatory domain .",
"In addition to CheA and CheY , an expanded set of molecules assist in the mechanics of signal reception , transmission , and regulation .",
"Specifically , bacteria utilize dedicated chemoreceptors ( also known as methyl-accepting chemotaxis proteins , MCPs ) to recognize ambient chemicals and transmit mechanical signals across the cell membrane to affect CheA kinase activity ( Ortega et al . , 2013; Parkinson et al . , 2015 ) .",
"The adaptor protein CheW universally participates in the coupling of conformational changes within receptors to kinase regulation ( Szurmant and Ordal , 2004; Liu and Parkinson , 1989 ) .",
"Bacteria , moreover , have evolved the ability to tune or adapt their chemotactic sensitivity to stimulus intensity , giving rise to short-term molecular memory and allowing an appropriate system response over wide ranges of chemical concentrations ( Hazelbauer and Lai , 2010; Parkinson et al . , 2015 ) .",
"In the case of the model organism , Escherichia coli , the adaptation mechanism involves the use of two enzymes , CheR and CheB , which reversibly modify specific residues in the receptor molecules ( Hazelbauer et al . , 2008; Hazelbauer and Lai , 2010; Goy et al . , 1977; Ortega et al . , 2013 ) .",
"The tunable control of chemotactic activity requires the assembly of collaborative core-signaling units , involving the chemoreceptor trimer of dimers ( TOD ) ( Amin and Hazelbauer , 2010; Li and Hazelbauer , 2011 ) , CheA dimer and CheW monomer ( Li and Hazelbauer , 2011; Falke and Piasta , 2014 ) .",
"Through the formation of large , highly organized clusters known as chemosensory arrays , thousands of core-signaling units establish a network of cooperative interactions that dramatically affect signal transmission and regulation and endow the basic two-component chemotaxis infrastructure with heightened information processing and control capabilities ( Hazelbauer and Lai , 2010; Falke and Piasta , 2014; Sourjik and Armitage , 2010; Bray et al . , 1998; Tu , 2013 ) .",
"Important progress has been made in the characterization of localized portions of array structure using a battery of genetic , biochemical , and biophysical techniques .",
"This progress includes the derivation of atomic structures of the individual core signaling components ( Kim and Yokota , 1999; Bilwes et al . , 1999; Park et al . , 2006; Li et al . , 2007; Griswold et al . , 2002 ) and several of their sub-complexes ( Park et al . , 2006; Li and Bayas , 2013; Briegel et al . , 2012 ) as well as the elucidation of key interactions between the core signaling components in soluble multi-protein complexes ( Bhatnagar et al . , 2010; Vu et al . , 2012; Wang et al . , 2012 ) and in reconstituted , attractant-regulated core complexes ( Li and Hazelbauer , 2011; Piasta et al . , 2013; Natale et al . , 2013; Falke and Piasta , 2014; Li and Hazelbauer , 2014 ) .",
"Recently , a global view of the extended structural organization of chemosensory arrays has emerged from cryo-electron tomography ( cryoET ) studies of native bacterial cells ( Briegel et al . , 2009; 2012; Liu et al . , 2012; Zhang et al . , 2007 ) .",
"Specifically , chemoreceptor TODs were observed to form hexagonal arrays with a 12 nm lattice spacing conserved across several , distantly related bacterial species including E . coli and T . maritima ( Briegel et al . , 2009; 2012; 2014a; Liu et al . , 2012; Zhang et al . , 2007 ) .",
"The conservation of this hexagonal organization has also been demonstrated in non-membrane spanning cytoplasmic chemosensory arrays ( Briegel et al . , 2014a; 2014b ) .",
"Additionally , studies using cryoET with sub-tomogram averaging , in tandem with crystallographic structures of portions of the core complex , have reported the extended structure of the array to consist of receptor TODs packed in a two-facing-two fashion about kinase-filled and kinase-empty rings ( Briegel et al . , 2012; 2014b; Liu et al . , 2012 ) .",
"However , due to the thickness of the cells as well as cellular crowding and heterogeneity , past cellular tomography studies have been limited to discerning only the overall arrangement of the core signaling components .",
"The lack of a high-resolution description of the intact and extended chemosensory array structure has hindered the development of a detailed understanding of molecular events occurring within the array during signaling .",
"To address this problem , we have taken a joint experimental-computational approach .",
"In particular , we have developed a novel reconstitution method yielding ultra-thin monolayer samples of core-signaling complex arrays , from which we derived a three-dimensional density map of the reconstituted core-signaling complex at 11 . 3 Å resolution using cryoET and sub-tomogram classification and averaging .",
"Through the computational synthesis of existing X-ray crystallography data and our new cryoET data , we have constructed an atomic model of the extended chemosensory array .",
"Our model highlights novel interaction interfaces between the receptor , CheA , and CheW and permits the use of large-scale , all-atom molecular dynamics ( MD ) simulations ( Perilla et al . , 2015 ) to further illuminate the molecular details of a key kinase-signaling event ."
],
[
"To overcome the limitations imposed by cellular tomography of native chemosensory arrays ( Briegel et al . , 2009; 2012; Liu et al . , 2012; Zhang et al . , 2007 ) , we elected to establish an in vitro reconstituted system for high-resolution structural analysis of the signaling complex .",
"Inspired by the template-directed method to assemble functional signaling complexes on lipid vesicles ( Montefusco et al . , 2007; Shrout et al . , 2003 ) , we designed a Ni2+-NTA lipid containing monolayer system ( Taylor et al . , 2007; Taylor and Taylor , 1999 ) to reconstitute the two-dimensional ( 2D ) arrays of signaling complexes for structural analysis .",
"To this end , we expressed and purified to high homogeneity E . coli chemotaxis proteins: CheA , CheW , and a His-tagged cytoplasmic signaling domain of the wild-type ( wt ) Tar receptor ( TarCF ) .",
"His-tagged TarCF can be readily incorporated into the Ni2+-NTA lipid monolayers , seen as homogeneous particles in the EM micrographs of negatively stained specimen ( Figure 1A ) .",
"Only in the presence of all three components ( TarCF , CheA , and CheW ) were ordered arrays evident ( Figure 1B ) , and even then , these microcrystalline 2D arrays were only formed under strictly constrained input ratios of the three components , a finding that is consistent with previous results indicating that the chemotactic function of the complex is diminished when one of the components is reduced or over-produced ( Zhang et al . , 2007 ) .",
"The optimal condition for array formation was established to be a mixture of TarCF , CheA and CheW with a molar ratio of 9:18:18 μM for TarCF:CheA:CheW in a lipid monolayer containing 2:1 DOPC:DOGS-NTA-Ni2+ lipids ( 33% Ni2+-NTA lipid ) .",
"Notably , the input molar ratio of the reconstitution mixture does not reflect the actual ratio of components incorporated into the monolayer , as illustrated in Figure 1C .",
"The resulting arrays are organized in hexagonal lattices with 12 nm spacing ( Figure 1B inset , white arrow ) , resembling the arrays formed in native cells ( Briegel et al . , 2012; Liu et al . , 2012 ) . 10 . 7554/eLife . 08419 . 003Figure 1 . Reconstitution of 2D arrays of the receptor signaling complex on lipid monolayers .",
"( A&B )",
"Negatively stained electron micrographs of reconstituted lipid monolayers with TarCF only ( A ) or with TarCF/CheA/CheW ( B ) .",
"Inset , Fourier transform of a region from the monolayer array , indicating a hexagonal lattice with a 12 nm repeat ( white arrow ) .",
"Scale bars , 100 nm .",
"( C ) SDS-PAGE gel analysis of the reconstituted monolayer sample ( lane",
"2 ) and the protein solution ( input mixture ) used to generate the monolayer arrays ( lane 3 ) .",
"Molecular weight markers are indicated ( lane 1 ) , and the input mixture contained TarCF:CheA:CheW in a ratio of 9:18:18 μM . DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 003 Compared to previous cellular tomography studies ( Briegel et al . , 2012; 2014b; Liu et al . , 2012; Zhang et al . , 2007 ) , the reconstituted monolayer system is ideal for high resolution structural analysis of chemosensory arrays by cryoET for several reasons:",
"1 ) the in vitro reconstituted monolayer array is thin ( 25 nm ) and pseudo-crystalline , compared to cells with thicknesses ranging from 500 nm to 1 µm;",
"2 ) the monolayer arrays are reconstituted with purified components , hence the system is well-defined , in contrast to native arrays in the crowded cellular environment;",
"3 ) the reconstituted system allows for control over which array components are present as well as manipulation of their signaling state;",
"4 ) the in vitro system provides large numbers of sub-tomogram volumes ( ~3000 core-signaling units/tomogram ) , thereby improving the noise statistics of the sub-tomogram averaging process central to achieving a high resolution structure .",
"Using cryoET , we collected and reconstructed , correcting for the contrast transfer function ( CTF ) of the microscope ( Fernández et al . , 2006 ) , 20 tomograms of monolayers containing reconstituted core-signaling complex arrays .",
"Figure 2A ( Video",
"1 ) shows a typical raw tomographic slice ( without CTF correction ) of a reconstituted monolayer , illustrating patches of 2D lattices with information extending beyond 22 Å ( inset , arrow ) .",
"By extracting and classifying CTF-corrected sub-tomograms , centered on each hexagon of receptor TODs ( Figure 2—figure supplement 1 , yellow circle ) , we obtained two major classes of the receptor hexagons: one containing a trimer of core-signaling units ( CheA2-trimer , Figure 2B and Figure 2—figure supplement 1 , cyan boxes ) and one containing a hexamer of core-signaling units ( CheA2-hexamer , Figure 2C and Figure 2—figure supplement 1 , orange box ) .",
"By mapping the individual sub-tomograms from the above two classes onto the original contributing tomograms , we were able to extract the extended lattice organization of the subunits in the monolayer ( Figure 2D ) , revealing an interlocking of the CheA2-trimer and CheA2-hexamer classes ( Figure 2E ) consistent with that seen in cellular tomograms ( Briegel et al . , 2012; 2014b; Liu et al . , 2012 ) . 10 . 7554/eLife . 08419 . 004Figure 2 . CryoET of monolayer arrays of TarCF/CheA/CheW ternary signaling complex .",
"( A ) A tomographic slice ( 1 . 2 nm thick ) through the reconstituted monolayer arrays of TarCF/CheA/CheW , without CTF correction .",
"Inset , The Fourier transform of a selected region , displaying Thon rings with information extended to at least 22 Å resolution ( arrow ) .",
"( B&C )",
"Averaged density maps of two sub-volume classes containing receptor hexagons ( 6 TODs ) ( red ) , one with a trimer of CheA dimers ( CheA2-trimer ) ( B ) and the other with a hexamer of CheA dimers ( CheA2-hexamer ) ( C ) .",
"Maps were generated following sub-tomogram volume classification and class-averaging , are contoured at 1 . 5σ , and are colored according to the height , from the receptor at the top ( red ) to CheA ( blue ) below .",
"( D ) Spatial arrangement of the CheA2-trimer ( cyan ) and CheA2-hexamer ( orange ) in the monolayer lattice array , after mapping the classified sub-volumes back onto the tomogram .",
"The array is formed by interlocking CheA2-trimer and CheA2-hexamer subunits .",
"( E ) A schematic lattice model for the chemosensory arrays .",
"Small circles represent receptor dimers; arrows represent CheA dimers ( CheA2 ) .",
"Dashed cyan and orange circles highlight a CheA2-trimer and CheA2-hexamer respectively .",
"The lattice unit cell is outlined in black .",
"Related to Figure 2—figure supplement 1 and 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 00410 . 7554/eLife . 08419 . 005Figure 2—figure supplement 1 . Classification of the sub-tomogram volumes . The sub-tomograms containing the receptor hexagon ( 6 TODs , yellow circles ) were subjected to alignment and classification .",
"Sections of the sub-tomogram classes are shown at the receptor region ( top row in each set ) and the CheA region ( bottom row in each set ) .",
"Two major configurations emerged , one with a trimer of CheA dimers ( CheA2-trimer , cyan boxes ) and the other with a hexamer of CheA dimers ( CheA2-hexamer , orange box ) .",
"All the CheA2-trimer classes were combined for final refinement of the density map . DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 00510 . 7554/eLife . 08419 . 006Figure 2—figure supplement 2 . Comparison of the chemotaxis arrays from native cells and from in vitro reconstituted monolayers .",
"( A ) A tomographic slice ( 5 . 2 nm thick ) of the native chemotaxis arrays from wild-type E . coli cells , overlaid with the positions of each sub-tomogram classified as either the CheA2-trimer unit ( cyan ) or the CheA2-hexamer unit ( orange ) .",
"The lattice organization with interlocking CheA2-trimer and CheA2-hexamer is the same as the reconstituted arrays ( see Figure 2A ) .",
"( B ) Sub-tomogram averaged density maps of the extended lattice ( CheA2-trimer surrounded by six interlocking CheA2-hexamers ) from native clusters ( light blue surface rendering , contoured at 2σ ) , overlaid with the extended lattice from reconstituted monolayer arrays ( mesh surface , contoured at 1 . 5σ ) .",
"The two lattices match very well .",
"( C ) Surface rendering of the sub-tomogram averaged extended lattice map from reconstituted monolayer arrays at 18 Å resolution , showing the interlocking CheA2-trimer ( cyan dashed circles ) and CheA2-hexamer ( orange dashed circles ) .",
"The map is contoured at 1 . 5σ and colored according to the height , from receptor ( red ) to CheA ( blue ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 00610 . 7554/eLife . 08419 . 007Video 1 . Tomographic slices of monolayer arrays . Related to Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 007 To directly compare the lattice organization in our reconstituted monolayer system with that in native E . coli cells , we obtained three CTF-corrected tomograms from wt E . coli cells that were partially lysed , using a phage-gene-induced instant lysis method that we developed recently ( Fu et al . , 2014 ) , to reduce the sample thickness .",
"Extracting and classifying sub-tomograms containing receptor hexagons from the native E . coli cells revealed the same two classes that were observed in the monolayer system .",
"As with the in vitro monolayer system , the two receptor hexagon classes observed in native E . coli cells also formed an interlocking lattice ( Figure 2—figure supplement 2A ) .",
"Extracting sub-tomograms with an extended unit that contained both the CheA2-trimer and CheA2-hexamer , we obtained an average density map for the in situ native chemosensory arrays that overlapped very well with the map from the monolayer system ( Figure 2—figure supplement 2B ) .",
"Therefore , the in vitro reconstituted monolayer system with purified E . coli proteins faithfully reproduces the lattice organization found in native E . coli cell membranes .",
"The 3D classification process further improved the resolution of the class-averaged sub-tomograms of CheA2-trimers ( Figure 2B ) to 11 . 3 Å and CheA2-hexamers ( Figure 2C ) to 17 . 5 Å resolution , as measured by gold-standard Fourier shell correlation ( FSC ) ( Figure 3—figure supplement 1A ) .",
"A uniform distribution of in-plane orientations of the sub-tomograms and a relatively well sampled , out-of-plane angle enhanced the quality of the averaged density maps ( Figure 3—figure supplement 1B&C ) .",
"Nevertheless , some resolution anisotropy exists , with 11 Å in X and Y directions and 15 . 8 Å in Z direction ( Figure 3—figure supplement 1A ) .",
"To take the effect of the anisotropic resolution into account , we low-pass filtered the density map according to the FSC of the Fourier conical shells along various directions ( Diebolder et al . , 2015 ) .",
"The resulting maps of the CheA2-trimer and CheA2-hexamer clearly delineate the density regions corresponding to the receptor , the CheA-P5/CheW ring at the receptor tip , and CheA kinase domain ( Figure 3B&C , Figure 3—figure supplement 2 and further display a number of new features .",
"In particular , the individual receptor dimers are thoroughly resolved , allowing the kinase and CheW/receptor interactions to be isolated to a specific receptor dimer ( Figure 3A–C ) .",
"Moreover , the position of the previously unobserved four-helix bundle of the CheA-P3 dimerization domain is clearly discerned to run parallel to the receptor and is positioned close to CheW-interacting receptor dimers ( Figure 3A–C ) .",
"In addition , our maps dramatically refine the area of density projecting below the CheA-P5 domain , suggesting that the CheA-P4 kinase domain alone occupies this density region ( Figure 2B , 3A , and Video 2 ) .",
"The CheA-P1 and CheA-P2 domains , on the other hand , are not resolved , likely due to their conformational flexibility . 10 . 7554/eLife . 08419 . 008Figure 3 . CheA2-trimer and CheA2-hexamer density maps with molecular dynamics flexible fitting ( MDFF ) of computationally constructed T . maritima subunit models .",
"( A ) Overall fitting of the CheA2-trimer density map contoured at 1 . 5σ .",
"The three core signaling complexes are colored in pink , blue and green .",
"( B ) A sectional view of the boxed region in A , rotated 90° .",
"The protein components are labeled at the indicated height of the complex ( gray boxes ) .",
"( C ) Sectional views of the gray-boxed regions in B at the receptor level ( top ) , the CheA-P3 and P5/CheW ring region ( middle ) , and CheA-P4 region ( bottom ) .",
"( D ) Overall fitting of the CheA2-hexamer density map contoured at 1 . 5σ .",
"( E ) A sectional view at the CheA-P3 and CheW-ring region of the CheA2-hexamer density map .",
"In ( A-E ) , CheA-P3 , P4 , P5 , CheW and receptor are labeled as P3 , P4 , P5 , W and R , respectively , and the CheA-P5/CheW interfaces 1 and 2 are indicated .",
"Related to Figure 3—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 00810 . 7554/eLife . 08419 . 009Figure 3—figure supplement 1 . Resolutions of the density maps .",
"( A ) Gold-standard Fourier shell correlation ( FSC ) of the CheA2-trimer ( left ) and CheA2-hexamer ( right ) density maps .",
"At FSC=0 . 143 , the overall resolution of the CheA2-trimer map is 11 . 3 Å and that of the CheA2-hexamer is 17 . 5 Å .",
"The FSC curves for the conical Fourier shells along the X , Y , and Z directions are in solid green , blue and dark-red , respectively , and along the 10 other directions are in dotted lines .",
"The CheA2-trimer map was calculated from two independent data sets of 3000 sub-volumes each , and the CheA2-hexamer was from two independent data sets of 300 sub-volumes each .",
"( B ) Angular distributions of contributing sub-tomograms for the in-plane rotation angle ( left ) and the maximum tilt angle relative to electron beam direction ( right ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 00910 . 7554/eLife . 08419 . 010Figure 3—figure supplement 2 . X-Z sectional views of the CheA2-trimer density map with MDFF model . The positions of the sections are indicated in the last panel with an orthogonal view ( X-Y plane ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 01010 . 7554/eLife . 08419 . 011Figure 3—figure supplement 3 . A metric for the goodness of fit for the docking of the CheA-P4 domain .",
"( A ) Distribution of 23 classes of fits for the P4 domain starting from random orientations .",
"( B ) The models from the top 9 highest cross-correlation classes are shown in panels1-9 , with the cross-correlation values and number of contributing fits ( % ) indicated below .",
"These 9 classes constitute 90 . 1% of total fits .",
"Panel 10 is an overlay of the #1 fit ( blue ) with the MDFF model ( orange ) .",
"The red and blue spheres indicate the N and C termini of P4 , respectively .",
"The corresponding connecting termini from P3 and P5 are in light blue and pink , respectively .",
"P3 and P5 domains are in gray . DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 01110 . 7554/eLife . 08419 . 012Video 2 . MDFF model fitting of the CheA2-trimer density map . Related to Figure 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 012 Regarding the CheA-P5/CheW ring , our density map clearly shows a pseudo three-fold symmetry ( Figure 2B ) in which the density at the CheA-P5/CheW interface between core-signaling units ( interface",
"2 ) is considerably weaker than the density at the CheA-P5/CheW interface within core-signaling units ( interface",
"1 ) ( Figure 3A&C ) ( Briegel et al . , 2012 ) .",
"This finding is in contrast to the previously described pseudo six-fold symmetry of the CheA-P5/CheW ring ( Li and Bayas , 2013 ) .",
"Most importantly , the previously described ‘empty hexagon’ that is surrounded by six CheA-occupied hexagons ( Briegel et al . , 2012 ) is not empty , but rather contains a well-ordered continuous ring of densities ( Figure 2C ) that we were able to unambiguously assign to individual CheW monomers ( Figure 3D&E ) .",
"This ring of CheW , as previously speculated ( Liu et al . , 2012 ) , provides additional interactions that couple neighboring receptor TODs and strengthens the interlocking baseplate .",
"Hence , our maps confirm the existence of the CheW ring and establish its participation in the structural foundation responsible for the ultra-stability of the chemosensory array ( Liu et al . , 2012; Erbse and Falke , 2009 ) and for the high cooperativity and extraordinary sensitivity measured in chemotaxis responses ( Goldman et al . , 2009 ) .",
"The resolution of our cryoET data permitted the unambiguous assignment of distinct regions of density to specific protein components , enabling the construction of all-atom models of the chemosensory array substructures and extended lattice ( Figure 4—figure supplement 1 ) .",
"A schematic overview of the modeling procedures carried out in this study is provided in Figure 4—figure supplement 2 with a more detailed discussion of these procedures located in the Methods section .",
"Briefly , we first constructed models of the receptor TOD , CheA-P3P4 dimer , CheA-P5/CheW ring , and CheW-only ring , taking advantage of existing high-resolution X-ray structures from the thermophilic bacterium Thermotoga maritima ( Kim and Yokota , 1999; Bilwes et al . , 1999; Park et al . , 2006; Li and Bayas , 2013 ) .",
"We then heuristically-arranged , using a 12 nm lattice constant , the resulting component models to produce models of the CheA2-trimer and CheA2-hexamer subunits identified by sub-tomogram classification ( Figure 4—figure supplement 1D&E ) .",
"To further refine the key protein-protein interfaces within our atomic models , we adopted a dual MD-based strategy , utilizing both unbiased MD and electron-density-biased molecular dynamics flexible fitting ( MDFF ) simulations ( Trabuco et al . , 2008 ) .",
"For the subject of our unbiased refinement simulations , we extracted from the CheA2-hexamer model a portion corresponding to the array unit cell , including six receptor TODs , three CheA dimers , and 12 CheW monomers all together arranged as three coupled core-signaling units ( Figure 4—figure supplement 1C , herein the 'unit-cell model' ) .",
"For our density-biased refinement simulations , we focused our efforts on the CheA2-trimer model , owing to the higher-resolution of its associated density map , and hence , better resolved MDFF biasing forces .",
"Because the CheA-P4 density is not as well defined as the other parts of the complex , likely due to its conformational flexibility , we carried out a rigid-body docking of the CheA-P4 domain , starting from 10 , 000 random angular orientations and up to 20 Å shifts from the center of the mass .",
"This fitting exercise resulted in 23 classes separated by 3° and 3 Å ( Figure 3—figure supplement 3A ) , generating a metric for the goodness of fit of the P4 domain positioning .",
"In addition to the class of 'best fit' ( Figure 3—figure supplement 3B , panel 1 ) , one other class , in which P4 is flipped relative to the best fit , was seemingly structurally possible ( Figure 3—figure supplement 3B , panel 5 ) .",
"However , compared to the best-fit class , this alternative class had a lower cross-correlation value , lower occupancy with only a third the number of contributing fits , and the positions of P4-N and C termini are reversed ( flipped ) , making it hard to connect the P3 and P5 termini with short linkers .",
"Thus , we have focused our efforts and resources on the highest ranking class of P4 position .",
"It should be noted , though , that use of the alternative P4 positioning might produce considerably different MD trajectories .",
"Solvation and ionization of the unit cell and CheA2-trimer models produced systems of size 1 . 25 million and 1 . 75 million atoms , respectively , which were subsequently energy minimized and equilibrated for 10 ns , as described in the Methods section .",
"The unit-cell model was then subjected to an 80 ns unconstrained production simulation ( Video 3 ) , while a 70 ns symmetry-constrained MDFF simulation was used to computationally bias the tertiary structure of the protein components within the CheA2-trimer model according to our 11 . 3 Å CheA2-trimer density map ( Figure 3 , Video 2 ) .",
"The resulting unit-cell and CheA2-trimer models agreed well with previous structural studies , in particular with respect to the residues participating in the CheA-P5/receptor and CheW/receptor interaction interfaces , as defined by NMR ( Vu et al . , 2012; Wang et al . , 2012; Ortega et al . , 2013 ) , crystallography ( Li and Bayas , 2013 ) , and disulfide mapping studies ( Piasta et al . , 2013; Natale et al . , 2013 ) .",
"For succinctness , specific residues participating in the various protein-protein interfaces within the array have been listed in Supplementary file 1 .",
"In addition , the equilibrated model of the T . maritima TOD maintained the conserved trimer-forming contacts observed in the X-ray structure of the E . coli serine receptor ( Tsr ) ( Kim and Yokota , 1999 ) and revealed two additional trimer-stabilizing salt bridges , namely E387/R389 ( conserved as E402/R404 in E . coli Tsr ) and E351/R403 ( structurally homologous to D363/R415 in E . coli Tsr ) ( Figure 4—figure supplement 3A ) .",
"Moreover , in both models , the CheA-P4 kinase domain was seen to stably occupy the region of density directly below the plane defined by the CheW and CheA-P5/CheW rings .",
"Finally , in tandem with the direct visualization of the CheA-P3 dimerization domain in our cryoET density maps , the all-atom model further revealed previously uncharacterized specific interactions between the P3 bundle and adjacent receptors , involving D333/K390 and D345/R379 contact pairs ( I304/N405 and D316/R394 in E . coli respectively ) ( Figure 4B , Supplementary file 1 ) . 10 . 7554/eLife . 08419 . 013Figure 4 . CheA dimer conformational switch .",
"( A ) Top and side views of the core-signaling unit , consisting of two receptor TODs ( red ) , a single CheA dimer ( blue ) , and four CheW monomers ( green ) .",
"( B ) Two distinct classes , undipped ( top ) and dipped ( bottom ) , of core-signaling unit structures are present in our MD simulations .",
"Classes differ especially in the orientation of CheA-P4 domain with respect to the rest of the CheA dimer and core signaling unit .",
"Specific contacts that stabilize either conformation are indicated for T . maritima and in parentheses for the corresponding residues in E . coli .",
"CheA-P5 and CheW have been removed for clarity .",
"( C ) Time series of CheA dimer conformations extracted from unit cell simulations .",
".",
"Traces track the projection of the conformations of 27 CheA dimers from the wt ( top ) and R297A mutant ( bottom ) unit cell simulations onto the first principal component of the 'dipping' motion .",
"Colored traces track CheA dimers that undergo an extended ( >10 ns ) 'dipping' motion .",
"Horizontal dashed lines visually demarcate the undipped and dipped CheA dimer classes .",
"Vertical dashed lines separate the initial 80 ns equilibration simulation from nine 450 ns production simulations .",
"Related to Figure 4—figure supplement 1 , 2 , 3 , 4 and 5 , and Supplementary file 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 01310 . 7554/eLife . 08419 . 014Figure 4—figure supplement 1 . Nomenclatures .",
"( A ) TOD: receptor trimer of dimers .",
"( B ) Core-signaling unit/complex: 2 TOD , 1 CheA dimer , 4 CheW .",
"( C ) Unit cell: 3 coupled core-signaling units .",
"( D ) CheA2-trimer: 3 core-signaling units , pseudo three-fold symmetry .",
"( E ) CheA2-hexamer: 6 core-signaling units , pseudo six-fold symmetry .",
"( F ) Extended lattice: interlocked CheA2-trimer and CheA2-hexamer . DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 01410 . 7554/eLife . 08419 . 015Figure 4—figure supplement 2 . Overview of molecular modeling and simulation strategy taken in this study .",
"( A ) High resolution X-ray structures from T . maritima were taken as inputs for the generation of models corresponding to the array’s core components , namely the receptor trimer-of-dimers , coupled CheA/CheW rings , and CheW-only ring .",
"( B ) The resulting core-component models were arranged heuristically , assuming a 12 nm lattice constant , to produce models of the CheA2-hexamer and CheA2-trimer array substructures .",
"For simplicity , only the CheA2-hexamer organization is shown .",
"( C ) A portion of the heuristically-constructed CheA2-hexamer model corresponding to the array unit cell was extracted for further study with all-atom MD simulations .",
"( D ) MDFF simulations were conducted to refine the CheA2-hexamer and CheA2-trimer models utilizing their respective density maps .",
"A core-signaling unit was taken from the MDFF-refined CheA2-trimer model and deposited in the PDB data bank under accession code 3JA6 .",
"The full MDFF-refined CheA2-trimer model was subjected to further investigation using all-atom MD simulations . DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 01510 . 7554/eLife . 08419 . 016Figure 4—figure supplement 3 . Computational modeling of the extended chemosensory array structure .",
"( A ) Molecular dynamics ( MD ) simulations show that T . maritima receptors form a stable trimer-of-dimers ( TOD ) .",
"Side view ( left ) and top view ( right ) of the highly conserved protein interaction tip , highlighting the inter-receptor salt bridge network formed by E351/R403 and E387/R389 .",
"Symmetry-related monomers within individual receptor dimers are distinguished by red and grey coloring .",
"( B ) MDFF-refined , all-atom model of the array subunits combining CheA2-trimer ( cyan circle ) and CheA2-hexamer ( orange circle ) maps .",
"( C ) All-atom model of the T . maritima lattice containing 3 x 3 unit cells .",
"A portion of the lattice corresponding to a single unit cell is outlined in black .",
"Top ( top left ) and side ( bottom left ) views of the array unit cell model arranged as three coupled core-signaling units .",
"Receptor TODs are shown in red , CheA dimers in blue , and CheW monomers in green . DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 01610 . 7554/eLife . 08419 . 017Figure 4—figure supplement 4 . Overview of key all-atom molecular dynamics simulations conducted in this study .",
"( A ) Table summarizing simulations for each molecular system with atom number reported roughly in millions ( M ) of atoms ( including protein , solvent , and ions ) and simulation duration in nanoseconds ( ns ) .",
"( B ) Schematic detailing the organization of unit cell simulations .",
"An initial equilibration simulation of 80 ns provided a base structure from which nine wild type ( purple ) and nine R297A mutant ( red ) unit cell simulations were subsequently launched in parallel .",
"Each of the 18 production simulations were 450 ns in length , totaling over 8 microseconds of sampling on the unit cell system . DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 01710 . 7554/eLife . 08419 . 018Figure 4—figure supplement 5 . Time series of CheA dimer conformations extracted from CheA2-trimer simulations . Traces track the projection of the conformations of 30 CheA dimers onto the first principal component of the 'dipping' motion .",
"Colored traces track CheA dimers that undergo an extended ( >10 ns ) 'dipping' motion .",
"Horizontal dashed line visually demarcates the undipped and dipped CheA dimer classes .",
"Vertical dashed line separates initial 70 ns MDFF simulation from ten , 120 ns production simulations . DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 01810 . 7554/eLife . 08419 . 019Video 3 . Molecular dynamics simulation of array unit cell .",
"Shown here is a 75 ns clip of a wild type unit cell trajectory , illustrating the dynamics of the 1 . 2 million atom model , including 6 receptor TODs ( red ) , 3 CheA dimers ( blue ) , and 12 CheW monomers ( green ) .",
"Periodic images , shown here with reduced opacity , enforce the boundary conditions of the extended array architecture but are not simulated explicitly .",
"Solvent and ions have been removed for clarity .",
"Related to Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 019 The construction of atomic models of the array unit cell and subunits permitted the use of equilibrium all-atom MD simulations to further investigate the molecular details of dynamic events potentially relevant to signaling .",
"An overview of the key MD simulations conducted in this study is given in Figure 4—figure supplement 4A .",
"The unit cell system contains the minimal set of components needed to represent the full receptor signaling array , which was made possible through the use of periodic boundary conditions to mimic the bulk symmetry of the chemosensory array , preventing the need to interpret potentially problematic effects due to unconstrained boundaries ( Figure 4—figure supplement 2C , Video 3 ) .",
"We conducted a series of nine simulations of 450 ns each , using the equilibrated unit-cell model; additionally , we ran ten , 120 ns simulations of the equilibrated CheA2-trimer system for comparison with the unit-cell simulations .",
"Intriguingly , our simulations of both models revealed an ensemble of distinct core-signaling unit conformations ( Figure 4 B&C ) , including structures in which the associated CheA dimer displayed either an undipped conformation ( Figure 4B , top ) or dipped conformation ( Figure 4B , bottom ) .",
"In the latter case , the P4 domain of one CheA monomer adopted a 'dipped' state through rotations about the P3-P4 and P4-P5 flexible linkers , significantly affecting its contacts with neighboring receptor dimers and the P5 domain ( Video 4 ) .",
"As many biochemical , biophysical , and mutational studies have implicated dynamic structural changes within these regions of the core-signaling unit during the propagation of signals ( Piasta et al . , 2013; Natale et al . , 2013; Wang et al . , 2014; Briegel et al . , 2013 ) , we systematically identified the distinct structural classes of core-signaling unit conformations present in our MD simulations and isolated them for comparative analysis .",
"Specifically , we used the UPGMC hierarchical clustering method ( Müllner , 2013 ) to assign the conformations of the 27 core-signaling units sampled in our unit cell simulations ( 3 units/unit cell ) to groups of similar structure based on their pairwise root-mean-square deviation ( RMSD ) .",
"Cross-examination of structures within the resulting core-signaling unit clusters revealed the formation of two new salt bridges stabilizing the 'dipped' state , namely R297/E397 ( R265/E368 in E . coli ) between the P3 and P4 domains and E390/R379 ( E361/R394 in E . coli ) between the P4 domain and nearby receptor tip ( Figure 4B , bottom ) .",
"Moreover , to accommodate the reorientation of the P4 domain , the P3 dimerization bundle was observed to break the receptor contacts ( D333/K390 and D345/R379 ) observed in the ‘undipped’ state ( Figure 4B , Video 4 ) , suggesting that the mobility of the P3 bundle plays a key role in the conformational dynamics of the CheA dimer . 10 . 7554/eLife . 08419 . 020Video 4 . Molecular dynamics simulations reveal conformational switch in CheA P4 domain .",
"Shown here is one of four 'dipping' events observed in the wild type unit cell simulations , leading to modified contacts between the CheA dimer and receptor TODs .",
"Strong contacts between P3 and neighboring receptor dimers ( D333/K390 shown here with licorice representation ) are disrupted in favor of new contacts between P3/P4 and P4/receptor stabilizing the dipped state ( R297/E397 and E390/R379 respectively , shown here with licorice representation ) .",
"Related to Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 020 We next sought to examine the temporal evolution of the dipping motion in each of the CheA dimers present in our simulations .",
"For this purpose , we used Principal Component Analysis ( PCA ) to systematically derive , from the trajectory of a single dipping CheA dimer , a pseudo reaction coordinate by which to easily monitor the progression towards the 'dipped' conformation .",
"A total of four 'dipping' events were observed in our unit cell simulations , as illustrated by projection of the conformations of the 27 CheA dimer time series onto the first principal component ( Figure 4C , top ) .",
"Importantly , an additional two dipping events were observed in the 30 CheA dimers of the relatively shorter simulations of CheA2-trimer model ( Figure 4—figure supplement 5 ) , demonstrating that the ability of the conformational change to occur is not an artifact of the particular choice of CheA P4 positioning during modeling .",
"Interestingly , the three extended 'dipping' events observed in the unit-cell simulations ( Figure 4C; red , blue , and green traces ) as well as the two events observed in the CheA2-trimer simulations were accompanied by the formation of the R297/E397 contact .",
"Notably , this contact was not formed in the one short dipping event , which returned to the 'undipped' bulk state ( Figure 4C; gold trace ) , suggesting that the R297/E397 contact may play a role in stabilizing the 'dipped' state .",
"To further investigate the significance of the R297/E397 contact for the conformational dynamics of CheA , we launched nine additional unit cell simulations with an R297A mutation to prevent the potential formation of the R297/E397 salt bridge .",
"Indeed , while two CheA dimers exhibited the dipping motion in these simulations , including one dimer that underwent two dips , the mutants quickly return to the bulk ( Figure 4C , bottom ) .",
"To determine if the CheA-P4 dipping motion observed in the MD simulations of the T . maritima chemosensory array is sampled in the native chemotactic response of E . coli , we carried out cysteine disulfide cross-linking experiments .",
"In particular , we tested the interaction interface for contacts existing in the undipped state ( I304/N405 and D316/R394 ) or only in the dipped state ( E361/R394 ) ( Figure 5B ) .",
"Notably , in the simulations , R394 of Tsr switches its contact with D316 of CheA-P3 to E361 of CheA-P4 during the transition of the CheA dimer from 'undipped' to 'dipped' ( Video 4 ) . 10 . 7554/eLife . 08419 . 021Figure 5 . Biochemical validation of alternative CheA conformations in E . coli .",
"( A ) Swimming ability of E . coli cells with mutations in the CheA-P3 and Tsr interface ( I304/N405 and D316/R394 ) and in the 'dipped' CheA-P4 and Tsr interface ( E361/R394 ) .",
"Swimming activities are normalized to the cysless CheA and wt Tsr , ± standard deviation ( n=6 ) .",
"Inset , representative images of soft agar plates for swimming ability , with specific constructs labeled in red .",
"( B ) Disulphide cross-linking of the CheA-P3 and Tsr interface ( I304C/N405C and D316C/R394C ) in the undipped CheA dimer conformation ( top panel of Figure 5B ) and the CheA-P4 and Tsr interface ( E361C/R394C ) occurring in the dipped CheA-P4 'dipped' conformation ( bottom panel of Figure 5B ) .",
"Non-reducing ( top ) and reducing ( bottom ) SDS-PAGE gels were analyzed by immunoblotting for Tsr and CheA .",
"Cross-linked species were indicated with blue arrows .",
"( C ) Swimming ability of E . coli cells with mutations at R265 of CheA-P3 domain , normalized to the wt , ± standard deviation ( n=8 ) .",
"Related to Figure 5—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 02110 . 7554/eLife . 08419 . 022Figure 5—figure supplement 1 . CryoEM images of plunge-frozen E . coli cells expressing WT Tsr and WT CheA ( A&B ) , R265A CheA ( C ) , R265C CheA ( D ) , R265S CheA ( E ) , and R265E CheA ( F ) .",
"The arrays are marked with white curved arrows .",
"Scale bars , 100 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 08419 . 022 Using soft-agar assays , it was seen that the chemotactic ability of the I304C/N405C double cysteine mutant is appreciably compromised compared to that of the control ( cysless CheA/wt Tsr ) , any of the single mutants ( I304C/wt Tsr , cysless CheA/N405C , cysless CheA/N405S ) , and when one half of the pair has been mutated to serine ( I304C/N405S ) ( Figure 5A ) , suggesting that dynamic interaction between CheA-P3 and the receptor is important for chemotactic function .",
"Moreover , in vivo cross-linking and western blot analysis showed a high molecular weight band present only in the double cysteine mutant , suggesting the presence of species formed by cross-linking between CheA-P3 and Tsr ( Figure 5B ) .",
"We also examined cross-linking residue pairs that involve Tsr-R394 interactions with CheA , one in the 'undipped' state ( CheA-E316C/Tsr-R394 ) and the other in the 'dipped' state ( CheA-E361C/Tsr-R394 ) .",
"When Tsr-R394 is replaced by a cysteine or serine , either as a single mutant ( cysless CheA/R394C , cysless CheA/R394S ) or in the context of a double mutant ( D316C/R394C , D316C/R394S , E361C/R394C , E361C/R394S ) , the chemotaxis function of E . coli is partially inhibited .",
"On the other hand , the chemotactic ability of CheA-E361C as a single mutant ( E361C/wt Tsr ) is also partially inhibited , while CheA-D316C ( E316C/wt Tsr ) mutation bears no effect on the function ( Figure 5A ) .",
"Furthermore , the cross-linking pattern of both Tsr-R394 mutant pairs showed two high molecular weight bands corresponding to distinct cross-linked species , one with a disulfide formed between Tsr and CheA ( Figure 5B , upper band , blue arrows ) and the other with a disulfide between two Tsr molecules with the R394C mutation ( lower band ) .",
"Interestingly , the cross-linking of CheA-P4/Tsr ( E361C/R394C ) in the predicted 'dipped' conformation is much weaker than the cross-linking of CheA-P3/Tsr ( D316C/R394C ) in the 'undipped' state , though both involve the same R394 residue of Tsr ( Video 4 ) .",
"The lower cross-linking efficiency could be due to the relatively infrequent occurrence of the CheA 'dipped' conformation , and/or because the residues are further apart in a dominant conformation , suggesting that the CheA-P4 'dipped' conformation observed in silico may have been sampled within the native chemosensory complex of E . coli .",
"Our MD simulations of the T . maritima unit cell further indicated that R297 on the CheA-P3 domain is potentially involved in the stabilization of the conformational transition of the CheA-P4 ( Figure 4C , Video 4 ) .",
"Indeed , substitution of the corresponding residue in E . coli ( R265 ) with several amino acids of different properties ( R265C/S/A/E ) were all detrimental to the chemotactic function of E . coli as measured by the soft-agar assay , without affecting the cluster formation ( Figure 5C , Figure 5—figure supplement 1 ) .",
"Since this residue is located at the N-terminus of the four-helix P3 dimerization motif , R265 could direct the P2-P3 linker away from the cis subunit and toward the trans subunit , thus anchoring CheA-P1P2 to CheA-P4’ for trans-interaction and phosphorylation ( Bilwes et al . , 1999 ) .",
"A more complete model of the core-signaling complex for E . coli may be necessary to fully interpret the drastic impact of this single CheA residue on the entire chemotactic machinery ."
],
[
"While much effort has been expended in the derivation of models to describe the transduction of ligand-binding events within the receptor proteins , including an established piston mechanism and a hypothesized alternating static–dynamic 'yin-yang' on-off switch model ( Falke and Piasta , 2014 ) , how the structure and dynamics of the CheA kinase are affected during signaling remains poorly understood .",
"In this study , we identified , using MD simulations , a dipping motion of the CheA P4 domain , which was functionally characterized using swim assay and cross-linking experiments .",
"While the role of the predicted conformational change in CheA is not immediately clarified in the preliminary biochemical experiments carried out here , our model highlights the importance of CheA dynamics for signaling and suggests that the dynamics of the P4 kinase domain , in particular , warrants special investigation .",
"More importantly , the atomic model presented here , in general , provides improved knowledge of the positioning of the P3 and P4 domains , incorporates the presence of the CheW only ring , and identifies probable novel side-chain contacts within the extended chemosensory architecture .",
"Further improving the resolution of our cryoET data to better than 8 Å using the novel lipid-monolayer system described above would allow generation of an atomic homology model of the E . coli chemosensory array , greatly facilitating the use of the wealth of existing biochemical and biophysical data and providing directly transferrable structural and dynamical predictions .",
"We hope that the findings presented here will inspire further experimental and computational studies towards the elucidation of a complete mechanistic description of signal transduction and amplification within this truly impressive biological sensory apparatus ."
],
[
"Plasmids and cell strains used in this study were gifts from Dr . Parkinson , University of Utah , except for plasmid pHTCF ( kind gift from Dr . Weis , University of Massachusetts , Amherst ) .",
"Plasmid pHTCF is an IPTG-inducible expression vector for the N-terminal His6-tagged cytoplasmic fragment of wt aspartate receptor ( TarCF ) that contains residues 257–553 .",
"Plasmids pKJ9 and PPA770 are IPTG-inducible for the expression of CheA and CheW , respectively .",
"Plasmid pRR53 is an IPTG-inducible expression vector ( ampR ) for the wt serine receptor ( Tsr ) .",
"Plasmid pGP26 is a sodium salicylate ( Na-S ) -inducible expression vector ( camR ) for cysteine-less CheA and wt CheW .",
"Plasmids pRR53 and pGP26 were used to generate mutations in Tsr and CheA , respectively .",
"E . coli strain RP3098 , which lacks all Che proteins and chemoreceptors , was transformed with plasmid pKJ9 or PPA770 for CheA or CheW expression , respectively .",
"CheA expression was induced at an OD600 of 0 . 6–0 . 8 , with 1 mM IPTG , overnight at 15°C .",
"CheA was purified using an Affi-gel Blue column ( Bio Rad , Hercules , CA ) followed by gel filtration on a Superdex 200 column .",
"Further purification with a Mono Q ion exchange column resulted in >99% homogeneity with an overall yield of 50 mg/L of cells .",
"CheW expression was induced by the addition of IPTG ( 0 . 5 mM ) , at an OD600 of 0 . 4–0 . 6 , at 37°C .",
"CheW was purified through 20%–40% ammonium sulfate precipitation , a DEAE column followed by a MonoQ ion exchange column and a Superdex 75 size exclusion column .",
"This procedure resulted in highly purified CheW with a yield of 6 mg/L of cells .",
"His6-tagged wt TarCF ( His6-TarCFQEQE ) was expressed in DH5alpha cells with plasmid pHTCF .",
"TarCF was induced by the addition of IPTG ( 0 . 5 mM ) at an OD600 of 0 . 4–0 . 6 at 37°C and purified with a Ni2+-NTA affinity column followed with a mono Q column for quick removal of imidazole , without dialyzing overnight .",
"The yield for TarCF was excellent ( 120 mg/L of cells ) .",
"A Ni2+ lipid containing monolayer system was used to reconstitute the chemotaxis core-signaling complex arrays .",
"A mixture of 9:18:18 µM of TarCF:CheA:CheW in a buffer containing 75 mM Tris-HCl , pH 7 . 4 , 100 mM KCl , 5 mM MgCl2 was applied to a Teflon well , over which we immediately lay a lipid monolayer containing 2:1 DOPC:DOGS-NTA-Ni2+ lipid mixture , at 2 mg/ml concentration .",
"The monolayer set up was left undisturbed in a humidity chamber overnight .",
"The monolayer specimen was picked up with holey carbon grids , stained with 1% uranyl acetate , and examined with an FEI T12 microscope operated at 120 KV .",
"Reconstituted monolayers using the best conditions identified by negative staining ( Figure 1B ) , were picked up with perforated R2/2 Quantifoil grids ( Quantifoil Micro Tools , Jena , Germany ) pre-coated with 10 nm fiducial gold beads on the backside of the grid and plunge-frozen using a manual gravity plunger .",
"This method prevents disruption of the monolayer by supporting single-side blotting which eliminates the contact between the blotting filter paper and the delicate monolayer .",
"The frozen-hydrated EM grids were loaded into FEI Polara cartridges and imaged under low-dose conditions using a Tecnai Polara microscope ( FEI Corp . , OR . ) operating at 200kv .",
"A series of low dose projection images were recorded with tilt angles ranging from 70° to -70° with a Gatan 4K × 4K CCD camera ( Gatan , Inc . , PA ) , at a nominal magnification of 39 , 000× , with a defocus value of 5–8 µm and an accumulated dose of ~60 e-/Å2 .",
"A total of 32 tomographic tilt series were collected using an FEI automated tomography software .",
"Of the 32 tilt series collected , 20 tilt series with negligible mechanical or physical artifacts were selected for image processing and tomographic volume reconstruction .",
"The monolayer produces an ideal EM specimen: it is thin ( 25 nm ) and also provides strong signals in power spectra , due to near-crystalline packing of the protein components ( Figure 2A inset ) , allowing accurate determination of the Contrast Transfer Function ( CTF ) using strip-based periodogram averaging in TomoCTF ( Fernández et al . , 2006 ) .",
"The tilted projection series were roughly aligned using IMOD ( Kremer et al . , 1996 ) , and the alignment parameters were further refined using fiducial-free Area Matching with Geometry Refinement as implemented in Protomo ( Winkler , 2007 ) .",
"Using the refined geometry parameters , the raw projections were centered and rotated so the tilt azimuth was coincident with the Y-axis using the IMOD 'newstack'function .",
"These rotated stacks were corrected for the CTF with phase flipping , and volume reconstructions were made using SIRT as implemented in IMOD .",
"These were calculated using a GPU , thereby removing an additional interpolation in the reconstruction step , by avoiding the use of cosine stretching of the input projections .",
"Reconstructed volumes calculated from 20 SIRT iterations , providing higher contrast , were used for the initial cycles of sub-tomogram extraction and alignment , while those from 60 SIRT iterations were used for the final cycles .",
"To extract sub-tomograms , initial positions of the receptor complexes , respective to a Cartesian grid defined by each tomogram , were approximated by using a template matching algorithm implemented in Matlab with a reference that emphasized the receptor dimers with little influence from CheA .",
"Both the template and tomograms were low-pass filtered to 4 nm and binned by 3 .",
"This resolution , as well as a coarse angular search , were chosen to eliminate any statistical correlation of high resolution information between half data sets in later image processing steps .",
"Following template matching sub-volume extraction , the data were randomly segregated into two groups , which were processed independently for all subsequent steps .",
"Sub-tomogram alignment and classification were carried out using Protomo's i3 image processing utilities ( Winkler , 2007 ) .",
"Using Multivariate Statistical Analysis and Hierarchical Ascendant Classification , eight class averages were produced from each half data set by focusing the analysis on the CheA portion of the complex .",
"Initial references for each half set were generated by choosing averages from eight classes .",
"These references were then used to align class averages chosen to each have ~50 contributing sub-volumes .",
"In the following cycle , the raw sub-tomograms were subject to multi-reference alignment , but only a small in-plane and translational adjustment was allowed .",
"This alignment by classification was repeated five times , while allowing the automatic exclusion of high variance outliers after the second cycle .",
"In addition to the CheA2-trimer and CheA2-hexamer classes ( Figure 2B , C ) , divergent organizations of CheA/receptor complex were also included as references ( Figure 2—figure supplement 1 ) .",
"After the final cycle , class averages containing either CheA2-trimer or CheA2-hexamer were manually selected and averaged together for each half data set , and the corresponding gold-standard FSC was calculated to evaluate the reliability of the data .",
"Soft cylindrical masks were used , rather than spherical masks , given the extended slab like nature of the specimen .",
"The final averages of CheA2-trimer or CheA2-hexamer from two half data sets of 3 , 000 sub-volumes or 300 sub-volumes , respectively , were combined and an empirical correction for the CTF envelope was applied for sharpening , which helped to further clarify the receptor dimers , as well as the P3 dimerization domain .",
"To access the degree of resolution anisotropy , conical Fourier shell correlations from the two independent half data sets of CheA2-trimer , along each of the principal axes , as well as the 10 axes bisecting them , were calculated ( Diebolder et al . , 2015 ) .",
"The averaged density map of CheA2-trimer was then low-pass filtered according the conical FSCs along three principle axes by using cones with a 42° half-angle , adjusted for any overlapping regions in reciprocal space .",
"A model of the cytoplasmic portion of the T . maritima receptor dimer was taken from the X-ray crystal structure of the TM1143 chemoreceptor ( PDB 2CH7 ) ( Park et al . , 2006 ) .",
"Using the E . coli receptor TOD ( PDB 1QU7 ) ( Kim and Yokota , 1999 ) as a reference , a T . maritima receptor TOD model ( Figure 4—figure supplement 1A , Figure 4—figure supplement 2A ) was obtained by arranging individual receptor dimer models from the previous step so that homologous trimer-forming contacts were preserved .",
"CheA-P34: An atomic model of the soluble T . maritima CheA dimer , including the dimerization ( P3 ) and kinase ( P4 ) domains , was based on atomic coordinates from the X-ray crystal structure PDB 1B3Q ( Bilwes et al . , 1999 ) .",
"CheA-P5/CheW and CheW rings: Atomic models for both the CheA-P5/CheW and CheW rings were based on the X-ray crystal structure of the Receptor/CheA-P5/CheW ternary complex , PDB 4JPB ( Li and Bayas , 2013 ) .",
"In the case of the CheW ring model , the P5 domains of the CheA-P5/CheW ring model were exchanged with CheW monomers , using the dual-SH3-like fold shared between by CheA-P5 and CheW , to obtain an appropriate placement and orientation with respect to the neighboring monomers .",
"Figure 4—figure supplement 3 schematically summarizes the modeling procedures described above .",
"All missing loops were added using MODELLER ( Sali , 1993 ) .",
"The TOD , CheA-P5/CheW , and CheW ring core component models were subjected to 150 ns of equilibration to ensure their structural integrity .",
"The CheA2-trimer and CheA2-hexamer subunits models ( Figure 4—figure supplement 1D&E ) were constructed heuristically; using as a visual reference the extended organization of kinase-filled and kinase-empty rings evident in our density maps to arrange the components , also assuming an approximate 12 nm lattice constant ( Figure 4—figure supplement 3B ) .",
"Next , we made use of the CheA-P5/receptor interface from the ternary complex structure PDB 4JPB ( Li and Bayas , 2013 ) to model the CheW/receptor interface , assuming a receptor-binding mode homologous to that of CheA-P5 .",
"Using the CheA-P5 and CheW monomer/receptor models from the previous step , positional constraints on the receptor TODs were set relative to the height and orientation of the protein rings .",
"Finally , CheA-P3 , 4 core component models were placed between adjacent TODs in accordance with the patterns observed in our density maps and joined to nearby ring-bound regulatory domains ( P5 ) at the P4-P5 flexible linker .",
"From the CheA2-hexamer model we then extracted a portion corresponding to the array unit cell ( Figure 4—figure supplement 2C , Figure 4—figure supplement 3C ) for further study with all-atom MD simulations .",
"In addition , symmetry-constrained molecular dynamics flexible fitting ( MDFF ) simulations ( Trabuco et al . , 2008 ) were used to refine the overlap between our experimental densities and heuristically constructed CheA2-trimer and CheA2-hexamer subunit models ( Figure 4—figure supplement 3D , Video 2 ) .",
"The Situs modeling package ( Wriggers , 2010 ) , was used to rigidly dock the subunit models into their respective cryoET maps to provide the initial overlap for our MDFF simulations .",
"The array unit cell model was hydrated with TIP3P water molecules using VMD’s solvate plugin ( Humphrey et al . , 1996 ) , producing a simulation box defined by hexagonal lattice parameters a=208 Å , b=208 Å , c=334 Å , α=90° , β=90° , γ=120° .",
"Using VMD’s autoionize plugin , the hydrated system was then neutralized and subsequently ionized with sodium and chloride ions to the physiological concentration of 150 mM , resulting in a model containing 1 , 153 , 756 atoms .",
"The unit cell model was then subjected to a series of conjugant gradient energy minimizations ( 300 , 000 steps in total ) and restrained NPT equilibration simulations ( 10 ns in total ) .",
"In the same fashion , the CheA2-trimer and CheA2-hexamer subunit models were hydrated and ionized to produce systems of size 1 , 751 , 375 atoms ( 245x245x310 Å ) and 4 , 588 , 588 atoms ( 385x405x310 Å ) respectively .",
"Each subunit model was then subjected to the same minimization ( 300 , 000 steps ) and restrained NPT equilibration ( 10 ns ) scheme as the unit cell model .",
"An outline of subsequent equilibration and production simulations is given in Figure 4—figure supplement 4A .",
"Production simulations of the unit cell and MDFF-refined CheA2-trimer models were conducted with weak ( spring constant = 0 . 1 kcal/mol*nm2 ) harmonic restraints placed on the alpha carbons of the first five membrane-proximal receptor residues to maintain TOD splay in the absence of membrane and crowding agents .",
"In the case of the post-MDFF production simulations of the CheA2-trimer , additional weak harmonic constraints were placed on the outermost CheW and CheA-P5 domains to enforce the bulk array boundary conditions , as the trimer organization does not permit the use of periodic boundary conditions to represent the necessary symmetry .",
"All molecular dynamics simulations were performed using the parallel molecular dynamics code , NAMD 2 . 9 ( Phillips et al . , 2005 ) and CHARMM22 force field ( MacKerell et al . , 1998 ) with CMAP corrections ( Mackerell et al . , 2004 ) .",
"Equilibrium simulations were conducted in the NPT ensemble with isobaric and isothermal conditions maintained at 1 atm and 330 K for equilibration , or 350 K for production using the Nosé-Hoover Langevin piston , with a period 200 femtoseconds ( fs ) and relaxation time of 50 fs , and the Langevin thermostat with a temperature coupling of 5 ps-1 .",
"The r-RESPA integrator scheme with an integration time step of 2 fs was used ( Phillips et al . , 2005 ) .",
"SHAKE constraints were applied to all hydrogen atoms .",
"Short-range , non-bonded interactions were calculated every 2 fs with a cutoff of 12 Å and long-range electrostatics were evaluated every 6 fs using the particle-mesh-Ewald ( PME ) method with a grid size of 1 Å .",
"Periodic boundary conditions with fixed cross-sectional area ( x-y plane ) were used .",
"MDFF simulations were performed in the NVT ensemble at 330 K using the settings described above with additional restraints applied to prevent loss of secondary structure , chirality errors , and the formation of cis-peptide bonds .",
"Visualization and extraction of raw trajectory data for analysis were performed using VMD .",
"Principal Components Analysis ( PCA ) was carried out using custom scripts ( source code file PCA . py ) involving the Numpy , Scipy , and MDAnalysis python packages ( Michaud-Agrawal et al . , 2011 ) .",
"For the PCA analysis , a single dip-exhibiting CheA dimer was isolated from one of our wild-type unit cell simulations , and each frame ( 23 , 331 frames in total ) was aligned to the initial CheA dimer model using the P5 domains ( residues 543–671 ) .",
"Principal components were computed using the alpha carbons of the P4 domains ( residues 352 to 542 ) .",
"The fractional variances accounted for by the top three modes were 41 . 8% , 31 . 1% , and 8 . 1% respectively .",
"Subsequently , the three CheA dimers from each replica of the wild-type unit cell model ( 27 dimers total ) , R297A unit cell model ( 27 dimers total ) , and CheA2-trimer model ( 30 dimers total ) simulations were extracted , aligned to the P5 domains , and projected on to the top principal component of the wild-type dip-exhibiting CheA dimer .",
"These projections were grouped according to model type to create Figure 4C ( top and bottom ) and Figure 4—figure supplement 5 .",
"Illustrations of the PCA results were produced using the python-plotting package , Matplotlib .",
"Clustering analysis was performed using custom scripts ( source code file clustering . py ) involving the python packages noted above as well as the implementation of the UPGMC hierarchical , agglomerative clustering algorithm from the fastcluster package ( Müllner , 2013 ) .",
"For the clustering analysis , we first extracted the three core-signaling units from each of the nine wild-type unit cell replica simulations , using 1500 frames/core-signaling unit for a total of 40 , 500 frames .",
"The RMSD distance matrix was then computed using the ‘rms_fit_trj’ function from the MDAnalysis package .",
"Our analysis identified four major clusters of structures within the above distance matrix with relative populations of 80% , 10% , 10% and 2% , representing the undipped and dipped CheA dimer states as well as two intermediate states respectively .",
"Specific mutations on CheA and Tsr were generated by site-directed mutagenesis on the background of cysteine-less CheA ( pGP26 ) ( Miller et al . , 2006; Zhao and Parkinson , 2006 ) and wt Tsr ( pRR53 ) , respectively .",
"Each mutation was introduced using a pair of primers ( Integrated DNA Technologies , Inc . , Coralville , Iowa ) , complementary to the template except for the site of mutation , and PfuUltra II Fusion HS DNA polymerase ( Agilent Technologies , Santa Clara , CA ) , following the manufacturer’s thermocycling parameters .",
"The presence of the mutations was confirmed by DNA sequencing .",
"Starter cultures were grown in LB broth ( 10% tryptone/5% yeast extract/10% NaCl ) , supplemented with appropriate antibiotics , overnight at 37°C with 250-rpm shaking .",
"Subsequently , the overnight cultures were diluted 1:50 into a 5-ml LB broth , supplemented with the appropriate antibiotics and allowed to grow at 37°C with 250-rpm shaking .",
"When the optical density at 600 nm of the cultures reached ~0 . 8 , cells were induced with 100 μM IPTG and 0 . 6 μM Na-S , in the presence of 100 μM serine , for 1 hr at 37°C .",
"After induction , cells were collected by centrifugation ( 3000 x g , 4°C for 10 mins ) and then re-suspended in cold PBS , in the presence of 100 μM serine .",
"Cross-linking was initiated by addition of 60 μM copper ( II ) sulfate and 200 μM phenanthroline ( 1 hr , RT ) and stopped by addition of 20 mM iodoacetamide and 3 . 7 μM neocuproin .",
"Cells were immediately mixed with 4× NuPAGE lithium dodecyl sulfate/PAGE sample buffer ( Invitrogen Corp . , Carlsbad , CA ) , with or without reducing agent ( dithiothreitol ) , and then boiled for 5 min before electrophoresis .",
"Samples were analyzed on 4–12% SDS-PAGE gels in MES running buffer ( ( Invitrogen Corp . , Carlsbad , CA ) ) .",
"Gels were transferred to nitrocellulose membranes , blocked , and immunoblotted by using antiserum against Tsr ( 1:2500 ) and CheA ( 1:1250 ) ( gifts from Dr . Subramaniam , NIH ) , followed by an alkaline phosphatase conjugated anti-rabbit antibody ( 1:50 , 000 , Sigma ) .",
"Bands were detected on the membrane using an NBT/BCIP kit ( Promega Corporation , Madison , WI ) following the manufacturer’s instructions .",
"The UU2682 strain does not express any of the chemoreceptors , CheA or CheW , rendering it non-chemotactic despite the presence of an intact flagellar system .",
"Presence of both pRR53 ( wt-Tsr ) and pGP26 ( cysless CheA and wt-cheW ) is required to rescue the chemotaxis of UU2682 , observed as formation of attractant rings on a soft-agar media .",
"To assess the effect of mutations on Tsr and/or CheA on the chemotactic ability of E . coli , the mutant plasmids were introduced into the UU2682 strain and assayed for formation of attractant rings .",
"The soft agar assay protocol used here is adapted from the Parkinson laboratory ( University of Utah ) .",
"Fresh colonies were plated on LB-agar media ( 10% tryptone/5% yeast extract/10% NaCl/10% agar ) , supplemented with carbenicillin ( 100 μg/ml ) and chloramphenicol ( 34 μg/ml ) , and grown overnight at 37°C .",
"Next day , using a fine-tip toothpick , colonies were picked from the fresh LB-agar plate and stabbed into a soft-agar media ( 10% Tryptone/5% yeast extract/5%NaCl/0 . 27% agar ) containing antibiotics ( carbenicillin 50 μg/ml , chloramphenicol 17 μg/ml ) , inducers ( 100 μM IPTG and 0 . 6 μM Na-S ) and 100 μM serine .",
"Plates were then incubated at 32°C for ~8 hr and the diameter of attractant rings immediately measured after incubation ."
]
] | [
"Chemotactic responses in bacteria require large , highly ordered arrays of sensory proteins to mediate the signal transduction that ultimately controls cell motility .",
"A mechanistic understanding of the molecular events underlying signaling , however , has been hampered by the lack of a high-resolution structural description of the extended array .",
"Here , we report a novel reconstitution of the array , involving the receptor signaling domain , histidine kinase CheA , and adaptor protein CheW , as well as a density map of the core-signaling unit at 11 . 3 Å resolution , obtained by cryo-electron tomography and sub-tomogram averaging .",
"Extracting key structural constraints from our density map , we computationally construct and refine an atomic model of the core array structure , exposing novel interfaces between the component proteins .",
"Using all-atom molecular dynamics simulations , we further reveal a distinctive conformational change in CheA .",
"Mutagenesis and chemical cross-linking experiments confirm the importance of the conformational dynamics of CheA for chemotactic function ."
] | [
"To survive , an organism must be able to collect and interpret information about its environment and behave accordingly .",
"Bacteria are able to do this via a process called “chemotaxis” .",
"Inside the bacteria are sensors that contain a two-dimensional network of proteins called a chemosensory array , which detect chemical changes in the environment and signal to motor proteins to allow the bacterium to move to a more favorable location .",
"Thousands of proteins make up the chemosensory array , and so a key question is how do these proteins interact with each other to work together as a team ?",
"Cassidy , Himes , Alvarez et al . have now used a technique called cryo-electron tomography to determine the three-dimensional structure of the chemosensory array in the bacteria species Escherichia coli in high detail .",
"This revealed the structures of several key components of the array , including some protein regions that are critical for signaling during chemotaxis .",
"Cassidy et al . were then able to use the electron tomography data to create a model of the array that details all of its individual atoms .",
"Supercomputer simulations of this model revealed that during chemotaxis , a key signaling protein changes shape in a way that is critical for signal processing .",
"This shape change was confirmed to be important for chemotaxis by chemical experiments and tests on mutant E . coli cells .",
"The next steps are to further improve the structure so that more details of the array organization can be distinguished , as well as to investigate the structure of other signaling states .",
"By assembling structural “snapshots” of these different states , in the long term Cassidy et al . aim to develop models that detail the atoms in every one of the components involved in the chemotaxis signaling pathway ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"plant biology",
"cell biology"
] | Orchestration of microtubules and the actin cytoskeleton in trichome cell shape determination by a plant-unique kinesin | elife-09351-v2 | [
[
"Plant cells assume an amazing diversity of cell shapes that enable these cells to execute unique physiological functions , and the study of plant cell shape determination has remained an intriguing part of plant biology ( Smith and Oppenheimer , 2005; Szymanski , 2009 ) .",
"The plant cytoskeletal system , composed of microtubules ( MTs ) and actin filaments ( F-actin ) , plays a central role in cell morphogenesis in both tip-growing and diffuse-growing cell types .",
"In particular , the cortical MTs play pivotal roles in plant cell growth and directionality , mainly by orienting the deposition of nascent cellulose microfibrils during biosynthesis of the cell wall ( Paradez et al . , 2006 ) .",
"F-actin plays central roles in polarized cell elongation , mainly by regulating intracellular transport ( Hussey et al . , 2006 ) .",
"However , despite emerging evidence that cortical MTs and F-actin coordinately regulate plant cell morphogenesis ( Petrasek and Schwarzerova , 2009; Sampathkumar et al . , 2011; Sambade et al . , 2014 ) , the molecular mechanisms underlying the crosstalk between these two cytoskeletal systems remain largely unknown .",
"The Arabidopsis thaliana leaf trichome , a single cell bearing three or four branches on top of a stalk , has long been a model system for investigating the role of the cytoskeleton in defining plant cell shape ( Smith , 2003; Szymanski , 2009 ) .",
"The trichome phenotype indicates the cytoskeletal homeostasis inside the plant cell .",
"For example , cortical MTs mainly affect initiation of trichome branches , and disruption of genes encoding MT-associated proteins usually produces trichomes with abnormal numbers of branches ( Oppenheimer et al . , 1997; Krishnakumar and Oppenheimer , 1999; Burk et al . , 2001; Buschmann et al . , 2009; Nakamura and Hashimoto , 2009; Kong et al . , 2010 ) .",
"By contrast , actin filaments mainly affect elongation of trichomes , and disruption of genes encoding actin-related proteins , such as components of the ARP2/3 actin nucleation complex and the upstream WAVE/SCAR regulatory complex ( W/SRC ) , usually results in impaired trichome elongation but with normal branch number ( Mathur et al . , 2003; Basu et al . , 2004; Deeks et al . , 2004; El-Assal Sel et al . , 2004; El-Din El-Assal et al . , 2004; Li et al . , 2004; Szymanski , 2005; Zhang et al . , 2005a; Djakovic et al . , 2006; Le et al . , 2006 ) .",
"Therefore , theoretically , mutation in a gene that participates in both processes would lead to trichome defects in both branching and elongation and would assist in the elucidation of the crosstalk between the MTs and the F-actin .",
"However , such a central player , which directly links and integrates MTs and F-actin and further establishes the required cytoskeletal systems for trichome cell shaping , remains to be identified .",
"KCBP ( kinesin-like calmodulin-binding protein ) occurs solely within the plant kingdom , and uniquely in the kinesin superfamily .",
"KCBP has a C-terminal motor head , beyond which locates a calmodulin-binding domain ( CBD ) at the extreme end of the C-terminus; besides KCBP , the CBD is only found in the sea urchin kinesin KinC .",
"KCBP also contains a MyTH4-FERM tandem , which only occurs in several myosin families outside the plant kingdom , in its tail region at the N-terminus ( NT ) ; thus , KCBP has long been regarded as a chimera of kinesin with a myosin ( Abdel-Ghany et al . , 2005 ) .",
"Using biotinylated calmodulin as a probe , KCBP was firstly isolated as a novel calmodulin-binding protein with a kinesin motor domain ( Reddy et al . , 1996 ) .",
"In vitro biochemical assays showed that the CBD of KCBP binds calcium/calmodulin , which negatively regulates KCBP motor activity , and that the N-terminal tail , including the MyTH4-FERM domain , could co-sediment with MTs ( Narasimhulu and Reddy , 1998 ) .",
"Interestingly , genetic analysis of the zwichel ( zwi ) mutants , which have a shortened stalk and only two branches , revealed that the ZWICHEL ( ZWI ) gene encodes the KCBP protein ( Hülskamp et al . , 1994; Oppenheimer et al . , 1997 ) .",
"KCBP was detected in mitotic MT arrays such as the preprophase band , the spindle , and the phragmoplast in various plants , indicating that it may play a role in cell division ( Bowser and Reddy , 1997; Smirnova et al . , 1998; Preuss et al . , 2003; Buschmann et al . , 2015 ) .",
"Nevertheless , loss of function in KCBP only produces a noticeable phenotype in trichomes , which are less-branched and contain shortened stalks and swollen , stunted branches ( Oppenheimer et al . , 1997 ) , providing a hint of clue that KCBP possibly plays the role involving in both cortical MTs and F-actin .",
"However , whether KCBP localizes to cortical MTs in trichome cells is still an open question , cellular basis of defects in kcbp trichomes needs to be examined , direct evidence linking KCBP and F-actin is currently missing , and the role of the mysterious MyTH4-FERM domain remains to be unraveled .",
"Here , we show that the N-terminal tail comprising the MyTH4 domain strongly binds to MTs , and the FERM domain physically binds to F-actin .",
"During trichome morphogenesis , KCBP localizes to cortical MTs , distributes in a specific gradient , and is concentrated at the branching sites and the apexes of elongating branches .",
"KCBP orchestrates the MT-actin interplay and is required to assemble the specific cytoskeletal configuration for trichome branching , elongation , and tip sharpening .",
"Our findings build the direct link between the MT-based KCBP motor and the actin cytoskeleton , unravel the cellular basis controlling trichome cell shaping , and provide significant insights into the mechanisms of cytoskeletal regulation of cell shape determination ."
],
[
"To determine whether KCBP localizes to cortical MTs , we performed live-cell imaging using a functional , GFP-tagged KCBP fusion under the control of its endogenous regulatory elements; this construct fully rescued the typical zwichel trichome defects in the kcbp-1 ( Salk_031704 in the Arabidopsis Biological Resource Center; see ‘Materials and methods’ ) mutant ( Figure 1—figure supplement 1A ) ( Humphrey et al . , 2015 ) , which was also designated as zwiA ( N531704 in the Nottingham Arabidopsis Stock Centre; see ‘Materials and methods’ ) ( Buschmann et al . , 2015 ) .",
"Notably , we found that GFP-KCBP localizes to puncta along cortical MTs in both pavement cells and hypocotyl cells ( Figure 1A , Figure 1—figure supplement 2B , Video 1 , Video 2 ) .",
"Most GFP-KCBP proteins dwell on or move along cortical MTs as discrete particles for a short time ( 15 . 9 s , n = 647 ) ; only a small portion of GFP-KCBP particles remain immobilized on MTs for longer times ( Video 1 , Video 2 ) .",
"Kymograph analysis further confirmed the short-lived and poorly processive nature of KCBP movement ( Figure 1B , Figure 1—figure supplement 2C ) , which is the characteristic of non-processive motors of the kinesin-14 subfamily ( Fink et al . , 2009 ) .",
"We then quantified the distributions of the velocity and the run-length observed for GFP-KCBP .",
"The distributions indicated that , upon fitting to an exponential line , the motor moved at an average velocity of 0 . 68 μm/min ( n = 647 ) ( Figure 1C , Figure 1—source data 1 ) , and at an average run length of 0 . 18 μm ( n = 647 ) ( Figure 1D , Figure 1—source data 1 ) .",
"Taken together , these results indicated that KCBP likely acts as a non-processive motor to facilitate crosslinking or bundling during MT organization . 10 . 7554/eLife . 09351 . 003Figure 1 . KCBP colocalizes with cortical MTs in vivo .",
"( A ) GFP-labeled kinesin-like calmodulin-binding protein ( KCBP ) localizes along cortical microtubules ( MTs ) ( mCherry-TUB6 ) in a punctate pattern in Arabidopsis epidermal pavement cells .",
"The yellow box highlights the area used to generate the kymograph and the yellow arrow marks the direction for the kymographic analysis in ( B ) .",
"See also Video",
"1 . ( B ) Kymograph showing the dynamicity of GFP-KCBP particles .",
"The bright dots indicate transient appearance of most GFP-KCBP particles and the linear tracks indicate the motility of GFP-KCBP particles either dwelling on ( marked by arrowheads ) or moving along MTs ( marked by arrows ) for a short time .",
"( C , D )",
"Distribution of the velocity ( C ) and the run length ( D ) of GFP-KCBP moving along cortical MTs .",
"The mean values are shown with standard deviations and examined sample sizes .",
"Dashed lines represent the trends derived from exponential fits .",
"Scale bars , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 00310 . 7554/eLife . 09351 . 004Figure 1—source data 1 . Distribution of the velocity and the run length of GFP-KCBP moving along cortical MTs . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 00410 . 7554/eLife . 09351 . 005Figure 1—figure supplement 1 . Genetic identification of the kcbp-1/zwiA mutant .",
"( A ) Gene structure of the KCBP gene .",
"Black rectangles indicate exons , gray rectangles indicate the 5′ and 3′ untranslated regions , and thick lines indicate introns .",
"The arrow indicates the location of the T-DNA insertion in the kcbp-1/zwiA mutant .",
"( B ) PCR-based identification of the T-DNA insertion .",
"LP and RP indicate a pair of KCBP specific primers .",
"BP indicates the T-DNA border primer .",
"( C ) RT-PCR-based identification of the kcbp-1/zwiA allele .",
"Red arrows indicate the position and the direction of primers , which are listed in the Supplementary file 1 . * Sequencing analysis of the PCR product amplified by the LBb1 . 3/RP primer pair revealed that the T-DNA insertion is in the third exon and located at the position of 957 bp downstream the ATP start codon of the KCBP gene and introduces a premature stop codon 61 bp downstream the insertion site by frame shift .",
"Thus , the kcbp-1/zwiA mutant is expected to produce a short peptide of 284 amino acids containing the first 263 amino acids of KCBP at a lower level .",
"The sequence of the PCR product is pasted below:CATCTGATCGATCTACGCCTCCCAGTTTAGATGAACGCATTGACCTCGTTGGAAAGCTCTTCAAAAAAACTTTGAAGCGTGTTGAACTCAGGGACGAACTTTTTGCCCAAATCTCCAAACAGACTAGACATAATCCTGACAGGCAATACTTGATCAAAGCTTGGGAATTGATGTACTTATGTGCCTCCTCTATGCCTCCTAGCAAAGATATCGGTGGATATCTATCTGAGTATATTCATAATGTCGCACACGATGCAACTATTGAACCGGATGCTCAGGTTCTTGCTGTTAACACTTTGAAAGCTTTAAAGCGCTCTATCAAAGCCAACATTAATAACACATTGCGGACGTTTTTAATGTACTGGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTCCGAAATCGGCAAAATNote: The 325-bp fragment , bolded , is the sequence of 633–957 bp downstream the ATG start codon of the KCBP gene; and the remaining 186-bp fragment is the T-DNA sequence .",
"The 21 bp , underlined and bolded , is the sequence of the primer 031704-RP; and the 19 bp , underlined , is the reverse complement sequence of the primer LBb1 . 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 00510 . 7554/eLife . 09351 . 006Figure 1—figure supplement 2 . KCBP colocalizes with cortical MTs in Arabidopsis hypocotyl cells .",
"( A ) The genomic GFP-KCBP fusion complements the trichome defects of the kcbp-1 mutant .",
"Scale bar , 1 mm . ( B ) GFP-labeled KCBP localizes along cortical MTs ( mCherry-TUB6 ) in a punctate pattern in Arabidopsis hypocotyl cells .",
"The yellow box highlights the area used to generate the kymograph and the yellow arrow marks the direction for the kymographic analysis in ( C ) .",
"See also Video",
"2 . Scale bar , 5 μm .",
"( C ) Kymograph showing the dynamicity of GFP-KCBP particles .",
"The bright dots indicate transient appearance of most GFP-KCBP particles and the linear tracks indicate the motility of GFP-KCBP particles either dwelling on ( marked by arrowhead ) or moving along MTs ( marked by arrow ) for a short time .",
"Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 00610 . 7554/eLife . 09351 . 007Video 1 . Localization and dynamicity of kinesin-like calmodulin-binding protein ( KCBP ) on cortical microtubules ( MTs ) in Arabidopsis epidermal pavement cells . Images were obtained at 3-s intervals .",
"A total of 30 time lapse images were applied to make the video .",
"Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 00710 . 7554/eLife . 09351 . 008Video 2 . Localization and dynamicity of KCBP on cortical MTs in Arabidopsis hypocotyl cells . Images were obtained at 3-s intervals .",
"A total of 35 time lapse images were applied to make the video .",
"Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 008 To gain further insights into the role of KCBP in regulating trichome morphogenesis , we examined the spatio-temporal dynamics of GFP-KCBP during trichome morphogenesis .",
"Indeed , we observed that KCBP largely colocalizes with cortical MTs in developing trichomes ( Figure 2—figure supplement 1 ) .",
"Intriguingly , KCBP forms a gradient in the elongating trichome branch , with the highest density at the extreme apex , which is devoid of MTs ( Figure 2—figure supplement 1 ) .",
"Because the signal of mCherry-labeled MTs is much weaker than that of GFP and undergoes rapid photobleaching , it is extremely hard to detect in trichomes due to their three-dimension geometry; therefore , to ensure an accurate comparison , we further observed KCBP and MTs using the GFP-KCBP-expressing line and the GFP-TUB6-expressing line , respectively .",
"We observed punctuate KCBP localization in the cortical gradient in developing trichomes , with the highest density at the extreme apex ( Figure 2A , D , G; Figure 2—figure supplement 2A; Video 3 , Video 4 ) .",
"In GFP-TUB6-expressing trichomes at identical developmental stages , we observed transversely aligned MT arrays ( transverse MT rings ) in a similar gradient with higher MT density towards the branch tip , but forming a MT-depleted zone at the extreme apex ( Figure 2B , E , H; Figure 2—figure supplement 2B; Video 3 ) .",
"In consistent with the result from the double-labeling imaging ( Figure 2—figure supplement 1 ) , KCBP largely colocalizes with cortical MTs along the trichome branch , but shows highest levels in the MT-depleted zone at the branch apex ( Figure 2A , B , G , H; Video 3 , Video 4 ) .",
"We also found a similar distribution at the branching site where KCBP accumulates , but which has a relatively sparse MT network ( Figure 2A , B , D , E; Video 3 ) .",
"To further validate the presence of the MT-depleted zone , we used GCP2 ( Liu et al . , 2014 ) , a component of the MT nucleation complex , as a control .",
"Interestingly , we observed that , similar to MTs , GCP2 shows a tip-directed gradient , but with a GCP2-depleted zone at the extreme apexes of elongating branches ( Figure 2—figure supplement 3; Video 5 ) . 10 . 7554/eLife . 09351 . 009Figure 2 . Spatio-temporal distribution of GFP-KCBP in developing wild-type trichomes .",
"( A–F )",
"Localization of KCBP and spatial organization of the cytoskeleton in stage 2/3 trichomes .",
"GFP-KCBP particles form a cortical gradient with the highest expression at the branching site and the tip region of the main stem ( A ) .",
"The arrow and the arrowhead highlight the concentration of GFP-KCBP at the extreme apex of the main stem and the apical region of the incipient primary branch by three-dimension ( 3-D ) reconstruction , respectively ( D ) .",
"Transverse MT arrays form rings encircling the elongating main stem , but leave a MT-depleted zone at the extreme apex .",
"The incipient primary branch is also encircled by transverse MT rings , with the apex colonized by sparse MT meshworks ( B ) .",
"The arrow and the arrowhead in ( E ) highlight the 3-D reconstruction of the MT-depleted zone at the extreme apex of the main stem and the sparse MT meshworks at the apical region of the incipient primary branch , respectively .",
"Cytoplasmic cables extend along the growth axis of the main stem , from the base to the tip region where they form a fine , cortical F-actin cap mainly in a transverse pattern , highlighted by the brackets ( C ) .",
"The arrow and the arrowhead in ( F ) highlight the 3-D reconstruction of the F-actin cap at the tip region of the main stem and actin bundles at the apical region of the incipient primary branch , respectively .",
"See also Video",
"3 . ( G–I ) Localization of KCBP and spatial organization of the cytoskeleton in stage 3/4 trichomes .",
"KCBP forms a transverse cortical punctate pattern , with the highest amounts ( indicated by arrows ) at the extreme apex of the elongating branches .",
"The tandem arrowhead highlights GFP-KCBP particles in the transverse linear pattern ( G ) .",
"Transverse MT rings display a tip-directed density gradient , but with a MT-depleted zone ( indicated by arrows ) at the extreme apex of the elongating branches ( H ) .",
"Cytoplasmic actin cables ( indicated by open arrows ) extend along the growth axis of elongating branches and reach near the tip , where there is a fine , transversely aligned F-actin cap ( highlighted by brackets ) ( I ) .",
"See also Video",
"4 . The maximum z-projection of image stacks at 0 . 2-μm intervals was applied to all figures .",
"Scale bars , 20 μm .",
"One grid unit in ( D–F ) indicates 7 . 31 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 00910 . 7554/eLife . 09351 . 010Figure 2—figure supplement 1 . Colocalization of KCBP with cortical MTs in stage 2 trichomes . GFP-KCBP particles form a cortical gradient with the highest expression at the branching site and the tip region of the main stem .",
"The arrowheads highlight the strongest accumulation of GFP-KCBP at the apex of the main stem and the apical region of the incipient primary branch .",
"Transverse MT arrays form rings encircling the elongating main stem , but leave a MT-depleted zone at the extreme apex .",
"The incipient primary branch is also encircled by transverse MT rings , with the apex colonized by sparse MT meshworks .",
"The arrows highlight the transverse MT rings .",
"The mCherry-labeled MTs are difficult to detect and undergo rapid photobleaching in trichomes .",
"The maximum z-projection of image stacks at 0 . 2-μm intervals was applied to all figures .",
"Scale bars , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 01010 . 7554/eLife . 09351 . 011Figure 2—figure supplement 2 . Localization of KCBP and spatial organization of MTs in stage 2/3 wild-type trichomes .",
"( A ) The GFP-KCBP images , which were used to make the z-projection in Figure 2A , were sequentially illustrated at 0 . 4-μm intervals .",
"GFP-KCBP was observed to strongly accumulate at apexes of elongating branches and branching sites in stage 2/3 trichome .",
"Scale bars , 5 μm .",
"( B ) The GFP-TUB6 images , which were used to make the z-projection in Figure 2B , were sequentially illustrated at 0 . 4-μm intervals .",
"A MT-depleted zone was observed at extreme apexes of elongating branches in stage 2/3 trichome .",
"Scale bars , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 01110 . 7554/eLife . 09351 . 012Figure 2—figure supplement 3 . Localization of GCP2 in stage 2/3 wild-type trichomes .",
"( A ) The Z-projection image , which was acquired from a high-resolution stack of 46 planes at 0 . 2-μm intervals , shows a tip-oriented cortical gradient of GCP2-3×GFP particles in the elongating main stem in a stage 2/3 trichome .",
"Absence of GCP2-3×GFP was observed at the extreme apexes of the main stems .",
"Scale bar , 10 μm .",
"( B ) Absence of GCP2-3×GFP at the extreme apexes of the main stems is highlighted by the 3-D reconstruction of stage 2/3 trichomes .",
"One grid unit indicates 5 . 63 μm .",
"( C ) The GCP2-3×GFP images , which were used to make the Z-projection in ( A ) , were sequentially illustrated at 0 . 4-μm intervals .",
"Absence of GCP2-3×GFP was shown at extreme apexes of elongating branches in stage 2/3 trichome .",
"Scale bars , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 01210 . 7554/eLife . 09351 . 013Video 3 . Spatio-temporal distribution of GFP-KCBP , MTs , and actin filaments is highlighted by 3-D reconstitution in stage 2/3 trichomes . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 01310 . 7554/eLife . 09351 . 014Video 4 . The spatio-temporal dynamics and distribution of GFP-KCBP in developing trichomes . Images were obtained at 3-s intervals .",
"A total of 8 time lapse images were applied to make the video .",
"Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 01410 . 7554/eLife . 09351 . 015Video 5 . Spatio-temporal distribution of GCP2-3XGFP is highlighted by 3-D reconstitution in stage 2/3 trichomes . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 015 The enrichment of KCBP at the MT-depleted zone induced us to compare the distribution of KCBP with the actin cytoskeleton in developing trichomes at identical developmental stages .",
"We observed that F-actin forms thick bundles parallel to the long axis of elongating branches and extend from the stalk to the region near branch tips , but also form a cap with fine , cortical F-actin mainly in a transverse pattern in the tip region ( Figure 2C , F , I; Video 3 ) .",
"Notably , the transverse cortical F-actin cap occupies a larger area than the MT-depleted zone , but coincides with the area where KCBP accumulates ( Figure 2A–I ) .",
"Together , our observations suggest that the specific distribution of KCBP is tightly associated with the assembly of the required cytoskeletal organization for trichome branching and elongation .",
"To further reveal the cellular mechanisms by which KCBP affects trichome morphogenesis , we monitored MT organization at various stages of trichome development in kcbp-1 mutants and the wild-type control .",
"The wild-type Arabidopsis leaf trichome initiates from a bulging protodermal cell and then grows out into a cylindrical cell , which exhibits random organization of the MT array ( Figure 3A ) .",
"The random MT network shifts into a transverse MT array ( rings ) encircling the elongating cylindrical cell ( Figure 3B ) .",
"Slightly later , an area near the tip bulges out and gradually forms the primary branch ( Figure 3B–D ) .",
"Most trichomes undergo two consecutive branching events to form the typical three-branch pattern ( Figure 3E , F , Figure 3—figure supplement 1A ) .",
"During trichome branch elongation , cortical MTs also shift to transverse rings with the typical tip-directed MT density gradient ( Figure 3C–F ) .",
"Intriguingly , a MT-depleted zone occurs at the extreme apex of the elongating branch ( Figure 3D; Video 6 ) .",
"At the maturation stage , fully elongated trichome branches form a fine and pointed tip , and cortical MTs reorient to an oblique or longitudinal configuration ( Figure 3—figure supplement 1A ) . 10 . 7554/eLife . 09351 . 016Figure 3 . Abnormal MT organization in kcbp-1 trichomes .",
"( A–F )",
"Spatio-temporal MT organization in wild-type trichomes during development .",
"The stage 1 trichomes exhibit random MT networks ( A ) .",
"In stage 2/3 trichomes , the random MT network shifts into transverse MT rings encircling the elongating main stem and the incipient primary branch ( B , C ) .",
"In stage 3/4/5 trichomes ( D–F ) , cortical MTs rings encircle the elongating branches but leave a MT-depleted zone at the extreme apex , which is highlighted by 3-D reconstruction ( the inset in D ) of the area outlined by the dotted box .",
"See also Video",
"6 . ( G–K ) Spatio-temporal MT organization in kcbp-1 trichomes during development .",
"The stage 1 kcbp-1 trichomes form shorter and rounder tubular cells with random MT networks ( G ) .",
"In stage 2/3 trichomes , formation of the transverse MT rings that encircle the incipient primary branch is impaired ( H , I ) .",
"The 3-D reconstruction ( the inset in I ) of the incipient primary branch tip ( outlined by the dotted box ) shows that the MT-depleted zone is not well defined .",
"In stage 3/4/5 trichomes ( J , K ) , transverse MT rings are present in a relatively loose distribution .",
"The circled B indicates the base of trichome stalks .",
"See also Video",
"7 . The maximum z-projection of image stacks at 0 . 2-μm intervals was applied to all figures .",
"One grid unit in the inset of ( D , I ) indicates 7 . 31 μm .",
"Scale bars , 20 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 01610 . 7554/eLife . 09351 . 017Figure 3—figure supplement 1 . Cortical MT organization in wild-type and kcbp-1 mature trichomes . In stage 6 wild-type mature trichomes ( A ) , fully elongated branches form a fine and pointed tip , and cortical MTs exhibit an oblique or longitudinal configuration .",
"By contrast , the stage 6 kcbp-1 mature trichomes ( B ) form one blunt tip and another relatively more-pointed tip , with oblique/longitudinal MTs .",
"The maximum z-projection of image stacks at 0 . 2-μm intervals was applied .",
"The photo stitching application of the Volocity ( Version 6 . 2 ) program was applied to acquire the image of the entire mature trichomes .",
"Scale bars are 60 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 01710 . 7554/eLife . 09351 . 018Video 6 . The 3-D reconstructed cortical MT configuration in stage 3/4 wild-type trichomes . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 018 By contrast , kcbp-1 trichomes initiate normally , but form a shorter and rounder tubular cell with a randomly oriented MT network ( Figure 3G ) .",
"Subsequently , the primary branching event takes place from the severely reduced stalk ( Figure 3H , I ) , but the secondary branching does not occur , yielding a two-branch trichome ( Figure 3J , K , Figure 3—figure supplement 1B ) .",
"Eventually , one branch elongates and forms a tip that is not as pointed as the wild type , whereas the growth of the other branch is impaired , producing a swollen and blunt tip ( Figure 3K , Figure 3—figure supplement 1B ) .",
"Notably , cortical MT organization in kcbp-1 trichomes is distinct from that in the wild type .",
"During branching and elongation of the primary branch of kcbp-1 trichomes , formation of the transverse MT rings that encircle the incipient branch is impaired , and the MT-depleted zone is never observed ( Figure 3H , I; Video 7 ) .",
"In the normally elongating branch ( stem ) , the transverse MT rings are seen in a relatively loose distribution , and the MT-depleted zone is not well defined ( Figure 3I–K; Video 7 ) . 10 . 7554/eLife . 09351 . 019Video 7 . The 3-D reconstructed cortical MT configuration in stage 3/4 kcbp-1 trichomes . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 019 Together , these observations indicated that KCBP is required to assemble the transverse MT rings encircling the elongating branch and formation of the MT-depleted zone , and that loss of KCBP function results in impairment of trichome branch initiation and elongation , and branch tip sharpening .",
"The coincidence of KCBP with the cortical actin network and the exclusion of MTs at the branch apex prompted us to examine whether the organization of F-actin is aberrant during the morphogenesis of kcbp-1 trichomes .",
"We therefore monitored global actin cytoskeleton changes during trichome development ( Figure 4A–H ) .",
"Strikingly , the parallel cytoplasmic actin cables , which extend along the long axis to the branch tip in wild-type trichomes ( Figure 4B–D ) , are disorganized in kcbp-1 trichomes , which instead have a curly , intertwined meshwork of thick actin bundles ( Figure 4E–H ) .",
"Moreover , the transverse cortical F-actin cap , which is observed at the branch tip of elongating wild-type trichomes ( Figure 4C ) , is disrupted in kcbp-1 trichomes ( Figure 4G , H ) . 10 . 7554/eLife . 09351 . 020Figure 4 . Aberrant organization of F-actin in kcbp-1 trichomes .",
"( A–D )",
"Spatio-temporal organization of F-actin in wild-type trichomes during development .",
"In stage 1/2 trichomes , a population of cytoplasmic actin cables align with the growth axis ( A , B ) .",
"In stage 3/4/5 trichomes , the cytoplasmic actin cables extend from the base to the branch tip , while a fine , cortical F-actin cap mainly in the transverse pattern is present in the tip region of elongating branches ( C , D ) .",
"The 3-D reconstructed image ( the inset in C ) generated from the area outlined by the dotted box highlights the F-actin .",
"White open arrows highlight the parallel cytoplasmic actin cables .",
"( E–H )",
"Spatio-temporal organization of F-actin in kcbp-1 trichomes during development .",
"The stage 1/2 trichomes display random meshworks of thick actin bundles ( E , F ) .",
"At stage 3/4/5 , the curly intertwined meshwork of thick actin bundles dominates inside trichomes , but parallel-aligned cytoplasmic actin cables and the fine F-actin cap at the branch apex as shown in wild-type are lost ( G , H ) .",
"The 3-D reconstructed image ( the inset in G ) generated from the area outlined by the dotted box highlights the disorganized F-actin in the tip region of the incipient primary branch .",
"The circled B indicates the base of trichome stalks .",
"The maximum z-projection of image stacks at 0 . 2-μm intervals was applied to all figures .",
"Scale bars , 20 μm .",
"One grid unit in the inset of ( D , E ) indicates 7 . 31 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 020 Taken together , these findings implicate KCBP in the assembly of parallel cytoplasmic actin cables along the growth axis and the transverse cortical F-actin cap at the branch apex , which are likely required for polarized branch elongation accompanied by tip sharpening , and further suggest that KCBP plays a critical role in regulating the MT-actin interaction .",
"To gain genetic insights into the in vivo functions of the individual domains of KCBP , we performed genetic complementation tests using genomic constructs with various domain truncations ( Figure 5A , B ) .",
"As expected , the construct without the motor domain could not rescue the trichome morphology of the kcbp-1 mutant ( Figure 5B ) , whereas the construct without the C-terminal CBD domain could perfectly rescue the kcbp-1 trichome phenotype ( Figure 5B ) , clearly indicating that the CBD domain is dispensable during trichome development .",
"Furthermore , we found that constructs with either the NT truncation or the MyTH4 truncation could rescue the kcbp-1 trichome morphology , but the construct with a truncation of both NT and MyTH4 could not rescue the kcbp-1 trichome phenotype ( Figure 5B ) .",
"In addition , constructs with either truncation of the FERM domain or the ( MyTH4+FERM ) domains could rescue the kcbp-1 trichome morphology , but the construct containing a truncation of the whole ( NT+MyTH4+FERM ) could not rescue the kcbp-1 trichome morphology ( Figure 5B ) .",
"Importantly , these findings indicated that the NT and the MyTH4 domains are essential for trichome cell shape determination . 10 . 7554/eLife . 09351 . 021Figure 5 . Genetic analyses on the role of individual domains of KCBP in trichome development .",
"( A ) Schematic diagram of the domain organization of KCBP .",
"( B ) Genetic complementation test using various truncated versions of KCBP .",
"The genotype column shows the individual constructs containing various truncations used for the genetic complementation in the kcbp-1 background .",
"The phenotype column shows leaf trichomes in various transformants .",
"Scale bars , 1 mm .",
"The asterisk indicates the GFP-KCBP genomic fusion used in the complementation test . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 021 In addition to the truncation tests , we introduced a point mutation into KCBP that produced a ‘rigor KCBP’ ( T982N ) that is defective in ATP ( Adenosine 5′-triphosphate ) hydrolysis , which is required to perform force-generating tasks ( Figure 6A ) .",
"Strikingly , the rigor-KCBP exerted specific dominant-negative effects that resulted in more-severe trichome phenotypes ( Figure 6D , G–N ) , resembling a class of trichome mutants with defects in the actin cytoskeleton ( Basu et al . , 2004 , 2005; Li et al . , 2004; Zhang et al . , 2005a ) .",
"Most trichomes in plants expressing rigor-KCBP contained two branches but had an extremely twisted and swollen branch ( Figure 6G–I ) , and some of them had extremely elongated branches , more than two times as long as the wild-type control ( Figure 6M , N ) .",
"Interestingly , a small fraction of trichomes formed three branches but still contained one or two swollen and twisted branches ( Figure 6J–L ) .",
"Moreover , we observed more-severe abnormalities in the organization of both cortical MTs and F-actin in rigor-KCBP trichomes when compared to the wild-type control ( Figure 6—figure supplement 1 , Figure 6—figure supplement 2 ) .",
"In addition , the typical , two-branched trichomes in zwichel mutants are aligned in a nearly parallel pattern on leaves , with the elongated and pointed branch toward the proximal part and another shortened and blunt-ended branch toward the distal part ( Figure 6C ) ( Hülskamp et al . , 1994; Oppenheimer et al . , 1997 ) .",
"Notably , in rigor-KCBP trichomes , the elongated and pointed branch toward the proximal part keeps unchanged , and the extremely swollen and twisting specifically takes place in another branch , which derives from the shortened and blunt-ended branch of the kcbp-1 background ( Figure 6D ) .",
"Collectively , these findings genetically supported the idea that KCBP participates in the organization of the actin cytoskeleton during trichome branching and elongation . 10 . 7554/eLife . 09351 . 022Figure 6 . The trichome phenotype of rigor-KCBP transformants .",
"( A ) A point mutation was introduced into genomic KCBP including its native regulatory elements generate the rigor-KCBP with a threonine 982-to-asparagine substitution in its ATP-binding motif .",
"The rigor-KCBP construct was introduced into the kcbp-1 mutant to allow us to detect specific dominant-negative effects .",
"( B , E )",
"The typical wild-type trichomes contain a well-extended stalk and three/four branches .",
"( C , F )",
"Most kcbp-1 trichomes contain an unextended stalk and two branches including one shortened branch , with a swollen and blunt tip .",
"( D , G–N )",
"Most rigor-KCBP trichomes contain two branches including one extremely twisted and swollen branch ( G–I ) ; a few of these trichomes show extremely elongated branches ( M , N ) .",
"A small portion of rigor-KCBP trichomes form three branches with one or two swollen and twisted branches ( J–L ) .",
"The arrowheads highlight unextended branches in a rigor-KCBP trichome .",
"The arrows highlight two elongated branches with a fine tip , but in an irregular pattern , in a rigor-KCBP trichome .",
"Scale bars are 1 mm in B–D , and 0 . 2 mm in E–N . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 02210 . 7554/eLife . 09351 . 023Figure 6—figure supplement 1 . Abnormal MT organization in rigor-KCBP trichomes .",
"( A ) The formation of the transverse MT rings that encircles the incipient primary branch ( indicated by the arrow ) is impaired in a stage 2/3 trichome .",
"( B ) Transverse MT rings and the MT-depleted zone are not well defined in the extremely swollen branch ( indicated by the arrow ) of a stage 3/4 trichome .",
"( C ) Transverse MT rings and the MT-depleted zone are not well defined in the extremely swollen branch ( indicated by the arrow ) of a stage 3/4 trichome , which contains other two short branches with relatively fine tip .",
"( D ) A stage 4/5 trichome contains three branches with relatively fine tips , but the branching pattern is irregular when compared to the wild type ( see Figure 3A–F ) .",
"Transverse MTs are perpendicular to the growth axis of elongating branches .",
"( E ) A stage 4/5 trichome contains only one extremely twisted branch with oblique MTs .",
"( F ) A stage 6 mature trichome contains two swollen branches , the longer one has a relatively fine tip and displays oblique MTs , another shorter one has a swollen , blunt tip and displays transverse MTs ( indicated by the arrow ) .",
"( G ) An irregular stage 6 mature trichome contains three branches , one is extremely long with a relatively fine tip and displays oblique MTs , the other two are seriously unextended with a blunt tip and shorter one has a swollen , blunt tip and displays oblique MTs ( indicated by the arrow ) .",
"The circled B indicates the base of trichome stalks .",
"The maximum z-projection of image stacks at 0 . 2-μm intervals was applied to all figures .",
"The photo stitching application of the Volocity ( Version 6 . 2 ) program was applied to acquire the image of the entire mature trichomes .",
"Scale bars are 20 μm in ( A–C ) and are 40 μm in ( D–G ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 02310 . 7554/eLife . 09351 . 024Figure 6—figure supplement 2 . Abnormal organization of actin filaments in rigor-KCBP trichomes .",
"( A ) The stage 1 trichomes display random meshworks of thick actin bundles .",
"( B–F )",
"The curly , intertwined , thick actin bundles dominate inside developing rigor-KCBP trichomes , and no parallel-aligned cytoplasmic actin cables and the fine F-actin cap at the branch apex shown in wild type ( see Figure 2C , I ) is observed .",
"A stage 2 trichome initiates the primary branch ( B ) .",
"A stage 3/4 trichome contains one relatively extended branch and another extremely swollen and unextended branch ( C ) .",
"A stage 3/4 trichome contains only one twisted , swollen branch ( D ) .",
"A stage 4/5 trichome contains only one extremely swollen and twisted branch ( E ) .",
"A stage 4/5 trichome contains three branches , one of which has a relatively fine tip , the others are extremely swollen with blunt tips .",
"The arrowhead indicates the fine tip , and arrows indicate blunt tips ( F ) .",
"The circled B indicates the base of trichome stalks .",
"The maximum z-projection of image stacks at 0 . 2-μm intervals was applied to all figures .",
"Scale bars , 20 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 024 To unravel the molecular and cellular basis of the essential role of the N-terminal tail of KCBP and its actin-related function , we purified various truncated versions of motorless KCBP tagged with GFP ( Figure 7—figure supplement 1 ) and mapped the specific domain that may physically bind to MTs or F-actin .",
"Single-molecule imaging showed that either the NT ( amino acids 1–115 ) or the MyTH4 domain could bind to MTs , but the FERM domain did not bind MTs; the ( NT+MyTH4 ) region and the ( NT+MyTH4+FERM ) region showed similar MT-binding activity to that of the single NT or MyTH4 domain , whereas the motorless KCBP ( NT+MyTH4+FERM+Coiled Coil ) exhibited enhanced MT-binding activity and promoted the formation of MT bundles ( Figure 7A ) .",
"Furthermore , the FERM domain directly bound to F-actin; the ( NT+MyTH4+FERM ) region showed similar F-actin-binding activity as the single FERM domain , whereas the motorless KCBP ( NT+MyTH4+FERM+Coiled Coil ) exhibited enhanced F-actin-binding and bundling activity ( Figure 7B ) . 10 . 7554/eLife . 09351 . 025Figure 7 . In vitro MT- and F-actin-binding activity of the motorless KCBP and a working model for KCBP during trichome morphogenesis .",
"( A ) The KCBP N-terminal tail containing the MyTH4 domain binds to MTs in vitro .",
"Rhodamine-labeled MTs were incubated with GFP-NT , GFP-MyTH4 , GFP-FERM , GFP-NT-MyTH4 , GFP-NT-MyTH4-FERM , GFP-NT-MyTH4-FERM-CC recombinant proteins , or control GFP , respectively .",
"GFP-NT-MyTH4-FERM-CC , GFP-NT-MyTH4-FERM , GFP-NT-MyTH4 , GFP-NT , and GFP-MyTH4 exhibited a punctate pattern along MTs .",
"Among them , GFP-NT-MyTH4-FERM-CC promoted the formation of densely packed MT bundles .",
"Scale bars , 10 μm .",
"( B ) The FERM domain binds to F-actin in vitro .",
"Actin filaments were visualized in the presence of Alexa561-phalloidin .",
"Alexa561-phalloidin labeled F-actin was incubated with GFP-NT , GFP-MyTH4 , GFP-FERM , GFP-NT-MyTH4 , GFP-NT-MyTH4-FERM , GFP-NT-MyTH4-FERM-CC recombinant proteins , or control GFP , respectively .",
"GFP-NT-MyTH4-FERM-CC , GFP-NT-MyTH4-FERM , and GFP-FERM exhibited a punctate pattern along actin filaments .",
"Among them , GFP-NT-MyTH4-FERM-CC promoted the formation of F-actin bundles .",
"Scale bars , 5 μm .",
"( C ) A working model for KCBP during trichome cell shaping ( right panel ) .",
"The three spheres on the left panel show the 3-D reconstructions of the KCBP localization , the MT configuration , and the F-actin configuration in developing trichomes ( at stage 2/3 ) , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 02510 . 7554/eLife . 09351 . 026Figure 7—figure supplement 1 . Coomassie blue-stained SDS-PAGE gel of the purified GFP-KCBP recombinant proteins with various truncations . KD , kiloDalton , indicates the mass of molecular markers . DOI: http://dx . doi . org/10 . 7554/eLife . 09351 . 026 Taken together , these results demonstrated that KCBP contains a second MT-binding domain spanning the NT and the MyTH4 region , and can also bind to F-actin via the FERM domain , co-ordinating the interplay between MTs and F-actin .",
"We propose that , during trichome morphogenesis , KCBP forms a cortical gradient and highly accumulates at trichome-branching sites and apical regions of elongating branches , thus , integrating MTs and F-actin to assemble the required cytoskeletal configuration for trichome branching , elongation , and tip sharpening ( see the model in Figure 7C ) ."
],
[
"Due to its unique nature , KCBP is one of the most studied kinesins within the plant kingdom; numerous studies have investigated its biochemical characteristics and potential regulatory roles in cell division .",
"However , since KCBP was cloned and linked to the trichome phenotype of the zwichel mutation in Arabidopsis , in-depth understanding the in vivo function of KCBP , especially of the roles of the mysterious MyTH4-FERM tandem , has been halted for over a decade .",
"Here , for the first time , we provided high-quality live-cell images showing KCBP's cortical MT localizations in Arabidopsis epidermal pavement cells , hypocotyl cells , and trichome cells ( Figures 1 , 2 , Figure 1—figure supplement 2B , Figure 2—figure supplement 1; Videos 1–4 ) ; thus , this work reveals the direct evidence linking the interphase KCBP function with trichome morphogenesis .",
"Furthermore , although KCBP has long been assumed to be part kinesin and part myosin , the direct evidence linking KCBP with the actin cytoskeleton is still lacking .",
"The MyTH4 domain and the FERM domain are usually found in a tandem in myosins of the Myosin VII , X , and XV families , which differ substantially from the Myosin VIII and XI families found in the plant lineage .",
"Myo VII contains a pair of MyTH4-FERM tandem domains , the second FERM domain of the Drosophila Myo VIIa binds to actin at high density ( Yang et al . , 2009 ) .",
"Unlike Myo VII , the MyTH-FERM tandem of Myo X , but not either of the isolated domains , was shown to bind to MTs ( Weber et al . , 2004; Kerber and Cheney , 2011 ) and also binds its cargo proteins , including β-integrins and the axonal guidance receptor DCC ( Zhang et al . , 2004; Zhu et al . , 2007; Wei et al . , 2011 ) .",
"Interestingly , in Drosophila myosin XV Sisyphus , the MyTH4 domain binds to MTs , whereas the FERM domain binds to various cargos including the MT-severing protein , Katanin p60 subunit ( Liu et al . , 2008 ) .",
"In the present study , by single-molecule imaging , we dissected the functional domain of the N-terminal tail and demonstrated that the NT plus the MyTH4 domain functions as a second MT-binding region besides the motor domain ( Figure 7A ) , and the FERM domain physically binds to F-actin ( Figure 7B ) .",
"Thus , this work provides the first direct evidence linking the MT-based KCBP motor with the actin cytoskeleton , and further indicates that KCBP functions as a hub protein to mediate the interplay between MTs and actin filaments .",
"Most importantly , our work solves the long-standing mystery of the function of the unique MyTH4-FERM domain in KCBP .",
"We propose that the introduction of the MyTH4-FERM domain into the N-terminal tail region of a Kinesin-14 motor during evolution conferred the novel functions of KCBP: the NT and the MyTH4 domain act synergistically to bind strongly to MTs , but still maintain the MT-binding activity of the individual domains , and the FERM domain carries out the novel actin-binding activity , thus , orchestrating the interaction between MTs and the actin cytoskeleton .",
"Our speculation is supported by the genetic evidence that the KCBP containing the NT-MyTH4 truncation could not rescue the kcbp-1 phenotype , but either the NT-truncated version of KCBP or the MyTH4-truncated version could fully complement the kcbp-1 phenotype ( Figure 5B ) .",
"These findings indicate that the second MT-binding domain ( NT+MyTH4 ) is essential for trichome morphogenesis , yet either the NT or the MyTH4 domain is fully functional , and compensate each other to bind to MTs .",
"Surprisingly , either the FERM-truncated KCBP or the MyTH4-FERM-truncated KCBP could rescue the kcbp-1 phenotype ( Figure 5B ) .",
"This unexpected finding indicates that the FERM domain is dispensable ( Figure 5B ) , despite its actin-binding activity .",
"However , our spatio-temporal observation revealed that loss-of-function mutation of KCBP results in defects in both MTs and F-actin ( Figures 3 , 4 ) .",
"Moreover , the interaction between KCBP and the actin cytoskeleton was genetically highlighted by the phenotype of plants expressing the dominant-negative rigor-KCBP construct , which show twisting , swollen trichome phenotype , similar to aberrant trichome morphology in a class of mutants of actin-related genes ( Figure 6 , Figure 6—figure supplement 1 , Figure 6—figure supplement 2 ) .",
"Therefore , we proposed that KCBP indeed regulates actin dynamics , yet its actin-related function is redundant with other proteins in a same pathway .",
"One explanation is that KCBP may function redundantly with KCH kinesins in the same kinesin-14 subfamily , which is extremely expanded in plants .",
"KCH kinesins not only are minus-end-directed kinesins as with KCBP but bind to both MTs and F-actin , acting as dynamic linkers between MTs and actin filaments ( Preuss et al . , 2004; Frey et al . , 2009; Petrasek and Schwarzerova , 2009; Schneider and Persson , 2015 ) .",
"So , further live-cell imaging and genetic analyses are required to investigate the potential functional redundancy between KCHs and KCBP .",
"Another possibility is that KCBP may be partially redundant with the actin nucleator ARP2/3 complex as well as the W/SRC complex .",
"The second scenario is supported by previous findings that KCBP acts in the same genetic pathway with DIS1/ARP3 , a member of the ARP2/3 complex , and IBT1/SCAR2 , a subunit of the W/SRC complex ( Schwab et al . , 2003; Zhang et al . , 2005a , 2005b ) , and that ARP2/3 partially colocalizes with MTs in pavement cells and the W/SRC subunit ABIL3 binds to cortical MTs when overexpressed ( Jorgens et al . , 2010; Zhang et al . , 2013 ) .",
"Interestingly , ARP2/3 and BRK1 , a subunit of the W/SRC complex , were shown to specifically accumulate at the branch apex ( Dyachok et al . , 2008; Yanagisawa et al . , 2015 ) .",
"Thus , most likely , KCBP functions redundantly with ARP2/3 and W/SRC complexes to organize the cortical F-actin at the branch apex .",
"Trichome morphogenesis represents a unique growth mode comprising trichome branching and highly polarized diffuse growth of branch elongation accompanied by a tip-sharpening process ( Beilstein and Szymanski , 2004; Sambade et al . , 2014; Yanagisawa et al . , 2015 ) .",
"However , the exact nature of the cytoskeletal organization that enables the unique growth mode has not yet been elucidated .",
"Here , we provided live-cell images in high quality , showing the spatio-temporal dynamics and 3-D reconstruction of cytoskeletal organization during trichome morphogenesis .",
"Our observations clearly showed that the transverse MT rings encircling elongating branches and the MT-depleted zone at the extreme apexes are required for trichome elongation and tip sharpening , consistent with previous reports ( Beilstein and Szymanski , 2004; Yanagisawa et al . , 2015 ) .",
"Moreover , our observations also revealed that parallel actin cables along the growth axis and the cortical F-actin cap are required for trichome elongation and tip sharpening .",
"Several previous reports also documented the cortical F-actin cap near the branch tips of elongating trichomes , but its configuration remains a matter of debate because of the technical challenges of live-cell imaging of trichome tips .",
"By immunostaining , despite the very dim signal , the cortical F-actin cap was detected as roughly showing a transverse pattern ( Zhang et al . , 2005a ) , but phalloidin staining showed a clear F-actin cap mainly in a longitudinal pattern ( Yanagisawa et al . , 2015 ) .",
"Here , we acquired high-quality images in live trichomes using ABD2-GFP labeling , and these clearly showed the F-actin cap mainly in a transverse pattern ( Figures 2 , 4; Video 3 ) .",
"We consider that live-cell imaging with ABD2-GFP gives a weaker signal , but should reflect the natural scenario .",
"We also note that the fine F-actin cap at trichome branch tips is distinct from the actin fringe that is seen in a longitudinal pattern at the subapical domain of pollen tubes , which undergo tip growth ( Lovy-Wheeler et al . , 2006; Cheung et al . , 2010; Wu et al . , 2010; Su et al . , 2012 ) .",
"Instead , the specific cytoskeletal configuration , especially for the MT-depleted zone and the cortical F-actin cap near the trichome branch tip , may be associated with the unique , polarized diffuse growth of trichome branch elongation accompanied by tip sharpening during trichome cell shaping .",
"Strikingly , in kcbp-1 trichomes , these specific cytoskeletal systems are compromised or disrupted ( Figures 3 , 4 ) , indicating the essential role of KCBP in assembly of the required cytoskeletal systems for trichome cell shape control .",
"This notion is further supported by our important discovery that KCBP exhibits a cortical gradient along elongating branches and concentrates at the extreme apexes , where are colonized by the MT-depleted zone and the cortical F-actin cap ( Figure 2 ) .",
"Our findings indicated that KCBP is required for establishing the transverse MT rings to promote branch elongation , and that , through the FERM domain , KCBP may dominantly bind to cortical F-actin .",
"This binding occurs at higher density at the extreme apex , as suggested by previous findings ( Yang et al . , 2009 ) , and plays a critical role in the assembly of the F-actin cap , which then facilitates the maintenance of the MT-depleted zone to promote rapid elongation of trichome branches and tip sharpening ( see model in Figure 7C ) .",
"Recent work showed that ARP2/3 and BRK1 , a subunit of the W/SRC complex , specifically accumulate at the branch apex , and plasma-membrane associated ARP2/3 and W/SRC complexes likely nucleate cortical F-actin at the branch apex ( Dyachok et al . , 2008; Yanagisawa et al . , 2015 ) .",
"Therefore , most likely , KCBP functions in concert with the ARP2/3 complex as well as the W/SRC complex to assemble the specific cytoskeletal configuration near the branch tip region during trichome cell shaping .",
"It would also be interesting to examine whether a local MT disassembly machinery occurs at the extreme branch apex to generate the MT-depleted zone , but an intriguing hypothesis is that KCBP might recruit the Katanin p60 subunit via its FERM domain , as suggested by the previous finding from the MyTH4-FERM-containing Sisyphus myosin motor ( Liu et al . , 2008 ) , to locally disassemble MTs .",
"During trichome branching , we propose that once the branching program initiates , by a yet-unknown mechanism , the branching site becomes an isotropic zone with randomized sparse MT network; then , KCBP is required to rapidly colonize that area and organize the new transverse MT rings and the cortical F-actin cap to prime the initiation and subsequent elongation of the incipient branch .",
"Previous studies showed that treatment with the MT stabilizing drug taxol can produce second branches in kcbp trichomes , albeit at the relatively low frequency of 5 . 5% , indicating that transient stabilization of MTs could compensate for the loss of KCBP activity ( Mathur and Chua , 2000; Buschmann et al . , 2009 ) .",
"Importantly , we revealed that KCBP exhibits non-processive movement by the live-cell , single-molecule ( particle ) motility assay ( Figure 1C , D ) , and that the second MT-binding domain is essential , based on the genetic complementation tests ( Figure 5 ) ; our data further support the bundling or crosslinking role of KCBP in the formation of the MT rings at the branching site .",
"KCBP may further function in concert with AN1 , a CtBP/BARS protein implicated in Golgi membrane trafficking and interacts with KCBP both physically and genetically ( Folkers et al . , 2002; Kim et al . , 2002; Minamisawa et al . , 2011 ) , to integrate cytoskeletal dynamics and membrane trafficking to promote trichome branch initiation .",
"In conclusion , KCBP acts as a central player or a hub integrating the MT and actin cytoskeleton and further orchestrates their interplay to generate a specific cytoskeletal configuration that is required for trichome branching , elongation , and tip sharpening ( see model in Figure 7C ) .",
"Our findings reveal the in vivo function of the plant unique kinesin KCBP and further provide significant insights into molecular and cellular mechanisms that will close the knowledge gap between the cytoskeletal regulation and cell shape control , especially for the shaping of leaf trichome cells , which represents a unique growth mode comprising trichome branching , polarized branch elongation , and tip sharpening ."
],
[
"A . thaliana ecotype Columbia ( Col-0 ) was used as the genetic background in this study .",
"Among the zwichel ( zwi ) alleles , the zwi-3 allele and zwi-w2 allele were frequently used in previous studies .",
"The zwi-3 allele in Columbia ecotype background , which is expected to produce a truncated ZWI protein ( KCBP ) at 522 amino acids position lacking the coiled-coil and motor domains , shows the typical , strong zwichel trichome phenotype ( Oppenheimer et al . , 1997; Krishnakumar and Oppenheimer , 1999 ) .",
"And the zwi-w2 allele in RLD ecotype background shows weaker zwichel trichome phenotype , containing a small portion ( about 15 . 9% ) of three-branched trichomes , because sequencing analysis revealed that although a C to T transition results in a stop codon at amino acid position 72 , re-initiation of translation likely occurs using the in-frame AUG ∼20 bp downstream from that mutation site as the start codon ( Folkers et al . , 2002 ) .",
"To find a strong or null KCBP/ZWICHEL allele in Columbia ecotype background , we searched the Salk collection in the Arabidopsis Biological Resource Center , and finally selected the accession of Salk_031704 , which contains a T-DNA insertion in the third exon ( 22 exons in the KCBP/ZWICHEL gene in total ) and shows typical , strong zwi trichome phenotype .",
"Coincidently , the Salk_031704 strong allele was used in recent studies and was designated either as kcbp-1 ( Humphrey et al . , 2015 ) or as zwiA ( accession is N531704 ) , which was ordered from the Nottingham Arabidopsis Stock Centre ( Buschmann et al . , 2015 ) .",
"The ABD2-GFP marker line is kindly provided by Prof . Shanjin Huang ( Tsinghua University ) .",
"The GFP-TUB6 marker line was described in our recent study ( Liu et al . , 2014 ) , and the mCherry-TUB6 marker line was generated using the identical method , only replacing the GFP-encoding sequence with the mCherry-encoding sequence .",
"Plasmid construction and generation of GFP-KCBP and rigor-KCBP lines can be found in the following parts , correspondingly .",
"Various cross combinations ( the GFP-KCBP line and the mCherry-TUB6 marker line; the kcbp-1 mutant and the GFP-TUB6 marker line; the rigor-KCBP line and the GFP-TUB6 marker line; the kcbp-1 mutant and the ABD2-GFP marker line; the rigor-KCBP line and the ABD2-GFP marker line ) were performed to obtain corresponding materials to observe the dynamics of KCBP , MTs , and F-actin , respectively .",
"Plant growth conditions and transformation procedures were as described previously ( Liu et al . , 2014 ) .",
"The tiny first or second true leaves in the seedlings at 10-day-old stage were dissected and used for live-cell imaging of trichomes at various developmental stages .",
"In general , the Phusion DNA polymerase with high-fidelity ( New England Biolabs , Beverly , MA , United States ) was used to amplify all the required gene products in this study , Fusion PCR was applied to get the GFP-KCBP fusion fragment and various constructs containing individual domain truncations ( Szewczyk et al . , 2006 ) , and the gateway-based technology was applied to get final expression constructs , please refer to our recent study for detailed description ( Liu et al . , 2014 ) .",
"To get the GFP-KCBP construct , the genomic fragment of the KCBP gene , including its coding sequence and the 761 bp upstream fragments from the translation initiation codon ATG , was amplified from the genomic DNA with primers of KCBPP-F and GA-KCBP-R .",
"The amplified fragment was cloned into the pENTR/D-TOPO vector by a TOPO-based cloning strategy to get the Entry 1 clone according to manufacturer's instruction ( Invitrogen , Carlsbad , CA , United States ) .",
"Then , a VisGreen version ( Teerawanichpan et al . , 2007; Liu et al . , 2014 ) of GFP tag was added to the NT of KCBP by the following manipulations .",
"The promoter region of the KCBP gene was amplified with primers of KCBPP-F and KCBPP-R; the GFP-encoding sequence was amplified with primers of PGFP-F and GFP-R; the first part of KCBP-coding sequence ( 1–2050 bp ) was amplified with primers of KCBP1X-F and KCBP1X-R .",
"The above-mentioned three PCR fragments were further linked together by Fusion PCR using primers of KCBPP-F and KCBP1X-R .",
"The resulting PCR fragment was subsequently cloned into the pENTR/D-TOPO vector to get the Entry 2 clone .",
"Finally , both the Entry 1 vector and Entry 2 vector were digested by Not I and Xba I , and further ligation reaction was conducted between the gel-purified fragment containing the latter part of KCBP ( 1951–6023 bp ) and the fragment containing the promoter , GFP , and the first part of KCBP genomic sequence , the resulting Entry 3 clone was delivered into pEarleyGate302 by recombination reaction to get the final GFP-KCBP construct .",
"A series of constructs containing various domain truncations were made by the following manipulations .",
"To get the KCBP-∆MyTH4 ( KCBP lacking 117–275 amino acids ) construct , the fragment containing promoter and 1–435 bp KCBP genomic sequence was amplified with primers of KCBPP-F and KCBP-∆MyTH4-R , and the 992–6023 bp of the KCBP genomic sequence was amplified with primers of KCBP-∆MyTH4-F and GA-KCBP-R , then the two PCR fragments were linked together by Fusion PCR using primers of KCBPP-F and GA-KCBP-R .",
"Finally , the resulting PCR fragment was cloned into pENTR/D-TOPO vector and was then delivered into pEarleyGate302 to get the final KCBP-∆MyTH4 construct .",
"The same strategy was used to generate constructs of KCBP-∆NT ( KCBP lacking 2–121 amino acids ) , KCBP-∆NT-MyTH4 ( KCBP lacking 2–275 amino acids ) , KCBP-∆FERM ( KCBP lacking 275–497 amino acids ) , KCBP-∆MyTH4-FERM ( KCBP lacking 116–505 amino acids ) , KCBP-∆NT-MyTH4-FERM ( KCBP lacking 2–505 amino acids ) , KCBP-∆CC-Motor-CBD ( KCBP lacking 532–1266 amino acids ) , and KCBP-∆CBD ( KCBP lacking 1210–1266 amino acids ) primers can be found in the Supplementary file 1 .",
"To get the rigor-KCBP construct , we introduced threonine ( ACT ) 982-to-asparagine ( AAC ) mutation by the PCR-based mutagenesis .",
"In detail , we designed a pair of overlapping primers ( KCBP-T982N-R and KCBP-T982N-F ) with the desired nucleotide changes at the target site , and amplified the front half with primers of KCBP2X-F and KCBP-T982N-R , and that latter half with primers of KCBP-T982N-F and GA-KCBP-R , then the two fragments were linked together by Fusion PCR using primers of KCBP2X-F and GA-KCBP-R .",
"The resulting PCR fragment was cloned into the pENTR/D-TOPO vector to get the Entry 4 clone .",
"Finally , both the Entry 1 vector and the Entry 4 vector were digested by Not I and Xba I , and further ligation reaction was conducted between the gel-purified fragment containing the latter part of KCBP containing the threonine 982-to-asparagine mutation and the fragment containing the promoter and the first part of KCBP genomic sequence , the resulting Entry 5 clone was delivered into pEarleyGate302 to get the final rigor-KCBP construct .",
"The KCBP ( At5G65930 ) genomic fragment comprising its endogenous promoter and coding region was used for genetic complementation tests .",
"Plasmid construction for the GFP-KCBP construct , the rigor-KCBP construct , and other constructs containing various domain truncations was all based on the above-mentioned KCBP fragment .",
"The constructs were introduced into the kcbp-1 mutant .",
"The T3 homozygous transgenic lines were used to observe trichome phenotype under a fully automated Stereo Microscope ( Leica M205 FA ) with Leica Application Suite V4 . 2 .",
"The images of trichomes were a maximum z-projection of image series acquired by the Leica LAS Multifocus program .",
"Live-cell imaging was carried out under a spinning disk confocal microscope ( UltraView VoX , Perkin Elmer , Beaconsfield , Buckinghamshire , UK ) equipped with the Yokogawa Nipkow CSU-X1 spinning disk scanner , Hamamatsu EMCCD 9100-13 , Nikon TiE inverted microscope with the Perfect Focus System as described previously ( Liu et al . , 2014 ) .",
"Acquired images were processed and analyzed using Volocity ( Perkin Elmer ) , Image J ( http://rsbweb . nih . gov/ij ) , MetaMorph ( Molecular Devices , Sunnyvale , CA , United States ) .",
"The run-length distribution and the velocity distribution of GFP-KCBP were calculated in Origin software ( OriginLab ) by frequency counts .",
"The mean values and 95% confidence interval were calculated in SAS ( SAS Software ) , as described previously ( Kong et al . , 2015 ) .",
"The full-length cDNA fragments of KCBP were amplified from the Arabidopsis cDNA with primers of KCBP1X-F and GA-KCBP-R , and the GFP-coding sequence amplified with primers of GFP-F and GFP-R , then the two PCR fragments were linked together by Fusion PCR using primers of GFP-F and GA-KCBP-R .",
"The resulting PCR fragment was cloned into the pENTR/D-TOPO vector to get the Entry 6 clone .",
"The cDNA fragments encoding polypeptide of GFP-NT-MyTH4-FERM-CC ( 1–749 amino acids ) , GFP-NT-MyTH4-FERM ( 1–614 amino acids ) , GFP-NT-MyTH4 ( 1–275 amino acids ) , GFP-MyTH4 ( 116–275 amino acids ) , GFP-FERM ( 276–503 amino acids ) , GFP-NT ( 1–115 amino acids ) , and GFP were generated based on the Entry 6 clone .",
"The truncated fragments were digested with Sal I and Not I and were reconstructed into the pET-28a vector to get final expression constructs .",
"Primers can be found in the Supplementary file 1 .",
"Finally , the expression constructs were transformed into Escherichia coli strain Transetta ( DE3 , TransGen Biotech , Beijing , China ) to induce expression .",
"The recombinant proteins were purified using nickel-nitrilotriacetic acid ( Ni-NTA ) resin following procedures described by the manufacturer ( Qiagen , Hilden , Germany ) .",
"Fractions containing the protein were collected , combined , and dialyzed overnight against PEM buffer ( 80 mM PIPES , 1 mM EGTA ( ethylene glycol tetraacetic acid ) , and 1 mM MgSO4 , pH 6 . 9 ) .",
"Protein concentration was determined by a Bio-Rad protein assay kit .",
"Protein samples of 3 μg were analyzed by SDS-PAGE ( Sodium Dodecyl Sulfate Polyacrylamide Gel Electropheresis ) .",
"The purified porcine brain tubulin labeled with NHS-rhodamine was kindly provided from Prof . Tonglin Mao ( China Agricultural University ) .",
"Taxol-stabilized NHS-rhodamine MTs were incubated with 1 μM GFP-NT , 1 μM GFP-MyTH4 , 1 μM GFP-FERM , 1 μM GFP-NT-MyTH4 , 1 μM GFP-NT-MyTH4-FERM , 1 μM GFP-NT-MyTH4-FERM-CC , and 1 μM control GFP , respectively , in PEM buffer at equal molar ratios for 30 min at room temperature , modified from a previous study ( Liu et al . , 2013 ) .",
"Fluorescent images of MTs and various GFP-KCBP truncated proteins were visualized using a Zeiss inverted fluorescence microscope ( Axio Observer Z1 ) with a Zeiss Plan-Apochromat 100× oil immersion objective ( NA = 1 . 4 ) .",
"The rabbit skeletal muscle actins were provided by Prof . Shanjin Huang ( Tsinghua University ) .",
"F-actin ( 3 µM ) was incubated with 1 μM GFP-NT , 1 μM GFP-MyTH4 , 1 μM GFP-FERM , 1 μM GFP-NT-MyTH4 , 1 μM GFP-NT-MyTH4-FERM , 1 μM GFP-NT-MyTH4-FERM-CC , and 1 μM control GFP at the indicated concentrations at room temperature for 30 min and then labeled with 3 µM Alexa561-phalloidin ( Invitrogen ) .",
"Actin filaments were subsequently diluted to a final concentration of 10 nM in fluorescence buffer ( 10 mM imidazole , pH 7 . 0 , 50 mM KCl , 2 mM MgCl2 , 1 mM EGTA , 100 mM DTT ( Dithiothreitol ) , 100 µg/ml Glucoxidase , 15 mg/ml Glucose , 20 µg/ml catalase , and 0 . 5% methylcellulose ) , modified from a previous study ( Wu et al . , 2010 ) .",
"The diluted samples were visualized using a Zeiss inverted fluorescence microscope ( Axio Observer Z1 ) with a Zeiss Plan-Apochromat 100× oil immersion objective ( NA = 1 . 4 ) .",
"A standard PCR-based method was used to identify the T-DNA insertion in kcbp-1 ( Humphrey et al . , 2015 ) , as described by Kong et al . ( 2015 ) .",
"Gene-specific primers ( 031704-LP and 031704-RP ) were used to amplify an approximate 1000 bp DNA fragment in KCBP .",
"The T-DNA insertion was detected using 031704-RP and the left-border primer ( LBb1 . 3 ) .",
"For RT-PCR analysis of transcription level of KCBP in the kcbp-1 mutant , total RNA was extracted using Trizol reagent Invitrogen and was used for first strand cDNA synthesis by the SuperScript III First-Strand Synthesis System ( Life Technologies , Carlsbad , CA , United States ) with oligo ( dT ) 18 primers .",
"Then , the cDNA was used as a template for PCR reactions using primers shown in the primer list .",
"UBQ5 was used as control ."
]
] | [
"Microtubules ( MTs ) and actin filaments ( F-actin ) function cooperatively to regulate plant cell morphogenesis .",
"However , the mechanisms underlying the crosstalk between these two cytoskeletal systems , particularly in cell shape control , remain largely unknown .",
"In this study , we show that introduction of the MyTH4-FERM tandem into KCBP ( kinesin-like calmodulin-binding protein ) during evolution conferred novel functions .",
"The MyTH4 domain and the FERM domain in the N-terminal tail of KCBP physically bind to MTs and F-actin , respectively .",
"During trichome morphogenesis , KCBP distributes in a specific cortical gradient and concentrates at the branching sites and the apexes of elongating branches , which lack MTs but have cortical F-actin .",
"Further , live-cell imaging and genetic analyses revealed that KCBP acts as a hub integrating MTs and actin filaments to assemble the required cytoskeletal configuration for the unique , polarized diffuse growth pattern during trichome cell morphogenesis .",
"Our findings provide significant insights into the mechanisms underlying cytoskeletal regulation of cell shape determination ."
] | [
"Within a cell , a structure called the cytoskeleton provides a scaffold that supports the cell's shape .",
"In both plant and animal cells , this scaffold is largely made of tube-like structures called microtubules and a web of filaments made of a protein called actin .",
"In a plant called Arabidopsis thaliana , specialized hair-like cells usually protrude from the surface of leaves .",
"These cells—called trichomes—are widely used to study how the cytoskeleton influences plant cell shape .",
"Microtubules are required to regulate the number of branches these trichomes have .",
"The actin filaments control the length of the trichomes , but it is not known how the two elements of the cytoskeleton interact to determine the overall shape of the trichomes .",
"One protein that might help to co-ordinate microtubules and actin filaments is called kinesin-like calmodulin-binding protein ( or KCBP for short ) .",
"This protein is a type of motor protein known as a kinesin and can move along microtubules , but it also contains a section called the MyTH4-FERM domain , which is found in another type of motor protein .",
"When KCBP is removed from A . thaliana , the leaf trichomes are shorter and blunter than normal and have fewer branches .",
"Here , Tian et al . investigated the role of KCBP in trichome development using a combination of genetic and microscopy techniques .",
"The experiments show that the MyTH4-FERM domain of KCBP allows the protein to bind to both actin filaments and microtubules .",
"During trichome formation , KCBP associates with the microtubules that are attached to the membrane that surrounds the cell .",
"Particularly , high levels of the protein accumulate at the sites where branches will form and at the tips of developing branches .",
"These branch tips have few microtubules but have many actin filaments .",
"Further experiments revealed that KCBP acts as a hub that brings together microtubules and actin filaments to modulate the cytoskeleton during trichome formation .",
"Tian et al . 's findings provide new insights into how the cytoskeleton influences plant cell shape .",
"A future challenge is to understand how KCBP prevents microtubules forming at the tips of growing branches during trichome formation ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression"
] | Co-transcriptional R-loops are the main cause of estrogen-induced DNA damage | elife-17548-v2 | [
[
"The hormone estrogen ( E2 , 17β-estradiol ) is essential for the development and function of mammary tissue ( Bieche et al . , 2001 ) , stimulating a transcriptional program that drives breast cell proliferation .",
"Paradoxically , E2 exposure is also associated with an elevated risk of breast carcinogenesis ( Liehr , 2000; Yager and Davidson , 2006 ) .",
"Specifically , higher E2 serum concentrations and longer lifetime E2 exposure are both positively correlated with an increased incidence of sporadic breast cancer ( Clemons and Goss , 2001; Colditz , 1998; Hilakivi-Clarke et al . , 2002 ) .",
"Breast cancers exhibit a large number of chromosomal abnormalities , including mutations and copy number alterations ( Nik-Zainal et al . , 2016 ) .",
"Moreover , E2 leads to DNA damage in breast epithelial cells that express the estrogen receptor ( ER ) ( Liehr , 2000; Williamson and Lees-Miller , 2011 ) , and in rat models , E2 stimulation is causally linked to chromosome instability and aneuploidy ( Li et al . , 2004 ) .",
"Despite strong links between estrogen and genomic instability , the molecular mechanism by which E2 causes this instability in breast cancer is unclear .",
"Functionally , E2 is a key transcriptional regulator that governs the expression of thousands of genes in breast cells ( Cheung and Kraus , 2010; Hah et al . , 2011 ) through its association with the nuclear hormone receptors ER-α and ER-β .",
"Upon translocation into the nucleus , the E2-ER complex binds to estrogen-response elements or to other transcription factors , thus altering gene expression ( Marino et al . , 2006 ) .",
"Among the genes induced by E2 are a number that are important for cell proliferation ( Gong et al . , 2014 ) .",
"Thus , one proposed model to explain E2-induced genome instability is that the uncontrolled proliferation driven by deregulation of genes such as Cyclin D1 causes replication stress and DNA damage ( Caldon , 2014; Halazonetis et al . , 2008 ) .",
"However , another relatively unexplored hypothesis is that the dramatic increase in transcription itself contributes to E2-induced DNA damage .",
"Co-transcriptional structures known as R-loops form upon hybridization of nascent RNA with the template DNA strand and are prevalent in mammalian genomes .",
"These structures are proposed to serve regulatory roles in the cell including the patterning of promoter chromatin and the facilitation of transcription termination ( Ginno et al . , 2012; Skourti-Stathaki et al . , 2011 ) .",
"In certain contexts , however , R-loops may serve as precursors for DSBs .",
"The coordinated cleavage of an R-loop at the switch region in B-cells facilitates recombination and generates antibody diversity ( Santos-Pereira and Aguilera , 2015; Yu et al . , 2003 ) .",
"Changes in R-loop levels , moreover , are clearly associated with increased DNA damage and genomic instability .",
"For example , the loss of RNA processing and R-loop regulatory factors results in an increase in R-loops and R-loop-dependent DNA damage in both yeast and human cells ( Garcia-Rubio et al . , 2015; Huertas and Aguilera , 2003; Li and Manley , 2005; Paulsen et al . , 2009; Sollier et al . , 2014; Tuduri et al . , 2009 ) .",
"Additionally , highly transcribed loci may be more prone to R-loop formation .",
"This is evidenced by the enrichment of R-loops at rDNA repeats ( El Hage et al . , 2010 ) and the binding of the R-loop suppressing factor Npl3 preferentially at the most highly transcribed genes ( Santos-Pereira et al . , 2013 ) .",
"While many studies have shown that the absence of RNA processing factors can lead to R-loop-dependent DNA damage , little is known about how more physiological changes , such as those induced by hormones or environmental stimuli , may influence R-loop formation and DNA damage in human cells .",
"Dramatic and rapid changes in gene expression could challenge co-transcriptional processing pathways resulting in R-loop formation .",
"Here , we tested whether the formation of R-loops following increased gene expression is a source of E2-induced DNA damage in ER-positive breast epithelial cells .",
"We report that E2 treatment leads to a dramatic increase in R-loops , most strongly at E2-responsive genes .",
"E2-responsive genes , moreover , are enriched in breast cancer rearrangements .",
"We find that E2 treatment also leads to a significant increase in DNA replication- and transcription-dependent DNA damage signaling and DSBs through an R-loop dependent process .",
"Collectively , our data indicate that physiological changes in transcription can drive R-loop formation to promote genomic instability in human cells ."
],
[
"We first sought to characterize the DNA damage that arises in response to E2 stimulation in ER-positive breast epithelial cells ( Williamson and Lees-Miller , 2011 ) .",
"To do so , we examined phosphorylation of the histone variant H2AX ( P-H2AX ) , a marker of DNA damage , after treatment of hormone-starved MCF7 cells with different doses of E2 .",
"We observed a clear , dose-dependent increase in P-H2AX intensity 24 hr after E2 addition ( Figure 1A , B ) .",
"E2 treatment also resulted in an increase in 53BP1 foci ( Figure 1—figure supplement 1 ) , another marker of damaged DNA .",
"Hormone-free culture conditions cause ER-positive cells to withdraw from the cell cycle , and low P-H2AX levels arise during normal DNA replication ( Mirzoeva and Petrini , 2003 ) .",
"Thus , we asked whether an increase in the percentage of cells in S phase following E2 stimulation could account for the increased P-H2AX signal .",
"Notably , cells treated with 1 or 100 nM E2 had similar cell cycle profiles ( Figure 1—figure supplement 2 ) , despite significantly different P-H2AX levels , suggesting that the increase in DNA damage was not due to differences in the S phase population .",
"Importantly , ER-negative MCF10A breast epithelial cells showed no significant change in P-H2AX signal when treated with 100 nM E2 ( Figure 1—figure supplements 3A , B ) , consistent with the previous finding that E2-induced DNA damage depends on the ER ( Williamson and Lees-Miller , 2011 ) .",
"We also asked if E2 treatment leads to an increase in DSBs or if the observed changes in DNA damage response ( DDR ) signaling are induced by some alternative mechanism .",
"The neutral comet assay provided direct evidence that E2 leads to DSB formation ( Figure 1C , D ) . 10 . 7554/eLife . 17548 . 003Figure 1 . Estrogen induces DNA damage and DSBs in a replication-dependent manner .",
"( A ) Immunostaining for P-H2AX in MCF7 cells treated with 0 , 1 , 10 , or 100 nM E2 for 24 hr . ( B ) Quantification of P-H2AX immunostaining for data shown in ( A ) , ****p<0 . 0001 .",
"n = 2 biological replicates .",
"( C ) Neutral comet assay in MCF7 cells treated with 0 , 10 , or 100 nM E2 for 24 hr . ( D ) Quantification of the neutral comet tail moment for data in ( C ) .",
"****p<0 . 0001 .",
"n = 4 biological replicates .",
"≥50 comets/condition .",
"( E ) Quantification of P-H2AX immunostaining per nucleus in cells treated with 0 , 10 or 100 nM E2 for indicated time prior to fixation .",
"min = minutes , h = hours .",
"****p<0 . 0001 .",
"n = 3 biological replicates .",
"( F ) Quantification of the percent of P-H2AX positive cells and EdU staining in cells treated with 0 or 100 nM E2 for the indicated time .",
"Cells were pulsed for 30 min with 10 μM EdU prior to fixation .",
"Error bars represent SD of 2 biological replicates .",
"( G ) Immunostaining for EdU and P-H2AX for the experiment described in ( F ) .",
"( H ) Quantification of P-H2AX immunostaining per nucleus in cells treated with 0 or 100 nM E2 concurrently with DMSO or 1 μM Cdc7 inhibitor PHA 767491 for 14 hr .",
"Cells were pulsed with 10 μM EdU 30 min prior to fixation .",
"n = 3 biological replicates .",
"( I ) Quantification of P-H2AX immunostaining in MCF7 cells treated with 0 or 100 nM E2 for 12 hr prior to the addition of 0 . 8 μM flavopiridol or DMSO for 2 hr .",
"Cells were pulsed with 10 μM EdU for 30 min prior to harvesting .",
"****p<0 . 0001 .",
"n = 3 biological replicates .",
"For all graphs: box and whiskers represent 25–75 and 10–90 percentiles , respectively .",
"The line represents the median value .",
"a . u . = arbitrary units .",
"Associated p-values are from non-parametric Mann-Whitney rank sum t-test .",
">1000 cells/condition unless noted . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 00310 . 7554/eLife . 17548 . 004Figure 1—figure supplement 1 . Immunostaining for 53BP1 in MCF7 cells either treated with 0 or 100 nM E2 for 24 hr . Nuclei stained with Hoechst . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 00410 . 7554/eLife . 17548 . 005Figure 1—figure supplement 2 . FACS profiles of MCF7 cells treated with 0 nM E2 , 1 nM E2 , or 100 nM E2 for 24 hr . Cells were pulsed with 25 μM BrdU for 30 min prior to fixation .",
"DNA content is marked by propidium iodide ( x-axis ) and BrdU incorporation is shown on the y-axis .",
"The percentage of cells in each of the four cell-cycle quadrants is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 00510 . 7554/eLife . 17548 . 006Figure 1—figure supplement 3 . Effect of E2 on MCF10A cells .",
"( A ) Quantification of P-H2AX immunostaining in MCF10A cells treated with 0 or 100 nM E2 for 24 hr . ns = not significant ( non-parametric Mann-Whitney rank sum t-test ) .",
"n = 3 biological replicates .",
">250 cells/condition .",
"a . u . = arbitrary units .",
"( B ) Western blot for ER-α in MCF10A and MCF7 cells . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 00610 . 7554/eLife . 17548 . 007Figure 1—figure supplement 4 . Quantification of the percent of cells positive for EdU incorporation after treatment with 0 or 100 nM E2 for the indicated length of time and then pulsed with 10 μM EdU for 30 min prior to fixation . h = hours .",
"Errors bars represent SD of 2 biological replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 00710 . 7554/eLife . 17548 . 008Figure 1—figure supplement 5 . Effects of PHA 767491 on EdU incorporation and Ser2 phosphorylation of RNA Pol II in MCF7 cells .",
"( A ) Quantification of the percent of cells positive for EdU incorporation for cells treated with 0 or 100 nM E2 concurrently with DMSO or 1 μM Cdc7 inhibitor PHA 767491 for 14 hrs .",
"Cells were pulsed with EdU 30 min prior to fixation .",
"Errors bars represent the SD of 3 biological replicates .",
"( B ) Western blot of Ser2 CTD phosphorylation ( pSer2 ) of RNA Pol II in MCF7 cells treated with 100 nM E2 concurrently with either control , 1 μM Cdc7 inhibitor PHA 767491 for 14 hr , or 0 . 8 μM flavopiridol for 2 hrs . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 00810 . 7554/eLife . 17548 . 009Figure 1—figure supplement 6 . Quantification of the percent of cells positive for EdU incorporation for cells treated with 0 or 100 nM E2 for 12 hr prior to the addition of 0 . 8 μM flavopiridol or DMSO for 2 hr . Cells were pulsed with 10 μm EdU for 30 min prior to harvesting .",
"Error bars represent the SD of 3 biological replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 009 Previous studies have shown that E2 induces the rapid and transient formation of DNA breaks necessary for transcriptional induction at E2 promoters , and these breaks have been reported to be dependent upon the activity of TopoIIβ and APOBEC3B ( Ju et al . , 2006; Periyasamy et al . , 2015 ) .",
"When we examined this early response to E2 , we found that the P-H2AX signal resulting from 24 hr of E2 stimulation was significantly greater than the signal resulting from very short ( 10–40 min ) treatments , raising the possibility that the later DNA breaks arise through a different mechanism ( Figure 1E ) .",
"Given that a significant portion of cells would have entered S-phase by the 24-hr time point , we asked whether DNA replication was associated with E2-induced damage by pulsing cells with EdU at various times after E2 addition and monitoring H2AX phosphorylation .",
"The percentage of P-H2AX positive cells increased dramatically after 16 and 24 hr , and the majority of these cells were EdU positive ( Figure 1 F , G , and Figure 1—figure supplement 4 ) .",
"This finding strongly suggests that E2-induced DNA damage may require DNA replication .",
"Indeed , when we treated E2-stimulated cells with a Cdc7 inhibitor ( PHA 767491 ) that largely blocks S phase entry ( Figure 1—figure supplement 5A ) , we found that the DNA damage was reduced ( Figure 1H ) .",
"At the 1 μM dose of PHA 767491 used , phosphorylation of RNA Pol II at Ser2 by Cdk9 was not inhibited , indicating the effect on P-H2AX is not a result of inhibiting RNA Pol II elongation ( Figure 1—figure supplement 5B ) .",
"We also asked if the late phase of E2-induced DNA damage is affected by blocking transcription coincident with S-phase entry .",
"Treatment with the transcription inhibitor flavopiridol abolished the late E2-induced increase in P-H2AX ( Figure 1I ) but did not significantly affect the percentage of cells in S phase ( Figure 1—figure supplement 6 ) .",
"Taken together , these results demonstrate that E2 induces DSBs in a replication- and transcription-dependent manner .",
"E2 stimulation causes a rapid burst of transcription at a large number of genes ( Hah et al . , 2011 ) .",
"As defects in co-transcriptional processing have been shown to promote R-loop formation ( Hamperl and Cimprich , 2014; Santos-Pereira and Aguilera , 2015; Sollier and Cimprich , 2015 ) , we asked whether E2 stimulation , which might saturate co-transcriptional processing pathways , leads to elevated R-loop levels .",
"To do so , we probed genomic DNA from mock- or E2-treated cells ( 24 hr ) with the anti RNA-DNA hybrid monoclonal antibody S9 . 6 ( Boguslawski et al . , 1986 ) .",
"We observed a dramatic increase in RNA-DNA hybrids upon E2 treatment ( Figure 2A ) .",
"Moreover , RNase H treatment , which specifically removes the RNA strand of an RNA-DNA hybrid , abolished the signal ( Figure 2A ) , indicating antibody specificity for RNA-DNA hybrids . 10 . 7554/eLife . 17548 . 010Figure 2 . Estrogen induces robust R-loop formation at E2-responsive genes .",
"( A ) Slot blot to detect global RNA-DNA hybrids with S9 . 6 antibody in MCF7 cells treated with 0 or 100 nM E2 for 24 hr .",
"Total denatured DNA is stained with a single-strand DNA antibody .",
"RNase H was added as indicated .",
"( B ) Meta-gene analysis for indicated DRIP signal over indicated genomic features .",
"Data are shown for DRIP-seq biological replicate # 3 .",
"An enrichment is seen in all data sets but relative read density at these sites varies between replicates .",
"( C ) DRIP-seq read counts normalized for total mapped reads from DRIP in 0 nM E2 conditions vs DRIP from cells treated with 100 nm E2 for 2 hr ( top ) or 24 hr ( bottom ) .",
"Graphs are from 3 biological experiments .",
"Black dots indicate DRIP peaks and red dots indicate induced DRIP peaks relative to 0 nM E2 .",
"( D ) Integrated Genome Viewer ( IGV ) display of DRIP-seq enrichment at XBP1 .",
"Scale = million mapped reads .",
"RNase H was performed prior to DRIP-seq on one replicate each from 0 nM E2 and 100 nM E2 24 hr .",
"Independent replicates are shown as 1–3 .",
"( E ) IGV display of CCND1 ( Cyclin D1 ) , as described in ( D ) .",
"( F ) DRIP-qPCR validation .",
"Cells were treated with 0 , 10 , or 100 nM E2 for 24 hr and harvested for DRIP .",
"MLKL and 83/84 are negative controls .",
"Error bars represent S . E . M . of 2 biological experiments .",
"RNase H treatment was performed for 24 hr prior to DRIP-qPCR where indicated .",
"( G ) Functional signatures by GREAT of E2-induced DRIP peaks found to be differentially induced in 100 nM E2 , 2 hr ( top ) or 100 nM E2 24 hr ( bottom ) than in 0 nM E2 treated cells .",
"The 7 highest enrichment scores are shown , with red highlighting E2-associated signatures .",
"( H ) Fold change in DRIP signal after 2 hr of 100 nM E2 relative to 0 nM E2 ( x-axis ) vs . fold change in GRO-seq signal after 160 min of 100 nM E2 relative to 0 nM E2 ( y-axis ) .",
"E2-induced DRIP peaks that show a positive ( red ) or negative ( blue ) fold change in GRO-seq upon E2 stimulation are highlighted .",
"Negative changes in DRIP upon E2 that correspond to a positive ( yellow ) or negative ( green ) fold change in GRO-seq are also shown .",
"GRO-seq data from ( Hah et al . , 2011 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 01010 . 7554/eLife . 17548 . 011Figure 2—source data 1 . Genomic coordinates for DRIP peaks identified as induced in MCF7 cells treated with 100 nM E2 for 2 hrs relative to MCF7 cells treated with 0 nM E2 . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 01110 . 7554/eLife . 17548 . 012Figure 2—source data 2 . Genomic coordinates for DRIP peaks identified as induced in MCF7 cells treated with 100 nM E2 for 24 hrs relative to MCF7 cells treated with 0 nM E2 . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 01210 . 7554/eLife . 17548 . 013Figure 2—figure supplement 1 . R-loops are induced with E2 prior to S phase and exhibit R-loop features .",
"( A ) Average read count in input ( x-axis ) versus S9 . 6 immunoprecipitation ( y-axis ) in MCF7 cells treated with 0 nM E2 ( left ) , 100 nM E2 for 24 hr ( middle ) or 100 nM E2 for 2 hr ( right ) .",
"Black dots above the diagonal represent DRIP peaks above input .",
"Red dots correspond to E2-induced peaks .",
"( B ) RNase H treatment .",
"Read per kilobase per million ( RPKM ) plots of DRIP peaks in 0 nM E2 versus DRIP peaks from 0 nM E2 sample treated with RNase H prior to IP ( left ) and of DRIP peaks from cells treated with 100 nM E2 for 24 hr versus DRIP peaks from cells treated with RNase H after 100 nM E2 for 24 hr ( right ) .",
"Red dots show identified E2-induced DRIP peaks and their sensitivity to RNase H . ( C ) GC skew density in DRIP peaks .",
"The strongest 3000 DRIP peaks from each indicated samples are shown .",
"GC skew obtained from Ginno et al . , 2012 .",
"( D ) G-quartet density in DRIP peaks .",
"The strongest 3000 DRIP peaks from each indicated samples are shown .",
"G-quartet data obtained from Chambers et al . , 2015 .",
"( E ) IGV display of DRIP-seq enrichment at SLC7A5 .",
"Scale = million mapped reads .",
"An RNase H control was performed prior to DRIP-seq from 0 or 100 nM E2-treated cells for 24 hrs .",
"Biological replicates are shown as 1–3 .",
"( F ) FACS profiles of MCF7 cells treated with 0 nM E2 ( left ) , or 100 nM E2 for 2 hr ( middle ) or 24 hrs ( right ) .",
"Cells were pulsed with 25 μM BrdU prior to fixation .",
"DNA content is marked by propidium iodide ( x-axis ) and BrdU incorporation is shown on the y-axis .",
"The percentage of cells in each of the four cell-cycle quadrants is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 01310 . 7554/eLife . 17548 . 014Figure 2—figure supplement 1—source data 1 . Genomic coordinates for all identified DRIP peaks from MCF7 cells treated with 0 nM E2 . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 01410 . 7554/eLife . 17548 . 015Figure 2—figure supplement 1—source data 2 . Genomic coordinates for all identified DRIP peaks from MCF7 cells treated with 100 nM E2 for 2 hrs . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 01510 . 7554/eLife . 17548 . 016Figure 2—figure supplement 1—source data 3 . Genomic coordinates for all identified DRIP peaks from MCF7 cells treated with 100 nM E2 for 24 hr . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 01610 . 7554/eLife . 17548 . 017Figure 2—figure supplement 2 . Sequence features and expression analysis associated with DRIP-seq .",
"( A ) GC skew density in E2-induced DRIP peaks that show a positive ( red ) or negative ( blue ) change in GRO-seq upon E2 .",
"( B ) G-quartet density in E2-induced DRIP peaks that show a positive ( red ) or negative ( blue ) change in GRO-seq upon E2 .",
"Error bands for ( A , B ) represent the 1st and 3rd quartile from 1000x bootstrap .",
"( C ) DRIP peak strength compared to expression level for DRIP performed with 0 nM E2 ( left ) and 100 nM E2 for 2 hr ( middle ) or 100 nM E2 for 24 hr ( right ) .",
"Expression levels in non-E2 treated or E2-treated cells for similar time periods obtained from publically available RNA-seq ( Honkela et al . , 2015 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 017 To directly determine the location and abundance of R-loops genome-wide , we performed DNA-RNA immunoprecipitation ( DRIP ) with this antibody followed by next generation sequencing on cells treated with no E2 or 100 nM E2 for 2 or 24 hr .",
"These times were chosen to assess R-loop formation prior to and/or concurrent with E2-transcriptional induction and DNA damage .",
"In mock-treated MCF7 cells , we observed 17 , 445 DRIP peaks across the genome ( Figure 2—figure supplement 1A , Figure 2—figure supplement 1—source data 1 ) , a number in line with the previous DRIP-seq experiments performed in human Ntera2 cells and primary human fibroblasts ( Ginno et al . , 2012; Lim et al . , 2015 ) .",
"Strikingly , however , in cells treated with E2 we observed a dramatic increase in DRIP peaks , with 33 , 458 or 24 , 781 peaks at 2 or 24 hr , respectively ( Figure 2—figure supplement 1A , Figure 2—figure supplement 1—source data 2 , 3 ) .",
"DRIP peaks from mock-treated cells covered 49 . 0 Mb of genome space , while those from E2-treated cells for 2 hr or 24 hr covered 81 . 9 MB and 63 . 2 Mb of genome space , respectively .",
"Importantly , the vast majority of peaks were abolished by RNase H treatment ( Figure 2—figure supplement 1B ) .",
"Next , we asked whether the RNA-DNA hybrids identified exhibit known R-loop features .",
"Indeed , DRIP peaks were enriched at promoters , 5’ gene regions and sites of transcriptional termination ( Figure 2B ) , consistent with their published distribution and roles at these regulatory regions ( Ginno et al . , 2013; Ginno et al . , 2012; Skourti-Stathaki et al . , 2014 ) .",
"DRIP peaks also exhibited GC skew ( Figure 2—figure supplement 1C ) and overlapped with in vitro mapped G-quartets ( Figure 2—figure supplement 1D ) ( Chambers et al . , 2015 ) , two sequence characteristics that are associated with R-loop formation ( Duquette et al . , 2004; Ginno et al . , 2013 ) .",
"Taken together , these observations suggest that E2 treatment is associated with a dramatic induction of R-loops at new genomic sites .",
"To further explore the effects of E2 on R-loop formation , we sought to identify the genomic regions in which DRIP signal was induced at 2 and 24 hr by differential peak calling .",
"Using triplicate data for each condition , we combined all DRIP-positive regions in at least one sample with a set of similarly-sized intervals with no significant enrichment and performed the differential analysis .",
"Differential peaks were only identified if they showed a significant change upon E2 induction in each biological replicate .",
"Analysis of RNA-DNA hybrids induced after E2 treatment revealed a dramatic increase throughout the genome , with 8 , 023 and 3 , 263 peaks significantly induced at 2 hr or 24 hr , respectively ( Figure 2C , Figure 2—figure supplements 1A , B , Figure 2C — source data 1 , 2 ) .",
"Of those hybrids induced at 24 hr , ~60% were also significantly induced at 2 hr , suggesting that the E2 treatment can result in a sustained increase in R-loop formation at many loci .",
"Loci that showed a robust increase following E2 addition include XBP1 , CCND1 ( Cyclin D1 ) , and SLC7A5 ( Figure 2D , E , Figure 2—figure supplement 1E ) , all targets of E2-transcription ( Honkela et al . , 2015 ) .",
"We validated our DRIP-seq data at several loci using DRIP-qPCR .",
"A dose-dependent increase in DRIP signal was observed upon both 10 nM and 100 nM E2 treatment at several E2-transcription targets ( Figure 2F ) , while two genomic regions ( MLKL and 83/84 ) devoid of DRIP peaks showed no change .",
"Using GREAT , a bioinformatic tool that annotates ontologies for cis-regulatory regions of the genome ( McLean et al . , 2010 ) , we next asked whether there was a common signature among these E2-induced RNA-DNA hybrids .",
"We found a dramatic enrichment of R-loops in E2-transcriptionally responsive genes at both time points ( Figure 2G , red highlighting ) .",
"These data clearly demonstrate that the induction of R-loops by E2 is a robust response that occurs quickly at E2-responsive genes , and persists at a subset of these genes .",
"Taken together with data shown in Figure 1 , these data also demonstrate that R-loop formation occurs prior to the onset of DNA damage , and in cells that have not yet entered S phase ( Figure 2—figure supplement 1F ) .",
"To systematically investigate the relationship between E2-responsiveness and R-loop induction , we also correlated DRIP signal enrichment with changes in expression level upon E2 stimulation .",
"We focused on transcriptionally engaged RNA polymerase by using publically available global run-on sequencing ( GRO-seq ) data from samples prepared under similar conditions ( Hah et al . , 2011 ) ( Figure 2H ) .",
"A modest correlation between the induction of DRIP signal and the induction of expression was observed , consistent with the requirement for transcription in R-loop formation .",
"Although the majority of induced DRIP peaks were found in E2-responsive genes ( red highlighting ) , some were also induced at sites that were not E2-responsive or that were negatively regulated by E2 ( blue highlighting ) .",
"Both sets of hybrid-forming sequences exhibit GC skew and enhanced G-quadruplex formation ( Figure 2—figure supplements 2A , B ) .",
"We also found no meaningful correlation between DRIP signal enrichment and overall expression level as measured by publically available RNA-seq data from similarly paired conditions .",
"This finding indicates that DRIP peaks do not form only on highly expressed genes ( Figure 2—figure supplement 2C ) .",
"We conclude that R-loop induction by E2 is correlated with E2-induced transcription , but that expression level is not the sole determinant of R-loop formation and other secondary structures or sequence elements may contribute .",
"Because E2-induced R-loops are strongly enriched in E2-transcriptionally responsive genes , we wondered if estrogen-responsive loci are also more prone to mutation in breast cancer .",
"To accomplish this , we utilized the somatic mutation data from the recent , large-scale whole-genome sequencing of 560 breast tumors ( Nik-Zainal et al . , 2016 ) .",
"Three groups of somatic mutations were used:",
"1 ) structural variants such as duplications , deletions , or inversions ,",
"2 ) translocations , or",
"3 ) simple somatic mutations such as substitutions and indels ( Nik-Zainal et al . , 2016 ) .",
"For each group of mutations , we determined whether the sites of mutation were enriched in E2-responsive loci , as measured by the Z-score from RNA-seq data ( Honkela et al . , 2015 ) .",
"For mutated genes in each of the three classifications , we examined enrichment relative to control gene sets matched for both expression level and replication timing ( Figure 3—figure supplements 1 , 2 , and 3 ) .",
"Notably , we observed a significant enrichment of both structural variants ( p<1 . 00*10−6 ) and translocations ( p<1 . 00*10−6 ) in genes that are transcriptionally-responsive to E2 ( Figure 3A , B ) .",
"In contrast , there were fewer simple somatic mutations in E2-responsive loci relative to the matched set ( p<1 . 00*10−6 ) ( Figure 3C ) .",
"These results demonstrate that genes whose expression is induced in response to E2 are enriched in genomic rearrangements in breast tumors and support the view that these sites are prone to certain types of DNA damage . 10 . 7554/eLife . 17548 . 018Figure 3 . Breast cancer rearrangements are enriched in E2-responsive genes .",
"( A ) Histogram of E2-induced expression changes ( Z-score ) for genes that overlap with breast cancer structural variants ( red bars ) compared to the distribution of E2-induced expression changes ( Z-score ) for a control gene set ( blue bars ) , matched for expression level and replicating timing .",
"Genes with structural variants are enriched in E2-responsive genes .",
"p<1 . 00*10−6 .",
"( B ) Histogram of E2-induced expression changes ( Z-score ) for genes with breast cancer translocations ( red bars ) compared to a control gene set ( blue bars ) , as described in ( A ) .",
"Genes with translocations are enriched in E2-responsive genes p<1 . 00*10−6 .",
"( C ) Histogram of enrichment of breast cancer simple somatic mutations in E2-responsive genes .",
"Data plotted as the log transformation of simple somatic mutations per kilobase for E2-induced genes ( red bars ) relative to a matched set ( blue bars ) , as described previously .",
"E2-responsive genes have fewer simple somatic mutations than the control gene set ( p<1 . 00*10–6 ) .",
"In A–C , p-values represent two-tailed bootstrap of medians .",
"All breast cancer mutation data from ( Nik-Zainal et al . , 2016 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 01810 . 7554/eLife . 17548 . 019Figure 3—figure supplement 1 . Comparison of ( A ) Replication timing based on RepliSeq signal across the gene body of regions containing structural variants in breast cancer ( x-axis ) relative to that of the matched regions ( y-axis ) , and ( B ) Mean expression level based on RNA-seq of regions associated with structural variants ( x-axis ) relative to that of the matched regions ( y-axis ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 01910 . 7554/eLife . 17548 . 020Figure 3—figure supplement 2 . Comparison of ( A ) Replication timing based on RepliSeq signal across the gene body of translocated regions ( x-axis ) relative to that of the matched regions ( y-axis ) , and ( B ) Mean expression level based on RNA-seq of translocations ( x-axis ) relative to that of the matched regions ( y-axis ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 02010 . 7554/eLife . 17548 . 021Figure 3—figure supplement 3 . Comparison of ( A ) Replication timing based on RepliSeq signal across the gene body of genes with simple somatic mutations ( x-axis ) relative to that of the matched regions ( y-axis ) , and ( B ) Mean expression level based on RNA-seq of genes with simple somatic mutations ( x-axis ) relative to that of the matched regions ( y-axis ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 021 Given that E2-responsive genes are strongly enriched in regions of genomic rearrangement in breast tumors and these mutations could arise through DNA DSB formation , we asked whether we could directly detect DNA damage in the vicinity of E2-induced R-loops .",
"To address this , we performed a proximity ligation assay ( PLA ) using the S9 . 6 and P-H2AX antibodies to visualize interactions between RNA-DNA hybrids and phosphorylated H2AX .",
"Notably , the percentage of cells that contain at least one PLA focus in the nucleus increased approximately 2-fold following E2 treatment ( Figure 4A , B ) .",
"The relatively small number of detectable interactions between the RNA-DNA hybrid and P-H2AX antibodies upon E2 addition may indicate that the majority of DNA damage occurs at a distance from the R-loop that is outside the limits of PLA detection ( <30–40 nM ) .",
"In order to confirm the interaction between R-loops and P-H2AX , we used the S9 . 6 antibody to precipitate RNA-DNA hybrids from cross-linked E2-treated cells .",
"We found that that both P-H2AX and another DDR target , P-KAP1 , interacted with RNA-DNA hybrid containing chromatin fragments ( Figure 4C ) .",
"This interaction was specific , as it did not occur with MCM3 , another chromatin-bound protein ( Figure 4C ) .",
"Taken together , these results suggest that DNA damage markers bind the chromatin environment near R-loops following E2 treatment . 10 . 7554/eLife . 17548 . 022Figure 4 . E2-induced R-loops occur on chromatin marked by DNA damage and RNase H reduces E2-induced DSBs .",
"( A ) Proximity ligation assay between S9 . 6 antibody and P-H2AX antibody in cells treated either with 0 or 100 nM E2 for 24 hr . ( B ) Quantification of the percentage of cells with ≥ 1 PLA focus per nucleus .",
"Single-antibody controls from cells treated with 100 nM E2 for 24 hr are shown .",
"Error bars represent the SEM from 4 biological replicates .",
"**p<0 . 01 ( Student’s t-test ) .",
"( C ) P-H2AX and P-KAP1 levels from co-IP of S9 . 6 or IgG from cross-linked and sonicated cells treated with 100 nM E2 for 24 hr .",
"Input is 60% of the IP .",
"( D ) Western blot with Flag antibody in MCF7 tetOn-RH cells .",
"1000 ng/mL doxycycline ( DOX ) was added for 48 hrs where indicated .",
"( E ) Immunostaining for P-H2AX and FLAG in MCF7 tetON-RH cells treated with increasing concentrations of DOX ( 100 , 250 , 500 1000 ng/mL ) for 24 hrs prior to the addition of either 0 or 100 nM E2 for 24 hr . ( F ) Quantification of P-H2AX intensity for the experiment described in ( E ) , where the triangle indicates increasing concentrations of DOX .",
"****p<0 . 0001 ( non-parametric Mann-Whitney rank sum t-test ) .",
"n = 3 biological replicates .",
">1000 cells/condition quantified .",
"( G ) Neutral comet assay in MCF7-tetOn-RH cells treated with or without 1000 ng/mL DOX for 24 hrs prior to 0 nM E2 or 100 nM for 24 hr . ( H ) Quantification of the neutral comet tail moment described in ( G ) .",
"**p<0 . 01 ( non-parametric Mann-Whitney rank sum t-test ) .",
"n = 3 biological replicates .",
">100 comets/condition .",
"a . u . = arbitrary units . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 02210 . 7554/eLife . 17548 . 023Figure 4—figure supplement 1 . Quantification of the fold change in the percent of cells with >5 P-H2AX foci per cell in MCF7-tetON-RH cells treated with indicated concentrations of DOX .",
"*p<0 . 05 . Error bars represent SD of 3 technical replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 02310 . 7554/eLife . 17548 . 024Figure 4—figure supplement 2 . Expression of RNase H prevents E2-induced DNA damage .",
"( A ) Quantification of P-H2AX intensity per nucleus in MCF7 tetON-RH clone 6 ( left ) or MCF7 tetON-RH clone 8 ( right ) treated with 0 or 1000 ng/mL DOX for 24 hr prior to the addition of 0 nM or 100 nM E2 for 24 hr .",
"****p<0 . 0001 ( non-parametric Mann-Whitney rank sum t-test ) .",
"n = 3 technical replicates .",
">500 cells/condition .",
"( B ) Western blot with Flag antibody in MCF7 tetOn-RH cells , clones 6 and 8 .",
"DOX was added at 1000 ng/mL MCF7 tetOn-RH cells for 48 hr . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 02410 . 7554/eLife . 17548 . 025Figure 4—figure supplement 3 . FACS profiles of MCF7 tetON-RH cells treated with or without 1000 ng/mL DOX for 24 hr prior to the addition of 100 nM E2 for 24 hr . Cells were pulsed with 25 μM BrdU for 30 min prior to fixation .",
"DNA content is marked by propidium iodide ( x-axis ) and BrdU incorporation is shown on the y-axis .",
"The percentage of cells in each of the four cell-cycle quadrants is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 02510 . 7554/eLife . 17548 . 026Figure 4—figure supplement 4 . EU incorporation following RNase H expression in MCF7 cells .",
"( A ) EU staining in MCF7 tetON-RH cells either treated with or without 1000 ng/mL DOX for 24 hr prior to the addition of 0 or 100 nM E2 for 24 hr ( 48 hr DOX total ) .",
"100 μM DRB was added for 2 hr .",
"Cells were pulsed with 100 μM EU for 30 min before fixation .",
"Hoechst is used to stain the nucleus .",
"( B ) Quantification of EU staining for the experiment described in ( A ) .",
"n = 2 biological replicates .",
">200 cells/condition .",
"a . u . = arbitrary units . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 026 We then asked whether the E2-induced accumulation of DSBs is a direct result of E2-induced R-loop formation .",
"If R-loops cause E2-induced DNA damage , expression of RNase H should abrogate DSB formation .",
"We tested this idea by monitoring P-H2AX levels in an MCF7 cell line stably expressing a doxycycline-inducible , Flag-tagged RNase H ( Figure 4D ) .",
"Strikingly , we observed a dramatic and dose-dependent reduction in P-H2AX intensity and foci formation upon increasing RNase H expression ( Figure 4E , F , Figure 4—figure supplement 1 ) .",
"Similar results were observed in two additional independent stable cell clones ( Figure 4—figure supplements 2A , B ) .",
"Importantly , expression of RNase H did not alter E2-driven cell cycle progression ( Figure 4—figure supplement 3 ) , nor did it reduce global levels of transcription , as measured by EU labeling ( Figure 4—figure supplements 4A , B ) .",
"Thus , the decreased P-H2AX signal is not a result of reduced transcription or a defect in cell cycle progression .",
"Finally , we found that RNase H expression resulted in a significant decrease in DSBs upon E2 stimulation , as measured by the neutral comet assay ( Figure 4G , H ) .",
"These findings clearly demonstrate that RNA-DNA hybrids are needed to form E2-induced DSBs .",
"We recently showed that R-loop-mediated DSB formation depends on factors specifically involved in transcription-coupled nucleotide excision repair ( TC-NER ) , but not factors specifically involved in global genome NER ( GG-NER ) ( Sollier et al . , 2014 ) .",
"In these studies , R-loops were induced by the knockdown of RNA processing genes and/or splicing factors .",
"To ask if the DNA damage resulting from E2-induced co-transcriptional R-loop formation arises by a similar mechanism , we assessed whether knockdown of NER factors altered the DDR following E2 .",
"Strikingly , we found that P-H2AX levels were significantly reduced following knockdown of XPG , an endonuclease involved in both forms NER ( Figure 5A , Figure 5—figure supplement 1 ) .",
"XPG knockdown also dramatically reduced the comet tail moment that forms in response to E2 treatment ( Figure 5B , C ) , signifying a significant reduction in E2-mediated DSBs .",
"Importantly , we also found that knockdown of the TC-NER specific factor Cockayne syndrome group B ( CSB ) significantly reduced P-H2AX levels induced by E2 ( Figure 5D ) .",
"In contrast , knockdown of XPC , a lesion-recognition factor specifically involved in GG-NER , caused little change in P-H2AX levels after E2 treatment ( Figure 5E ) .",
"These findings suggest that R-loops induced by E2 are processed by TC-NER factors .",
"Taken as a whole , our findings demonstrate that replication-dependent E2-induced DNA damage results from the induction of R-loops triggered by E2-mediated transcription . 10 . 7554/eLife . 17548 . 027Figure 5 . Knockdown of NER and R-loop processing factors reduces E2-induced DNA damage and DSBs .",
"( A ) P-H2AX intensity based on immunostaining of MCF7 cells transfected with indicated siRNA 64 hrprior to the addition of 0 or 100 nM E2 for 24 hr .",
"****p<0 . 0001 .",
"Western blot shows the level of XPG .",
"( B ) Neutral comet assay in cells transfected with indicated siRNA 64 hr prior to the addition of 0 or 100 nM E2 for 24 hr . ( C ) Quantification of neutral comet tail moment described in ( B ) .",
"****p<0 . 0001 .",
">100 comets/condition .",
"( D , E )",
"P-H2AX intensity based on immunostaining in MCF7 cells treated as in ( A ) with the indicated siRNA .",
"****p<0 . 0001 .",
"Western blot shows the level of CSB or XPC .",
"The siGL3 control in the quantification shown in ( D ) is the same as shown in ( A ) .",
"a . u . = arbitrary units .",
"For all data , associated p-values are from non-parametric Mann-Whitney rank sum t-test .",
"n=3 biological replicates .",
"Quantification from >1000 cells/condition unless noted . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 02710 . 7554/eLife . 17548 . 028Figure 5—figure supplement 1 . XPG knockdown does not alter E2-induced cell cycle progression .",
"( A ) FACS profiles of MCF7 cells transfected with siXPG or siGL3 and grown for 64 hr prior to the addition of 0 or 100 nM E2 for 24 hr , with DNA content marked by propidium iodide ( x-axis ) , and cell content shown on the y-axis . DOI: http://dx . doi . org/10 . 7554/eLife . 17548 . 028"
],
[
"Given the clear link between E2 exposure and breast cancer incidence , there is significant interest in understanding the molecular mechanisms by which E2 promotes DNA damage and genome instability , which can drive tumorigenesis .",
"Accordingly , several mechanisms for E2-induced DNA damage have been elucidated .",
"For example , TopoIIβ and APOBEC3B cause the rapid and transient formation of DNA damage in a process necessary for E2-induced transcriptional activation ( Ju et al . , 2006; Periyasamy et al . , 2015 ) .",
"Additionally , E2 metabolites can cause oxidative DNA damage ( Lavigne et al . , 2001; Yager , 2014 ) .",
"Here , we report the first R-loop-dependent mechanism of E2-induced DNA damage .",
"Specifically , we show that R-loops induced by E2 treatment in ER-positive breast cancer cells lead to DSB formation in the vicinity of the R-loop .",
"Our findings suggest that increased transcription due to a physiologically relevant stimulus may result in R-loop-mediated genomic instability .",
"Intriguingly , the R-loop-dependent DNA damage we observe requires the initiation of DNA replication ( Figure 1F , H ) .",
"Although E2 causes a dramatic increase in R-loops after 2 hr ( Figure 2C ) , no significant change in DNA damage is observed at that time ( Figure 1F ) .",
"Thus , although R-loops are present , they do not cause DNA damage until S phase entry .",
"Consistent with this finding , R-loop-dependent DNA damage has been previously associated with DNA replication ( Alzu et al . , 2012; Gan et al . , 2011; Houlard et al . , 2011 ) .",
"Moreover , a few common fragile sites correspond to late-replicating , long genes where R-loops form , and early-replicating fragile sites are associated with highly transcribed genes ( Barlow et al . , 2013; Helmrich et al . , 2011 ) .",
"This suggests that genes that are highly transcribed or R-loop prone may pose the greatest challenge to genome integrity when they meet the replication fork .",
"Interestingly , we find that TC-NER factors are also involved in generating the replication-associated DNA damage observed ( Figure 5 ) .",
"This raises the possibility that these factors may be acting in a non-canonical manner in S phase , and there are several possible mechanisms by which their activity may be coordinated with replication activities that have been previously discussed ( see Sollier et al . , 2014 ) .",
"Importantly , we find that R-loops are induced predominantly at E2-responsive genes ( p<10−233 ) .",
"In agreement with this , we see a correlation between the induction of expression by E2 , as measured by GRO-seq , and the induction of R-loops by E2 ( Figure 2H ) .",
"The modest strength of the correlation likely indicates that factors distinct from the transcription status play an important role in dictating R-loop formation .",
"Interestingly , a small subset of genomic regions exist that show a significant increase in R-loops despite no increase in expression ( Figure 2H , blue highlighting ) .",
"Similarly , RPL13A , the expression of which is unaffected by E2 ( Shah and Faridi , 2011 ) , showed a significant increase in R-loops upon E2 ( Figure 2F ) .",
"One possibility is that upon a burst in transcription , the cell’s normal splicing and R-loop modulating factors initially become saturated .",
"Thus , some loci that are prone to form R-loops may accumulate these structures and become vulnerable to DNA damage , regardless of their response to the transcription stimulus .",
"We also observe that breast cancer rearrangements , but not simple somatic mutations , are significantly enriched in E2-responsive genes ( Figure 3 ) .",
"Both structural variants and translocations have been suggested to occur due to erroneous repair of a DSB ( Kasparek and Humphrey , 2011 ) and these mutation types have been shown to be more prevalent in breast tumors relative to other tumor tissues ( Yang et al . , 2013 ) .",
"Intriguingly , some cancer-associated genes are found among genes forming E2-induced R-loops .",
"For example , we observed a dramatic increase in R-loops at Cyclin D1 ( Figure 2E ) , which is amplified in up to 20% of breast cancers , the majority of which are ER-positive ( Arnold and Papanikolaou , 2005; Osborne et al . , 2004 ) .",
"Similarly , E2 induces robust R-loop formation at ZNF703 .",
"This locus has been implicated in driving oncogenesis in luminal B breast tumors ( Holland et al . , 2011; Sircoulomb et al . , 2011 ) .",
"Both Cyclin D1 and ZNF703 were among the genes identified as breast cancer drivers , as were an additional 27 genes with E2-induced R-loops ( Nik-Zainal et al . , 2016 ) .",
"Our findings raise the possibility that E2-induced R-loop formation could lead to DNA damage and genome instability at some of these and other R-loop prone loci .",
"Intriguingly , testosterone ( DHT ) stimulation in prostate cells has been shown to lead to DSBs and recurrent translocations at androgen-receptor ( AR ) target loci ( Haffner et al . , 2010; Lin et al . , 2009 ) .",
"While these breaks are TOP2B-dependent and necessary for the induction of transcription , DHT may also induce DNA damage in an R-loop and replication dependent manner .",
"Notably , in prostate cancer , recurrent rearrangements are frequently observed at androgen-receptor ( AR ) target genes ( Kumar-Sinha et al . , 2008 ) .",
"In summary , our findings have uncovered a role for R-loops in E2-induced DNA damage and reveal a new mechanism by which genome instability may arise in ER-responsive breast cancer cells .",
"Notably , transcription factor responses vary between cell types and following different physiological and environment stimuli .",
"Thus , the induction of potentially toxic R-loops at sites of induced transcription may offer insight into the tissue-specific DNA damage and mutation patterns observed in some cancers ."
],
[
"MCF7 cells were obtained from ATCC ( HTB-22 , RRID:CVCL_0031 ) , where they were authenticated by STR profiling and tested negative for mycoplasma .",
"These MCF7 cells were cultured in DMEM ( GIBCO ) supplemented with 10% FBS in 5% CO2 at 37°C .",
"For estrogen stimulation , MCF7 cells were plated in DMEM with 10% FBS for at minimum of 16 hr before being washed 3 times in 1X PBS .",
"Phenol-red free DMEM ( GIBCO ) with 10% charcoal-stripped FBS ( Invitrogen ) was then added for 48 hr prior to the addition of E2 dissolved in EtOH or the vehicle control ( 100% EtOH ) .",
"MCF10A cells were obtained from ATCC ( CRL-10317 , RRID:CVCL_0598 ) , where they were authenticated by STR profiling and tested negative for mycoplasma .",
"MCF10A cells were cultured in a 1:1 mixture of DMEM and F12 medium ( GIBCO ) supplemented with 5% horse serum , hydrocortisone ( 0 . 5 mg/ml ) , insulin ( 10 μg/ml ) , epidermal growth factor ( 20 ng/ml ) , cholera toxin ( 100 ng/ml ) and penicillin-streptomycin ( 100 μg/ml each ) .",
"For estrogen stimulation , MCF10A cells were washed 3 times in 1X PBS and then media containing a 1:1 mixture of phenol-red free DMEM and F12 medium ( GIBCO ) supplemented with 5% charcoal-stripped FBS ( Invitrogen ) , hydrocortisone ( 0 . 5 mg/ml ) , insulin ( 10 μg/ml ) , and cholera toxin ( 100 ng/ml ) was added and cells were grown for 48 hr before stimulation .",
"The MCF7-TetON-RH cell line was generated from MCF7 cells ( ATCC HTB-22 , RRID:CVCL_0031 , as described above ) that were first transduced with a pLVX-EF1α-Tet3G-Hygro transactivator-containing vector and selected with 500 μg/mL hygromycin .",
"Hygromyocin-resistant cells were then transduced with a pLVX-Tight-Puro vector , expressing a FLAG-tagged truncated version of RNase H1 ( pLVX-Tight-Puro-RH-Flag ) and selected with 1 μg/mL puromycin .",
"Cells were maintained in 500 μg/mL hygromyocin and 1 μg/mL puromycin .",
"Antibodies to single-stranded DNA ( EMD Millipore , #MAB3868 ) , P-H2AX ( Cell Signaling , 9718S ) , P-KAP1 ( Bethyl , A300-767A ) , MCM3 ( Abcam , ab4460 ) , XPG ( Santa Cruz , 393004 ) , CSB ( Bethyl , A301-345A ) , XPC ( Abcam , ab6264 ) , 53BP1 ( BD , 612523 ) , BrdU ( BD , 347580 ) ALPHA-TUB ( Sigma , T9026 ) , FLAG ( Sigma , F1804 ) , GAPDH ( Abcam , ab8245 ) , ER-α ( Santa Cruz , HC-20 ) , and RNA Pol II phospho S2 H5 ( Abcam , ab24758 ) were used .",
"The S9 . 6 antibody was purified from a S9 . 6 hybridoma cell line ( ATCC , HB-8730; RRID: CVCL_G144 ) .",
"Hybridoma supernatant was applied to a 1 mL HiTrap Protein G HP column ( GE Healthcare ) .",
"The antibody was eluted with 100 mM glycine pH 2 . 5 , in 0 . 5 mL fractions .",
"Fractions were screened for antibody by SDS-PAGE and antibody-containing fractions were pooled and dialyzed in PBS overnight followed by dialysis in 50% glycerol for 6 hr .",
"The antibody concentration was measured against a BSA standard and aliquots were made at 1 μg/mL .",
"The pLVX-Tight-Puro-RH-Flag was made by inserting a truncated FLAG-tagged human RNase H1 lacking the first 27 amino acids .",
"siRNAs , purchased from ThermoFisher , were: siGL3 ( D-001400-01 ) , siXPG ( D-006626-02 ) , siCSB2 ( D-00488-04 ) , siCSB4 ( D-00488-06 ) , siXPC1 ( D-016040-01 ) , siXPC2 ( D-016040-02 ) , and siXPC3 ( D-016040-04 ) .",
"All siRNA transfections were performed using Dharmafect1 ( ThermoFisher ) according to the manufacturer’s protocol and 20 nM siRNA unless otherwise indicated .",
"For siRNA transfections , cells were reverse transfected with 20 nM siRNA in antibiotic free DMEM with 10% FBS .",
"16 hr later , the media was removed , cells were washed 3 times with 1X PBS and phenol-red free DMEM with 10% charcoal-stripped FBS was added .",
"64 hr after transfection , cells were transfected again with 10 nM of the siRNA and E2 or vehicle control ( 100% EtOH ) was added to the media for 24 hrs .",
"17ß-Estradiol ( E2 ) ( Sigma , AC00006 ) was dissolved in 200-proof EtOH ( Goldshield ) .",
"Flavopiridol ( Tocris , 3094 ) and the Cdc7 inhibitor PHA 767491 ( Sigma , PZ0178 ) were prepared in DMSO .",
"Doxycycline ( DOX ) ( Sigma ) was added every 24 hr when indicated .",
"Human RNaseH purified from MBP-RNaseH1 was a gift from the Chedin lab and was used with commercially available RNase H buffer ( NEB , M0297 ) .",
"The neutral comet assay was performed using the CometAssay Reagent Kit for Single Cell Gel Electrophoresis Assay ( Trevigen ) according to the manufacturer’s protocol with 1X TBE running buffer .",
"Electrophoresis was performed at 4°C in the CometAssay Electrophoresis System II .",
"SYBR-Gold ( Invitrogen ) was used to stain DNA .",
"Images were taken on an epifluorescence microscope at 10x magnification .",
"Comet tail moments were quantified using OpenComet software ( Gyori et al . , 2014 ) .",
"In box and whisker plots , box and whiskers indicate 25–75 and 10–90 percentiles , respectively , with lines representing median values .",
"Cells were fixed with 4% PFA/PBS ( EMS ) for 15 min , permeabilized with 0 . 25% Triton-X 100 for 15 min , washed 3 times in 1X PBS , and blocked in 2% BSA/PBS for 1 hr at RT .",
"The primary antibody was incubated overnight at 4°C .",
"Antibodies were used at the following dilutions: Rabbit P-H2AX antibody ( 1:500 , Cell Signaling ) , FLAG , mouse FLAG antibody ( 1:500 , Sigma ) , mouse 53BP1 ( 1:500 , BD ) .",
"Cells were then washed 3 times in 1X PBS and stained with Hoechst ( 1:1000 ) and anti-rabbit AlexaFluoro-488-conjugated secondary ( 1:1000 ) .",
"For co-staining , anti-mouse AlexaFluoro-594-conjugated secondary ( 1:1000 ) was used .",
"Cells were imaged on a fully automated Molecular Devices ImageXpress Micro microscope .",
"Images were captured at either 20X or 40X .",
"Analysis of P-H2AX intensity was performed MetaXpress quantification software , where Hoechst is used as a mask for the nucleus and the intensity and/or number of foci per nucleus is determined .",
"In box and whisker plots , box and whiskers indicate 25–75 and 10–90 percentiles , respectively , with lines representing median values .",
"For EdU staining , cells were pulsed for 30 min with 10 μM EdU from the Click-iT Edu Alexa Fluor 488 imaging kit ( ThermoFisher ) .",
"Cells were then fixed by 4% PFA/PBS for 15 min , and permeabilized with 0 . 25% Triton/PBS for 15 min .",
"The Click-It reaction was then performed according to manufacturer’s instructions .",
"Immunostaining was carried out as described .",
"Images were acquired on a fully automated Molecular Devices ImageXpress Micro microscope and intensity analysis was performed using MetaXpress quantification software .",
"For EU staining , cells were pulsed for 1 hr with 100 μM EU from the Click-iT RNA Alexa Fluor 488 imaging kit ( ThermoFisher ) .",
"When indicated , 100 μM DRB ( Cayman Chemical Company ) was added for 2 hrs with 100 μM EU being added for the last hour .",
"Cells were then fixed by 4% PFA/PBS for 15 min , and permeabilized with 0 . 25% Triton/PBS for 15 min .",
"The Click-It reaction was then performed according to manufacturer’s instructions .",
"Cells were then incubated in Hoechst ( 1:1000 ) for 15 min before the slides were mounted with Pro-Long Gold Antifade reagent and imaged on a Zeiss Axioscope at 40X .",
"EU signal intensity was calculated using ImageJ ( v 1 . 47 ) .",
"Total nucleic acid was extracted by a standard SDS/Proteinase K lysis followed by phenol/chloroform extraction and EtOH/sodium acetate precipitation .",
"DNA ( 1 μg ) from each sample was spotted on Nylon membrane ( Amersham ) using a slot blot apparatus and vacuum suction .",
"For RNase H treatment , 5 μg of DNA was treated with RNase H at 37°C overnight , and then purified by standard phenol/chloroform EtOH precipitation .",
"For total DNA control , the membrane was denatured for 10 min in 0 . 5 M NaOH , 1 . 5 M NaCl , and neutralized for another 10 min in 1 M NaCl , 0 . 5 M Tris-HCl pH7 . 0 .",
"Membranes were then UV-crosslinked ( 0 . 12J/m2 ) , blocked in 5% milk/TBST , and incubated overnight at 4°C with mouse S9 . 6 ( 1:500 ) or single-strand DNA antibody ( 1:10 , 000 ) .",
"Blots were washed 3 times with TBST and secondary antibody ( 1:10 , 000 goat anti-mouse HRP ) was added for 1 hr at RT .",
"DRIP was performed as described in Ginno et al . ( 2012 ) .",
"Briefly , DNA was extracted carefully by phenol/chloroform extraction in phase lock tubes , precipitated with EtOH/sodium acetate , washed with 70% EtOH , and resuspended in TE .",
"DNA was digested with a cocktail of restriction enzymes ( Bsrg1 , EcoR1 , HindIII , SspI , XbaI ) overnight at 37˚C .",
"For RNase H-treated samples , 8 μg of DNA was treated with RNase H overnight at 37˚C .",
"DNA was purified by standard methods described above .",
"4 μg of DNA was bound with 10 μg of S9 . 6 antibody in 1 X binding buffer ( 10 mM NaPO4 pH 7 , 140 mM NaCl , 0 . 05% Triton X-100 ) overnight at 4˚C .",
"Protein A/G sepharose beads were added for 2 hr .",
"Bound beads were washed 3 times in binding buffer and elution was performed in elution buffer ( 50 mM Tris pH 8 , 10 mM EDTA , 0 . 5% SDS , Proteinase K ) for 45 min at 55˚C .",
"DNA was purified as described .",
"qPCR was performed on a Roche LightCycler 480 Instrument II using SYBR-Green master mix ( Biorad ) .",
"qPCR primers are described in Supplementary file 1 .",
"DNA libraries were prepared from 3 pooled DRIPs by sample .",
"Briefly , input and DRIP DNA was sonicated to approximately 300–700 bp on a Bioruptor ( Diagenode ) .",
"End repair ( NEB E6050S ) , A-tailing ( NEB M0212S ) , ligation of adapters ( NEB M2200S; Affymetrix Prep2Seq Adapters 79800 ) , and PCR amplification were performed by standard methods as described ( Ginno et al . , 2012 ) .",
"Ampure XP beads ( Beckman Coulter , A63880 ) were used for size selection .",
"Libraries were pooled and sequenced on an Illumina HiSeq machine with single-end 50 bp reads by Elim BioPharm ( Hayward , CA ) .",
"Raw reads from the DRIP experiments were aligned to the reference genome hg19/GRCh37 using bowtie2 .",
"An interval file of restriction fragments for the enzyme cocktail used in DRIP was obtained by verbose search of restriction site sequences with bowtie .",
"Aligned reads with Q score over 10 were counted over the intervals separated by restriction enzyme cut sites using bedtools .",
"Intervals with lower coverage ( less than 10 counts per interval in any sample ) were removed from the data set .",
"Regions enriched over background were discovered with DESeq2 .",
"To perform differential analysis using triplicate data , we combined the set of regions with positive DRIP signal in at least one condition , together with the matched set of negative intervals and performed differential analysis with DESeq2 .",
"Read counts were normalized to the total number of mapped reads .",
"To perform analysis of functional term enrichment , we ran GREAT analysis ( McLean et al . , 2010 ) on sets of differential DRIP regions .",
"RNA-seq data from GEO , accession number GSE62789 , was obtained from MCF7 cells that were hormone starved and treated with 0 nM E2 or 10 nM E2 for 160 min ( used to match 2 hr DRIP ) or 1280 min ( used to match 24 hr DRIP ) ( Honkela et al . , 2015 ) .",
"DRIP peaks were sorted for those that are within 1 KB of a gene .",
"7628 gene-proximal regions that form DRIP peaks in any sample were identified .",
"XY plots were generated in R for both control and E2 conditions showing the relative expression distribution of all genes relative to Log2Fold change in DRIP peak signal over input .",
"GRO-seq data from MCF7 cells was obtained from ( Hah et al . , 2011 ) , where cells were treated with vehicle or 100 nM E2 for 160 min .",
"The bedGraph data from accession GSE27463 was converted to coverage files over the DRIP restriction intervals using bedtools .",
"As GSE27463 is aligned to hg18 , liftOver was used to convert the intervals to hg18 before determining the coverage .",
"Fold changes between the mock and 160 min E2 GRO-seq were determined using DESeq2 .",
"Intervals that had an adjusted p value < 0 . 1 in the DRIP and an adjusted p value < 0 . 5 in the GRO-seq were called as significant , and were split into categories on the basis of having positive or negative log2 fold changes in DESeq2 .",
"The criteria used when in Figure 2H are: ( 1 ) Up in DRIP: log2FoldChange ( 2 hr IP vs Mock IP ) > 0 , adjusted p<0 . 1 ( 2 ) Down in DRIP: log2FoldChange ( 2 hr IP vs Mock IP ) < 0 , adjusted p<0 . 1 ( 3 ) Up in GRO-seq: log2Fold Change ( 2 hr GRO-seq vs Mock GRO-seq ) > 0 , adjusted p<0 . 9 ( 4 ) Down in GRO-seq: log2Fold Change ( 2 hr GRO-seq vs Mock GRO-seq ) < 0 , adjusted p<0 . 9 .",
"Cells were pulse-labeled with 25 μM BrdU for 30 min .",
"Cells were washed with 1X PBS , and the cell pellet was resuspended in 500 μL 1X PBS .",
"Cells were fixed in 5 mL ice-cold 70% EtOH , permeabilized with 0 . 25% TritonX-100/PBS for 15 min on ice , blocked in 2% BSA/PBS for 15 min , and incubated in primary BrdU antibody ( BD Bioscience 1:100 ) for 3 hr .",
"Cells were then washed with 1X PBS 3 times and incubated for 1 hr in a 1:400 solution of AlexaFluoro-488 secondary .",
"Propidium iodide ( PI , 0 . 1 mg/mL , Sigma ) and RNase A ( Qiagen ) were added and cells were run on a FACS Caliber ( BD Biocience .",
"Cell cycle profiles were determined using FlowJo software .",
"For all genes in the MCF7 mock and E2-induced RNA-seq dataset ( Honkela et al . , 2015 ) , E2-induction was defined as the z-score between the mock-treated ( 0 hr ) and E2-induced samples following 160 min E2 .",
"The z-score was calculated using the provided means and standard deviations from the dataset , using the formula ( µE2 - µMock ) /sqrt ( σ2E2 + σ2Mock ) .",
"Breast cancer structural variants , translocations , and simple somatic mutations were obtained from ( Nik-Zainal et al . , 2016 ) , accession number EGAS00001001178 .",
"For the translocation data , genes were included on the list if they had at least one translocation in any patient sample .",
"Similarly , for the structural variant data , genes were included on the list if they had at least one structural variant in any patient sample .",
"For each mutation data set , a background data set made up of genes with similar replication timing and expression was used .",
"Expression was defined as the average expression from the mock , 160 min E2-treated and 1280 min E2-treated RNA-seq samples .",
"Replication timing was determined from the mean wavelet smoothed RepliSeq signal across each gene body , using ENCODE MCF7 RepliSeq data ( accession number wgEncodeEH002247 ) .",
"We used custom python scripts to match each translocated gene to a similar counterpart ( scripts: https://github . com/cimprichlab/stork_et_al_analysis ) .",
"Briefly , the replication and expression data were studentized , and the Euclidean distance between each mutated and non-mutated gene was calculated .",
"For every gene in the mutated set , we picked the closest gene by Euclidean distance that was not currently in the matched set .",
"We then compared the Z-score of estrogen induction between the test and matched sets .",
"Simple somatic mutation data were obtained from the ICGC commons ( Nik-Zainal et al . , 2016 ) .",
"Using the previously-calculated z-scores for estrogen induction from the RNA-seq dataset , genes with z-score greater than 2 were categorized as induced , while genes with z-score less than 2 were categorized as non-induced .",
"The raw numbers of simple somatic mutation events were counted over each gene in the RNA-seq dataset , and the counts were normalized to the length of the gene in kilobases as defined by the UCSC gene associated with the gene symbol .",
"Genomic regions matched for replication timing and expression were calculated as described above .",
"Data were log transformed before plotting .",
"Significance was assessed by a two-tailed bootstrap of the median , where the data were re-sampled into sets with the same size as the test and matched sets 1 , 000 , 000 times , and the median was calculated for both sets .",
"The p-value was calculated as the fraction of instances that the absolute value of the difference in medians between the resampled test and matched set was greater than the absolute value of the difference in median between the true test and matched sets .",
"For the proximity ligation assay ( PLA ) , cells were pre-extracted with cold 0 . 5% NP-40 for 3 min on ice .",
"Cells were then fixed with 4% PFA/PBS for 15 min , washed 3 times with 1X PBS and blocked for 1 hr at RT with 2% BSA/PBS .",
"Cells were then incubated with primary antibody overnight at 4°C ( 1:200 mouse S9 . 6 antibody alone; 1:500 rabbit P-H2AX alone; or 1:200 mouse S9 . 6 with 1:500 rabbit P-H2AX ) .",
"Cells were washed 3 times in 1X PBS and incubated in a pre-mixed solution of PLA probe anti-mouse minus and PLA probe anti-rabbit plus ( Sigma ) for 1 hr at 37˚C .",
"The Duolink In Situ Detection Reagents ( Green ) were then used to perform the PLA reaction according to the manufacturer’s instructions .",
"Slides were mounted in Duolink In Situ Mounting Medium with DAPI and imaged on a Zeiss Axioscope at 40X .",
"The number of PLA foci was quantified using Image J . Cells were trypsinized , washed in 1X PBS , and resuspended in 25 mL of 1X PBS .",
"Cells were crosslinked in 1% formaldehyde ( Pierce ) , quenched with 0 . 125 M glycine , and washed 2 times in 1X PBS containing protease inhibitor ( PI; Roche ) .",
"The cells were lysed in lysis buffer ( 50 mM HEPES pH 7 . 9 , 140 mM NaCl , 1 mM EDTA , 10% glycerol , 0 . 5% NP-40 , 0 . 25% Triton X-100 ) with PI and chromatin was sonicated in shearing buffer ( 0 . 1%SDS , 1 mM EDTA , 10 mM Tris pH 8 . 1 ) on a Covaris to an average size of 1 kb .",
"Washed Protein A/G sepharose beads ( Pierce ) were used to pre-clear chromatin for 2 hr . 10 μg of chromatin and 20 μg of S9 . 6 antibody or 20 μg mouse IgG were incubated overnight at 4°C .",
"Pre-washed protein A/G sepharose beads were then added to chromatin/antibody mixture for 2 hr .",
"After washing three times in binding buffer ( 10 mM NaPO4 pH 7 , 140 mM NaCl , 0 . 05% Triton X-100 ) , bound beads were boiled in 30 μL 5X sample buffer and loaded on a 4–20% gradient gel ( Biorad ) .",
"In this study , biological replicates indicate replicates of the experiment performed with separate passages of a cell line and independently prepared reagent mixes , most often at different times .",
"Technical replicates indicate distinct measurements from distinct wells or coverslips from the same biological experiment , processed at the same time .",
"Box and whiskers in all graphs represent 25–75 and 10–90 percentiles , respectively .",
"The line represents the median value .",
"All box and whisker plots show representative experiments .",
"Prism v6 ( GraphPad Software ) was used to perform student’s t-test and non-parametric Mann-Whitney rank sum t-test , where noted .",
"Error bars represent standard deviation ( SD ) unless otherwise noted as standard error of the mean ( SEM ) .",
"*p<0 . 05 , **p<0 . 01 , ***p<0 . 001 , and ****p<0 . 0001 ."
]
] | [
"The hormone estrogen ( E2 ) binds the estrogen receptor to promote transcription of E2-responsive genes in the breast and other tissues .",
"E2 also has links to genomic instability , and elevated E2 levels are tied to breast cancer .",
"Here , we show that E2 stimulation causes a rapid , global increase in the formation of R-loops , co-transcriptional RNA-DNA products , which in some instances have been linked to DNA damage .",
"We show that E2-dependent R-loop formation and breast cancer rearrangements are highly enriched at E2-responsive genomic loci and that E2 induces DNA replication-dependent double-strand breaks ( DSBs ) .",
"Strikingly , many DSBs that accumulate in response to E2 are R-loop dependent .",
"Thus , R-loops resulting from the E2 transcriptional response are a significant source of DNA damage .",
"This work reveals a novel mechanism by which E2 stimulation leads to genomic instability and highlights how transcriptional programs play an important role in shaping the genomic landscape of DNA damage susceptibility ."
] | [
"The hormone estrogen controls the development of breast tissue .",
"However too much estrogen can damage the DNA in human cells and may be linked to an increased risk of breast cancer .",
"In breast cells , estrogen activates many genes via a process called transcription .",
"The transcription process results in the production of an RNA molecule that contains a copy of the instructions encoded within the gene .",
"Previous studies have found that , in certain cases , a new RNA molecule can stick to the matching DNA from which it was made .",
"This creates a structure known as an R-loop , which can lead the DNA to break .",
"DNA breaks are particularly harmful because they can dramatically alter the cell’s genome in ways that allow it to become cancerous .",
"However , it was not clear if the large increase in transcription triggered by estrogen causes an increase in R-loops , which could help to explain the DNA damage that has been reported to occur when cells are treated with estrogen .",
"Now , Stork et al . show that treating human breast cancer cells with estrogen causes an increase in R-loops and DNA breaks .",
"The R-loops occurred particularly in regions of the genome that contain estrogen-activated genes .",
"Stork et al . also found that regions of estrogen-activated transcription were more frequently mutated in breast cancers , and further experiments confirmed that the R-loops were responsible for many of the DNA breaks that occurred following estrogen treatment .",
"Taken together , these findings demonstrate that the changes in transcription due to estrogen lead to increased R-loops and DNA breaks , which may make the cells vulnerable to becoming cancerous .",
"The next challenge is to determine precisely where these DNA breaks that result from estrogen occur on the DNA .",
"Knowing the location of the DNA breaks will be useful in determining what additional factors or genomic features make an R-loop more prone to being broken .",
"This in turn might help explain how the R-loops lead to DNA damage .",
"In addition , further studies are also needed to determine if tumor samples from breast cancer patients also contain increased levels of R-loops ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Auditory cortex shapes sound responses in the inferior colliculus | elife-51890-v2 | [
[
"Information processing is typically studied as a set of computations ascending along a hierarchy of sensory areas , often overlooking the role of descending feedback between these nuclei .",
"In the auditory system , the auditory cortex ( AC ) sends extensive feedback to nuclei earlier in the auditory pathway , including the auditory thalamus ( Alitto and Usrey , 2003; Rouiller and Durif , 2004; Winer et al . , 2001 ) and the inferior colliculus ( IC ) ( Bajo and Moore , 2005; Bajo et al . , 2007; Coomes et al . , 2005; Doucet et al . , 2003; Saldaña et al . , 1996; Williamson and Polley , 2019 ) .",
"Previous studies have demonstrated the importance of cortical feedback to IC in auditory behaviors ( Bajo et al . , 2010; Xiong et al . , 2015 ) , however , the mechanisms by which information processing is shaped via the descending feedback pathway remain poorly characterized .",
"In this study , we selectively modulated activity in AC , targeting either excitatory projections or inhibitory interneurons , to quantify how AC shapes spontaneous activity and sound-evoked responses in IC .",
"Previous studies demonstrated that neuronal responses to sounds in IC are altered by focal electrical stimulation and inactivation of AC .",
"Cortical stimulation shifted tuning properties of IC neurons toward those of the stimulated neurons in frequency ( Jen et al . , 1998; Jen and Zhou , 2003; Ma and Suga , 2001a; Yan et al . , 2005; Yan and Suga , 1998; Zhou and Jen , 2007 ) , amplitude ( Jen and Zhou , 2003; Yan et al . , 2005; Zhou and Jen , 2007 ) , azimuth ( Zhou and Jen , 2007; Zhou and Jen , 2005 ) , and duration ( Ma and Suga , 2001b ) .",
"Stimulation of AC had mixed effects on sound-evoked responses in IC , increasing and decreasing responses in different subpopulations of neurons ( Jen et al . , 1998; Zhou and Jen , 2005 ) .",
"Consistent with this effect , different patterns of direct cortico-collicular activation enhanced or suppressed white noise-induced responses in IC ( Vila et al . , 2019 ) .",
"AC inactivation studies , on the other hand , found less consistent effects on IC responses .",
"Whereas one study found that inactivation of AC caused a shift in best frequency in IC neurons ( Zhang et al . , 1997 ) , several other studies showed that inactivation of AC had no effect on frequency selectivity in IC ( Jen et al . , 1998 ) , but rather modulated sound-evoked and spontaneous activity ( Gao and Suga , 1998; Popelár et al . , 2003; Popelář et al . , 2016 ) .",
"Cortico-collicular feedback is critical to auditory learning , specifically learning to adapt to a unilateral earplug during sound localization ( Bajo et al . , 2010 ) .",
"Pairing electrical leg stimulation with a tone induced a shift in best frequency of IC neurons , while presentation of a tone alone was insufficient ( Gao and Suga , 1998; Gao and Suga , 2000 ) .",
"Furthermore , cortico-collicular feedback was necessary to induce running in response to a loud noise ( Xiong et al . , 2015 ) .",
"In AC , modulation of sound responses is not a monophasic process , but instead is shaped by interactions between excitatory and inhibitory cortical neurons .",
"Modulating activity of different inhibitory interneuron subtypes in AC narrowed frequency tuning and attenuated tone-evoked responses of excitatory neurons , while suppression had the opposite effect ( Aizenberg et al . , 2015; Hamilton et al . , 2013; Phillips and Hasenstaub , 2016; Seybold et al . , 2015 ) .",
"Electrical stimulation of AC , cooling or pharmacological inactivation affected the amplitude of sound-evoked responses and shifted the best frequency of neurons in IC , but it remains unknown how specific these effects are to direct feedback , and whether the effects of intra-cortical inhibition propagate to the IC .",
"The goal of the present study is to examine the role of cortico-collicular projections in shaping sound responses in IC .",
"IC receives glutamatergic ( Feliciano and Potashner , 1995 ) inputs from neurons originating predominantly in layer 5 of AC ( Bajo and Moore , 2005; Bajo et al . , 2007; Coomes et al . , 2005; Doucet et al . , 2003; Saldaña et al . , 1996; Winer et al . , 1998 ) .",
"We used viral transfection methods to selectively drive excitatory or inhibitory opsin expression in AC-IC projections .",
"We then recorded neuronal activity in IC and tested how activation or suppression of AC-IC projections affected spontaneous activity and sound-evoked responses in IC .",
"To better understand whether and how intra-cortical network interactions propagated to IC , we manipulated the activity of the two most common inhibitory neuronal subtypes in AC , Parvalbumin- ( PV ) and Somatostatin- ( SST ) positive interneurons ( Rudy et al . , 2011 ) ."
],
[
"Our first goal was to characterize the effects of activating the direct cortico-collicular projections on tone-evoked responses in IC .",
"A combination of cortical anterograde and collicular retrograde viral transfections was used in order to achieve specific viral transfection of cortico-collicular neurons .",
"We delivered either an excitatory opsin , ChannelRhodopsin2 ( ChR2 ) or an inhibitory opsin , ArchaerhodopsinT ( ArchT ) , bilaterally , specifically to the neurons in the auditory cortex which project to the inferior colliculus ( Figure 1A ) .",
"To achieve such specificity , we injected a retrograde virus that encoded Cre recombinase ( Retro2 AAV . Cre ) in IC ( Figure 1B ) .",
"This retrograde viral construct ensured that neurons projecting to IC expressed Cre recombinase a few weeks later .",
"At the same time , we injected a virus that encoded ChR2 or ArchT in reversed fashion under the FLEX cassette in AC ( AAV . Flex . ChR2 , AAV . Flex . ArchT ) ( Figure 1C ) .",
"This strategy ensured that only neurons expressing Cre recombinase in the auditory cortex would express ChR2 or ArchT in AC .",
"Therefore , opsin was expressed exclusively in AC-IC projecting neurons , which terminate across all regions of IC , including central nucleus ( CNIC ) and dorsal cortex of IC ( DCIC ) , from which we recorded ( Figure 1D ) .",
"Shining light over AC of these mice would therefore directly activate or suppress only these cortico-collicular feedback projections to IC .",
"First , we measured and quantified neuronal spiking in IC in response to stimulation or suppression of cortico-collicular projections using ChR2 or ArchT .",
"To activate this projection , we shone blue laser over AC , recorded neuronal activity in IC , and quantified the effects of manipulating feedback in the absence of sound ( Figure 2A , C , D ) .",
"We measured the spiking activity in IC as we varied the duration of laser manipulation ( 1 ms , 5 ms , 25 ms , 250 ms ) .",
"As exected , activation of cortico-collicular neurons resulted , on average , in an increase in firing rate of IC neurons .",
"This effect persisted at the 5 ms , 25 ms , and 250 ms laser duration ( Figure 2C; 1 ms: p=5 . 3e-4 , ON = 5 . 6 ± 0 . 6 Hz , OFF = 4 . 9 ± 0 . 5 Hz; 5 ms: p=0 . 0027 , ON = 6 . 9 ± 0 . 7 Hz , OFF = 4 . 8 ± 0 . 5 Hz; 250 ms: p=0 . 005 , ON = 5 . 6 ± 0 . 6 Hz , OFF = 4 . 8 ± 0 . 5 Hz ) .",
"Whereas the direction of the effect was consistent with our prior expectations , the magnitude of the effect was unexpectedly small .",
"This suggests that AC targets a small subpopulation of IC neurons and that the effect of activation does not spread far within IC .",
"We tested the effect of suppressing AC-IC neurons on firing responses in IC and , surprisingly , we detected little difference in firing in IC neurons ( Figure 2D; 25 ms: p=0 . 039 , ON = 9 . 5 ± 0 . 7 Hz , OFF = 9 . 5 ± 0 . 7 Hz; 250 ms: p=0 . 02 , ON = 9 . 5 ± 0 . 8 Hz , OFF = 9 . 5 ± 0 . 8 Hz ) .",
"This result suggests that AC-IC inputs at rest do not contribute to IC responses , but rather modulate collicular activity only when activated .",
"Next , our goal was to characterize modulation of sound-evoked responses in IC by cortical feedback .",
"We first tested the effects of feedback modulation on acoustic click responses .",
"We chose clicks as the initial stimulus because they drive fast responses in both AC and IC .",
"Laser stimulation began 250 ms prior to click train onset to allow for the response to the laser to come to a steady state , and continued activation throughout the clicks ( Figure 2B ) .",
"Activating the feedback using ChR2 had a weak suppressive effect on the firing rate of IC neurons in response to clicks .",
"Consistent with previous experiments , we observed an overall increase in spontaneous activity ( Figure 2E , bottom; p=0 . 0031 , spont ON = 5 ± 0 . 87 Hz , spont OFF = 4 . 3 ± 0 . 92 Hz ) .",
"Activating feedback caused a small decrease in click-evoked response ( Figure 2E , left; p=0 . 001 , click ON = 10 . 4 ± 1 . 2 Hz , click OFF = 10 . 7 ± 1 . 1 Hz ) .",
"By contrast , suppressing cortico-collicular feedback using ArchT had no effect on IC click responses ( Figure 2F ) .",
"Because attenuating the input does not affect the activity in IC during clicks , this result further suggests that the strength of the baseline signal from AC to IC , even in the presence of cortical activity , is not sufficient to modulate IC responses .",
"We also tested whether the effects of cortico-collicular modulation differed across tone frequency range .",
"We presented a stimulus that consisted of tones of 50 frequencies ranging from 3 to 70 kHz .",
"To modulate the feedback from AC , we tested three different laser onsets ( −100 ms , −20 ms , +8 ms ) relative to tone onset to isolate the effect of timing on affecting IC responses .",
"These delays were chosen for the following reasons: −100 ms delay would allow for the laser effect on cortical activity to come to a steady state , allowing to quantify the effect throughout the tone pip; +8 ms is set up to mimic the time scale of cortical response to a tone , effectively amplifying the onset of the cortical response; −20 ms delay is an intermediate value .",
"We found that activating cortico-collicular feedback using ChR2 increased spontaneous activity of IC neurons ( Figure 3A , left; −100 ms: p=0 . 022 , ON = 4 . 3 ± 0 . 47 Hz; OFF = 3 . 6 ± 0 . 38; −20 ms: p=8 . 6e-6 , ON = 5 . 6 ± 0 . 63 , OFF = 3 . 7 ± 0 . 47 ) .",
"However , overall the feedback decreased tone-evoked response magnitude in IC , which we defined as the difference between spontaneous and tone-evoked response , at all laser onsets ( Figure 3A , right; −100 ms: p=3 . 2e-5 , ON = 14 . 2 ± 0 . 98 Hz; OFF = 15 . 5 ± 1 . 04 Hz; −20 ms: p=1 . 4e-6 , ON = 11 . 8 ± 1 . 08 Hz , OFF = 13 . 9 ± 1 Hz; +8 ms: p=0 . 0034 , ON = 13 . 03 ± 1 . 03 , OFF = 13 . 7 ± 1 . 03 ) .",
"This suggests that the broad activation of feedback upregulates the baseline activity of IC neurons , but decreases tone-evoked response ( Figure 6C , −100 ms: p=0 . 018 , ON = 19 . 01 ± 1 . 2 Hz , OFF = 18 . 5 ± 1 . 2 Hz; +8 ms: p=0 . 007 , ON = 17 . 3 ± 1 . 2 Hz; OFF = 16 . 9 ± 1 . 2 Hz ) .",
"By contrast , suppressing the feedback using ArchT had no effect on either spontaneous activity or tone-evoked response magnitude ( Figure 3B ) , suggesting that at baseline AC does not provide strong modulation of IC activity , as removing it does not affect sound-evoked effects in IC .",
"We then examined the effect of feedback on frequency tuning of IC units .",
"Activation of feedback using ChR2 decreased frequency selectivity in the subsets of IC units that exhibited a decrease in tone-evoked response magnitude or increase in spontaneous activity ( Figure 4A ) , but not in units that exhibited an increase in tone-evoked response magnitude or decrease in spontaneous activity ( Figure 4B , mag decrease: −20 ms , p=0 . 00031 , sparse ON = 0 . 49 ± 0 . 027 , sparse OFF = 0 . 55 ± 0 . 025; +8 ms , p=0 . 00029 , sparse ON = 0 . 46 ± 0 . 027 , sparse OFF = 0 . 55 ± 0 . 024; spont increase: −100 ms , p=0 . 011 , sparse ON = 0 . 49 ± 0 . 032 , sparse OFF = 0 . 56 ± 0 . 031; −20 ms , p=0 . 00018 , sparse ON = 0 . 46 ± 0 . 034 , sparse OFF = 0 . 56 ± 0 . 029; +8 ms , p=2 . 2e-6 , sparse ON = 0 . 41 ± 0 . 032 , sparse OFF = 0 . 55 ± 0 . 029 ) .",
"Interestingly , these changes were observed almost exclusively in cells located in DCIC rather than CNIC ( Figure 5 ) .",
"Specifically , activation of feedback increased spontaneous activity ( Figure 5A , left; −100 ms: p=0 . 0204 , ON = 3 . 5 ± 0 . 5 Hz , OFF = 2 . 5 ± 0 . 3 Hz; −20 ms: p=2 . 8e-7 , ON = 5 . 2 ± 0 . 7 Hz , OFF = 2 . 3 ± 0 . 3 Hz ) and decreased tone-evoked response magnitude ( Figure 5A , right , −100 ms: p=3 . 3e-6 , ON = 14 . 8 ± 1 . 4 Hz , OFF = 16 . 5 ± 1 . 5 Hz; −20 ms: p=6 . 3e-7 , ON = 11 . 8 ± 1 . 6 Hz , OFF = 14 . 7 ± 1 . 4 Hz ) in cells located in DCIC , but not in CNIC ( Figure 5B ) .",
"Similarly , activation of feedback decreased frequency selectivity ( Figure 5C; mag decrease: −20 ms , p=6 . 3e-4 , sparse ON = 0 . 46 ± 0 . 03 , sparse OFF = 0 . 53 ± 0 . 03; +8 ms , p=4 . 7e-5 , sparse ON = 0 . 41 ± 0 . 03 , sparse OFF = 0 . 53 ± 0 . 03; spont increase: −100 ms , p=0 . 0044 , sparse ON = 0 . 43 ± 0 . 04 , sparse OFF = 0 . 52 ± 0 . 04; −20 ms , p=0 . 00013 , sparse ON = 0 . 39 ± 0 . 04 , sparse OFF = 0 . 53 ± 0 . 04; +8 ms , p=5 . 2e-6 , sparse ON = 0 . 33 ± 0 . 03 , OFF = 0 . 53 ± 0 . 04 ) in cells located in DCIC , but not in CNIC ( Figure 5D ) .",
"This is consistent with the enhanced density of AC projections in DCIC as compared to CNIC ( Figure 1D ) .",
"To verify that these results were not due to a select few high firing rates of units in DCIC , we repeated the analysis comparing only units with spontaneous activity in the laser OFF condition less than 40 Hz .",
"With these matched firing rates we still observed the same statistically significant effects ( DCIC – mag decrease −100 ms: p=3 . 3e-6 , ON = 14 . 8 Hz , OFF = 16 . 6 Hz; −20 ms: p=6 . 3e-7 , ON = 12 . 1 Hz , OFF = 12 . 6 Hz; spont increase −100 ms: p=0 . 02 ON=3 . 5 Hz , OFF = 2 . 5 Hz; −20 ms: p=2 . 8e-7 , ON = 5 . 1 Hz , OFF = 4 . 9 Hz; CNIC – mag decrease +8 ms: p=0 . 0039 , ON = 11 . 7 Hz , OFF = 12 . 6 Hz ) .",
"To elucidate changes in the shape of the frequency response functions in IC with activation of cortico-cortical feedback beyond the general changes in selectivity we observed with changes in sparseness , we fitted linear regressions to light off versus light on firing rates .",
"This analysis can reveal whether changes in frequency selectivity are linear/subtractive , multiplicative/divisive , or some combination of these transformations .",
"For example , a slope of 1 and a positive y-intercept would represent a linear increase in frequency response ( equal increase in response across all frequencies ) .",
"Over the population of IC neurons with decreased tone-evoked response magnitude and/or increased spontaneous activity , the median slopes and y-intercepts of ranked linear fits to frequency responses are less than 1 and above zero , respectively ( Figure 6A ) .",
"These results , in combination with the decreased tone-evoked activity observed ( Figure 6A; Figure 6B; Figure 6C , −100 ms: p=0 . 018 , ON = 19 . 01 ± 1 . 2 Hz , OFF = 18 . 5 ± 1 . 2 Hz; +8 ms: p=0 . 007 , ON = 17 . 3 ± 1 . 2 Hz; OFF = 16 . 9 ± 1 . 2 Hz ) , indicate that the decrease in frequency selectivity was due to a decrease in response to tones at preferred frequencies , not non-preferred frequencies .",
"This result suggests that the suppressive effect of the cortico-collicular feedback is preferential for higher firing responses .",
"Suppressing feedback resulted in very small ( <0 . 04% ) change in sparseness , therefore it did not affect frequency selectivity in IC ( Figure 4B ) .",
"Correlations in trial-to-trial responsiveness of neurons can affect the information encoded by a neuronal population ( Abbott and Dayan , 1999; Averbeck et al . , 2006; Nirenberg and Latham , 2003; Shadlen and Newsome , 1998; Zohary et al . , 1994 ) .",
"To test whether activation of cortico-collicular feedback affected correlations in trial-to-trial variability of response strength between units recorded simultaneously we measured noise correlations .",
"We found that activating cortico-collicular feedback using ChR2 had no effect on noise correlations during spontaneous activity or tone response ( Figure 3—figure supplement 1 ) .",
"To better understand the effect of modulation of cortical activity on spectro-temporal receptive field properties of IC neurons , we presented a continuous signal comprised of dynamic random chords ( DRCs ) sampled from a uniform distribution of loudness values per frequency bin .",
"The unbiased nature of this stimulus allowed us to estimate the spectro-temporal receptive field of neurons ( STRF ) , which quantifies the dynamics of sound waveform in time and frequency that lead to a neuronal response .",
"During the DRC stimulus , we turned on the laser every other second for 250 ms to activate cortico-collicular projections with ChR2 .",
"We found that a subset of IC units reduced their mean DRC-evoked firing rates ( N = 56 ) , whereas another subset of IC units increased their mean DRC-evoked firing rate when we activated cortico-collicular feedback ( N = 47 ) .",
"We next separately computed the receptive fields for each neuron when laser was off and when laser was on and compared the STRFs for activation with ChR2 ( Figure 7A ) .",
"To quantify those changes , we identified the positive ( activation ) and negative ( suppressive ) regions in the STRFs and compared them for laser ON and laser OFF conditions .",
"In the subset of neurons whose firing rate increased with laser STRFs changed: only 42% of positive lobes , and 63% of negative lobes persisted with the laser ( Figure 7B , left ) .",
"Of those lobes that persisted , for positive lobes , there was on average a decrease in temporal width ( p=0 . 00098 , ON = 0 . 0303 ± 0 . 0018 s , OFF = 0 . 037 ± 0 . 0023 s ) , frequency selectivity ( p=0 . 00042 , ON = 6 . 7 ± 1 . 1 Hz , OFF = 9 . 6 ± 1 . 8 Hz ) and STRF size ( p=0 . 00036 , ON = 46 . 05 ± 6 . 9 pixels , OFF = 77 . 5 ± 12 . 04 pixels ) , whereas for negative lobes , we did not detect any changes ( Figure 7C , left ) .",
"For IC units whose firing rate was decreased , there was a much smaller change in the lobes with cortico-collicular activation , with 78% and 79% of positive and negative lobes persisting , respectively ( Figure 7B , right ) .",
"For both positive lobes and negative lobes , the only difference was an increase in the temporal width when laser activated cortico-collicular feedback ( Figure 7C , right; Positive lobes: p=0 . 026 , ON = 0 . 034 ± 0 . 0025 s , OFF = 0 . 032 ± 0 . 0025 s; Negative lobes: p=0 . 02 , ON = 0 . 037 ± 0 . 0028 s , OFF = 0 . 032 ± 0 . 0024 s ) .",
"The decrease in STRF size in units with increased DRC-evoked response and decrease in sparseness across the population is consistent with the interpretation that the effect of the cortico-collicular feedback leads to a decrease in responsiveness to tones that evoke the greatest responses ( in the center of the receptive field ) and an increase to stimuli that evoke weaker activity .",
"In other words , neuronal firing increases overall , but selective responses to specific frequency bands decrease .",
"Because the majority of the units were recorded as multi-units , it is possible that this change in selectivity is due to the change in firing rate of differentially tuned single units .",
"Auditory responses in AC are shaped by interactions between excitatory and inhibitory neurons ( Wood et al . , 2017 ) .",
"To determine how modulating frequency selectivity in AC might affect tone-evoked responses in IC in a frequency-selective fashion , we perturbed the excitatory-inhibitory interactions by modulating two different classes of inhibitory interneuron known to contribute to sound responses in AC: PV and SST inhibitory interneurons ( Figure 8A ) .",
"We found that modulating PV interneuron activity in AC had no significant effects on spontaneous and tone-evoked activity or frequency selectivity in IC ( Figure 8B–E ) despite modulating frequency selectivity , spontaneous activity , and tone-evoked response magnitude in AC .",
"Specifically , in AC , activating PVs decreased spontaneous activity and tone-evoked response magnitude ( Figure 8F; spontaneous activity: p=2 . 2e-9 , ON = 0 . 88 ± 0 . 16 Hz , OFF = 2 . 7 ± 0 . 34 Hz; tone-evoked response magnitude: p=8 . 02e-7 , ON = 6 . 9 ± 1 . 06 Hz , OFF = 11 . 5 ± 1 . 3 Hz ) and increased frequency selectivity ( Figure 8G; p=3 . 3e-12 , ON = 0 . 59 ± 0 . 022 , OFF = 0 . 42 ± 0 . 02 ) , while suppressing PVs increased spontaneous activity ( Figure 8I; p=5 . 2e-5 , ON = 4 . 08 ± 0 . 54 Hz , OFF = 3 . 06 ± 0 . 55 Hz ) and decreased frequency selectivity ( Figure 8J; p=3 . 8e-5 , ON = 0 . 34 ± 0 . 027 , OFF = 0 . 41 ± 0 . 031 ) .",
"This suggests that the changes in cortical activity driven by manipulation of PV activity does not propagate to the inferior colliculus .",
"Different interneuron classes may function in distinct networks , so we also tested the effects of modulating SST interneurons in AC .",
"Modulating SST interneurons had no effect on tone-evoked activity ( Figure 9B , D , right ) or frequency selectivity ( Figure 9C , E ) , but suppressing SST interneurons increased spontaneous activity in IC ( Figure 9D , left; p=0 . 029 , ON = 6 . 9 ± 0 . 66 Hz , OFF = 6 . 7 ± 0 . 63 Hz ) , a change that we also observed with activation of the direct feedback projections ( Figure 2A , left ) .",
"Similar to PV interneurons , activating SST interneurons decreased spontaneous activity and tone-evoked response magnitude in AC ( Figure 9F; spontaneous activity: p=1 . 4e-12 , ON = 0 . 97 ± 0 . 25 Hz , OFF = 2 . 9 ± 0 . 37 Hz; tone-evoked response magnitude: p=1 . 3e-15 , ON = 3 . 3 ± 0 . 59 Hz , OFF = 11 . 4 ± 1 . 2 Hz ) and increased frequency selectivity ( Figure 9G; p=1 . 6e-13 , ON = 0 . 69 ± 0 . 024 , OFF = 0 . 47 ± 0 . 019 ) .",
"In AC , suppressing SSTs reduced spontaneous activity , but had no significant effect on tone-evoked response magnitude or frequency selectivity ( Figure 9I , J; p=8 . 1e-4 , ON = 3 . 6 ± 0 . 41 Hz , OFF = 2 . 2 ± 0 . 38 Hz ) .",
"This lack of effect was not due to the relatively small effect of light penetrating to the deep layers .",
"In fact , to confirm that modulating PV and SST activity in AC affected activity of units in L5/6 where the feedback projections we computed changes in spontaneous activity at each tetrode which spanned the entire auditory cortex .",
"We found that activity was modulated across the layers ( Figures 8–9 H , K ) .",
"Whereas modulating PVs did not have an effect on IC activity , SST suppression resulted in an increase in spontaneous , but not tone-evoked activity in IC , which suggests that inhibitory modulation of sound responses in AC does not propagate to IC ."
],
[
"Auditory cortex sends extensive projections to IC ( Bajo and Moore , 2005; Bajo et al . , 2007; Coomes et al . , 2005; Doucet et al . , 2003; Saldaña et al . , 1996; Winer et al . , 1998 ) .",
"Our results demonstrate that activation of this cortico-collicular feedback modulates sound responses in IC by upregulating spontaneous IC activity , decreasing frequency selectivity and reducing spectro-temporal receptive field size in IC ( Figures 2 , 3 , 4 ) .",
"This effect was localized to DCIC , corresponding to the sites with largest density of AC projections ( Figure 5 ) .",
"Interestingly , suppressing cortico-collicular feedback had little effect on IC activity ( Figures 2 , 3 ) , which suggests that at baseline and during passive tone presentation the feedback does not affect activity of IC neurons .",
"We also found that SST , but not PV inhibitory interneurons modulated IC spontaneous activity , suggesting that the effects of modulation of cortical activity by PVs does not back-propagate to IC , whereas SST-driven modulation affects spontaneous activity , but not tone-evoked responses ( Figures 8 , 9 ) .",
"Overall our findings imply that direct cortico-collicular feedback can modulate responses to simple ( pure tones ) and more complex ( DRCs ) auditory stimuli by reducing , rather than increasing , sound selectivity .",
"This modulation occurs independently of the activity of cortical inhibitory interneurons .",
"Whereas optogenetic manipulation allows for temporally precise control and cell-type specificity , the technique lacks the spatial specificity of electrical stimulation .",
"Since both AC and cortico-collicular projections are tonotopically organized ( Barnstedt et al . , 2015; Lim and Anderson , 2007; Markovitz et al . , 2013; Straka et al . , 2015 ) , spatially specific stimulation can more accurately mimic frequency-specific responses by activating specific regions in the tonotopic map .",
"Previous studies found that electrical stimulation of AC caused best frequencies in IC to shift towards the best frequencies of the stimulated site in AC ( Gao and Suga , 1998; Gao and Suga , 2000; Ma and Suga , 2001a; Yan et al . , 2005; Yan and Suga , 1998; Zhou and Jen , 2007 ) .",
"These results suggest that cortico-collicular feedback may be important for increasing the representation of behaviorally relevant stimuli in IC .",
"However , when activating direct feedback , we did not observe consistent changes in best frequency of IC neurons ( Figure 6D ) .",
"This may be due to activation of feedback projections across cortex , and therefore across the tonotopic map , in this paradigm .",
"Previous studies found no effect of activating projection terminals on responses in the CNIC , only shell regions of IC ( Xiong et al . , 2015 ) , which are the predominant targets of cortico-collicular feedback .",
"In our study we were able to record from a larger neuronal population , revealing a subset of neurons in central IC that were modulated by feedback .",
"This is consistent with another study which observed enhanced white noise-induced activity in a small subset of neurons in the CNIC with activation of direct cortico-collicular feedback ( Vila et al . , 2019 ) .",
"The effects in central IC may be due to direct cortico-collicular feedback , intra-collicular circuits ( Malmierca et al . , 1995; Miller et al . , 2005; Saldaña and Merchań , 1992; Sturm et al . , 2014 ) , or a combination of these mechanisms .",
"Previous inactivation studies have shown mixed effects on sound-evoked responses in IC , but consistently demonstrated no effect on frequency selectivity , agreeing with our results .",
"Specifically , suppression of AC increased or decreased IC sound responses in distinct subsets of cells ( Popelár et al . , 2003; Popelář et al . , 2016 ) while suppression of direct cortico-collicular feedback terminals in IC decreased sound-evoked responses ( Xiong et al . , 2015 ) .",
"It is possible that we saw little effect from suppression of cortico-collicular projections because inactivation through the auditory cortex suppressed only a subset of feedback projections to IC .",
"Combined , these studies suggest that at baseline the cortex provides only weak modulation of IC activity .",
"Understanding how AC modulates sound responses in central IC may provide insight into how AC drives changes in auditory behavior .",
"Previously , we found that behavioral frequency discrimination acuity changed after differential auditory fear conditioning and these changes were driven by the auditory cortex ( Aizenberg and Geffen , 2013; Aizenberg et al . , 2015 ) .",
"The behavioral task used to test frequency discrimination acuity was a modified pre-pulse inhibition ( PPI ) of the acoustic startle reflex task .",
"Although AC can modulate this behavior , PPI is still observed after decerebration ( Li and Frost , 2000 ) and the underlying circuit is believed to be subcortical ( Fendt et al . , 2001 ) .",
"The IC is a critical structure in the PPI circuit ( Fendt et al . , 2001; Leitner and Cohen , 1985; Li et al . , 1998 ) , with the central nucleus of IC receiving ascending auditory projections and the external nucleus of IC acting as the output station ( Fendt et al . , 2001; Li and Yue , 2002 ) .",
"The broad frequency tuning in the shell regions of the IC ( Barnstedt et al . , 2015; Syka et al . , 2000 ) made the central nucleus of IC , which has sharper tuning and a tonotopic organization ( Barnstedt et al . , 2015; Ehret et al . , 2003; Malmierca et al . , 2008; Syka et al . , 2000 ) , a good candidate for how AC may drive changes in frequency discrimination acuity .",
"Limited evidence for a descending pathway from AC to the pedunculopontine tegmental nucleus ( Schofield and Motts , 2009 ) suggests another alternative pathway by which AC modulates frequency discrimination .",
"We activated cortico-collicular neurons by shining light in the cortex , thus initiating spikes at the level of the cell body as would occur in response to a sound .",
"An important consideration with this method , however , is that these neurons do not only send collaterals to IC , but also to the auditory thalamus ( Asokan et al . , 2018; Williamson and Polley , 2019 ) , amygdala ( Asokan et al . , 2018 ) , and striatum ( Asokan et al . , 2018 ) .",
"There is also evidence of descending projections to IC from thalamus ( Winer et al . , 2002 ) and amygdala ( Marsh et al . , 2002 ) .",
"When interpreting our results , it is important to consider that the effects we observe from activating cortico-collicular feedback may also be due , at least in part , to these secondary pathways .",
"One caveat is that we used a square pulse for optogenetic manipulations , whereas a more complex stimulation pattern potentially can achieve stronger activation ( Vila et al . , 2019 ) .",
"One caveat of our study is that our experiments were performed in passively listening mice .",
"It is possible that the lack of effect we observe with suppression of cortico-collicular feedback is due to the lack of task engagement .",
"Previous studies have found that inactivation of this pathway affects innate responses to sound ( Xiong et al . , 2015 ) and auditory learning ( Bajo et al . , 2010 ) .",
"Furthermore , studies have found that task engagement modulates neuronal responses to sounds ( Downer et al . , 2017; Fritz et al . , 2003; Kuchibhotla et al . , 2017; Lakatos et al . , 2013; McGinley et al . , 2015 ) .",
"Thus , we would expect suppression of this pathway to affect sound processing in IC during a behavioral task .",
"Future studies should explore how activity changes with an animal actively engaged in a behavioral task .",
"We observed little effect of modulating AC inhibitory interneurons on activity in IC despite the changes observed in AC ( Figures 8 and 9 ) , which suggests there may be another neuron subtype that plays a modulatory role during cortico-collicular plasticity in driving feedback modulation .",
"One possibility that was not explored here are cholinergic inputs from the nucleus basalis ( NB ) .",
"NB , a cholinergic nucleus in the basal forebrain , has been implicated as a key structure in learning-induced auditory plasticity .",
"Studies have found that pairing NB stimulation with presentation of a tone has been sufficient to induce receptive field plasticity in AC ( Kilgard and Merzenich , 1998; Kilgard et al . , 2001; Weinberger , 2003 ) and that blocking muscarinic receptors in AC impairs this plasticity ( Miasnikov et al . , 2008 ) .",
"Of greater interest is the receptive field plasticity also observed in IC .",
"Acetylcholine applied to AC increased spontaneous and tone-evoked activity in both AC and IC ( Ji et al . , 2001 ) .",
"Furthermore , pairing AC stimulation with nucleus basalis stimulation ( Ma and Suga , 2003 ) or pairing tone presentation with nucleus basalis stimulation ( Zhang et al . , 2005 ) shifted best frequencies in both AC and IC and application of atropine in AC inhibited this IC plasticity ( Zhang et al . , 2005 ) .",
"It is plausible that NB cholinergic inputs may be responsible for driving responses of IC neurons rather than PV or SST inhibitory interneurons .",
"However , SSTs in AC are depolarized by application of cholinergic agonist ( Kuchibhotla et al . , 2017 ) .",
"While the lack of effect we observe with interneuron inhibition might suggest that SST and PV interneurons are not involved in this circuit , we must also consider that direct activation of feedback is not exactly equivalent to disinhibition .",
"Direct activation causes synchronized activation of cells , while disinhibition allows for asynchronous activation .",
"This difference may explain the different results .",
"Furthermore , the lack of effect we observe with inhibitory interneuron activation is consistent with our results from suppressing cortico-collicular feedback .",
"Thus , while another neuronal subtype may be responsible for driving feedback activity in a behavioral context , inhibitory interneurons may still play a role in shaping responses of feedback once active ."
],
[
"All experiments were performed in adult male and female mice ( supplier: Jackson Laboratories; age , 12–15 wk; weight , 22–32 g; wild-type C57BL/6J RRID:IMSR_JAX:000664; Pvalb-Cre mice , strain: B6;129P2-Pvalbtm1 ( cre ) Arbr/J RRID:IMSR_JAX:008069; Sst-Cre mice , strain: Ssttm2 . 1 ( cre ) Zjh/J RRID:IMSR_JAX:013044; Cdh23 mice , strain: Cdh23tm2 . 1Kjn/J RRID:IMSR_JAX:018399 , or Pvalb-Cre x Cdh23 or Sst-Cre x Cdh23 crosses ) .",
"Mice were housed at 28°C on a 12 hr light–dark cycle with water and food provided ad libitum , less than five animals per cage .",
"In Pvalb-Cre mice Cre recombinase ( Cre ) was expressed in parvalbumin-positive interneurons , and in Sst-Cre , Cre was expressed in somatostatin-positive interneurons .",
"All animal work was conducted according to the guidelines of University of Pennsylvanian IACUC ( protocol number 803266 ) and the AALAC Guide on Animal Research .",
"Anesthesia by isoflurane and ketamine and euthanasia by CO2 were used .",
"All means were taken to minimize the pain or discomfort of the animals during and following the experiments .",
"All experiments were performed during the animals' dark cycle .",
"Original spike data and code are available on Dryad ( http://doi . org/10 . 5061/dryad . 1t61c80 ) .",
"Modified AAVs encoding ArchT ( AAV9-CAG-FLEX-ArchT-GFP or AAV9-CAG-FLEX-ArchT-tdTomato; UNC Vector Core ) or ChR2 ( AAV9-CAG-FLEX-ChR2-tdTomato; Penn Vector Core ) were used for selective suppression or excitation , respectively .",
"Retrograde AAV virus encoding Cre ( retro AAV-hSyn-Cre-GFP ) was custom made in our laboratory .",
"Briefly , RetroAAV2 hSyn Cre-GFP was packaged using the Helper-Free system ( Agilent ) and the retrograde trafficking plasmid , Retro2 , which bears capsid mutations in serotype 2 .",
"At least 21 days prior to electrophysiological recordings , mice were anesthetized with isoflurane to a surgical plane .",
"The head was secured in a stereotactic holder .",
"The mouse was subjected to a small craniotomy ( 2 × 2 mm ) over AC under aseptic conditions .",
"Viral particles were injected ( 750 nl ) bilaterally using a syringe pump ( Pump 11 Elite , Harvard Apparatus ) targeted to AC ( coordinates relative to bregma: −2 . 6 mm anterior , ±4 . 3 mm lateral , +1 mm ventral ) .",
"Fiber-optic cannulas ( Thorlabs , Ø200 μm Core , 0 . 22 NA ) were implanted bilaterally over the injection site at depth of 0 . 5 mm from the skull surface .",
"For a subset of mice , to target direct feedback , the mouse was also subjected to a craniotomy over IC ( 1 × 4 mm ) .",
"Retro AAV viral construct was injected ( 3 × 200 nl ) via glass syringe ( 30–50 um diameter ) using a syringe pump ( Pump 11 Elite , Harvard Apparatus ) bilaterally in IC .",
"Craniotomies were covered with a removable silicon plug .",
"A small headpost was secured to the skull with dental cement ( C and B Metabond ) and acrylic ( Lang Dental ) .",
"For postoperative analgesia , slow release Buprenex ( 0 . 1 mg/kg ) and Bupivicane ( 2 mg/kg ) were injected subcutaneously .",
"An antibiotic ( 5 mg/kg Baytril ) was injected subcutaneously daily ( for 4 days ) at the surgical site during recovery .",
"Virus spread was confirmed in all mice postmortem by visualization of the fluorescent protein expression in fixed brain tissue , and its colocalization with PV or SST or , for feedback cohort , expression in AC layer 5/6 , following immuno-histochemical processing with the appropriate antibody .",
"To confirm the location of viral injection sites , tissue sections containing the IC or AC were cut at 40 μm using a cryostat .",
"Sections were mounted onto glass slides and imaged using Zeiss LSM 800 confocal microscope .",
"20X images were taken throughout the entire IC or AC , and tiles were stitched together to form a composite image using Zen software .",
"Masks were drawn around the edge of the tissue to hide the embedding medium using Photoshop .",
"Stimuli were delivered via a magnetic speaker ( Tucker-Davis Technologies ) directed toward the mouse’s head .",
"Speakers were calibrated prior to the experiments to ± 3 dB over frequencies between 3 and 70 kHz by placing a microphone ( Brüel and Kjaer ) in the location of the ear contralateral to the recorded AC hemisphere , recording speaker output and filtering stimuli to compensate for acoustic aberrations ( Carruthers et al . , 2013 ) .",
"Free-standing speaker was used to approximate the conditions under which the mouse typically experiences sounds in awake state .",
"To obtain a quick assessment of auditory onset responses we used click trains .",
"Click trains were composed of six 50 ms clicks at 70 dB sound pressure level relative to 20 microPascals ( SPL ) with a 50 ms ISI .",
"Click trains were repeated 120 times with a 450 ms ISI between trains .",
"Alternating click trains were also paired with 1 s laser stimulation beginning 250 ms prior to click train onset .",
"To measure tuning for direct feedback cohorts , a train of 50 pure tones of frequencies spaced logarithmically between 3 and 70 kHz , at 70 dB SPL in pseudo-random order was presented 20 times .",
"Each tone was 50 ms duration ( 5 ms cosine squared ramp up and down ) with an inter-stimulus interval ( ISI ) of 450 ms . Alternating tones were paired with continuous 250 ms laser pulse at either −100 ms , −20 ms , or +8 ms onset relative to tone onset .",
"For Sst-Cre and Pvalb-Cre cohorts , a train of pure tones of 35 frequencies spaced logarithmically between 3 and 70 kHz and 8 uniformly spaced intensities from 0 to 70 dB SPL were presented 10 times in a pseudo-random order .",
"Alternating tones were paired with continuous 250 ms laser pulse at −100 ms relative to tone onset .",
"To measure spectro-temporal receptive fields ( STRFs ) we constructed DRCs from 20 ms chords ( with 1 ms ramp ) of 50 frequencies spaced logarithmically between 5 and 40 kHz with average intensity of 50 dB SPL and 20 dB SPL standard deviation selected from a uniform distribution for each chord .",
"Total duration was 40 min with a 250 ms continuous laser pulse presented every 1 s .",
"All recordings were carried out inside a double-walled acoustic isolation booth ( Industrial Acoustics ) .",
"Mice were placed in the recording chamber , and a headpost was secured to a custom base , immobilizing the head .",
"Activity of neurons in AC were recorded via a custom silicon multi-channel probe ( Neuronexus ) , lowered in the area targeting AC via a stereotactic instrument following a craniotomy at a 35-degree angle .",
"The electrode tips were arranged in a vertical fashion that permits recording the activity of neurons across the depth of the auditory cortex and the inferior colliculus .",
"Activity of neurons in IC were recorded via the same custom probes , lowered in the area targeting IC via a stereotactic instrument following either a craniotomy ( Sst-Cre and Pvalb-Cre cohorts ) or removal of the silicon plug , vertically .",
"Recording sites in IC were identified based on anatomical markers and stereotaxic coordinates as dorsal cortex ( <0 . 5 mm of midline ) or central nucleus of IC ( center of central nucleus of IC ~1 mm lateral of midline ) .",
"Recordings were made in multiple locations across IC .",
"Electro-physiological data from 32 channels were filtered between 600 and 6000 Hz ( spike responses ) , digitized at 32 kHz and stored for offline analysis ( Neuralynx ) .",
"Spikes belonging to single neurons and multi-units were detected using commercial software ( Plexon ) .",
"Original spike data and code are available on Dryad ( http://doi . org/10 . 5061/dryad . 1t61c80 ) .",
"We examined the experimental groups described in Table 1 ( note: we do not distinguish here between Sst-Cre/Pvalb Cre and Sst-Cre x Cdh23 and Pvalb-Cre x Cdh23 ) .",
"Initial experiments were performed under anesthesia to control for stability or recordings .",
"For cohorts with awake and anesthetized recordings data were analyzed separately , but we observed no difference in our results so data were combined ( Figure 8—figure supplement 1 ) .",
"We used power analysis for effect size of 25% and expected variance in firing rate ( 50% ) to determine the minimum total number of units for each experimental group at N = 44 .",
"All groups included a greater number of units ( single- and multi-units were combined in analysis ) .",
"The number of units in measuring tone-evoked responses are specified in Table 1 .",
"Mice that did not show effect of laser activation or suppression in auditory cortex were excluded .",
"We recorded from AC in Cdh23+ChR2 mice and Cdh23+ArchT mice to verify an effect in AC ( Figure 3—figure supplement 2 ) in all mice used in our analysis .",
"Neurons were stimulated by application of continuous light pulse delivered from either blue ( 473 nm , BL473T3-150 , used for ChR2 stimulation ) or green DPSS laser ( 532 nm , GL532T3-300 , Slocs lasers , used for ArchT stimulation ) through implanted cannulas .",
"Laser power measured through cannulas was 3 mW .",
"Timing of the light pulse was controlled with microsecond precision via a custom control shutter system , synchronized to the acoustic stimulus delivery .",
"Noise correlations were calculated as the pairwise Pearson correlation coefficient between the spike counts of units recorded simultaneously .",
"For spontaneous activity spikes were counted in a window 20 ms before tone onset and for tone response spikes were counted in a window 75 ms after tone onset .",
"Significant differences and P values were calculated using paired Wilcoxon sign-rank test ( unless noted otherwise ) with standard MATLAB routine .",
"For the laser alone data , to compare distributions to standard normal distribution data were normalized by mean and standard deviation and then significant differences and P values were calculated by Kolmogorov-Smirnoff test with standard MATLAB routine .",
"Mean ± standard error of the mean was reported unless stated otherwise .",
"* indicates p<0 . 05 , ** indicates p<0 . 01 , *** indicates p<0 . 001 ."
]
] | [
"The extensive feedback from the auditory cortex ( AC ) to the inferior colliculus ( IC ) supports critical aspects of auditory behavior but has not been extensively characterized .",
"Previous studies demonstrated that activity in IC is altered by focal electrical stimulation and pharmacological inactivation of AC , but these methods lack the ability to selectively manipulate projection neurons .",
"We measured the effects of selective optogenetic modulation of cortico-collicular feedback projections on IC sound responses in mice .",
"Activation of feedback increased spontaneous activity and decreased stimulus selectivity in IC , whereas suppression had no effect .",
"To further understand how microcircuits in AC may control collicular activity , we optogenetically modulated the activity of different cortical neuronal subtypes , specifically parvalbumin-positive ( PV ) and somatostatin-positive ( SST ) inhibitory interneurons .",
"We found that modulating the activity of either type of interneuron did not affect IC sound-evoked activity .",
"Combined , our results identify that activation of excitatory projections , but not inhibition-driven changes in cortical activity , affects collicular sound responses ."
] | [
"How do we hear the world around us ?",
"Hearing begins when hair cells in the inner ear translate incoming sound waves into electrical signals .",
"These signals travel via the auditory nerve and the brainstem to the midbrain , where an area called the inferior colliculus processes them .",
"The inferior colliculus then passes the signals on to another area deep within the brain , the thalamus , which processes the signals further before it too passes them on to an area of the brain’s outer layer called the auditory cortex .",
"At each stage of the auditory pathway , the signals undergo more complex processing than at the previous stage .",
"Researchers have tended to think of this pathway as a one-way route from the ear to the brain .",
"But in reality , feedback occurs at various points along the pathway , enabling areas that do higher processing to shape the responses of areas earlier in the pathway .",
"This feedback is particularly prevalent in the auditory system , where one such strong feedback route is from the auditory cortex to the inferior colliculus .",
"This reverse connection helps animals learn new behavioral responses to sounds , for example , to run away from a loud noise .",
"By manipulating the activity of this pathway in mice using a technique called optogenetics , Blackwell et al . provide further clues to how the auditory pathway works .",
"Optogenetics involves introducing light-sensitive ion channels into neurons , and then using light to activate or inhibit those neurons on demand .",
"Blackwell et al . show that activating the feedback pathway from the auditory cortex to the inferior colliculus in awake mice changes how the inferior colliculus responds to sounds .",
"By contrast , inhibiting the pathway has no effect on inferior colliculus responses .",
"This suggests that the feedback pathway is not active all the time , but instead influences inferior colliculus activity only during specific behavior , for example , perhaps when we are listening for a specific sound like the ringing of a phone .",
"Understanding how the brain processes sound is important for understanding how we communicate and why we appreciate music .",
"It could also help in treating hearing loss .",
"Stimulating the inferior colliculus using a device implanted in the brainstem can improve hearing in people with certain types of deafness .",
"Strengthening or weakening the feedback pathway from the auditory cortex to the inferior colliculus could make these implants more effective .",
"In the future , it may even be possible that stimulating the pathway directly could restore hearing without any implant being required ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"biochemistry and chemical biology"
] | Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling | elife-20304-v2 | [
[
"Cholesterol , which makes up nearly half of the lipid molecules in the plasma membrane of animal cells , can influence many signal transduction events at the cell surface .",
"It plays an important role in modulating the function of cell-surface receptors , including G-protein coupled receptors ( GPCRs ) , the largest class of receptors that transduce signals across the plasma membrane , and antigen receptors on immune cells ( Burger et al . , 2000; Pucadyil and Chattopadhyay , 2006; Swamy et al . , 2016 ) .",
"The structures of several GPCRs reveal cholesterol molecules tightly associated with the heptahelical transmembrane domain ( 7TMD ) ( Cherezov et al . , 2007; Ruprecht et al . , 2004; Wu et al . , 2014 ) .",
"Cholesterol can influence GPCR stability , oligomerization and ligand affinity ( Fahrenholz et al . , 1995; Gimpl et al . , 1997; Gimpl and Fahrenholz , 2002; Prasanna et al . , 2014; Pucadyil and Chattopadhyay , 2004 ) .",
"Cholesterol also organizes membrane microdomains , or ‘rafts , ’ containing proteins and lipids that function as platforms for the detection and propagation of extracellular signals ( Lingwood and Simons , 2010 ) .",
"In all of these cases cholesterol plays a permissive role; however , it is not sufficient to trigger signaling on its own .",
"Could cholesterol play a more instructive role— is it sufficient , not just necessary , to initiate signaling from the plasma membrane ?",
"We find that cholesterol can indeed play an instructive signaling role in the Hedgehog ( Hh ) pathway , an iconic signaling system that plays roles in development , regeneration , and cancer .",
"Multiple seemingly unrelated links have been described between cholesterol and Hh signaling ( summarized in [Eaton , 2008; Incardona and Eaton , 2000] ) .",
"While the best-defined role for cholesterol is in the biogenesis of Hh ligands ( Porter et al . , 1996 ) , it also plays an independent role in the reception of Hh signals .",
"Pharmacological or genetic depletion of cholesterol reduces cellular responses to Hh ligands , which has led to the view that cholesterol is permissive for Hh signaling ( Blassberg et al . , 2016; Cooper et al . , 1998; Cooper et al . , 2003; Incardona et al . , 1998; Incardona and Roelink , 2000 ) .",
"Distinct from these previous observations , we find that an acute increase in plasma membrane cholesterol is sufficient to activate Hh signaling .",
"Thus , cholesterol can initiate signals from the cell surface by acting as an activating ligand for a GPCR family protein ."
],
[
"While testing the effect of a panel of sterol lipids on Hh signaling in cultured fibroblasts , we made the serendipitous observation that cholesterol could induce the transcription of Hh target genes .",
"Since cholesterol is very poorly soluble in aqueous media , we delivered it to cultured cells as an inclusion complex ( hereafter called MβCD:cholesterol ) with the cyclic oligosaccharide Methyl-β–cyclodextrin ( MβCD ) ( Zidovetzki and Levitan , 2007 ) .",
"Throughout this paper , we state the concentration of MβCD in the MβCD:cholesterol complexes , since this concentration is known exactly; for saturated complexes , the molar concentration of cholesterol is predicted to be ~8–10-fold lower than that of MβCD ( Christian et al . , 1997; Klein et al . , 1995 ) .",
"MβCD:cholesterol complexes have been shown to be the most effective way to rapidly increase cholesterol in the plasma membrane , the subcellular location for most transmembrane signaling events ( Christian et al . , 1997 ) .",
"MβCD:cholesterol activated Hh signaling in NIH/3T3 cells and Mouse Embryonic Fibroblasts ( MEFs ) , cultured cell lines that have been extensively used for mechanistic studies of the Hh pathway ( Figure 1 ) .",
"MβCD:cholesterol treatment activated the transcription of Gli1 ( Figure 1A and B ) , a direct Hh target gene used as a measure of signal strength , and also reduced protein levels of the repressor form of the transcription factor GLI3 , a consequence of signaling known to be independent of transcription ( Figure 1B ) .",
"MβCD:cholesterol induced a concentration-dependent , bell-shaped Hh signaling response ( Figure 1A ) .",
"Low doses of MβCD:cholesterol , which have only a minor effect on signaling , also increased the potency of the native ligand SHH , as seen by a leftward shift in the SHH dose-response curve ( Figure 1C ) . 10 . 7554/eLife . 20304 . 003Figure 1 . Cholesterol is sufficient to activate Hh target genes in NIH/3T3 cells .",
"( A ) Gli1 mRNA , encoded by a direct Hh target gene , was measured by quantitative real-time reverse-transcription PCR ( qRT-PCR ) and normalized to mRNA levels of the housekeeping gene GAPDH after treatment ( 12 hr ) with various doses of naked MβCD or a saturated MβCD:cholesterol ( 8 . 8:1 molar ratio ) complex .",
"In both cases , the concentration of MβCD is plotted on the abscissa .",
"( B ) Immunoblotting was used to measure protein levels of GLI1 , full-length GLI3 and the repressor fragment of GLI3 after treatment ( 12 hr ) with various concentrations ( in mM ) of MβCD:cholesterol .",
"Dotted lines demarcate non-contiguous regions of the same immunoblot that were juxtaposed for clarity .",
"( C ) Gli1 induction in response to various doses of SHH in the presence or absence of a low dose of MβCD:cholesterol .",
"Inset shows non-linear curve fits to the data after a normalization in which the Gli1 mRNA level in the absence of SHH was set to 0% and at the maximum dose of SHH was set to 100% .",
"( D ) Time course of Gli1 induction ( left y-axis ) after treatment with SHH ( 265 nM ) or the MβCD:cholesterol complex ( 2 . 5 mM ) .",
"The gray circles ( right y-axis ) show the kinetics of increase in unesterified cholesterol ( normalized to total protein ) after the addition of MβCD:cholesterol .",
"In all graphs , circles depict mean values from 3 replicates and error bars show the SD . DOI: http://dx . doi . org/10 . 7554/eLife . 20304 . 00310 . 7554/eLife . 20304 . 004Figure 1—figure supplement 1 . MβCD:cholesterol treatment increases the free cholesterol content of NIH/3T3 cells .",
"( A ) Mean ( ±SD , n = 4 ) mRNA levels of Gli1 or of two genes , encoding HMG-CoA reductase and synthase , in the cholesterol biosynthetic pathway that are negatively regulated by cellular cholesterol levels are shown after treatment with the indicated concentrations of MβCD or the MβCD:cholesterol complex .",
"Asterisks denote statistical significance for difference from the untreated sample using two-way ANOVA with a Holm-Sidak post-test .",
"( B ) Levels of free cholesterol in the plasma membrane were assessed by staining with fluorescently labeled Perfringolysin O ( PFO ) , a toxin that preferentially binds to the accessible ( or chemically active ) pool of cholesterol in membranes . DOI: http://dx . doi . org/10 . 7554/eLife . 20304 . 004 Cholesterol can influence multiple cellular processes at short and long timescales , so we compared the kinetics of MβCD:cholesterol-induced activation of Gli1 to ( 1 ) the kinetics of MβCD:cholesterol-mediated delivery of cholesterol to cells and to ( 2 ) the kinetics of SHH-induced Gli1 expression .",
"Cholesterol loading of cells by MβCD:cholesterol was nearly complete by 2 hr , as determined by a standard enzymatic assay for free ( unesterified ) cholesterol ( Figure 1D ) .",
"The increase in cellular levels of free cholesterol was also confirmed by the transcriptional suppression of genes encoding enzymes in the pathway for cholesterol biosynthesis ( Figure 1—figure supplement 1A ) .",
"Importantly , there was a significant increase in the accessible or chemically active ( Radhakrishnan and McConnell , 2000 ) pool of cholesterol in the plasma membrane , as shown by increased cell-surface labeling with a cholesterol-binding toxin ( Perfringolysin O ( PFO ) , Figure 1—figure supplement 1B ) ( Das et al . , 2013 ) .",
"The initial activation of Gli1 by MβCD:cholesterol coincided with the loading of cells with cholesterol , starting at 2 hr ( Figure 1D ) .",
"The kinetics of Gli1 induction by MβCD:cholesterol paralleled those of Gli1 induction by the native ligand SHH , despite the fact the absolute levels of signaling were higher in response to SHH .",
"The rapid Hh signaling response to cholesterol , temporally correlated with the acute increase in cholesterol levels in the plasma membrane , is unlikely to be mediated by indirect or secondary transcriptional effects .",
"It was important to distinguish signaling effects caused by MβCD from those caused by cholesterol itself , especially because MβCD has been proposed to enhance Hh signaling by extracting an inhibitory sterol from cells ( Sever et al . , 2016 ) .",
"Following a previously-described protocol ( Christian et al . , 1997 ) , we treated fibroblasts with a series of MβCD complexes in which the MβCD concentration was held constant at 1 . 25 mM while the cholesterol concentration was varied .",
"Under these conditions , Hh signaling activity increased in proportion to the amount of cholesterol in the MβCD:cholesterol complexes ( Figure 2A ) .",
"Thus , cholesterol must be the active ingredient in these complexes that activates Hh signaling . 10 . 7554/eLife . 20304 . 005Figure 2 . The cholesterol in MβCD:cholesterol complexes activates Hedgehog signaling .",
"( A ) Mean ( ±SD , n = 3 ) Gli1 mRNA levels after 12 hr of treatment of NIH/3T3 cells with a series of inclusion complexes in which the MβCD concentration was clamped at 1 . 25 mM while the cholesterol concentration was varied to yield MβCD:cholesterol molar ratios of 12:1 , 9:1 , 7:1 and 6:1 .",
"( B ) Structures of cholesterol analogs tested for Hh signaling activity as inclusion complexes with MβCD .",
"Structural differences from cholesterol are highlighted in red: ent-cholesterol is the mirror-image of cholesterol with inverted stereochemistry at all 8 stereocenters; epi-cholesterol is a diastereomer with inverted stereochemistry only at the 3 carbon postion; 7-dehydrocholesterol , lathosterol and desmosterol are naturally occurring cholesterol precursors .",
"( C ) Mean ( ±SD , n = 4 ) Gli1 mRNA levels after treatment ( 12 hr ) with inclusion complexes of MβCD ( 1 . 25 mM ) with the indicated sterols ( see B for structures ) .",
"Asterisks denote statistical significance for difference from the untreated sample using one-way ANOVA with a Holm-Sidak post-test . DOI: http://dx . doi . org/10 . 7554/eLife . 20304 . 005 To define the structural features of cholesterol required to activate Hh signaling , we used MβCD to deliver a panel of natural and synthetic analogs ( Figure 2B ) .",
"This experimental approach was inspired by previous studies of the cholesterol sensor SREBP cleavage-activating protein ( SCAP ) ( Brown et al . , 2002 ) .",
"The Hh signaling activity of cholesterol was exquisitely stereoselective— neither its enantiomer ( ent-cholesterol ) nor an epimer with an inverted configuration only at the 3-hydroxy position ( epi-cholesterol ) could activate Hh target genes ( Figure 2C ) .",
"Enantioselectivity was consistent with cholesterol acting through a chiral binding pocket on a protein target , rather than by altering membrane properties ( Covey , 2009 ) .",
"Hh signaling activity was also lost when either the number or the position of double bonds in the tetracyclic sterol nucleus were altered in 7-dehydrocholesterol ( 7-DHC ) and lathosterol , two endogenous biosynthetic precursors of cholesterol .",
"Interestingly , desmosterol , another immediate biosynthetic precursor of cholesterol that contains an additional double-bond in the iso-octyl chain , retained signaling activity .",
"This structure-activity relationship points to the tetracyclic ring , conserved between cholesterol and desmosterol , as the critical structural element required for activity .",
"We cannot exclude the possibility that desmosterol activated signaling because it was rapidly converted to cholesterol in cells .",
"These strict structural requirements suggest a specific , protein-mediated effect of cholesterol on the Hh signaling pathway and further exclude the possibility that signaling activity is due to extraction of an inhibitor from cells by MβCD ( present at the same concentration in all the sterol complexes tested in Figure 2C ) .",
"MβCD:sterol inclusion complexes have been suggested to potentiate Hh signaling by depleting an inhibitory molecule through an exchange reaction ( Sever et al . , 2016 ) .",
"This model cannot explain our results because the concentration ( Figure 2A ) and structure ( Figure 2C ) of the sterol in the inclusion complex , despite an unchanging MβCD concentration , can modulate Hh signaling activity .",
"A simplified schematic of the Hh signaling pathway is provided in Figure 3A ( Briscoe and Thérond , 2013 ) .",
"The receptor for Hh ligands , Patched 1 ( PTCH1 ) , inhibits signaling by suppressing the activity of SMO , a member of the GPCR superfamily .",
"SHH binds and inhibits PTCH1 , thereby allowing SMO to adopt an active conformation and transmit the Hh signal across the plasma membrane .",
"Cytoplasmic signals from SMO overcome two negative regulators of the pathway , protein kinase A ( PKA ) and suppressor of fused ( SUFU ) , ultimately leading to the activation and nuclear translocation of the GLI family of Hh transcription factors . 10 . 7554/eLife . 20304 . 006Figure 3 . Smoothened activity is necessary for cholesterol to activate Hh signaling .",
"( A ) Schematic of the Hh signaling pathway showing the sequence in which core components function to transmit the signal from the cell surface to the nucleus .",
"SAG and 20 ( S ) -OHC are agonists and SANT-1 , vismodegib , and cyclopamine are antagonists that bind and modulate the activity of SMO .",
"Forskolin blocks signaling by elevating cAMP levels , which increases the activity of Protein Kinase A . ( B ) Mean ( ±SD , n = 3 ) Gli1 mRNA levels after treatment with MβCD:cholesterol ( 1 . 25 mM , 12 hr ) in the presence of vismodegib ( 1 μM ) , cyclopamine ( 10 μM ) or forskolin ( 10 μM ) .",
"( C ) Mean ( ±SD , n = 3 ) Gli1 mRNA levels after addition of agonists ( 12 hr ) to Smo-/- cells , in which both Smo alleles have been genetically inactivated , or Smo-/- cells stably expressing a wild-type ( WT ) SMO protein or a variant SMO protein carrying an inactivating mutation ( V333F ) in its 7TMD ( Byrne et al . , 2016 ) .",
"SHH was used at 265 nM , 20 ( S ) -OHC at 5 μM , and MβCD:cholesterol at 1 . 25 mM . ( D ) Mean ( ±SD , n = 4 ) Gli1 mRNA levels in Ptch1-/- cells after treatment with cyclopamine alone or cyclopamine in the presence of SAG ( 100 nM ) , MβCD ( 1 . 25 mM ) or MβCD:cholesterol ( 1 . 25 mM ) .",
"Asterisks denote statistical significance for differences from the “no inhibitor” sample in B , the V333F-expressing cell line in C , and the “no treatment” sample in D using one-way ( B , D ) or two-way ( C ) ANOVA with a Holm-Sidak post-test . DOI: http://dx . doi . org/10 . 7554/eLife . 20304 . 00610 . 7554/eLife . 20304 . 007Figure 3—figure supplement 1 . MβCD:cholesterol fails to drive SMO accumulation in the ciliary membrane .",
"( A ) SMO protein levels in primary cilia were determined by immunostaining NIH/3T3 cells after treatment ( 12 hr ) with SHH ( 265 nM ) or the indicated concentrations of MβCD:cholesterol .",
"The kinetics of SMO accumulation in cilia were measured after treatment with SHH ( B , 265 nM ) or MβCD:cholesterol ( C , 1 . 2 mM ) .",
"Each point depicts SMO fluorescence at a single cilium and the red bars show the median and interquartile range of measurements from ~250 cilia per condition for A and ~100 cilia per condition for B and C . DOI: http://dx . doi . org/10 . 7554/eLife . 20304 . 007 To pinpoint the site of cholesterol action within this sequence of signaling events , we conducted a series of epistasis experiments ( Figure 3 ) .",
"The addition of forskolin ( Fsk ) , which leads to an increase in the activity of PKA , blocks Hh signaling at a step between SMO and the GLI proteins .",
"Fsk inhibited MβCD:cholesterol-mediated signaling , placing the site of cholesterol action at the level of or upstream of PKA ( Figure 3B ) .",
"Two direct SMO antagonists , the steroidal natural product cyclopamine and the anti-cancer drug vismodegib , blocked Gli1 activation by MβCD:cholesterol ( Figure 3B ) ( Sharpe et al . , 2015 ) .",
"This pharmacological profile established that MβCD:cholesterol requires SMO activity to promote signaling .",
"Indeed , MEFs completely lacking SMO ( Smo-/- cells ) failed to respond to MβCD:cholesterol , and the stable re-expression of wild-type ( WT ) SMO , but not a point mutant locked in an inactive conformation ( Smo-V333F ) , rescued signaling ( Figure 3C ) ( Varjosalo et al . , 2006; Wang et al . , 2014 ) .",
"Thus , cholesterol must activate the Hh pathway at the level of PTCH1 , SMO or an intermediate step .",
"We evaluated the possibility that MβCD:cholesterol interferes with the function of PTCH1 by using Ptch1-/- MEFs , which completely lack PTCH1 protein and have high levels of Hh target gene induction driven by constitutively activated SMO ( Taipale et al . , 2002 ) .",
"MβCD:cholesterol activated signaling in Ptch1-/- cells treated with cyclopamine to partially suppress SMO activity , showing that cholesterol signaling activity did not depend on the presence of PTCH1 ( Figure 3D ) .",
"MβCD:cholesterol behaved much like the direct SMO agonist SAG , since both could overcome SMO inhibition by cyclopamine in the absence of PTCH1 .",
"Our epistasis experiments pointed to SMO as the target of cholesterol .",
"However , compared to treatment with the native ligand SHH , SMO did not accumulate to high levels in primary cilia in cells treated with MβCD:cholesterol ( Figure 3—figure supplement 1A–C ) , an observation that may explain the lower signaling efficacy of cholesterol compared to SHH .",
"SMO contains two physically separable binding sites capable of interacting with steroidal ligands ( Figure 4A ) ( Nachtergaele et al . , 2012; Sharpe et al . , 2015 ) .",
"Agonistic oxysterols , such as 20 ( S ) -hydroxycholesterol ( 20 ( S ) -OHC ) , engage a hydrophobic groove on the surface of the extracellular cysteine-rich domain ( CRD ) of SMO ( Myers et al . , 2013; Nachtergaele et al . , 2013; Nedelcu et al . , 2013 ) .",
"We recently reported that cholesterol could also occupy this CRD groove ( Byrne et al . , 2016 ) .",
"A cholesterol molecule was resolved in this groove in a crystal structure of SMO .",
"Furthermore , purified SMO bound to beads covalently coupled to cholesterol and this interaction could be blocked by free 20 ( S ) -OHC , consistent with the view that both 20 ( S ) -OHC and cholesterol occupy the same binding site ( Byrne et al . , 2016 ) .",
"In addition , the extracellular end of the SMO 7TMD binds to the steroidal alkaloid cyclopamine , as well as to several non-steroidal synthetic agonists and antagonists ( Chen et al . , 2002a; Chen et al . , 2002b; Frank-Kamenetsky et al . , 2002; Khaliullina et al . , 2015 ) . 10 . 7554/eLife . 20304 . 008Figure 4 . The Smoothened cysteine-rich domain is required for cholesterol-mediated activation of Hh signaling .",
"( A ) Structure of human SMO ( PDB 5L7D ) , with the CRD in orange , the 7TMD in blue , the linker domain in pink , and the cholesterol ligand bound to the CRD in green .",
"The Cα positions of the gatekeeper residues in the two ligand binding sites are highlighted as yellow spheres and numbered , with the mouse numbering shown in parenthesis .",
"The inset shows a close-up of the cholesterol-binding site .",
"D95 and Y130 form part of a hydrogen-bonding network ( dotted lines ) with the 3-hydroxyl of cholesterol , G111 abuts the iso-octyl chain of cholesterol , and D473 is a critical residue in the 7TMD binding-site .",
"( B , C and D )",
"Dose-response curves for the indicated agonists in Smo-/- cells stably expressing WT SMO ( always solid black circles ) or the indicated SMO variants ( open circles ) carrying mutations in the 7TMD ligand-binding site ( B ) or at two opposite ends of the CRD binding groove ( C and D ) .",
"All agonists were applied to cells for 12 hr and mean ( ±SD ) values for Gli1 mRNA are plotted based on 3 replicates .",
"In C and D , values on the abscissa represent Log ( [Agonist] in M ) and the ordinate for all four graphs is only shown once at the left . DOI: http://dx . doi . org/10 . 7554/eLife . 20304 . 00810 . 7554/eLife . 20304 . 009Figure 4—figure supplement 1 . Role of the cysteine-rich domain of Smoothened in responses to cholesterol and side-chain oxysterols .",
"( A and B )",
"Dose-response curves for MβCD:cholesterol in Smo-/- cells stably expressing WT SMO ( solid black circles ) or the indicated SMO mutants .",
"D477R ( A ) is an activating mutation in the 7TMD , Y134F ( A ) is a mutation in the CRD ( see Figure",
"5 ) that abrogates cholesterol and oxysterol responses , and ΔCRD ( B ) is an activating N-terminal truncation mutant that lacks the entire CRD .",
"( C ) Gli1 induction in Smo-/- cells expressing SMO-WT or SMO-G115F ( see Figure 4 and associated discussion ) treated with the indicated side-chain oxysterols , each applied at 5 μM as an inclusion complex with 44 μM MβCD .",
"The activity of MβCD:cholesterol ( 1 . 2 mM ) in both cell lines is shown in ( D ) for comparison .",
"( E ) Structures of the various side-chain oxysterols used in C , with differences from cholesterol highlighted in red . DOI: http://dx . doi . org/10 . 7554/eLife . 20304 . 009 In order to distinguish if the activating effect of cholesterol is mediated by the cholesterol binding groove in the SMO CRD or the cyclopamine binding site in the 7TMD , we asked whether MβCD:cholesterol could activate signaling in Smo-/- cells stably reconstituted with wild-type SMO ( SMO-WT ) or SMO variants carrying mutations in gatekeeper residues that have been shown to disrupt these two ligand-binding sites .",
"The Asp477Gly mutation in the 7TM binding-site of SMO ( Figure 4A ) , initially isolated from a patient whose tumor had become resistant to vismodegib , reduces binding and responsiveness to a subset of 7TM ligands , including SAG and vismodegib ( Yauch et al . , 2009 ) .",
"In the CRD , Asp99Ala/Tyr134Phe and Gly115Phe are mutations at opposite ends of the shallow sterol-binding groove that block the ability of 20 ( S ) -OHC to both bind SMO and activate Hh signaling ( Figure 4A ) ( Nachtergaele et al . , 2013 ) .",
"The Asp99Ala and Tyr134Phe mutations disrupt a hydrogen-bonding network with the 3β-hydroxyl group of sterols ( Figure 4A , inset ) ( Byrne et al . , 2016 ) .",
"The Asp477Gly mutation in the 7TMD domain had no effect on the ability of MβCD:cholesterol to activate Hh signaling ( Figure 4B ) .",
"SMO bearing a bulkier , charge-reversed mutation at this site ( Asp477Arg ) that increases constitutive signaling activity also remained responsive to MβCD:cholesterol ( Figure 4—figure supplement 1A ) ( Dijkgraaf et al . , 2011 ) .",
"In contrast , the Asp99Ala/Tyr134Phe mutation in the CRD reduced the ability of MβCD:cholesterol to activate Hh signaling ( Figure 4C ) .",
"The Asp99Ala/Tyr134Phe SMO mutant was also impaired in its responsiveness to SHH and to 20 ( S ) -OHC , but remained responsive to the 7TMD ligand SAG ( Figure 4C ) .",
"A complete deletion of the CRD ( SMO-△CRD ) , which increased basal SMO signaling activity like the Asp477Arg mutation , also abolished signaling responses to MβCD:cholesterol ( Figure 4—figure supplement 1B ) ( Myers et al . , 2013; Nedelcu et al . , 2013 ) .",
"This mutational analysis supports the model that the CRD binding-site , rather than the 7TMD binding-site , mediates the effect of cholesterol on SMO activity and thus on Hh signaling .",
"Interestingly , a mutation in Gly115 , which is located on the opposite end of the CRD ligand-binding groove ( Figure 4A ) , did not alter the response to MβCD:cholesterol , even though it diminished the response to 20 ( S ) -OHC as previously noted ( Figure 4D ) ( Nachtergaele et al . , 2013 ) .",
"The SMO-Gly115Phe mutant also responded normally to the native ligand SHH ( Figure 4D ) .",
"Gly115 is located near the iso-octyl chain of cholesterol in the SMO structure ( Figure 4A ) .",
"The introduction of a bulky , hydrophobic phenyl group at residue 115 may prevent the hydroxyl in the iso-octyl chain of 20 ( S ) -OHC from being accommodated in the binding groove , but not disrupt binding of the purely hydrophobic iso-octyl chain of cholesterol .",
"The ability of mutations to segregate 20 ( S ) -OHC responses from cholesterol responses is consistent with solution-state small-angle X-Ray scattering data showing distinct conformations for SMO bound to these two steroidal ligands ( Byrne et al . , 2016 ) .",
"The ability of the Gly115Phe mutation to distinguish between cholesterol and 20 ( S ) -OHC responses allowed us to address an important outstanding question: could cholesterol activate SMO only after being oxidized to a side-chain oxysterol ?",
"In addition to 20 ( S ) -OHC , oxysterols carrying hydroxyl groups on the 25 and 27 positions can bind and activate SMO ( Corcoran and Scott , 2006; Dwyer et al . , 2007; Myers et al . , 2013; Nachtergaele et al . , 2012 ) .",
"However , 20 ( S ) -OHC , 25-OHC and 27-OHC , when delivered to cells as MβCD conjugates , were all significantly compromised in their ability to activate Hh signaling in cells expressing SMO-Gly115Phe ( Figure 4—figure supplement 1C ) .",
"In contrast , cholesterol-induced signaling was unaffected ( Figure 4—figure supplement 1D ) ; therefore , cholesterol must not be activating signaling by being metabolized to one of these side-chain oxysterols .",
"Instead , our data suggests that cholesterol can directly activate Hh signaling through the CRD of SMO .",
"Our mechanistic experiments in cultured fibroblasts led us to ask whether cholesterol could also promote Hh-dependent cell differentiation decisions .",
"In the developing vertebrate spinal cord , the Hh ligand Sonic Hedgehog ( SHH ) acts as a morphogen to specify the dorsal-ventral pattern of progenitor subtypes ( Figure 5A ) ( Jessell , 2000 ) .",
"This spatial patterning process can be recapitulated in vitro .",
"Mouse neural progenitors exposed to increasing concentrations of SHH will express transcription factors that mark differentiation towards progressively more ventral neural subtypes: low , medium and high Hh signaling will generate progenitor subtypes positive for Nkx6 . 1 , Olig2 , and Nkx2 . 2 , respectively ( Dessaud et al . , 2008; Gouti et al . , 2014; Kutejova et al . , 2016 ) .",
"MβCD:cholesterol induced the formation of both Nkx6 . 1+ and Olig2+ progenitor subtypes at a low frequency in cultures of mouse spinal cord progenitors ( Figure 5B and C ) and also activated the transcription of Gli1 ( Figure 5D ) .",
"The activation of both Gli1 induction and ventral neural specification by MβCD:cholesterol was significantly less than that produced by a saturating concentration of SHH .",
"However , we note that MβCD:cholesterol inclusion complexes could not be delivered at higher concentrations due to deleterious effects on the adhesion and viability of neural progenitors .",
"Taken together , these observations suggest that MβCD:cholesterol is sufficient to activate low-level Hh signals in neural progenitors and consequently to direct differentiation towards neural cell types that depend on such signals . 10 . 7554/eLife . 20304 . 010Figure 5 . Cholesterol induces the differentiation of neural progenitors .",
"( A ) A schematic illustrating the relationship between marker proteins used to assess differentiation and progenitor cell populations in the embryonic neural tube ( taken from ( Niewiadomski et al . , 2014 ) ) .",
"FP – floor plate progenitors , MN – motor neuron progenitors , p0 , p1 , p2 , p3 – ventral interneuron progenitors .",
"( B ) Differentiation of neural progenitors was assessed by immunostaining for Nkx6 . 1+ and Olig2+ expression ( see A ) after treatment ( 48 hr ) with Retinoic Acid ( RA , 100 nM ) alone or RA plus SHH ( 25 nM ) , MβCD ( 2 mM ) or the saturated MβCD:cholesterol inclusion complex ( 2 mM ) .",
"The percentage of nuclei ( stained with DAPI ) positive for four differentiation markers ( see A ) in 15 different images is plotted in ( C ) , with each point representing one image of the type shown in ( B ) and the red line drawn at the median value .",
"Asterisks denote statistical significance ( unpaired t-test , Holm-Sidak correction , n = 15 ) for the comparison between cells treated with RA+MβCD and RA+MβCD:cholesterol .",
"( D ) Gli1 mRNA ( mean ± SD , n = 3 ) after 48 hr of the indicated treatments .",
"Asterisks denote statistical significance for difference from the RA-treated sample using one-way ANOVA with a Holm-Sidak post-test . DOI: http://dx . doi . org/10 . 7554/eLife . 20304 . 010"
],
[
"To establish a causal or regulatory role for a component in a biological pathway , experiments should demonstrate that the component is both necessary and sufficient for activity .",
"Cholesterol has been shown to be necessary for SMO activation , based on experiments using inhibitors of cholesterol biosynthesis and high concentrations of naked MβCD to strip the plasma membrane of cholesterol ( Cooper et al . , 2003 ) .",
"Impaired SMO activation caused by cholesterol deficiency has also been noted in Smith-Lemli-Opitz syndrome ( SLOS ) , a congenital malformation syndrome caused by defects in the enzyme that converts 7-dehydrocholesterol to cholesterol ( Blassberg et al . , 2016; Cooper et al . , 2003 ) .",
"In contrast to our results , the SMO CRD is dispensable for this permissive role of cholesterol .",
"The depletion of cholesterol reduces signaling by SMO mutants lacking the entire CRD ( Myers et al . , 2013 ) or carrying mutations in the CRD binding-groove ( Blassberg et al . , 2016 ) .",
"By analogy with other GPCRs , these permissive effects are likely to be mediated by the SMO 7TMD .",
"We now find that cholesterol is also sufficient to activate Hh signalling in a dose-dependent manner .",
"This instructive effect is mediated by the Class F GPCR SMO and maps to its extracellular CRD .",
"Cholesterol engages a hydrophobic groove on the surface of the CRD , a groove that was previously shown to mediate the activating influence of oxysterols ( Myers et al . , 2013; Nachtergaele et al . , 2013; Nedelcu et al . , 2013 ) and represents an evolutionarily conserved mechanism for detecting hydrophobic small-molecule ligands ( Bazan and de Sauvage , 2009 ) .",
"An analogous mechanism is present in the Frizzled family of Wnt receptors , where the Frizzled CRD binds to the palmitoleyl group of Wnt ligands , an interaction that is required for Wnt signaling ( Janda et al . , 2012 ) .",
"Thus , the instructive effects of cholesterol revealed in our present study and the permissive effects of cholesterol reported previously map to distinct , separable SMO domains .",
"Our observation that MβCD:cholesterol is sufficient to activate SMO through its CRD is in agreement with a report published recently during the review process of our manuscript ( Huang et al . , 2016 ) .",
"There are many reasons why this activating effect of cholesterol on Hh signalling may not have been appreciated previously despite the fact that the activating effects of side-chain oxysterols have been known for a decade ( Corcoran and Scott , 2006; Dwyer et al . , 2007 ) .",
"First , the method of delivery , as an inclusion complex with MβCD , is critical to presenting cholesterol , a profoundly hydrophobic and insoluble lipid , in a bioavailable form capable of activating Smo .",
"Even clear solutions of cholesterol in the absence of carriers like MβCD contain microcrystalline deposits or stable micelles that sequester cholesterol ( Haberland and Reynolds , 1973 ) .",
"In contrast , side-chain oxysterols , which harbor an additional hydroxyl group , are significantly more hydrophilic and soluble in aqueous solutions , shown by their ~ 50 fold faster transfer rates between membranes ( Theunissen et al . , 1986 ) .",
"Second , cholesterol levels in the cell are difficult to manipulate because they are tightly controlled by elaborate homeostatic signalling mechanisms ( Brown and Goldstein , 2009 ) .",
"MβCD:cholesterol inclusion complexes have been shown to be unique in their ability to increase the cholesterol content of the plasma membrane rapidly at timescales ( ~1–4 hr ) at which cytoplasmic signaling pathways operate ( Christian et al . , 1997; Yancey et al . , 1996 ) .",
"Other methods of delivery using low density lipoprotein particles and lipid dispersions , or mutations in genes regulating cholesterol homeostasis , function on a much slower time scale and are thus more likely to be confounded by indirect effects given the myriad cellular processes affected by cholesterol ( Christian et al . , 1997 ) .",
"Finally , the bell-shaped Hh signal-response curve ( Figure 1A ) implies that MβCD:cholesterol must be delivered in a relatively narrow , intermediate concentration range ( 1–2 mM ) to observe optimal activity , with higher ( >5 mM ) concentrations commonly used to load cells with cholesterol producing markedly lower levels of signaling activity .",
"Our results are particularly informative in light of the recently solved crystal structure of a SMO protein containing the CRD , linker domain and entire 7TMD but lacking the cytoplasmic tail ( hereafter called SMO△C ) ( Figure 4A ) ( Byrne et al . , 2016 ) .",
"SMO△C was unexpectedly found to contain a cholesterol ligand in its CRD groove .",
"Cholesterol also made key contacts with the linker domain and third extracellular loop of the 7TMD ( Figure 4A ) , and molecular dynamics simulations showed that cholesterol can stabilize these extracellular regions of SMO ( Byrne et al . , 2016 ) .",
"However , the function of this bound cholesterol , whether it is an agonist , antagonist or co-factor , remains an important unresolved question in SMO regulation .",
"Structure-guided point mutations in CRD residues that form hydrogen-bonding interactions with the 3β-hydroxyl of cholesterol , reduced signaling by cholesterol ( Figure 4C ) , making it likely that cholesterol activates SMO by binding to the CRD in the pose revealed in the structure ( Figure 4A ) .",
"Thus , the cholesterol-bound SMO structure recently reported by our groups may very well represent an active-state conformation of the CRD .",
"A comparison of this cholesterol-bound structure with a structure of inactive SMO bound to the potent 7TMD antagonist vismodegib ( which lacks cholesterol in the CRD groove ) revealed a conformational change that may drive SMO activation ( Figure 6 ) ( Byrne et al . , 2016 ) .",
"Cholesterol binding is predicted to induce a clockwise rotation of the CRD on the 7TMD pedestal , perhaps driving SMO activation by a rearrangement of contacts between the CRD and the 7TMD .",
"A caveat to this model is that it depends on a structural comparison with SMO△C bound to a synthetic antagonist and not with un-liganded SMO△C , which has thus far eluded crystallization . 10 . 7554/eLife . 20304 . 011Figure 6 . Conformational changes in Smoothened induced by cholesterol binding . A comparison of the structures of SMO△C bound to vismodegib ( PDB ID 5L7I , left ) , representing an inactive state , and cholesterol ( PDB ID 5L7D , right ) , highlights a rotation of the CRD and the helical extracellular loop 3 relative to the 7TMD .",
"This rotation may communicate ligand binding at the CRD to conformational changes in the 7TMD . DOI: http://dx . doi . org/10 . 7554/eLife . 20304 . 01110 . 7554/eLife . 20304 . 012Figure 6—figure supplement 1 . A comparison of all Smoothened CRD structures .",
"( A ) Superposition of four isolated CRD structures ( turquoise , gray , blue , green ) and two CRD structures ( orange , pink ) solved in the context of a complete SMO△C molecule also containing the linker domain and the 7TMD .",
"The dotted circle highlights the location of the unique conformation and associated disorder seen in unliganded , Xenopus apo-CRD ( turquoise ) .",
"‘x’ denotes Xenopus laevis , ‘z’ Danio rerio , and ‘h’ human SMO .",
"‘apo’ denotes un-liganded structures; otherwise , the specific ligands are noted in the legend .",
"PDB IDs used to generate this superposition are as follows: 5KZZ ( xSMO-CRD-apo ) , 5KZV ( xSMO-CRD-20 ( S ) -OHC ) , 5KZY ( xSMO-CRD-cyclopamine ) , 4C79 ( zSMO-CRD-apo ) , 5L7D ( hSMO△C-cholesterol ) and 5L7I ( hSMO△C-vismodegib ) .",
"( B ) Superposition of the CRDs in xSMO-CRD-apo ( turquoise ) and hSMO△C-cholesterol ( orange ) is shown along with a symmetry partner ( yellow ) of xSMO-CRD-apo seen in the crystal .",
"The region of altered conformation is partially disordered and forms a zinc-stabilized contact between the two symmetry related xSMO-CRD-apo molecules . DOI: http://dx . doi . org/10 . 7554/eLife . 20304 . 012 Based on structures of the isolated Xenopus laevis CRD , either alone ( apo-CRD ) or in complex with 20 ( S ) -OHC ( but notably not cholesterol ) , a recently published report proposed that sterols drive SMO activation by inducing a conformational change within the CRD itself ( Huang et al . , 2016 ) .",
"We disagree with this model for several reasons .",
"First , these structures do not contain the linker domain and the entire 7TMD , both of which make critical contacts with cholesterol and the CRD , and hence cannot reveal changes in orientation between the CRD and the 7TMD that are essential to understanding how CRD ligands communicate with the 7TMD .",
"Second , all structures of the SMO CRD ( with the exception of the Xenopus apo-CRD ) are conformationally identical ( Figure 6—figure supplement 1A ) , regardless of whether they contain a bound ligand ( cholesterol-bound SMO△C , cyclopamine-bound CRD or 20 ( S ) -OHC-bound CRD ) or not ( Danio rerio apo-CRD or vismodegib-bound SMO△C ) and regardless of whether they were crystallized by standard vapor diffusion ( the CRD structures ) or lipidic cubic phase methods ( the SMO△C structures ) .",
"Hence this conserved conformation , seen in both the isolated CRD and the more physiological SMO△C molecule , is unlikely to be an artefact of crystal packing as suggested by these authors ( Huang et al . , 2016 ) .",
"Finally , a careful inspection of the crystal lattice contacts in the Xenopus apo-SMO structure ( PDB ID 5KZZ ) revealed that the region of the proposed conformational change is partially disordered ( Figure 6—figure supplement 1A ) and involved in the coordination of a zinc ion together with a symmetry-related molecule in the crystal , a very tight , near-covalent interaction that was likely driving crystal formation ( Figure 6—figure supplement 1B ) .",
"Since the crystallization solution for the Xenopus apo-SMO , but not the solutions used to crystallize the sterol-bound CRDs , contained 200 mM zinc acetate , the altered conformation observed may have been induced by a non-physiological , zinc-promoted crystal contact ( Huang et al . , 2016 ) .",
"A surprising feature of the structure is that CRD-bound cholesterol is located at a considerable distance ( ~12 Å ) away from the membrane , which would require a cholesterol molecule to desolvate from the membrane and become exposed to water in order to access its CRD binding pocket ( Byrne et al . , 2016 ) ( Figure 7 ) .",
"The kinetic barrier , or the activation energy ( ΔG‡ ) for this transfer reaction is predicted to be high ( ~20 kcal/mole ) , based on the ΔG‡ for cholesterol transfer between two acceptors through an aqueous environment ( Yancey et al . , 1996 ) .",
"The unique ability of MβCD to shield cholesterol from water while allowing its rapid transfer to acceptors would allow it to bypass this kinetically unfavorable step by delivering it to the CRD binding site ( Figure 7 ) .",
"These considerations present a regulatory puzzle for future research: how does cholesterol gain access to the CRD-binding pocket without MβCD and is this process regulated by native Hh ligands ?",
"Indeed , the kinetic barrier for cholesterol transfer to the CRD pocket makes it an ideal candidate for a rate-limiting , regulated step controlling SMO activity in cells . 10 . 7554/eLife . 20304 . 013Figure 7 . Models for how cholesterol may gain access to its binding-site in the SMO cysteine-rich domain . The structure of SMO bound to cholesterol ( PDB 5L7D ) is shown embedded in a lipid bilayer composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine ( POPC ) and cholesterol in a ratio of 3:1 ( Byrne et al . , 2016 ) .",
"The SMO CRD is colored orange; the linker domain and 7TMD are colored blue .",
"Two molecules of MβCD ( PDB QKH , shown as green sticks ) form an inclusion complex with each molecule of cholesterol ( PDB CLR , colored yellow in stick representation with the 3-hydroxyl shown red ) .",
"MβCD could deliver cholesterol directly to the CRD binding pocket ( left ) or to the outer leaflet of the plasma membrane ( right ) , which would subsequently require a second transfer step from the membrane to the CRD .",
"The activation energy for the direct delivery mechanism on the left ( <10 kcal/mole ) is much lower than for the mechanism on the right ( ~20 kcal/mole ) , where cholesterol has to desolvate from the membrane without a carrier to access the CRD site ( Lopez et al . , 2011; Yancey et al . , 1996 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 20304 . 013 MβCD:cholesterol was consistently less active than the native ligand SHH in our assays ( Figures 1D , 5C and D ) .",
"Comparing the doses of MβCD:cholesterol to the doses of SHH delivered to cells is difficult .",
"SHH was used at saturating concentrations; however , we could not assess the effects of MβCD:cholesterol at saturating doses , because the downward phase of the bell-shaped dose-response curve ( in cultured fibroblasts , Figure 1A ) and cell toxicity ( in neural progenitors ) proved to be dose-limiting .",
"Aside from these technical considerations related to delivery , other possibilities for lower activity include the observation that MβCD:cholesterol did not induce the high-level accumulation of SMO in primary cilia ( Figure 3—figure supplement 1 ) and the possibility that a different ligand regulates high-level signaling by SMO .",
"Mutations in the 7TMD binding-site do not alter the constitutive or SHH-induced signaling activity of SMO , which has led to view that this site does not regulate physiological signaling ( Myers et al . , 2013; Yauch et al . , 2009 ) .",
"In contrast , mutations in the cholesterol-binding site impaired responses to SHH ( Byrne et al . , 2016 ) .",
"Hence , a putative alternate ligand would have to engage a third , undefined site .",
"Lastly , the presence of active PTCH1 is a major difference between SHH- and MβCD:cholesterol-induced signaling .",
"The biochemical activity of PTCH1 ( which is inactivated by SHH ) may oppose the effects of MβCD:cholesterol , limiting signaling responses .",
"Interestingly , MβCD:cholesterol was able to restore maximal Hh responses in the absence of PTCH1 ( Figure 3D ) .",
"Our results may have implications for understanding how PTCH1 inhibits SMO , a longstanding mystery in Hh signaling .",
"The necessity and sufficiency of cholesterol for SMO activation , mediated through two different regions of the molecule , means that SMO activity is likely to be highly sensitive to both the abundance and the accessibility of cholesterol in its membrane environment .",
"Furthermore , PTCH1 has homology to a lysosomal cholesterol transporter , the Niemann-Pick C1 ( NPC1 ) protein ( Carstea et al . , 1997 ) , and PTCH1 has been purported to have cholesterol binding and transport activity ( Bidet et al . , 2011 ) .",
"Thus , our work supports a model where PTCH1 may inhibit SMO by reducing cholesterol content or cholesterol accessibility ( or chemical activity ) in a membrane compartment that also contains SMO , leading to alterations in SMO conformation or trafficking ( Bidet et al . , 2011; Incardona et al . , 2002; Khaliullina et al . , 2009 ) .",
"Since cholesterol is a ubiquitous component of cellular membranes that affects many cellular processes , PTCH1-induced changes in cholesterol are likely to be confined to a specific membrane compartment .",
"The base of the cilium is a good candidate for such a compartment because PTCH1 is localized most prominently at the ciliary base in Hh-responsive tissues in the mouse embryo ( Rohatgi et al . , 2007 ) and because most Hh pathway components , from PTCH1 to the GLI transcription factors , are found in and around primary cilia ( Briscoe and Thérond , 2013 ) .",
"Two key questions must be answered before endogenous cellular cholesterol can be considered the elusive second messenger that communicates the Hh signal from PTCH1 to SMO: Do Hh ligands alter cholesterol abundance or activity ?",
"and Is cholesterol a substrate for the predicted transporter activity of PTCH1 ?",
"Answering these questions will require developing or adapting tools to measure and perturb cholesterol in specific cellular compartments and an assessment of the biochemical activities of purified SMO and PTCH1 reconstituted into cholesterol-containing membranes .",
"While cholesterol is an abundant lipid , clearly critical for maintaining membrane biophysical properties and for stabilizing membrane proteins , our work suggests that it may be also used as a second messenger to instruct signaling events at the cell surface through GPCRs and perhaps other cell-surface receptors ."
],
[
"Each experiment shown in the paper was repeated at least three independent times with similar results .",
"All data was analyzed using GraphPad Prism .",
"All points reflect mean values calculated from at least 3 replicates and error bars denote standard deviation ( SD ) .",
"The statistical tests used to evaluate significance are noted in the figure legends .",
"Statistical significance in the figures is denoted as follows: ns: p>0 . 05 , *p≤0 . 05 , **p≤0 . 01 , ***p≤0 . 001 , ****p≤0 . 0001 .",
"For maintenance , MM13 mouse embryonic stem cells ( mESCs ) ( Wichterle et al . , 2002 ) were plated on irradiated primary mouse embryonic fibroblasts ( pMEFs ) and cultured in mESC media ( Dulbecco’s Modified Eagle’s Medium high glucose ( Hyclone; Pittsburgh , PA ) and 15% Optima FBS ( Atlanta Biologicals ) supplemented with 1% MEM non-essential amino acids ( Thermo Fisher Scientific ) , 1% penicillin/streptomycin ( Gemini Bio-Products; West Sacramento , CA ) , 2 mM L-glutamine ( Gemini Bio-Products ) , 1% EmbryoMax nucleosides ( Millipore ) , 55 µM 2-mercaptoethanol ( Thermo Fisher Scientific ) , and 1000 U/ml ESGRO LIF ( Millipore ) .",
"The mESCs were differentiated as previously described with minor modifications ( Gouti et al . , 2014; Ying et al . , 2003 ) .",
"Briefly , the pMEFs were removed from the mESCs by dissociating the cells with 0 . 25% Trypsin/EDTA and then incubating the cells on tissue culture plates for two short successive periods ( 20 min each ) .",
"To induce differentiation , the cells were plated on Matrigel ( BD Biosciences; San Jose , CA ) coated glass coverslips ( 12 mm diameter , placed in a 24-well plate ) at a density of 2 . 4 × 104 cells per coverslip in N2B27 media ( Dulbecco’s Modified Eagle’s Medium F12 ( Gibco ) and Neurobasal Medium ( Gibco ) ( 1:1 ratio ) supplemented with N-2 Supplement , B-27 Supplement , 1% penicillin/streptomycin ) , 2 mM L-glutamine , 40 µg/ml Bovine Serum Albumin , and 55 µM 2-mercaptoethanol ) .",
"On Day 0 and Day 1 , cells were cultured in N2B27 with 10 ng/ml bFGF ( R&D Scientific ) .",
"On Day 2 , the media was changed and the cells were cultured in N2B27 with 10 ng/ml bFGF ( R&D Scientific ) and 5 µM CHIR99021 ( Axon; Netherlands ) .",
"On Day 3 , the media was changed and the cells were cultured in 1 ml of N2B27 supplemented with 100 nM Retinoic Acid ( RA ) , 100 nM RA and 25 nM SHH , 100 nM RA and 2 mM MeβCD , or 100 nM RA and 2 mM MβCD + 0 . 23 mM cholesterol .",
"On Day 4 , 1 ml N2B27 with 100 nM RA was added to each well , thus diluting each treatment condition by half .",
"On Day 5 the cells were rinsed and fixed for further analysis .",
"NIH/3T3 cells were cultured in Dulbecco's modified Eagle's Medium ( DMEM ) containing 10% Fetal Bovine Serum ( FBS , Optima Grade , Atlanta Biologicals ) in 24-well plates at an initial density of 7 . 5 × 104 on acid-washed glass cover-slips that were pre-coated with poly-L-lysine .",
"Confluent cells were exchanged into 0 . 5% FBS DMEM to induce ciliogenesis for 24 hr .",
"Ciliated cells were treated with the indicated drugs each dissolved in 0 . 5% FBS DMEM .",
"Samples were fixed using 4% paraformaldehyde in phosphate buffered saline ( PBS ) for 10 min and washed three times with PBS .",
"For SMO localization studies , cells were blocked and permeabilized in 1% donkey serum , 10 mg / mL bovine serum albumin ( BSA ) , 0 . 1% triton X-100 , and PBS .",
"Primary antibodies were administered in block buffer for 2 hr at room temperature .",
"Cover-slips were washed three times with wash buffer containing PBS and 0 . 1% triton X-100 .",
"Secondary antibodies were administered in block buffer for 1 hr .",
"Cover-slips were washed three more times in wash buffer and mounted on glass slides using Pro-Long Diamond Antifade Mountant with DAPI ( Thermo Fisher Scientific ) .",
"For PFO staining , cells were fixed in 4% PFA , washed three times with PBS and stained with PFO in PBS without detergent .",
"Cover-slips were washed three times with PBS and mounted on glass slides using Pro-Long Diamond Antifade Mountant with DAPI ( Thermo Fisher Scientific ) .",
"Images were acquired on an inverted Leica SP8 laser scanning confocal microscope with a 63X oil immersion objective ( NA 1 . 4 ) using a HyD hybrid detector .",
"Z-stacks were acquired with identical acquisition settings ( gain , offset , laser power , frame format ) within a given experiment .",
"The following primary antibodies were used: rabbit anti-Smo ( 1:500 ) ( Rohatgi et al . , 2007 ) , guinea pig anti-Arl13b ( Pusapati et al . , 2014 ) , goat anti-GFP ( 1:2000 ) ( Rockland; Limerick , PA ) , mouse anti-Nkx2 . 2 ( 1:100 ) ( 74 . 5A5 , Developmental Studies Hybridoma Bank , Iowa City , IA ) , mouse anti-Nkx6 . 1 ( 1:100 ) ( F55A10 , Developmental Studies Hybridoma Bank ) , guinea pig anti-Olig2 ( 1:20 , 000 ) ( Novitch et al . , 2001 ) , rabbit anti-Pax6 ( 1:1000 ) ( AB2237 , Millipore ) .",
"The following secondary antibodies were used: Alexa Fluor 488 , Alexa Fluor 594 , and Alexa Fluor 647 ( Thermo Fisher Scientific ) .",
"Image processing for ciliary SMO levels was carried out using maximum projection images of the acquired Z-stacks using ImageJ .",
"For quantification of ciliary Smo , first a mask was constructed using the Arl13b image ( primary cilia marker ) , and then the mask was applied to the corresponding Smo image where the integrated fluorescence was measured .",
"An identical region outside the cilia was measured to determine background fluorescence .",
"Background correction was applied on a per cilia basis by subtracting the background fluorescence from the cilia fluorescence .",
"For neural differentiation experiments , fluorescent images were collected on a Leica TCS SP8 confocal imaging system equipped with a 40x oil immersion objective using the Leica Application Suite X ( LASX ) software .",
"For each experiment , coverslips from each condition were grown , collected , and processed together to ensure that the cells were fixed and stained for the same duration of time .",
"To ensure uniformity in imaging , the gain , offset , and laser power settings on the microscope were held constant for each antibody .",
"At least 15 images were taken per condition .",
"To ensure all cells were represented , z-stacks were acquired and counts were performed on the compressed images .",
"Cell counts were conducted using the NIH ImageJ software suite with cell counter plugin .",
"In total , 5000–6000 cells were analyzed per condition .",
"The experiment was conducted independently a total of three times .",
"Representative images shown in Figure 5 were processed equally using Adobe Photoshop , Adobe illustrator , and CorelDraw software .",
"Cells were cultured in Dulbecco's modified Eagle's Medium ( DMEM ) containing 10% Fetal Bovine Serum ( FBS , Optima Grade , Atlanta Biologicals ) in 6-well plates at an initial density of 3 × 105 cells / well .",
"Confluent cells were switched into 0 . 5% FBS DMEM to induce ciliogenesis for 24 hr .",
"Cells were treated with indicated drugs dissolved in 0 . 5% FBS DMEM in duplicate .",
"One sample was used to measure total protein by bicinchoninic acid assay ( BCA ) , and the second for total lipid extraction and subsequent cholesterol quantification .",
"Cells were washed once with Phosphate Buffered Saline ( PBS ) , and harvested using a Corning cell lifter in PBS .",
"The cell suspension was transferred to a 1 . 5 mL ependorf tube , centrifuged at 1000 x g and the PBS aspirated .",
"Total lipids were extracted from the cell pellet by the addition of 200 μL of chloroform-methanol ( 2:1 vol/vol ) .",
"To induce phase separation , 100 μL of PBS was added to the lipid extract and the sample was centrifuged at 5000 x g for 5 min .",
"The organic layer was transferred to a fresh 1 . 5 mL eppendorf tube and the solvent removed under reduced pressure .",
"Relative total free cholesterol was measured using the Amplex Red Cholesterol Assay Kit ( Thermo Fisher Scientific ) following the manufacturer's instructions .",
"Lysis buffer containing 50 mM Tris pH 7 . 4 , 150 mM NaCl , 2% Nonidet P-40 , 0 . 5% sodium deoxycholate , 0 . 1% sodium dodecyl sulfate , 1 mM dithithreitol , and Sigma Fast protease inhibitor cocktail ( Sigma-Aldrich ) was used to disrupt the cell pellet .",
"A ratio of total free cholesterol to total protein was used as a normalization method ."
]
] | [
"Cholesterol is necessary for the function of many G-protein coupled receptors ( GPCRs ) .",
"We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog ( Hh ) pathway , a prominent cell-cell communication system in development .",
"Cholesterol influences Hh signaling by directly activating Smoothened ( SMO ) , an orphan GPCR that transmits the Hh signal across the membrane in all animals .",
"Unlike many GPCRs , which are regulated by cholesterol through their heptahelical transmembrane domains , SMO is activated by cholesterol through its extracellular cysteine-rich domain ( CRD ) .",
"Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation , both in response to cholesterol and to native Hh ligands .",
"Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility ."
] | [
"Cells must communicate with each other to coordinate the development of most tissues and organs .",
"Damage to these communication systems is often seen in degenerative disorders and in cancer .",
"The Hedgehog signaling pathway is one of a handful of these critical systems .",
"Reduced Hedgehog signals can lead to birth defects , while excessive Hedgehog signals can lead to skin and brain cancers .",
"Cells transmit the Hedgehog signal by releasing a protein into their surroundings , where it can influence neighboring cells .",
"Despite years of study , it is not understood how the Hedgehog signal is transmitted from the outside to the inside of a receiving cell .",
"Studies first done in flies and subsequently confirmed in humans have shown that a protein called Smoothened is needed to transmit the Hedgehog signal across the membrane of receiving cells .",
"But it was not known how Smoothened carries out this critical signaling step to influence gene activation inside the cell and consequently to change cell behavior .",
"Now , Luchetti , Sircar et al . find that cholesterol , an important component of the cell membrane , directly binds to Smoothened and changes its shape so that it can activate Hedgehog signaling components inside cells .",
"The experiments made use of mouse cells , and the discovery shows that cholesterol may play a previously underappreciated role in cell-to-cell communication .",
"This newly discovered role for cholesterol has implications for diseases , including a unique set of developmental disorders caused by abnormalities in pathways that produce cholesterol in human cells .",
"Furthermore , this unexpected insight into Smoothened’s activity may be clinically important , because Smoothened can cause cancer when mutated and is the target of anti-cancer drugs that are being used in the clinic .",
"Following on from these findings , a major step will be to uncover if and how Hedgehog signals regulate cholesterol to allow Smoothened to transmit these signals across the cell membrane ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"cell biology"
] | Physical association between a novel plasma-membrane structure and centrosome orients cell division | elife-16550-v1 | [
[
"The orientation of the mitotic division axis has been proposed to control tissue morphogenesis as well as cell fate determination , thus playing an important role in shaping embryonic forms ( Lu and Johnston , 2013; Moorhouse and Burgess , 2014; Siller and Doe , 2009 ) .",
"The mechanism determining the orientation of the mitotic spindle has been extensively studied in both cultured and embryonic cells and precise molecular processes are well understood ( Bell et al . , 2015; Cao et al . , 2010; Delaval et al . , 2011; Kiyomitsu and Cheeseman , 2012; Woolner and Papalopulu , 2012; Zheng et al . , 2010 ) .",
"One strategy to achieve this is the control of centrosome dynamics .",
"Centrosome works as a microtubule-organizing center ( MTOC ) in animal cells and consists of a pair of mother and daughter centrioles , which are distinct in both structure and age ( Azimzadeh and Bornens , 2007 ) .",
"Following duplication and migration , the two centrosomes become aligned to serve as spindle poles during mitosis .",
"Thus , the axis of centrosome alignment is frequently consistent with mitotic spindle orientation unless additional constraints such as cell shape exist to alter the spindle orientation ( Gibson et al . , 2011; Minc et al . , 2011 ) .",
"Specific mother-daughter centrosome inheritance coupled with asymmetric cell division is a highly conserved phenomenon ( Yamashita , 2009 ) .",
"It has been first reported in budding yeast that the 'old' spindle pole body corresponding to the mother centrosome , segregates into the bud ( Pereira et al . , 2001 ) .",
"Drosophila melanogaster male germline stem cells and neuroblasts have contributed to our understandings of the molecular mechanisms underlying the asymmetric migration of the duplicated centrosomes during interphase .",
"In male germline cells , membrane localized Adenomatous polyposis coli 2 ( Apc2 ) and the Drosophila Par-3 homolog , Bazooka , associated with E-cadherin , tethers one centrosome adjacent to the niche , called the hub , and consequently ensures spindle orientation and asymmetric stem cell division ( Inaba et al . , 2015a; Yamashita et al . , 2003 ) .",
"In male germline cells , it is the mother centrosome with stable astral microtubules which is anchored near the hub ( Yamashita et al . , 2007 ) .",
"In neuroblasts , the centrosome with the higher MTOC activity remains in the neuroblast following asymmetric cell division ( Rebollo et al . , 2007 ) .",
"In contrast to the male germ line , it is the daughter centrosome that is retained in the stem cell ( Conduit and Raff , 2010; Januschke et al . , 2011 ) .",
"Centrobin , associated with the daughter centrosome , was found to be responsible for this oriented cell division ( Januschke et al . , 2013 ) .",
"In both cell systems , the centrosome with a higher MTOC activity is less motile and is inherited by the stem cell ( Pelletier and Yamashita , 2012 ) .",
"In addition to a role in spindle orientation , the centrosome also has an important role in cilia formation .",
"During ciliogenesis , the mother centriole converts into the basal body in a quiescent ( G0 phase ) or interphase ( G1 phase ) cell to nucleate a primary cilium .",
"Following re-entry or progression of the cell cycle , the primary cilium is disassembled and the basal body/mother centriole is reused for mitotic spindle formation ( Kobayashi and Dynlacht , 2011 ) .",
"It is unclear how the centrosome transition is coordinated between cilia and spindle .",
"In this study , we use embryos of ascidian , belonging to the phylum Tunicata , a sister group of the vertebrates ( Satoh et al . , 2014 ) .",
"Ascidian embryos are ideally suited to study mechanisms of cell division because of their invariant cleavage pattern and the small number of cells that form their bodies ( Conklin , 1905; Nishida , 1986 ) .",
"The pattern of cell division is highly conserved among different ascidian species ( Conklin , 1905; Lemaire et al . , 2008; Zalokar and Sardet , 1984 ) .",
"This implies robust mechanistic constraints on the cell division patterns of ascidian development .",
"Several studies , including our own , have reported unique mechanisms of spindle orientation in ascidian embryos ( Kumano et al . , 2010; Nakamura et al . , 2005; Negishi and Yasuo , 2015; Negishi et al . , 2007; Nishikata et al . , 1999; Prodon et al . , 2010 ) .",
"In this study , we focused on embryonic epidermal cells in the cosmopolitan ascidian , Ciona intestinalis .",
"It was previously reported that almost all epidermal cells cleave along the anterior-posterior ( A–P ) axis at the last ( 11th ) division when the Ciona embryo starts shaping into a tadpole larval form ( Ogura et al . , 2011 ) .",
"We describe here a novel membrane structure that may control centrosome dynamics including ciliary positioning and spindle orientation during this last cell division of the epidermal cells ."
],
[
"The epidermal cell lineage of ascidians is known to divide alternately along the A-P and medial-lateral ( M– L ) axes during early cleavage stages ( Nishida , 1994 ) .",
"The perpendicular shift of the cell division axis during successive rounds of cell division is thought to result from a 90° translocation of the duplicated centrosomes around the nucleus to the opposite directions ( Sach’s rule ) ( Mardin and Schiebel , 2012; Strome , 1993 ) .",
"This alternating 90° shift of the division axis tends to follow the long-axis rule based on cell shape ( Hertwig’s rule ) ( Hertwig , 1984; Minc et al . , 2011 ) .",
"With live imaging analysis of epidermal cell division , we confirmed that almost all epidermal cells divide along the A–P axis at the last ( 11th ) division as reported previously ( Ogura et al . , 2011 ) .",
"This oriented cell division occurs regardless of the cell shape and whether the 10th cell division occurred along the A–P or M-L axis ( Figure 1A–C , Video 1 ) .",
"Additionally , we found , during the 11th ( but not the 10th ) cell cycle , that the nucleus of the epidermal cells gradually shifts toward the posterior side ( Figure 1D , E and Video 1 ) . 10 . 7554/eLife . 16550 . 003Figure 1 . Ciona intestinalis epidermal cell mitosis and the posterior nuclear positioning prior to the final cell division .",
"( A ) Representative epidermal cell divisions ( white arrows ) from the 10th to the 11th cell cycle in an embryo expressing PH-GFP/H2B-mCherry; frames are from Video 1 .",
"Elapsed time: 32'–43' shows the 10th cell division , while 150'–157' shows the 11th cell division .",
"During this process , the embryo changes into a tadpole-shape consisting of a head and tail .",
"Anterior: left .",
"Dorsal: upper .",
"Bar: 30 µm .",
"( B , C )",
"In the final mitotic division , ascidian epidermal cells do not divide following the Sachs’s and Herwig’s rules .",
"( B ) Rose diagram showing the angle of the cell division axis relative to the embryonic A-P axis in the 11th cell division .",
"Following the 10th cell division , we selected daughter cells that were produced via an A-P oriented cell division with less than 30° of the cell division axis relative to the embryonic A-P axis and then measured their cell division angle at the 11th cell division .",
"n = 95; cells from three embryos were used .",
"( C ) Rose diagram showing the angle of the cell division axis relative to the long axis of the cell in the last cell division .",
"n = 160 cells from three embryos .",
"( D ) Representative frames of a 4D confocal dataset imaging epidermal cells of an embryo expressing PH-GFP/H2B-mCherry .",
"Nuclei show a posteriorly biased positioning prior to M-phase .",
"Numbered arrows indicate the same nucleus in the sequence .",
"Time elapsed from the start of recording is shown in orange .",
"Bar: 10 µm .",
"( E ) Bee-swarm plots indicating the nuclear position relative to the centre of the cell along the embryonic A-P axis , measured just before the breakdown of the nuclear membrane , in the 10th and 11th cell-division cycles .",
"n = 106 cells each at the 10th and 11th division cycles from three embryos .",
"Black lines show the average nuclear position relative to the centre of the cell ( blue dotted line ) ; the average positions were 0 . 18 and 1 . 0 µm toward the posterior side at the 10th and 11th division cycle , respectively .",
"p-values were obtained using the Welch's t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 00310 . 7554/eLife . 16550 . 004Video 1 . ( 10 fps ) A low-magnification , low-resolution time-lapse movie of an embryo expressing PH-GFP ( green ) and H2B-mCherry ( magenta ) , made with the maximum-intensity projection of the confocal microscopy data . Lateral view: facing; anterior: left .",
"Ten minutes are compressed to one second .",
"White arrows: representative cells .",
"This video is related to Figure 1A . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 00410 . 7554/eLife . 16550 . 005Video 2 . ( 7 fps ) A high-magnification , high-resolution time-lapse movie of an embryo expressing PH-GFP , made with the maximum-intensity projection of the confocal microscopy data . Four minutes and 40 s are compressed to one second .",
"Anterior: left .",
"This video is related to Figure 2B . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 005 To analyse the epidermal cell morphology during the 10th and 11th cell-division cycles , we used GFP-conjugated Pleckstrin homology domain of PLC1δ1 ( PH-GFP ) to visualise the plasma membrane ( Audhya et al . , 2005; Carroll et al . , 2003; Hurley and Meyer , 2001 ) .",
"We found a unique structure ( membrane invagination ) that formed in almost all of the epidermal cells specifically during the interphase of the last ( 11th ) division cycle ( Figure 2A , B , magenta and blue arrows ) .",
"Time-lapse observations of cells that had committed to cell division revealed that the invaginating membrane did not adopt a stable , solid form , but rather appeared to be a highly dynamic filamentous structure ( Video 2 ) .",
"To confirm the structure of the invaginating membranes , we used another membrane binding probe using a domain of c-Ha-Ras , memGFP ( Morita et al . , 2012 ) or membrane-staining fluorescent dye , FM4-64 ( Figure 2C ) , which demonstrated that the invaginations were not an artefact of a specific protein or GFP staining itself .",
"The filamentous structures are located near the apical surface approximately 1 . 5 μm beneath the apical membrane ( base: 1 . 33 ± 0 . 56 µm , tip: 0 . 98 ± 0 . 37 µm n = 71 , Figure 2D ) .",
"The length of the invaginations changed over time , ranging from roughly one-third to half of the cell diameter ( 2 . 0–5 . 0 μm ) ( Figure 2E ) .",
"Interestingly , some cells that formed a vertex with posterior cells had two or more invaginations ( Figure 2B , magenta arrows ) .",
"Quantitative analysis showed that most of the invaginations projected from the posterior or lateral side toward the anterior ( Figure 2F ) , at a nearly perpendicular angle to the cell membrane ( Figure 2G ) , although transient , randomly projected filamentous membranes were also observed ( See Videos 6 and 8 ) .",
"These results suggest that the membrane structure forms near the apical cortex and elongates along the A-P axis .",
"All of the invaginations disappeared just before spindle formation became visible , suggesting that the formation of the invagination is temporally regulated .",
"In fact , the invaginations were observed without exception during the interphase of the 11th cell cycle in epidermal cells .",
"These results prompted us to investigate whether this novel membrane structure is involved in the spindle orientation . 10 . 7554/eLife . 16550 . 006Figure 2 . Characterisation of the invaginating membrane structure .",
"( A ) Low-magnification image of epidermal cells in a normal Ciona intestinalis embryo expressing PH-GFP , during the 11th cell division cycle .",
"A maximum-intensity projection image of the confocal stack is shown .",
"Anterior: left; ventral side: facing .",
"Black bar: 30 µm .",
"( B ) High-magnification images of epidermal cells in a normal embryo expressing PH-GFP , during the 11th cell-division cycle .",
"Images are from Video 2; elapsed times are indicated .",
"Anterior: left .",
"Both blue and red arrows indicate membrane invaginations; red arrows show invaginations forming a wedge shape .",
"( C ) Membrane invaginations in epidermal cells in a membraneGFP-expressing embryo ( upper panel ) and a FM4-64 stained embryo ( lower panel ) .",
"Anterior: left; ventral: facing .",
"Bars: 10 µm .",
"Blue arrows: invaginations .",
"( D ) A membrane invagination formed near the apical cortex in an epidermal cell expressing PH-GFP/H2B-mCherry .",
"XY-projection panel shows a maximum-intensity projection of the confocal stack; the YZ panel was reconstructed from the same confocal data set .",
"Blue arrows: invaginations .",
"Orange dotted lines indicate the same invagination in both panels .",
"Bar: 10 µm .",
"( E ) Histogram showing the distribution of invagination length , calculated from confocal images .",
"n = 118 invaginations counted from three embryos .",
"( F ) Rose diagram showing the angle of the invagination relative to the embryonic A–P axis .",
"Almost all of the invaginations had an angle <90° , meaning they formed toward the anterior .",
"n = 104; invaginations counted in three embryos .",
"( G ) Rose diagram showing the angle of invagination relative to the plasma membrane from which it arises , indicating that the invaginations extends perpendicular to the lateral membrane .",
"n = 103 invaginations counted in three embryos . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 006 We next used Serial block-face ( SBF ) scanning electron microscopy ( SEM ) ( Denk and Horstmann , 2004 ) , an advanced 3D electron-microscope technique , to examine the detailed structure of the membrane invaginations .",
"For this analysis , ascidian embryos were fixed at the 11th cell-division cycle .",
"In a series of several hundred sections , we observed fragmented membrane structures directed toward the centrioles ( Figure 3A , blue arrows , Video 3 ) .",
"Notably , 3D reconstructions from serial sections ( Figure 3B , Videos 4 and 5 ) showed the tip of the invagination approaching the centrioles as if to capture the organelle .",
"These observations raised the interesting possibility that the invagination might physically capture the centrosome and pull it toward the posterior of the cell .",
"SEM images showed that the invaginations were formed of a double-bilayer plasma membrane and that adjacent posterior cell membrane also contributed to this structure ( Figure 3A , Videos 3–5 ) .",
"To confirm this observation , we expressed green ( GFP ) and red ( tdTomato ) fluorescence proteins conjugated with the PH domain in cells of the anterior and posterior epidermal lineages , respectively , and observed the membranes of anterior and posterior juxtaposed cells ( Figure 3C ) .",
"At the border between two differently labelled cells , the invaginating membranes were labelled with both green and red fluorescence , indicating that the layers of the invaginating membrane were derived from the both of the neighbouring cells ( Figure 3C , white arrowheads ) . 10 . 7554/eLife . 16550 . 007Video 3 . A video made of serial sections of SBM-SEM images . Blue arrow: invagination .",
"Magenta arrowhead: centrosome .",
"The depth between each frame is 50 nm .",
"This video is related to Figure 3A . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 00710 . 7554/eLife . 16550 . 008Video 4 . A movie integrating serial sections of SBF-SEM images and segmentations of the structures . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 00810 . 7554/eLife . 16550 . 009Figure 3 . The invaginations consist of two plasma membranes and elongate toward the centrosome .",
"( A ) Selected z-sections from the SBF-SEM sequence in Video 3; ΔZ indicates the depth below the top panel .",
"Blue arrows: invaginations .",
"Magenta arrowhead: centriole .",
"The posterior cell is coloured in red .",
"Bar: 2 µm .",
"( B ) A segmentation figure of 3D-reconstructed SBF-SEM data derived from Video 5 .",
"Lower panel: view from the perspective of the black arrow in the upper panel .",
"Individual cells are labelled in the same colour code in both images .",
"Red and blue balls indicate the position of a pair of centrioles .",
"Bar: 10 µm .",
"( C ) Two-colour labelling of the anterior ( PH-GFP ) and posterior ( PH-tdTomato ) lineage epidermal cells showing that the invaginations are derived from the plasma membranes of both neighbouring cells .",
"White arrowheads: invaginations on the border that were labelled with both colours .",
"Bar: 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 00910 . 7554/eLife . 16550 . 010Video 5 . 3D-segmentations from serial sections of SBM-SEM images: pairs of red and blue balls indicate the positions of centrioles in the cell . This video is related to Figure 3B . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 01010 . 7554/eLife . 16550 . 011Video 6 . ( 10 fps ) A time-lapse movie of an embryo expressing PH-GFP ( green ) and ensconsin-tdTomato ( magenta ) , made with the maximum-intensity projection of the confocal microscopy data . The video starts at the end of the 10th cell division .",
"Five minutes are compressed to one second .",
"Anterior: left .",
"This video is related to Figure 4ADOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 011 We further explored the relationship between the invaginations and centrosomes , which act as a MTOC orchestrating the microtubule dynamics during interphase and mitotic spindle formation .",
"MTOCs in epidermal cells were fluorescently labelled with the microtubule-binding protein ensconsin ( E-MAP-115 ) tagged with tdTomato ( Bulinski et al . , 2001; Dong et al . , 2011 ) ( Figure 4A and Video 6 ) .",
"We found that the interphase microtubule array was aligned with the A-P axis with its nucleation site localised at the posterior side , indicating that a stable MTOC was positioned to the posterior of the cells .",
"To record the dynamics of centrosome behaviour in epidermal cells , we decided to use end-binding 3 ( EB3 ) , which marks the plus end of the microtubule as it grows away from the organising centre ( Akhmanova and Hoogenraad , 2005 ) and highlights the MTOC activity at the centrosome .",
"Before the formation of the invagination at the early interphase of the 11th cell cycle , EB3-labelled centrosomes were located beneath the apical surface of the epidermal cells ( Figure 4B , white arrowheads and Video 7 ) .",
"We then co-visualised the centrosomes and membrane invaginations by time-lapse imaging of EB3-mCherry/PH-GFP ( Figure 4C and Video 8 ) .",
"These analyses showed that the invaginations appeared to extend toward highly concentrated EB3-mCherry signals over time ( Figure 4C , 33'–79' ) .",
"Importantly , the invagination and the centrosome remain associated during posterior displacement .",
"Although during the 10th division cycle , centrosomes were distributed equally along the A-P axis of epidermal cells ( Figure 4D ) , in the 11th division cycle following the emergence of the membrane invagination , the distribution of centrosome positioning was strongly biased to the posterior of the cell ( Figure 4C , D ) .",
"By the time the invagination disappeared , the centrosome had duplicated; the two centrosomes then migrated asymmetrically until they were aligned with the axis of invagination ( Figure 4C , white brackets in 79' ) .",
"Indeed , our quantitative analysis indicated that the posterior-most centrosome migrated a shorter distance than the anterior one in the 11th division cycle , while this is not the case in the previous cell cycle ( Figure 4E ) .",
"After the centrosome migration , we measured the angle between the axis of aligned centrosomes and the direction of invagination",
"( s ) .",
"When there were multiple invaginations , we calculated the composition of these structures as a vector .",
"The analysis revealed that the migrated centrosomes were well-aligned along the axis of membrane invagination ( Figure 4F ) .",
"Mitotic spindles then form with the two spindle poles aligned along the axis of the invagination ( Figure 4A , 104'; Figure 4C , 79' and 96' ) .",
"Taken together , these observations suggest that the formation of the invagination may be correlated with the apical and posterior positioning of the centrosomes and the subsequent asymmetric migratory behaviour of the duplicated centrosomes . 10 . 7554/eLife . 16550 . 012Figure 4 . Quantitative description of centrosome behaviour in relation to the membrane invaginations .",
"( A ) Epidermal cells in the 11th cell cycle showing the interphase microtubule array along the A-P axis and membrane invaginations toward the microtubule nucleation site in an embryo expressing PH-GFP/Ensconsin-tdTomato .",
"Blue arrows: invaginations .",
"Panels: frames from Video 6; elapsed time is indicated .",
"( B , C )",
"Centrosome dynamics in the 11th cell cycle epidermal cells in an embryo expressing EB3-GFP/H2B-mCherry or PH-GFP/EB3-mCherry; elapsed time is indicated in each panel .",
"Bars: 10 µm .",
"( B ) XZ-view of epidermal cells in transition from late 10th cell cycle to early 11th cell cycle in an embryo expressing EB3-GFP/ H2B-mCherry .",
"Frames were selected from Video",
"7 . The focus is on the left daughter cell .",
"Anterior: left .",
"The centrosome ( white arrowhead ) was located lateral to the nucleus after mitotic division ( 15' ) but moved toward the apical cortex ( dotted line ) .",
"( C ) XY-projection images of 11th cell cycle epidermal cells in an embryo expressing PH-GFP/EB3-mCherry , highlighting the elongation of membrane invaginations toward the MTOC .",
"Anterior: left .",
"Each frame was selected from Video",
"8 . White arrowheads: microtubule nucleation sites .",
"Blue arrows: invaginations .",
"White brackets: pairs of centrosomes .",
"( D ) Centrosome position just before duplication during the 10th ( n = 78 cells ) and 11th ( n = 109 cells ) cell cycles .",
"Black bars show the average centrosome position relative to the centre of the cell ( blue dotted line ) ; the average centrosome position was 0 . 03 and 1 . 7 µm toward the posterior side at the 10th and 11th cell cycles , respectively .",
"p-values were obtained using the Welch's t-test .",
"( E ) Migration distance of anterior and posterior centrosomes determined by recording centrosome positions just before duplication and after the migration ceased during the 10th ( n = 78 cells from three embryos ) and 11th ( n = 109 cells from three embryos ) cell cycles .",
"Histograms are presented as the mean ± SD , p-values were obtained using the Welch's t-test .",
"Inset: illustration showing how the centrosome behaviours were measured .",
"( F ) Relationship between the centrosome axis and the invagination axis .",
"To measure the angle between these axes , we measured the angle of the invagination just before centrosome duplication and the angle of the centrosome axis just after migration ( n = 109 cells from three embryos ) , and calculated the difference between the two angles .",
"For multiple invaginations , a composite vector was used as the angle of the invaginations .",
"Left: illustration showing how two angles were measured .",
"Black dotted line: the centrosome axis .",
"Pink dotted line: the direction of the invagination",
"( s ) .",
"The bold black arc shows the angle . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 01210 . 7554/eLife . 16550 . 013Video 7 . ( 7 fps ) A time-lapse movie of an embryo expressing EB3-GFP ( green ) and H2B-mCherry ( magenta ) , made with the reconstructed cross-sections of confocal microscopy data . The video starts at the M phase of the 10th cell division .",
"Four minutes and 40 s are compressed to one second .",
"Apical side: up .",
"This video is related to Figure 4B . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 01310 . 7554/eLife . 16550 . 014Video 8 . ( 10 fps ) A time-lapse movie of an embryo expressing PH-GFP ( green ) and EB3-mCherry ( magenta ) , made with the maximum-intensity projection of the confocal microscopy data . The video starts at the end of the 10th cell division .",
"Five minutes are compressed to one second .",
"Anterior is left .",
"This video is related to Figure 4C . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 014 Interestingly , serial SBF-SEM sections revealed that primary cilia were located near the centrioles associated with the invaginations ( Figure 5A and Video 9 ) .",
"We also confirmed the posterior localization of cilia in 11th cell cycle epidermal cells in embryos immunostained with anti-acetylated tubulin ( Figure 5B , C ) .",
"Posterior cilium positioning was highly reminiscent of the posterior centrosome positioning ( Figure 5D ) .",
"We speculate that the primary cilia associated with the membrane invaginations in this study are the same cilia reported previously ( Thompson et al . , 2012 ) .",
"Live imaging of a GFP-tagged ciliary protein , ADP-ribosylation factor-like ( ARL ) family of small GTPases , Arl13b ( Duldulao et al . , 2009; Paridaen et al . , 2013 ) , showed that almost all cilia are associated with invaginations ( 97 . 8% , 221/226 cells in three embryos , Figure 5D ) .",
"All Arl13b-GFP labelled structures disappeared in the mitotic phase ( n = 165 cells ) .",
"Measuring the distance between the tip of invagination and two centrioles ( basal body and daughter ) in 3D images reconstructed from SBF-SEM sections revealed that the invagination tip comes in closer proximity to the basal body derived from the mother centriole ( Kobayashi and Dynlacht , 2011 ) than to the daughter centriole ( 9/10 invaginations ) ( Figure 6A , B ) .",
"These results suggest that the basal body as well as the mother centriole are targets of the invagination . 10 . 7554/eLife . 16550 . 015Figure 5 . The centriole associated with the membrane invaginations carries the primary cilium .",
"( A ) Selected z-sections from the SBF-SEM sequence in Video 9; ΔZ indicates the depth starting from the z-level of the top panel .",
"Blue arrow: invagination .",
"Orange arrowhead: the centriole and cilium complex .",
"The posterior cell is tinted in red .",
"Bar: 1 µm .",
"( B ) Acetylated tubulin immunofluorescence counterstained with DAPI , showing that the epidermal cells of the ascidian embryo contained a primary cilium at the 11th cell cycle stage .",
"A maximum-intensity projection of the confocal z-stack is shown .",
"Black bar: 50 µm .",
"Area in the white square is enlarged in the inset; cell contours were manually outlined in orange .",
"White bar: 10 µm .",
"( C ) Analysis of the position of cilia in the ascidian epidermal cells during the last cell cycle , showing a tendency to localise to the posterior side , consistent with previous observations ( Thompson et al . , 2012 ) .",
"We measured 113 cilia from three embryos .",
"The black bar shows that , relative to the centre of the cell ( blue dotted line ) , the average cilium position was 2 . 1 µm toward the posterior side of the cell .",
"( D ) Membrane invaginations and cilia were observed simultaneously in an embryo expressing PH-tdTomato and ADP-ribosylation factor-like 13b ( Arl13b ) -GFP; Arl13b labels primary cilia ( Duldulao et al . , 2009; Paridaen et al . , 2013 ) .",
"The XY-projection panel shows a representative epidermal cell from another embryo .",
"The XZ panel was reconstructed from the same z-stack data used for the XY-projection panel .",
"Blue arrows: invaginations .",
"Orange dotted lines indicate the same invagination in both panels .",
"Bar: 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 01510 . 7554/eLife . 16550 . 016Video 9 . A video made with serial sections of SBM-SEM images . Blue arrow: the invagination .",
"Orange arrowhead: the cilia and centriole .",
"The depth between each frame is 50 nm .",
"This video is related to Figure 5A . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 01610 . 7554/eLife . 16550 . 017Video 10 . ( 12 fps ) In a cytochalasin-treated embryo expressing PH-GFP/EB3-mCherry , membranes invaginated from the anterior side and the number of invaginations increased . Anterior: left .",
"Five minutes are compressed to one second .",
"This video is related to Figure 7A . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 01710 . 7554/eLife . 16550 . 018Figure 6 . The membrane invagination approaches closer to the basal body than to the daughter centriole .",
"( A ) A representative segmented cell from SBF-SEM data .",
"Green: nucleus .",
"Blue and red balls: the basal body and daughter centriole , respectively .",
"Upper panel: top view of the whole cell .",
"Bar: 10 µm .",
"The lower panel shows a closer view at the tip of the invagination .",
"Distances between 3D objects were measured in AMIRA .",
"We recognized the mother centrioles/basal bodies by the presence of cilia in the serial sections of SBF-SEM .",
"( B ) Distance of the tip of the individual invagination to the basal body or daughter centriole .",
"The X- and Y-axis show the distance to the basal body and daughter centriole , respectively .",
"We measured ten invaginations and found that nine of ten invaginations approached the basal body more closely than the daughter centriole . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 018 Next , we investigated the involvement of the cytoskeleton in forming the invaginations .",
"Microtubule depolymerisation with nocodazole blocked the formation of invaginations completely ( Figure 7B ) , indicating that MTOC activity induces the invaginations and that , in turn , the invaginations relocate the MTOC to the posterior of cells .",
"On the other hand , a treatment of embryos with cytochalasin , which perturbs actin polymerisation , increased the number of invaginations , which now originated from all lateral plasma membrane domains ( Figure 7A–C and Video 10 ) .",
"Thus , the tubular membrane structure of ascidian epidermal cells depends on microtubule function , reminiscent of the 'nanotubes' described in Drosophila male germ cells ( Inaba et al . , 2015b ) , rather than the actin-based membrane tubes described in other systems ( ex . some cytonemes and tunneling nanotubes ) ( Gerdes and Carvalho , 2008; Hsiung et al . , 2005; Rustom et al . , 2004; Sanders et al . , 2013 ) .",
"Drosophila microtubule-based nanotubes ( MT-nanotubes ) are filled with microtubules ( Inaba et al . , 2015b ) .",
"During the course of live imaging analyses , we did not observe microtubules within the membrane invaginations .",
"By contrast , we often observed that the plus end of the microtubule labeled with EB3-mCherry reached to the tip of the invagination ( Figure 7D ) .",
"Further , we explored the topological relationship between the membrane invagination and microtubules by serial transmission electron microscopy ( serial-TEM ) observation ( Figure 7E , Video 11 ) .",
"In serial-TEM , the membrane invaginations were visualized as double bilayer fragments ( Figure 7Ea–f ) extending toward the basal body ( Figure 7Eh–k ) .",
"Abundant microtubules were also observed between the tip of invagination and the basal body ( Figure 7Ef–h ) , but no microtubules were observed within the invagination itself .",
"These results indicate that the membrane invagination in ascidian epidermal cell is distinct from microtubule-filled nanotubes although both tubular structures are sensitive to microtubule depolymerization .",
"Our results also indicate that the membrane invagination may associate with the centrosome via microtubules . 10 . 7554/eLife . 16550 . 019Video 11 . A video made of sections of serial-TEM images shows the invagination toward the basal body . This video is related to Figure 7E . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 01910 . 7554/eLife . 16550 . 020Video 12 . ( 10 fps ) A time-lapse movie of an embryo expressing PH-GFP ( green ) and EB3-mCherry ( magenta ) , made with a time series of a single confocal plane . Laser irradiation occurs at the third frame .",
"Ten seconds are compressed to one second .",
"Anterior: left .",
"This video is related to Figure 8A . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 02010 . 7554/eLife . 16550 . 021Figure 7 . The role of cytoskeletal elements in the formation of the membrane invagination and the implication of microtubule function .",
"( A ) Representative epidermal cells expressing PH-GFP/EB3-mCherry in control and cytochalasin-treated embryos .",
"The cytochalasin panel is from Video 10 .",
"Blue arrows: membrane invaginations .",
"Bars: 10 µm .",
"Anterior: left .",
"( B ) The number of invaginations after inhibitor treatment: cytochalasin treatment ( 160 cells from three embryos ) increased the number of invaginations , while nocodazole treatment ( 121 cells from three embryos ) decreased the number of invaginations compared to control ( 113 cells from three embryos ) .",
"Histograms are presented as the mean ± SD .",
"p-values were obtained using the Welch's t-test .",
"( C ) In cytochalasin-treated cells , unlike normal cells , invaginations also formed from the anterior side and extended toward the posterior of the cell .",
"We counted 177 and 476 invaginations , respectively , in three control and three cytochalasin-treated embryos .",
"( D ) A high time-resolution timelapse recording of a representative cell expressing PH-GFP/EB3-mCherry .",
"A blue arrowhead indicates the membrane invagination .",
"Time elapsed from the start of recording is shown in orange .",
"Bar: 10 µm .",
"Anterior: left .",
"( E ) A series of images from the serial TEM observation .",
"TEM images ( upper panels ) and the corresponding schematic drawings ( lower panels ) are shown with the microtubules as green tubes , the membrane invagination as black double-line in a–e , or its tip as a hexagon filled by gray in f , and the basal body as an orange structure in h–k . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 021 Based on these observations , we speculated that the membrane invagination is physically connected to the centrosome and may have a role in positioning it to the posterior side of the cell .",
"To examine whether a pulling force was generated between the membrane invaginations and the posteriorly located centrioles , we employed laser ablation ( Rauzi et al . , 2008 ) .",
"The junction of the fluorescently labelled invagination and the EB3-mCherry-labelled centrosome was cut by UV laser irradiation , and the cells were observed by time-lapse imaging ( Figure 8A , white cross ) .",
"Interestingly , the end of the cut edge of the invaginations regressed immediately to the basal position , suggesting that the invaginations are normally under tension ( Figure 8A , Video 12 ) .",
"Therefore , the displacement of the nucleus during the 11th cell-division cycle could be explained by a posteriorly directed force ( Figure 1D , E ) .",
"In the series of laser ablation experiments ( Figure 8B ) , we noted that the longer invaginations had a tendency to regress more rapidly ( Figure 8C ) .",
"The results show a positive correlation between the initial length and both average and max speed of regression ( recoiling ) .",
"When we cut one of two invaginations that cooperatively support a single centrosome , a significant recoil occurred toward the intact ( uncut ) invagination ( Video 13 ) .",
"This finding strongly suggests that the centrosome was balanced by two invaginations that generate pulling forces .",
"Interestingly , soon ( 15 min ) after the cutting , we found that the plasma membrane re-invaginated toward the centrosome ( Figure 8D ) . 10 . 7554/eLife . 16550 . 022Video 13 . ( 10 fps ) A time-lapse movie of an embryo expressing PH-GFP ( green ) and EB3-mCherry ( magenta ) , made with a time series of a single confocal plane . Laser irradiation on the lower invaginations occurs at the 10th frame .",
"This clearly shows a bounce toward the intact invagination after the ablation of the other .",
"Ten seconds are compressed to one second .",
"Anterior: left .",
"This video is related to Figure 8A . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 02210 . 7554/eLife . 16550 . 023Video 14 . ( 10 fps ) A time-lapse movie of an embryo expressing PH-GFP ( green ) and EB3-mCherry ( magenta ) , made with the maximum-intensity projection of the confocal data . This shows that the radially formed invatginations affected the centrosome dynamics .",
"Five minutes are compressed to one second .",
"Anterior is left .",
"This video is related to Figure 10A ( lower panels ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 02310 . 7554/eLife . 16550 . 024Figure 8 . Laser ablation and regeneration of the membrane invagination .",
"( A , B )",
"Effect of UV laser ablation on membrane invagination in a cell expressing PH-GFP/EB3-mCherry .",
"( A ) Image shows frames from Video 12 , with the elapsed time indicated .",
"In the 2'' frame , the white 'X' indicates the point of laser ablation .",
"The membrane recoiled rapidly after ablation .",
"Bar: 10 µm .",
"( B ) Graph showing measurements of invagination length following laser ablation .",
"The black line , indicated with purple arrow head , shows the time of ablation .",
"A colour code was used to highlight the difference of initial length of the membrane invaginations when the laser ablation was conducted: orange , >4 µm; green , 4 µm to 3 µm; blue , <3 µm .",
"n = 31 invaginations .",
"( C ) The correlation between the initial length and average ( left ) or maximum ( right ) speed .",
"Coloured dots correspond to the lines in B . The correlation coefficient",
"( r ) is 0 . 52 or 0 . 44 for the average speed or the maximum speed , respectively ( Pearson correlation ) .",
"( D ) The plasma membrane re-invaginated after UV laser ablation .",
"We observed this regeneration event at least five independent experiments .",
"The white 'X' indicates the point of laser ablation .",
"Time elapsed from the start of recording is shown in orange .",
"Frames for 0'-14' correspond to single confocal planes while those for 5'10'–9'30' are max intensity projections .",
"Bars: 10 µm .",
"Anterior: left . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 024 The regeneration potential of the membrane invagination obstructed the functional analysis by laser ablation .",
"Thus , we decided to indirectly alter the direction of invaginations by disruption of the planar cell polarity ( PCP ) pathway , which establishes polarity in multicellular tissues ( Adler , 2002; Gubb and Garcia-Bellido , 1982; Wallingford , 2012 ) .",
"To disrupt the PCP pathway , we depleted Dishevelled ( Dsh ) ( Hotta et al . , 2003; Theisen et al . , 1994; Wallingford and Habas , 2005 ) , a core component of PCP , by injection of antisense morpholino oligo ( MO ) .",
"This resulted in a radial formation of multiple membrane invaginations ( Figure 9A , B , Figure 10A , blue arrows , and Video 14 ) .",
"Disruption of directional membrane invaginations was strongly correlated with randomization of mitotic spindle orientation ( Figure 9A , B , Figure 10A and Video 14 ) .",
"Co-injection of Dsh mRNA with Dsh MO rescued both the direction of invaginations and cell division orientation along the A-P axis ( Figure 9C ) .",
"These results suggest that the posterior distribution of the invaginations is tightly associated with the direction of mitotic divisions .",
"We also found that the nuclei were distributed around the center of the cells with the omni-directional invaginations , as opposed to the posterior localisation observed in the cells with directional invaginations ( Figure 10B ) .",
"Moreover , the centrosomes were also positioned in the center of the cells with the radial formation of invaginations ( Figure 10A; Omni-directional 86' , Figure 10C and Video 14 ) .",
"Following this , the two duplicated centrosomes exhibited equivalent motility in the cells with the omni-directional invaginations ( Figure 10A; Omni-directional 105' , Figure 10D and Video 14 ) , in contrast to the asymmetric centrosome migration found in the cells displaying directional invaginations ( Figure 10A; Directional 83' , Figure 10D ) .",
"Finally , epidermal cilia form in the center of the cells with omni-directional invaginations , instead of at a posterior position ( Figure 10E , F ) .",
"These results suggest that the centrosome dynamics in ascidian epidermal cells is tightly correlated with the direction of membrane invaginations . 10 . 7554/eLife . 16550 . 025Figure 9 . Depletion of Dsh resulted in the radial formation of multiple membrane invaginations and randomized the cell division axis in the 11th cell cycle epidermal cells .",
"( A ) The directional invaginations labeled by PH-GFP in the control MO-injected cells .",
"The bar represents 10 µm ( left ) .",
"Rose diagrams showing the angle of the invagination relative to the embryonic A–P axis , n = 166 invaginations from three embryos ( middle ) , and the angle of cell division relative to the embryonic A–P axis , n = 108 cells from three embryos ( right ) .",
"The results are almost same as the normal embryos in Figure 1 and Figure 2 .",
"( B ) The omini-directional invaginations labeled by PH-GFP in the Dsh MO-injected cells .",
"The bar represents 10 µm ( left ) .",
"Rose diagrams showing the angle of the invagination relative to the embryonic A-P axis , n = 182 invaginations from three embryos ( middle ) , and the angle of cell division relative to the embryonic A-P axis , n = 109 cells from three embryos ( right ) .",
"In the embryos with radially formed invaginations , the orientation of cell division is randomized .",
"( C ) Co-injection of Dsh MO and Dsh mRNA restores directional invaginations .",
"The bar represents 10 µm ( left ) .",
"Rose diagrams showing the angle of the invagination relative to the embryonic A-P axis , n = 213 invaginations from three embryos ( middle ) , and the angle of cell division relative to the embryonic A–P axis , n = 137 cells from three embryos ( right ) .",
"The results are reminiscent of normal and the control MO-injected embryos . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 02510 . 7554/eLife . 16550 . 026Figure 10 . The centrosome dynamics is highly correlated with the directionality of the membrane invagination .",
"( A ) Centrosome dynamics with the directional ( upper ) or the omni-directional ( lower; frames from Video 14 ) membrane invaginations in embryos injected with MOs and expressing PH-GFP/EB3-mCherry .",
"Blue arrows indicate the invaginations .",
"Elapsed time is indicated in each panel .",
"Anterior is left .",
"Bars: 10 µm .",
"( B ) Bee-swarm plots showing the nuclear position relative to the centre of the cell along the embryonic A–P axis with the directional ( upper; control MO , n = 114 cells from three embryos and lower; Dsh MO + Dsh mRNA , n = 89 cells from three embryos ) and the omini-directional ( middle; Dsh MO , n = 113 cells from three embryos ) invaginations .",
"Black lines show the average nuclear position relative to the centre of the cell ( blue dotted line ) ; the average position was 0 . 53 , −0 . 03 and 0 . 6 µm toward the posterior side in the embryos injected with control MO , Dsh MO and Dsh MO + Dsh mRNA , respectively .",
"p-values were obtained using the Welch's t-test .",
"( C ) Centrosome position relative to the centre of the cell along the embryonic A–P axis just before duplication with the directional ( upper; control MO , n = 110 cells from three embryos and lower; Dsh MO + Dsh mRNA , n = 146 cells from three embryos ) and the omini-directional ( middle; Dsh MO , n = 115 cells from three embryos ) invaginations .",
"Black lines show the average centrosome position relative to the centre of the cell ( blue dotted line ) ; the average centrosome position was 1 . 54 , 0 . 18 and 1 . 46 µm toward the posterior side in the embryos injected with control MO , Dsh MO and Dsh MO + Dsh mRNA , respectively .",
"p-values were obtained using the Welch's t-test .",
"( D ) Histograms showing migration distance of anterior and posterior centrosomes over the period from centrosome duplication to the end of migration in epidermal cells with the directional ( upper; control MO , n = 110 cells from three embryos and lower; Dsh MO + Dsh mRNA , n = 146 cells from three embryos ) and the omini-directional ( middle; Dsh MO , n = 115 cells from three embryos ) invaginations .",
"Blue and orange columns show the migration of the anterior and posterior centrosomes , respectively .",
"The data are presented as the mean ± SD .",
"p-values were obtained using the Welch's t-test .",
"( E ) Cilium positioning in epidermal cells with the directional ( upper ) or the omini-directional ( lower ) membrane invaginations in embryos expressing Arl13b-GFP .",
"( F ) Bee-swarm plots showing cilium position relative to the centre of the cell along the embryonic A-P axis in epidermal cells with the directional ( upper; control MO , n = 127 cells from three embryos and lower; Dsh MO + Dsh mRNA , n = 99 cells from three embryos ) and the omini-directional ( middle; Dsh MO , n = 118 cells from three embryos ) invaginations .",
"Black lines show the average cilium position relative to the centre of the cell ( blue dotted line ) ; the average cilium positions were 1 . 82 , 0 . 00 and 1 . 42 µm toward the posterior side in embryos injected with control MO , Dsh MO and Dsh MO + Dsh mRNA , respectively .",
"p-values were obtained using the Welch's t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 026"
],
[
"We report here that an unusual membrane structure , which forms during interphase of a specific embryonic cell cycle , is correlated with the dynamics of centrosomes .",
"This includes positioning of centrosomes and cilia and determination of the cell division axis ( graphical summary and model in Figure 11A , B ) .",
"We propose a model whereby these events are coupled by physical interaction between a centriole and plasma membrane , which results in the generation of a tensile force in the latter .",
"Although we could not directly demonstrate that the membrane invagination is required for mitotic spindle orientation , our data is consistent with our model whereby the centrosome is captured by a unique membrane structure and is subsequently tethered to the posterior side of cells .",
"In the Drosophila stem cell systems , duplicated centrosomes generated from an eccentrically positioned centrosome show differential motilities ( Yamashita and Fuller , 2008 ) .",
"In the ascidian epidermal cells in the 11th cell cycle , the duplicated centrosomes also exhibit differential motilities with the less motile centrosome remaining associated with the membrane invagination .",
"Although some microtubule ( MT ) -filled nanotubes resist nocodazole treatment ( Önfelt et al . , 2006 ) , the formation of the ascidian membrane invagination , like the MT-nanotubes in Drosophila male germ cells ( Inaba et al . , 2015b ) , depends on microtubule function .",
"However , the distribution of the microtubule is distinct in the latter two tubular membrane structures and it is not known whether MT-nanotubes protrude toward the centrosome .",
"Interestingly , a similar membrane invagination to the one we observed in ascidian embryos was previously reported in the C . elegans zygote , and it was suggested that the actomyosin cortex counteracts pulling forces mediated by microtubules , resulting in the posterior displacement of the spindle and the unequal cell division ( Redemann et al . , 2010 ) .",
"Our pharmacological tests with either microtubule or actin inhibitors resulted in similar observations to those described for the C . elegans membrane invagination .",
"Importantly , we report here for the first time that membrane invaginations take place in normal ( untreated ) cells in a multicellular tissue ( ascidian embryonic epidermis ) .",
"Our observations collectively indicate that these invaginations ( generally one or two per cell ) arising from two adjacent plasma membranes may control centrosome dynamics and the orientation of the cell division axis .",
"It should be also noted that a similar membrane invagination is observed during the formation of immunological synapses between T cells and antigen-presenting cells .",
"During this process , T cells rapidly reposition their centrosome to the center of the immunological synapse .",
"Importantly , the centrosome repositioning is coupled with the formation of a membrane invagination originating from the synaptic interphase and reaching towards the T-cell centrosome in a mirotubule-dependent manner ( Yi et al . , 2013 ) .",
"Therefore , the microtubule-dependent mechanism in which a membrane invagination captures and repositions the centrosome may be a general mechanism that transcends species and cell types for polarised centrosome repositioning .",
"In ascidian epidermal cells , this process appears to be also involved in the posterior positioning of the cilia .",
"In this study , we revealed that the tip of the membrane invagination is found in close vicinity of the basal body , indicating that the membrane invagination might associate preferentially with the mother centriole .",
"Since the mother and daughter centrioles inherit distinct components ( Pelletier and Yamashita , 2012; Pereira et al . , 2001 ) , specific molecules of the mother centriole might be involved in establishing the proposed association with the membrane invagination .",
"Posteriorly localized cilia are found in several systems and it will be interesting to determine if their position depends on similar mechanism ( Antic et al . , 2010; Borovina et al . , 2010; Hashimoto et al . , 2010; Momose et al . , 2012; Thompson et al . , 2012 ) . 10 . 7554/eLife . 16550 . 027Figure 11 . A proposed model for mitotic spindle orientation driven by membrane invaginations .",
"( A ) Graphical summary of the spatial relationship of each component .",
"Anterior is left .",
"Interphase: nuclei ( purple ) , cell membrane ( green ) , centrosome ( magenta ) , microtubule ( red ) and cilium ( orange ) .",
"Mitotic phase: neither invaginations nor cilia are found .",
"( B ) Schematic drawing of the course of 11th epidermal cell division in relation to the invagination .",
"Apical view of cells .",
"Upper panel: ( 1 ) Nuclei ( purple ) and centrosomes ( magenta ) just after the 10th cytokinesis; ( 2 ) After the centrosome migrates toward the apical cortex , the membrane invaginates toward the centrosome from the posterior plasma membrane; ( 3 ) The invagination shrinks , pulling the centrosome and nucleus toward the posterior; ( 4 ) The centrosomes duplicate and exhibit distinct migratory activities with the less motile centrosome remaining associated with the membrane invagination; ( 5 ) The mitotic spindle forms aligned along the A-P axis .",
"In the lower panel , the assumed forces involved in the invagination are shown .",
"MTOC activity on the centrosome causes the 'approaching' invagination from the plasma membrane , likely to be depending on microtubule function .",
"After the centrosome is associated with the tip of membrane invagination , a tensile force acting on the invagination brings the centrosome toward the posterior . DOI: http://dx . doi . org/10 . 7554/eLife . 16550 . 027 In this study , we found the positive correlation between the initial length of the membrane invagination and both the average and maximum speed of regression following laser ablation .",
"This observation implies that the membrane invagination has an elastic restoring force rather than an active force generator like cell-cell junctions ( Ishihara and Sugimura , 2012; Rauzi et al . , 2008 ) .",
"Some questions remain to be addressed—for example , which molecule ( s ) acts as the force generator to invaginate the plasma membrane and establish a link with the centrosome .",
"The microtubule-dependent invagination process targeting the centrosome implies the involvement of plus-end-directed motor .",
"Indeed , the generation of membrane invaginations in C . elegans one-cell embryos involves cortical dynein motors ( Redemann et al . , 2010 ) .",
"Therefore , this plus-end motor protein would be one of the promising candidates for the formation of invagination in ascidian epidermal cells .",
"The rigidity of the plasma membrane ( Ramanathan et al . , 2015; Stewart et al . , 2011 ) should be measured during the cell cycle to clarify the mechanism of the phase transition of the membrane invagination , from 'approaching' to 'pulling' the centrosome .",
"Furthermore , by disrupting a core PCP component in ascidian epidermal cells , we have shown a tight correlation between the direction of membrane invagination and centrosome dynamics including mitotic spindle orientation .",
"However , in this experiment , we cannot rule out the possibility that the PCP pathway affects centrosome behavior independently of this membrane structure .",
"Hence , it should be further addressed how the PCP pathway participates in the regulation of centrosome dynamics and the polarization of the membrane invagination .",
"It will also be intriguing to investigate whether the PCP pathway regulates the asymmetric distribution of the actomyosin network and/or plus-end motors in ascidian epidermal cells .",
"Nevertheless , our present work uncovers a previously unknown mechanism associating the centrosome and the plasma membrane , and may open new avenues of investigation into the hidden mechanisms of oriented cell division that underlie embryogenesis and organogenesis ."
],
[
"Ciona intestinalis adults were supplied by the National Bio Resource Project ( NBRP , Japan ) or purchased from the Station Biologique de Roscoff ( France ) .",
"Eggs , embryos , and microinjections were handled following conventional protocols ( Sardet et al . , 2011 ) .",
"We injected mRNAs into unfertilized eggs or eight cell stage embryos .",
"The embryos were cultured and observed at 20°C .",
"For inhibitor treatment , embryos that had just reached the 10th cell division were placed in artificial seawater methylcellulose ( ASWM ) containing nocodazole ( 1 nM final concentration; 1:200 , 000 dilution of DMSO stock ) or cytochalasin ( 10 µg/ml final concentration; 1:1000 dilution of ethanol stock ) , or 0 . 1% ethanol ( control ) .",
"PH-GFP was a gift from Dr . A . McDougall; H2B-mCherry was a gift from Dr . H . Nishida .",
"We constructed pRN3-membraneGFP ( Morita et al . , 2012 ) by PCR-amplifying the ORF from pCS2+ membraneGFP ( forward primer CAACTTTGGCAGATCTGGATCCCATCGATTCGAA; reverse primer GCCCTATAGTGAGTCGTATTACGTAGCGGCCGCGGATCTGGT ) and subcloning it into the pRN3 vector with an In-Fusion HD cloning system ( Clontech , Mountain View , CA ) .",
"The pRN3 was digested by BglII and NotI .",
"To construct PH-tdTomato , we made pRN3-RfA-tdTomato from pCX3-RfA-tdTomato , a gift from Dr . T . Momose .",
"The pCX3-RfA-tdTomato was digested by Acc65I and blunt-ended .",
"RfA-tdTomato was cut out by the restriction enzyme BglII and subcloned into the pRN3 vector .",
"The ORF of the PH domain was PCR-amplified from PH-GFP ( forward primer AAAGGATCCACCATGGACTCGGGCCGGGACTTCCT; reverse primer TTTGAATTCCCCGGGGATGTTGAGCTCCTTCAGGA ) and subcloned into the pENTR vector , which was mixed with pRN3-RfA-tdTomato in a Gateway LR reaction ( Life Technologies , Carlsbad , CA ) .",
"We constructed EB3-mCherry from EB3-GFP , a gift from Dr . A . Akhmanova .",
"The EB3-GFP was digested by EcoRI and XhoI and subcloned into pRN3 between the EcoRI and NotI sites to make pRN3-EB3-GFP .",
"Next , the mCherry ORF was removed from H2B-mCherry by BamHI digestion and inserted into the BamHI site of pRN3-EB3-GFP .",
"To construct ensconsin-tdTomato , the ensconsin ORF was amplified by PCR ( forward primer CAACTTTGGCAGATCTACCATGGAGCAGAAGCTCATCTC; reverse primer CTTGCTCACCATGATATCGACCGGTGGATCCGAAGA ) from pHTB-ensconsin-3xVenus ( Negishi et al . , 2013 ) and subcloned with an In-Fusion HD cloning system into pRN3-tdTomato , which was created by removing the PH domain from pRN3-PH-tdTomato by BglII and EcoRV .",
"To construct pRN3-Arl13b-GFP , the Ciona Arl13b ( NCBI accession number: XP_002129357 ) ORF was cloned by PCR ( forward primer CAACTTTGGCAGATCTACCATGATCGGTCAAATGGGG; reverse primer CATGAATTCAGATCTAACAACAATGTCTTCCTCAGAAT ) from a cDNA library ( a gift from Dr . Miho Suzuki ) , and was inserted with the In-Fusion HD cloning system into pRN3-GFP , which was created by digesting the PH domain out from pRN3-PH-GFP by BglII .",
"Ciona Dsh ( Aniseed Gene model: KH2012:KH . L141 . 37 ) MO ( 5’-AACAATTTTCGTTTCATCCGACATT-3’ , Gene Tools , Philomath , OR ) and standard control MO ( Gene Tools , Philomath , OR ) were injected at 0 . 3 mM .",
"The rescuing Dsh construct was generated by changing the nucleotides at the MO target site as follows , ( −1 ) 5’-catgGAgACaAAgATcGTgTAT-3’ ( +24 ) , small letters indicate the replaced nucleotides with PCR ( forward primer CAACTTTGGCAGATCTACCATGGAGACAAAGATCGTGTATTATCTTGGCGATGAACAAA; reverse primer ACCAGATCCGCGGCCCATGACGTCAACAAAATAATCAC ) .",
"Dsh mRNA was injected at 1 . 0 µg/µl .",
"The mRNAs were synthesised using an mMESSAGE mMACHINE kit ( Life Technologies , Carlsbad , CA ) .",
"Ascidian embryos were prepared for live imaging as described previously ( Negishi et al . , 2013 ) .",
"We injected mRNAs as follows; PH-GFP ( 1 . 8 µg/µl ) , PH-tdTomato ( 2 . 0 µg/µl ) , H2B-mCherry ( 1 . 2 µg/µl ) , EB3-GFP ( 2 . 0 µg/µl ) , EB3-mCherry ( 2 . 5 µg/µl ) , membraneGFP ( 2 . 0 µg/µl ) , and ensconsin-tdTomato ( 2 . 5 µg/µl ) .",
"For FM4-64 ( Life Technologies ) staining , the embryo was incubated in artificial seawater containing the dye ( 10 µg/ml ) for three hours , and then the stained embryo was mounted in ASWM .",
"The embryos were observed with an Olympus IX 81 ( 60x / 1 . 20 NA water immersion lens , Olympus , Japan ) with a Yokogawa CSU-X1 spinning-disk confocal unit and an iXon3 897 EM-CCD camera ( Andor , UK ) .",
"For laser ablation , an N2 Micropoint laser ( 16 Hz , 365 nm wave length , Photonic Instruments , US , Arlington Heights , IL ) attached to a CSU microscopy system was used .",
"Images were obtained with Andor IQ2 software .",
"Embryos were also observed with a Leica SP5 ( 40x / 1 . 30 NA oil immersion lens ) or Leica SP8 ( 63x/1 . 20 NA water immersion lens ) , or Nikon A1 ( 60x / 1 . 20 NA water immersion lens ) scanning confocal microscopy system .",
"Acetylated tubulin immunofluorescence was previously described ( Hudson and Lemaire , 2001 ) .",
"The stained embryos were mounted in Fluoro-KEEPER with DAPI ( Nacalai Tesque , Japan ) .",
"Acquired data were processed with ImageJ software ( http://imagej . nih . gov/ij/ ) .",
"The angle , length , and centroid of the cell and the nucleus were manually measured with ImageJ .",
"The length and direction of the invagination were measured in the maximum-intensity projection of time-lapse movies of embryos expressing PH-GFP at the time point when an invagination reached its maximum length ( Figure 1B , Figure 2E–G and Figure 7C ) .",
"The cell division axis relative to the embryonic A-P axis was also measured in the maximum-intensity projection of time-lapse movies of embryos expressing PH-GFP ( Figure 1B and Figure 9 ) .",
"The long axis of the cell was determined and measured just before the nuclear membrane breakdown , and we calculated the angle of the cell division axis relative to the long axis ( Figure 1C ) .",
"In embryos expressing PH-GFP/H2B-mCherry , we measured the position of individual nuclei just before the nuclear membrane breakdown .",
"We measured the centroid of the nucleus and the cell , and calculated the distance of both centroids along the A-P axis ( Figure 1E and Figure 10B ) .",
"We also measured the centrosome position just before the separation of the duplicated centrosomes during the 10th and 11th cell division cycles in embryos expressing PH-GFP/EB3-mCherry , and normalized the centrosome position to the centroid of the cell ( Figure 4D and Figure 10C ) .",
"To measure the migration distance of duplicated centrosomes , we calculated for each centrosome the distance between the position just before migration started and that when migration ceased in embryos expressing PH-GFP/EB3-mCherry; both positions were normalized to the centroid of the cell ( Figure 4E and Figure 10D ) .",
"We also used embryos expressing PH-GFP/EB3-mCherry to measure the angle of the centrosome axis relative to the invagination axis; the line between the two duplicated centrosomes just after their separation during the 11th division cycle was used as the centrosome axis ( Figure 4F ) .",
"When there were multiple invaginations , we calculated the composition of these structures as a vector ( see also Figure 4F ) .",
"To quantify the cilium’s position in DAPI/acetylated tubulin-immunostained embryos , we traced the cell contours , calculated the centre of the cell , and then measured the distance between the cilium and the centre of the cell along the A–P axis ( Figure 5C ) .",
"We also quantified cilia positions at 30 min before the 11th cytokinesis started with Arl13b-GFP labelled cilia in MO-injected embryos .",
"In the laser ablation experiments , we measured the length of invagination in each frame from a single confocal plane for more than 10 min ( Figure 8 ) .",
"To calculate the average speed of regression after UV laser ablation , we divided the length of invagination by the total time and in the case of maximum speed , we measured the change of length between each time frame ( one second ) .",
"All measurements were completed manually with ImageJ ( Schneider et al . , 2012 ) .",
"All results showing statistically significant difference were supported by power analysis with R-package compute . es ( http://cran . r-project . org/web/packages/compute . es ) and pwr ( https://cran . r-project . org/web/packages/pwr ) .",
"All data were obtained from at least three different batches of embryos .",
"Ciona intestinalis tailbud embryos were washed with washing buffer [50% artificial seawater containing 0 . 1 M sodium cacodylate buffer ( pH 7 . 4 ) ] and fixed in prefix buffer [2 . 5% glutaraldehyde and 2 . 0% paraformaldehyde in 0 . 1 M sodium cacodylate buffer ( pH 7 . 4 ) and 50% artificial seawater] overnight at 4°C .",
"The prefixed embryos were then triple-washed with washing buffer and postfixed with 2% osmium tetroxide and 1 . 5% potassium ferrocyanide in 0 . 1 M sodium cacodylate ( pH 7 . 4 ) and 50% artificial seawater for 1 hr at 4°C .",
"The samples were then washed with distilled water , incubated with 1% thiocarbohydrazide in distilled water for 1 hr at 60°C , washed again with distilled water , and postfixed again with 2% osmium tetroxide in distilled water for 30 min at room temperature .",
"After washing with distilled water , the fixed embryos were stained en bloc with 1% uranyl acetate in distilled water overnight at 4°C , and then with 0 . 2 M lead aspartate ( pH 5 . 5 ) for 30 min at 60°C .",
"The embryos were washed with distilled water and dehydrated by a graded series of ethanol ( 50–100% ) at 4°C and acetone at room temperature .",
"Finally , they were infiltrated with durcupan resin , and the resin was polymerized at 60°C for 3 days .",
"The resin blocks containing the embryos were manually trimmed with a razor blade and glued onto an aluminum SBF-SEM rivet with conductive epoxy resin ( SPI Conductive Silver Epoxy; SPI Supplies and Structure Prove , Inc . , West Chester , PA , USA ) .",
"Specimens on the rivet were further trimmed with a razor blade to as small a size as possible to include only one embryo ( about 100–200 μm ) .",
"All lateral surfaces of the specimens were ion-coated with gold to a thickness of 20 nm to dissipate the electric charge caused by electron-beam irradiation during SEM imaging .",
"In this study , we used a system originally developed at the Max Planck Institute for Medical Research , Heidelberg , Germany ( Denk and Horstmann , 2004 ) ; in this system , a scanning electron microscope ( MERLIN , Carl Zeiss Microscopy , Jena , Germany ) equipped with an in-chamber ultramicrotome system ( 3View; Gatn Inc . , Pleasanton , CA , USA ) was used for slicing and imaging the SBF-SEM stacks .",
"Specimens mounted on SBF-SEM rivets were placed on the specimen stage in the SEM chamber , and the block surface was sliced by the in-chamber ultramicrotome and imaged each time with a back-scattered electron detector .",
"The serial image stacks were acquired automatically as reported previously ( Miyazaki et al . , 2014 ) .",
"The SBF-SEM images were recorded with an accelerating voltage of 1 . 5 kV with a dwell time of 1 . 0 μs .",
"The image size was 8192 × 8192 pixels .",
"The slice thickness was 50 nm .",
"After 2× binning of the images , the image stack was automatically aligned using ‘Register Virtual Stack Slices’ in the Fiji/ImageJ software package ( http://fiji . sc/Fiji ) ( Schindelin et al . , 2012 ) .",
"Individual cells and organelle structures were manually segmented using the AMIRA software package ( FEI Visualization Science Group , Burlington , MA , USA ) .",
"This software package was also used to generate the figures .",
"Ciona tailbud embryos were fixed in prefix buffer [2% gultaraldehyde] in 0 . 1 M phosphate buffer ( pH7 . 2 ) for 3 hr at room temperature .",
"After washing with 0 . 1 M phosphate buffer , the embryos were postfixed with 1% osmium tetroxide in 0 . 1 M phosphate buffer for 1 hr at room temperature .",
"The samples were washed with distilled water and dehydrated by a graded series of ethanol ( 50–100% ) at 4°C .",
"Finally , the embryos were embedded in durcupan resin and cut into ultrathin sections ( 70 nm thickness ) .",
"The serial sections were collected on pioloform-coated single slot copper grids and stained with uranyl acetate and lead citrate .",
"They were then observed with a transmission electron microscope ( JEM1010; JEOL Co . , Japan ) .",
"The serial section images were aligned with the IMOD software package ( Kremer et al . , 1996 ) as described previously ( Miyazaki et al . , 2014 ) ."
]
] | [
"In the last mitotic division of the epidermal lineage in the ascidian embryo , the cells divide stereotypically along the anterior-posterior axis .",
"During interphase , we found that a unique membrane structure invaginates from the posterior to the centre of the cell , in a microtubule-dependent manner .",
"The invagination projects toward centrioles on the apical side of the nucleus and associates with one of them .",
"Further , a cilium forms on the posterior side of the cell and its basal body remains associated with the invagination .",
"A laser ablation experiment suggests that the invagination is under tensile force and promotes the posterior positioning of the centrosome .",
"Finally , we showed that the orientation of the invaginations is coupled with the polarized dynamics of centrosome movements and the orientation of cell division .",
"Based on these findings , we propose a model whereby this novel membrane structure orchestrates centrosome positioning and thus the orientation of cell division axis ."
] | [
"An animal develops from a single fertilized egg cell .",
"Several rounds of cell division then occur to create new cells and form an embryo .",
"Often , the direction of cell division is oriented , rather than random .",
"In other words , the positioning of the two new daughter cells is highly organized during cell division .",
"This orientation of the direction of cell division is also important for shaping the body’s tissues .",
"Animal cells contain a structure called the centrosome that helps to regulate cell division ( amongst other roles ) .",
"Just before a cell divides , the centrosome duplicates itself and the copies move toward opposite ends of the cell .",
"A structure called the mitotic spindle then forms out of the centrosomes and ensures that the newly forming cells contain the correct amount of genetic material .",
"The orientation of the spindle specifies where the cell splits into two , and this orientation is ultimately governed by the position of the centrosomes inside the cell .",
"However , it is not fully understood how cells position their centrosomes .",
"Sea squirts are simple marine animals that are well suited for studying cell division , in part because their embryos consist of a small number of cells .",
"Negishi et al . have now studied the final cell division cycle of the outer cells of sea squirt embryos , during which nearly all the cells divide in the same direction – along an axis that stretches from the embryo’s head to its tail .",
"This revealed that before a spindle forms in these cells , the cell membrane at the rear end of each cell is pulled into the cell , forming an “invagination” that elongates along the head-to-tail axis .",
"The finger-like membrane invagination captures the centrosome and pulls it towards the rear end of the cell .",
"Following this , the centrosome duplicates and the new centrosomes move until they are aligned with the membrane invagination .",
"Once both centrosomes are aligned correctly , the spindle forms .",
"Thus , membrane invaginations serve to position centrosomes .",
"The next steps are to identify the molecules that allow membrane invaginations and centrosomes to interact with each other and to determine the forces that place centrosomes in their correct location ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"cell biology"
] | The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ | elife-05615-v2 | [
[
"Adipose tissue is a major storehouse for excess energy and has recently been shown to be an endocrine tissue critical for the generation of hormones and cytokines involved in energy metabolism ( Galic et al . , 2010 ) .",
"Abnormalities in adipocyte differentiation or functions may result in the development of metabolic syndrome by inducing insulin resistance , and these abnormalities are likely to be induced in part by inappropriate regulation of gene expression required for adipocyte differentiation or functions .",
"The conversion of preadipocytes to mature adipocytes is a strictly controlled process regulated by a series of transcriptional activators .",
"Both in vivo and in vitro studies have shown that peroxisome proliferator-activated receptor γ ( PPARγ ) and three CCAAT/enhancer family members ( α , β , and δ ) are essential regulators of adipogenesis ( Tontonoz et al . , 1994b; Yeh et al . , 1995; Barak et al . , 1999; Imai et al . , 2004; Sugii et al . , 2009 ) .",
"The adipogenic program is generally composed of two steps .",
"The first step is initiated by adipogenic stimuli that induce early adipogenic activators including C/EBPβ , C/EBPδ and Krüppel-like factors ( KLFs ) and lead to the formation of early enhanceosomes ( Mori et al . , 2005; Oishi et al . , 2005 ) .",
"In the second step , these early enhanceosomes induce late adipogenic activators including PPARγ and C/EBPα that lead to terminal differentiation by inducing genes necessary for the characteristics of mature adipocytes ( Rosen et al . , 2002; Lefterova et al . , 2008 ) .",
"Recent studies using high-throughput sequencing have shown that transcriptional activators do not function individually but rather cooperate with other activators and form hotspots at specific genomic regions ( Moorman et al . , 2006; Chen et al . , 2008; He et al . , 2011; Siersbaek et al . , 2011; Boergesen et al . , 2012; Gerstein et al . , 2012 ) .",
"In addition , the existence of super-enhancers has been recently reported ( Loven et al . , 2013; Whyte et al . , 2013 ) .",
"Super-enhancers are large genomic domains occupied by master transcriptional activators and mediators , which induce expression of genes that define cell identity , and are especially characterized by a large amount of localization of Mediator subunit 1 ( MED1 ) .",
"During the early phase of adipogenic differentiation , hotspots are central constituents in super-enhancers and have been suggested to cooperate with super-enhancers to drive the differentiation process ( Siersbaek et al . , 2014 ) .",
"A previous study showed that C/EBPβ , C/EBPδ , CREB1 and PPARγ function as candidate master transcriptional activators in adipose tissue ( Hnisz et al . , 2013 ) .",
"Of those master transcriptional activators , PPARγ plays a central role in adipogenesis , whereas other factors cannot induce adipocyte differentiation in the absence of PPARγ ( Farmer , 2006; Rosen and MacDougald , 2006 ) .",
"PPARγ has two isoforms , PPARγ1 and PPARγ2 .",
"These two isoforms are generated from alternate promoter usage and splicing , and PPARγ2 contains 30 additional amino acids at the amino-terminus ( Zhu et al . , 1995; Fajas et al . , 1997; Tontonoz and Spiegelman , 2008 ) .",
"PPARγ1 is ubiquitously expressed and PPARγ2 is strictly expressed in adipose tissues , while both isoforms are strongly induced during adipocyte differentiation .",
"Although ectopic expression of either of the PPARγ isoforms can induce adipocyte differentiation , PPARγ2 is thought to play a more central role in this process ( Mueller et al . , 2002; Zhang et al . , 2004 ) .",
"It has been reported that PPARγ expression and activity are regulated at different levels such as transcription , protein degradation and post-translational modification ( van Beekum et al . , 2009; Eeckhoute et al . , 2012; Ahmadian et al . , 2013; Lee and Ge , 2014 ) .",
"Tripartite motif-containing ( TRIM ) proteins ( also known as E3 ubiquitin–protein ligases ) are characterized by the presence of a RING finger , one or two zinc-binding motifs called B-boxes , and an associated coiled-coil region ( RBCC ) , and there are currently known to be 77 TRIM proteins in humans .",
"TRIM family proteins are involved in a broad range of biological processes , and their alterations result in diverse pathological conditions ( Meroni and Diez-Roux , 2005; Ozato et al . , 2008; Hatakeyama , 2011 ) .",
"TRIM23 is a member of the TRIM family and possesses carboxy-terminal ARF ( ADP ribosylation factor ) domains .",
"A recent study has shown that TRIM23 mediates atypical lysine 27 ( K27 ) -linked polyubiquitin conjugation to NEMO , which plays an important role in the NFκB pathway , and this conjugation is essential for TLR3- and RIG-I/MDA5-mediated antiviral innate and inflammatory responses ( Arimoto et al . , 2010 ) .",
"In this study , we identified TRIM23 as a novel factor that regulates PPARγ protein stability , possibly via atypical ubiquitin conjugation to PPARγ .",
"TRIM23 knockdown caused reduction of PPARγ protein levels , which were restored by treatment with a proteasome inhibitor , leading to a severe defect in adipogenic differentiation .",
"TRIM23 is dispensable for the early adipogenic program but is indispensable for the late adipogenic program .",
"By controlling PPARγ abundance , TRIM23 functions as a regulator for a critical link between early and late enhanceosomes ."
],
[
"To determine whether TRIM23 is involved in adipocyte differentiation , we first examined the expression of TRIM23 during differentiation of 3T3-L1 cells and in mouse adipose tissue .",
"Real-time PCR analysis and immunoblot analysis revealed that TRIM23 was expressed in preadipocytes and that mRNA and protein levels slightly increased during adipogenesis ( Figure 1A , B ) .",
"Consistent with these results , TRIM23 was found to be predominantly expressed in the preadipocyte-containing stromal vascular fraction ( SVF ) at a level as high as that in the mature adipocyte fraction ( Figure 1C ) .",
"We also measured Trim23 expression in a model of diet-induced obesity , and we found no significant difference between the expression levels in mice receiving the high-fat diet and those receiving the control chow ( Figure 1C ) .",
"We next examined the subcellular localization of TRIM23 .",
"We fractionated 3T3-L1 cells into nuclear extracts and cytoplasmic S100 fraction , and we found that a large amount of TRIM23 was distributed in the cytoplasm ( Figure 1D ) . 10 . 7554/eLife . 05615 . 003Figure 1 . TRIM23 is expressed during adipocyte differentiation .",
"( A ) Real-time PCR analysis of mRNA expression of Trim23 during 3T3-L1 differentiation .",
"Total RNA was isolated from 3T3-L1 cells on the indicated days .",
"Trim23 mRNA was normalized to that of Gtf2b .",
"( B ) Immunoblot analysis of TRIM23 and PPARγ protein during 3T3-L1 cell differentiation .",
"( C ) Real-time PCR analysis of mRNA expression of Trim23 in mouse adipose tissue .",
"Trim23 mRNA was normalized to that of Gtf2b .",
"( D ) Subcellular localization of TRIM23 .",
"Nuclear extracts and cytoplasmic S100 fraction were prepared from 3T3-L1 cells , and immunoblot analysis of TRIM23 , HDAC1 , and GAPDH was performed . DOI: http://dx . doi . org/10 . 7554/eLife . 05615 . 003 We next examined whether TRIM23 expression was necessary for adipocyte differentiation .",
"Two different shRNAs ( shTRIM23a and shTRIM23b ) and a non-targeting control shRNA ( shControl ) were introduced into 3T3-L1 preadipocytes using retroviral vectors .",
"One shRNA ( shTRIM23a ) efficiently depleted and the other ( shTRIM23b ) weakly depleted TRIM23 expression levels in 3T3-L1 preadipocytes relative to shControl ( Figure 2A ) .",
"These cells were stimulated to differentiate , and the ability to undergo differentiation to mature adipocytes was evaluated by determination of lipid accumulation using Oil Red O staining , direct measurement of intracellular triglyceride contents and determination of relative mRNA levels of adipocyte-specific genes including Fabp4 , Cidec , Klf15 , Adipoq , and Retn .",
"Remarkably , TRIM23 knockdown significantly decreased lipid accumulation and markedly impaired the induction of adipocyte-specific genes ( Figure 2B–D and Figure 2—figure supplement 1 ) .",
"To test whether this regulatory network is relevant to human adipocytes , we introduced siRNA into human primary visceral preadipocytes and differentiated them to mature adipocytes .",
"Consistent with the results for mouse 3T3-L1 cells , TRIM23 knockdown significantly decreased lipid accumulation ( Figure 2B and Figure 2—figure supplement 2 ) .",
"These findings indicate that TRIM23 is required for efficient conversion of preadipocytes to mature adipocytes .",
"It has been shown that most adipocyte-specific genes are PPARγ target genes ( Lefterova et al . , 2008; Nielsen et al . , 2008 ) .",
"We next examined the occupancy of PPARγ at a well-described PPARγ target gene , Fabp4 , during adipocyte differentiation .",
"PPARγ forms heterodimers with RXRα , which specifically bind to PPAR response elements ( PPREs ) .",
"It has been reported that PPARγ is recruited to two PPREs located 5500 bp upstream from the transcriptional start site ( TSS ) of the Fabp4 gene , and this recruitment mediates activation of the Fabp4 gene ( Figure 2E ) ( Tontonoz et al . , 1994a; Nielsen et al . , 2006 ) .",
"We found that TRIM23 depletion decreased the occupancy of PPARγ on the enhancer PPRE at day 4 using chromatin immunoprecipitation ( ChIP ) -qPCR analysis ( Figure 2F ) .",
"It has been shown that some subunits of Mediator complex are necessary for the adipogenic process .",
"MED14 is required for full activation of PPARγ-mediated transcription and adipocyte differentiation in vitro , and MED23 is required for the early transcriptional events during adipogenesis ( Wang et al . , 2009; Grontved et al . , 2010 ) .",
"MED1 is also required for adipogenesis in vitro ( Ge et al . , 2002 ) .",
"We tested the occupancy of MED1 , one of the Mediator subunits , and found that TRIM23 depletion reduced the occupancy of MED1 at the Fabp4 gene at day 4 ( Figure 2G ) .",
"We also found reduced occupancy of Pol II at the Fabp4 gene in TRIM23 knockdown cells ( Figure 2H ) .",
"These findings suggest that the adipogenic defect by TRIM23 knockdown is caused by reduced PPARγ recruitment and subsequent reduced transcriptional activation on the target genes . 10 . 7554/eLife . 05615 . 004Figure 2 . TRIM23 is required for 3T3-L1 adipocyte differentiation .",
"( A ) Immunoblot analysis of TRIM23 knockdown before induction of adipogenesis is shown .",
"( B ) Cells were stained with Oil Red O to visualize the accumulation of lipid droplets at day 6 .",
"( C ) The amounts of intracellular triacylglyceride ( TG ) were quantified at day 6 .",
"( D ) RNA levels of Gtf2b and Fabp4 were determined by real-time PCR at days 0 , 2 , 4 and 6 .",
"Expression level of each gene was normalized to that of the Gtf2b .",
"( E ) Schematic representation of the Fabp4 gene .",
"The locations of the sequences amplified in the ChIP are shown at the bottom in base pairs relative to the Fabp4 transcriptional start site .",
"( F , G and H )",
"ChIP analysis of PPARγ ( F ) , MED1 ( G ) , and Pol II ( H ) on the Fabp4 gene during adipocyte differentiation .",
"Ct values of each ChIP were normalized to that of input .",
"All data represent means ± s . d . from three independent experiments .",
"The p values for the indicated comparisons were determined by Student's t test ( * , p < 0 . 05; ** , p < 0 . 01 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 05615 . 00410 . 7554/eLife . 05615 . 005Figure 2—figure supplement 1 . Quantitative analysis of Trim23 , Cidec , Klf15 , Adipoq and Retn mRNA during 3T3-L1 differentiation . mRNA levels of Trim23 , Cidec , Klf15 , Adipoq and Retn were determined by real-time PCR at days 0 , 2 , 4 , and 6 .",
"Expression level of each gene was normalized to the level of Gtf2b . DOI: http://dx . doi . org/10 . 7554/eLife . 05615 . 00510 . 7554/eLife . 05615 . 006Figure 2—figure supplement 2 . TRIM23 is required for human visceral preadipocyte differentiation . Short interfering RNAs targeting TRIM23 ( siTRIM23 ) and a non-targeting control siRNA ( siControl ) were introduced into human primary visceral preadipocytes , and the preadipocytes were differentiated to mature adipocytes .",
"Adipocytes were stained with Oil Red O to visualize the accumulation of lipid droplets at day 10 . DOI: http://dx . doi . org/10 . 7554/eLife . 05615 . 006 We showed that TRIM23 knockdown reduces PPARγ recruitment to the enhancer and subsequent Pol II recruitment to the promoter at the target genes .",
"To elucidate mechanisms of decreased PPARγ-mediated gene activation in TRIM23 knockdown 3T3-L1 cells , we investigated mRNA levels of several adipogenic transcription factors during adipogenesis .",
"Real-time PCR analysis revealed that induction of early transcriptional factors , Cebpb and Cebpd , was largely unaffected during adipocyte differentiation and that the expression of late adipogenic activators , Pparg and Cebpa , was equally induced at day 2 but was significantly reduced from day 4 by TRIM23 depletion ( Figure 3 ) .",
"These findings suggest that an adipogenic defect by TRIM23 knockdown is caused by the decreased expression of late adipogenic activators . 10 . 7554/eLife . 05615 . 007Figure 3 . TRIM23 is required for induction of Pparg1 , Pparg2 and Cebpa but not for induction of Cebpb and Cebpd during adipogenesis . RNA levels of Pparg , Cebpa , Cebpb and Cebpd were determined by real-time PCR at days 0 , 2 , 4 and 6 .",
"Expression level of each gene was normalized to that of the Gtf2b .",
"The data represent means ± s . d . from three independent experiments .",
"The p values for the indicated comparisons were determined using Student's t-test ( * , p < 0 . 05; ** , p < 0 . 01 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 05615 . 007 It has been reported that hormonal treatment of 3T3-L1 cells acutely induces the expression of C/EBPβ and C/EBPδ within a few hours after stimulation .",
"These early transcriptional activators have also been shown to be recruited to their target sites including the Pparg locus ( Steger et al . , 2010; Siersbaek et al . , 2011 ) .",
"C/EBPβ marks a subset of early transcription factor hotspots and forms early enhanceosomes with other early adipogenic transcription factors ( Siersbaek et al . , 2011 ) .",
"These early enhanceosomes facilitate epigenetic modification and chromatin remodeling .",
"To determine whether TRIM23 affects PPARγ induction , we investigated the expression levels of Cebpb and Cebpd and subsequent recruitment of C/EBPβ and C/EBPδ to the Pparg locus at the early phase of adipogenesis .",
"As shown in Figure 4A , there were little differences in Cebpb and Cebpd mRNA levels between TRIM23 knockdown and controls .",
"We next examined the possibility that TRIM23 affected the occupancy of C/EBPβ and C/EBPδ at the Pparg promoter during adipogenesis using ChIP-qPCR analysis ( Figure 4B , C ) .",
"As previously reported , C/EBPβ and C/EBPδ were enriched to their target sites , including Pparg , Iqck , and Cav2 loci ( Siersbaek et al . , 2011 ) , but TRIM23 depletion had no significant effect on this enrichment .",
"These results suggest that TRIM23 functions at the downstream stage after recruitment of early adipogenic activators . 10 . 7554/eLife . 05615 . 008Figure 4 . TRIM23 knockdown does not affect the induction and occupancy of C/EBPβ and C/EBPδ at the Pparg promoter during adipogenesis .",
"( A ) RNA levels of Trim23 , Cebpb and Cebpd were determined by real-time PCR at 0 , 6 , 12 , 24 and 48 hr .",
"Expression level of each gene was normalized to that of Gtf2b .",
"( B ) Schematic representation of the Pparg promoters .",
"The locations of the sequences amplified in the ChIP are shown at the bottom in base pairs relative to the Pparg1 and Pparg2 transcriptional start sites .",
"( C ) Occupancy of C/EBPβ and C/EBPδ on a subset of target genes at 4 hr .",
"Ct values of each ChIP were normalized to that of input .",
"( D ) Cell proliferation analysis of 3T3-L1 cells during adipocyte differentiation .",
"3T3-L1 cells were counted at each time point .",
"The data represent means ± s . d . from three independent experiments .",
"The p values for the indicated comparisons were determined by Student's t test ( * , p < 0 . 05; ** , p < 0 . 01 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 05615 . 008 During differentiation , growth-arrested 3T3-L1 preadipocytes synchronously re-enter the cell cycle and undergo several rounds of clonal expansion , called mitotic clonal expansion ( MCE ) ( Tang et al . , 2003b ) .",
"MCE is required for expression of adipogenic proteins and progression of terminal differentiation .",
"C/EBPβ has been shown to play a pivotal role in MCE ( Tang et al . , 2003a ) .",
"To evaluate MCE in TRIM23-knockdown cells , we counted cell numbers during adipocyte differentiation ( Figure 4D ) .",
"However , there was no difference between TRIM23-knockdown cells and the corresponding control .",
"These findings also support the idea that TRIM23 does not affect the activities of C/EBPβ .",
"Taken together , we concluded that PPARγ2 expression by TRIM23 is not regulated through induction and occupancy of C/EBPβ and C/EBPδ at the Pparg2 promoter .",
"Activators such as C/EBPβ and C/EBPδ promote the recruitment of general transcriptional machinery composed of general transcription factors ( GTFs ) and RNA Pol II to form the pre-initiation complex ( PIC ) ( Lemon and Tjian , 2000; Thomas and Chiang , 2006 ) .",
"To achieve this goal , activators recruit GTFs and Pol II directly or indirectly through binding to many cofactors .",
"The transcription cofactors can be largely classified into two groups .",
"The first group is composed of covalent histone-modifying enzymes and ATP-driven nucleosome remodelers to reorganize the chromatin architecture ( Belotserkovskaya and Berger , 1999; Jaenisch and Bird , 2003; Margueron et al . , 2005 ) .",
"These factors create an open chromatin environment suitable for transcription .",
"The second group consists of general cofactors such as the Mediator complex and the SAGA complex ( Timmers and Tora , 2005 ) .",
"The Mediator complex is an evolutionarily conserved coregulatory complex that typically works in communication between activators and general transcription machinery ( Malik and Roeder , 2010 ) .",
"It has been shown that some subunits of the Mediator complex are necessary for the adipogenic process ( Ge et al . , 2002; Wang et al . , 2009; Grontved et al . , 2010 ) .",
"To clarify the steps of Pparg transcription at which TRIM23 acts after activator recruitment , we observed epigenetic changes at the Pparg promoters during adipogenesis by ChIP-qPCR analysis .",
"Trimethylation of histone H3 lysine 4 ( H3K4me3 ) , acetylation of histone H3 lysine 27 ( H3K27ac ) , and monomethylation of histone H4 lysine 20 ( H4K20me1 ) correlate with gene activation and increase at the Pparg gene early in the differentiation process , whereas dimethylation of histone H3 lysine 9 ( H3K9me2 ) correlates with gene repression and decreases during adipogenesis ( Fujiki et al . , 2009; Tie et al . , 2009; Wakabayashi et al . , 2009; Mikkelsen et al . , 2010; Wang et al . , 2013 ) .",
"H3K4me3 and H3K27ac levels at Pparg gene increased during adipocyte differentiation , and there was little change caused by TRIM23 depletion ( Figure 5A , B ) .",
"There were no differences in H4K20me1 and H3K9me2 levels at day 2 between TRIM23-knockdown 3T3-L1 cells and corresponding control cells ( Figure 5—figure supplement 1 ) .",
"These results indicate that TRIM23 does not affect epigenetic marks at the Pparg promoter .",
"It has also been reported that the Pparg promoter is reorganized by ATP-driven nucleosome remodelers and that C/EBPβ binding is required for its chromatin opening .",
"Therefore , we monitored chromatin opening using formaldehyde-assisted isolation of regulatory elements ( FAIRE ) analysis ( Giresi et al . , 2007 ) .",
"We performed FAIRE analysis at days 0 and 2 , and we observed increased chromatin opening around the C/EBPβ binding sites on the Pparg promoter at day 2 , while there was little difference caused by TRIM23 knockdown ( Figure 5C ) .",
"Since PPARγ by itself enhances the expression of PPARγ2 by binding to the PPRE at the Pparg promoter , we performed ChIP-qPCR analysis with a PPARγ antibody and found that the occupancy of PPARγ at the Pparg promoter was significantly decreased at day 4 by TRIM23 depletion ( Figure 5D ) .",
"In accordance with the data for PPARγ , the occupancy of Pol II at the Pparg promoter was equally increased at day 2 but was decreased at day 4 , although there was little change in the occupancy of MED1 at day 2 and 4 by TRIM23 depletion ( Figure 5E , F ) .",
"Taken together , knockdown of TRIM23 did not affect formation of early enhanceosomes , epigenetic changes , chromatin remodeling , or recruitment of Pol II at the Pparg promoter during early adipocyte differentiation , but knockdown of TRIM23 reduced the formation of late enhanceosomes and the recruitment of Pol II during late adipogenic differentiation . 10 . 7554/eLife . 05615 . 009Figure 5 . TRIM23 knockdown does not affect the epigenetic marks for activated genes and chromatin opening but decreases the occupancy of Pol II at the Pparg promoters .",
"( A and B )",
"ChIP analysis of H3K4me3 ( A ) and H3K27ac ( B ) at the Pparg promoters during adipocyte differentiation .",
"Ct values of each ChIP were normalized to that of input .",
"( C ) Formaldehyde-assisted isolation of regulatory elements ( FAIRE ) analysis during adipocyte differentiation .",
"The enrichment of fragmented genomic DNA in the FAIRE samples was analyzed and normalized to that of input .",
"( D , E and F )",
"ChIP analysis of PPARγ ( D ) , MED1 ( E ) , and Pol II ( F ) at the Pparg promoter during adipocyte differentiation .",
"Ct values of each ChIP were normalized to that of input .",
"All data represent means ± s . d . from three independent experiments .",
"The p values for the indicated comparisons were determined by Student's t test ( * , p < 0 . 05; ** , p < 0 . 01 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 05615 . 00910 . 7554/eLife . 05615 . 010Figure 5—figure supplement 1 . Depletion of TRIM23 does not affect H3K9me2 and H4K20me1 marks at the Pparg promoter .",
"( A ) Control IgG ChIP does not occupy a subset of gene promoters during adipogenesis .",
"Ct values of each ChIP were normalized to that of input .",
"( B and C )",
"ChIP analysis of H3K9me2 ( B ) and H4K20me1 ( C ) at the Pparg promoter during adipocyte differentiation .",
"Ct values of each ChIP were normalized to that of input .",
"All data represent means ± s . d . from three independent experiments .",
"The p values for the indicated comparisons were determined by Student's t test ( * , p < 0 . 05; ** , p < 0 . 01 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 05615 . 010 It has been reported that a positive feedback loop between C/EBPα and PPARγ expression supports the maintenance of the differentiated stage ( Rosen et al . , 2002 ) .",
"Both C/EBPα and PPARγ binding sites exist at the Pparg promoter , and PPARγ binding sites also exist at the Cebpa locus ( Farmer , 2006; Nielsen et al . , 2008 ) .",
"PPARγ activates its own expression via direct targeting and/or via C/EBPα binding to the Pparg promoter .",
"If transcriptional activity of PPARγ is directly enhanced by TRIM23 , knockdown of TRIM23 should lead to decreased PPARγ expression .",
"This hypothesis is important to clarify a discontinuity in PPARγ expression between early and late adipocyte differentiation .",
"To examine whether TRIM23 mediates PPARγ expression via the transcriptional activity of PPARγ , we measured the activity using a dual luciferase system .",
"TRIM23 showed slight suppression of PPARγ2-mediated transcriptional activity but did not show a dose-dependent relationship ( Figure 6A ) .",
"It has been reported that the transcriptional activity of PPARγ was modulated by SUMO-1 modification and that some TRIM proteins have SUMO E3 activities ( van Beekum et al . , 2009; Chu and Yang , 2011 ) .",
"We therefore investigated whether TRIM23 mediates SUMOylation of PPARγ2 .",
"Although PPARγ2 was definitely SUMOylated in cells , TRIM23 did not affect this modification ( Figure 6B ) .",
"These findings suggest that changes in the transcriptional activity of PPARγ were not critical for impairment of PPARγ induction in TRIM23-knockdown cells . 10 . 7554/eLife . 05615 . 011Figure 6 . TRIM23 does not affect PPARγ2 transcriptional activity .",
"( A ) TRIM23 does not affect PPARγ-mediated transcriptional activity in HEK293T cells .",
"The peroxisome proliferator-activated receptor response element firefly luciferase reporter vector ( PPRE–Luc ) , pGL4 . 74 renilla luciferase reporter plasmid , and expression vectors encoding TRIM23 and Pparg were transfected into HEK293T cells and the cells were incubated in culture medium containing 10% charcoal-treated fetal bovine serum for 24 hr .",
"Cells were incubated with or without troglitazone ( 2 μM ) for 24 hr , harvested , and quantified firefly luciferase and renilla luciferase mRNA with real-time PCR .",
"The data represent means ± s . d . from three independent experiments .",
"( B ) TRIM23 does not promote SUMOylation of PPARγ2 .",
"An in vivo assay for SUMOylation of PPARγ2 by TRIM23 was performed .",
"Expression vectors encoding FLAG-PPARγ2 , Myc-TRIM23 , and HA-SUMO1 were transfected into HEK293T cells .",
"Cell lysates were immunoprecipitated with anti-FLAG antibody and then immunoblot analysis was performed to detect modifications of PPARγ2 . DOI: http://dx . doi . org/10 . 7554/eLife . 05615 . 011 To elucidate the mechanism of reduction of PPARγ expression by TRIM23 knockdown during late adipogenesis , we examined the expression of adipogenic activators during adipocyte differentiation at the protein level .",
"Immunoblot analysis revealed that TRIM23 knockdown did not affect induction of early adipogenic activators but that TRIM23 knockdown impaired induction of late adipogenic activators ( Figure 7A ) .",
"Protein levels of PPARγ1 and PPARγ2 were already decreased at day 2 by TRIM23 knockdown ( Figure 7B ) , whereas mRNA level of PPARγ1 was hardly reduced and that of PPARγ2 was moderately reduced in TRIM23-knockdown cells ( Figure 3 ) .",
"Since the amount of PPARγ protein was more greatly reduced than the amount of Pparg mRNA in TRIM23-knockdown cells , we speculated that the stability of PPARγ protein was impaired by TRIM23 knockdown .",
"It has been reported that the stability of PPARγ protein is regulated by the ubiquitin proteasome systems ( Floyd and Stephens , 2002 ) .",
"To determine whether reduction of PPARγ protein in TRIM23-knockdown cells depends on proteasome activity , we observed the levels of PPARγ protein in the presence and absence of a proteasome inhibitor , MG132 .",
"3T3-L1 cells were differentiated for 48 hr and subsequently treated with MG132 .",
"Immunoblot analysis showed that administration of MG132 blocked the reduction of both PPARγ1 and PPARγ2 protein levels in TRIM23-knockdown cells ( Figure 7C ) .",
"We also examined protein levels of PPARγ in preadipocytes and found that administration of MG132 blocked the reduction of PPARγ in TRIM23-knockdown cells ( Figure 7—figure supplement 1 ) .",
"These findings indicated that the presence of TRIM23 is sufficient to inhibit degradation of PPARγ not only in a differentiating state but also in an undifferentiated state .",
"Next , we examined the protein stability of PPARγ1 and PPARγ2 .",
"TRIM23-knockdown 3T3-L1 cells and the corresponding control cells were differentiated by the differentiation cocktail for 48 hr , and were subsequently treated with MG132 for 6 hr and with cycloheximide ( CHX ) for the indicated times .",
"TRIM23 knockdown promoted the degradation of PPARγ1 and PPARγ2 ( Figure 7D , E ) .",
"These results suggest that TRIM23 stabilized PPARγ1 and PPARγ2 proteins during adipocyte differentiation . 10 . 7554/eLife . 05615 . 012Figure 7 . TRIM23 stabilizes PPARγ2 . ( A ) Immunoblot analysis of C/EBPα , C/EBPβ , C/EBPδ and PPARγ proteins during 3T3-L1 cell differentiation .",
"( B ) The intensity of the immunoreactive bands of PPARγ2 obtained by immunoblot analysis with anti-PPARγ antibody was determined relative to that obtained with anti-β-actin antibody .",
"( C ) Immunoblot analysis of PPARγ protein in the absence and presence of MG132 .",
"3T3-L1 cells with stable knockdown of TRIM23 or the corresponding control cells were differentiated by the differentiation cocktail for 48 hr and subsequently treated with 10 μM of MG132 for 6 hr ( D ) 3T3-L1 cells with stable knockdown of TRIM23 or the corresponding control cells were differentiated by the differentiation cocktail for 48 hr and subsequently treated with 10 μM of MG132 for 6 hr , followed by cycloheximide ( CHX ) treatment ( 5 μM ) for 0 , 1 , 2 or 4 hr .",
"The cell lysates were subjected to immunoblot analysis with an anti-PPARγ , anti-TRIM23 or anti-β-actin antibody .",
"β-actin is shown as a loading control .",
"The result is representative of two independent experiments .",
"( E ) The intensity of the PPARγ1 and PPARγ2 bands was normalized to that of the corresponding β-actin bands shown in ( D ) and is indicated as a percentage of the normalized value at 0 hr . DOI: http://dx . doi . org/10 . 7554/eLife . 05615 . 01210 . 7554/eLife . 05615 . 013Figure 7—figure supplement 1 . The presence of TRIM23 is sufficient to inhibit basal degradation of PPARγ .",
"Immunoblot analysis of PPARγ protein in the absence and presence of MG132 .",
"3T3-L1 preadipocytes with stable knockdown of TRIM23 or the corresponding control cells were treated with MG132 ( 10 mM ) for 6 hr . DOI: http://dx . doi . org/10 . 7554/eLife . 05615 . 013 Since it has been shown that TRIM23 is a putative E3 ubiquitin ligase and mediates atypical polyubiquitin conjugation to NEMO , we hypothesized that TRIM23 was involved in the stability of PPARγ protein via its E3 ligase activity ( Arimoto et al . , 2010 ) .",
"We next examined whether TRIM23 binds to and ubiquitinates PPARγ2 .",
"Using immunoprecipitation assays in HEK293T cells , we detected overexpressed HA-PPARγ2 in a protein complex with FLAG-TRIM23 ( Figure 8A ) .",
"This interaction was not affected by the treatment of troglitazone ( Figure 8B ) .",
"We also found that endogenous PPARγ2 was co-precipitated with FLAG-TRIM23 ( Figure 8C ) .",
"Furthermore , we found that TRIM23 facilitated ubiquitination of PPARγ2 in vivo and in vitro ( Figure 8D , E ) .",
"We next examined which domain of TRIM23 was required for adipocyte differentiation .",
"3T3-L1 cells were infected with a retrovirus expressing shRNA for TRIM23 ( shTRIM23a ) or a non-targeting control shRNA ( shControl ) and were subsequently infected with a retrovirus expressing control or shRNA-resistant FLAG-TRIM23 deletion mutants ( Figure 8F ) .",
"Expression levels of TRIM23 were verified by immunoblot analysis ( Figure 8G ) .",
"These cells were induced to differentiate , and the ability to undergo differentiation to mature adipocytes was evaluated by determination of lipid accumulation using Oil Red O staining .",
"As expected , even a small amount of wild-type TRIM23 expression rescued lipid accumulation; however , a larger amount of TRIM23 deletion mutants failed to rescue lipid accumulation , indicating that both an amino-terminal RING finger domain , which is a ubiquitin ligase catalytic domain , and a carboxy-terminal ARF domain were required for adipocyte differentiation ( Figure 8H ) .",
"These results suggest that ubiquitin conjugation to PPARγ by TRIM23 plays an important role in adipocyte differentiation . 10 . 7554/eLife . 05615 . 014Figure 8 . TRIM23 interacts with PPARγ2 and promotes ubiquitination of PPARγ2 . ( A ) In vivo assay for interaction between TRIM23 and PPARγ2 .",
"FLAG-TRIM23 and HA-PPARγ2 were transfected into HEK293T cells .",
"Lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-FLAG and anti-HA antibodies .",
"( B ) In vivo assay for interaction between TRIM23 and PPARγ2 with or without troglitazone ( 1 μM ) .",
"FLAG-TRIM23 and HA-PPARγ2 were transfected into HEK293T cells .",
"Coimmunoprecipitation assays were performed using the cell extract from these cells with anti-FLAG antibody in the presence or absence of 1 μM troglitazone .",
"( C ) In vivo assay for interaction between TRIM23 and PPARγ2 .",
"3T3-L1 cells stably expressing FLAG-TRIM23 were generated , differentiated , and harvested at day 6 .",
"Whole cell lysates were immunoprecipitated with anti-FLAG antibody and then immunoblot analysis was performed with anti-FLAG and anti-PPARγ antibodies .",
"( D ) In vivo assay for ubiquitination of PPARγ2 by TRIM23 .",
"FLAG-PPARγ2 , Myc-TRIM23 , and HA-ubiquitin ( HA-Ub ) were transfected into HEK293T cells .",
"Cell lysates were immunoprecipitated with anti-FLAG antibody and then immunoblot analysis was performed to detect ubiquitination of PPARγ2 .",
"( E ) Promotion of in vitro PPARγ2 polyubiquitination by TRIM23 .",
"An in vitro ubiquitination assay was performed with the indicated combinations of ATP , HA-Ub , His6-E1 , His6-E2 ( UbcH5B ) , His6-GST-TRIM23-FLAG , and GST-PPARγ2 .",
"Reaction mixtures were subjected to immunoblot analysis with anti-PPARγ ( top ) , anti-HA ( middle ) or anti-FLAG ( bottom ) .",
"The positions of GST-PPARγ2 or His6-GST-TRIM23-FLAG modified by various numbers of HA-Ub moieties are indicated .",
"( F ) Schematic representation of TRIM23 deletion mutants is shown .",
"Protein motifs are indicated .",
"RING , ring-finger domain; B-box , B-box domain; CC , coiled-coil domain; ARF , ADP ribosylation factor domain .",
"( G ) Immunoblot analysis of ectopic expression of TRIM23 deletion mutants in TRIM23-knockdown 3T3-L1 cells before induction of adipogenesis .",
"( H ) Cells were stained with Oil Red O to visualize the accumulation of lipid droplets at day 6 . DOI: http://dx . doi . org/10 . 7554/eLife . 05615 . 014 Degradative polyubiquitin chains are targeted to proteasomes through ubiquitin receptors including S5a/Rpn10 and Rad23 .",
"We next examined the binding of ubiquitinated PPARγ to GST-ubiquitin receptors and found that ubiquitinated PPARγ purified from cells exogenously expressing TRIM23 bound less efficiently to S5a than did that from cells without exogenous expression of TRIM23 .",
"In contrast , HR23B bound to ubiquitinated PPARγ from both cells with and those without exogenous expression of TRIM23 ( Figure 9A ) .",
"These findings indicate that TRIM23-dependent modification of PPARγ in vivo decreased its recognition by 26S proteasome in a manner dependent on the proteasome subunit S5a/Rpn10 .",
"It is well known that K48-linked ubiquitin chains are responsible for proteasomal degradation and that K63-linked chains are involved in various cell signaling processes , though the roles of other atypical ubiquitin linkages through M1 , K6 , K11 , K27 , K29 or K33 or mixed linkages within the same chain remain unclear ( Kulathu and Komander , 2012 ) .",
"We hypothesized that TRIM23 mediates atypical ubiquitination of PPARγ to inhibit its degradation .",
"We performed an in vitro ubiquitination assay using methylated ubiquitin or various ubiquitin mutants ( all Lys mutated to Arg except the indicated Lys residue ) to study the TRIM23-mediated ubiquitin-linkages of PPARγ .",
"Intriguingly , TRIM23 mediated ubiquitin conjugation to PPARγ in the presence of methylated ubiquitin and amino-terminally His6-tagged no Lys ( K0 ) ubiquitin ( Figure 9B ) .",
"Considering that methylated ubiquitin is unable to form polyubiquitin chains and that amino-terminally His6-ubiquitin mutants interfere with linear ubiquitin conjugation , these findings indicated that TRIM23 ubiquitinated PPARγ at multiple sites .",
"Furthermore , TRIM23 more efficiently mediated ubiquitin conjugation in the presence of untagged K0 ubiquitin or His6-K27 ubiquitin ( Figure 9B ) , suggesting that TRIM23 can conjugate linear and K27-linked polyubiquitin chains to PPARγ .",
"To confirm that this atypical ubiquitination of PPARγ is responsible for reduced recognition by 26S proteasome , we performed an in vitro binding assay using PPARγ ubiquitinated by TRIM23 and GST-ubiquitin receptors including HR23B and S5a .",
"Consistent with the results shown in Figure 9A , although GST-HR23B efficiently bound to M1- and K27-Ub conjugates on PPARγ2 , GST-S5a poorly pulled down the conjugates ( Figure 9C , D ) .",
"Taken together , these findings suggested that atypically ubiquitinated PPARγ by TRIM23 decreased its recognition by 26S proteasome , leading to resistance to proteasomal degradation . 10 . 7554/eLife . 05615 . 015Figure 9 . TRIM23 mediates atypical polyubiquitin conjugation of PPARγ2 , leading to reduced recognition of ubiquitinated PPARγ2 by the proteasomal ubiquitin receptor S5a .",
"( A ) PPARγ2 polyubiquitination by TRIM23 leads to reduced recognition by the proteasomal ubiquitin receptor S5a .",
"HEK293T cells transiently transfected with plasmids encoding FLAG-PPARγ2 and/or HA-TRIM23 were lysed .",
"GST , GST-HR23B , and GST-S5a were resuspended with cell lysates , followed by pull-down with glutathione Sepharose beads .",
"Samples were separated by SDS-PAGE , followed by immunoblotting using the indicated antibodies .",
"( B ) In vitro PPARγ2 polyubiquitination by TRIM23 .",
"An in vitro ubiquitination assay was performed with the indicated combinations of ATP , various ubiquitin mutants , His6-E1 , E2 ( UbcH5C ) , His6-GST-TRIM23-FLAG , and GST-PPARγ2 .",
"Reaction mixtures were subjected to immunoblot analysis with anti-PPARγ ( top ) , anti-TRIM23 ( middle ) or anti-Ub ( bottom ) antibodies .",
"The positions of GST-PPARγ2 or His6-GST-TRIM23-FLAG modified by various numbers of ubiquitin moieties are indicated .",
"( C and D )",
"Conjugation of PPARγ2 with M1- and/or K27-linked polyubiquitin chains leads to reduced recognition by the proteasomal ubiquitin receptor S5a .",
"An in vitro ubiquitination assay was performed with the indicated combinations of ATP , ubiquitin mutants , His6-E1 , E2 ( UbcH5C ) , TRIM23-FLAG , and PPARγ2 .",
"Reaction mixtures were subjected to GST pull-down assay .",
"PPARγ2-ubiquitin conjugates were incubated with GST-S5a ( C ) or GST-HR23B ( D ) prebound to glutathione Sepharose beads .",
"Samples were separated by SDS-PAGE , followed by immunoblotting using a PPARγ antibody .",
"Equivalent amounts of bound and unbound fractions were loaded in each lane . DOI: http://dx . doi . org/10 . 7554/eLife . 05615 . 015 Our data suggest that the defect in adipogenesis by TRIM23 knockdown was caused at least through the decreased expression of PPARγ2 .",
"Therefore , we expected that overexpression of PPARγ2 at an abundance that is sufficient to overcome the instability of PPARγ2 protein should rescue the adipogenic defect by TRIM23 knockdown .",
"To confirm this hypothesis , 3T3-L1 cells were infected with a retrovirus expressing shRNA for TRIM23 ( shTRIM23a ) or a non-targeting control shRNA ( shControl ) and were subsequently infected with a retrovirus expressing HA-PPARγ2 or control .",
"Expression levels of PPARγ2 and TRIM23 were verified by immunoblot analysis ( Figure 10A and Figure 10—figure supplement 1 ) .",
"TRIM23 knockdown was maintained in spite of ectopic expression of PPARγ2 during adipocyte differentiation ( Figure 10B ) .",
"These cells were induced to differentiate , and the ability to undergo differentiation to mature adipocytes was evaluated by the determination of lipid accumulation using Oil Red O staining ( Figure 10C ) and determination of relative mRNA levels of adipocyte-specific genes ( Figures 10D and Figure 10—figure supplement 1 ) .",
"Ectopic expression of PPARγ2 remarkably rescued lipid accumulation in TRIM23-knockdown cells and the induction of adipocyte-specific genes .",
"The induction of early transcriptional factors , C/EBPβ and C/EBPδ , in TRIM23-depleted cells with ectopic expression of PPARγ2 was largely rescued to levels comparable to those in control 3T3-L1 cells during adipocyte differentiation , except for sustained high expression level of C/EBPβ after day 4 ( Figure 10—figure supplement 1 ) .",
"These findings suggest that TRIM23 functions in the upstream stage of PPARγ induction during adipogenesis . 10 . 7554/eLife . 05615 . 016Figure 10 . PPARγ2 expression rescues the adipogenesis defect in TRIM23-knockdown 3T3-L1 cells .",
"( A ) Immunoblot analysis of ectopic PPARγ2 expression before induction of adipogenesis is shown .",
"( B ) Changes in Trim23 mRNA during adipocyte differentiation in 3T3-L1 cells .",
"Total RNA was isolated from 3T3-L1 cells on the indicated days of differentiation .",
"Trim23 mRNA was determined by real-time PCR and normalized to that of Gtf2b .",
"( C ) Cells were stained with Oil Red O to visualize the accumulation of lipid droplets at day 6 .",
"( D ) RNA levels of Fabp4 , Cebpa , and Pparg were determined by real-time PCR at days 0 , 2 , 4 and 6 of differentiation .",
"Expression level of each gene was normalized to the level of Gtf2b .",
"The data represent means ± s . d . from three independent experiments .",
"The p values for the indicated comparisons were determined using Student's t-test ( *p < 0 . 05; **p < 0 . 01 ) .",
"( E ) Model for TRIM23 function in PPARγ abundance during adipocyte maturation .",
"Several adipogenic stimuli activate early adipogenic activators such as C/EBPβ , C/EBPδ , glucocorticoid receptor ( GR ) and signal transducer and activator of transcription 5a ( STAT5a ) .",
"These activators then induce late adipogenic activators including PPARγ and C/EBPα .",
"TRIM23 mediates atypical polyubiquitin conjugation to PPARγ and may stabilize the PPARγ protein .",
"TRIM23 plays a critical role in the switching from early to late adipogenic enhanceosomes by regulating the abundance of PPARγ .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 05615 . 01610 . 7554/eLife . 05615 . 017Figure 10—figure supplement 1 . PPARγ2 expression rescues the adipogenesis defect that occurred in TRIM23-knockdown 3T3-L1 cells .",
"( A ) Immunoblot analysis of PPARγ and C/EBPα protein during 3T3-L1 differentiation .",
"( B ) RNA levels of Cidec , Klf15 , Cebpb , Adipoq , Retn and Cebpd , were determined by real-time PCR at days 0 , 2 , 4 , and 6 .",
"Expression level of each gene was normalized to that of Gtf2b .",
"The data represent means ± s . d . from three independent experiments .",
"The p values for the indicated comparisons were determined using Student's t-test ( *p < 0 . 05; **p < 0 . 01 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 05615 . 017"
],
[
"Adipocyte differentiation is tightly regulated by a complicated transcriptional cascade .",
"PPARγ plays a central role in the late phase of adipogenesis .",
"Thus , elucidation of the mechanisms that regulate PPARγ expression is essential for understanding adipocyte differentiation .",
"In this study , we showed that TRIM23 knockdown in preadipocytes results in decreased expression of PPARγ and a severe defect in late adipogenic differentiation .",
"Ectopic expression of PPARγ2 rescues the adipogenic defect induced by TRIM23 knockdown .",
"Therefore , we further investigated the mechanism by which TRIM23 regulates the expression of PPARγ .",
"A considerable number of studies have shown the importance of transcriptional regulation of Pparg gene expression .",
"Early adipogenic activators such as C/EBPβ and C/EBPδ provoked by adipogenic stimuli induce low expression levels of PPARγ1 , PPARγ2 and C/EBPα ( Yeh et al . , 1995; Ishibashi et al . , 2012 ) .",
"Then PPARγ and C/EBPα directly induce each other's high expression level that supports the maintenance of the differentiated stage ( Rosen et al . , 2002 ) .",
"The binding of activators to enhancer DNA elements promotes the recruitment of GTFs and Pol II to the promoter DNA in order to form a PIC ( Lemon and Tjian , 2000; Thomas and Chiang , 2006 ) .",
"Activators also mediate the recruitment of covalent histone-modifying enzymes and ATP-driven nucleosome remodeling complexes to the promoter to reorganize the chromatin architecture to be competent for transcription , leading to transcription initiation ( Belotserkovskaya and Berger , 1999; Jaenisch and Bird , 2003; Margueron et al . , 2005 ) .",
"To elucidate the mechanism of reduced PPARγ expression by TRIM23 knockdown , we investigated the occupancy of activators such as C/EBPβ and C/EBPδ , epigenetic marks by ChIP-qPCR analysis and the degree of chromatin opening by FAIRE analysis at the Pparg promoters .",
"However , there were no substantial changes in these during the early phase of differentiation , and we therefore concluded that recruitment of early activators and PIC formation were not affected by TRIM23 .",
"An apparent change observed in TRIM23-knockdown cells was a decrease in the occupancy of PPARγ and Pol II at the Pparg locus during the late phase of differentiation , during which PPARγ and C/EBPα support the high expression level of each other through a positive feedback loop .",
"Therefore , this change caused by TRIM23 knockdown was likely to be provoked by impaired positive-feedback regulation of late adipogenic activators .",
"We examined the expression of adipogenic activators in detail , and we found that the protein level of PPARγ2 was already decreased at day 2 by TRIM23 knockdown ( Figure 7B ) , whereas the mRNA level of Pparg was moderately reduced in TRIM23-knockdown cells ( Figure 3 ) .",
"We therefore speculated that TRIM23 regulates PPARγ expression at the protein level .",
"Although numerous studies have focused on the regulation of Pparg expression at the transcriptional level , information on regulation of PPARγ at the protein level remains limited .",
"The stability of PPARγ protein is regulated by the ubiquitin proteasome pathway , and PPARγ is actually an unstable protein ( t½ = 2 hr ) ( Waite et al . , 2001; Christianson et al . , 2008 ) .",
"Seven-in-absentia homolog 2 ( Siah2 ) facilitates ubiquitination and degradation of PPARγ in vivo , but it is unclear whether Siah2 directly ubiquitinates PPARγ in vitro ( Kilroy et al . , 2012 ) .",
"It has also been reported that PPARγ by itself acts as an E3 ubiquitin ligase and mediates K48-linked polyubiquitination and degradation of p65 , suggesting that PPARγ as an E3 ligase is critical to terminate NFκB signaling ( Hou et al . , 2012 ) .",
"PPARγ forms polyubiquitin chains without a substrate using UBCH3 as E2 in vitro , while it has not been determined whether PPARγ is autoubiquitinated or not .",
"In this study , we showed that TRIM23 conjugates atypical polyubiquitin chains including M1- and K27-linked ubiquitin chains to PPARγ , leading to reduced binding to the proteasomal ubiquitin receptor S5a .",
"Functions regulated by K48- and K63-linked ubiquitin chains have been extensively studied .",
"Past studies have established that K48-linked chains are responsible for proteasomal degradation and that K63-linked chains are involved in various cell signaling processes .",
"Recently , roles of other atypical ubiquitin linkages through K6 , K11 , K27 , K29 , K33 , M1 , or mixed linkages within the same chain have been shown ( Kulathu and Komander , 2012 ) .",
"It has been reported that E3 ubiquitin ligase Smad ubiquitination regulatory factor 1 ( Smurf1 ) prevents orphan nuclear receptor Nur77 degradation through mediating its atypical ubiquitination via K6 and K27 linkage ( Lin et al . , 2014 ) .",
"It has also been shown that RING type E3 ligase SCFβ-TrCP stabilizes the oncoprotein Myc by ubiquitination that requires K33 , K48 and K63-linked ubiquitin chains , which antagonizes SCFFbw7-mediated K48-linked polyubiquitin chain formation ( Popov et al . , 2010 ) .",
"Moreover , Ring1B generates self-atypical mixed K6- , K27- , and K48-linked non-proteolytic polyubiquitin chains on itself , whereas E6AP leads to its degradation via ubiquitination of the same residues of Ring1B ( Ben-Saadon et al . , 2006 ) .",
"In this study , we showed that TRIM23 mediates atypical polyubiquitin conjugation including M1- and K27-linked ubiquitin chains to PPARγ and that ubiquitination of PPARγ by TRIM23 causes reduced recognition of PPARγ by 26S proteasome .",
"Although the detailed mechanism remains to be elucidated , these findings suggest that M1- and/or K27-linked polyubiquitin chains to PPARγ lead to resistance to degradation via reduced recognition by 26S proteasome .",
"In conclusion , TRIM23 is a novel positive regulator of adipocyte maturation via control of switching from early to late adipogenic enhanceosomes through regulating the abundance of PPARγ ( illustrated in Figure 10E ) .",
"Results of further studies on TRIM23 may be useful for revealing the abnormalities in adipocyte differentiation and for providing a potential therapeutic target for obesity and diabetes mellitus ."
],
[
"All experiments were performed according to the guidelines laid down by the Animal Welfare Committee of Hokkaido University and under institutional approval .",
"For diet-induced obesity , male C57BL/6 mice were fed high-fat diet containing a 57-kcal% fat high-fat diet ( Clea Japan , Inc . , Tokyo , Japan ) for 27 weeks starting at 8 weeks of age ( n = 3 ) .",
"Simultaneously , a separate group of male C57BL/6 mice were fed a diet of normal chow ( n = 3 ) containing 5 kcal% fat and served as lean controls .",
"The epididymal fat pads were dissected and minced and then placed in 1 mg/ml collagenase ( Sigma–Aldrich , St . Louis , Missouri ) in PBS and incubated at 37°C with shaking for 30 min .",
"The samples were then centrifuged at 300×g for 5 min at room temperature .",
"The floating fraction was collected as adipocytes , whereas the precipitated fraction was collected as the SVF .",
"HEK293T cells were cultured under an atmosphere of 5% CO2 at 37°C in Dulbecco's modified Eagle's medium ( Sigma–Aldrich ) supplemented with 10% fetal bovine serum ( FBS ) ( Gibco BRL , Paisley , UK ) .",
"3T3-L1 cells were cultured under the same conditions in DMEM with 10% calf serum ( CS ) ( Equitech-Bio Inc . , Kerrville , TX ) .",
"Differentiation into mature adipocytes was induced by exposing the confluent cells to DMEM with 10% FBS for 2 days and to a conditioned medium supplemented with 0 . 5 mM IBMX , 1 μM dexamethasone , and 10 μg/ml insulin ( induction medium ) for 2 days .",
"Cells were then incubated in a medium containing 5 μg/ml insulin .",
"After 2 days , the medium was replaced with a conditioned medium .",
"Subsequently , the conditioned medium was changed regularly every 2 days .",
"Intracellular lipid accumulation was evaluated using Oil Red O staining .",
"3T3-L1 cells cultured on six-well plates were washed with PBS , fixed for 10 min with 10% formaldehyde in PBS , and stained for 30 min with Oil Red O solution ( 0 . 5% Oil Red O [Sigma–Aldrich] in isopropanol with Milli Q [3:2 vol/vol] ) .",
"After the cells had been washed twice with 60% isopropanol and twice with PBS , lipid accumulation was evaluated .",
"Intracellular triglyceride content was determined by Lab Assay Triglyceride ( WAKO , Osaka , Japan ) and normalized to the amounts of total cellular protein determined by a Bio-rad protein assay ( Bio-Rad Laboratories Inc . , Hercules , CA ) according to each manufacturer's instructions .",
"Culture and differentiation of human primary visceral preadipocytes ( Poietics human visceral preadipocytes , Lonza Walkersville Inc . , Walkersville , MD , USA ) into adipocytes were performed according to the manufacturer's protocol .",
"Human TRIM23 and mouse Pparg2 cDNAs were amplified by PCR from HEK293T and 3T3-L1 cDNAs , respectively , by polymerase chain reaction ( PCR ) with KOD plus ( Toyobo , Osaka , Japan ) .",
"The primers used for the amplification are listed in Table 1 .",
"TRIM23 and Pparg2 cDNAs were ligated into the p3×FLAG vector ( Invitrogen , Carlsbad , CA ) and the pCGN-HA vector . 10 . 7554/eLife . 05615 . 018Table 1 . List of SYBR Green primers for real-time PCRDOI: http://dx . doi . org/10 . 7554/eLife . 05615 . 018GeneForward primerReverse primerPCR primers for cloning of cDNA Trim23AGGATGGCTACCCTGGTTGTAAACAAATCAAGCAACATCCAATACTCC Pparg2GTTATGGGTGAAACTCTGGGACTGCTAATACAAGTCCTTGTAOligonucleotides for shRNA shTRIM23aGATCCCCGAAGAAATGGCTCTAAGTGTTCAAGAGACACTTAGAGCCATTTCTTCTTTTTAAGCTTAAAAAGAAGAAATGGCTCTAAGTGTCTCTTGAACACTTAGAGCCATTTCTTCGGG shTRIM23bGATCCCCGGTAGATGTTAAATCGCATTTCAAGAGAATGCGATTTAACATCTACCTTTTTAAGCTTAAAAAGGTAGATGTTAAATCGCATTCTCTTGAAATGCGATTTAACATCTACCGGGqRT-PCR primers for gene expression AdipoqGCACTGGCAAGTTCTACTGCAAGTAGGTGAAGAGAACGGCCTTGT CebpaTGCGCAAGAGCCGAGATAACGGTCATTGTCACTGGTCAACT CebpbCAAGCTGAGCGACGAGTACAAGCTGCTCCACCTTCTTCTG CebpdATCGACTTCAGCGCCTACATGCTTTGTGGTTGCTGTTGAA CidecAGCTAGCCCTTTCCCAGAAGTCAGGCAGCCAATAAAGTCC Fabp4CATCAGCGTAAATGGGGATTGTCGTCTGCGGTGATTTCAT Klf15CCCAATGCCGCCAAACCTATGAGGTGGCTGCTCTTGGTGTACATC Pparg1 +2TGCAGGAGCAGAGCAAAGAGCGGCTTCTACGGATCGAAAC Pparg1TGAAAGAAGCGGTGAACCACTGTGGCATCTCTGTGTCAACCATG Pparg2TGGCATCTCTGTGTCAACCATGGCATGGTGCCTTCGCTGA RetnTTTTCTTCCTTGTCCCTGAACTGGATCTTCTTGTCGATGGCTTCAT Gtf2bGTTCTGCTCCAACTTTTGCCTTGTGTAGCTGCCATCTGCACTT Trim23TTGGAATGGCTCACACAGAACACATGGGCATCAACAACACqPCR primers for ChIP Pparg1 −1 . 0 kbCTGTCTATCATGTGGGCTTCAGACCTTACACATAGGGTGGAGA Pparg1 −0 . 4 kbACAAACTTCTCCATGACAGACACGCCTTGCTCCTCACAG Pparg1 +0 . 3 kbCTGCGTAACTGACAGCCTAACACTTGGTCACTCTCCGTCCT Pparg2 −1 . 0 kbGATACACTGCCCTGTGTAAGGGAGCAGCCCTTGTCACATAA Pparg2 −0 . 2 kbGAACAGTGAATGTGTGGGTCACTGACTGAGAGCCAGTTGTGA Pparg2 +0 . 5 kbGTGAGCATTTCAGAACACTTGGGCCTGAAGAAGAACAGAAATTCTAC Negative controlTGGTAGCCTCAGGAGCTTGCATCCAAGATGGGACCAAGCTG MyogGGGTCTCTTCCTCTTACCCGATACCTTGCTGGCCATGGAC IqckGAAACAAAGCCTTCCCATCCTCCTTTCTTGCTGTGGCTTC Cav2CTCAGAAAAGGCAGGGAAAGCCCAGTCATGACAACACCAA Fabp4 -10 , 000 bpCCATGAGGAAATTCGCTACACCCTTCCACCCTTATCTCACAC Fabp4 −5500 bpGAGAGCAAATGGAGTTCCCAGATTGGGCTGTGACACTTCCAC Fabp4 −200 bpCATTGCCAGGGAGAACCAATCCTTCATGACCAGACCCTGT Fabp4 +500 bpCAGGTGAACCCGCAAGAAAGGCTTGGCAAAGAAGGCCAC HEK293T cells were transfected by the calcium phosphate method .",
"After 48 hr , the cells were harvested and lysed in a solution containing 50 mM Tris-HCl ( pH 7 . 4 ) , 150 mM NaCl , 1% Triton X-100 , leupeptin ( 10 μg/ml ) , 1 mM phenylmethylsulfonyl fluoride , 400 μM Na3VO4 , 400 μM EDTA , 10 mM NaF , and 10 mM sodium pyrophosphate .",
"The cell lysates were centrifuged at 16 , 000×g for 20 min at 4°C , and the resulting supernatant was incubated with antibodies for 2 hr at 4°C .",
"Protein A-Sepharose ( GE Healthcare , Uppsala , Sweden ) that had been equilibrated with the same solution was added to the mixture , which was then rotated for 1 hr at 4°C .",
"The resin was separated by centrifugation , washed 5 times with ice-cold lysis buffer , and then boiled in SDS sample buffer .",
"Immunoblot analysis was performed with primary antibodies , horseradish peroxidase-conjugated antibodies to mouse or rabbit immunoglobulin G ( 1:20 , 000 dilutions , Promega Corporation , Madison , WI ) and an enhanced chemiluminescence system ( ECL , Thermo Fisher Scientific , Rockford , IL ) .",
"The following primary antibodies were used: anti-FLAG M2 ( Sigma–Aldrich ) , anti-HA ( Covance , Princeton , NJ ) , anti-PPARγ ( sc-7273 , Santa Cruz Biotechnology , Santa Cruz , CA ) , anti-TRIM23 ( HPA039605 , Sigma–Aldrich ) , anti-β-actin AC15 ( Sigma–Aldrich ) , anti-GAPDH ( Ambion , Austin , TX ) , anti-HDAC1 ( 10E2 , Cell Signaling Technology , Beverly , MA ) , anti-GST ( sc-138 , Santa Cruz ) , anti-Ub ( P4D1 , sc-8017 , Santa Cruz ) , anti-C/EBPα ( sc-61 , Santa Cruz ) , anti-C/EBPβ ( sc-150 , Santa Cruz ) and anti-C/EBPδ ( sc-151 , Santa Cruz ) .",
"Nuclear extracts and cytoplasmic S100 fraction were prepared from 3T3-L1 cells according to the method of Dignam et al . ( 1983 ) .",
"A complementary DNA encoding mouse Pparg2 containing an HA-tag at its amino terminus was subcloned into pMX-neo , and cDNAs encoding shRNA-resistant human TRIM23 deletion mutants containing a 3×FLAG-tag at their amino-terminus were subcloned into pMSCV-neo ( Takara ) .",
"The resulting vectors were used to transfect Plat-E cells and thereby generate recombinant retroviruses .",
"3T3-L1 cells were infected with the recombinant retroviruses and selected in a medium containing G418 ( 0 . 4 mg/ml , Nacalai Tesque , Kyoto , Japan ) .",
"Cells were seeded in 24-well plates at 5 × 104 cells per well ( HEK293T ) and then incubated at 37°C with 5% CO2 overnight .",
"The peroxisome proliferator-activated receptor response element firefly luciferase reporter plasmid ( PPRE–Luc , a kind gift from Bruce M Spiegelman ) and pGL4 . 74 renilla luciferase ( Promega ) reporter plasmid were transfected with Pparg and/or TRIM23 expression vectors into HEK293T cells using Fugene HD reagent ( Roche , Branchburg , NJ ) .",
"Transfected cells were incubated in DMEM ( Invitrogen ) supplemented with 10% charcoal-treated FBS ( Equitech-Bio ) for 24 hr and then incubated with 2 μM troglitazone for 24 hr and harvested , and firefly luciferase and renilla luciferase mRNA were quantified by real-time PCR .",
"pSUPER-retro-puro vector was purchased from OligoEngine ( OligoEngine , Seattle , WA ) .",
"The sequences of short hairpin RNA ( shRNA ) oligonucleotides are listed in Table 1 shRNA for a negative control with no significant homology to any known gene sequences in human and mouse genomes was designed according to a previous report ( Gou et al . , 2004 ) .",
"Approximately 50% confluent Plat-E cells in 100-mm dishes were transfected with 10 μg pSUPER-retro-puro-shTRIM23-a , shTRIM23-b or control shRNA vector using Fugene HD reagent ( Roche ) .",
"Culture supernatant containing the retrovirus was collected 48 hr after transfection , and retroviral supernatant was added to 3T3-L1 cell lines in 100-mm dishes with polybrene ( 8 μg/ml; Sigma–Aldrich ) .",
"Cells were cultured with puromycin ( 5 μg/ml ) for 1 week .",
"Human primary visceral preadipocytes in six-well tissue culture plates ( ∼3 . 3 × 105 cells per well ) were transfected with 12 nM siRNA targeting human TRIM23 ( On-TARGET plus SMART pool , L-006523-00-0005 , Dharmacon/Thermo Scientific , Lafayette , CO , USA ) or with 12 nM ON-TARGETplus Non-targeting Pool ( D-001810-10-20 , Dharmacon ) using Lipofectamine RNAiMAX Transfection Reagent ( Invitrogen ) .",
"Total cell number per dish was counted during a period of 6 days .",
"3T3-L1 cells were harvested from the 10-cm dish using 0 . 25% trypsin ( Sigma–Aldrich ) and 0 . 1 mM EDTA , and they were resuspended in a condition medium , followed by staining with 0 . 4% trypan blue ( Sigma–Aldrich ) .",
"Unstained cells were counted under a light microscope .",
"Total RNA was isolated from 3T3-L1 cells using an ISOGEN ( Nippon Gene , Tokyo , Japan ) , with removal of contaminating DNA by a TURBO DNA-Free Kit ( Invitrogen ) , and reverse transcription ( RT ) was performed by ReverTra Ace ( Toyobo , Osaka , Japan ) .",
"The resulting cDNA was subjected to real-time PCR with a StepOne machine and Power SYBR Green PCR master mix ( Applied Biosystems , Foster City , CA ) .",
"The primers used for the amplification are listed in Table 1 .",
"Gtf2b transcript was used as an internal control to normalize the mRNA levels of each gene .",
"Reaction mixtures ( each 30 μl ) each containing 40 mM HEPES-NaOH ( pH 7 . 9 ) , 60 mM potassium acetate , 1 mM ATP , 5 mM MgCl2 , 0 . 5 mM EDTA , 2 mM DTT , E1 ( 0 . 1 μg; Enzo Life Sciences , Farmingdale , New York ) , E2 ( 0 . 5 μg of His6-UbcH5B or 0 . 5 μg of His6-UbcH5C ) , ubiquitin ( 4 μg of HA-Ub or 10 μg of other Ub mutants ) , TRIM23 ( 1 μg of His6-GST-TRIM23-FLAG or 0 . 1 μg of TRIM23-FLAG ) , and PPARγ ( 1 μg of His6-GST-PPARγ or 0 . 1 μg of PPARγ ) were incubated for 2 hr at 30°C .",
"The reaction was terminated by the addition of SDS sample buffer containing 4% 2-mercaptoethanol and heating at 95°C for 5 min .",
"Immunoblot analysis was performed with mouse monoclonal antibodies to Anti-FLAG M2 ( Sigma–Aldrich ) , anti-HA ( Covance ) and anti-PPARγ ( sc-7273 , Santa Cruz Biotechnology ) .",
"HEK293T cells transiently transfected with plasmids encoding FLAG-PPARγ2 and/or HA-TRIM23 were harvested and lysed .",
"PPARγ2-ubiquitin conjugates were prepared by an in vitro ubiquitination assay .",
"GST pull-down assays were performed according to the instructions of the manufacturer ( Glutathione Sepharose 4B , GE Healthcare ) .",
"Briefly , 5 μg of GST ( UBPBio , Aurora , CO , USA ) , GST-S5a ( UBPBio ) or GST-HR23B ( UBPBio ) was bound to Glutathione Sepharose beads ( 20 μl for each sample ) for 1 hr , and the beads were then washed 3 times and resuspended in ice-cold lysis buffer .",
"HEK293T cell lysates or PPARγ2-ubiquitin conjugates were incubated with the beads prebound by GST-tagged proteins for 1 hr at 4°C on a rotating platform .",
"The supernatant was collected as an unbound fraction , and nonspecific binding was removed by washing 5 times with ice-cold lysis buffer .",
"The bound proteins were eluted by 10 mM reduced glutathione .",
"Samples were separated by SDS-PAGE , followed by immunoblotting using indicated antibodies .",
"Equivalent amounts of bound and unbound fractions were loaded in adjacent lanes .",
"Cells ( 1 × 106 ) were cross-linked with 0 . 5 M DSG in PBS for 45 min and 1% formaldehyde in PBS for 10 min at room temperature .",
"Cells were resuspended in a hypotonic buffer ( 10 mM HEPES-NaOH pH 7 . 9 , 1 . 5 mM MgCl2 , 10 mM KCl , and 0 . 5 mM DTT ) and were homogenized using a dounce homogenizer .",
"Crude nuclei were lysed in lysis buffer ( 0 . 1% SDS , 1% Triton X-100 , 10 mM EDTA , 150 mM NaCl , 50 mM Tris-HCl pH 8 . 0 ) and were sonicated with a Bioruptor Sonicator ( Diagenode , Sparta , NJ ) 30 times for 30 s each time at the maximum power setting to generate DNA fragments of ∼150–400 bps .",
"Sonicated chromatin was incubated for 3 hr at 4°C with 5–15 μg of normal IgG or specific antibodies .",
"Antibodies used were as follows: normal rabbit IgG ( sc-2027 , Santa Cruz ) , normal mouse IgG ( sc-2025 , Santa Cruz ) , anti-Pol II total Rpb1 ( F-12 , sc-55492 , Santa Cruz ) , anti-C/EBPβ ( sc-150 , Santa Cruz ) , anti-C/EBPδ ( sc-636 , Santa Cruz ) , anti-PPARγ ( sc-1984 , Santa Cruz ) , anti-MED1 ( sc-5334 , Santa Cruz ) , H3K4me3 ( 07-473 , EMD Millipore , Billerica , MA , USA ) , H3K27ac ( ab4729 , Abcam , Cambridge , MA ) , H3K9me2 ( ab1220 , Abcam ) and H4K20me1 ( ab9051 , Abcam ) .",
"Then protein A agarose ( GE Healthcare ) preblocked with BSA and salmon sperm DNA ( BioDynamics Laboratory , Tokyo , Japan ) was added and incubated for 1 hr at 4°C .",
"Beads were washed once with IP buffer ( 20 mM Tris-HCl pH 8 . 0 , 150 mM NaCl , 2 mM EDTA , 1% Triton X-100 ) , 2 times with high-salt buffer ( 20 mM Tris-HCl pH 8 . 0 , 500 mM NaCl , 2 mM EDTA , 1% Triton X-100 ) , 2 times with LiCl buffer ( 250 mM LiCl , 20 mM Tris-HCl pH 8 . 0 , 1 mM EDTA , 1% Triton X-100 , 0 . 1% NP40 and 0 . 5% NaDOC ) , and 2 times with TE buffer .",
"Bound complexes were eluted from the beads with 100 mM NaHCO3 and 1% SDS by incubating at 50°C for 30 min with occasional vortexing .",
"Crosslinking was reversed by overnight incubation at 65°C .",
"Immunoprecipitated DNA and input DNA were treated with RNase A and Proteinase K by incubation at 45°C .",
"DNA was purified using a QIAquick PCR purification kit ( 28106 , QIAGEN , Valencia , CA ) or MinElute PCR purification kit ( 28006 , QIAGEN ) .",
"Immunoprecipitated and input material was analyzed by quantitative PCR .",
"ChIP signal was normalized to total input .",
"3T3-L1 cells ( 1 × 106 ) were cross-linked , and crude nuclei were isolated and sonicated as described for the ChIP assay ( Simon et al . , 2012 ) .",
"Samples were centrifuged and DNA in the supernatants was isolated by three extractions with phenol/chloroform/isoamyl alcohol ( 25:24:1 ) .",
"After the final extraction , the FAIRE samples were reverse cross-linked as described for the ChIP assay .",
"The enrichment of fragmented genomic DNA in the FAIRE samples and input material was analyzed by quantitative PCR .",
"FAIRE signal was normalized to total input .",
"We used the unpaired Student's t test to determine the statistical significance of experimental data ."
]
] | [
"Adipocyte differentiation is a strictly controlled process regulated by a series of transcriptional activators .",
"Adipogenic signals activate early adipogenic activators and facilitate the transient formation of early enhanceosomes at target genes .",
"These enhancer regions are subsequently inherited by late enhanceosomes .",
"PPARγ is one of the late adipogenic activators and is known as a master regulator of adipogenesis .",
"However , the factors that regulate PPARγ expression remain to be elucidated .",
"Here , we show that a novel ubiquitin E3 ligase , tripartite motif protein 23 ( TRIM23 ) , stabilizes PPARγ protein and mediates atypical polyubiquitin conjugation .",
"TRIM23 knockdown caused a marked decrease in PPARγ protein abundance during preadipocyte differentiation , resulting in a severe defect in late adipogenic differentiation , whereas it did not affect the formation of early enhanceosomes .",
"Our results suggest that TRIM23 plays a critical role in the switching from early to late adipogenic enhanceosomes by stabilizing PPARγ protein possibly via atypical polyubiquitin conjugation ."
] | [
"The world is facing a global epidemic of obesity , which also increases the risk for diabetes and heart disease .",
"Obesity is caused when excess fat is stored in fat cells , and overweight individuals have larger fat cells compared to healthy weight people .",
"Therefore understanding how fat cells are created in the body can provide new ways to combat obesity .",
"Fat cells , also known as adipocytes , arise from precursor cells via a process called adipogenesis .",
"This requires the activity of proteins called transcription factors that bind to DNA and switch on the expression of genes .",
"PPARγ is an important transcription factor that drives the expression of the genes that are needed to convert a precursor cell to a mature adipocyte .",
"For adipogenesis to proceed , cells have to maintain the appropriate levels of PPARγ .",
"If the amount of PPARγ bound to DNA is too low , then it is unable to activate gene expression .",
"However , the mechanisms by which cells maintain the correct levels of PPARγ activity remain poorly understood .",
"Watanabe et al . analyzed this process in mouse cells and identified a protein called TRIM23 that is produced in precursor cells .",
"Cells in which the levels of TRIM23 were artificially lowered failed to mature into fat cells; this suggests that this protein is necessary for adipogenesis .",
"Furthermore , in the absence of TRIM23 , the amount of PPARγ that occupied regions of DNA was also markedly reduced .",
"A direct consequence of this was a decline in the expression of several genes that are required for the later steps in the adipogenesis process .",
"Watanabe et al . next analyzed the mechanism through which TRIM23 had an effect on the levels of PPARγ .",
"It is known from previous work that TRIM23 belongs to a family of enzymes that attach a small molecular tag called ubiquitin onto other proteins .",
"This ubiquitin tag typically marks these proteins for rapid destruction by a large molecular machine called the proteasome .",
"Watanabe et al . found that TRIM23 also modified PPARγ with ubiquitin , but that it did so in an unusual manner that instead prevented the proteasome from recognizing PPARγ and destroying it .",
"As such , TRIM23 stabilizes the levels of PPARγ in cells .",
"By providing new insights into how adipogenesis is regulated , these findings suggest that TRIM23 may be a potential therapeutic target in the treatment of diabetes and disorders related to obesity ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"genetics and genomics"
] | Repeated losses of PRDM9-directed recombination despite the conservation of PRDM9 across vertebrates | elife-24133-v2 | [
[
"Meiotic recombination is a fundamental genetic process that generates new combinations of alleles on which natural selection can act and , in most sexually-reproducing organisms , plays critical roles in the proper alignment and segregation of homologous chromosomes during meiosis ( Coop and Przeworski , 2007; de Massy , 2013; Lam and Keeney , 2014 ) .",
"Meiotic recombination is initiated by a set of double strand breaks ( DSBs ) deliberately inflicted throughout the genome , whose repair leads to crossover and non-crossover recombination events ( Lam and Keeney , 2014 ) .",
"Most of the molecular machinery involved in this process in vertebrates has been conserved since the common ancestor of plants , animals and fungi ( de Massy , 2013 ) .",
"Notably , in all sexually reproducing species that have been examined , the SPO11 protein generates DSBs , which localize to histone H3 lysine K4 trimethylation marks ( H3K4me3 ) along the genome ( Borde et al . , 2009; Buard et al . , 2009; Lam and Keeney , 2014 ) .",
"Yet not all features of meiotic recombination are conserved across species .",
"As one example , in many species , including all yeast , plant and vertebrate species studied to date , recombination events are localized to short intervals ( of hundreds to thousands of base pairs; Lange et al . , 2016 ) known as recombination hotspots , whereas in others , such as in flies or worms , the recombination landscape seems more uniform , lacking such hotspots ( Rockman and Kruglyak , 2009; Chan et al . , 2012; Heil et al . 2015 ) Among species with recombination hotspots , there are at least two mechanisms directing their location .",
"In mammalian species , including apes , mice and likely cattle , the locations of recombination hotspots are specified by PRDM9 binding ( Baudat et al . , 2010; Myers et al . , 2010; Parvanov et al . , 2010; Sandor et al . , 2012; Great Ape Genome Project et al . , 2016 ) .",
"In these species , PRDM9 has four major functional domains: a KRAB , SSXRD and PR/SET domain ( referred to as the SET domain in what follows ) , followed by a C2H2 zinc finger ( ZF ) array ( Figure 1 ) .",
"During meiosis , PRDM9 binds sequences throughout the genome , as specified by its ZF array ( reviewed in Ségurel et al . , 2011 ) , and the SET domain of PRDM9 makes H3K4me3 and H3K36me3 marks nearby ( Eram et al . , 2014; Powers et al . , 2016 ) .",
"These actions ultimately serve to recruit SPO11 to initiate DSBs , by a mechanism that remains unknown but is associated with the presence of both histone marks ( Grey et al . , 2017; Getun et al . , 2017 ) and may involve KRAB and SSXRD domains ( Parvanov et al . , 2017 ) . 10 . 7554/eLife . 24133 . 003Figure 1 . Phylogenetic distribution and evolution of PRDM9 orthologs in vertebrates . Shown are the four domains: KRAB domain ( in tan ) , SSXRD ( in white ) , PR/SET ( in light green ) and ZF ( in gray/dark green; the approximate structure of identified ZFs is also shown ) .",
"The number of unique species included from each taxon is shown in parenthesis .",
"Complete losses are indicated on the phylogeny by red lightning bolts and partial losses by gray lightning bolts .",
"Lightning bolts are shaded dark when all species in the indicated lineage have experienced the entire loss or same partial loss .",
"Lightning bolts are shaded light when it is only true of a subset of species in the taxon .",
"ZF arrays in dark green denote those taxa in which the ZF shows evidence of rapid evolution .",
"White rectangles indicate cases where we could not determine whether the ZF was present , because of the genome assembly quality .",
"For select taxa , we present the most complete PRDM9 gene found in two examplar species .",
"Within teleost fish , we additionally show a PRDM9 paralog that likely arose before the common ancestor of this taxon; in this case , the number of species observed to have each paralog is in paranthesis .",
"Although the monotremata ZF is shaded gray , it was not included in our analysis of rapid evolution because of its small number of ZFs . DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 00310 . 7554/eLife . 24133 . 004Figure 1—figure supplement 1 . Phylogenetic approach to identifying PRDM9 orthologs and related gene families . A maximum likelihood phylogeny built with RAxML , using an alignment of SET domains , distinguishes between genes that cluster with mammalian PRDM9 and PRDM11 with 100% bootstrap support .",
"Genes shown in black , which are orthologous to both PRDM9 and PRDM11 , are only found in jawless fish . DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 00410 . 7554/eLife . 24133 . 005Figure 1—figure supplement 2 . Neighbor-joining ( NJ ) guide tree based on the SET domain . A NJ guide tree analysis on SET domains identified in our RefSeq , whole genome assembly , and transcriptome datasets was used as an initial step to identify sequences clustering with human PRDM9/7 or PRDM11 .",
"These sequences ( in red ) were selected for phylogenetic analysis with RAxML; they included all RefSeq genes in our dataset that have been previously annotated as PRDM9/7 or PRDM11 ( in yellow ) .",
"Genes more closely related to known PRDM genes other than PRDM9 or PRDM11 ( in black ) were excluded from further analysis . DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 00510 . 7554/eLife . 24133 . 006Figure 1—figure supplement 3 . Expression levels of genes with a known role in meiotic recombination in testes of three exemplar species: human , swordtail fish and bearded dragon ( a lizard ) .",
"For three swordtails ( X . malinche ) and one bearded dragon , the FPKM per individual is plotted for each transcript .",
"For humans , the point represents the average expression of 122 individuals from the gene expression atlas ( see Materials and methods ) .",
"For bearded dragons , PRDM9 and RAD50 were represented by multiple transcripts ( two and three respectively ) , and the average expression level is shown .",
"Dashed lines show the point estimate or average expression level of PRDM9 to highlight that several genes in each species have expression levels comparable to or lower than PRDM9 in testes . DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 00610 . 7554/eLife . 24133 . 007Figure 1—figure supplement 4 . Amino acid diversity as a function of amino acid position in the ZF alignment for six examplar species . Each plot shows the 95% range of diversity levels at that site for all C2H2 ZFs from a species of that taxon ( gray ) ; the values at PRDM9 are show in red or blue .",
"Turtles , snakes and coelacanth show a pattern of diversity that is similar to those in mammalian species with a complete PRDM9 ortholog , with higher diversity at DNA-binding sites ( residues 11 , 12 , 15 and",
"18 ) and reduced diversity at most other sites .",
"In bony fish , this pattern is not observed in PRDM9β genes ( blue ) or in partial PRDM9α genes ( shown for A . mexicanus ) , where PRDM9 ZF diversity is more typical of other C2H2 ZFs . DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 00710 . 7554/eLife . 24133 . 008Figure 1—figure supplement 5 . Examples of differences in computationally predicted PRDM9 binding motifs for species from three taxa . Shown are two mouse from the same species ( Mus musculus subspecies; Genbank: AB844114 . 1; FJ899852 . 1 ) , two pythons from the same species ( Python bivittatus; the genome sequence and a Sanger resequenced individual; see Materials and methods ) , and two species of swordtail fish ( X . birchmanni and X . malinche; genome sequences ) .",
"The position weight matrix was obtained using C2H2 prediction tools available at http://zf . princeton . edu . DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 008 A common feature of the recombination landscape in these species is that recombination tends to be directed away from PRDM9-independent H3K4me3 peaks ( Brick et al . , 2012 ) and , in particular , only a small proportion of hotspots occurs at transcription start sites ( TSSs; Myers et al . , 2005; Coop et al . , 2008 ) .",
"In contrast , in yeasts , plants , and vertebrate species ( such as birds and canids ) that lack functional PRDM9 orthologs , recombination events are concentrated at or near promoter-like features , including TSSs and CpG islands ( CGIs ) , perhaps because they are associated with greater chromatin accessibility ( Lichten and Goldman , 1995; Auton et al . , 2013; Choi et al . , 2013; Hellsten et al . , 2013; Lam and Keeney , 2015; Singhal et al . , 2015 ) .",
"Similarly , in mouse knockouts for PRDM9 , recombination events appear to default to promoter-like features that carry H3K4me3 peaks ( Brick et al . , 2012; Narasimhan et al . , 2016 ) .",
"The two mechanisms by which recombination events are targeted to the genome are associated with dramatic differences in the evolution of recombination hotspots .",
"When recombination is directed by PRDM9 , hotspot locations are not shared between closely related ape species or between mouse subspecies and differ even among human populations ( Ptak et al . , 2004; Myers et al . , 2005; Ptak et al . , 2005; Coop et al . , 2008; Hinch et al . , 2011; Auton et al . , 2012; Great Ape Genome Project et al . , 2016 ) .",
"This rapid evolution appears to be driven by two phenomena .",
"First , the binding specificity of the PRDM9 ZF leads to the existence of ‘hotter’ and ‘colder’ alleles , that is , sequences that are more or less likely to be bound by PRDM9 ( Myers et al . , 2008 ) .",
"In heterozygotes carrying a colder and a hotter allele , this asymmetry in binding leads to the hotter alleles more often experiencing a DSB ( Baker et al . , 2015; Davies et al . , 2016 ) .",
"Since repair mechanisms use the intact , colder allele as a template , the sequences to which PRDM9 binds are preferentially lost ( Boulton et al . , 1997; Kauppi et al . , 2005 ) .",
"This process of under-transmission of the hotter allele in hot/cold heterozygotes acts analogously to selection for the colder allele ( Nagylaki and Petes , 1982 ) and is thus expected to drive the rapid loss of hotspots from the population ( leading to the ‘hotspot paradox’; Pineda-Krch and Redfield , 2005; Coop and Myers , 2007 ) , consistent with empirical observations in humans and mice ( Berg et al . , 2010; Myers et al . , 2010; Baker et al . , 2015; Smagulova et al . , 2016 ) .",
"In addition to this loss of hotspots in cis , changes in the PRDM9 binding domain can also lead to the rapid loss—and gain—of whole sets of hotspots .",
"Interestingly , PRDM9 has the fastest evolving C2H2 ZF array in the mouse and human genomes ( Oliver et al . , 2009; Myers et al . , 2010 ) .",
"More generally , mammalian PRDM9 genes show strong evidence of positive selection at known DNA-binding sites of ZFs ( Oliver et al . , 2009 ) .",
"Thus , in mammals carrying PRDM9 , individual hotspots are lost quickly over evolutionary time , but changes in the PRDM9 ZF generate novel sets of hotspots , leading to rapid turnover in the fine-scale recombination landscape between populations and species .",
"The mechanism driving the rapid evolution of the PRDM9 ZF is unclear .",
"One hypothesis is that the under-transmission of hotter alleles eventually leads to the erosion of a sufficient number of hotspots that the proper alignment or segregation of homologs during meiosis is jeopardized , strongly favoring new ZF alleles ( Coop and Myers , 2007; Myers et al . , 2010; Ubeda and Wilkins , 2011 ) .",
"Whether hotspot loss would exert a sufficiently strong and immediate selection pressure to explain the very rapid evolution of the PRDM9 ZF remains unclear .",
"An alternative explanation has emerged recently from the finding that in mice , widespread asymmetric binding by PRDM9 on the two homologs is associated with hybrid sterility ( Davies et al . , 2016; Smagulova et al . , 2016 ) .",
"Since older PRDM9 motifs are more likely to have experienced erosion and hence to be found in heterozygotes for hotter and colder alleles , there may be an immediate advantage to new ZFs that lead to greater symmetry in PRDM9 binding ( Davies et al . , 2016 ) .",
"Regardless of the explanation , the rapid evolution of the PRDM9 ZF is likely tied to its role in recombination .",
"Conversely , in species that do not use PRDM9 to direct meiotic recombination events , the rapid evolution of recombination hotspots is not seen .",
"In birds that lack an ortholog of PRDM9 , the locations of recombination hotspots are conserved over long evolutionary time scales .",
"Similarly , both the location and heats of recombination hotspots are conserved across highly diverged yeast species , in which H3K4me3 marks are made by a single gene without a DNA binding domain ( Lam and Keeney , 2015 ) .",
"In these taxa , it remains unknown whether the coincidence of recombination with functional genomic elements , such as TSSs and CGIs , is facilitated by specific binding motifs or simply by greater accessibility of the recombination machinery to these genomic regions ( Brick et al . , 2012; Auton et al . , 2013; Choi et al . , 2013; Lam and Keeney , 2015; Singhal et al . , 2015 ) .",
"Even if there are specific motifs that increase rates of recombination near functional genomic elements , they are likely to have important , pleiotropic consequences ( Nicolas et al . , 1989 ) .",
"Thus , there may be a strong countervailing force to the loss of hotspots by under-transmission of hotter alleles in these cases , leading to the evolutionary stability of hotspots .",
"These observations sketch the outline of a general pattern , whereby species that do not use PRDM9 to direct recombination target promoter-like features and have stable fine-scale recombination landscapes , whereas those that employ PRDM9 tend to recombine away from promoters and experience rapid turnover of hotspot locations .",
"This dramatic difference in the localization of hotspots and their evolutionary dynamics has important evolutionary consequences for genome structure and base composition , for linkage disequilibrium ( LD ) levels along the genome , as well as for introgression patterns in naturally occurring hybrids ( Fullerton et al . , 2001; McVean et al . , 2004; Duret and Galtier , 2009; Janoušek et al . , 2015 ) .",
"It is therefore important to establish the generality of these two mechanisms and characterize their distribution across species .",
"To date , studies of fine-scale recombination rates are limited to a handful of organisms .",
"In particular , although it has been previously reported that the PRDM9 gene arose early in metazoan evolution ( Oliver et al . , 2009 ) , direct evidence of its role in recombination is limited to placental mammals ( mice , primates and more circumstantially cattle ) .",
"It remains unknown which species carry an intact ortholog and , more broadly , when PRDM9-directed recombination is likely to have arisen .",
"To address these questions , we investigated the PRDM9 status of 225 species of vertebrates , using a combination of genome sequences and RNAseq data ."
],
[
"In order to identify which species have PRDM9 orthologs , we searched publically available nucleotide and whole genome sequences to create a curated dataset of vertebrate PRDM9 sequences .",
"To this end , we implemented a blastp-based approach against the RefSeq database , using human PRDM9 as a query sequence ( see Materials and methods for details ) .",
"We supplemented this dataset with 44 genes strategically identified from 30 whole genome assemblies and seven genes identified from de novo assembled transcriptomes from testis of five species lacking genome assemblies ( see Materials and methods for details ) .",
"Neighbor joining ( NJ ) and maximum likelihood trees were built using identified SET domains to distinguish bona fide PRDM9 orthologs from members of paralagous gene families and to characterize the distribution of PRDM9 duplication events ( Figure 1—figure supplement 1 and 2 ) .",
"Since the placement of the major taxa used in our analysis is not controversial , in tracing the evolution of PRDM9 orthologs , we assumed that the true phylogenetic relationships between taxa are those reported by several recent papers ( synthesized by the TimeTree project; Hedges et al . , 2015 ) .",
"This approach identified 227 PRDM9 orthologs ( Supplementary file 1A , B ) , found in jawless fish , cartilaginous fish , bony fish , coelacanths , turtles , snakes , lizards , and mammals .",
"We confirmed the absence of PRDM9 in all sampled birds and crocodiles ( Oliver et al . , 2009; Singhal et al . , 2015 ) , the absence of non-pseudogene copies in canids ( Oliver et al . , 2009; Muñoz-Fuentes et al . , 2011 ) , and additionally were unable to identify PRDM9 genes in amphibians ( Figure 1 ) , despite targeted searches of whole genome sequences ( Supplementary file 1B ) .",
"We further inferred an ancient duplication of PRDM9 in the common ancestor of teleost fish , apparently coincident with the whole genome duplication that occurred in this group ( Figure 1 , Figure 2 ) .",
"We used both phylogenetic methods and analysis of the ZF structure to distinguish these copies ( see Figure 2—figure supplement 1 , Materials and methods ) and refer to them as PRDM9α and PRDM9β in what follows .",
"While PRDM9β orthologs were identified in each species of teleost fish examined , we were unable to identify PRDM9α type orthologs within three major teleost taxa , suggesting at minimum three losses of PRDM9α type orthologs within teleost fish ( Figure 2 , Supplementary file 1A ) .",
"Several additional duplication events appear to have occurred more recently in other vertebrate groups , including in jawless fish , cartilaginous fish , bony fish , and mammals ( Supplementary file 1A ) . 10 . 7554/eLife . 24133 . 009Figure 2 . Phylogenetic distribution and functional domains of PRDM9α orthologs in teleost fish and in holostean fish that are outgroups to the PRDM9α/PRDM9β duplication event . Shown are the four domains: KRAB domain ( in tan ) , SSXRD ( in white ) , PR/SET ( in light green ) and ZF ( in gray/dark green; the approximate structure of identified ZFs is also shown ) .",
"The number of unique species included from each taxon is shown in parenthesis .",
"Complete losses are indicated on the phylogeny by red lightning bolts and partial losses by gray lightning bolts .",
"Lightning bolts are shaded dark when all species in the indicated lineage have experienced the loss .",
"Lightning bolts are shaded light when it is only true of a subset of species in the taxon .",
"ZF arrays in dark green denote those taxa in which the ZF shows evidence of rapid evolution .",
"White rectangles indicate cases where we could not determine whether the ZF was present , because of the genome assembly quality .",
"While many taxa shown have more than one PRDM9α ortholog , the genes identified from each species generally have similar domain architectures .",
"Exceptions include Clupeiformes , Esociformes , and Holostean fish , for which two alternative forms of PRDM9α paralogs are shown .",
"Based on this distribution , we infer that the common ancestor of ray-finned fish likely had a rapidly evolving and complete PRDM9α ortholog . DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 00910 . 7554/eLife . 24133 . 010Figure 2—figure supplement 1 . Section of maximum-likelihood phylogeny of the SET domain showing bony fish PRDM9 orthologs α and β .",
"The reciprocal monophyly of PRDM9 orthologs α and β is reasonably well supported and in particular bootstrap support for the monophyly of PRDM9α genes is 75% .",
"The ZF domains for representative PRDM9 orthologs of each type are shown to the right , with each gray pentagon indicating the location of a ZF .",
"In swordtail fish , the complete ZF array is found within a single exon , and the last tandem array of six ZFs forms a minisatellite structure .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 01010 . 7554/eLife . 24133 . 011Figure 2—figure supplement 2 . Analysis of ZF evolution in PRDM9β .",
"Red lines show the median ( solid ) and first and third quantiles ( dashed lines ) for all 48 complete PRDM9 orthologs identified in vertebrates that have four or more ZFs .",
"Blue lines show the median ( solid ) and first and third quantiles ( dashed lines ) for all other C2H2 ZF genes from X . maculatus ( 157 genes ) .",
"Results about the rate of ZF evolution in the PRDM9β gene from X . maculatus are qualitatively similar regardless of our choice of which cluster of individual ZF domains to include in our analysis , indicating that our ability to detect evidence of positive selection at DNA-binding residues in these arrays , or lack thereof , is unlikely to be influenced by this choice . DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 011 Since a necessary condition for PRDM9 to play a role in meiotic recombination is for it to be expressed in the germline , we looked for PRDM9 in expression data from testis tissues in order to confirm its presence .",
"We focused on testis expression rather than ovaries because although both obviously contain germline cells , preliminary analyses suggested that meiotic gene expression is more reliably detected in testes ( see Materials and methods ) .",
"We selected 23 representative species , spanning each major vertebrate group , with publically available testis expression or testis RNA-seq ( Supplementary file 2A ) ; we also generated testis RNA-seq data for two species of bony fish ( see Materials and methods ) .",
"In teleost fish with both PRDM9α and PRDM9β genes , we were able to detect either the expression of both orthologs or only expression of PRDM9α orthologs .",
"In species of teleost fish with only PRDM9β genes , we consistently identified expression of PRDM9β genes .",
"More generally , we were able to identify PRDM9 expression in nearly all RNA-seq datasets from species in which the genome carried a putative ortholog , the elephant shark ( Callorhinchus milii ) being the sole exception ( Supplementary file 2B , C ) .",
"Concerned that absences of PRDM9 observed in some species could reflect lower quality genome assemblies rather than true loss events , we also used testis RNAseq data to investigate putative losses of PRDM9 in amphibians and fish ( PRDM9α ) .",
"To this end , we relied on the fact that when PRDM9 is present , it is detectable in RNAseq data from the whole testis of vertebrates ( see above ) .",
"Our approach was to analyze testis transcriptome data from species lacking PRDM9 sequences in their genome assemblies , using an analysis that is not biased by the genome assembly ( see Materials and methods ) .",
"For each species , we confirmed that the dataset captured the appropriate cell populations and provided sufficient power to detect transcripts that are expressed during meiosis at levels comparable to PRDM9 in mammals ( Figure 1—figure supplement 3 , Supplementary file 2B , D ) .",
"With this approach , we were able to find support for the loss of PRDM9 in salamanders ( Cynops pyrrhogaster , Ambystoma mexicanum ) and frogs ( Xenopus tropicalis ) .",
"Because of the paucity of amphibian genomes , however , it is not clear whether or not these examples represent a widespread loss of PRDM9 within amphibians or more recent , independent losses .",
"Within bony fish , we were able to confirm the three independent losses of PRDM9α type orthologs in one species each of percomorph ( Xiphophorus birchmanni ) , cypriniform ( Danio rerio ) and osteoglossomorph fish ( Osteoglossum bicirrhosum ) .",
"Thus , in all cases with sufficient power to detect expression of PRDM9 in testes data , our findings were consistent with inferences based on genome sequence data .",
"PRDM9 orthologs identified in jawless fish , some bony fish , coelacanths , lizards , snakes , turtles , and placental mammals have a complete domain structure , consisting of KRAB , SSXRD and SET domains , as well as a C2H2 ZF array .",
"The phylogenetic relationships between these species suggest that a complete PRDM9 ortholog was present in the common ancestor of vertebrates ( Figure 1 ) .",
"Despite its widespread taxonomic distribution , however , the complete domain structure was not found in several of the 149 sampled lineages with PRDM9 orthologs ( Figure 1; in addition to the complete losses of the gene described above ) .",
"Instances include the absence of the SSXRD domain in some cartilaginous fish ( see Materials and methods ) , absence of both KRAB and SSXRD domains in PRDM9β orthologs ( Figure 1 ) and in PRDM9α orthologs found distributed throughout the teleost fish phylogeny ( Figure 2 , Figure 2—figure supplement 1 ) , and the absence of the KRAB domain in monotremata ( Ornithorhynchus anatinus ) and marsupial mammals ( Sarcophilus harrisii , Figure 1; Supplementary file 1A ) .",
"Because these frequent N-terminal losses could be the result of assembly or gene prediction errors , we sought to confirm them by systematically searching genomes and transcriptomes for evidence of these missing domains ( see Materials and methods ) .",
"We required not only that missing domains homologous to PRDM9 be absent from the genome in a whole genome search , but also that the missing domain not be present in the transcriptome , when other domains of PRDM9 were .",
"This approach necessarily limits our ability to verify putative losses when there are no suitable transcriptome data , but nonetheless allowed us to confirm the losses of the KRAB and SSXRD domains in a PRDM9 ortholog from holostean fish ( Lepisosteus oculatus ) , in all PRDM9β orthologs from teleost fish ( Figure 1 ) , in PRDM9α orthologs that lost their complete domain structure in several taxa of teleost fish ( Gadus morhua , Astyanax mexicanus , Ictalurus punctatus , Esox lucius; Supplementary file 2C ) , as well as losses of the KRAB domain in two PRDM9 orthologs identified in monotremata ( both in O . anatinus , Supplementary file 2C ) , indicating a minimum of six N-terminal domain losses within vertebrates .",
"For representative cases where we were able to confirm missing N-terminal domains , we further investigated whether the truncated genes had become pseudogenes by testing whether the ratio of nonsynonymous to synonymous substitutions in the SET domain is significantly different than 1 ( see Materials and methods ) .",
"In all cases of N-terminal truncation , the partial PRDM9 shows evidence of functional constraint ( i . e . , dN/dS <1 , where dN is the rate of amino-acid substitutions and dS of synonymous substitutions; see Materials and methods for more details ) .",
"This conservation is most strikingly seen in teleost fish , in which a partial PRDM9 ortholog has been evolving under constraint for hundreds of millions of years ( Figure 1 , Figure 2—figure supplement 1 , Supplementary file 3A ) .",
"These observations suggest that in these species , PRDM9 has an important function that it performs without KRAB or SSXRD domains .",
"Moreover , these cases provide complementary observations to full PRDM9 knockouts in amphibians and archosaurs , allowing the roles of specific domains to be dissected .",
"Rapid evolution of the PRDM9 ZF array has been reported previously in all species with evidence for PRDM9-directed recombination , including cattle , apes and mice .",
"While it is not known whether this rapid evolution is a necessary consequence of its role in recombination , plausible models suggest it is likely to be ( see Introduction ) .",
"If so , we expect species with PRDM9-directed recombination to show evidence for rapidly-evolving PRDM9 ZF arrays and can use this feature to hone in on the subset of PRDM9 orthologs most likely to play a role in recombination .",
"To this end , we characterized the rapid evolution of the PRDM9 ZF in terms of the proportion of amino acid diversity within the ZF array that occurs at DNA-binding sites ( using a modification of the approach proposed by Oliver et al . , 2009 ) .",
"This summary statistic is sensitive to both rapid amino acid evolution at DNA binding sites and concerted evolution between the individual ZFs ( see Materials and methods ) .",
"Using this statistic , placental mammals that have PRDM9-directed recombination show exceptionally high rates of evolution of the PRDM9 ZF compared to other ZFs ( Table 1; Baudat et al . , 2010; Myers et al . , 2010; Parvanov et al . , 2010 ) .",
"Moreover , two of six cattle PRDM9 orthologs that we identified were previously associated with interspecific variation in recombination phenotypes ( Supplementary file 3B; Sandor et al . , 2012; Ma et al . , 2015 ) , and both are seen to be rapidly evolving ( Table 1 , Supplementary file 3B ) . 10 . 7554/eLife . 24133 . 012Table 1 . Evolution of the ZF in PRDM9 orthologs with different domain architectures .",
"PRDM9 orthologs for which an empirical comparison dataset is available are ordered by their domain structures: from the top , we present cases of complete PRDM9 orthologs with KRAB-SSXRD-SET domains; partial orthologs putatively lacking KRAB or SSXRD domains or partial orthologs lacking both; then those containing only the SET domain .",
"A row is shaded green if the ZF is in the top 5% most rapidly evolving C2H2 ZF in the species , as summarized by the proportion of amino-acid diversity at DNA-binding sites , and is blue if it is ranked first .",
"A complete PRDM9 ortholog from dolphins ( Balaenoptera acuforostrata scammoni ) is shaded in gray because there is no amino acid diversity between ZFs of the tandem array .",
"The empirical rank is also shown , as are the number of PRDM9 orthologs identified in the species .",
"Asterisks indicate PRDM9 orthologs known to play a role in directing recombination .",
"For PRDM9 genes from teleost fish , under major group , we additionally indicate whether or not the gene is a PRDM9α or PRDM9β gene . DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 012OrganismMajor groupPRDM9 structureProportion AA diversity at DNA-binding sitesRankNumber of PRDM9 genes from speciesNumber of ZF genes evaluated from speciesBalaenoptera acutorostrata scammoniplacentalKRAB-SSXRD-SETNANA1272Bison bison bisonplacentalKRAB-SSXRD-SET0 . 66711285Bos taurus* ( chr1 ) placentalKRAB-SSXRD-SET0 . 68413313Bos taurus ( chrX ) placentalKRAB-SSXRD-SET0 . 41463313Bos taurus* ( chrX ) placentalKRAB-SSXRD-SET0 . 41473313Bubalus bubalisplacentalKRAB-SSXRD-SET0 . 66711268Chelonia mydasturtleKRAB-SSXRD-SET0 . 414111235Chlorocebus sabaeusplacentalKRAB-SSXRD-SET0 . 50011344Chrysemys picta belliiturtleKRAB-SSXRD-SET0 . 47811308Cricetulus griseusplacentalKRAB-SSXRD-SET0 . 78131259Dasypus novemcinctusplacentalKRAB-SSXRD-SET0 . 61411289Dipodomys ordiiplacentalKRAB-SSXRD-SET0 . 56711194Esox luciusteleost fish ( α ) KRAB-SSXRD-SET0 . 45514234Fukomys damarensisplacentalKRAB-SSXRD-SET0 . 43031227Homo sapiens*placentalKRAB-SSXRD-SET0 . 68711357Latimeria chalumnaecoelacanthKRAB-SSXRD-SET0 . 54521227Loxodonta africanaplacentalKRAB-SSXRD-SET0 . 61711381Macaca fascicularisplacentalKRAB-SSXRD-SET0 . 68011364Macaca mulattaplacentalKRAB-SSXRD-SET0 . 64511366Marmota marmota marmotaplacentalKRAB-SSXRD-SET0 . 48311277Microcebus murinusplacentalKRAB-SSXRD-SET1 . 00011326Mus musculus*placentalKRAB-SSXRD-SET0 . 91011224Nannospalax galiliplacentalKRAB-SSXRD-SET1 . 00011307Octodon degusplacentalKRAB-SSXRD-SET0 . 33353227Octodon degusplacentalKRAB-SSXRD-SET0 . 33163227Ovis ariesplacentalKRAB-SSXRD-SET0 . 61512252Ovis ariesplacentalKRAB-SSXRD-SET0 . 39842252Ovis aries musimonplacentalKRAB-SSXRD-SET0 . 353121285Papio anubisplacentalKRAB-SSXRD-SET0 . 58511404Pelodiscus sinensisturtleKRAB-SSXRD-SET0 . 69211221Peromyscus maniculatus bairdiiplacentalKRAB-SSXRD-SET1 . 00011243Protobothrops mucrosquamatussquamataKRAB-SSXRD-SET0 . 46251195Python bivittatussquamataKRAB-SSXRD-SET0 . 57111206Rattus norvegicusplacentalKRAB-SSXRD-SET0 . 57011255Rousettus aegyptiacusplacentalKRAB-SSXRD-SET0 . 74211258Salmo salarteleost fish ( α ) KRAB-SSXRD-SET0 . 53894510Salmo salarteleost fish ( α ) KRAB-SSXRD-SET0 . 500114510Sus scrofaplacentalKRAB-SSXRD-SET0 . 54211248Thamnophis sirtalissquamataKRAB-SSXRD-SET0 . 45931179Tupaia chinensisplacentalKRAB-SSXRD-SET1 . 00011249Tursiops truncatusplacentalKRAB-SSXRD-SET0 . 93911233Myotis lucifugusplacentalSSXRD-SET0 . 52412308Myotis lucifugusplacentalSSXRD-SET0 . 310682308Octodon degusplacentalSSXRD-SET0 . 282463227Sarcophilus harrisiimarsupialSSXRD-SET0 . 2242772344Callorhinchus milliicartilaginous fishKRAB-SET0 . 3146163Astyanax mexicanusteleost fish ( α ) SET0 . 258602158Astyanax mexicanusteleost fish ( β ) SET0 . 1671522158Clupea harengusteleost fish ( α ) SET0 . 27964118Clupea harengusteleost fish ( α ) SET0 . 27874118Clupea harengusteleost fish ( α ) SET0 . 274104118Clupea harengusteleost fish ( β ) SET0 . 1581144118Cynoglossus semilaevisteleost fish ( β ) SET0 . 182801107Danio rerioteleost fish ( β ) SET0 . 1793451367Esox luciusteleost fish ( α ) SET0 . 295324234Esox luciusteleost fish ( β ) SET0 . 1921764234Esox luciusteleost fish ( β ) SET0 . 1921774234Fundulus heteroclitusteleost fish ( β ) SET0 . 1891581206Haplochromis burtoniteleost fish ( β ) SET0 . 1801481168Ictalurus punctatusteleost fish ( α ) SET0 . 320148140Ictalurus punctatusteleost fish ( α ) SET0 . 319158140Ictalurus punctatusteleost fish ( α ) SET0 . 306248140Ictalurus punctatusteleost fish ( α ) SET0 . 303258140Ictalurus punctatusteleost fish ( α ) SET0 . 286338140Ictalurus punctatusteleost fish ( α ) SET0 . 276398140Ictalurus punctatusteleost fish ( α ) SET0 . 253558140Ictalurus punctatusteleost fish ( β ) SET0 . 1791278140Larimichthys croceateleost fish ( β ) SET0 . 192701115Lepisosteus oculatusholostei fishSET0 . 223481106Maylandia zebrateleost fish ( β ) SET0 . 1731611176Neolamprologus bricharditeleost fish ( β ) SET0 . 1731411152Nothobranchius furzeriteleost fish ( β ) SET0 . 1802451266Notothenia coriicepsteleost fish ( β ) SET0 . 16783187Oreochromis niloticusteleost fish ( β ) SET0 . 1731731190Oryzias latipesteleost fish ( β ) SET0 . 2131041191Otolemur garnettiiplacentalSET0 . 2661211285Poecilia formosateleost fish ( β ) SET0 . 1911841242Poecilia latipinnateleost fish ( β ) SET0 . 1911751235Poecilia mexicanateleost fish ( β ) SET0 . 1911871244Poecilia reticulatateleost fish ( β ) SET0 . 1911621212Pundamilia nyerereiteleost fish ( β ) SET0 . 1731341147Pygocentrus nattereriteleost fish ( α ) SET0 . 331122142Pygocentrus nattereriteleost fish ( β ) SET0 . 1791242142Salmo salarteleost fish ( β ) SET0 . 1884114510Salmo salarteleost fish ( β ) SET0 . 1804544510Sinocyclocheilus anshuiensisteleost fish ( β ) SET0 . 1852242284Sinocyclocheilus anshuiensisteleost fish ( β ) SET0 . 1852252284Sinocyclocheilus grahamiteleost fish ( β ) SET0 . 1852111271Sinocyclocheilus rhinocerousteleost fish ( β ) SET0 . 1852082269Sinocyclocheilus rhinocerousteleost fish ( β ) SET0 . 1852092269Takifugu rubripesteleost fish ( β ) SET0 . 18866198Xiphophorus maculatusteleost fish ( β ) SET0 . 1911171158 In addition to placental mammals , PRDM9 orthologs in jawless fish , some bony fish ( Salmoniformes , Esociformes , Elopomorpha ) , turtles , snakes , lizards , and coelacanths show similarly elevated values of this statistic ( Figure 1—figure supplement 4 ) .",
"In fact , PRDM9 is the most rapidly evolving ZF gene genome-wide in most species in these taxa and all PRDM9 orthologs with the complete domain structure were in the top 5% of the most rapidly evolving ZFs in their respective genomes ( Table 1 , Supplementary file 3B ) .",
"In contrast , evidence of such rapid evolution is absent from other taxa of bony fish , including all PRDM9β orthologs and partial PRDM9α orthologs , as well as from the putatively partial PRDM9 orthologs found in the elephant shark , the Tasmanian devil , and in several species of placental mammals ( see Materials and methods for details ) .",
"We only observed one instance ( little brown bat , Myotis lucifugus ) in which a partial PRDM9 ortholog was evolving unusually rapidly ( Table 1 ) ; in this case , we were unable to confirm the loss of the missing KRAB domain ( see Materials and methods ) , so it remains possible this ortholog is in fact intact .",
"In summary , with one possible exception , species show evidence of rapid evolution of the ZF binding affinity if and only if they carry the intact PRDM9 ortholog found in placental mammals .",
"This concordance of rapid evolution with the complete domain structure is highly unlikely by chance ( taking into account the phylogenetic relationship between orthologs , p<10−6; see Materials and methods ) .",
"Assuming that rapid evolution of the ZF is indicative of PRDM9-directed recombination , these observations carry two implications: KRAB and SSXRD domains are required for this role and non-mammalian species such as turtles or snakes also use PRDM9 to direct recombination .",
"While partial orthologs of PRDM9 have lost one or both of their N-terminal domains , they retain the SET and ZF domains known to play a role in recombination , are under purifying selection , and are expressed in testis .",
"In principle then , these partial orthologs could still play a role in directing recombination .",
"To evaluate this possibility , we started by examining whether the catalytic activities of the SET domains of partial PRDM9 orthologs are conserved .",
"We did so because the catalytic specificities of PRDM9 are believed to be important to its role in directing recombination: two marks made by the SET domain of PRDM9 , H3K4me3 and H3K36me3 , are associated with hotspot activity in mammals ( Powers et al . , 2016; Grey et al . , 2017; Yamada et al . , 2017; Getun et al . , 2017 ) and the human PRDM9 is unusual in being able to add methyl groups to different lysine residues of the same nucleosomes , when most other methyltransferase genes are responsible for only a single mark ( Eram et al . , 2014; Powers et al . , 2016 ) .",
"Specifically , we focused on three tyrosine residues shown to be important for the catalytic specificities of the human PRDM9 gene ( Y276 , Y341 and Y357; see Materials and methods and Supplementary file 1A; Wu et al . , 2013 ) and asked if those residues were conserved across vertebrates .",
"Loss of individual residues is not necessarily evidence for loss of catalytic activity , as compensatory changes may have occurred .",
"For example , a substitution at Y357 of PRDM7 has led to the loss of H3K36me3 specificity , but H3K4me3 activity appears to have been retained through compensatory substitutions ( Blazer et al . , 2016 ) .",
"Nonetheless , PRDM9 orthologs with substitutions at these residues are unlikely to utilize the same catalytic mechanisms as human PRDM9 for any methyltransferase activity that they retain .",
"We found that each of the three residues is broadly conserved across the vertebrate phylogeny , with substitutions observed in only 57 of 227 PRDM9 orthologs , including 11 genes from placental mammals and 46 genes from bony fish .",
"Strikingly , however , none of these substitutions occur in a complete PRDM9 ortholog containing KRAB , SSXRD , SET and ZF domains .",
"Within mammals , the majority of PRDM9 orthologs that experienced these substitutions lack the ZF array entirely , including eight PRDM7 genes from primates , which share a substitution at Y357 , and one PRDM9 ortholog from a bat ( Miniopterus natalensis ) that carries a substitution at Y276 .",
"Others are lacking the KRAB domain , including PRDM9 orthologs identified from a lemur ( Galeopterus variegatus ) and a rodent ( Octodon degus ) , which carry substitutions at Y276 and Y357 , respectively .",
"Within bony fish , we identified 46 PRDM9 orthologs with substitutions at one or more of these residues , including the partial PRDM9 ortholog from holosteans ( see above ) and all PRDM9β orthologs in teleosts ( Supplementary file 1A ) .",
"The distribution of substitutions at these residues within PRDM9β genes suggests that numerous independent substitution events have occurred in this gene family following the loss of KRAB and SSXRD domains ( Figure 3 ) .",
"In contrast , no substitutions were observed at these residues in any PRDM9α ortholog , regardless of their domain architecture .",
"These observations could be consistent with a lack of constraint on the ancestral methyltransferase activities of PRDM9 in PRDM9β genes after the PRDM9α/PRDM9β duplication event ( or conceivably an indication that there has been convergent evolution towards a new functional role ) .",
"Thus , PRDM9β genes not only lack KRAB and SSXRD domains , they likely lack some methyltransferase activity of the SET domain . 10 . 7554/eLife . 24133 . 013Figure 3 . Substitutions at SET domain catalytic residues in bony fish PRDM9 genes .",
"( a ) Lineages within bony fish carrying substitutions at each of three tyrosine residues involved in H3K4me3 catalysis in human PRDM9 are shown in blue , yellow and red .",
"( b ) Lineages carrying substitutions at one , two or three of these residues are shown in red , pink and blue respectively .",
"All PRDM9β genes as well as a partial PRDM9 ortholog from holostean fish carry one or more substitutions at these residues .",
"The PRDM9β gene from Xiphophorus is indicated by the presence of asterisk . DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 013 To more directly test the hypothesis that the partial ortholog of PRDM9 does not direct recombination , we examined patterns of crossing-over in naturally-occurring swordtail fish hybrids ( X . birchmanni x X . malinche; see Materials and methods ) .",
"Like other percomorphs , swordtail fish have testis-specific expression of a PRDM9β type gene that lacks the KRAB and SSXRD domains , and has a slowly evolving ZF array ( Figure 4; Figure 4—figure supplement 1 ) ; they further carry substitutions at two catalytic residues of the SET domain ( Y341F and Y357P ) , as well as at residues of the SET domain implicated in H3K4me2 recognition ( see Materials and methods ) .",
"Based on these features , we predicted that they should behave like a PRDM9 knockout , with no increase in recombination around the PRDM9 motif . 10 . 7554/eLife . 24133 . 014Figure 4 . Patterns of recombination and PRDM9 evolution in swordtail fish .",
"( a ) The ZF array of PRDM9 appears to be evolving slowly in Xiphophorus , with few changes over 1 million years of divergence ( Cui et al . , 2013; Jones et al . , 2013 ) .",
"( b ) PRDM9 is upregulated in the germline relative to the liver in X . birchmanni ( circles ) and X . malinche ( squares; panel shows three biological replicates for each species ) .",
"( c ) The computationally-predicted PRDM9 binding sites is not unusually associated with H3K4me3 peaks in testes",
"( d ) Crossover rates increase near H3K4me3 peaks in testis",
"( e ) Crossover rates increase near CGIs",
"( f ) Crossover rates do not increase near computationally-predicted PRDM9 binding sites ( see Figure 4—figure supplement 3 for comparison ) .",
"Crossover rates were estimated from ancestry switchpoints in naturally occurring hybrids between X . birchmanni and X . malinche ( see Materials and methods ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 01410 . 7554/eLife . 24133 . 015Figure 4—figure supplement 1 . Expression levels of meiosis-related genes in swordtail fish tissues . In general , the seven meiosis-related genes surveyed had higher expression in tissues containing germline cells than liver tissue , but this pattern was much more pronounced in testis tissue ( compared to ovary tissue ) .",
"As a result , we focused our analysis of meiosis related genes on RNAseq data generated from testis .",
"Results shown are based on analysis of three male and female biological replicates from each swordtail species ( X . birchmanni and X . malinche ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 01510 . 7554/eLife . 24133 . 016Figure 4—figure supplement 2 . Recombination frequency in swordtails as a function of distance to the TSS . Partial correlation analyses suggest that the association between the TSS and recombination rate in swordtails is explained by H3K4me3 peaks and CGIs . DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 01610 . 7554/eLife . 24133 . 017Figure 4—figure supplement 3 . Recombination rates show a peak near the computationally predicted PRDM9A binding motif in humans and gor-1 allele in gorillas . Most work investigating relationships between PRDM9 motifs and recombination rates have focused on the PRDM9 motif empirically inferred from recombination hotspots , but the empirical motif is unknown for many species .",
"To generate results comparable to those we present for swordtails in Figure 4F , we therefore determined recombination rate ( using the map based on LD patterns in the CEU; Frazer et al . , 2007 ) as a function of distance to computationally predicted binding sites for the PRDM9A motif in humans and as a function of distance to computationally predicted binding sites for the gor-1 PRDM9 allele ( Schwartz et al . , 2014 ) in gorillas ( using the LD-based map from Great Ape Genome Project et al . , 2016 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 01710 . 7554/eLife . 24133 . 018Figure 4—figure supplement 4 . Higher observed recombination rate at testis-specific H3K4me3 peaks than liver-specific H3K4me3 peaks . H3K4me3 peaks found only in the testis and not in the liver of X . birchmanni have higher observed recombination rates in X . birchmanni – X . malinche hybrids .",
"This pattern supports the conclusion that H3K4me3 peaks are associated with recombination in swordtails . DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 01810 . 7554/eLife . 24133 . 019Figure 4—figure supplement 5 . MEME prediction of sequences enriched in testis-H3K4me3 peaks relative to liver-specific H3K4me3 peaks . Results shown in A-E are from five replicate runs of 2000 testis-specific sequences using liver-specific sequences as a background comparison set .",
"The swordtail computationally-predicted PRDM9 binding motif is shown for comparison . DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 019 To test these predictions , we collected ~1X genome coverage from 268 natural hybrids and inferred crossover events from ancestry switchpoints between the two parental species using a hidden Markov model ( see Materials and methods ) .",
"By this approach , we found recombination rates to be elevated near TSSs and CGIs , two promoter-like features ( Figure 4; Figure 4—figure supplement 2 ) .",
"Moreover , and in contrast to what is observed in species with PRDM9-mediated recombination ( Figure 4—figure supplement 3 ) , there is no elevation in recombination rates near computationally-predicted PRDM9 binding sites ( Figure 4F ) .",
"These patterns resemble those previously reported for birds lacking PRDM9 ( Singhal et al . , 2015 ) .",
"In addition , we performed native chromatin Chip-seq with an H3K4me3 antibody in X . birchmanni testis and liver tissue .",
"Consistent with a role for H3K4me3 in inducing DSBs , recombination is increased around H3K4me3 peaks ( testing the association with distance , rho = −0 . 072 , p=2 . 3e-69; Figure 4 ) , an effect that remains significant after correcting for distance to TSSs and CGIs ( rho = −0 . 026 , p=5 . 4e-10 ) .",
"In fact , the increase in recombination rate near the TSS is almost completely explained by the joint effects of proximity to H3K4me3 peaks and CGI ( TSS with both: rho = −0 . 009 , p=0 . 02 ) .",
"Windows that contain testis-specific H3K4me3 peaks have significantly higher recombination rates than those that contain liver-specific H3K4me3 peaks ( Figure 4; Figure 4—figure supplement 4 ) .",
"However , H3K4me3 peaks in the testis are not enriched for the computationally predicted PRDM9 motifs ( Figure 4 ) , nor do they overlap with PRDM9 motifs in the testis more than the liver ( see Methods ) .",
"Conversely , sequence motifs associated with testis-specific H3K4me3 peaks do not resemble the predicted PRDM9 motif ( Figure 4; Figure 4—figure supplement 5 ) .",
"Thus , there is no evidence that PRDM9 lays down the H3K4me3 marks associated with an increase in recombination in swordtails .",
"To put the genomic patterns of recombination in swordtail fish in an explicit comparative framework , we re-analyzed patterns of recombination near TSSs and CGIs in previously published genetic maps based on LD data from three species without functional PRDM9 genes ( dog , zebra finch and long-tailed finch ) and three species known to use PRDM9-mediated recombination ( human , gorilla and mouse ) , as well as using a pedigree-based genetic map for one species with a complete PRDM9 ortholog , but for which no direct evidence of PRDM9’s role in recombination has yet been reported ( sheep; see Materials and methods for details and references ) .",
"Among species with complete PRDM9 genes , recombination rates are either weakly reduced near TSSs and CGIs or similar to what is seen in nearby windows ( Figure 5; see Figure 5—figure supplement 1 for results with genetic maps based on pedigrees or admixture switches instead of LD data in humans and dogs ) .",
"In contrast , in all species lacking PRDM9 and in swordtail fish , the recombination rate is notably increased in windows overlapping either a TSS or CGI relative to nearby windows .",
"Quantitative comparisons are difficult because of the varying resolution of the different genetic maps .",
"Nonetheless , these results indicate that patterns of recombination near TSSs and CGIs differ between species carrying complete PRDM9 orthologs and species lacking PRDM9 altogether , and that swordtail fish exhibit patterns of recombination similar to species that completely lack PRDM9 , despite the presence of a partial ortholog . 10 . 7554/eLife . 24133 . 020Figure 5 . Patterns of recombination near TSSs and CGIs in species with and without complete PRDM9 orthologs . For each species , recombination rates were binned in 10 kb windows along the genome; curves were fit using gaussian loess smoothing .",
"The fold change in recombination rates shown on the y-axis is relative to recombination rates at the last point shown .",
"Species shown in the top row have complete PRDM9 orthologs ( mouse , human , gorilla and sheep ) , whereas species in the bottom row have no PRDM9 ortholog ( dog , zebra finch , long-tailed finch ) , or a partial PRDM9 ortholog ( swordtail fish ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 02010 . 7554/eLife . 24133 . 021Figure 5—figure supplement 1 . Dependence of patterns of recombination near TSSs and CGIs in dog and human on the type of genetic map .",
"( a ) Recombination rates near the TSS and CGI in dogs are shown using recombination maps inferred either from LD patterns or pedigree data .",
"The magnitude of the peak near these features is lower in the map with lower resolution .",
"This observation raises the possibility that a higher resolution map in swordtail fish would result in a higher peak near these features .",
"( b ) Recombination rates near the TSS and CGI in humans are shown using recombination maps inferred either from LD patterns or ancestry switches in African-American samples .",
"Recombination rates near the TSS and CGI in human do not seem to be strongly influenced by the choice of genetic map , though peaks at these features are slightly reduced in admixture- and pedigree-based methods . DOI: http://dx . doi . org/10 . 7554/eLife . 24133 . 021"
],
[
"Based on our reconstruction of 227 PRDM9 orthologs across the vertebrate phylogeny , we inferred that the ancestral domain architecture of PRDM9 consisted of KRAB , SSXRD and SET domains followed by a C2H2 ZF array , and that this complete architecture was likely already in place in the common ancestor of vertebrates .",
"Moreover , even though to date only the functions of the SET domain and C2H2 ZF array have been connected to the role of PRDM9 in directing recombination , the evolutionary patterns uncovered here suggest that all four domains are important .",
"The first line of evidence is that there is no observation of rapid evolution of the ZF domains in PRDM9 orthologs from which KRAB and SSXRD domains have apparently been lost ( including a subset of species in which the catalytic activity of the SET domain is seemingly conserved ) , suggesting that there has not been rapid evolution of binding specificity .",
"In contrast , there is evidence of rapid evolution of the PRDM9 ZF in all species that have KRAB , SSXRD , SET , and ZF domains .",
"Since plausible models suggest that the rapid evolution of PRDM9 binding affinity is a consequence of the role of this gene in directing recombination ( see Introduction ) , this observation suggests that all four domains are required for this role .",
"The second piece of evidence is that swordtail fish with a truncated copy of PRDM9 that is missing KRAB and SSXRD domains behave like PRDM9 knockouts in their fine-scale recombination patterns .",
"It is unclear if this behavior can be attributed to loss of the N terminal domains , since two key catalytic residues within the SET domain were also substituted in this species .",
"However , substitutions at catalytic residues are only seen in PRDM9 genes that have lost KRAB and/or SSXRD domains or have lost the ZF entirely .",
"When the ZF is lost , PRDM9 obviously cannot induce DSBs by binding DNA and its new role may not require the same methyltransferase specificities .",
"We speculate that the absence of KRAB and SSXRD domains in a PRDM9 ortholog may similarly signify that PRDM9 is no longer used to direct recombination and lead to reduced constraint on the catalytic activities of the SET domain .",
"Consistent with this hypothesis , a recent paper suggests that the KRAB domain may play a role in recruiting the recombination machinery ( Parvanov et al . , 2017 ) .",
"If the partial ortholog of PRDM9 is not used to direct recombination at all , then the overall conservation of the protein points to another role of the gene .",
"In that regard , we note that partial PRDM9 orthologs share their domain architecture with other members of the PRDM gene family , many of which act as transcription factors ( Hayashi et al . , 2005; Vervoort et al . , 2016 ) .",
"Conversely , if the presence of all four domains , conservation of catalytic residues , and the rapid evolution of the ZF array are sufficient indications of PRDM9-directed recombination , then the role of PRDM9 in directing recombination appears to have originated before the diversification of vertebrates .",
"It would follow that many non-mammalian vertebrate species , such as snakes , use the gene to determine the location of recombination hotspots .",
"One hint in that direction is provided by the high allelic diversity seen in the ZF within a python species ( Python bivittatus ) , reminiscent of patterns observed in apes ( Schwartz et al . , 2014; Figure 1—figure supplement 5 ) .",
"Assessing the role of PRDM9 in directing recombination in these species is a natural next step in understanding the evolution of recombination mechanisms .",
"It further appears that the intact PRDM9 has often been duplicated , with more than one copy associated with recombination rate variation in cattle ( Sandor et al . , 2012; Ma et al . , 2015 ) .",
"Based on the RAxML tree of the SET domain , we count 55 independent cases of duplications .",
"How commonly more than one copy of PRDM9 retains a role in directing recombination remains to be investigated .",
"More generally , the distribution of PRDM9 across vertebrates raises the question of why species switch repeatedly from one recombination mechanism to another .",
"Although PRDM9-directed recombination clearly confers enough of an advantage for it to be widely maintained in vertebrates , at least six taxa of vertebrates carry only partial PRDM9 orthologs and the gene has been lost entirely at least three times ( based on 227 orthologs; Figure 1 , Figure 2 ) .",
"Thus , PRDM9 is not essential to meiotic recombination in the sense that SPO11 is , for example ( Lam and Keeney , 2014 ) .",
"Instead , the role of PRDM9 is perhaps best envisaged as a classic , trans-acting recombination rate modifier ( Otto and Barton , 1997 , Otto and Lenormand , 2002; Coop and Przeworski , 2007 ) , which was favored enough to be adopted at some point in evolution , but not so strongly or stably as to prevent frequent losses .",
"In this regard , it is worth noting that in mammalian species studied to date , recombination rates are lower near promoters than in species lacking PRDM9 ( Myers et al . , 2005; Coop et al . , 2008 ) .",
"Because recombination hotspots have higher rates of point mutations , insertions and deletions , and experience GC-biased gene conversion , there may be an advantage conferred by directing recombination to non-genic regions .",
"Recombination at the TSS could have the further disadvantage of uncoupling coding and regulatory variants , potentially uncovering negative epistasis , and therefore leading to indirect selection for decreased recombination at the TSS .",
"Alternatively ( but non mutually-exclusively ) , because PRDM9 binding motifs are strongly associated with certain transposable element classes in mammals ( Myers et al . , 2008 ) , the role of PRDM9 in recombination could be related to the regulation of certain families of transposable elements .",
"With a more complete picture of recombination mechanisms and their consequences across the tree of life , these hypotheses can start to be tested in an evolutionary context ."
],
[
"As a first step in understanding the distribution of PRDM9 in vertebrates , we identified putative PRDM9 orthologs in the RefSeq database .",
"We used the blastp algorithm ( Altschul et al . , 1990 ) using the Homo sapiens PRDM9 sequence , minus the rapidly evolving tandem ZF array , with an e-value threshold of 1e-5 .",
"We downloaded GenPept files and used Batch Entrez to retrieve the corresponding GenBank files ( September 2016 ) .",
"The longest transcript for each locus and amino acid and DNA sequences corresponding to the KRAB , SSXRD and SET domains of these sequences ( as annotated by the Conserved Domain Database; Marchler-Bauer et al . , 2015 ) , were downloaded using a R script ( Supplementary file 4 ) .",
"The retrieved SET domain sequences , an additional 44 retrieved from whole genome assemblies , as well as seven retrieved from RNAseq datasets for five species without sequenced genomes ( see Predicting PRDM9 orthologs from whole genome sequences ) were input into ClustalW2 ( Larkin et al . , 2007 ) , in order to generate a neighbor-joining ( NJ ) guide tree ( see Figure 1—figure supplement 2 ) .",
"This approach was used to identify and remove genes that cluster with known PRDM family genes from humans and that share the SET domain of PRDM9 but were previously reported to have diverged from PRDM9 before the common ancestor of vertebrates ( Vervoort et al . , 2016; see Phylogenetic Analysis of PRDM9 orthologs and related gene families ) .",
"There were a number of groups not included in the RefSeq database or for which we were unable to identify PRDM9 orthologs containing the complete domain architecture .",
"For 33 representative species from these groups , we investigated whether we could find additional PRDM9 orthologs in their whole genome assemblies ( see Supplementary files 1 , 2 ) ( Song et al . , 2015; Xiong et al . , 2016; Bradnam et al . , 2013; Georges et al . , 2015 ) .",
"To this end , we ran tblastn against the whole genome assembly , using the PRDM9 ortholog from the most closely related species that contained a KRAB domain , a SET domain , and at least one ZF domain ( Supplementary file 1B ) .",
"The number of hits to each region was limited to ten , and gene models were only predicted when a blast hit to the SET domain was observed with an e-value threshold of 1e-10 .",
"When a single contig was identified containing an alignment to the full length of the query sequence , this contig was input into Genewise , along with the PRDM9 protein sequence from a species with a high quality ortholog ( using a closely related species where possible ) , in order to create a new gene model .",
"When PRDM9 domains were found spread across multiple contigs , we needed to arrange them in order to generate the proper sequences of the genomic regions containing PRDM9 orthologs from each species .",
"When linkage information was available and we observed the presence of PRDM9 domains on linked contigs , we arranged the sequences of these contigs accordingly , with gaps padded with 100 Ns , before inputting them into Genewise .",
"In cases where linkage information was not available , our approach differed depending on whether or not we identified more than one hit to each region of the query sequence .",
"In species where there appeared to be only one PRDM9 ortholog , we arranged the contigs according to the expected arrangements of the domains , though did not include any ZF arrays unless they were found on the same contig as the complete SET domain because the repeat structure of these domains makes homology difficult to infer .",
"In species with more than one PRDM9 ortholog , we did not attempt to construct any gene models not supported by linkage or by transcripts identified from the same species ( see Confirming the expression or absence of PRDM9 in the testes of major phylogenetic groups; Supplementary file 1B for details ) .",
"The positions of KRAB , SSXRD and SET domains for each gene model were annotated using CD-blast ( Domain Accessions smart00317 , pfam00856 , cl02566 , pfam09514 , pfam01352 , cd07765 , smart00349 ) .",
"This approach resulted in the identification of additional PRDM9 orthologs containing at minimum the SET domain , in two jawless fish , two cartilaginous fish , nine bony fish , one monotreme , two marsupials , one turtle , four lizards , and eight snakes ( Supplementary file 1A ) .",
"We were unable to detect PRDM9 orthologs in one lizard ( Anolis carolinenesis ) , or in any of three amphibian species ( Supplementary file 1B ) .",
"We used RNA-seq data to investigate whether these negative findings are due to genome assembly quality or reflect true losses ( see below ) .",
"To understand the evolution of PRDM9 within vertebrates , we used a phylogenetic approach .",
"We first built an alignment of the amino acid sequences of putative PRDM9 and PRDM11 SET domains using Clustal Omega ( Sievers et al . , 2011 ) .",
"We included genes clustering with PRDM11 because it had been reported that PRDM11 arose from a duplication event of PRDM9 in the common ancestor of bony fish and tetrapods ( Vervoort et al . , 2016 ) , and we were interested in identifying any PRDM9 orthologs carried by vertebrate species that may precede this duplication event .",
"The alignment coordinates were then used to generate a nucleotide alignment , which was used as input into the program RAxML ( v7 . 2 . 8; Stamatakis , 2006 ) .",
"We performed 100 rapid bootstraps followed by maximum likelihood estimation of the tree under the General Reversible Time substitution model , with one partition for each position within a codon .",
"The resulting phylogeny contained monophyletic groups corresponding to the PRDM9 and PRDM11 duplication event , with 100% bootstrap support ( Figure 1—figure supplement 1 ) .",
"These groups were used to label each putative ortholog as PRDM9 or PRDM11 .",
"Only jawless fish have PRDM9 orthologs basal to this duplication event , suggesting PRDM11 arose from PRDM9 before the common ancestor of cartilaginous fish and bony vertebrates .",
"We observed at least one PRDM11 ortholog in each of the other vertebrate species examined .",
"Within teleost fish , we identified two groups of PRDM9 orthologs , which we refer to as PRDM9α and PRDM9β ( Figure 2—figure supplement 1 ) .",
"While the bootstrap support for the monophyly of the two groups is only 75% for PRDM9α and 54% for PRDM9β , the potential duplication event suggested by this tree is coincident with the whole genome duplication event known to have occurred in the common ancestor of teleost fish ( Taylor et al . , 2003 ) .",
"Moreover , the phylogenetic grouping based on the SET domain is concordant with general differences in the domain architectures between the two orthologs: In contrast to PRDM9α , PRDM9β genes have derived ZF array structures , containing multiple tandem ZF arrays spread out within the same exon ( Figure 2—figure supplement 1 ) and are always found without the KRAB and SSXRD domains , whereas PRDM9α genes generally have a single tandem array of ZFs consistent with the inferred ancestral domain architecture , and occasionally have KRAB and SSXRD domains ( Figure 2 ) .",
"A necessary condition for PRDM9 to be involved in recombination is its expression in meiotic cells .",
"For groups of taxa in which we detected a PRDM9 ortholog , we evaluated whether this ortholog was expressed in the testes , using a combination of publically available RNAseq data and RNAseq data that we generated .",
"Additionally , in groups of species where PRDM9 appeared to be absent from the genome , we used publically available RNAseq data to confirm the absence of expression of PRDM9 .",
"In both cases , we used a stringent set of criteria to try to ensure that the absence of expression was not due to data quality issues ( see details below ) .",
"We downloaded data for jawless fish , cartilaginous fish , bony fish , coelacanth , reptile , marsupial and monotreme species for which Illumina RNAseq data were available ( Supplementary file 2A , C , D ) .",
"We additionally generated RNAseq data for two percomorph fish species , Xiphophorus birchmanni and X . malinche ( see below ) .",
"Downloaded reads were converted to fastq format using the sratoolkit ( v2 . 5 . 7; Leinonen et al . , 2011 ) and trimmed for adapters and low quality bases ( Phred <20 ) using the program cutadapt ( v1 . 9 . 1; https://cutadapt . readthedocs . io/en/stable/ ) .",
"Reads shorter than 31 bp post-quality trimming were discarded .",
"The program interleave_fastq . py was used to combine mate pairs in cases where sequence data were paired-end ( Crawford , 2014; https://gist . github . com/ngcrawford/2232505 ) .",
"De-novo transcriptome assemblies were constructed using the program velvet ( v1 . 2 . 1; Zerbino and Birney , 2008 ) with a kmer of 31; oases ( Schulz et al . , 2012; v0 . 2 . 8 ) was used to construct transcript isoforms .",
"Summaries of these assemblies are available in Supplementary file 2A .",
"In order to identify potential PRDM9 transcripts in each of 24 assembled transcriptomes , we implemented tblastn using the human PRMD9 sequence , minus the ZF domain , as the query sequence , with an e-value threshold of 1e-5 .",
"The identified transcripts were extracted with a custom script and blasted to our dataset of all PRDM genes ( Supplementary file 5 ) .",
"If the best blast hit was a PRDM9 ortholog , we considered PRDM9 expression in the testis to be confirmed ( see results in Supplementary file 2C ) .",
"For five species lacking genome assemblies , we extracted PRDM9 orthologs with best blast hits to human PRDM9/7 and included these in our phylogenetic analyses ( see Phylogenetic Analysis of PRDM9 orthologs and related gene families ) .",
"Failure to detect PRDM9 could mean that PRDM9 is not expressed in that tissue , or that data quality and sequencing depth are too low to detect its expression .",
"To distinguish between these possibilities , we used other recombination-related genes as positive controls , reasoning that if expression of several other conserved recombination-related genes were detected , the absence of PRDM9 would be more strongly suggestive of true lack of expression .",
"Eight recombination-related genes are known to be conserved between yeast and mice ( Lam and Keeney , 2014 ) .",
"We used the subset of seven that could be reliably detected in whole genome sequences , and we asked which transcriptomes had reciprocal best tblastn ( e-value <1e-5 ) hits to all of these proteins , using query sequences from humans ( Supplementary file 2A; Supplementary file 2D ) .",
"In addition , in order to assess whether PRDM9 expression might simply be lower than that of other meiotic genes , we quantified absolute expression of PRDM9 and the seven conserved recombination-related proteins in whole testes , using data from three major taxa ( bony fish , mammals , and reptiles ) ; see Analysis of PRDM9 expression levels and expression levels of other conserved recombination-related genes for more details .",
"Together , these results suggest that not detecting PRDM9 in whole testes transcriptomes provides support for its absence .",
"Three Xiphophorus birchmanni and three X . malinche were collected from the eastern Sierra Madre Oriental in the state of Hidalgo , Mexico .",
"Fish were caught using baited minnow traps and were immediately euthanized by decapitation ( Texas A&M AUP# - IACUC 2013–0168 ) .",
"Testis , ovaries , and liver were dissected and stored at 4°C in RNAlater .",
"Total RNA was extracted from testis , ovary and liver tissue using the Qiagen RNeasy kit ( Valencia , CA , USA ) following the manufacturer’s protocol .",
"RNA was quantified and assessed for quality on a Nanodrop 1000 ( Nanodrop technologies , Willmington , DE , USA ) and approximately 1 μg of total RNA was used input to the Illumina TruSeq mRNA sample prep kit .",
"Samples were prepared following the manufacturer’s protocol with minor modifications .",
"Briefly , mRNA was purified using manufacturer’s beads and chemically fragmented .",
"First and second strand cDNA was synthesized and end repaired .",
"Following A-tailing , each sample was individually barcoded with an Illumina index and amplified for 12 cycles .",
"The six libraries were sequenced on the HiSeq 2500 at the Lewis Sigler Institute at Princeton University to collect single-end 150 bp reads , while single-end 100 bp data was collected on the HiSeq 4000 at Weill Cornell Medical College for all other samples ( SRA Accessions: SRX2436594 and SRX2436597 ) .",
"Reads were processed and a de novo transcriptome assembled for the highest coverage testis library following the approach described above for publicly available samples .",
"Details on assembly quality are available in Supplementary file 2A .",
"Other individuals were used in analysis of gene expression levels ( see next section ) .",
"To determine whether some of the genes in our conserved recombination-related gene set were expressed at similar levels to PRDM9 , implying similar detection power , we examined expression levels of these genes in three species representing the bony fish , reptilian , and mammalian taxa ( Xiphophorus malinche , Pogona vitticeps , and Homo sapiens ) .",
"To quantify expression in X . malinche , we mapped trimmed reads from testes RNAseq libraries that we generated from three individuals to the X . maculatus reference genome ( v4 . 4 . 2; Schartl et al . , 2013; Amores et al . , 2014 ) using bwa ( v0 . 7 . 10; Li and Durbin , 2009 ) .",
"The number of trimmed reads per individual ranged from 9 . 9 to 27 . 5 million .",
"We used the program eXpress ( v1 . 5 . 1; Roberts et al . , 2011 ) to quantify fragments per kilobase of transcript per million mapped reads ( FPKM ) for each gene , and extracted the genes of interest from the results file based on their ensembl gene id . eXpress also gives confidence intervals on its estimates of FPKM .",
"For the bearded lizard Pogona vitticeps , we only had access to one publically available testis-derived RNAseq library .",
"We followed the same steps used in analysis of swordtail FPKM except that we mapped to the transcriptome generated from the data and identified transcripts belonging to recombination-related gene sets using the reciprocal best blast hit approach described above .",
"Several publically available databases already exist for tissue specific expression in humans .",
"We downloaded the ‘RNA gene dataset’ from the Human Protein Atlas ( v15 , http://www . proteinatlas . org/about/download ) .",
"This dataset reports average FPKM by tissue from 122 individuals .",
"We extracted genes of interest from this data file based on their Ensembl gene id .",
"Examination of these results demonstrated that other meiotic genes ( 2-5 ) in each species had expression levels comparable to PRDM9 ( Figure 1—figure supplement 3 ) .",
"This finding suggests that these genes are appropriate positive controls , in that detecting their expression but not that of PRDM9 provides evidence against expression of PRDM9 in testes .",
"In addition to complete losses of PRDM9 , we were unable to identify one or more functional domains of PRDM9 in orthologs identified from the platypus , Tasmanian devil , elephant shark , all bony fish and several placental mammals .",
"To ask whether the missing PRDM9 domains were truly absent from the genome assembly , we first used a targeted genome-wide search .",
"To this end , we performed a tblastn search of the genome against the human PRDM9 ortholog with an e-value of 1e-10 .",
"For all blast hits , we extracted the region and 2 Mb flanking in either direction , translated them in all six frames ( http://cgpdb . ucdavis . edu/DNA_SixFrames_Translation/ ) , and performed an rpsblast search of these regions against the CDD ( database downloaded from NCBI September 2016 ) with an e-value of 100 to identify any conserved domains , even with weakly supported homology .",
"We extracted all rpsblast hits to the missing functional domain ( SET CDD id: smart00317 , pfam00856 , cl02566; SSXRD CDD id: pfam09514; KRAB domains pfam01352 , cd07765 , smart00349 ) and used them as query sequences in a blastp search against all KRAB , SSXRD and SET containing proteins in the human genome .",
"If PRDM9 or PRMD7 was the top blast hit in this search , we considered that the missing domain could be a result of assembly or gene model prediction error ( if not , we investigated the potential loss of these domains further ) .",
"This approach allowed us to rule out genome-wide losses of PRDM9 domains in nine out of 14 species of mammals for which our initial approach had failed to identify complete PRDM9 orthologs .",
"In each case , we checked whether or not the identified domains were found adjacent to any of our predicted gene models and adjusted the domain architecture listed for these RefSeq genes accordingly in our dataset ( see Supplementary file 1A ) .",
"In five species of mammals ( Tasmanian devil , three bat species , and the aardvark ) , we only identified a partial PRDM9 ortholog , but we were unable to confirm the loss of domains using RNAseq data ( see next section ) .",
"Within bats , each partial gene model starts within 500 bp of an upstream gap in the assembly .",
"Moreover , we were able to identify a KRAB domain corresponding to PRDM9 from a closely related species of bat ( Myotis brandtii ) .",
"Thus , we believe that in the case of bats , these apparent domain losses are due to assembly errors or gaps .",
"For species with available RNAseq data from taxa in which we predicted PRDM9 N-terminal truncation based on our initial analyses , we sought to confirm the domain structure observed in the genome with de novo transcriptome assemblies from testis RNAseq ( described above ) .",
"As before , we only considered transcriptomes that passed our basic quality control test ( Supplementary file 2D ) .",
"Because RNAseq data are not available for all species with genome assemblies , we were only able to perform this stringent confirmation in a subset of species ( Supplementary file 2C ) .",
"As a result , we consider cases where N-terminal losses are confirmed in the genome as possible losses but are most confident about cases where N-terminal losses are observed both in the genome and transcriptome .",
"To examine the transcripts of PRDM9 orthologs from the transcriptome assemblies ( Supplementary file 2A ) , for each domain structure , we translated each transcript with a blast hit to the human PRDM9 in all six frames and used rpsblast against all of these translated transcripts , with an e-value cutoff of 100 ( as described above ) .",
"Finally , we performed a reciprocal nucleotide blast ( blastn; e-value cutoff 1e-20 ) to confirm that these transcripts were homologous to the PRDM9 ortholog identified using phylogenetic methods in these taxa .",
"Results of this analysis can be found in Supplementary file 2C .",
"In summary , there were two cases where the transcriptomes supported additional domain structures not found in the whole genome sequence ( Supplementary file 2C ) : a PRDM9 ortholog from the spotted gar ( Lepisosteus oculatus ) that was observed to have a KRAB domain not identified in the genome sequence , and a PRDM9α ortholog from the Atlantic salmon ( Salmo salar ) that was observed to have both KRAB and SSXRD domains not identified in the genome search .",
"In all other cases , we confirmed the losses of either the KRAB or SSXRD domains , including:",
"( i ) PRDM9β orthologs missing KRAB and SSXRD domains in all species of teleost fish expressing these orthologs ( Supplementary file 2B , C )",
"( ii ) PRDM9α orthologs missing KRAB and SSXRD domains identified from Astyanax mexicanus , Esox lucius , Gadus morhua , and Ictalurus punctatus , and",
"( iii ) loss of the KRAB domain from one PRDM9 ortholog in monotremata ( O . anatinus ) and both KRAB and SSXRD domains from the other ortholog in this species .",
"For all groups in which we confirm that there is only a partial PRDM9 ortholog based on the above analyses , we asked whether the PRDM9 gene in question has likely become a pseudogene ( as it has , for example , in canids; Oliver et al . , 2009; Muñoz-Fuentes et al . , 2011 ) , in which case the species can be considered a PRDM9 knockout .",
"Though such events would be consistent with our observation of many losses of PRDM9 , they would not be informative about the role of particular PRDM9 domains in recombination .",
"For this analysis , we aligned the SET domain of the PRDM9 coding nucleotide sequence to a high-quality PRDM9 sequence with complete domain structure from the same taxon using Clustal Omega ( see Supplementary file 3A ) , except for the case of PRDM9β in bony fish and the PRDM9 ortholog from cartilaginous fish , where such a sequence was not available .",
"In the case of PRDM9β , we compared the sequence between X . maculatus and A . mexicanus , sequences that are >200 million years diverged ( Hedges et al . , 2015 ) .",
"In the case of cartilaginous fish , we used the sequence from R . typus and C . milii , which are an estimated 400 million years diverged ( Hedges et al . , 2015 ) .",
"We analyzed these alignments with codeml , comparing the likelihood of two models , one with a fixed omega of 1 and an alternate model without a fixed omega , and performed a likelihood ratio test .",
"A significant result for the likelihood ratio test provides evidence that a gene is not neutrally-evolving ( Supplementary file 3A ) .",
"In all cases of N-terminal truncation analyzed , dN/dS is significantly less than one ( Supplementary file 3A ) .",
"While it is possible that some of these cases represent very recently pseudogenized genes , the widespread evidence for purifying selection on the SET domain strongly suggests that these PRDM9 orthologs are functionally important .",
"We also investigated constraint in all mammalian Ref-seq orthologs that appear to lack only an annotated KRAB or SSXRD domain; for this larger number of genes , we did not confirm all domain losses , due to the large number of genome searches that would be required and lack of RNAseq data for most species .",
"We found evidence of purifying selection in all cases except for five PRDM7 orthologs from primates , for which we had been unable to identify a KRAB domain ( Supplementary file 3C ) .",
"PRDM7 is thought to have arisen from a primate specific duplication event and to have undergone subsequent losses of the C2H2 ZF array and of some catalytic specificity of its SET domain ( Blazer et al . , 2016 ) .",
"Thus , PRDM7 orthologs are unlikely to function in directing recombination .",
"Our findings further suggest they are evolving under very little constraint , and may even be non-functional .",
"More generally , within placental mammals , the majority of partial PRDM9 orthologs that we identified lack the ZF array completely or have truncated arrays ( notably , there are fewer than four tandem ZFs in 24 of 28 orthologs ) , in sharp contrast to other taxa in which partial orthologs to PRDM9 lack the N terminal domains , yet have conserved ZF arrays and are constrained .",
"Moreover , the paralogs lacking a long ZF tend to be found in species that already carry a complete PRDM9 ortholog ( 21 of 24 ) .",
"Thus , some of these cases may represent recent duplication events in which one copy of PRDM9 is under highly relaxed selection , similar to PRDM7 in primates .",
"The SSXRD domain is the shortest functional domain in the PRDM9 protein .",
"One species of cartilaginous fish ( Rhincodon typus ) , and several species of bony fish ( Anguilla anguilla , A . rostrata , A . japonica , Salmo trutta , S . salar ) have weakly predicted SSXRD domains ( e-values >10 , see Supplementary file 1B and 2C ) .",
"This observation is potentially suggestive of functional divergence or loss of this domain .",
"Unfortunately , because the domain is so short , there is little power to reject dN/dS = 1; though the estimate of dN/dS was 0 . 10 and 0 . 11 between cartilaginous fish and eel and salmon orthologous regions , respectively , the difference between models was not significant in either case .",
"Based on these findings , we tentatively treat the weakly predicted SSXRD domain in Rhincodon typus and in the above species of bony fish as evidence that this domain is present in these species , but note that we were unable to identify a similar region in predicted gene models from another species of cartilaginous fish ( Callorhinchus milii ) .",
"We performed Sanger sequencing of Python bivittatus PRDM9 from a single individual to collect additional data on within species diversity of the ZF array ( Figure 1—figure supplement 5 ) .",
"Primers were designed based on the Python bivattatus genome ( Castoe et al . , 2013 ) to amplify the ZF containing exon of PRDM9 and through a gap in the assembly .",
"Primers were assessed for specificity and quality using NCBI Primer Blast ( http://www . ncbi . nlm . nih . gov/tools/primer-blast/ ) against the nr reference database and were synthesized by IDT ( Coralville , IA , USA ) .",
"DNA was extracted from approximately 20 mg of tissue using the Zymo Quick-DNA kit ( Irvine , CA , USA ) following the manufacturer’s protocol .",
"PCR was performed using the NEB Phusion High-Fidelity PCR kit ( Ipswich , MA , USA ) .",
"Reactions were performed following manufacturer’s instructions with 60 ng of DNA and 10 μM each of the forward ( ZF: 5’TTTGCCATCAGTGTCCCAGT’3; gap: 5’ GCTTCCAGCATTTTGCCAGTT’3 ) and reverse ( ZF: 5’ TTGATTCACTTGTGAGTGGACAT’3; gap: 5’ GAGCTTTGCTGAAATCGGGT’3 ) primers .",
"Products were inspected for non-specific amplification on a 1% agarose gel with ethidium bromide , purified using a Qiagen PCR purification kit ( Valencia , CA , USA ) and sequenced by GeneWiz ( South Plainfield , NJ , USA ) .",
"In species in which PRDM9 is known to play a role in recombination , the level of sequence similarity between the individual ZFs of the tandem array is remarkably high , reflective of high rates of ZF turnover due to paralogous gene conversion and duplication events ( Oliver et al . , 2009; Myers et al . , 2010; Jeffreys et al . , 2013 ) .",
"It has further been observed that DNA-binding residues show high levels of amino acid diversity , suggestive of positive selection acting specifically at DNA-binding sites , that is , on binding affinity ( e . g . Oliver et al . , 2009; Schwartz et al . , 2014 ) .",
"These signals have been previously studied by comparing site specific rates of synonymous versus non-synonymous substitutions ( dN/dS ) between paralogous ZFs in PRDM9’s tandem ZF array ( Oliver et al . , 2009 ) .",
"Assessing statistical significance using this approach is problematic , however , because the occurrence of paralogous gene conversion across copies means that there is no single tree relating the different ZFs , in violation of model assumptions ( Schierup and Hein , 2000; Wilson and McVean , 2006 ) .",
"Here , we used a statistic sensitive to both rapid evolution at DNA-binding sites and high rates of gene conversion: the total proportion of amino acid diversity observed at DNA-binding sites within the ZF array .",
"We then assessed significance empirically by comparing the value of this statistic to other C2H2 ZF genes from the same species ( where possible ) .",
"To this end , for each species with a PRDM9 ortholog , we downloaded the nucleotide and protein sequences for all available RefSeq genes with a C2H2 ZF motif annotated in Conserved Domain Database ( pfam id# PF00096 ) .",
"To simplify alignment generation , we only used tandem ZF arrays with four or more ZFs matching the 28 amino acid long C2H2 motif ( X2-CXXC-X12-HXXXH-X5 where X is any amino acid ) .",
"In all of our analyses , if a gene had multiple tandem ZF arrays that were spatially separated , only the first array of four or more adjacent ZFs was used for the following analysis ( Supplementary file 3B ) .",
"However , an alternative analysis using all ZFs or different subsets of ZFs led to qualitatively similar results for the PRDM9β orthologs from bony fish , where ZFs are commonly found in multiple tandem arrays separated by short linker regions in the predicted amino acid sequence ( Figure 2—figure supplement 1; Figure 2—figure supplement 2 ) .",
"For species with PRDM9 orthologs with fewer than five ZFs , we implemented blastn against the whole genome sequence using the available gene model as a query sequence , in order to determine whether or not there was a predicted gap within the ZF array , and , if there was , to identify any additional ZFs found in the expected orientation at the beginning of the adjacent contig .",
"This approach was able to successfully identify additional ZF sequences on contigs adjacent to PRDM9 in the genome assembly for two species ( Latimeria chalumnae and Protobothrops mucrosquamatus ) .",
"These ZFs were included in subsequent analysis ( Supplementary file 1A ) .",
"Using the alignments generated above , we determined the amino acid diversity along the ZF domains of PRDM9 genes and all other C2H2 ZFs from the same species ( Table 1 , Supplementary file 3B ) , and calculated the proportion of the total amino acid diversity at canonical DNA-binding residues of the ZF array .",
"Specifically , we calculated the heterozygosity xk at position k across the aligned ZFs from a single tandem array as:xk=1− ∑i=1mfi2 where m is the number of unique amino acids found at position k across the fingers , and fi is the frequency of the ith unique amino acid across the fingers .",
"The total proportion P of amino acid diversity assigned to DNA-binding residues is the sum of xk at DNA-binding sites over the sum of xk at all sites in the ZF array .",
"To compare results to those for other genes , we ranked PRDM9 by the value P compared to all other C2H2 ZF genes from the same species ( Table 1 , Supplementary file 3B ) .",
"We used the R package phylotools ( Zhang et al . , 2012; https://cran . r-project . org/web/packages/phylotools/index . html ) to calculate a p-value for the correlation between complete domain structure and rapid evolution of the PRDM9 ZF array , taking into account phylogenetic relationships between PRDM9 orthologs .",
"We coded these variables using a binary approach with ‘00’ for incomplete domain structure and no evidence of rapid evolution and ‘11’ for complete domain structure and evidence of rapid evolution .",
"To describe the phylogenetic relationships between orthologs , we used the RAxML tree that we constructed from the SET domain for all PRDM9 orthologs .",
"Species with missing ZF information , including species where PRDM9 has been lost , were excluded from this analysis using the drop . tip function of the ape package ( Paradis et al . , 2004 ) , resulting in a tree with 91 tips .",
"We used the phyloglm command to perform a logistic regression evaluating the relationship between domain structure and the odds of rapid evolution of the ZF array .",
"In order to investigate whether the catalytic function of the SET domain is conserved in the PRDM9 orthologs identified above , we asked whether any PRDM9 orthologs in our dataset carried substitutions at three catalytic residues shown to mediate the methyltransferase activity of human PRDM9 ( Wu et al . , 2013 ) .",
"To this end , we used Clustal Omega to create an amino acid alignment of the SET domain with 15 amino acids of flanking sequence for each PRDM9 ortholog in our dataset and asked whether the gene had substitutions to tyrosine residues at positions aligning to Y276 , Y341 and Y357 in human PRDM9 ( Supplementary file 1A ) .",
"Domain alignments deposited at Baker et al . , 2017 .",
"In total , 57 genes were identified as having substitutions in at least one of these residues , including 11 from placental mammals and 46 from bony fish ( Supplementary file 1A ) .",
"To visualize the distribution of these substitution events within bony fish , we mapped these substitutions onto the phylogeny of PRDM9 orthologs generated above ( Figure 3 ) .",
"Percomorph fish have a partial ortholog of PRDM9 that lacks the KRAB and SSXRD domains found in mammalian PRDM9 .",
"As a result , we hypothesized that they would behave like PRDM9 knockouts , in that the predicted PRDM9 binding motif would not co-localize with recombination events , and functional genomic elements such as the TSS and CGIs would be enriched for recombination events .",
"To build a hybrid recombination map , we generated low coverage sequence data for 268 individuals from a natural hybrid population ( ‘Totonicapa’ ) formed between the percomorph species X . birchmanni and X . malinche ( RRID:SCR_008340 ) and sampled between 2013–2015 .",
"The two parental species are closely related , with pairwise sequence divergence <0 . 5% ( Schumer et al . , 2014 ) .",
"Interestingly , in sharp contrast to what is seen in placental mammals , the ZF is slowly evolving between X . birchmanni and X . malinche ( dN/dS = 0 . 09; Figure 4A ) .",
"DNA was extracted from fin clips for the 268 individuals and libraries were prepared following Stern , 2016 .",
"Briefly , three to ten nanograms of DNA was mixed with Tn5 transposase enzyme pre-charged with custom adapters and incubated at 55 C for 15 min .",
"The reaction was stopped by adding 0 . 2% SDS and incubating at 55 C for an additional seven minutes .",
"One of 96 custom indices were added to each sample in a plate with an individual PCR reaction including 1 ul of the tagmented DNA; between 13–16 PCR cycles were used .",
"After amplification , 5 ul of each reaction was pool and purified using Agencourt AMPpure XP beads .",
"Library size distribution and quality was visualized on the Bioanalyzer 1000 and size selected by the Princeton Lewis Sigler Core Facility to be between 350–750 basepairs .",
"Libraries were sequenced on the Illumina HiSeq 4000 at Weill Cornell Medical Center across three lanes to collect paired-end 100 bp reads .",
"Ancestry assignment in hybrids was performed using the Multiplexed Shotgun Genotyping ( ‘MSG’ ) pipeline ( Andolfatto et al . , 2011 ) .",
"This approach has been previously validated for genome-wide ancestry determination in late generation X . birchmanni x X . malinche hybrids ( Schumer et al . , 2014; Schumer et al . , 2016b ) .",
"Briefly , raw data was parsed by barcode and trimmed to remove low-quality basepairs ( Phred quality score <20 ) .",
"Reads with fewer than 30 bp after trimming were discarded .",
"Because of prohibitively long computational times , reads from individuals with more than 16 million reads were subsampled to 16 million before running the MSG pipeline .",
"The minimum number of reads for an individual to be included was set to 300 , 000 , since ancestry inference with fewer reads is predicted to have lower accuracy based on simulations ( Schumer et al . 2015 ) .",
"This procedure resulted in 239 individuals for our final analysis , with an average coverage of 8 . 3 million reads , or ~1X genome-wide coverage .",
"The parameters used in the MSG run were based on previous work on this hybrid population ( Schumer et al . in review ) .",
"The expected number of recombination events per chromosome ( recRate ) was set to 8 , based on a prior expectation of approximately 30 generations of admixture and assuming initial admixture proportions of 75% of the genome derived from X . birchmanni and 25% derived from X . malinche .",
"Similarly , priors for each ancestry state were set based on these mixture proportions ( par1 = 0 . 5625 , par1par2 = 0 . 375 and par2 = 0 . 0625 ) .",
"The recombination rate scaling factor was set to the default value of 1 .",
"Ancestry transitions were identified as the interval over which the posterior probability changed from ≥0 . 95 in support of one ancestry state to ≥0 . 95 for a different ancestry state .",
"Breakpoint intervals that occurred within 10 kb of a contig edge were excluded from the analysis due to concerns that false breakpoints may occur more frequently near the edges of contigs .",
"The identified recombination intervals varied significantly in their lengths , that is , in the resolution of the crossover event .",
"The median resolution was 13 kb , with 75% of breakpoints resolved within 35 kb or less .",
"To evaluate the relationship between recombination frequency and genomic elements such as the TSS , CGIs , and computationally predicted PRDM9 binding sites , we needed to convert the observed recombination events into an estimate of recombination frequency throughout the genome .",
"To this end , we considered the proportion of events observed in a particular 10 kb window; we note that this rate is not equivalent to a rate per meiosis .",
"We filtered the data to remove windows within 10 kb of a contig boundary .",
"Because the majority of events span multiple 10 kb windows , we randomly placed events that spanned multiple windows into one of the windows that they spanned .",
"We used the closest-feature command from the program bedops v2 . 4 . 19 ( Neph et al . , 2012 ) to determine the minimum distance between each 10 kb window and the functional feature of interest .",
"For the TSS , we used the Ensembl annotation of the Xiphophorus maculatus genome with coordinates lifted over to v . 4 . 4 . 2 of the linkage group assembly ( Amores et al . , 2014; Schumer et al . , 2016a ) http://genome . uoregon . edu/xma/index_v1 . 0 . php ) .",
"For CGIs , we used the annotations available from the UCSC genome browser beta site ( http://genome-test . cse . ucsc . edu/cgi-bin/hgTables ? hgsid=391260460_COev5GTglYu74K2t24uaU4UcaTvP&clade=vertebrate&org=Southern+platyfish&db=xipMac1&hgta_group=allTracks&hgta_track=cpgIslandExt&hgta_table=0&hgta_regionType=genome&position=JH556661%3A3162916-4744374&hgta_outputType=primaryTable&hgta_outFileName= ) .",
"To identify putative PRDM9 binding sites , we used the ZF prediction software available at zf . princeton . edu with the polynomial SVM settings to generate a position weight matrix for the X . malinche and X . birchmanni PRDM9 orthologs ( Persikov and Singh , 2014 ) .",
"This approach yielded identical predicted binding motifs in the two species ( Figure 4A ) .",
"We used this position weight matrix to search the X . malinche genome ( Schumer et al . , 2014 ) for putative PRDM9 binding sites with the meme-suite program FIMO ( v4 . 11 . 1; Grant et al . , 2011 ) .",
"We selected all regions with a predicted PRDM9 binding score of ≥5 .",
"Since the individuals surveyed are interspecific hybrids , and the two species may differ in the locations of predicted PRDM9 binding sites , we repeated the FIMO search against the X . birchmanni genome , obtaining qualitatively identical results .",
"After determining the minimum distance between each 10 kb window and the features of interest , we calculated the average recombination frequency in hybrids as a function of distance from the feature of interest in 10 kb windows ( Figure 4; Figure 4—figure supplement 2 ) .",
"To estimate the uncertainty associated with rates at a given distance from a feature , we repeated this analysis 500 times for each feature , bootstrapping windows with replacement .",
"Because we found a positive correlation between distance from the TSS and CGIs in 10 kb windows with recombination frequency , we checked that power ( i . e . , the proportion of ancestry informative sites ) was not higher near these features .",
"Most work in humans and mice has focused on the empirical PRDM9 binding motif rather than the computationally predicted motif .",
"Since we expect the computationally predicted motif to be a poorer predictor of PRDM9 binding , we checked how its use would affect the analyses , by repeating the analysis described above for the computational prediction obtained for the human PRDM9A allele , using recombination rates in 10 kb windows estimated from the CEU LD map ( Frazer et al . , 2007 ) ; downloaded from: http://www . well . ox . ac . uk/~anjali/AAmap/ ) .",
"We also repeated this analysis for the gor-1 PRDM9 allele in Gorilla gorilla , using recombination rates in 10 kb windows estimated from a recent LD map ( Schwartz et al . , 2014; Great Ape Genome Project et al . , 2016; downloaded from Stevison , 2016: https://github . com/lstevison/great-ape-recombination ) .",
"To investigate whether patterns of recombination rates near the TSS and CGI systematically distinguish between species that do and do not use PRDM9-directed recombination , we compared available data across species .",
"We downloaded previously published recombination maps for three species without PRDM9 genes ( dog , Auton et al . , 2013 , zebra finch and long-tailed finch , Singhal et al . , 2015 ) and four species with complete PRDM9 orthologs ( human , Frazer et al . , 2007; Hinch et al . , 2011; gorilla , Great Ape Genome Project et al . , 2016; sheep , Johnston et al . , 2016; and mouse , Brunschwig et al . , 2012 ) .",
"For each species , we binned recombination rate into 10 kb windows along the genome , excluding the sex chromosomes and windows overlapping with assembly gaps from all analyses .",
"For each species , we downloaded annotations of assembly gaps , TSSs and CGIs from the UCSC genome browser website .",
"For CGI positions in the gorilla genome , we used the LiftOver tool ( http://genome . ucsc . edu/cgi-bin/hgLiftOver ) to convert the available coordinates for the GorGor4 genome assembly to the GorGor3 assembly .",
"For zebra finch and long-tailed finches , we used the coordinates of CGIs and TSSs as annotated for the TaeGut3 . 2 genome assembly , noting that these coordinates are consistent with the TaeGut3 . 1 assembly for all chromosomes for which genetic distances were inferred in ( Singhal et al . , 2015 ) .",
"For each map , we calculated the distance to the nearest TSS and to the nearest CGI by from the midpoint of each 10 kb window .",
"To visualize these patterns , we fit a Gaussian loess curve using the distance to nearest TSS or CGI and recombination rate for each species , using only windows within 100 kb of a representative element .",
"For visual comparison , we scaled the resulting curves by setting the y-value ( recombination rate ) of the last point to one .",
"A caveat is that other than for swordtail and sheep , we relied on LD based genetics maps , which estimate population recombination rates 4Ner , where Ne is the effective population size and r the recombination rate per meiosis .",
"Because estimates of Ne decrease near genes as a consequence of diversity-reducing linked selection ( e . g . , Wright and Andolfatto , 2008; Hernandez et al . , 2011 ) , a decrease in estimated population recombination rates near genes may not reflect a reduction in the recombination rate r .",
"To explore the potential importance of this caveat , we considered two species where both LD maps and pedigree or admixture maps were available: dogs and humans .",
"In both cases , the qualitative results were the same as for the LD-based maps ( Figure 5—figure supplement 1 ) .",
"Since diversity-reducing linked selection should give rise , if anything , to a trough in diversity levels , it cannot explain the observed peaks at these features in species lacking PRDM9 or swordtail fish; in fact , since these species also experience this form of selection ( e . g . , Singhal et al . , 2015 ) , the true peaks in recombination rates near promoter-like features are likely somewhat more pronounced .",
"We note further that although the peak in recombination rate at these features in swordtail fish appears to be less prominent than in dog or birds , quantitative comparisons of different species are difficult because these maps differ in their resolution .",
"Whole testis and liver were dissected from two X . birchmanni adults and stored in HypoThermosol FRS ( BioLife Solutions , Bothell , WA ) buffer on ice until processing .",
"Native chromatin ChIP was performed as described previously ( Markenscoff-Papadimitriou et al . , 2014 ) .",
"Briefly , tissue was homogenized and lysed; the lysate was spun through a sucrose cushion ( to pellet nuclei ) .",
"Nuclei were resuspended in 500 ul MNase digestion buffer and digested with 1 unit of micrococcal nuclease ( MNase , Sigma N5386 , St . Louis , MO ) for 2 min at 37 C , then inactivated with 20 ul 0 . 5M EDTA and chilled on ice .",
"The first soluble chromatin fraction was recovered by spinning for 10 min at 10 , 000 rcf at 4C and collecting the supernatant .",
"To isolate the second soluble chromatin fraction , the pellet was resuspended in 500 µl dialysis buffer , rotated overnight at 4 C , then centrifuged for 10 min at 10 , 000 rcf at 4 C to pellet insoluble material .",
"The digestion quality of each fraction was evaluated on an agarose gel .",
"The two soluble fractions were combined for chromatin immunoprecipitation with 1 µg of H3K4me3 antibody ( Millipore 04–745 , Billerica , MA ) ; 1/10 vol was retained as an input control .",
"Antibody was bound to the remaining chromatin overnight while rotating at 4 C . The next day blocked Protein A and Protein G beads were added , and rotated for 3 hr .",
"The bound beads were then washed a total of 7 times with chilled wash buffers and immunoprecipitated chromatin was eluted in elution buffer for 30 min at 37 C and cleaned up with ChIP DNA Clean and Concentrator kit ( Zymo Research , Irvine , CA ) .",
"Libraries were prepared for sequencing using the NuGEN ultralow library prep kit ( NuGEN , San Carlos , CA ) following manufacturer’s instructions and sequenced on an Illumina HiSeq 2500 at Hudson Alpha to collect 10 . 3–10 . 5 and 12 . 2–14 . 5 paired-end 50 bp reads for pull-down and input samples respectively .",
"Raw reads were trimmed to remove adapter sequences and reads with fewer than 18 bp after adapter trimming using the program cutadapt .",
"These trimmed reads were then mapped to the X . maculatus reference genome with bowtie2 ( Langmead and Salzberg , 2012 ) and the resulting bam file was sorted with samtools ( Li et al . , 2009 ) .",
"Homer ( Heinz et al . , 2010 ) was used to generate bigWig files and call peaks using the option style –factor .",
"We also performed the analysis using the option style –histone and found that the results were qualitatively similar .",
"Peak files were converted to bed files and bedtools2 ( Quinlan and Hall , 2010 ) was used to analyze overlap between the locations of H3K4me3 peaks and predicted PRDM9 binding motifs in the swordtail genome ( see above ) .",
"Based on Homer analysis , which identified 20 , 662 peaks in the testis and 15 , 050 in the liver , the IP efficiency was estimated to be 38% for the testis sample and 40% for the liver sample; the peak width was estimated to be 229 bp for the testis sample and 238 for the liver sample .",
"Having identified H3K4me3 peaks in testis and liver tissue , we next asked about the relationship between these peaks and predicted PRDM9 binding sites ( see above ) .",
"If PRDM9 is generating H3K4me3 peaks during meiosis , we expect to see an association between predicted PRDM9 binding motifs in the swordtail genome and H3K4me3 peaks .",
"To test for such an association , we generated 500 null motifs by randomly shuffling without replacement the position weight matrix of the X . birchmanni PRDM9 and re-running FIMO as described above .",
"We then asked how frequently randomly shuffled PRDM9 motifs overlap H3K4me3 peaks compared to the real motif .",
"We found that no evidence that the real motif overlapped H3K4me3 peaks more frequently than the shuffled versions of the motif ( Figure 4C ) .",
"As a secondary approach , we compared H3K4me3 peaks that are specific to the testis to H3K4me3 peaks that are specific to the liver , defined as peaks in the testis where there is no overlapping peak in the liver .",
"Using a Chi-squared test , we asked whether H3K4me3 peaks found only in the testis are more likely to overlap a PRDM9 binding motif than those that are liver specific ( where the definition is analogous ) ( Figure 4 ) .",
"Because the size of H3K4me3 peaks will impact the expected overlap with PRDM9 binding motifs , we also constrained the size of the H3K4me3 peaks in the liver analysis to be the same as that inferred from the testis using the –size flag in homer ( 229 bp ) .",
"Results were not qualitatively different with the original analysis , using liver H3K4me3 peaks that were inferred to be 238 bp .",
"Counterintuitively liver-specific H3K4me3 peaks appear to overlap predicted PRDM9 motifs more often than testes-specific peaks ( χ = 14 . 8; p=1 . 2e-4 ) .",
"However , performing this same analysis with the 500 null motifs ( generated as described above ) , we found that liver-specific peaks were significantly enriched in shuffled motifs in 85% of simulations ( at the 0 . 05 level ) .",
"This analysis suggests that base composition differences between liver and testes-specific H3K4me3 peaks explain the difference in overlap results .",
"We also repeated the above analysis for clusters of three ZFs in the swordtail PRDM9 ZF array , using a smaller number of shuffled sequences ( n = 20 ) .",
"We observed the same qualitative patterns for each of the ZF clusters as reported above .",
"Finally , we used a third approach to ask about the association of H3K4me3 peaks and PRDM9 binding sites .",
"We generated five replicate datasets of H3K4me3 sequences and their flanking 250 bp regions from both the testis and the liver .",
"We ran the program MEME to predict motifs enriched in the testis-specific H3K4me3 peaks using the liver as a background sequence set on these five replicate datasets .",
"We then examined the top ten predicted motifs to ask whether any of these motifs resembled the computationally predicted PRDM9 binding motifs ( Figure 4—figure supplement 5 ) .",
"The above analyses suggest that in swordtail fish , PRDM9 does not make H3K4me3 marks but they do not indicate whether H3K4me3 peaks are associated with recombination events in swordtails .",
"We therefore verified that recombination rates in 10 kb windows are significantly correlated with the distance of that window to the nearest H3K4me3 peak ( rho = −0 . 072 , p=2 . 3e-69; Figure 4 ) .",
"This relationship weakens but remains significant when accounting for distance both to TSSs and CGIs by a partial correlation analysis ( rho = −0 . 026 , p=5 . 4e-10 ) .",
"Furthermore , windows that contain a testis-specific H3K4me3 peak have a higher recombination rate than windows that contain a liver-specific peak ( Figure 4—figure supplement 4 ) .",
"Finally , there is a significant positive correlation between the number of bp in a 10 kb window overlapping an H3K4me3 peak and the number of recombination events observed in that window in the testis but not in the liver ( testis: rho = 0 . 044 , p=2 . 8e-29; liver: rho = 0 . 002 , p=0 . 66 ) .",
"Together , these analyses suggest that a relationship between H3K4me3 peaks and recombination exists in swordtails , but not one mediated through PRDM9 binding ."
]
] | [
"Studies of highly diverged species have revealed two mechanisms by which meiotic recombination is directed to the genome—through PRDM9 binding or by targeting promoter-like features—that lead to dramatically different evolutionary dynamics of hotspots .",
"Here , we identify PRDM9 orthologs from genome and transcriptome data in 225 species .",
"We find the complete PRDM9 ortholog across distantly related vertebrates but , despite this broad conservation , infer a minimum of six partial and three complete losses .",
"Strikingly , taxa carrying the complete ortholog of PRDM9 are precisely those with rapid evolution of its predicted binding affinity , suggesting that all domains are necessary for directing recombination .",
"Indeed , as we show , swordtail fish carrying only a partial but conserved ortholog share recombination properties with PRDM9 knock-outs ."
] | [
"The genetic information of Eukaryotic organisms ( animals , plants and fungi ) is encoded on strands of DNA called chromosomes .",
"In animals that sexually reproduce , most cells carry two copies of each chromosome , with one inherited from each of their parents .",
"Sex cells such as sperm and egg cells are the exception , and contain only a paternal or maternal set respectively .",
"These chromosomes are not exact copies of the parental chromosomes , but are instead combinations of both of them , generated by a process called meiotic recombination .",
"Meiotic recombination begins by breaking the chromosomes , and the repair of those breaks shuffles DNA segments between the two chromosomes .",
"This shuffling is known as a “recombination event” .",
"In humans , apes and mice , the location of recombination events depends on where a protein called PRDM9 binds to the DNA .",
"Over the course of evolution , this binding location has changed relatively rapidly so that even closely related species such as humans and chimpanzees localize recombination events to different DNA regions .",
"In contrast , closely related species that do not produce PRDM9 tend to direct recombination events to similar DNA regions .",
"It remains unclear when PRDM9 first evolved its role in recombination , or why different methods of directing recombination have developed .",
"To begin answering these questions , Baker , Schumer et al . investigated whether 225 species of vertebrates ( backboned animals ) have a gene that encodes PRDM9 .",
"This analysis revealed that even distantly related animals have genes that produce equivalents of the complete PRDM9 protein .",
"However , several species have independently lost the ability to produce PRDM9 .",
"In certain other species , particular regions of the gene have been removed or shortened .",
"Notably , only species that carry genes that contain regions called the KRAB and SSXRD domains show relatively rapid evolution of where PRDM9 binds in the DNA .",
"To investigate this phenomenon further , Baker , Schumer et al . constructed a map of recombination events in swordtail fish , which carry a version of the gene that lacks the KRAB and SSXRD domains .",
"The PRDM9 protein produced by this gene does not direct where recombination events occur .",
"Overall , it appears that the KRAB and SSXRD domains are necessary for PRDM9 to direct meiotic recombination .",
"Furthermore , Baker , Schumer et al . predict that those species that have complete versions of PRDM9 use this protein to localize recombination events .",
"Knowing which species use PRDM9 in this way is the first step towards understanding why recombination mechanisms change in evolution , and with what consequences ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"genetics and genomics"
] | Constraint and divergence of global gene expression in the mammalian embryo | elife-05538-v2 | [
[
"Multicellular animals develop via complex pattern formation processes that unfold during embryogenesis , with gene regulation playing a central role .",
"Surprisingly , the impact of genetic variation on gene regulation and the relative importance of the genetic mechanisms underlying regulatory variation in mammalian embryogenesis remain undefined .",
"There are four broad categories of genetic mechanisms of gene expression regulation: imprinting , parental effects , cis-regulation , and trans-regulation .",
"All of these are potentially subject to natural selection and provide genetic windows into the forces that shape gene regulatory evolution .",
"Here , we quantify their impact and relevance in mammalian embryogenesis by asking which of these genetic mechanisms reveal evolutionary divergence or constraint since the last common ancestor of two distantly related mouse strains , C57BL/6 ( B6 ) and Cast/Ei ( Cast ) .",
"Genomic imprinting is a well-studied form of parent-of-origin-dependent gene expression that produces strongly biased allelic expression patterns in offspring according to the parental source of the inherited allele .",
"The progeny's imprinted gene expression patterns result from epigenetic modifications put in place in the previous generation and reflect the particular genetic architecture at these specialized loci .",
"A recent genome-wide survey estimated that fewer than 200 genes are imprinted in mice and humans ( Babak et al . , 2008 ) .",
"Imprinting is thought to play a critical role in early embryonic and placental development in mammals and has been shown to influence post-natal health and behavior [Reviewed in Peters ( 2014 ) ] .",
"Related to but distinct from imprinting is the concept of parental effects .",
"Parental effects produce phenotypic variation that depends , directly or indirectly , on the genotype of either the mother or father , rather than that of the offspring .",
"Therefore , the resulting phenotypic patterns lag a generation behind the genetic transmission of the causal variants .",
"The most well-studied parental genetic effects are caused by deposition of maternal transcripts into the egg prior to fertilization , resulting in differences in early embryonic development depending on the genotype of the mother .",
"Certain genes have also been shown to respond to maternal influence after birth through genetically defined maternal behaviors ( Weaver et al . , 2004 ) .",
"Because of the small contribution , through the sperm , of the paternal transcriptome to the fertilized zygote , and because of the stronger maternal contribution to child rearing in most model organisms , parental effects are typically thought of as synonymous with maternal effects , although true paternal effects are known to exist ( Rando , 2012 ) .",
"Maternal effects have been shown to be important during embryonic development , leading to differences in the birth weight of mice depending on the genotype of the mother ( Cowley et al . , 1989; Wolf et al . , 2011 ) .",
"However , neither the causal molecular mechanism for these effects in the mother nor the responding genes in the embryo have been identified and few studies to date have discriminated between prenatal and postnatal effects .",
"Maternal effects likely contribute to the heritability of complex traits but have been confounded with family structure in mouse as well as human studies ( Mott et al . , 2014 ) .",
"Genetic variants affecting gene expression are classified into cis or trans , depending on whether they act on the gene copy on the same chromatid ( cis ) or on both chromatids equally ( trans ) .",
"Regulatory variants near their target genes usually act in cis , by modifying the activity of a promoter or enhancer , but very occasionally act in trans if they affect a gene product that regulates a nearby locus .",
"One approach to quantifying cis and trans regulation in model organisms involves assaying gene expression in two parental lines compared to allele-specific expression ( ASE ) in the hybrids ( Wittkopp et al . , 2004 ) and asking whether alleles are regulated differently in the parentals compared to the hybrids .",
"In adult flies and mice , these analyses have demonstrated that cis and trans regulatory variants frequently target the same genes , often with opposite , compensatory effects on the target's expression ( McManus et al . , 2010; Goncalves et al . , 2012 ) .",
"Recent genome-wide studies of ASE patterns across many individuals have demonstrated the ability of high-throughput sequencing methods to directly identify genes with cis-regulatory variants ( Montgomery et al . , 2010; Pickrell et al . , 2010 ) .",
"Another method to quantify cis- and trans-regulatory variation is analysis of expression quantitative trait loci ( eQTL ) ( Schadt et al . , 2003 ) .",
"eQTL are identified as genetic loci whose genotypes correlate with gene expression changes across a number of genetically heterogeneous individuals .",
"Genome-wide eQTL studies have demonstrated that the strongest genetic variation in expression regulation generally occurs in variants located proximally to their target genes , presumably acting in cis .",
"While typically fewer in number , trans-regulatory variants affect both alleles of each target gene and can affect expression of many genes simultaneously , resulting in a large aggregate effect .",
"ASE and eQTL approaches have been applied widely in adult tissues in humans and model organisms .",
"However , these approaches have not yet been applied to understand the global relationship between genetic variation and gene expression during mammalian embryogenesis , and while imprinting has been studied extensively , the contribution of maternal effects on mammalian embryonic gene expression has never been addressed .",
"For several reasons , it is likely that patterns of regulatory divergence differ between embryos and adults .",
"First , eQTL studies have demonstrated dynamic trans-regulatory variation during Caenorhabditis elegans larval development ( Francesconi and Lehner , 2014 ) .",
"Second , recent studies in flies and fish have demonstrated higher expression of more ancient conserved genes as well as more conserved gene expression patterns during embryogenesis compared to the adult ( Domazet-Lošo and Tautz , 2010; Kalinka et al . , 2010 ) .",
"This suggests that the number of cis- as well as trans-eQTL should be lower in embryos compared to adult .",
"Here , we quantify evolutionary gene regulatory divergence between B6 and Cast during embryogenesis , addressing each of the four regulatory architectures .",
"We focus on embryonic day 11 . 5 ( E11 . 5 ) , which is equivalent to roughly 40 days of gestation in human .",
"It is a time characterized by organogenesis , rapid growth of the brain and limbs , increasingly complex and localized pattern formation , and consequently substantial constraints on allowable phenotypic variation .",
"An ASE/eQTL hybrid approach facilitates quantifying the different types of regulation and their effects on expression variation , revealing unique and important features of gene regulatory architecture during mammalian embryonic development ."
],
[
"For this study , we chose two inbred strains derived from geographically separate subspecies of Mus musculus: C57BL/6J ( B6 ) , the classic inbred mouse reference strain , which was derived from European mice , and CAST/EiJ ( Cast ) , which was derived from the Southeast Asian Mus musculus castaneus .",
"Inbreeding caused random subsets of alleles segregating in their ancestral populations to become fixed , and as a consequence the two strains exhibit one single nucleotide polymorphism ( SNP ) difference every 120 bp on average ( Keane et al . , 2011 ) .",
"We collected 16 E11 . 5 F1 embryos obtained from reciprocal crosses between B6 and Cast ( Figure 1 ) .",
"We profiled genome-wide gene expression using a 3′-biased RNA sequencing method , 3SEQ ( Beck et al . , 2010 ) , in order to maximize SNP coverage in the 3′ UTRs , where the average SNP density is ∼1 SNP per 170 bp ( compared to 1 SNP per 265 bp in the coding sequence ) .",
"Mapping of reads separately against the Cast ( Keane et al . , 2011 ) and B6 genomes allowed us to eliminate reference mapping bias ( Figure 2—figure supplement",
"1 ) as the average fraction of reads supporting the B6 allele ( B6 allele fraction , ‘BAF’ ) was 0 . 502 .",
"Overall , out of a possible 6917 genes with sufficient reads covering SNPs , 1594 ( 23% ) showed significant ASE ( p < 0 . 01 , paired t-test , Bonferroni corrected; Figure 2A , B ) .",
"We next backcrossed F1 mice ( derived from female B6 by male Cast crosses ) with B6 parentals to obtain N2 embryos .",
"We harvested 154 N2s at E11 . 5 , performed 3SEQ , and quantified ASE in the approximately 50% of embryos that were heterozygous at each gene .",
"There was excellent agreement between average BAF estimates from F1s and N2s ( Figure 2C; rho = 0 . 77 ) . 10 . 7554/eLife . 05538 . 003Figure 1 . Experimental design .",
"( A ) Reciprocal crosses of B6 and Cast parents were used to generate F1 hybrid embryos .",
"( B ) F1 hybrid adults were backcrossed to B6 to generate N2 embryos via reciprocal crosses of B6 females with F1 males and F1 females with B6 males .",
"Chromosomal regions inherited from a B6 parent are black and Cast regions are red .",
"( C ) Example genotypes at a controlling genetic locus , its corresponding target gene , and resulting gene expression levels for different types of gene regulatory variation assayed .",
"Allele-specific expression ( ASE ) and imprinting are measured by allele-specific read counts , whereas other types measure total gene expression levels .",
"X: X chromosome; Y: Y chromosome; m: mitochondrial DNA . DOI: http://dx . doi . org/10 . 7554/eLife . 05538 . 00310 . 7554/eLife . 05538 . 004Figure 2 . ASE . B6 allele fraction ( BAF ) for ( A ) non-ASE gene Stmn4 , ( B ) ASE gene Ascc1 .",
"Each point represents a single embryo , grouped by cross ( F1 embryos on left , N2 embryos on right ) .",
"Cross mother is listed first .",
"Bars are mean BAF and 95% confidence intervals .",
"( C ) Correlation of average BAF between F1 and N2 embryos .",
"Significant ASE genes ( called using union of F1 and N2 data ) are red .",
"Point size corresponds to relative expression level .",
"( D ) Transition from fetal ( Hbb-bh1 , Hbb-y ) to adult ( Hbb-b2 , Hbb-b1 ) beta-globins , shown as fraction of total expression of all beta-globins , averaged across individuals by time point ( somite number ) .",
"( E and F )",
"Divergent temporal regulation for B6 and Cast alleles of Hbb-b1 ( E ) and Hbb-b2 ( F ) in heterozygous N2 mice .",
"( G and H )",
"BAF for imprinted genes Igf2 ( G ) and H13 ( H ) .",
"( I ) Significantly imprinted genes ( red , with 95% confidence intervals; n = 31 ) clearly separate from non-imprinted genes ( gray , n = 8261; Figure 2—figure supplement 2 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 05538 . 00410 . 7554/eLife . 05538 . 005Figure 2—figure supplement 1 . B6 allele fractions . Histogram of BAF across genes with SNP coverage , in 0 . 01 increments . DOI: http://dx . doi . org/10 . 7554/eLife . 05538 . 00510 . 7554/eLife . 05538 . 006Figure 2—figure supplement 2 . Age-dependent ASE . ASE time courses for top eight genes ( A-H ) exhibiting significant dependence of ASE on somite number .",
"Left panel , BAF; right panel , normalized expression .",
"Only heterozygous embryos are shown in the left panels . DOI: http://dx . doi . org/10 . 7554/eLife . 05538 . 00610 . 7554/eLife . 05538 . 007Figure 2—figure supplement 3 . Significantly imprinted genes .",
"( A ) Magnification of the clusters close to points ( 1 , 0 ) and ( 0 , 1 ) in the context of the same plot shown in Figure 1I , where BAF is plotted as a function of the direction of the cross .",
"Mother's strain is listed first according to convention .",
"( B ) Plot of the average difference in BAF between the two reciprocal crosses , for each of the F1 and N2 crosses .",
"Each gene is a point .",
"For each gene , for F1 embryos , average BAF of embryos from CastxB6 cross was subtracted from average BAF of embryos from B6xCast cross .",
"For N2 embryos , only embryos heterozygous at the assayed gene are considered; for each gene , average BAF of embryos from F1xB6 cross is subtracted from average BAF of embryos from B6xF1 cross .",
"Significantly imprinted genes , where the two crosses give a large difference , are in red near ( 1 , 1 ) or ( −1 , −1 ) .",
"If imprinting were not usually Boolean ( either fully on or off ) but rather a quantitative phenomenon in which different tissues respond differently to the imprint or change the imprint , the significant genes would reside in the tails of a continuous distribution and not be so clearly separated from the bulk of the non-imprinted genes . DOI: http://dx . doi . org/10 . 7554/eLife . 05538 . 007 To quantify developmental progression for each embryo at the time of harvest , we counted the number of somites , which form every 90–120 min ( Saga and Takeda , 2001 ) and ranged from 42 to 57 in our embryos .",
"Of the 7712 genes with moderate expression of at least 5 reads per million ( RPM ) , 1796 genes showed significant correlation between total expression and somite number ( q < 0 . 05 , Spearman correlation ) .",
"Genes changing over this developmental time course were enriched for a number of gene ontology ( GO ) categories indicative of important developmental processes occurring at E11 . 5 , including synaptogenesis ( q = 2 . 2 × 10−8 , GOrilla ) and eye lens formation ( q = 5 . 8 × 10−7 ) .",
"A third significant GO category , blood microparticles ( q = 9 . 6 × 10−7 ) , includes the globins , which are beginning to undergo the transition from fetal to adult form over the developmental time period we assayed ( Noordermeer and de Laat , 2008 ) ( Figure 2D ) .",
"By correlating ASE with somite number , we identified twelve genes that show significant differential regulation between B6 and Cast alleles as a function of developmental timing ( q < 0 . 10 , Spearman rho ) .",
"Globins and functionally related genes were well represented in this set , which included both major ( Hbb-b1 ) and minor ( Hbb-b2 ) adult beta globins .",
"Both genes increase in absolute level and relative expression compared to fetal beta globins ( Figure 2D; Figure 2—figure supplement 2 ) , but the B6 allele of the major form is delayed compared to the Cast allele ( Figure 2E; rho = 0 . 86 , q < 10−14 ) , whereas the opposite occurs for the minor form ( Figure 2F; rho = −0 . 51 , q = 0 . 02 ) .",
"By the end of our time course , the alleles are equally expressed ( Figure 2E , F ) .",
"Transferrin also increases in absolute expression but the Cast allele is delayed compared to the B6 allele ( rho = −0 . 46 , q = 0 . 02; Figure 2—figure supplement 2 ) .",
"These results suggest that compensatory mechanisms have evolved in one or both strains to keep the overall regulatory dynamic of oxygen physiology stable during this important switching period .",
"Comparison of embryos from either side of the reciprocal cross allowed us to identify parent-of-origin dependent ASE patterns indicative of imprinting .",
"Consistent with previous studies in mouse embryos ( Babak et al . , 2008 ) , we found a total of 31 genes with significant imprinting ( Figure 2G–I; p < 0 . 01 , binomial test , Bonferroni corrected ) , all of which had been previously identified .",
"We note that the clear separation of most of the imprinted loci from the noise of the bulk of all expressed genes , with few exceptions ( Figure 2I; Figure 2—figure supplement 3 ) , suggests that most imprinting generally acts uniformly across the embryo , as expected from the methylation-based mechanism that sets the expression state of the regulated allele pre-zygotically ( Reik et al . , 1987 ) .",
"Imprinting is not the only molecular mechanism that results from the mother–offspring resource allocation conflict in mammals ( Moore and Haig , 1991 ) .",
"To explore other genetically-encoded parental effects , we took advantage of the backcross design , in which N2 offspring have genotypically distinct parents .",
"We identified a large number ( n = 331 ) of embryonically expressed genes whose total expression levels differed significantly depending on the genetic background of the mother ( Mann Whitney U test , p < 0 . 01 , Bonferroni corrected ) , irrespective of the genotype of the embryo ( Figure 3A , B; Supplementary file 1 ) .",
"For example , Angptl4 expression increases 52% from an average of 107 RPM in embryos with an F1 mother to an average of 163 RPM in embryos with a B6 mother ( Figure 3C ) .",
"As for the magnitude of the effect , these maternal effect target genes exhibited up to a 3 . 5-fold ( e . g . , Polm: 2 . 5–11-fold at a 95% confidence interval ) difference in total expression level depending on genotype of the mother . 10 . 7554/eLife . 05538 . 008Figure 3 . Maternal effect target genes .",
"( A ) Quantile–quantile ( qq ) plot for maternal effect target genes ( black circles ) , compared to cis-expression quantitative trait loci ( eQTL ) ( gray circles ) .",
"( B ) Expression by embryo for the top 20 maternal effect target genes , separated by maternal genotype .",
"For each gene , samples are grouped by F1 mother ( left ) and B6 mother ( right ) .",
"( C ) Total expression for Angptl4 for embryos grouped by embryonic genotype and maternal genotype .",
"Error bars indicate mean and 95% confidence intervals . DOI: http://dx . doi . org/10 . 7554/eLife . 05538 . 008 We were able to exclude a number of alternative mechanisms to explain these maternal effects .",
"All F1s used for the backcross were derived from B6 female by Cast male matings , and therefore all N2 embryos carry the B6 mitochondrial genome , ruling out a confounding mitochondrial effect .",
"The maternal effects were unchanged when considering sex , X chromosome genotype , litter , and developmental stage .",
"Furthermore , for the vast majority of genes with SNP coverage , we were able to verify that maternal effect target genes were not imprinted or the result of contamination by maternally expressed transcripts .",
"We found that maternal genotype by itself was a better predictor of the maternal effects than maternal genotype in conjunction with embryonic genotype at any of the imprinted loci , indicating that the maternal effects were not the result of trans effects downstream of imprinted genes .",
"To exclude the possibility of a batch effect in our 3SEQ data , we used quantitative RT-PCR ( qPCR ) to confirm 4 genes as maternal effect target genes .",
"The most significant of the maternal effect target genes , Angptl4 ( Figure 3C; p = 8 × 10−13 by 3SEQ , p = 1 × 10−11 by qPCR ) , functions in lipid metabolism and energy homeostasis , suggestive of a role in responding to the placental nutrient supply ( Yoshida et al . , 2002 ) .",
"Similarly , Gpx3 ( Figure 3B; p = 5 × 10−12 by 3SEQ , p = 6 . 6 × 10−4 by qPCR ) , a glutathione peroxidase , is an antioxidant enzyme that acts in fat and glucose metabolism and has been associated with obesity and type 2 diabetes in mouse and human ( Lee et al . , 2008; Chung et al . , 2009 ) .",
"Both of these genes are transcriptionally regulated by the peroxisome proliferator-activated receptor PPARγ .",
"To examine how genetic variation carried by the embryo itself affects gene expression phenotype , we performed eQTL analyses by correlating expression levels of each measurably expressed gene ( ‘target’ ) with N2 embryo genotypes ( either B6/Cast or B6/B6 ) at each recombination-defined locus ( ‘controlling locus’ ) .",
"Genotyping and recombination analysis based on the expressed SNPs ( Figure 4A ) divided the autosomes into 986 marker region blocks , each separated by one or more recombination events that define candidate controlling loci .",
"We tested every controlling locus for correlation with gene expression of every gene .",
"At a genome-wide significance level of q < 0 . 01 , we identified a total of 1034 eQTL . 10 . 7554/eLife . 05538 . 009Figure 4 . eQTL .",
"( A ) Example N2 embryo data used to call recombination events for chromosome 2 .",
"Each point represents BAF for a single gene .",
"The HMM calls green regions as heterozygous , blue regions as homozygous , with two recombination events occurring in the short intervening white regions .",
"Only genes with sufficient read coverage of single nucleotide polymorphisms ( SNPs ) are shown as points; genes with under 20 SNP reads are still genotyped , based on neighboring genes .",
"( B ) Overview of all eQTL , arranged by genomic position , shaded by log10 p-value .",
"cis-eQTL , along the diagonal , show the only appreciable signal .",
"( C ) Predicted BAF from transformed expression levels in heterozygotes and homozygotes ( x-axis ) correlate with BAF measured by ASE ( y-axis ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 05538 . 00910 . 7554/eLife . 05538 . 010Figure 4—figure supplement 1 . Random forest analysis . qq plots from random forest model that simultaneously considers age ( number of somites ) and genotype .",
"Left , all gene–marker pairs; right , gene–marker pairs only in trans . DOI: http://dx . doi . org/10 . 7554/eLife . 05538 . 010 1017 of these eQTL ( 98% ) mapped close to the target gene ( Figure 4B ) , suggesting cis-regulation as the underlying mechanism .",
"A gene's difference in expression between heterozygotes and homozygotes could accurately predict its allelic ratio as assayed by ASE ( Figure 4C ) .",
"This high correlation ( rho = 0 . 58 , n = 4821; rho = 0 . 70 when considering only the n = 1000 genes with the highest allele-specific read coverage ) provides a systematic validation of the large effect of embryonic cis-regulatory polymorphisms on expression phenotype .",
"Note that our study has sufficient power to identify substantial numbers of eQTL when performing this all–loci–by–all–genes analysis .",
"If , instead , we were focused specifically on only studying cis-regulation , we could have increased our power to identify cis-eQTL by only comparing genes to their nearby loci , thereby reducing the number of statistical tests being performed .",
"In contrast to the large number of cis-eQTL , we found few trans-eQTL regardless of FDR cutoff ( Figure 5A ) .",
"After the top 3 ( each comprising a controlling locus–target gene pair , e . g . , Figure 5B ) , significance drops off rapidly compared to background , unlike for the cis-eQTL ( Figure 5C ) .",
"To explore this lack of trans effects further , we reasoned that a set of genes with a high prior probability of having downstream effects might be enriched for trans-controlling loci .",
"Our cis-eQTL/ASE loci comprise a large set of such candidates because differences in expression levels of a gene product may propagate to downstream targets ( Yvert et al . , 2003 ) .",
"However , we found that neither strong cis-eQTL nor ASE loci are enriched for trans-eQTL .",
"Finally , application of a random forest model ( Michaelson et al . , 2010; Francesconi and Lehner , 2014 ) that simultaneously considers developmental progression ( number of somites ) and genotype also did not enrich for trans-eQTL ( Figure 4—figure supplement 1 ) .",
"We estimate that the number of comparable trans-eQTL is about 1% that of cis-eQTL . 10 . 7554/eLife . 05538 . 011Figure 5 . Trans-eQTL .",
"( A ) Number of cis-eQTL ( black ) and trans-eQTL ( red ) as a function of FDR .",
"Dashed line indicates number of false positive eQTL expected .",
"( B ) eQTL for Pop1 , the most significant trans-eQTL target ( vertical blue line ) .",
"Candidate controlling loci are shown across the x-axis in genome order; only the peak on Chr14 is significant .",
"Chromosomes are shaded in alternating background colors .",
"( C ) qq plot for all gene–marker pairs ( black ) and gene–marker pairs only in trans ( red ) .",
"Gray straight lines are y = x , x axis is expected p-values based on permutations .",
"( D ) Trans-eQTL hotspots , shown as the distribution of significant target genes per marker region ( p < 0 . 005 ) across all marker regions ( red ) or only marker regions containing the top 50% of ASE genes ( blue ) .",
"Gray shows the expected distribution if there were no hotspots .",
"( E and F )",
"Comparison of our study with that of eQTL in adult tissues as a function of FDR .",
"( E ) , Trans-eQTL as a proportion of total eQTL .",
"( F ) , total number of eQTL .",
"GeneNetwork sample ID indicated in parentheses .",
"( G ) Evolutionary constraint ( GERP score ) in coding regions ( CDS ) for the most significant ( red; n = 1347 ) and non-significant ( blue; n = 3726 ) cis-eQTL genes . DOI: http://dx . doi . org/10 . 7554/eLife . 05538 . 011 Because a trans-regulatory effect is potentially distributed over several target loci , we asked whether there was evidence for ‘hotspots’ , or controlling loci whose genotypes correlate with small expression differences at numerous target genes ( Brem et al . , 2002; Schadt et al . , 2003 ) .",
"Permutation tests revealed a significant signal ( p < 10−24 KS test , Figure 5D ) in support of hotspots , but no specific candidates .",
"Reinforcing the above result that ASE genes do not act as trans-controlling loci , strong ASE genes were not substantially more likely to act as hotspots than weaker ASE genes ( Figure 5D; p = 0 . 20 , KS test ) .",
"Therefore , at this stage of embryogenesis , we found no evidence for downstream propagation of genetically controlled expression level differences .",
"We hypothesized that the paucity of trans-eQTL was due to increased evolutionary constraint of regulatory networks during embryonic development .",
"Since trans-eQTL have previously been found to have weaker effects than cis-eQTL ( Montgomery and Dermitzakis , 2011 ) , an alternative hypothesis is that we were simply underpowered to find trans-eQTL .",
"To distinguish these possibilities , we obtained genotype and expression data from previously published eQTL studies performed in adult mouse tissues ( Wang et al . , 2006; Yang et al . , 2006; Chen et al . , 2008 ) .",
"After sampling individuals to match the same number of total eQTL found at an FDR of 0 . 05 , we found that all previously published studies conducted in adult tissues showed a substantially higher proportion of trans-eQTL than did our embryos ( Figure 5E , F ) .",
"This result was robust to varying the sample size from the previously published data , to whether p-values or FDR was used to assess significance , and to varying the thresholds for comparing proportions of trans-eQTL across the ranked eQTL .",
"Based on this result and our high power to detect cis-eQTL even at low FDR , we conclude that embryonic trans-eQTL are subject to elevated selective constraint compared to adult tissues .",
"If cis-regulatory divergence in embryos is strongly buffered and does not lead to downstream trans-regulatory effects , why does embryogenesis tolerate such a large number of cis-effects in the first place ?",
"We hypothesized that divergent cis-regulation between strains occurs preferentially in genes with lower evolutionary constraint .",
"In support of this hypothesis , ASE genes were significantly depleted of GO categories related to organ development ( p = 6 × 10−5 , q = 0 . 05 ) , as well as gene regulation ( p = 1 × 10−4 , q = 0 . 07 ) , and transcription factors ( p = 3 × 10−5 , q = 0 . 03 ) , demonstrating constraints on variation in core regulatory genes .",
"Furthermore , we found that evolutionary constraint at the DNA sequence level ( Cooper et al . , 2005; Davydov et al . , 2010 ) was lowest in the most significant cis-eQTL ( Figure 5G; rho = −0 . 14 , p = 6 × 10−13; n = 2788 ) .",
"Similarly , ASE magnitude of effect correlated negatively with evolutionary constraint ( rho = −0 . 10 , p = 9 × 10−8 , n = 2612 ) , whereas cis-eQTL in adult mouse tissues showed no correlation ( |rho| < 0 . 05 ) .",
"Thus , the strong enrichment for cis-eQTL/ASE in less conserved genes is unique to embryos ."
],
[
"We have performed the first , to our knowledge , comprehensive global analysis of the effects of genetic variation on mammalian gene expression during embryogenesis .",
"Our experimental design allowed us to simultaneously assay the effects of genetic variation on imprinting , maternal effects , cis-regulation , and trans-regulation .",
"Supporting the high quality of our data and analytical methods , we found good agreement between related measurements of cis-regulatory variation when comparing ASE in F1 embryos ( sequenced at high coverage ) with ASE in N2 embryos ( sequencing many individuals ) , and when comparing ASE with cis-eQTL .",
"Our two mouse strains exhibit SNPs at 17 . 7 million sites , about sixfold more than the heterozygosity in African humans ( 1000 Genomes Project Consortium , 2012 ) .",
"The vast majority of these polymorphisms have accumulated as a result of genetic drift ( Kimura , 1983 ) over the time since the present-day European and Southeast Asian M . musculus populations were separated from their common ancestral population .",
"Of the small subset of polymorphisms that affect molecular function , some may produce an advantageous phenotype subject to positive selection , some are deleterious to organismal fitness and are therefore subject to purifying selection , and yet others are selectively neutral ( subject to drift ) .",
"Missense polymorphisms are severely underrepresented genome-wide and exhibit lower derived allele frequencies relative to other genomic sites ( 1000 Genomes Project Consortium , 2012 ) , reflecting the global predominance of purifying selection .",
"This signal of purifying selection is weaker but still substantial for noncoding gene regulatory sites ( ENCODE Project Consortium , 2007 ) .",
"Given these known dynamics , our study sheds light on the microevolution of gene regulation .",
"Gene expression divergence of the type we quantified in this work occurs as a result of polymorphisms in regulatory regions or in expressed regulatory gene products .",
"The former manifests as cis-effects , the latter as trans-effects .",
"Because we focused on embryonic gene expression , two competing hypotheses as to what patterns we might find were plausible .",
"Under the first hypothesis , the substantial DNA sequence divergence between mouse strains would be reflected in many expression differences .",
"The alternative hypothesis is that few expression changes would be detected between strains .",
"This hypothesis is suggested by the developmental hourglass model ( Domazet-Lošo and Tautz , 2010; Kalinka et al . , 2010 ) , which posits that gene expression patterns are most highly conserved in the middle of embryonic development ( the so-called ‘phylotypic stage’ ) .",
"In support of the first hypothesis , we found substantial numbers of significant eQTL , demonstrating that a sizeable subset of all segregating polymorphisms causes expression changes .",
"However , we found that nearly all acted in cis , rather than trans .",
"This depletion of trans-regulatory variation is substantially stronger than that observed across several adult mouse tissues .",
"In contrast to adult tissues , eQTL acting in our embryos were correlated with lower DNA sequence conservation , supporting the conclusion that evolutionary constraint on gene expression regulation is markedly stronger during mammalian embryogenesis than in the adult .",
"In addition , the identified cis-expression changes do not themselves cause detectable trans-effects .",
"These findings support the idea of the developmental hourglass model ( Domazet-Lošo and Tautz , 2010; Kalinka et al . , 2010 ) while suggesting that substantial neutral divergence of molecular phenotype ( gene expression ) can accumulate in the form of cis-regulatory variation even under strong evolutionary constraint at the organismal ( embryonic ) level .",
"Previous studies have shown enrichment for trans-regulation when using an eQTL model that considers both genotype and stage during C . elegans larval development .",
"Perhaps because of the nearness of our embryos to the mouse phylotypic stage , or because of fundamental differences between mice and worms , we found that incorporating developmental timing ( i . e . , somite number ) into our eQTL model did not alter the balance between cis- and trans-regulation .",
"Somite number was , however , correlated with significant changes in ASE , indicating differential developmental timing of gene expression between the Cast and B6 strains .",
"In particular , we found that alleles of several hemoglobin-related genes , including the adult beta globins and transferrin , were expressed at differing levels between the strains at early stages of the fetal-to-adult hemoglobin transition , but then equalize by the end of our developmental time course .",
"These results demonstrate our ability to identify subtle tissue-specific expression differences between strains , despite sequencing from whole embryos containing a mix of tissues .",
"It is formally possible that some of the significant eQTL identified here act in trans , even though their target genes are located in genomic proximity .",
"However , the good agreement between eQTL and ASE suggests that the majority of our local eQTL are indeed cis-eQTL .",
"Furthermore , because there is no reason to believe that the proportion of locally-situated but trans-acting eQTL is higher in embryos than in adults , it does not materially alter our conclusions if a small number of our local eQTL do act in trans .",
"Because of the large number of embryos collected and individually sequenced for our study , we were able to accurately quantify ASE mean and standard deviation for thousands of genes .",
"When stratifying embryos by side of cross , we found that nearly all imprinted genes showed very strong allelic bias , meaning that the epigenetically silenced allele is expressed at only low levels across the embryo .",
"Consistent with previous studies that found few genes exhibiting tissue-specific imprinting [apart from those found in the extra-embryonic tissues; reviewed in Prickett and Oakey ( 2012 ) ] , we observed no weak allelic bias that might result from averaging effects across tissues .",
"In addition to the clear signature of parent-of-origin-dependent allelic expression , we also found strong maternal effects on total gene expression levels for hundreds of genes .",
"Whereas most previous studies have employed F1 intercrosses in which parental genotypes are indistinguishable , we used a backcross design , enabling us to track the potentially important sources of genetic variance contributed by the parents .",
"The observed maternal effects are restricted to pre-natal influences and separate from imprinting and mitochondrial effects .",
"Our results demonstrate that the genotype of the mother plays an important role in regulating gene expression , not just after birth , as has been previously suggested , but also during embryogenesis , affecting the expression of hundreds of genes ( Bult and Lynch , 1997; Weaver et al . , 2004; Wolf et al . , 2011 ) .",
"The implications of this finding are significant for human genetic twin , familial , and genome-wide association studies .",
"What is usually thought of as familial environment [e . g . , Grundberg et al . ( 2012 ) ] may have a substantial heritable genetic component exerting an effect not on the generation carrying it but on the offspring .",
"Parental genetic influences on offspring phenotype have been implicated in growth regulation and adult-onset obesity- and diabetes-related diseases but the gene targets of such effects have not been identified ( Jarvis et al . , 2005 ) .",
"While we cannot rule out an epigenetic or RNA-mediated mechanism underlying this regulation , it is more likely acting through the placenta's role of transmitting nutrients , hormones or other factors to the embryo .",
"This hypothesis is supported by the known functions of the most significant target gene , Angptl4 , which responds to lipid metabolism and energy homeostasis , and participates in angiogenesis ( Koster et al . , 2005; Scott et al . , 2012 ) .",
"We hypothesize that these maternal effect target genes continue to be expressed differently depending on maternal genotype , and may be at least partly responsible for the previously observed effect of maternal genotype on pre-natal growth ( Cowley et al . , 1989; Wolf et al . , 2011 ) .",
"In summary , using our hybrid ASE/eQTL approach we were able to assay all four types of genetic variation in gene expression regulation .",
"We found that cis-regulatory variation caused the largest divergence in overall gene expression in mouse embryos , affecting over 1000 genes with up to sevenfold differences in expression levels between the strains .",
"These cis-regulatory variants did not translate into trans-regulatory divergence , and we identified only ∼10 trans-eQTL genomewide .",
"Imprinting affected the allelic balance of dozens of genes , generally resulting in complete or nearly complete silencing of the inactive allele .",
"Finally , we identified over 300 maternal effect target genes .",
"The number of maternal effect targets , and their overall significance , are only slightly more modest than cis-regulatory effects , but nonetheless substantial , with approximately 1/3 as many genes involved , and maximal effect size of up to 3 . 5-fold difference or approximately 1/2 as large .",
"Given this context , it is clear that maternal effects can play a substantial genetic role in determining embryonic gene expression patterns .",
"We speculate that maternal effects contribute to evolutionary divergence , allowing expression heterogeneity in the embryo to build as a result of differences in the adult mother , where gene regulatory networks show less evolutionary constraint than in the developing embryo ."
],
[
"6-week old mice were purchased from The Jackson Laboratory .",
"All ‘Materials and methods’ were carried out in accordance with the Administrative Panel on Laboratory Animal Care protocol and the institutional guidelines set by the Veterinary Service Center at Stanford University .",
"The inbred strain C57BL/6J and wild-derived inbred strain CAST/EiJ were used in reciprocal crosses ( Figure 1 ) .",
"C57BL/6J females were crossed with CAST/EiJ males to generate ( B6 × Cast ) F1 hybrid embryos ( n = 8 ) , and CAST/EiJ females were crossed with C57BL/6J males to generate ( Cast × B6 ) F1 hybrid embryos ( n = 8 ) .",
"( B6 × Cast ) F1 progeny were backcrossed to C57BL/6J in reciprocal crosses to generate 154 N2 embryos ( F1 × B6 = 65 and B6 × F1 = 89 ) .",
"Individual embryos from 2 litters from each side of the F1 cross ( 2 B6 × Cast and 2 Cast × B6 ) , and 12 F1 × B6 litters and 18 B6 × F1 litters from the N2 cross were analyzed .",
"For all crosses , following identification of a vaginal plug on the morning after mating ( E0 . 5 ) , males were removed; pregnant females were housed separately .",
"Whole embryos were harvested at E11 . 5 and dissected to remove all embryonic membrane and placental material .",
"Somite numbers were recorded for each embryo at the time of dissection .",
"To isolate total RNA , individual embryos were homogenized in Trizol reagent ( GibcoBRL/Invitrogen , Grand Island , NY ) and RNA was extracted per manufacturer's directions .",
"Poly ( A ) + RNA was isolated using the Qiagen Oligotex Mini kit and 3SEQ libraries were constructed as described ( Beck et al . , 2010 ) .",
"For the N2 embryos , oligo ( dT ) and PCR primers were barcoded with Illumina multiplexing sequences .",
"F1 data were produced in several sequencing batches , first on an Illumina GAIIx sequencer producing 76 bp or 80 bp reads , then on an Illumina HiSeq 2000 sequencer producing 101 bp reads .",
"N2 data were produced only on the HiSeq 2000 .",
"Libraries from 12 individually barcoded N2 samples were pooled in equimolar amounts and sequenced in a single flow-cell lane to produce single-end 101 bp reads .",
"Prior to read mapping , 3′ poly ( A ) sequences likely to be derived from the mRNA poly ( A ) tail were trimmed using a procedure that tolerated some amount of sequencing error: we computed a score si for each position i in the read which was equal to the sum of the quality scores of each A after position i minus three times the sum of the quality scores of each non-A after position i .",
"All bases after the highest-scoring position were trimmed if this would result in trimming at least 3 bases .",
"The first 18 bases and the last 7 bases of the HiSeq F1 reads were trimmed to make them the same length as the GAIIx reads .",
"This was done to avoid read length being a confounding factor when analyzing ASE in F1 .",
"Only poly ( A ) stretches were trimmed from the N2 data .",
"A M . m .",
"castaneus genome was created in silico by applying the high-quality Cast SNPs ( Keane et al . , 2011 ) to the reference C57BL/6 genome mm9 .",
"For B6 , we used the mm9 genome .",
"For each of these two reference genomes , a transcriptome was computed using RefSeq annotations , including spliced transcripts of all genes separated by 200 nucleotides , and each spliced transcriptome was added to its reference genome sequence .",
"Bowtie2 ( Langmead and Salzberg , 2012 ) was used to map all reads to the composite genomes ( Cast or B6 including spliced transcripts ) with settings ‘-k 200 –score-min L , 0 , -0 . 10 –rdg 2 , 2 –rfg 2 , 2 –mp 1 , 1’ .",
"These settings recovered up to 200 sub-optimal genomic hits , allowing us to stringently filter reads derived from repetitive regions of the genome as follows: If the difference in edit distance ( 1 for each mismatch and 2 for each indel ) between a read's best alignment and its second-best alignment was within 3 , the read was discarded .",
"Reads were also filtered out unless all best alignments in the composite genome corresponded to the same genomic position ( e . g . , if a read aligned equally well to an intergenic region and to a splice junction in the transcriptome ) .",
"Each aligned read was then consolidated into a single genomic alignment , including intronic splice gaps .",
"After filtering , each F1 dataset had an average of 15 . 8 million mapped reads , and each N2 dataset had an average of 1 . 2 million reads .",
"Gene expression was quantified using only reads mapping to exons .",
"Read counts were typically normalized by the total number of sequenced reads per library , multiplied by 1 × 106 , resulting in RPM .",
"For the eQTL analyses , genes were removed if over 5% of the mapped reads were filtered based on the second-best hit criteria above .",
"Genes were also discarded if they matched the retrotransposed genes list compiled by UCSC ( Kent et al . , 2002 ) .",
"In the F1s , there were 13 , 574 genes with detectable expression ( 13 , 469 across N2s ) , 7554 of which passed the analysis threshold of 5 RPM ( 7712 in N2s ) .",
"We found Xist expression followed a bimodal distribution , and used this to identify females ( robust Xist expression ) and males ( little or no Xist expression ) .",
"All 5 genes significantly different between males and females , including Xist and its antisense transcript Tsix , were located on the X or Y chromosomes .",
"At E11 . 5 , embryos are just beginning sexual differentiation , so the small number of sex-specifically expressed genes is unsurprising .",
"Because of the small number of affected genes , as well as the repetitiveness of the sex chromosomes , subsequent analyses were restricted to genes and marker loci on autosomes .",
"Genes were assigned a preliminary genotype based on the proportion of B6-supporting reads , either ‘unknown’ if <20 SNP-covering reads were found , or homozygous or heterozygous otherwise .",
"For each chromosome , preliminary genotypes were then fed through a Hidden Markov Model ( HMM ) that penalized transitions between the heterozygous and homozygous states .",
"The HMM in effect smoothed the genotypes and allowed us to infer genotypes for unknown genes falling between confidently genotyped genes .",
"A post-processing step assigned the ‘unknown’ genotype to genes with low read coverage falling near putative recombination sites , typically affecting only a small number of genes for each chromosome .",
"We combined adjacent genes with identical genotypes across all embryos into a total of 986 marker regions across the 19 autosomes .",
"A more aggressive combining , allowing up to 5 recombination events across all N2 embryos , resulted in 286 marker regions .",
"Downstream analyses used the consensus genotype in each embryo , assigning the unknown genotype where the number of homozygous and heterozygous embryos was close to equal .",
"GO enrichment analyses were performed with GOrilla ( Eden et al . , 2009 ) , which enables easy visual inspection of results , and depletion analyses were performed using a hypergeometric test .",
"Only expressed genes ( or those with allele-specific read coverage ) were considered .",
"To avoid the possibility of small insertions and deletions causing misalignment around SNPs , ASE was quantified using only high quality annotated SNPs that were at least 50 bp from an annotated Cast indel of any quality , based on the published mouse polymorphism resource ( Keane et al . , 2011 ) .",
"For reads covering multiple SNPs , each SNP received 1/n votes for a given genotype ( Cast , B6 or other ) , to account for the low percentage of SNP positions with sequencing errors .",
"For example , a read covering 3 SNPs , 2 of which match B6 alleles and 1 that matches neither the B6 nor Cast allele , would contribute 0 . 67 read votes to the B6 expression total .",
"A read covering only a single SNP matching the Cast allele , would contribute a full 1 read vote to the Cast expression .",
"Because F1 and N2 ASE estimates largely agreed , ASE significance was estimated from the 170 pooled samples .",
"Significance was assessed by subtracting the B6 read votes from the Cast read votes , and then normalizing the differences based on library-size .",
"This was necessary because the F1 samples were sequenced to substantially more depth than the N2 samples .",
"A paired t-test was then applied to test for significance of these differences against 0 .",
"This simple approach takes full advantage of the magnitude of difference between the alleles as well as the variance in allelic expression across the large number of samples sequenced in this study .",
"We found that p-values calculated from the paired t-test correlated well with those derived by resampling , but were somewhat conservative .",
"Hence , our estimates of ASE are likely to be slight underestimates .",
"Additionally , genes were only called significant if their average BAF was less than 0 . 45 or greater than 0 . 55 .",
"Maternal effect target genes were identified by comparing expression normalized by library size from embryos with B6/B6 mothers to expression values from embryos with B6/Cast F1 mothers .",
"We used a Mann–Whitney U test to calculate the significance of this difference .",
"Because all F1 mothers used for the N2 backcross were derived from the B6 × Cast side of the cross , all N2 embryos had B6 mitochondria .",
"Maternal effects are similar in significance and direction when restricting analyses to only female embryos with two B6 X chromosomes , or when averaging expression across individuals in each litter .",
"Furthermore , the effects are consistent across somite numbers , even for genes that change expression during development .",
"To exclude batch effects as a potential cause of maternal effects , we used qPCR to validate candidate maternal effect target genes on an orthogonal platform .",
"0 . 5 μg total RNA per sample was reverse-transcribed into cDNA using Oligo ( dT ) 20 primer and Superscript III ( Invitrogen ) in 20 μl reactions , according to the manufacturer's instructions .",
"Following reverse transcription , each sample was treated with RNase H . 48 . 48 microfluidic dynamic array IFC chips ( Fluidigm ) were used to analyze the expression of 26 candidate maternal effect target genes and 15 control reference genes in 93 N2 samples .",
"1 μl of cDNA was pre-amplified using 2× Taqman PreAmp Master Mix ( Lifetech ) and 50 nM of each primer pair in 5 μl reaction volume , according to the manufacturer's instructions .",
"The cycling program was 10 min at 95°C followed by 10 cycles of 15 s at 95°C and 1 min at 60°C .",
"Following pre-amplification , each reaction was diluted fivefold .",
"RT-qPCR on the dynamic array chips was conducted on the BioMark system ( Fluidigm ) .",
"5 μl sample pre-mix containing 2 . 5 μl of SsoFast EvaGreen Supermix with Low ROX ( Bio-Rad ) , 0 . 25 μl of DNA Binding Dye Sample Loading Reagent ( Fluidigm ) and 2 . 25 μl of diluted pre-amplification samples , as well as 5 μl assay mix containing 2 . 5 μl of Assay Loading Reagent ( Fluidigm ) , 2 . 25 μl EB Buffer ( Qiagen ) and 0 . 25 μl of 100 μM primer pairs ( 500 nM in the final reaction ) were mixed on the chip using the IFC controller MX ( Fluidigm ) .",
"The thermal cycle was 60 s at 95°C followed by 30 cycles of 5 s at 96°C and 20 s at 60°C .",
"A dissociation curve was also drawn for each primer pair .",
"The following eight control genes were chosen based on high consistency between Fluidigm chips: Smarcc2 , Rnf216 , Nxf1 , Cnpy3 , Pmvk , Trmt1 , Fam149b , Farp1 .",
"These genes were used to normalize expression of the candidate maternal effect target genes using the standard curve method .",
"Significance of the maternal effect was calculated using the Mann–Whitney U test on the normalized expression values using a Bonferroni-corrected p = 0 . 05 cutoff .",
"Because of low consistency between Fluidigm chips , we were only able to validate four maternal effect candidate genes .",
"Between-sample read count comparisons showed some evidence of batch effects across N2 embryos , primarily at the level of sequencing lane .",
"To control for these batch effects , we used PEER ( Stegle et al . , 2012 ) to normalize for both unknown covariates as well as the following known batch effects: embryo sex , sequencing lane , and somite count .",
"For each gene and for each marker region , we performed a Wilcoxon rank-sum test comparing PEER-normalized expression in heterozygous and homozygous embryos .",
"We found that the total number of significant eQTL was substantially higher using PEER-normalized read counts compared to simple library-normalized read counts , although the fraction of trans-eQTL was not significantly different .",
"Results were also consistent when replacing the non-parametric ranksum test with the parametric t-test .",
"To remove the effects of correlated markers , downstream analyses used the most significant marker region on each chromosome for each target gene .",
"To estimate false discovery rates , marker–sample assignments were permuted and eQTL were called using an identical procedure to the unpermuted data .",
"Cis-eQTL were defined as those eQTL for which the marker region resides on the same chromosome as the target gene , and trans-eQTL were thus defined as the eQTL for which the marker region is on a different chromosome as the target gene .",
"While this is a conservative definition of trans-eQTL , we found no evidence of trans-eQTL residing on the same chromosome as the target gene .",
"We estimated expected ASE ( BAFpred ) from total expression levels using the following transformation for each gene:BAFpred=BAFexp2× ( 1−BAFexp ) , where BAFexp=μB6/B6μB6/B6+μB6/BCast , and μg are the mean total expression levels across all mice with genotype g at the gene of interest .",
"The BAFpred transformation is required to estimate μCast/Cast from μB6/B6 and μB6/Cast .",
"Because of variance in the estimation of μg , the predicted BAF can be larger than 1 . 0 .",
"We reanalyzed six previously published mouse eQTL datasets available from GeneNetwork ( Wang et al . , 2003 ) .",
"Array-normalized data were batch-normalized using PEER , then compared to genotype data provided by GeneNetwork .",
"Because these previous studies used an intercross model with three possible genotypes , eQTL were called using a linear regression , analogous to the t-test in our backcross experiment .",
"eQTL were also mapped using a random forest method ( Michaelson et al . , 2010 ) .",
"We used the normalized read counts as input to the random forest , and included marker genotype , sex , side of the cross , sequencing lane , and number of somites as predictors .",
"For 5 N2 embryos , no somite count was recorded , and hence the total number of input samples is 149 instead of 154 .",
"Genotypes were coded as 0 . 5 for heterozygous B6/Cast , 1 . 0 for homozygous B6/B6 , and 0 . 75 for missing genotypes .",
"False discovery rates were calculated as above using permuted input data after identifying the best markers per chromosome .",
"We found results were similar when using the variable selection frequency method ( Michaelson et al . , 2010 ) or the mean decrease in prediction accuracy method ( Francesconi and Lehner , 2014 ) .",
"We found GERP scores broadly correlated among upstream , UTR , coding and downstream gene regions , and therefore show only the average coding sequence GERP score .",
"We performed a Spearman correlation test to examine the hypothesis that GERP score is associated with cis-eQTL p-value and ASE effect size , although for clarity Figure 5G focuses only on the most extreme genes .",
"Because the set of very lowly expressed genes is enriched for low GERP scores , we filtered out the lowest expressed genes in each tissue .",
"We used RNA-seq data previously published ( Merkin et al . , 2012 ) for muscle , liver and brain to filter out lowly expressed genes in those tissue comparisons .",
"The strength of the associations with GERP scores for our ASE and cis-eQTL as well as previously published cis-eQTL is robust to all but the most extreme changes in filtering cutoff ."
]
] | [
"The effects of genetic variation on gene regulation in the developing mammalian embryo remain largely unexplored .",
"To globally quantify these effects , we crossed two divergent mouse strains and asked how genotype of the mother or of the embryo drives gene expression phenotype genomewide .",
"Embryonic expression of 331 genes depends on the genotype of the mother .",
"Embryonic genotype controls allele-specific expression of 1594 genes and a highly overlapping set of cis-expression quantitative trait loci ( eQTL ) .",
"A marked paucity of trans-eQTL suggests that the widespread expression differences do not propagate through the embryonic gene regulatory network .",
"The cis-eQTL genes exhibit lower-than-average evolutionary conservation and are depleted for developmental regulators , consistent with purifying selection acting on expression phenotype of pattern formation genes .",
"The widespread effect of maternal and embryonic genotype in conjunction with the purifying selection we uncovered suggests that embryogenesis is an important and understudied reservoir of phenotypic variation ."
] | [
"The way that the embryo of a mammal , such as a mouse or a human , develops from a fertilized egg is a complicated process that relies on controlling: which genes are active; when these genes activate; and for how long they are active .",
"In broad terms , there are four ways that this control can be achieved: First , inside the sperm or egg , genes can be marked with small chemical tags that flag these genes to be activated ( or remain inactive ) after fertilization , depending on whether the modification was made by the father ( in the sperm ) or the mother ( in the egg ) ; this process is known as ‘imprinting’ .",
"Second , the mother can alter the gene activity in her offspring via the placenta; this process is known as ‘maternal effect’ .",
"Third , instructions encoded within the embryo's DNA can directly control if , and when , a nearby gene becomes activated; this is known as ‘cis-regulation’ .",
"Finally , similar instructions can also control genes that are situated elsewhere in the embryo's DNA through indirect mechanisms; this is known as ‘trans-regulation’ .",
"Now , Spies , Smith et al . have investigated these four processes in the offspring of two different strains of mice , one originally from Europe and the other from Southeast Asia .",
"The two strains were crossbred and the resulting embryos were analyzed to see which of the four processes affected gene activity .",
"This analysis revealed 31 imprinted genes and 331 genes that exhibited a maternal effect—which sometimes changed gene activity by as much as 52% .",
"Spies , Smith et al . also found over a thousand DNA instructions in the embryo's DNA that could directly influence the activity of nearby genes , but fewer instructions that could indirectly control genes that were further away .",
"These results suggest that direct control of genes , which affects only the genes closest to the DNA instruction , can vary a lot between individual embryos of the same species .",
"However , indirect control of embryonically active genes , which affects many genes across the genome at the same time , appears much more tightly constrained by evolutionary forces .",
"Which genes in the mother are responsible for the molecular signals that drive the maternal effect is an important question for future work , with implications for the genetic basis of embryonic development and disease ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression"
] | Circular RNA biogenesis can proceed through an exon-containing lariat precursor | elife-07540-v2 | [
[
"Pervasive expression of circular RNA is a recently discovered feature of eukaryotic gene expression; these circular isoforms are ubiquitously expressed in humans and a number of highly diverged eukaryotic organisms and can exceed the level of the cognate mRNA ( Salzman et al . , 2012; Memczak et al . , 2013; Salzman et al . , 2013; Wang et al . , 2014 ) .",
"Rare examples of circular RNA expression had been known to exist for decades , but with a handful of exceptions , only known to exist at levels suggesting they were transcriptional noise or in vitro artifacts ( Nigro et al . , 1991; Cocquerelle et al . , 1992; Zaphiropoulos , 1996 ) .",
"The basic mechanism responsible for the biogenesis of circular RNA involves a ‘backsplicing’ reaction in which a branch point upstream of a circularized exon attacks a downstream splice donor , positioning the 3′ end of the exon to attack its own 5′ end in the second step ( Figure 1 ) ( Braun et al . , 1996; Pasman et al . , 1996; Schindewolf et al . , 1996 ) . 10 . 7554/eLife . 07540 . 003Figure 1 . Models for the production of circular RNA .",
"( Left )",
"Complementary sequences facilitate the biogenesis of circular RNA .",
"( Right )",
"Circular RNA biogenesis proceeds through a lariat precursor . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 00310 . 7554/eLife . 07540 . 004Figure 1—figure supplement 1 . mrps16 pre-mRNA predicted pair probabilities . Dot plot representation of the predicted minimum free energy ( MFE ) mrps16 pre-mRNA secondary structure .",
"This structure was predicted using the NUPACK web server .",
"The area highlighted by the orange box represents the region where interactions between flanking sequences would be predicted .",
"The nature of the nearby long-range interactions between exon 2 and intron 2/exon 3 is illustrated in the MFE structure of the mrps16 pre-mRNA . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 00410 . 7554/eLife . 07540 . 005Figure 1—figure supplement 2 . mrps16 pre-mRNA predicted MFE structure . Long-range interactions between exon 2 and intron 2/exon 3 are highlighted above .",
"The blue line traces the path of exon 2 , the orange line traces the path of intron 2 , and the green line traces the path of the first 17 nucleotides of exon 3 .",
"The vertical lines demarcate the start and end of each segment .",
"Note , these predicted long-range interactions do not extend to regions beyond exon 2 and thus , no extensive base-pairing interactions are predicted to form between the segments of the gene that flank the exon ( also depicted in the dot plot shown in Figure 1—figure supplement 1 ) .",
"The pink oval marks a highly structured region of exon 2 , which is noted later in our discussion of exonic sequence deletions . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 005 Until recently , one of the only known examples of an abundant circular RNA was discovered in the mouse sex-determining gene , SRY ( Capel et al . , 1993 ) .",
"This exon is flanked by long ( ∼15 kb ) inverted sequence whose complementarity is required for circularization ( Dubin et al . , 1995 ) .",
"Early in vitro studies also demonstrated that complementary sequences flanking an exon can promote circularization , though they did not appear necessary ( Pasman et al . , 1996 ) .",
"Modeled after work on SRY , recent studies in mammalian cell culture have found similar results in human genes ( Liang and Wilusz , 2014; Zhang et al . , 2014b ) , and additional genome-wide computational sequence analysis suggests this complementary sequence-mediated mechanism may be widespread and associated with Alu elements ( Jeck et al . , 2013; Zhang et al . , 2014b; Ivanov et al . , 2015 ) .",
"Although widespread in mammalian genomes , repeat sequences are far less common in the genomes of simple eukaryotes , indicating that an alternative mechanism may be at play in these organisms and in human genes that lack inverted repeats .",
"In addition , a recently published study in Drosophila fails to find a remarkable relationship between secondary structure and circular RNA biogenesis , noting that there are no noticeable sequence motifs in the introns flanking circularized exons besides canonical splice site sequences ( Westholm et al . , 2014 ) .",
"An alternative mechanism that has long been proposed in the literature involves an exon-containing lariat , which can serve as a precursor to the circularized exon ( Figure 1 , right ) ( Zaphiropoulos , 1996; Surono et al . , 1999; Burd et al . , 2010; Jeck et al . , 2013; Jeck and Sharpless , 2014 ) .",
"An early study noted a correlation between exon-skipping events and circular RNA isoforms in the human cytochrome P450 2C18 gene , describing four alternative circularization events and their corresponding exon-skipped transcripts ( Zaphiropoulos , 1996 ) ; a similar correspondence was noted in the dystrophin gene ( Surono et al . , 1999 ) .",
"Recently , a study in human cell culture posits a correlation between exon skipping and circular RNA biogenesis in a model gene , but treats the exon-skipping event as a byproduct rather than a precursor of the backsplicing event , regarding inverted sequences as the determinant of circularization ( Zhang et al . , 2014b ) .",
"Indeed , the only current evidence for this model is based on correlation between exon skipping and circular RNA production in a handful of genes .",
"No biochemical evidence of the activity of this pathway has been provided , and no global relationship exists between exon skipping and circular RNA biogenesis .",
"Due to the large size of human genes , cell culture studies have been unable to ectopically express entire circle-forming genes on a plasmid and therefore have not been able to completely recapitulate splicing from the endogenous locus .",
"Instead , expressed minigenes containing circle-forming exons and immediately flanking sequence have been used as surrogates ( Ashwal-Fluss et al . , 2014; Liang and Wilusz , 2014; Zhang et al . , 2014b; Starke et al . , 2015 ) .",
"These expression vectors may produce results that are due to characteristics of transcription in the minigene and , in some cases , generate large amounts of off-target splice products .",
"The ability to recapitulate the splicing of an endogenous locus is a powerful tool for studying the mechanism of circular RNA production .",
"For this reason , we turned to Schizosaccharomyces pombe as our model system for studying this process .",
"With a simple and small gene structure and relatively high expression of some circular RNAs , S . pombe is an ideal system for studying circular RNA biogenesis .",
"Because the second exon of mrps16 is the most highly expressed circular RNA in this organism ( Wang et al . , 2014 ) , we have adopted mrps16 as our model RNA for studying circular RNA production in the present work .",
"Utilizing the lariat debranching mutant ( dbr1∆ ) , we provide direct evidence of an exon-containing lariat precursor and the double lariat resulting from lariat re-splicing ( Figure 1 , right ) .",
"Furthermore , by expressing mrps16 on a plasmid , we completely recapitulate the quantitative relationship between circular and linear RNA expression in this gene , and through systematic splice site deletions of this plasmid , we demonstrate the necessity of a lariat precursor for circular RNA biogenesis .",
"However , lariat production is not sufficient for circularization , as demonstrated by the existence of several exon-containing lariats that do not give rise to circular RNAs , indicating an additional parameter regulating their production .",
"Overall , circularized exons are generally much larger than these skipped , uncircularized exons .",
"To test if the length of circularized exons directly affects their circularization efficiency ( CE ) , we utilized high-throughput and comprehensive mutagenesis of the mrps16 exon , which revealed that exon size is a significant determinant of RNA circularization ."
],
[
"We considered two models for the production of circular RNA in mrps16: that circular RNA is generated by ‘direct backsplicing , ’ perhaps facilitated by complementary sequence ( Figure 1 , left ) , or through a lariat precursor ( Figure 1 , right ) .",
"Each model predicts a distinct set of biochemical intermediates and byproducts: a Y-shaped intermediate in the case of direct backsplicing; and a lariat precursor and double lariat in the case of the lariat model .",
"These structurally distinct species discriminate the models , and we reasoned that determining which of these products exists could provide initial evidence for one or both of these models .",
"Before performing biochemical identification of these intermediates , we computationally predicted the structure of the mrps16 pre-mRNA to determine if there were any base-pairing interactions across the circularized exon .",
"We found that mrps16 lacks any apparent base pairing between the regions flanking the circularized exon ( Figure 1—figure supplements 1 , 2 ) ; thus , we hypothesized that the biogenesis of this circular RNA might proceed through a lariat precursor generated by exon skipping .",
"As canonical lariats are of low abundance and difficult to detect in wild-type fission yeast , we used a strain of yeast ( dbr1∆ ) lacking the debranching enzyme , which catalyzes the hydrolysis of the branch point 2′–5′ linkage , the first step of lariat decay ( Ruskin and Green , 1985 ) .",
"In this mutant , branched species can reach a much higher steady state concentration ( Nam et al . , 1997 ) .",
"We attempted to detect each of the products described above by reverse transcription-PCR ( RT-PCR ) , Sanger sequencing all major products to determine their identities .",
"Using outward-facing primers , we detected the canonical lariat byproducts from linear splicing , formed by the introns of mrps16 in dbr1∆ yeast ( Figure 2A , Lanes 3–6 ) along with the circularized form of exon 2 in both strains ( Figure 2A , Lanes 7–8 ) .",
"Although mrps16 is not known to have exon skipping , we easily detected the exon-containing lariat resulting from exon skipping ( i . e . , the lariat precursor ) ( Figure 2A , Lane 10 ) and verified the product's sequence through Sanger sequencing ( Figure 2B , top ) .",
"Using inward-facing primers that would amplify a linear exon 1–3 splice , we did not detect an exon-skipped transcript in either strain ( Figure 2A , Lanes 1–2 ) .",
"This result is in agreement with recent evidence from deep-sequencing experiments , which revealed that while exon skipping is quite pervasive in S . pombe , most exon-skipped transcripts are subject to nuclear decay and are thus detectable only in mutants deficient in decay ( Bitton et al . , 2015 ) .",
"Specifically , exon-skipped transcripts for mrps16 can compose up to 5% of all detected mrps16 transcripts in S . pombe mutants deficient in Dis3 , a nuclear exosome component ( Bitton et al . , 2015; See Supp . Table S5 ) .",
"This represents a >30-fold increase over wild-type S . pombe . 10 . 7554/eLife . 07540 . 006Figure 2 . RT-PCR analysis of circular RNA biogenesis intermediates .",
"( A ) reverse transcription-PCR ( RT-PCR ) of splice products and byproducts of mrps16 pre-mRNA in wild-type and dbr1∆ S . pombe .",
"Primers used are indicated above each lane of the gel .",
"Red dots represent the location of branch points .",
"The observed products are shown below the gel .",
"Laddering of bands in lanes 7 and 8 is due to rolling-circle reverse transcription of the 181 nt mrps16 circular RNA .",
"( B ) Sequencing trace for product in Figure 2A , lane 10 ( Top ) and Figure 2D lane 12 ( Bottom ) ( each marked with * on respective gels ) .",
"Due to the 2′–5′ linkage at the branch point , there is base misincorporation or nucleotide skipping at the branch point by the reverse transcriptase ( highlighted in trace ) .",
"Note , the top trace is mirrored for consistency with the orientation in the graphic representation .",
"( C ) Strategy to enrich for the byproduct of circular RNA backsplicing event .",
"Since the circularized exon of mrps16 contains a DraI restriction site , we can deplete PCR products containing the exon .",
"( D ) RT-PCR results after RNase R and/or DraI treatment ( treatment indicated above the gel ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 00610 . 7554/eLife . 07540 . 007Figure 2—figure supplement 1 . qPCR analysis of mrps16 splice isoforms . The relative proportions of the species computed by subtracting mean Ct values from mean Ct values of the linear RNA .",
"Error bars represent standard deviations from replicate experiments .",
"Percentages are computed assuming perfect qPCR primer efficiency for all primers and are noted beneath the bars . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 00710 . 7554/eLife . 07540 . 008Figure 2—figure supplement 2 . Figure 2D , Lane 12 cloning and sequencing results . The products from Figure 2D , Lane 12 ( reproduced above ) were cloned , and 16 colonies were chosen for PCR analysis .",
"Four distinct groups of products were observed , labeled A , B/C , D , and ‘undetected’ .",
"All products were sequenced and the results of each group of bands are described here .",
"Products B/C correspond to the ‘doublet’ product observed in lane 12; the alignment of these sequences is shown in Figure 3—figure supplement 3 .",
"‘Undetected’ refers to a PCR product that is not visible in the gel representing the original PCR reaction . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 00810 . 7554/eLife . 07540 . 009Figure 2—figure supplement 3 . Alignment of backsplice byproduct PCR products . The group of products labeled B/C in the lower gel in Figure 2—figure supplement 2 was sequenced and aligned using ClustalW2 .",
"The lane numbers to the left of the sequences correspond to the lower gel shown in Figure 2—figure supplement 2 .",
"The length of each product is shown at the end of each sequence . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 00910 . 7554/eLife . 07540 . 010Figure 2—figure supplement 4 . Additional DraI restriction digest PCR for Y-shaped intermediate product . Panel 2D with additional lanes indicating attempted amplification of the Y-shaped intermediate .",
"As in 2D , primers used are indicated above the gel and observed products are illustrated below .",
"The off-target product is the higher molecular weight species ( lane 13 ) and represents mispriming of the reverse primer to a downstream site in exon 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 010 To quantify the relative abundance of the mprs16 linear , circular , and exon-containing lariat species , we performed qPCR on RNA from both wild-type and dbr1∆ yeast ( Figure 2—figure supplement 1 ) .",
"We observed that the circular RNA is expressed at ∼one-eighth the expression of the linear mRNA , and in dbr1∆ yeast , the exon-containing lariat is expressed at ∼1/30th the expression of the linear RNA .",
"To test for the branched sequence indicative of the double lariat and Y-shaped intermediate ( see Figure 1 ) , we performed a PCR using inward-facing primers in the introns of mrps16 ( Figure 2A , Lanes 11–12 ) , but we were only able to recover an unspliced product containing exon 2 , which could be derived from genomic DNA , pre-mRNA , and the exon 2-containing lariat; the high abundance of these species impedes the detection of the putative double lariat/Y-shaped intermediate products .",
"To deplete these species prior to PCR amplification , we used the restriction enzyme DraI , which cuts uniquely within exon 2 of mrps16 ( see schematic in Figure 2C ) .",
"In parallel , prior to cDNA synthesis , we treated RNA with RNase R , a 3′–5′ exonuclease , which degrades linear species but cannot degrade circular RNA or lariats , or with a mock treatment not containing RNase .",
"We tested the sensitivity of the exon-containing lariat and linear mRNA to DraI and RNase R treatment as controls: RNase R decreases the abundance of the mRNA only , while treatment with DraI decreases the abundance of both mRNA and the exon-containing lariat ( Figure 2D , Lanes 1–8 ) .",
"Because of its high expression , there was still a significant amount of linear mRNA after each of these treatments , and treating with both DraI and RNase R had an additive effect on its depletion ( Figure 2D , Lanes 1–4 ) .",
"Using this combined depletion , we again attempted to amplify the branched sequence indicative of the backsplicing event with inward-facing primers in the introns of mrps16 .",
"As before , we amplified a dominant product containing exon 2 after treating with DraI or RNase R separately .",
"However , after the combined treatment , we discovered a new product at the expected size ( 518 bp ) for the branched byproduct diagnostic of backsplicing ( Figure 2D , Lane 12 ) ; this product is also faintly visible in singly-treated lanes ( Figure 2D , Lanes 10 and 11 ) .",
"Sanger sequencing revealed the expected sequence for the product with a misincorporation event at the branch point , which can arise during reverse transcription of lariats ( Figure 2B , bottom ) ( Vogel et al . , 1997 ) .",
"We noted the ‘doublet’ appearance of this product , and through thorough characterization by cloning and sequencing , we determined that the two bands do not represent distinct splice isoforms and are likely a technical artifact due to anomalous migration on our gel ( Figure 2—figure supplements 2 , 3 ) .",
"The enrichment of this product after RNase R treatment is strong evidence that this PCR product was derived from the double lariat rather than the Y-shaped intermediate , as the latter would be degraded by RNase R . In addition , by moving the forward primer upstream by only ∼100 bp into exon 1 , we were unable to detect a PCR product indicative of the Y-shaped intermediate ( predicted size 634 bp ) after treatment with DraI , RNase R , or both ( Figure 2—figure supplement 4 , Lanes 13–16 ) .",
"With evidence for the existence of the putative lariat precursor and double lariat , we performed genetic tests to determine if this model is the dominant pathway for circular RNA biogenesis in mrps16 .",
"We cloned the mrps16 gene in its entirety , along with 1 kb upstream and downstream into a plasmid , which allows the gene to be expressed using its natural promoter and terminator .",
"Because mrps16 is an essential gene , we expressed the plasmid copy of the gene in addition to its genomic copy .",
"In dbr1∆ S . pombe , the plasmid expression of the gene is roughly 10 fold higher than the expression from genomic copy , likely due to the high-copy number of the plasmid .",
"The ratio of the dominant splice isoforms of mrps16 ( i . e . , circular , linear , and lariat precursor ) is consistent between the genomic and plasmid copy of the gene , indicating expression from the plasmid does not bias the splicing pattern in a significant way ( Figure 3A ) .",
"In addition , the circular RNA produced from the plasmid is RNase R resistant and , notably , exhibits the same level of RNase R resistance as the genomic copy ( Figure 3B ) , strong evidence that the plasmid generates a circle rather than a spliced linear concatamer of exons . 10 . 7554/eLife . 07540 . 011Figure 3 . Quantitative analysis of mrps16 splice isoforms in dbr1∆ S . pombe reveals circular RNA can be produced through an exon-containing lariat precursor .",
"( A ) qPCR quantification of splice isoforms produced by ectopically expressed mrps16 relative to background expression of each isoform .",
"( B ) qPCR quantification of RNase R resistance of the endogenously and ectopically expressed mrps16 circular and linear RNA isoforms .",
"Positive values represent enrichment , while negative values represent depletion after RNase R treatment .",
"( C–D ) qPCR quantification of ectopically expressed mrps16 circular RNA ( C ) and lariat precursor RNA ( D ) for splice site and branch point mutants relative to background .",
"Cryptic splicing of the 3′ ss in intron 1 generates a circular RNA whose junction differs by 5 nt , and cryptic splicing of the 5′ ss in intron 1 generates a larger lariat precursor using a 5′ ss positioned 74 nt upstream of the original splice site .",
"( E ) Simplified kinetic framework for the production and decay of circular RNA .",
"Under this model , the values presented in ( F ) represent log2 ( k2mut/k2wt ) .",
"( F ) Ratio of circular to lariat precursor RNA for splice site and branch point mutants relative to background ratios .",
"Red ( predicted value < 0 ) and green ( predicted value = 0 ) coloring represents predictions under the lariat model .",
"These predictions neglect any effects due to cryptic splicing .",
"In all cases , error bars represent standard deviations from replicate experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 01110 . 7554/eLife . 07540 . 012Figure 3—figure supplement 1 . Agarose gel electrophoresis of mrps16 qPCR products . Primers used are indicated above the gel , and the splice site mutants tested are indicated directly below the gel using a number key .",
"Cryptic splicing of a large lariat precursor using a 5′ ss in exon 1 is responsible for the size shift observed in lane 11 . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 01210 . 7554/eLife . 07540 . 013Figure 3—figure supplement 2 . Effect of 5′ ss and BP double deletion on mrps16 circularization . qPCR quantification of ectopically expressed mrps16 circular RNA .",
"Splice site and branch point mutations are noted as in 3C .",
"Values for EV , wt mrps16 , and 5′ ss deletion are repeated here for reference .",
"In all cases , error bars represent standard deviations from replicate experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 01310 . 7554/eLife . 07540 . 014Figure 3—figure supplement 3 . Analysis of S . pombe pub1 circular RNA splicing .",
"( A ) Architecture of the pub1 gene along with sequencing traces for exon-skipped lariats .",
"Traces were derived from Sanger sequencing of RT-PCR products from dbr1∆ S . pombe .",
"( B ) RT-PCR for pub1 circular and linear RNA .",
"Isoforms of the pub1 ( wt and ∆BP ) are indicated below the gel .",
"( C ) qPCR quantification of circular RNA isoforms .",
"Expressed form of pub1 is indicated on the bars , and circular isoform tested is shown to the left .",
"Experiments shown in ( B ) and ( C ) were both performed in a pub1∆ background with pub1 expressed ectopically on a plasmid .",
"Error bars represent standard deviations from technical replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 014 Using the plasmid copy of mrps16 , we systematically deleted each of the splice sites and branch points in the introns of the gene and transformed these mutants into dbr1∆ S . pombe .",
"As predicted by both models ( Figure 1 ) , qPCR confirmed that the branch point upstream of the circularized exon and the 5′ ss in intron 2 are both necessary for circularization to occur ( Figure 3C ) .",
"Some of our mutagenesis experiments were confounded by activation of cryptic splice sites in mrps16 .",
"For example , deletion of the 3′ ss upstream of the circularized exon has almost no impact on circular RNA production ( Figure 3C ) .",
"This is due to cryptic splicing at a nearby 3′ ss located 5 nt downstream of the original splice site ( determined by Sanger sequencing , data not shown ) .",
"Both the 5′ ss of intron 1 and branch point of intron 2 are uniquely necessary for circular RNA biogenesis under the lariat model , which predicts that deleting either of these sites should disrupt circularization .",
"On the other hand , neither of these sites play necessary roles in the direct-backsplice model; if anything , their deletion might be expected to increase circularization by decreasing competition from linear splicing .",
"As predicted by the lariat model , deletion of the downstream branch point abolishes circular RNA biogenesis from the plasmid ( Figure 3C ) , and indeed this is the only mutant in which no lariat precursor is produced above background ( Figure 3D ) .",
"In contrast , deletion of the 5′ ss of intron 1 results in cryptic splicing of a larger lariat species ( Figure 3D ) , containing 74 nt of the upstream exon ( Figure 3—figure supplement 1 , Lane 11; product validated by sequencing ) and also results in circular RNA production ( Figure 3C ) .",
"Using steady state , first-order kinetics ( Figure 3E ) to model circular RNA production in each of the mutants , we tested whether our qPCR data were consistent with the kinetic predictions of the lariat model .",
"This model predicts that the fold change in rate of the second round of splicing corresponding to a given splice site deletion ( k2mut/k2wt ) is equal to the relative circle:lariat ratio ( plotted for each mutant in Figure 3F ) .",
"Under this model , deletions which do not perturb the kinetics of the second round of splicing will have an unchanged circle:lariat ratio .",
"Neglecting cryptic splicing , deletion of the 5′ ss of intron 1 and the branch point and 3′ ss of intron 2 should have a constant circle:lariat ratio , and generally this association holds ( Figure 3F , green bars ) .",
"Another prediction of the lariat model is that deletions that disrupt the second round of splicing should have a dramatically lower circle:lariat ratio .",
"As predicted , deletions of the branch point in intron 1 and flanking splice sites drastically reduce the rate of circular RNA biogenesis from the lariat precursor , which accumulates relative to the circular RNA ( Figure 3F , red bars ) .",
"We observed that both mutants impacting the first round of splicing in the lariat model produce slightly more circular RNA than would be predicted under this simple kinetic model .",
"Notably , the 5′ ss deletion in intron 1 fails to behave as a naive model would predict due to cryptic splicing that generates a variant exon 2-containing lariat .",
"To determine if the lariat precursor remains the major pathway for circular RNA biogenesis in this mutant , we created an mrps16 mutant containing both deletion of the upstream 5′ ss and downstream branch point ( to halt production of the lariat precursor ) , and , indeed , circular RNA production is strongly abated ( Figure 3—figure supplement 2 ) .",
"Still , for this double mutant and the single downstream branch point mutation ( both of which are unable to produce a lariat precursor ) , circular RNA production is slightly higher than predicted under a model relying exclusively on a lariat .",
"Thus , we cannot rule out that there may be a small population of circular RNA generated via a direct backsplice mechanism , as previously reported in vitro ( Pasman et al . , 1996; Schindewolf et al . , 1996 ) .",
"To determine whether the lariat model is unique to mrps16 , we mutagenized another circle-forming gene , pub1 .",
"pub1 contains four exons and produces two alternative circular isoforms: one composed only of exon 3 ( Wang et al . , 2014 ) and another , much less abundant isoform , consisting of exon 2 and exon 3 along with the intervening intron .",
"From dbr1∆ S . pombe , we detected the corresponding lariat precursors to each circular RNA isoform by RT-PCR and sequenced the products to confirm ( Figure 3—figure supplement 3A ) .",
"To test whether the lariat precursor is necessary for circularization in this gene , we created a plasmid containing the pub1 gene , with and without a deletion in the downstream branch point .",
"Because pub1 is non-essential , we performed these experiments in a pub1∆ strain to eliminate linear and circular RNA production from the genomic locus .",
"RT-PCR and qPCR analysis revealed a large decrease in circular RNA expression after branch point deletion ( Figure 3—figure supplement 3B , C ) , but circular RNA and fully spliced mRNA could still be detected , as a weak cryptic branch point was activated in the intron as evidenced by expression of the fully spliced linear isoform ( Figure 3—figure supplement 3B , Lane 4 ) .",
"Together , the existence of exon-containing lariats and their necessity for efficient circle production suggest that the dominant pathway for circular RNA biogenesis in S . pombe is through a lariat precursor .",
"Our findings raise the question: is production of an exon-containing lariat sufficient to produce circular RNA in S . pombe ?",
"There are only 14 reported examples of validated , stable exon-skipping events in wild-type S . pombe ( Awan et al . , 2013 ) , which would be predicted to produce such lariats .",
"Stable exon skipping could be sufficient for circular RNA production , but it is not required; as is the case for mrps16 , the exon-skipped transcripts may be quite unstable yet produce exon-containing lariats competent for circle formation .",
"We have chosen to test genes with stable exon-skipped transcripts because they are known to produce exon-containing lariats .",
"To assess the sufficiency of exon-containing lariats for circular RNA production , we used RT-PCR to test for these lariats and their putative corresponding circular RNAs ( except in two cases where the exons were under 40 nt long ) .",
"With both pairs of outward-facing primers , we observed bands indicative of exon-containing lariats based on their predicted migration and , in some cases , we observed larger PCR products consistent with rolling-circle reverse transcription of these lariats ( examples shown in Figure 4 ) ; however , no circular RNA from any of these loci in either wild-type or dbr1∆ S . pombe was detected ( Figure 4 , asterisks ) , demonstrating that exon skipping is not sufficient for detectable levels of circular RNA . 10 . 7554/eLife . 07540 . 015Figure 4 . Production of exon-containing lariats is not sufficient to generate circular RNA . RT-PCR analysis of a number of genes with validated exon-skipping events in S . pombe .",
"The primers pairs used are indicated below the gel .",
"Primer pair ‘C’ should amplify the circular RNA or the lariat precursor if the circle is absent , whereas primer pair ‘LP’ should specifically amplify the lariat precursor .",
"For each gene , asterisks indicate the expected product size for the circular RNA .",
"In each case , only exon-containing lariats from the exon-skipping events could be detected . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 015 We observed that skipped exons that did not produce circular RNA ( Figure 4 ) were significantly shorter compared to the sizes of the predicted circularized exons in S . pombe ( Figure 5A ) .",
"Previous genome-wide reports in human cells have demonstrated that large exons appear to be preferentially circularized in human cells ( Zhang et al . , 2014b ) .",
"However , these associations could be due to technical biases or confounding factors that are correlated with exon length ( e . g . , small , skipped exons may also contain exonic splicing silencers that promote exon skipping but restrict RNA circularization ) . 10 . 7554/eLife . 07540 . 016Figure 5 . Exon size predicts circular RNA production .",
"( A ) Notched box plots depicting the sizes of internal exons ( n = 2831 ) , single exon circular RNAs predicted from RNA-seq ( n = 86 ) , and skipped uncircularized exons ( n = 11 ) in S . pombe .",
"The notches represent the 95% confidence interval of the median ( specifically , ± 1 . 57 × IQR/√n , where IQR is interquartile range , and n is sample size ) .",
"p-values , denoted above the plots , were calculated using a Wilcoxon rank sum test .",
"Note only single exon circular RNAs were included in this analysis .",
"( B ) qPCR analysis of circular RNA production from an mrps16 isoform lacking 90 nucleotides from the center of its second exon ( mrps16 ∆90 ) .",
"Total circular RNA was measured using outward-facing primers outside the bounds of the deletion and normalized to total circular RNA produced by the endogenous locus .",
"Error bars represent standard deviations from replicate experiments .",
"( C ) Schematic outlining the production of mrps16 exon plasmid deletion library and quantitative RNA-seq ( qRNA-seq ) method for determining isoform-specific circular RNA expression .",
"( D ) Circularization efficiency ( CE ) vs exon length as determined by qRNA-seq experiment .",
"Outliers are noted on the plot using a parenthetical notation ( x , y ) , where x and y represent the first and last nucleotide of deletion , respectively , with 1 representing the first nucleotide of the exon .",
"Error bars represent two standard deviations from the mean ( see ‘Materials and methods’ for derivation ) .",
"Experiments shown in ( B ) and ( D ) were performed in wild-type S . pombe . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 01610 . 7554/eLife . 07540 . 017Figure 5—figure supplement 1 . RT-PCR analysis of circular RNA produced by endogenous mrps16 and mrps16 ∆90 . Primer pairs are indicated above the gel .",
"Primer pair wt is a set of primers specific to the wt mrps16 circular RNA , while primer pair ∆90 is specific to the deleted form of the exon .",
"Expressed form of mrps16 is shown below the gel and is expressed in addition to the endogenous copy . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 01710 . 7554/eLife . 07540 . 018Figure 5—figure supplement 2 . Measurement of RNA extraction size bias by qPCR . qPCR analysis of circular RNA production from mrps16 ∆90 .",
"Total circular RNA was measured using outward-facing primers outside the bounds of the deletion , while ∆90 circular RNA was measured using primers specific to the deletion isoform ( see Figure 5—figure supplement 1 ) .",
"All bars are normalized to total circular RNA produced by endogenous circular RNA ( first data point; empty vector ) .",
"Error bars represent standard deviations from technical replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 01810 . 7554/eLife . 07540 . 019Figure 5—figure supplement 3 . qPCR analysis of splice isoforms in mrps16 ∆90 . ( A ) qPCR quantification of mrps16 linear junctions , measured for both wt mrps16 and mrps16 ∆90 in wild-type S . pombe .",
"( B ) qPCR quantification of mrps16 circular RNA and lariat precursor in dbr1∆ S . pombe .",
"Error bars represent standard deviations from technical replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 01910 . 7554/eLife . 07540 . 020Figure 5—figure supplement 4 . qPCR analysis of splice isoform representation in exonic deletion library .",
"( A ) qPCR quantification of circular , linear , and lariat precursor abundance in exonic deletion library cDNA .",
"Primers indicated above plot and relative contribution of each species to the library are shown as percentages below each bar .",
"( B ) Quantification of circular RNA biogenesis from a wt mrps16 plasmid with three different primer pairs , each of which was used to construct a sequencing library .",
"In the absence of RNase R , inward-facing primers lead to much higher levels of PCR amplification due to amplification from linear RNA .",
"In contrast , with RNase R , the three primer pairs give essentially identical Ct values , indicating that the linear RNA is not largely contributing .",
"Note , we chose not to perform this experiment with our actual exonic deletion library , as variations in Ct values between the libraries will likely only reflect variability in the deletion isoforms detected and the expression levels of those isoforms , rather than contributions of various splice isoforms under the given treatment . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 02010 . 7554/eLife . 07540 . 021Figure 5—figure supplement 5 . Primary sequence analysis of mrps16 exonic deletion library . CE is plotted with respect to the position of each exonic deletion , depicted as horizontal bars extending from the start to end of the deletion . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 02110 . 7554/eLife . 07540 . 022Figure 5—figure supplement 6 . mrps16 exon 2 predicted structures . Structures represent the predicted MFE secondary structures at 30°C .",
"Deletion isoform is indicated above each structure along with its end-to-end length .",
"These structures were predicted using the NUPACK web server .",
"Parenthetical notation as in Figure 5D . DOI: http://dx . doi . org/10 . 7554/eLife . 07540 . 022 In order to test this directly , we made a 90 nt deletion in mrps16 and found that this deletion had a dramatic effect on circular RNA abundance ( total circular RNA does not differ significantly from background , Figure 5B ) .",
"Due to the unique sequence of the deletion , we designed primers to specifically amplify the ∆90 nt mrps16 circular species using a primer that spans the junction of the deletion ( Figure 5—figure supplement 1 ) .",
"Using this primer , we quantified the levels of the ∆90 nt circle and showed the expression of this isoform was ∼eightfold below endogenous circular RNA ( Figure 5—figure supplement 2 ) .",
"Given the high-copy number of the plasmid ( >10 per genome ) , this represents a >80 fold reduction in circular RNA produced by this deletion .",
"We reasoned that this large effect could be due to a bias against retaining small RNA in our RNA extraction procedure .",
"To address this concern , we subjected the purified RNA to a second round of extraction and purification .",
"If this procedure is biased against small RNAs , a second round of extraction would decrease the apparent ratio of ∆90 nt ( ectopic ) circle to full-length ( endogenous ) circle .",
"However , there was no relative depletion of small species after the second purification , supporting the conclusion that this deletion has a biological underpinning rather than being a technical artifact ( Figure 5—figure supplement 2 ) .",
"To determine whether this effect was simply caused by an increase in competitive splicing of linear mRNA in this mutant , we quantified the expression of both mRNA splice junctions but did not find a significant difference in their expression compared to wt mrps16 ( Figure 5—figure supplement 3A ) .",
"Another potential model is that this 90 nt deletion causes a decrease in the production of the lariat precursor , leading to a decrease in observed circular RNA .",
"In order to test this , we quantified both the abundance of the circular RNA and the lariat precursor in dbr1∆ S . pombe .",
"Contrary to this model , we observe an increase in the abundance of the lariat precursor relative to the circular RNA , consistent with this mutation causing a decrease in the rate of the second round of splicing ( Figure 5—figure supplement 3B ) .",
"Two non-mutually exclusive models could explain the decreased abundance of circular RNA from the 90 nt deletion: ( 1 ) small circles in mrps16 do not efficiently circularize due to their size ( e . g . , physical constraints ) ; ( 2 ) our deletion has removed a binding site for a trans-acting factor responsible for circularization .",
"To achieve a rapid and high-throughput test of these models , we used a quantitative RNA-seq ( ‘qRNA-seq’ ) protocol ( Figure 5C ) .",
"We generated a pooled library of varying length deletions within exon 2 of mrps16 by combinatorial inverse PCR and blunt ligation , using the wt mrps16 plasmid as a template , and transformed this pool of mutants into wild-type S . pombe .",
"Next , we extracted RNA and plasmid DNA from the population .",
"The RNA was enriched for circular isoforms by DNase and RNase R treatment and then reverse transcribed .",
"The cDNA and plasmid DNA were PCR amplified using the same pair of inward-facing primers and cycle numbers to control for any potential sequence-specific PCR biases; barcoded-sequencing adapters were further added by PCR .",
"Separate sequencing libraries were created at different cycle numbers to allow analysis of biases introduced by PCR .",
"To ensure that circular RNA species generated the vast majority of RNA-seq reads , we performed qPCR and demonstrated that:",
"( a ) the linear RNA was represented ∼40 fold below the expression of the circular RNA due to RNase R treatment and",
"( b ) the lariat precursor was represented at ∼300 , 000 fold below circular RNA , as expected since this experiment was performed using wild-type yeast ( Figure 5—figure supplement 4A ) .",
"We also showed that inward- and outward-facing primer pairs gave very similar qPCR values when used on RNase R-treated RNA , indicating both quantitate mainly circular isoform ( Figure 5—figure supplement 4B ) .",
"As an additional control , we created RNA-seq libraries with outward-facing primers , which are unable to amplify linear species .",
"In our library , each plasmid with a deletion contains a unique sequence within exon 2 corresponding to the deletion junction ( Figure 5C ) , allowing the abundance of each circular RNA isoform to be quantified ( from the RNA-seq libraries ) and normalized by the representation of each plasmid in the population ( from the DNA-seq libraries ) .",
"By quantifying the wild-type levels of circularization , we represented our quantitative results as relative circularization efficiency ( CE ) for each mutant ( where the value 1 represents wild-type levels of circularization from the plasmid ) .",
"We tested for an effect of deletion length on CE and found a strong systematic relationship between exon length and circularization ( Figure 5D ) .",
"This trend was maintained across sequencing libraries , which vary in cycles of library amplification and primer pairs ( data not shown ) .",
"Note , this plot does not include the 90 nt deletion discussed above , which we analyzed separately by clonal analysis .",
"While exon length is significant in our model , it is not completely predictive of CE , indicating the existence of other factors that could potentially affect circularization .",
"This library can contain multiple representatives for any given deletion size and for any given deletion of a primary sequence motif .",
"This allows us to rapidly test first order predictions that efficient circle production relies on the presence of a trans-acting factor binding site .",
"However , our data revealed no relationship between the location of a deletion ( i . e . , its inclusion of any primary sequence motif ) , and its representation in the library .",
"Thus , we were unable identify a primary sequence required for circularization ( see Figure 5—figure supplement 5 ) .",
"Analysis of outliers in our mrps16 mutagenesis data revealed that essentially all data points with higher-than-expected rates of circularization represented deletions in a region at the 3′ end of the exon that is predicted to be rich in secondary structure .",
"A hairpin , spanning from 135 to 159 , is predicted to form with high probability , and deletions that remove half of this hairpin are responsible for the most significant outliers ( Figure 5—figure supplement 6 ) .",
"These outliers suggest a model in which secondary structures may limit the flexibility of the exon: removing pieces of these secondary structures can destabilize them , increasing the effective length of the circularized exon .",
"We predicted the minimum free energy ( MFE ) structures of exon 2 for several of these deletions and found that these deletions maintained the apparent , end-to-end length of the exon and in some cases actually increased the apparent length ( Figure 5—figure supplement 6 ) .",
"We also found that deleting the entire hairpin ( 89 , 171 ) negated the enhancement in circularization ( not shown ) , and in agreement , the predicted structure for this deletion contains far fewer free nucleotides in this case ( 17 vs 37 for wt ) ( Figure 5—figure supplement 6 ) .",
"This indicates that outliers marked in Figure 5D do not simply remove a repressor's-binding site .",
"Despite this suggestive data that exonic structure is important for circular RNA production in mrps16 , we were unable to predict CE by computational structure prediction alone ( not shown ) .",
"This may reflect an inability of structure prediction methods to accurately predict in vivo RNA secondary structure or , instead , may reflect additional yet-to-be discovered regulatory features ."
],
[
"Circular RNA is a pervasive aspect of eukaryotic gene expression , and recent work has begun to shed light on the regulation and biogenesis of these still largely mysterious RNAs ( Ashwal-Fluss et al . , 2014; Liang and Wilusz , 2014; Zhang et al . , 2014b; Conn et al . , 2015 ) .",
"Current models for the production of circular RNAs posit that RNA secondary structures formed by inverted sequences in flanking introns are necessary elements for circularization , but this model lacks completeness , as circular RNAs can be found in genes that lack extensive complementary sequences .",
"This is abundantly clear in lower eukaryotes whose genomes are almost devoid of repetitive sequences ( Richard et al . , 2008 ) .",
"Using S . pombe as a model system , we provide thorough mechanistic evidence that circular RNA can be generated through an exon-containing lariat precursor , a longstanding , yet unsubstantiated model in the field ( Zaphiropoulos , 1996; Surono et al . , 1999 ) .",
"Based on splice site mutagenesis experiments , we find that lariat production is important for detectable production of mrps16 circular RNA .",
"However , a lariat precursor may not be strictly necessary , as suggested by our data and in vitro work in mammalian and yeast splicing extracts that have shown it is possible to directly backsplice a circular RNA in the absence of competing splice sites ( Pasman et al . , 1996; Schindewolf et al . , 1996 ) .",
"Rather , it is more likely that the lariat production contributes to backsplicing catalysis by positioning splice sites .",
"In addition to this catalytic function , lariat production may also serve to prevent competing splice sites from operating .",
"In other words , within the context of an exon-containing lariat , the only available splice sites are those bordering the circularized exon , and the upstream branch point , now ‘orphaned’ by lariat formation , must attack the downstream splice donor .",
"In this way , the exon-containing lariat may provide a microenvironment for the splicing of circular RNA .",
"A hypothesis generated by a model in which circular RNA is spliced from an exon-containing lariat precursor is that inhibiting lariat degradation should lead to increases in circular RNA expression .",
"Recently , Bähler and colleagues published a broad RNA-seq study of several whole transcriptome profiles of wild-type and dbr1∆ S . pombe , and the authors note a ∼3–4 fold global increase in circular RNA in dbr1∆ S . pombe ( Bitton et al . , 2015 ) .",
"However , upon analyzing mrps16 specifically , we observed significant variation in the mrps16 circular:total splice isoform ratios within the wild-type RNA-Seq biological replicates due to the very low number of reads ( Bitton et al . , 2015 , Supp . Table S11 ) .",
"Thus , while there appears to be a modest increase in the global levels of circular RNA in the debranching mutant , the effects on particular genes fail to reach statistical significance .",
"We have also found that production of an exon-containing lariat is not sufficient for circularization , just as inverted repeats are not sufficient for circular RNA production in mammals ( Zhang et al . , 2014b ) , suggesting other regulatory mechanisms responsible for its production .",
"Supporting the idea that factors beyond the presence of an exon-containing lariat are required for circular RNA production , we have found that , in general , uncircularized skipped exons are much smaller than the circular exons , hinting that exon size may have a direct effect on the efficiency with which an exon circularizes .",
"Using mrps16 as a model gene , we tested this directly with a library of exonic deletions and found a systematic decrease in circularization with a decrease in exon size .",
"Although there is a strong effect of exon size , circularization is presumably influenced by additional factors such as topological effects due to intronic sequence and combinatorial effects of RNA-binding proteins .",
"Indeed , we have found that intronic deletions can have a pronounced effect on RNA circularization .",
"None of these deletions ( which range from 133 to 329 bp long ) affect circularization as dramatically as deleting the branch point in either intron ( SB + JS , unpublished data ) .",
"We have previously identified circular RNAs in genes in Saccharomyces cerevisiae ( Wang et al . , 2014 ) that are known to generate exon-skipped linear isoforms ( Hossain et al . , 2011; Egecioglu et al . , 2012 ) , lending support to the possibility that this mechanism extends beyond S . pombe to other eukaryotes .",
"As an example , the three exon gene SUS1 has an exon-skipped linear transcript that is rapidly decayed ( Egecioglu et al . , 2012 ) .",
"Substitution of the SUS1 gene with a linear cDNA copy results in temperature sensitivity and histone H2B deubiquitination defects ( Hossain et al . , 2011 ) .",
"This was suggested to be due to a functional role of an alternatively spliced linear isoform , but could be due to a circularized isoform derived from the seemingly non-functional and rapidly-decayed exon-skipping event .",
"Indeed , it may be a general theme that short-lived exon-skipping events may give rise to long-lived circularized exons , which may play a productive role in cells .",
"As intron size increases , it may be that exon-containing lariats no longer have a strong topological effect .",
"This may limit the utility of this mechanism in higher eukaryotes , which may have adopted inverted sequences in an effort to produce local topological changes .",
"However , as alluded to earlier , lariat production may serve an additional function by isolating exons for circularization , implying that these mechanisms may work in tandem .",
"The existence of the lariat precursor model in lower eukaryotes suggests a possible mechanism in which circular RNA arose as a byproduct of exon skipping and that , as intronic architecture changed , so did mechanisms for maintaining expression of these molecules ."
],
[
"The following S . pombe strains were obtained from Bioneer Inc . ( Alameda , CA ) : ‘wild-type’ strain ED668 ( h+ ade6-M216 ura4-D18 leu1–32 ) and its deletion derivatives dbr1∆ and pub1∆ .",
"Strains were grown in yeast extract with supplements ( YES ) media when grown without selection .",
"Selection was performed in Edinburgh Minimal Media ( EMM ) in the absence of leucine ( EMM-Leu ) .",
"The base vector pW1336 was created from pREP41/GFP-Osh2 ( gift of Masayuki Onishi ) by removal of the nmt1 promoter and insert gene by SphI-SacI digest and replacement with an oligonucleotide polylinker .",
"It contains ARS1 from S . pombe , LEU2 from S . cerevisiae , and ampicillin-resistance .",
"S . pombe genomic DNA was amplified using the primers TTTCGGCGCGCCCTCTTTTTTTAAGAAAATTTCGTTGCTTG and GACGTTAATTAAATCCAGGAATGTTTTTGATGAGCACTAG ( for pub1 ) , and TCTAGGCGCGCCTAATTCCGTATTGATGGAG and AGGATTAATTAAGCTCTCTTGGTTGTGCAATCAG ( for mrps16 ) , and cloned as AscI-PacI fragments into pW1336 .",
"The inserts were validated by Sanger sequencing .",
"Plasmid deletions were generated using QuikChange Lightning Mutagenesis Kit ( Agilent Technologies , Santa Clara , CA ) according to the manufacturer's instructions ( primers shown in Supplementary file 1 ) .",
"Mutations were validated by Sanger sequencing .",
"For generating the exonic deletion library , inverse PCR was performed using ∼1 ng of plasmid template per 50 μl PCR reaction using Phusion polymerase ( New England Biolabs [NEB] , Ipswich , MA ) with high-fidelity buffer according to the manufacturer's instructions ( primers shown in Supplementary file 1 ) .",
"Prior to PCR , the primers were phosphorylated using T4 polynucleotide kinase ( PNK ) ( NEB ) at 10 μM primer concentration in 1× T4 DNA ligase buffer .",
"After PCR , these products were pooled , digested with Dpn1 for 4 hr at 37°C to remove vector template , and concentrated by isopropanol precipitation .",
"The concentrated products were then run on a 1% agarose gel for size selection .",
"The 12 kb was visualized using blue light and extracted using a clean razor blade .",
"The product was purified using Qiagen Gel Extraction kit according to manufacturer's instructions .",
"50 ng of the purified products was self-ligated using 400 U T4 DNA ligase ( NEB ) to circularize the vectors in 20 μl at 16°C overnight .",
"4 μl of the reaction products were transformed into Escherichia coli TOP10 chemically competent cells ( Life Technologies , Carlsbad , CA ) according to manufacturer's instructions , and transformed cells were plated on LB + carbenicillin plates .",
"After 17 hr of growth , 5 ml of LB was added to plates and colonies were pooled by scraping ( ∼1500 colonies obtained ) .",
"The bacteria were pelleted and plasmids were isolated using the Qiaprep Spin Miniprep kit according to the manufacturer's instructions ( Qiagen , Venlo , Netherlands ) .",
"Total RNA was isolated from log-phase yeast using an acid phenol-chloroform extraction .",
"After extraction and ethanol precipitation , RNA was treated with TURBO DNase ( Life Technologies ) and purified and concentrated using the Purelink RNA-mini kit ( Ambion , Austin , TX ) .",
"1 μg of total RNA was treated with 5 U RNase R ( Epicentre , Madison , WI ) in 10 μl total reaction volume at 37°C for 30 min .",
"The RNA was then reverse-transcribed without purification with the addition of 4 μl 5× supplement buffer ( 250 mM Tris pH 8 , 125 mM KCl , 15 mM MgCl2 ) and 2 μl 0 . 1 M DTT to provide necessary conditions for the RT reaction .",
"In all cases , 1 μg of total RNA was reverse transcribed with random hexamers using 100 U Maxima H-minus reverse transcriptase ( ThermoFisher Scientific , Waltham , MA ) according to the manufacturer's instructions .",
"PCRs were performed using Platinum Taq hot-start polymerase ( Life Technologies ) .",
"For all PCR reactions , 1 . 5 μl of the unpurified RT-reaction was used per 50 μl reaction volume .",
"All RT-PCR reactions were performed using the recommended cycling protocol for 35 cycles .",
"RT-DraI-PCRs were assembled as normal RT-PCRs , but an initial PCR phase ( 3 cycles ) was used to produce double-stranded cDNA .",
"The samples were then removed from the thermocycler and placed on ice .",
"20 U of DraI ( NEB ) was added directly to the 50 μl PCR reaction , and the reaction was allowed to incubate at 37°C for 1 hr .",
"From there , the PCR was resumed for 35 additional cycles without purification .",
"qPCR reactions were assembled as 10 μl reactions using AccuPower 2X GreenStar qPCR Master Mix ( Bioneer ) and Power SYBR Green Master Mix ( Life Technologies ) with 0 . 3 μl of template used per reaction .",
"We performed qPCRs on an ABI 7900HT using following cycling protocol: 50°C for 20 min , 95°C for 10 min , ( 95°C for 15 s and 60°C for 30–60 s ) × 45 cycles , followed by a dissociation stage .",
"RNA structures and dotplots are the predicted MFE structures determined by NUPACK ( Dirks and Pierce , 2004; Dirks et al . , 2007; Zadeh et al . , 2011 ) with default RNA settings at a temperature of 30°C .",
"Adapters were trimmed from reads using CutAdapt ( Martin , 2011 ) .",
"Using our custom pipeline ( Szabo et al . , 2015 ) , we aligned the reads from ( Bitton et al . , 2014 ) ( NCBI Gene Expression Omnibus accession number GSE50246 ) to the S . pombe transcriptome and determined the circular junctional reads derived from single exons ( analysis shown in Figure 5A ) .",
"Only junctions with a probability of greater than 0 . 7 were considered here .",
"Wild-type S . pombe was transformed with the plasmid library ( see ‘Expression vectors and mutagenesis’ for details on the creation of the plasmid library ) according to standard protocol , and transformants were plated on EMM-Leu to select .",
"∼2000 colonies were obtained and scraped using 5 ml EMM-Leu .",
"Cells were mixed to homogenize the sample and 2 ml of cells were used for RNA isolation and 2 ml for DNA isolation .",
"RNA was isolated by acid-phenol chloroform extraction and was treated with both RNase R ( Epicentre ) and Turbo DNase ( Ambion ) prior to cDNA synthesis .",
"Plasmid DNA was isolated using NucleoSpin Plasmid Miniprep Kit ( Macherey–Nagel , Bethlehem , PA ) using a published supplementary protocol for purification of plasmid DNA from yeast .",
"To enrich for plasmid DNA in the sample , we also treated with Exonuclease V ( NEB ) to degrade linear DNA fragments .",
"Sequencing libraries were produced using a PCR approach based on a previously reported method ( Zhang et al . , 2014a ) with some key differences .",
"In our protocol , PCRs were performed using Phusion polymerase ( NEB ) for 5 cycles in an initial amplification stage using tagged gene specific primers followed by n-5 cycles for the addition of the sequencing adapters and barcodes , where n = 25 , 30 , or 35 cycles ( each library receiving a unique barcode in order to reference cycle number ) .",
"Primers for the latter PCR stage were exactly as reported in Zhang et al . , and the primers were obtained directly from the Li Lab ( Sanford University; Stanford , CA ) .",
"Libraries were pooled and spiked with PhiX to a concentration of 25% .",
"Sequencing was performed on an Illumina MiSeq .",
"Reads were trimmed using CutAdapt ( Martin , 2011 ) and only reads greater than 40 nucleotides after trimming were used for analysis .",
"We aligned reads , using Bowtie 1 ( Langmead et al . , 2009 ) , first to an index containing an unmutated mrps16 sequence , and then the unaligned reads were aligned using a custom-built index containing all possible deletions of the mrps16 exon that could be generated using our combinatorial , outward-facing PCR strategy .",
"In both cases , we limited the alignments to at most 3 mismatches using the Bowtie ‘–v 3’ setting .",
"Since the primers were not purified after synthesis or before our combinatorial PCR , incomplete primer synthesis could generate mutants in our plasmid library , and we accounted for this in generating our index .",
"This leads to a number of variants differing by only a few nucleotides .",
"After aligning to these indexes , we determined the number of reads derived from each deletion isoform in each sequencing library , filtering out isoforms with less than 200 reads derived from the plasmid-based DNA-seq library .",
"We converted these read counts to circularization efficiency ( CE ) using the following equation:CE=nc/ ( nwt×s ) nv/nvtot , where nc is the number of reads derived from a given circular RNA isoform , nwt is the number of reads derived from wild-type circular RNA ( from background genomic expression ) , and s is a scaling factor that corrects for the increased average copy number of the plasmid , which we assume is not altered by the exonic deletion .",
"This value is determined from qPCR data from cells transformed with a wt mrps16 plasmid ( s = 19 in wild-type S . pombe ) .",
"This value is then normalized by the representation of the given vector isoform in the plasmid library ( nv/nvtot ) , where nv is the number of reads derived from a given plasmid isoform , and nvtot is the total number of reads in the plasmid-derived library ( after removing reads that align to the wild-type sequence ) .",
"Error bars in Figure 5D were generated using the relationship that nwt and nc are independent Poisson random variables and therefore , X: = nc| ( nwt + nc ) is bionomial with parameters probability of success B:= nc/ ( nc+nw ) and total trials nc+nw .",
"Standard binomial confidence intervals were assigned to this quantity , and the 95% CI was propagated to A: = nv/nvtot ( expression A ) using the transformation f ( A ) = 1/A .",
"Expression B can be converted to nc/ ( s * nwt ) using the transformation g ( B ) = B/ ( 1 − B ) /s .",
"Note that , CE = f ( A ) * g ( B ) .",
"The total variance of ln ( CE ) is obtained by combining a normal approximation for B ( corresponding to p^ in a binomial model ) and the delta method , which states that for sufficiently regular functions h , var ( h ( x ) ) = var ( x ) h′ ( x ) ^2 .",
"Therefore , var ( ln ( CE ) ) =var ( ln ( f ( A ) g ( B ) ) ) =var ( ln ( f ( A ) ) ) +var ( ln ( g ( B ) ) ) , and applyingvar ( ln ( f ( A ) ) ) =var ( ln ( 1/A ) ) = var ( ln ( A ) ) =var ( A ) *[ln ( A ) ]2=var ( A ) *1/A2 , where var ( A ) is estimated as A ( 1−A ) /nvtot . var ( ln ( g ( B ) ) ) =var ( ln ( B/ ( 1−B ) /s ) ) =var ( B ) *[ln ( B/ ( 1−B ) /s ) ]2=var ( B ) *1/ ( B ( 1−B ) ) 2 , where var ( B ) is estimated as B ( 1−B ) / ( nwt + nc ) .",
"Using these relationships , we computed the sd = sqrt ( var ( ln ( CE ) ) ) .",
"Finally , we transformed the approximate 95% CI for ln ( CE ) ( its point estimate plus or minus 2 sd ) to the 95% CI for CE by exponentiating the upper and lower bounds of the confidence interval for ln ( CE ) , resulting in the error bars on the plot ."
]
] | [
"Pervasive expression of circular RNA is a recently discovered feature of eukaryotic gene expression programs , yet its function remains largely unknown .",
"The presumed biogenesis of these RNAs involves a non-canonical ‘backsplicing’ event .",
"Recent studies in mammalian cell culture posit that backsplicing is facilitated by inverted repeats flanking the circularized exon ( s ) .",
"Although such sequence elements are common in mammals , they are rare in lower eukaryotes , making current models insufficient to describe circularization .",
"Through systematic splice site mutagenesis and the identification of splicing intermediates , we show that circular RNA in Schizosaccharomyces pombe is generated through an exon-containing lariat precursor .",
"Furthermore , we have performed high-throughput and comprehensive mutagenesis of a circle-forming exon , which enabled us to discover a systematic effect of exon length on RNA circularization .",
"Our results uncover a mechanism for circular RNA biogenesis that may account for circularization in genes that lack noticeable flanking intronic secondary structure ."
] | [
"DNA contains the instructions to make proteins .",
"These instructions are first copied into a molecule of RNA , which often has sections that code for protein ( called exons ) interrupted by non-coding sections ( called introns ) .",
"A process called splicing removes the introns and joins the exons to form a mature RNA molecule that is then translated to make proteins .",
"The standard splicing process joins the first exon to the second , and the second to the third , and so on to produce a liner RNA molecule .",
"Occasionally , one or more of the exons can be skipped in a process known as ‘alternative splicing’ .",
"In recent years , scientists have discovered that another type of alternative splicing , which creates circular RNA molecules , is common in animal cells .",
"It was suggested that circular RNAs might form when the splicing machinery joins the two ends of the same exon via a process referred to as ‘backsplicing’ .",
"Standard splicing first converts an intron into a looped structure called a ‘lariat’ ( which resembles a lasso ) ; the lariat is then removed by the splicing machinery and destroyed .",
"When exons are skipped during alternative splicing , a large lariat containing the skipped exon is produced .",
"Now , Barrett et al . have used yeast cells to investigate how circular RNAs are formed .",
"Yeast is a good model to study this process because it can be manipulated easily in the laboratory and expresses circular RNAs .",
"The experiments show that lariat structures containing exons ( as would be expected from exon-skipping ) are a common intermediate step before the production of a circular RNA .",
"However , some exon-containing lariats do not go on to form circular RNAs , suggesting that additional factors are important for the production of circular RNAs .",
"Barrett et al . next investigated the reasons why certain exons formed circular RNAs while others did not and revealed that longer exons formed circular RNAs more readily than shorter exons .",
"Further work is now required to understand the molecular basis of this result and identify other factors that contribute to the formation of circular RNAs ."
] | 2015 |
[
"Introduction",
"Results and discussion",
"Materials and methods"
] | [
"short report",
"structural biology and molecular biophysics",
"computational and systems biology"
] | Antiparallel protocadherin homodimers use distinct affinity- and specificity-mediating regions in cadherin repeats 1-4 | elife-18449-v2 | [
[
"Protocadherins ( Pcdhs ) encompass about 70% of the cadherin superfamily in mammals , and are involved in cell adhesion in the nervous system of higher animals ( Hulpiau and van Roy , 2011; Hulpiau et al . , 2013; Keeler et al . , 2015; Sotomayor et al . , 2014 ) .",
"Pcdhs segregate into two groups: clustered ( expressed from a large gene cluster ) and non-clustered .",
"Clustered Pcdhs , comprising the α , β , and γ subfamilies , mediate neuronal survival , self-avoidance and self/nonself discrimination , and promote dendritic arborization in neuronal cells ( Emond and Jontes , 2008; Garrett et al . , 2012; Kostadinov and Sanes , 2015; Ledderose et al . , 2013; Lefebvre et al . , 2012; Molumby et al . , 2016; Suo et al . , 2012; Wang et al . , 2002; Weiner et al . , 2005 ) .",
"Clustered Pcdhs thus function analogously to insect Dscam isoforms to mediate neuronal identity ( Zipursky and Sanes , 2010 ) .",
"Clustered Pcdhs are also broadly involved in synapse formation and maintenance , neuronal connectivity and neuronal survival ( Hayashi and Takeichi , 2015; Keeler et al . , 2015 ) .",
"Non-clustered Pcdhs play key roles in neuronal development ( Keeler et al . , 2015; Kim et al . , 2011 ) .",
"For example , Pcdh7 is involved in germ layer differentiation ( Rashid et al . , 2006; Yoshida , 2003 ) , and Pcdh1 and Pcdh8 mediate cell sorting and migration during gastrulation ( Kim et al . , 1998; Kuroda et al . , 2002 ) .",
"Both clustered and non-clustered Pcdhs control these phenotypes through homophilic interactions of their extracellular cadherin ( EC ) repeat domains ( Hirano et al . , 1999; Hoshina et al . , 2013; Kim et al . , 1998; Kuroda et al . , 2002; Schreiner and Weiner , 2010; Thu et al . , 2014; Yamagata et al . , 1999; Yoshida , 2003 ) .",
"Clustered Pcdh subfamilies show distinct phenotypes .",
"In zebrafish , α and γ Pcdhs express in overlapping but distinct brain regions ( Biswas et al . , 2012 ) .",
"In mammals , α Pcdhs regulate sorting of olfactory sensory neuron axons into glomeruli , serotonergic axon maturation , and dendritic patterning in CA1 pyramidal neurons ( Hasegawa et al . , 2012 , 2008; Katori et al . , 2009; Suo et al . , 2012 ) .",
"The γ subfamily is important for self/non-self discrimination in retinal starburst amacrine cells and Purkinje neurons ( Kostadinov and Sanes , 2015; Lefebvre et al . , 2012 ) .",
"Thus , available data suggest that the different Pcdh subfamilies may function independently or cooperatively , perhaps depending on the brain region and/or neuronal cell type .",
"Our recent PcdhγA1 and PcdhγC3 EC1-3 structures revealed dimer interactions between EC2 and EC3 ( Nicoludis et al . , 2015 ) , consistent with previous biochemical and bioinformatics data ( Schreiner and Weiner , 2010; Wu , 2005 ) .",
"Using sequence co-evolution analysis , we predicted intersubunit EC1-EC4 interactions , and proposed that clustered Pcdhs form extended antiparallel homodimers engaging EC1-4 .",
"A complementary biochemical and structural study arrived at a very similar docking model ( Rubinstein et al . , 2015 ) , which was recently confirmed for α and β clustered Pcdhs ( Goodman et al . , 2016 ) .",
"We determined the crystal structure of PcdhγB3 EC1-4 , the first full antiparallel dimer for a γ isoform .",
"We analyzed the clustered Pcdhs structures in light of biological , biochemical and evolutionary data to further resolve how clustered Pcdhs encode specificity .",
"We describe how structural differences between the α , β and γ subfamilies generate distinct modes of specificity encoding .",
"We also provide evidence that the EC1/EC4 and EC2/EC3 interfaces are functionally different: EC1/EC4 provides nonselective dimerization affinity while EC2/EC3 is generally responsible for enforcing specificity .",
"Finally , we extend our sequence coevolution analysis to the non-clustered Pcdhs and provide evidence that the EC1-4 interaction is broadly used by Pcdhs ."
],
[
"The in vitro-refolded recombinantly-expressed PcdhγB3 EC1-4 ( 47 kDa ) yielded two peaks on size exclusion chromatography ( SEC; Figure 1—figure supplement 1 ) .",
"Based on multi-angle light scattering ( MALS; Figure 1—figure supplement 1 ) , peak 1 was wide and polydisperse ( ~200–300 kDa ) .",
"Peak 2 was monodisperse at 80 kDa – consistent with a dimer – and readily yielded a crystal structure ( Figure 1—source data 1 ) .",
"As expected , each EC forms a seven-stranded Greek key β-sandwich motif ( Figure 1A ) , similarly to other clustered Pcdh structures ( Goodman et al . , 2016; Nicoludis et al . , 2015; Rubinstein et al . , 2015 ) .",
"Notably , EC4 has a unique β-strand arrangement compared to EC1-EC3 ( Figure 1B , C ) and all known cadherin repeat structures .",
"Strand β1a is extended by 4–5 residues , while β1b is correspondingly shortened .",
"Additionally , while in EC1-3 strand β2 splits into β2a and β2b , interacting with strands β5 and β1a , respectively , in EC4 it forms a continuous strand interacting with both simultaneously .",
"This distinct structural feature contributes to intersubunit EC1/EC4 interactions ( see below ) . 10 . 7554/eLife . 18449 . 003Figure 1 . PcdhγB3 EC1-4 extended antiparallel dimer relies on unusual EC4 β-strand arrangement and is similar to other clustered Pcdh dimers .",
"( A ) Structure of the PcdhγB3 EC1-4 antiparallel dimer , with each EC a different shade of blue and the Ca2+ ions in grey .",
"( B ) Superposition of PcdhγB3 EC2 and EC4 highlighting the differences in β-strands 1 and 2 .",
"( C ) Comparison of the canonical cadherin ( top ) and EC4 ( bottom ) β-strand arrangement .",
"( D ) The structures of Pcdh dimers α4 EC1-4 , α7 EC1-5 , β6 EC1-4 , and β8 EC1-4 ( grey ) were superimposed using the dimeric EC2-3 region onto γB3 EC1-4 ( blue ) , illustrating variations in twist/corkscrew .",
"( E ) The EC4 domains of clustered Pcdh structures ( colored as labeled ) were superimposed , highlighting EC1 position differences that correlate with subfamily .",
"Point of view ( eye symbol ) shown in ( D ) .",
"See Figure 1—figure supplements 1–4 . DOI: http://dx . doi . org/10 . 7554/eLife . 18449 . 00310 . 7554/eLife . 18449 . 004Figure 1—source data 1 . Statistics for PcdhγB3 EC1-4 structure . DOI: http://dx . doi . org/10 . 7554/eLife . 18449 . 00410 . 7554/eLife . 18449 . 005Figure 1—figure supplement 1 . PcdhγB3 refolding yields two species , one of which corresponds to monodisperse dimeric protein .",
"( A ) SEC profile of refolded PcdhγB3 run on a Superdex 200 16/60 column , with the two collected peak fractions indicated .",
"( B ) SEC-MALS profile of Peak 1 run on a Superdex S200 10/300 column was broad and polydisperse .",
"( C ) SEC-MALS profile of Peak 2 run on a Superdex S200 10/300 column was monodisperse at a molecular weight of ~80 kDa , consistent with a dimer ( monomeric molecular weight 47 kDa ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 18449 . 00510 . 7554/eLife . 18449 . 006Figure 1—figure supplement 2 . Protocadherins and non-classical cadherins have a distribution of orientation between repeat pairs that is distinct from classical cadherins . Distribution of tilt and azimuthal angles from adjacent EC repeat pairs of classical ( black ) , Pcdh15 and Cdh23 ( blue ) and clustered Pcdh ( red ) .",
"( Inset )",
"The orientation of adjacent EC repeats was defined by the tilt and azimuthal rotation of the EC domain principal axes . DOI: http://dx . doi . org/10 . 7554/eLife . 18449 . 00610 . 7554/eLife . 18449 . 007Figure 1—figure supplement 3 . EC1 and EC3 use the same face for intersubunit contacts , as do EC2 and EC4 . ( A ) Sequence alignment of clustered Pcdhs for which dimer interface structures are available .",
"EC1 and EC4 of PcdhγA1 are grey because their interaction interface is unknown .",
"Residues highlighted orange have a BSA > 10 Å2 in those respective structures .",
"We selected as conserved interface residues ( boxed ) those that have a BSA > 10 Å2 in 5 of 6 structures for EC2/EC3 or 4 of 5 structures for EC1/EC4 .",
"The interface regions are notably similar in EC1 and EC3 , as well as in EC2 and EC4 .",
"( B ) Superposition of the first half of PcdhγB3 EC1-4 with the second half yields an RMSD of 2 . 24 Å over 142 Cα atoms .",
"( C ) The two superimposed chains at the top ( B ) are rotated 90° , highlighting how similar surfaces of EC1 and EC3 , and EC2 and EC4 , form the extended antiparallel interface of clustered Pcdhs .",
"In ( B ) and ( C ) , the conserved interface positions identified in ( A ) are marked by Cα atom spheres . DOI: http://dx . doi . org/10 . 7554/eLife . 18449 . 00710 . 7554/eLife . 18449 . 008Figure 1—figure supplement 4 . HEPES molecule near the EC2/EC3 interface . Final 2Fo-Fc electron density contoured at 1σ , with the final structural model shown as sticks .",
"Nearby side chains from EC2 ( lighter blue ) and EC3 ( darker blue ) are labeled . DOI: http://dx . doi . org/10 . 7554/eLife . 18449 . 008 Although the asymmetric unit contains a single PcdhγB3 molecule , a crystallographic two-fold axis generates an antiparallel dimer with intersubunit EC1/EC4 and EC2/EC3 interactions ( Figure 1A ) .",
"This dimer is consistent with the PcdhγA1 EC1-3 crystal structure , validating the previously predicted interface ( Nicoludis et al . , 2015; Rubinstein et al . , 2015 ) , and with recent α and β Pcdhs structures ( Goodman et al . , 2016 ) , confirming that this interaction mechanism is conserved among all clustered Pcdh subfamilies ( Figure 1D ) .",
"The structures do differ noticeably in overall twist , including subfamily-specific differences in relative EC1/EC4 orientation ( Figure 1E ) .",
"The linear architecture of clustered Pcdhs enables extended antiparallel dimer interfaces .",
"Overall , the tilt and azimuthal angles between adjacent clustered Pcdh repeats are distinct from those of classical cadherins ( Figure 1—figure supplement 2 ) ( Nicoludis et al . , 2015 ) .",
"Classical cadherins , which typically dimerize through EC1/EC1 interfaces , exhibit smaller tilt angles and thus an overall curved structure ( Boggon et al . , 2002 ) .",
"Notably , the clustered Pcdh repeat orientation is such that EC1 and EC3 use the same face for intersubunit contacts , as do EC2 and EC4 ( Figure 1—figure supplement 3 ) , suggesting that longer cadherins could readily form even more extended interfaces .",
"Clustered Pcdh subfamilies control different phenotypes in vivo and have discrete expression patterns ( Biswas et al . , 2012; Keeler et al . , 2015 ) , suggesting that they encode specificity using distinct modes , which may relate to subfamily-specific structural features .",
"To investigate this hypothesis , we calculated the isoform conservation ratio ( ICR ) within individual subfamilies , which quantifies the extent to which individual residue positions are conserved among orthologs ( same isoform in different species ) and diversified in paralogs ( different isoforms in the same species ) ( Nicoludis et al . , 2015 ) , resulting in three ICR value sets for the α , β and γ subfamilies , respectively ( Figure 2—figure supplement 1 ) .",
"To account for subfamily differences in sequence conservation , we normalized the ICR values by dividing by the subfamily average .",
"We then mapped them onto the Pcdhα7 EC1-5 , Pcdhβ8 EC1-4 and PcdhγB3 EC1-4 structures ( Figure 2A ) .",
"Comparing the structures and isoform-specific conservation in the different subfamilies allowed us to identify key specificity determinant regions for individual subfamilies .",
"We illustrate three examples of how the subfamilies have encoded specificity using unique structural features . 10 . 7554/eLife . 18449 . 009Figure 2 . Isoform-specific conservation and structural differences reveal subfamily differences in diversity generation .",
"( A ) Subfamily-specific ICR values mapped onto the surfaces of Pcdhα7 ( top , green ) , Pcdhβ8 ( middle , magenta ) and PcdhγB3 ( bottom , blue ) .",
"The black outline marks the dimer interface footprint .",
"( B , C , D )",
"Unique structural features of the α ( left ) , β ( center ) , and γ ( right ) structures ( colored according to Figure 1 ) .",
"ICR values for highlighted residues shown below and normalized amino acid frequencies for these positions shown on the right .",
"See Figure 2—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 18449 . 00910 . 7554/eLife . 18449 . 010Figure 2—figure supplement 1 . Clustered Pcdh subfamilies have distinct patterns of isoform-specific conservation .",
"( A ) Subfamily-specific normalized ICR values as a sliding average with a 5-residue window size ( α = green , β = magenta , γ = cyan ) .",
"Loop regions illustrated in Figure 2B–D are indicated at the bottom .",
"( B ) ICR values for interface residues ( BSA > 10 Å2 ) for Pcdh α4 EC1-4 ( yellow ) , α7 EC1-5 ( green ) , β6 EC1-4 ( salmon ) , β8 EC1-4 ( magenta ) , γB3 EC1-4 ( cyan ) , and γA1 EC1-3 ( dark blue ) .",
"In black are the average and standard error .",
"Residues with high isoform-specific conservation localize to EC2 and EC3 surfaces . DOI: http://dx . doi . org/10 . 7554/eLife . 18449 . 010 In α isoforms , the EC2 β4-β5 loop is enriched in high-ICR and chemically diverse residues , and differs in conformation in the Pcdhα4 and Pcdhα7 structures ( Figure 2B ) : the Pcdhα4 EC2 β4-β5 loop contacts β1b of EC3 , while the corresponding loop in Pcdhα7 does not , suggesting variable interactions in other isoforms .",
"In comparison , the EC2 β4-β5 loop residues in both β and γ isoforms have lower ICR values , more similar loop structure , and do not contact β1b of EC3 .",
"Thus , this loop may have evolved to generate diversity within α isoforms , but not in other subfamilies .",
"In β isoforms , the Phe-X10-Phe loop between β3 and β4 of EC3 has limited diversity compared to α and γ isoforms and wedges between the EC2 β4-β5 loop and β2b strand ( Figure 2C ) .",
"In contrast , the Phe-X10-Phe loop of α and γ isoforms has a helical conformation , and has residues with higher ICR values and greater chemical diversity .",
"Therefore alterations in secondary structure can affect how specificity is encoded within the subfamilies .",
"The short loop following the extended β1a strand in EC4 contacts the EC1 β6-β7 loop ( Figure 2D ) , and there are large structural differences in the EC1/EC4 interaction between subfamilies ( Figure 1E ) .",
"α Isoforms have low-ICR residues at this interface , whereas β and γ isoforms have higher ICR value residues .",
"This thus suggests that the large structural differences drive inter-subfamily specificity , on which may be layered additional isoform specificity .",
"In all cases , sequence regions with high isoform-specific conservation correlate with interface contacts , revealing the interplay between dimer structure and how subfamilies encode specificity .",
"Diversity in the composition and conformation of loop regions provides distinct specificity mechanisms to subfamilies .",
"Phylogenetic analysis indicates that isoforms are more similar within than across subfamilies ( Sotomayor et al . , 2014; Wu , 2005 ) , and the available structures show that the interface architecture is more similar within subfamilies as well ( Figure 1E ) ( Goodman et al . , 2016; Nicoludis et al . , 2015 ) .",
"With this insight , the dimer interface seen in the PcdhγC3 EC1-3 crystal structure may represent a unique dimer architecture for C-type isoforms ( Nicoludis et al . , 2015 ) , as these isoforms are transcriptionally , functionally and evolutionarily distinct from the subfamilies in which they reside ( Chen et al . , 2012; Frank et al . , 2005; Kaneko et al . , 2006 ) .",
"Distinct expression of the clustered Pcdh subfamilies in different tissues and at different developmental stages supports the necessity for intra-subfamily specificity ( Biswas et al . , 2012; Frank et al . , 2005 ) .",
"Differences in subfamily structure and isoform-specific conservation suggest that homophilic specificity mechanisms emerged independently in each subfamily through diversification of subfamily-specific interface contacts .",
"The EC2/EC3 interaction is integral to clustered Pcdh dimerization specificity , as evidenced by bioinformatics and cell-aggregation assays ( Nicoludis et al . , 2015; Rubinstein et al . , 2015; Schreiner and Weiner , 2010; Thu et al . , 2014; Wu , 2005 ) .",
"We sought to understand the functional purpose of the EC1/EC4 interaction , and made three observations .",
"First , for all isoforms with available structures , fewer EC1/EC4 interface residues have high isoform-specific conservation compared to the EC2/EC3 interface residues ( Figure 2A , Figure 2—figure supplement 1 ) .",
"Second , interface residues shared by most isoforms are more hydrophobic in EC1/EC4 than in EC2/EC3 ( Figure 3A ) .",
"Third , the PcdhγB3 EC1/EC4 interface is much larger ( BSA = 976 Å2 per protomer ) than the EC2/EC3 interface ( 555 Å2 per protomer ) .",
"The lack of isoform specificity , the hydrophobic nature , and large interface area together suggest that the EC1/EC4 interface promotes binding with little specificity . 10 . 7554/eLife . 18449 . 011Figure 3 . The EC1/EC4 interface is enriched in affinity-driving hydrophobic residues , while the EC2/EC3 interface contains high-ICR residues driving specificity .",
"( A ) Amino acid frequencies in clustered Pcdhs of conserved interface residues ( see Figure 1—figure supplement 2 ) .",
"( B ) Plot of ICR value and ∆∆Gcalc of interface residues of Pcdh α4 EC1-4 ( yellow ) , α7 EC1-5 ( green ) , β6 EC1-4 ( salmon ) , β8 EC1-4 ( magenta ) , γB3 EC1-4 ( blue ) .",
"Two subsets of interface residues segregate from the main cluster: high-∆∆Gcalc and low-ICR residues ( ‘affinity’; black box ) and low-∆∆Gcalc and high-ICR residues ( ‘specificity’; crimson box ) .",
"Residue F86 from PcdhγB3 EC1-4 is labeled .",
"( C ) High-∆∆Gcalc and low-ICR residues ( black ) map primarily to EC1 and EC4 , while low-∆∆Gcalc and high-ICR residues ( crimson ) primarily map to EC2 and EC3 .",
"N253 ( * ) is found in the ‘specificity’ region for γB3 and in the ‘affinity’ region for β6 and β8 .",
"( D ) The EC1/EC4 interface features a hydrophobic cluster , with EC1 F86 near its center .",
"( E ) SEC-MALS profiles of WT PcdhγB3 EC1-4 ( blue; molecular weight 82 kDa ) and F86A ( black; molecular weight 52 kDa ) run on a Superdex S200 10/300 column , are consistent with dimeric and monomeric proteins , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 18449 . 011 To probe this hypothesis , we used the Computational Interface Alanine Scanning Server to assess each interface residue’s contribution to the free energy of complex formation ( ΔΔGcalc ) when computationally mutated to alanine ( Kortemme and Baker , 2002; Kortemme et al . , 2004 ) .",
"Using the five available EC1-4 interfaces , two residue groups emerged from comparing the ICR values to ΔΔGcalc: one group with low ICR values and high ΔΔGcalc , the other with high ICR values and low ΔΔGcalc ( Figure 3B ) .",
"These residue groups can be regarded as contributing to the affinity and specificity of the complex , respectively .",
"When mapped on PcdhγB3 , predicted affinity residues concentrated on EC1 and EC4 , and predicted specificity residues on EC2 and EC3 , corroborating the distinction between EC1/EC4 and EC2/EC3 interactions ( Figure 3C ) .",
"In the PcdhγB3 EC1/EC4 interface , F86 , one of the predicted affinity-driving residues from EC1 , wedges into a cavity created by hydrophobic EC4 residues ( Figure 3D ) .",
"A PcdhγB3 EC1-4 F86A mutant indeed disrupted dimerization , resulting in a monomeric protein as measured by MALS ( Figure 3E ) .",
"Thus , the hydrophobic interactions between EC1 and EC4 are crucial to dimerization .",
"Analogously , purified EC1-3 constructs failed to dimerize in vitro whereas EC1-4 constructs did ( Nicoludis et al . , 2015; Rubinstein et al . , 2015 ) , and K562 cells expressing ΔEC1 or ΔEC4-6 constructs did not aggregate while cells expressing chimeras in which EC1 and EC4 derived from different paralogs did ( Schreiner and Weiner , 2010; Thu et al . , 2014 ) .",
"Together , these results indicate that the EC1/EC4 interaction is not strictly required for the specificity of dimerization but it drives dimerization affinity through non-specific hydrophobic interactions .",
"The antiparallel EC1-4 interaction architecture can encode diverse specificities within the clustered Pcdh family .",
"Is this architecture unique to clustered Pcdhs or is it ancestral , and thus also found in non-clustered Pcdhs ?",
"These include the δ-1 ( Pcdh1 , Pcdh7 , Pcdh9 , Pcdh11 ) and δ-2 ( Pcdh8 , Pcdh10 , Pcdh17 , Pcdh18 , Pcdh19 ) families that are integral to the development and maintenance of the nervous system ( Keeler et al . , 2015; Kim et al . , 2011 ) .",
"We used sequence coevolution analysis , which successfully predicted the clustered Pcdh interface ( Nicoludis et al . , 2015 ) ( Figure 4—figure supplement 1 ) , to look for evidence of an antiparallel interface in non-clustered Pcdhs ( Figure 4A ) .",
"As in clustered Pcdhs , most covarying residue pairs in non-clustered Pcdhs were intra-domain structural contacts of the well-conserved cadherin fold .",
"Additionally , several covarying pairs are found between EC2 and EC3 , or EC1 and EC4 , similar to those observed for the clustered Pcdhs .",
"When mapped onto the PcdhγB3 dimer , these covarying pairs are somewhat further apart than true interface contacts ( Figure 4B ) , which could be due to differences in dimerization interfaces , as we observe between the clustered Pcdh families , or in the δ-1 or δ-2 Pcdhs secondary structure , for which there are no available structures .",
"This analysis thus predicts an antiparallel EC1-4 interaction in members of the non-clustered Pcdhs .",
"Notably , we cannot determine whether all members or only a subset – and if so , which – likely use this architecture .",
"However maximum parsimony suggests that the ancestral Pcdh used the antiparallel EC1-4 dimer interaction , and Pcdh members which do not show this interaction mechanism either diverged before it evolved or lost it subsequently . 10 . 7554/eLife . 18449 . 012Figure 4 . Evolutionary couplings in non-clustered Pcdhs predict an antiparallel interface engaging EC1-EC4 . ( A ) The top 38 covarying pairs are shown in black , and include a number of EC1-EC4 and EC2-EC3 covarying residue pairs .",
"The intramolecular contact maps of PcdhγB3 EC1-4 , Pcdhα4 EC1-4 , Pcdhα7 EC1-4 , Pcdhβ6 EC1-4 , Pcdhβ8 EC1-4 and PcdhγA1 EC1-3 are in gray for reference .",
"The observed interface contact residues are also mapped ( α4 , yellow; α7 , green; β6 , salmon; β8 , magenta; γB3 , blue; γA1 , dark blue ) .",
"( B ) Covarying residue pairs across EC1-EC4 or EC2-EC3 are mapped onto the PcdhγB3 EC1-4 structure with a line between coupled residues .",
"Alignments and evolutionary couplings in Figure 4—source data 1 and 2 .",
"( C ) Amino acid frequencies at non-clustered Pcdh alignment positions corresponding to the conserved interface residue positions identified in clustered Pcdhs ( Figure 1—figure supplement 2 ) .",
"See Figure 4—figure supplements 1 and 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 18449 . 01210 . 7554/eLife . 18449 . 013Figure 4—source data 1 . Alignment of non-clustered Pcdhs EC1-4 . DOI: http://dx . doi . org/10 . 7554/eLife . 18449 . 01310 . 7554/eLife . 18449 . 014Figure 4—source data 2 . Evolutionary couplings from the non-clustered Pcdh alignment . DOI: http://dx . doi . org/10 . 7554/eLife . 18449 . 01410 . 7554/eLife . 18449 . 015Figure 4—source data 3 . Alignment of clustered Pcdhs EC1-4 . DOI: http://dx . doi . org/10 . 7554/eLife . 18449 . 01510 . 7554/eLife . 18449 . 016Figure 4—source data 4 . Evolutionary couplings from the clustered Pcdh alignment . DOI: http://dx . doi . org/10 . 7554/eLife . 18449 . 01610 . 7554/eLife . 18449 . 017Figure 4—figure supplement 1 . Evolutionary couplings in clustered Pcdhs are consistent with all available EC1-EC4 antiparallel homodimeric interfaces . The top 83 covarying pairs are shown in black .",
"The intramolecular contact maps of PcdhγB3 EC1-4 , Pcdhα4 EC1-4 , Pcdhα7 EC1-4 , Pcdhβ6 EC1-4 , Pcdhβ8 EC1-4 and PcdhγA1 EC1-3 are in gray for reference .",
"The observed interface contact residues are also mapped ( α4 , yellow; α7 , green; β6 , salmon; β8 , magenta; γB3 , blue; γA1 , dark blue ) .",
"Alignments and evolutionary couplings in Figure 4—source data 3 and 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 18449 . 01710 . 7554/eLife . 18449 . 018Figure 4—figure supplement 2 . Phylogenetic tree distinguishes clustered from non-clustered Pcdhs . Based on this phylogeny , evolutionary couplings were obtained for the two groups labeled .",
"The clustered and non-clustered Pcdh alignments had effective sequences numbers of 2660 and 405 . 5 , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 18449 . 018 Finally , we looked at the composition of a predicted non-clustered Pcdh interface , by selecting residues homologous to those found at clustered Pcdh interfaces .",
"The predicted EC1/EC4 interface residues are predominantly hydrophobic , while EC2/EC3 residues have more polar and ionic character ( Figure 4C ) .",
"Notably , positions 41 and 77 in EC1 and 320 , 321 , 371 and 373 in EC4 are more hydrophobic in non-clustered than clustered Pcdhs , indicating that these may form contacts in some non-clustered Pcdhs .",
"Thus , like in the clustered Pcdhs , the EC1/EC4 interaction may promote dimer affinity while the EC2/EC3 interaction provides specificity .",
"Recently , we and others predicted that clustered Pcdhs form homophilic antiparallel EC1-EC4 complexes based on crystal structures , mutagenesis and bioinformatics ( Nicoludis et al . , 2015; Rubinstein et al . , 2015 ) .",
"Our structure of PcdhγB3 EC1-4 confirms our sequence coevolution analysis , demonstrating the robustness of the analysis and revealing the molecular details of this interaction .",
"Here we extended this prediction to other non-clustered Pcdhs using sequence coevolution analysis .",
"Analysis of the PcdhγB3 EC1-4 structure in comparison to α or β subfamily dimers revealed structural differences that correlated with differences in isoform-specific conservation , indicating distinct specificity mechanisms .",
"Unlike the Dscams , where isoforms vary at specific alternatively-spliced regions ( Li et al . , 2016; Meijers et al . , 2007; Sawaya et al . , 2008; Wojtowicz et al . , 2007 ) , the clustered Pcdh subfamilies are structurally diverse , and thus can encode specificity in different ways .",
"We identified a hydrophobic interaction between EC1 and EC4 that contributes to dimerization affinity , whereas its conservation among clustered Pcdh isoforms suggests that this interaction is not a driver of specificity .",
"Overall , our data support a general role for conserved hydrophobic EC1/EC4 interactions in affinity , and for highly diversified polar EC2/EC3 contacts in specificity , and sequence analyses suggest that this is conserved in at least some non-clustered Pcdhs ."
],
[
"Human PcdhγB3 EC1-4 ( residues 1–414 , not counting the signal peptide ) was cloned into pET21 with a C-terminal hexahistidine tag , expressed in BL21 Gold ( DE3 ) Escherichia coli cells in terrific broth .",
"Cells were induced at OD600 = 0 . 8 with 0 . 5 mM isopropyl β-D-1-thiogalactopyranoside ( IPTG ) at 37°C for 4 hr , harvested and lysed by sonication in 8 M guanadinium hydrochloride ( GuHCl ) , 50 mM HEPES pH 7 . 5 , 2 mM CaCl2 , and 20 mM imidazole .",
"Cell lysates were diluted to 5 M GuHCl and loaded onto Ni-Sepharose , washed with 50 mM HEPES pH 7 . 5 , 250 mM NaCl , 10 mM CaCl2 , and 25 mM imidazole and eluted with 250 mM imidazole .",
"Eluted protein was refolded at 1 mg/mL in 12 hr dialysis steps reducing the GuHCl concentration from 2 . 5 M to 1 . 25 M and finally 0 M in refolding buffer ( 100 mM Tris pH 8 . 5 , 10 mM CaCl2 , 1 mM EDTA , 5 mM dithiothreitol ( DTT ) , and 0 . 5 M L-arginine ) .",
"Concentrated refolded protein was purified by size-exclusion chromatography ( SEC ) on a Superdex 200 16/60 column ( GE Healthcare , Pittsburgh , PA ) in 20 mM Tris pH 8 . 5 , 200 mM NaCl , and 2 mM CaCl2 ( SEC buffer ) .",
"Two peaks were isolated and each peak was run again separately by SEC before being concentrated for crystallization .",
"Approximate molecular mass of PcdhγB3 EC1-4 protein ( WT or F86A mutant ) was determined using a Superdex S200 10/300 column ( GE Healthcare , Pittsburgh , PA ) with in-line Wyatt Dawn Heleos II and Optilab T-rex refractive index detectors .",
"Protein ( 100 μL at 4 mg/mL ) was injected and run at 0 . 4 mL/min in SEC buffer .",
"Signals were aligned , normalized and band-broadened using bovine serum albumin as a standard .",
"Crystals were obtained by vapor diffusion at room temperature in 0 . 1 M HEPES pH 7 , 4% ethylene glycol , and 5% polyethylene glycol monomethyl ether 500 in a 0 . 3 μL protein ( 12 mg/mL ) to 0 . 3 μL reservoir drop , then cryoprotected with reservoir with 20% glycerol before flash cooling in liquid N2 .",
"Diffraction data ( Figure 1—source data 1 ) were processed in HKL2000 ( Otwinowski and Minor , 1997 ) .",
"The PcdhγB3 EC1-4 structure was determined by an iterative molecular replacement search with single domains of the PcdhγA1 EC1-3 structure ( PDBID 4zi9 ) in PHENIX ( Adams et al . , 2010 ) .",
"Model building was done in Coot ( Emsley and Cowtan , 2004 ) and refinement in PHENIX ( Adams et al . , 2010 ) .",
"We analyzed the physicochemical properties of the dimer interface using PISA ( Krissinel and Henrick , 2007 ) .",
"In the structure , we found a HEPES molecule near the EC2/EC3 interface that forms a salt bridge with N253 and N155 ( Figure 1—figure supplement 4 ) .",
"MALS data were collected with Tris as the buffer ( Figure 1—figure supplement 1 ) , indicating that HEPES is not required for dimerization .",
"Overall percent identity of the most common residue at each position was used to calculate ICR values , dividing the percent identity across subfamily orthologs by the percent identity across subfamily paralogs .",
"ICR values were then normalized by dividing by the whole sequence ICR average within each subfamily .",
"The alignment and identity data are provided here ( Nicoludis et al . , 2015 ) .",
"Pcdhα4 EC1-4 ( 5dzw ) , Pcdhα7 EC1-5 ( 5dzv ) , Pcdhβ6 EC1-4 ( 5dzx ) , Pcdhβ8 EC1-4 ( 5dzy ) , and PcdhγB3 EC1-4 ( 5k8r ) dimer structures were submitted to the Computational Interface Alanine Scanning Server using default settings ( Kortemme and Baker , 2002; Kortemme et al . , 2004 ) .",
"Previously , we generated an alignment of clustered Pcdhs using mouse PcdhγC3 and manually filtered by phylogeny , using FastTree 2 . 1 ( Price et al . , 2010 ) , to eliminate non-clustered Pcdhs ( Figure 4—figure supplement 2 ) ( Nicoludis et al . , 2015 ) .",
"Both this clustered Pcdh and the now separated non-clustered Pcdh alignment were filtered to remove sequences with more than 50% gaps and columns with more than 30% gaps , and trimmed to contain only EC1-EC4 .",
"The clustered and non-clustered Pcdh alignments had 2660 and 405 . 5 effective non-redundant sequences , respectively; sequences were considered redundant and downweighted when more than 90% identical over their full length ( Hopf et al . , 2014 ) .",
"Evolutionary couplings ( Hopf et al . , 2014; Marks et al . , 2011 ) were computed using the updated ‘PLMC’ algorithm ( Weinreb et al . , 2015 ) available on https://github . com/debbiemarkslab/plmc , which uses a pseudo maximum likelihood approximation ( Balakrishnan et al . , 2011; Ekeberg et al . , 2013; Kamisetty et al . , 2013 ) .",
"Alignments and all EC scores are provided ( Figure 4—source data 1–4 ) .",
"We used the precision of the intra-domain evolutionary couplings to determine whether the inter-domain evolutionary couplings are likely to be true .",
"For the clustered Pcdh alignment , 83 non-local ( more than five residues apart ) contacts fall above a threshold of 80% precision of the intra-domain evolutionary couplings .",
"Intra-domain evolutionary couplings are considered true if they correspond to structural contact ( minimum atom distance < 8 Å ) in any structure ( Figure 4—figure supplement 1 ) .",
"Based on the same 80% precision threshold , the top 38 non-local ECs are significant in the non-clustered Pcdh alignment .",
"We exclude couplings between residues greater than 400 in this analysis due to the false signal from gaps in this region ."
]
] | [
"Protocadherins ( Pcdhs ) are cell adhesion and signaling proteins used by neurons to develop and maintain neuronal networks , relying on trans homophilic interactions between their extracellular cadherin ( EC ) repeat domains .",
"We present the structure of the antiparallel EC1-4 homodimer of human PcdhγB3 , a member of the γ subfamily of clustered Pcdhs .",
"Structure and sequence comparisons of α , β , and γ clustered Pcdh isoforms illustrate that subfamilies encode specificity in distinct ways through diversification of loop region structure and composition in EC2 and EC3 , which contains isoform-specific conservation of primarily polar residues .",
"In contrast , the EC1/EC4 interface comprises hydrophobic interactions that provide non-selective dimerization affinity .",
"Using sequence coevolution analysis , we found evidence for a similar antiparallel EC1-4 interaction in non-clustered Pcdh families .",
"We thus deduce that the EC1-4 antiparallel homodimer is a general interaction strategy that evolved before the divergence of these distinct protocadherin families ."
] | [
"As the brain develops , nerve cells or neurons connect with one another to form complex networks .",
"These connections form between branch-like structures , called dendrites , that project from the cell body of each neuron .",
"To prevent unneeded connections from forming , dendrites that belong to the same neuron need a way to recognize and avoid one another .",
"A family of proteins called protocadherins supports this process of self-avoidance .",
"Protocadherins have three main parts or domains: an extracellular domain that faces outwards away from the cell , a transmembrane domain that sits within the cell’s surface membrane and an intracellular domain that faces into the cell’s interior .",
"There are two major groups of protocadherins – clustered and non-clustered – and the former are responsible for the self-avoidance behavior between dendrites .",
"Clustered protocadherins in turn comprise three subfamilies , each of which consists of multiple variants with slightly different structures ( known as isoforms ) .",
"The particular set of protocadherin isoforms that a neuron displays on its surface distinguishes that neuron from all others , a little like a barcode .",
"When two dendrites meet , the protocadherins in their membranes come into contact with one another .",
"If both dendrites come from the same neuron and therefore possess identical sets of protocadherins , then all protocadherins can form two-subunit complexes containing one copy of the same isoform from each dendrite .",
"These complexes are called homodimers and their formation acts as a signal that informs the cell that it has encountered one of its own dendrites and should therefore not establish a connection .",
"By using X-rays to determine the structure of a crystallized protocadherin fragment down to the level of its individual atoms , Nicoludis et al . now reveal exactly how clustered protocadherins form homodimers .",
"The results show that each protocadherin subfamily uses a slightly different type of interaction due to differences in the structure of their extracellular domains .",
"The next challenge is to identify the signaling cascade that is triggered by the formation of clustered protocadherin homodimers , and to work out how activation of this cascade prevents a permanent connection from forming .",
"In addition , the results of Nicoludis et al . predict that some non-clustered protocadherins form dimers with a similar architecture to that of clustered protocadherins .",
"This possibility should also be tested experimentally ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"structural biology and molecular biophysics"
] | Importin-9 wraps around the H2A-H2B core to act as nuclear importer and histone chaperone | elife-43630-v2 | [
[
"Eukaryotic chromatin is organized into nucleosomes , which are structural and functional units that are composed of 147 base pairs of DNA wrapped around two H3-H4 dimers and two H2A-H2B dimers ( Luger et al . , 1997 ) .",
"Nucleosomes are assembled in the nucleus during S-phase as new H2A , H2B , H3 and H4 proteins are synthesized in the cytoplasm ( Adams and Kamakaka , 1999; Annunziato , 2012; Verreault , 2000 ) .",
"Newly translated histones are folded and assembled into H2A-H2B and H3-H4 dimers , which are then imported into the nucleus for deposition onto replicating chromatin .",
"Despite their small sizes , histones do not diffuse into the nucleus but are transported by nuclear import receptors of the Karyopherin-β family termed importins ( Baake et al . , 2001; Jäkel et al . , 1999; Johnson-Saliba et al . , 2000; Mosammaparast et al . , 2002b; Mosammaparast et al . , 2001; Mühlhäusser et al . , 2001 ) .",
"Importins usually recognize their protein cargos by binding nuclear localization signals ( NLSs ) in their polypeptide chains .",
"Importins bind nucleoporins to traverse the permeability barrier of the nuclear pore complex ( NPC ) ( reviewed in Chook and Süel , 2011; Cook et al . , 2007; Görlich and Kutay , 1999; Izaurralde et al . , 1997; Kim et al . , 2018; Kosinski et al . , 2016; Lin et al . , 2016; Soniat and Chook , 2015 ) .",
"The small GTPase Ran controls direction of transport .",
"Binding of cargos and RanGTP to importins is mutually exclusive .",
"In the nucleus , where Ran is kept in the GTP state by guanine nucleotide exchange factor RCC1 , importins bind RanGTP with high affinity , resulting in cargo release ( Chook and Süel , 2011; Izaurralde et al . , 1997; Soniat and Chook , 2015 ) .",
"Studies in importin-deletion yeast strains identified Kap114 ( S . cerevisiae homolog of Importin-9 or Imp9 ) as the primary H2A-H2B importer , and Kap121 ( homolog of Importin-5 ) and Kap123 ( homolog , Importin-4 ) as secondary importers ( Mosammaparast et al . , 2002b; Mosammaparast et al . , 2001 ) .",
"Pull-down binding from cytosolic HeLa extract and proteomics tracking nuclear-cytoplasmic localization in human cells also identified core histones as Imp9 cargos ( Jäkel et al . , 2002a; Kimura et al . , 2017 ) .",
"The use of multiple backup importin systems is also seen in human cells , as many previous studies have shown that H2A and H2B can bind and be imported into nuclei of digitonin-permeabilized cells by several human importins ( such as Importin-β , Karyopherin-β2 , Importin-4 , Importin-5 , Importin-7 ) in addition to Importin-9 ( Baake et al . , 2001; Johnson-Saliba et al . , 2000; Mosammaparast et al . , 2002b; Mosammaparast et al . , 2001; Mühlhäusser et al . , 2001 ) .",
"Core histones H2A , H2B , H3 and H4 all contain disordered N-terminal tails followed by small histone-fold domains; H2A also has a disordered C-terminal tail ( Luger et al . , 1997 ) .",
"The N-terminal tails of histones contain many basic residues , somewhat resembling classical NLS motifs ( Blackwell et al . , 2007; Ejlassi-Lassallette et al . , 2011; Johnson-Saliba et al . , 2000; Marchetti et al . , 2000; Greiner et al . , 2004; Mosammaparast et al . , 2001; Moreland et al . , 1987 ) .",
"H2A and H2B tails are able to target heterologous proteins into the nucleus ( Mosammaparast et al . , 2001 ) , but removal of the tails does not abolish localization of H2A-H2B in the nucleus ( Thiriet and Hayes , 2001 ) .",
"Furthermore , analysis of seven different importins binding to H3 and H4 tails vs . full-length H3-H4 vs . H3-H4•Asf1 chaperone complex suggested that specificities for importin-binding reside not only in the tail ‘NLSs’ but also in the histone folds and the bound chaperone ( Soniat et al . , 2016 ) .",
"Here , we solved the crystal structure of Imp9 bound to the full-length H2A-H2B dimer to understand how histones are recognized for nuclear import .",
"The superhelical Imp9 wraps around the histone dimer .",
"Most of the N-terminal tails of both H2A and H2B are disordered , and only five residues of the H2B tail contact Imp9 .",
"Binding of Imp9 blocks DNA and H3-H4 sites on H2A-H2B , and Imp9 prevents H2A-H2B from aggregating on DNA , consistent with a histone chaperone-like activity for Imp9 .",
"Unlike other importin-cargo complexes , RanGTP does not dissociate Imp9•H2A-H2B but binds the complex and enhances its dissociation by DNA .",
"The Ran•Imp9•H2A-H2B complex is also able to promote H2A-H2B assembly into nucleosomes .",
"Formation of the Ran•Imp9•H2A-H2B complex appears to modulate importin-histone interactions to facilitate histone deposition to nuclear targets such as the assembling nucleosome ."
],
[
"The major nuclear importer for H2A-H2B in S . cerevisiae is Kap114 ( Mosammaparast et al . , 2002b; Mosammaparast et al . , 2001 ) .",
"Imp9 , the human homolog of Kap114 , was previously shown to bind and import H2A-H2B ( Jäkel et al . , 2002a; Kimura et al . , 2017; Mühlhäusser et al . , 2001 ) .",
"We show Imp9-histone interactions in immunoprecipitation from the cytoplasmic fraction of a stable HeLa cell line expressing mCherry-H2B ( Figure 1A ) .",
"We also show by fluorescence microscopy that Imp9 in these cells localizes mostly to the cytoplasm ( Figure 1B ) .",
"Similar cytoplasmic localization of Imp9 was reported in the Human Protein Atlas ( Thul et al . , 2017; Uhlen et al . , 2017 ) .",
"To understand how Imp9 recognizes histones for nuclear import , we solved the crystal structure of human Imp9 bound to full-length X . laevis H2A-H2B ( dissociation constant , KD = 30 nM; Table 1 and Figure 1—figure supplement 1 ) by single wavelength anomalous dispersion to 2 . 7 Å resolution ( Figure 1—source data 1 ) .",
"Imp9 is made up of twenty tandem HEAT repeats , each containing two antiparallel helices A and B that line the convex and concave surfaces of superhelical-shaped protein , respectively ( Figure 1C and Figure 1—figure supplement 2A , B ) .",
"The concave surface of Imp9 is mostly acidic , with a few small basic patches ( Figure 1—figure supplement 2B ) .",
"This charged concave surface of Imp9 wraps around H2A-H2B , burying 1352 Å2 ( 26% of the H2A-H2B surface ) at three distinct interfaces 1–3 ( Figure 2A–D and Figure 2—figure supplements 1–3 ) .",
"The Imp9-bound H2A-H2B has a canonical histone-fold as in nucleosomes ( 151 Cα atoms aligned , r . m . s . d . 0 . 505 Å; PDB ID 1AOI ) ( Luger et al . , 1997 ) .",
"In our structure , the N-terminal and C-terminal tails of H2A ( residues 1–16 , 101–130 ) and H2B ( 1-27 , 125-126 ) , the first 14 residues of Imp9 and its H19loop ( residues 936–996 ) were not modeled due to missing electron density .",
"The N- and C-terminal HEAT repeats of Imp9 ( Interfaces 1 and 3 ) clamp the histone-fold domain while the inner surface of central HEAT repeats 7–8 ( Interface 2 ) interacts with a five-residue segment of the H2B N-terminal tail ( Figure 2 ) .",
"Interface 1 on Imp9 comprises the loop that follows helix 2B and the last turns of helices 3B , 4B and 5B ( Figure 2B , Figure 2—figure supplements 1A , 2A and 3A ) .",
"Hydrogen-bonding with H2A-H2B residues caps the C-terminal ends of these Imp9 B helices ( Figure 2—figure supplement 1D ) .",
"Of note is the end-to-end capping of the last turn of Imp9 helix 4B by the first turn of histone H2B helix α2 .",
"Interface 1 on the histones involves α2-L2-α3 of H2A and α1-L1-α2 of H2B , which constitute a significant portion of the basic DNA-binding surface found in nucleosomes .",
"Although histones and Imp9 surfaces at this interface are electrostatically complementary ( Figure 1—figure supplement 2B ) , interactions also involve many hydrogen bonds , hydrophobic interactions and main chain interactions ( Figure 2—figure supplement 1A , D ) .",
"Interface 2 involves Imp9 helices 7B , 8B and the H8loop ( connects helices 8A to 8B ) binding to the short 28KKRRK32 segment of the H2B N-terminal tail ( Figure 2C and Figure 2—figure supplements 1B , 2B–C and and 3B ) .",
"Electron densities for H2B 28KKRRK32 are weak ( see Figure 2—figure supplement 2B–C ) and atomic displacement parameters ( ‘B-factors’ ) for H2B residues 28–32 are also high ( >100 Å2 ) , suggesting dynamic interactions .",
"Charged H2B side chains make electrostatic interactions with several acidic Imp9 residues , while the aliphatic part of these basic side chains and their backbone participate in hydrophobic interactions .",
"Interface 3 involves the last three HEAT repeats of Imp9 , specifically the last turn of helix 18A and the short loop that follows , the H18-19loop , the C-terminal half of helix 19A and the first turn of helix 20B ( Figure 2D , Figure 2—figure supplements 1C , 2D and 3C ) .",
"Instead of the typical basic H2A-H2B residues interacting with the acidic Imp9 residues , charges at Interface 3 are reversed ( Figure 1—figure supplement 2B ) .",
"A basic patch formed by the Imp9 H18-19loop and nearby helices complement an acidic surface on the histones formed by residues from H2A helices α2 and αC , and the C-terminal half of H2B that comprises α2-α3-αC .",
"Of note here are salt bridges between Imp9 residue Arg898 and several acidic residues of H2A ( Figure 2D ) .",
"Many hydrophobic contacts are also found at this interface , and several helices ( Imp9 H18A , H19A and histone H2B α2 ) are capped through hydrogen-bonding with partner proteins ( Figure 2—figure supplements 1C and 3C ) .",
"We analyzed the distribution of binding energy of the extensive Imp9-H2A-H2B interface through mutagenesis of the N-terminal histone tails and several long Imp9 loops and determined KDs of the mutants using isothermal titration calorimetry ( ITC; Table 1 and Figure 1—figure supplement 1 ) .",
"Imp9 binds full-length H2A-H2B with high affinity ( KD = 30 nM ) .",
"We did not make mutations to Interface 1 because of the many main-chain interactions found there ( Figure 2—figure supplement 1D ) .",
"Interface 2 involves the H8 loop of Imp9 and the N-terminal tail of H2B , both of which are convenient for deletion mutagenesis .",
"Similarly , two long Imp9 loops ( H18-19loop and H19loop ) in Interface 3 are convenient for deletion mutagenesis .",
"H2A-H2B mutant assembled with the core of H2A ( residues 14–119 ) and full-length H2B , hence named H2AΔTail-H2B , has similar binding affinity ( KD = 40 nM ) as full-length H2A-H2B .",
"This result is consistent with structural observations that H2A residues in its N- and C-terminal tails are disordered and likely do not contact Imp9 .",
"Removal of the H2B tail ( deleting residues 1–35 ) , generating mutant H2A-H2BΔ ( 1-35 ) , also did not affect binding affinity ( KD = 40 nM ) .",
"This is not surprising given the weak electron density and high B-factors of H2B 28KKRRK32 bound to Imp9 in Interface 2 ( Figure 2—figure supplement 2 ) .",
"A H2A-H2B mutant dimer with only the core domain ( H2A residues 14–119 complexed with H2B residues 25–123; named H2AΔTail-H2BΔTail ) also bind as tightly to Imp9 as the full-length histones ( KD = 40 nM ) .",
"Removal of the Imp9 H8loop ( Imp9ΔH8loop ) , which forms part of the binding site for H2B 28KKRRK32 , also did not decrease binding ( KD , Imp9ΔH8loop = 10 nM; Table 1 , Figure 1—figure supplement 1E ) .",
"The histone tails thus do not contribute much binding energy for interactions with Imp9 .",
"At Interface 3 , the basic H18-19loop of Imp9 contacts the acidic patch of the histones while the nearby H19loop is mostly disordered and its contribution to histone binding is uncertain .",
"Removal of the H18-19loop reduced the affinity 15-fold ( KD = 450 nM; Table 1 , Figure 1—figure supplement 1F ) .",
"We note the endothermic binding reaction that occurred upon truncation of this 40-residue loop .",
"This result suggests substantial contribution of Interface three to the total binding energy .",
"Removal of the H19loop did not affect affinity ( KD = 40 nM; Table 1 , Figure 1—figure supplement 1G ) , suggesting that this disordered loop does not participate in H2A-H2B binding .",
"A large portion of the Imp9 interface on H2A-H2B overlaps with the DNA-binding and H3-H4-binding interfaces used in nucleosomes ( Figure 3A–C ) .",
"This feature of Imp9 occluding interfaces used in the nucleosome is common to many H2A-H2B histone chaperones of H2A-H2B ( Hammond et al . , 2017 ) .",
"Imp9 in fact buries more surface area on H2A-H2B ( 1352 Å2 ) than well-characterized H2A-H2B chaperones such as Nap1 ( 387 Å2 ) , Swr1 ( 488 Å2 ) , Anp32e ( 533 Å2 ) , Chz1 ( 906 Å2 ) , Spt16 of FACT ( 185 Å2 ) and YL1 ( 883 Å2 ) ( Hondele et al . , 2013; Hong et al . , 2014; Kemble et al . , 2015; Luger et al . , 1997; Mosammaparast et al . , 2002a; Obri et al . , 2014; Zhou et al . , 2008 ) .",
"Histone chaperones are a class of functionally , structurally and mechanistically diverse histone-binding proteins that ‘chaperone’ histones to protect them from promiscuous DNA-histone interactions ( Elsässer and D'Arcy , 2012; Mattiroli et al . , 2015 ) in many different contexts surrounding the formation of nucleosomes ( Laskey et al . , 1978 ) .",
"The observation that Imp9 buries more surface area on H2A-H2B than well-characterized histone chaperones raises the question of whether Imp9 might also function as a histone chaperone .",
"This function is manifested biochemically by the protein outcompeting DNA from non-nucleosomal DNA•H2A-H2B complexes ( Andrews et al . , 2010; Andrews et al . , 2008; Hondele et al . , 2013; Hong et al . , 2014 ) .",
"To test if Imp9 can compete H2A-H2B from DNA like histone chaperone Nap1 , we performed native gel-based competition assays .",
"Titration of Nap1 or Imp9 against DNA•H2A-H2B complexes leads to the release of free DNA as Nap1 or Imp9 binds H2A-H2B ( Figure 3D , E ) .",
"These results suggest that Imp9 can act as a histone chaperone by shielding H2A-H2B from promiscuous interactions while it accompanies the histones from the cytoplasm to the nucleus .",
"RanGTP generally binds importins with high affinity to dissociate Importin-cargo complexes and release cargos into the nucleus .",
"However , this appears not to be the case with Imp9•H2A-H2B .",
"When increasing concentrations of RanGTP ( 5–30 molar equivalents S . cerevisiae Ran ( 1–177/Q71L ) ) are added to an immobilized MBP-Imp9•H2A-H2B complex , the histones are not released ( Figure 4A; controls shown in Figure 4—figure supplement 1A , C ) .",
"The RanGTP protein used in these experiments is fully active as it easily dissociates a cargo/NLS from Kapβ2 ( Figure 4B and Figure 4—figure supplement 1B ) .",
"In a separate experiment , the Imp9•H2A-H2B complex also remains intact when added to immobilized MBP-RanGTP ( Figure 4C ) .",
"MBP-RanGTP binds to H2A-H2B-bound Imp9 to form what seems to be a heterotetrameric MBP-RanGTP•Imp9•H2A-H2B complex ( Figure 4C ) .",
"We examined the interactions of Imp9•H2A-H2B with RanGTP in solution using electrophoretic mobility shift assays and size exclusion chromatography .",
"Electrophoretic mobility shift assays ( EMSA ) show the formation of a 1:1 complex between Imp9 and H2A-H2B ( Figure 4D ) as well as between Imp9 and RanGTP ( Figure 4E , lanes 3–6 ) .",
"A complex containing equimolar amounts of Imp9 , H2A-H2B and RanGTP can also form ( Figure 4E , lanes 7–10 ) .",
"Size exclusion chromatography of Imp9•H2A-H2B in the presence of excess RanGTP also shows a large complex that contains Imp9 , H2A-H2B and Ran ( Figure 4—figure supplement 2 ) .",
"We used analytical ultracentrifugation to rigorously and quantitatively assess the formation of a heterotetrameric RanGTP•Imp9•H2A-H2B complex .",
"We examined individual Imp9 , H2A-H2B and RanGTP proteins , equimolar mixes of Imp9+H2A-H2B and Imp9+RanGTP , and a 1:1:3 molar ratio mix of Imp9 , H2A-H2B and RanGTP by analytical ultracentrifugation ( protein concentrations 3–10 μM; Figure 4F ) .",
"Sedimentation coefficient values of the individual proteins estimated from the sedimentation velocity experiments are consistent with their molecular weights: Imp9 ( 3 . 7S ) , H2A-H2B ( 1 . 3S ) and RanGTP ( 1 . 4S ) .",
"The binary complexes of Imp9•H2A-H2B and Imp9•RanGTP are both larger , at 4 . 3S and 4 . 2S , respectively .",
"The mixture of Imp9 , H2A-H2B and RanGTP gave peaks at 1 . 4S ( excess RanGTP ) and 4 . 6S .",
"The 4 . 6S assembly is larger than either Imp9•H2A-H2B or Imp9•RanGTP and is likely the quaternary RanGTP•Imp9•H2A-H2B complex .",
"We also studied Imp9 , Imp9•RanGTP , Imp9•H2A-H2B , and RanGTP•Imp9•H2A-H2B ( protein concentrations 31–43 μM ) by small angle X-ray scattering ( SAXS ) .",
"SAXS profiles for the four Imp9-containing samples were analyzed to calculate radius of gyration ( Rg ) , maximum particle size ( Dmax ) and pair distribution function ( P ( r ( Figure 4G , Figure 4—figure supplement 3 , Figure 4—source datas 1 and 2 ) .",
"The linearity of the Guinier plots confirms a high degree of homogeneity for each of the SAXS samples ( Figure 4—figure supplement 3A–D ) .",
"Molecular weight of the RanGTP•Imp9•H2A-H2B complex was estimated to be 161 . 1 KDa by using SAXS MOW ( Fischer et al . , 2010 ) a value nearly identical to the expected molecular weight of 163 . 2 kDa from the sequence thus confirming stability of the 4-polypeptide chain RanGTP•Imp9•H2A-H2B complex in solution ( Figure 4G ) .",
"We compared the Imp9•H2A-H2B structure with the structures of different importins bound to RanGTP , to predict the Ran-binding site on Imp9 .",
"In these structures , RanGTP is always sandwiched between N-terminal and either central or C-terminal HEAT repeats of the importins ( Figure 4—figure supplement 4 ) .",
"Importin-RanGTP interactions at the first four HEAT repeats of importins ( binding Switch 1 , Switch two and α3 of RanGTP ) appear to be structurally conserved even though the interface on the opposite side of RanGTP involves different central or C-terminal HEAT repeats in different importins ( Chook and Blobel , 1999; Kobayashi and Matsuura , 2013; Lee et al . , 2005; Tsirkone et al . , 2014; Vetter et al . , 1999 ) .",
"Structural alignment of HEAT repeats 1–4 of Imp9 with HEAT repeats 1–4 of Importin-β ( 1-462 ) •RanGTP ( PDB ID 1IBR ( Vetter et al . , 1999 ) ; r . m . s . d . of 152 Cαs in the alignment is 3 . 27 Å ) , Kap95•RanGTP ( 2BKU ( Lee et al . , 2005 ) r . m . s . d . of 152 Cαs in the alignment is 3 . 20 Å ) , Kapβ2•RanGTP ( 1QBK ( Chook and Blobel , 1999 ) r . m . s . d . of 152 Cαs in the alignment is 4 . 02 Å ) , Kap121•RanGTP ( 3W3Z ( Kobayashi and Matsuura , 2013 ) ; r . m . s . d . of 144 Cαs in the alignment is 2 . 51 Å ) , Transportin-SR2•RanGTP ( 4C0Q; ( Maertens et al . , 2014 ) r . m . s . d . of 144 Cαs in the alignment is 5 . 02 Å ) and Importin-13•RanGTP ( 2 × 19 ( Bono et al . , 2010 ) ; r . m . s . d . of 144 Cαs in the alignment is 3 . 29 Å ) , and examination of the six structures at a single orientation of Imp9 , show that RanGTP binds in very similar orientations to very similar locations at the N-terminus of these importins ( Figure 4—figure supplement 4A–F ) .",
"Examination of the interactions between the N-terminal HEAT repeats of Kap95 , Kap121 , Importin-β , Importin-13 and Transportin-SR2 with RanGTP , together with the sequence alignment of this region of the importins show positional/structural conservation of many interacting and potentially interacting ( in Imp9 ) residues ( Figure 4—figure supplement 5A–G ) .",
"These analyses suggest that the N-terminal HEAT repeats of Imp9 are likely to be important in binding RanGTP .",
"Structural alignment of HEAT repeats 1–4 of Imp9 and Kap121•RanGTP allows us to predict the RanGTP binding site at the N-terminus of Imp9 ( Figure 4—figure supplement 6A , B ) .",
"The prediction is supported by an Imp9 mutant with HEAT repeats 1–3 removed that no longer binds RanGTP ( Figure 4—figure supplement 6C–E ) .",
"This likely Ran-binding site at the N-terminus of Imp9 appears separate from but adjacent to the H2A-H2B binding site ( Figure 4—figure supplement 6A , B ) .",
"The GTPase can most likely access Imp9 without dislodging H2A-H2B but proximity of RanGTP to the histones could modulate Imp9-histones interactions especially the kinetics of binding .",
"We performed native gel-based competition assays to titrate DNA against Imp9•H2A-H2B or RanGTP•Imp9•H2A-H2B .",
"DNA is unable to compete H2A-H2B from Imp9•H2A-H2B ( Figure 3D–E and Figure 5A , lanes 5–7 ) but can compete H2A-H2B from RanGTP•Imp9•H2A-H2B to produce Imp9•RanGTP and DNA•H2A-H2B ( Figure 5A–B , lanes 8–10 ) .",
"Unlike Imp9 , which efficiently displaces DNA from the DNA•H2A-H2B complex ( Figure 5C–D , lanes 4–6 ) , Imp9•RanGTP does not displace DNA from the DNA•H2A-H2B complex ( Figure 5C–D , lanes 8–10 ) .",
"These results show that the interaction between Imp9 and H2A-H2B is altered by RanGTP .",
"We next tested the ability of Imp9 and Imp9•RanGTP to assemble and disassemble nucleosomes ( Figure 5E , F ) .",
"Like Nap1 , Imp9 and Imp9•RanGTP do not influence the stability of the tetrasome ( Figure 5—figure supplement 1A ) .",
"To monitor nucleosome assembly , we titrated H2A-H2B alone or with Nap1 , Imp9 or Imp9 +RanGTP against tetrasome and assayed the formation of nucleosomes ( Figure 5E ) .",
"Nucleosomes form from H2A-H2B alone or with H2A-H2B and Nap1 or Imp9 +RanGTP ( Figure 5E , lanes 4–5 and 8–9 ) but not with Imp9 alone ( Figure 5E , lanes 6–7 ) .",
"Imp9 will bind H2A-H2B preventing its deposition on tetrasomes to make a nucleosome ( Figure 5—figure supplement 1B , lanes 6–7 ) .",
"Notably , in the presence of RanGTP , Imp9 is better at promoting H2A-H2B deposition than either Nap1 or no chaperone .",
"To monitor nucleosome disassembly , we titrated Nap1 , Imp9 , or Imp9 +RanGTP against nucleosomes ( Figure 5F ) .",
"We see that Imp9 can extract H2A-H2B from the nucleosome to produce tetrasome and Imp9•H2A-H2B ( Figure 5F , lanes 5–6; Figure 5—figure supplement 1C ) , while Nap1 and Imp9 +RanGTP have no effect ( Figure 5F , lanes 3–4 and 7–8 ) .",
"These data reinforce the chaperone-like activity of Imp9 and show that Ran influences the interaction between Imp9 and H2A-H2B , possibly through an allosteric mechanism as comparative analysis with other importin•RanGTP complexes suggests that the RanGTP binding site does not overlap with the H2A-H2B binding site .",
"The RanGTP binds the Imp9•H2A-H2B complex to modulate importin-histones interactions to facilitate release of the histones for nucleosome assembly ."
],
[
"The solenoid-shaped Imp9 wraps around the folded globular domain of the H2A-H2B dimer , leaving most of the N-terminal tails of H2A , H2B and the C-terminal tail of H2A disordered in the complex .",
"Only the 5-residue 28KKRRK32 segment of the H2B tail contacts Imp9 even though weak electron density and high atomic displacement parameters of the H2B tail suggests that these interactions are dynamic .",
"Our structural observations that Imp9 binds mostly to the globular domain of the H2A-H2B are also consistent with the lack of effect in Imp9 binding when either or both histone tails are deleted ( Table 1 ) , and with the previously reported nuclear localization of a mutant of H2A-H2B that lacks both its N-terminal tails ( Thiriet and Hayes , 2001 ) .",
"However , very weak dynamic/fuzzy long-range electrostatic interactions between Imp9 and histones tails may still exist - we had previously reported very weak and dynamic interactions between an importin-cargo pair by NMR that could not be observed by X-ray crystallography or detected in mutagenesis/ITC experiments ( Yoshizawa et al . , 2018 ) .",
"Nevertheless , H2A-H2B thus belongs to a small category of nuclear import cargos that mostly use surfaces of folded domains rather than extended linear nuclear import/localization motifs to bind their importins ( Aksu et al . , 2016; Bono et al . , 2010; Cook et al . , 2009; Grünwald and Bono , 2011; Grünwald et al . , 2013; Matsuura and Stewart , 2004; Okada et al . , 2009 ) .",
"Imp9-binding blocks both the nucleosomal DNA- and H3-H4-binding sites of H2A-H2B in a manner that is reminiscent of histone chaperone-H2A-H2B interactions .",
"Interestingly , the Imp9•H2A-H2B binding interface is much larger than any known complexes of H2A-H2B or H2A . Z/H2B bound to histone chaperones ( Elsässer and D'Arcy , 2012; Mattiroli et al . , 2015 ) .",
"Imp9 also acts biochemically like a histone chaperone to prevent H2A-H2B from aggregating with DNA in vitro .",
"The ability of Imp9 to structurally sequester H2A-H2B from promiscuous interactions with DNA and its function in trafficking the histones fit with the broadly defined class of histone-binding proteins known as histone chaperones ( Elsässer and D'Arcy , 2012; Mattiroli et al . , 2015 ) .",
"It is also generally thought that there is little to no free histones in the cell as they are either bound in nucleosomes or by histone chaperones ( Elsässer and D'Arcy , 2012; Mattiroli et al . , 2015 ) .",
"Imp9 binds H2A-H2B in the cytoplasm , acts as a storage chaperone in the cytoplasm and a nuclear import receptor to take histones through the NPC ( Jäkel et al . , 2002a; Kimura et al . , 2017; Mosammaparast et al . , 2002a; Mosammaparast et al . , 2001; Mühlhäusser et al . , 2001 ) .",
"Görlich and colleagues proposed in 2002 that negatively charged importins act as chaperones toward positively charged cargo proteins like histones ( Jäkel et al . , 2002b ) .",
"Others also suggested importins acting as chaperones ( Lusk et al . , 2002 ) .",
"We provide structural evidence to support this proposal as the mostly negatively charged Imp9 indeed shields the mostly positively charged histone-fold domain of H2A-H2B , and perhaps also dynamically shields the extended basic histone tails .",
"The ability of Imp9 to chaperone H2A-H2B , however , goes beyond charge shielding .",
"Despite overall charge complementarity , there are only a few salt bridges at the Imp9•H2A-H2B interface , which also employs hydrophobic interactions and hydrogen bonds , many involving main chains of both proteins .",
"Imp9 also shields many hydrophobic patches on H2A-H2B .",
"The interaction further involves a charge reversal where a basic surface at the C-terminal end of Imp9 interacts with the acidic patch of H2A-H2B .",
"The extensive and persistent interactions that allows Imp9 to surround and shield H2A-H2B also differ significantly from the recently revealed chaperoning interactions of another importin , that of Kapβ2 ( or Transportin-1 ) with the Fused in Sarcoma protein ( FUS ) .",
"Kapβ2-FUS interactions are anchored through high affinity binding at the 26-residue PY-NLS linear motif of FUS that then enable weak , distributed and dynamic interactions with multiple mostly intrinsically disordered regions of FUS , to block formation of higher-order FUS assemblies and liquid-liquid phase separation ( Yoshizawa et al . , 2018 ) .",
"The way the Imp9 solenoid wraps around H2A-H2B leaves the predicted N-terminal Ran-binding site of Imp9 accessible and ready to bind RanGTP .",
"We showed by pull-down , electrophoretic mobility shift , size exclusion chromatography , analytical ultracentrifugation and SAXS experiments that Imp9 binds both the histones and RanGTP simultaneously and stably , suggesting that unlike most importin-cargo complexes , Imp9•H2A-H2B is unlikely to be dissociated by RanGTP alone upon entering the nucleus .",
"This finding is not without precedence as Pemberton and colleagues previously showed an assembly that contains Kap114 , H2A-H2B , RanGTP and the histone chaperone Nap1 ( Mosammaparast et al . , 2002a ) .",
"Unlike the Pemberton study , which found the complex containing RanGTP , histones and importin to be intact in the yeast nucleus , we do not detect interactions between Imp9 and H2A-H2B in the nucleus even though that interaction is easily observed in the cytoplasm ( Figure 1A , B ) .",
"Imp9 is likely dissociated from histones soon after the complex enters the nucleus .",
"We showed that RanGTP changes the interactions between Imp9 and H2A-H2B as it forms the RanGTP•Imp9•H2A-H2B complex .",
"DNA competes effectively with RanGTP•Imp9•H2A-H2B to produce Imp9•RanGTP and DNA•H2A-H2B even though it is unable to extract H2A-H2B from Imp9•H2A-H2B .",
"Furthermore , RanGTP•Imp9•H2A-H2B is better at promoting H2A-H2B deposition to assemble nucleosome than either Nap1•H2A-H2B or no chaperone , while Imp9 alone cannot deposit H2A-H2B .",
"The GTPase in the RanGTP•Imp9•H2A-H2B complex appears to modulate Imp9-H2A-H2B interactions to facilitate histone release and nucleosome assembly .",
"Accessibility of the N-terminal HEAT repeats of Imp9 in the histones complex may allow formation of the RanGTP•Imp9•H2A-H2B complex , but proximity of the Ran and histones binding sites coupled with the flexibility of the HEAT repeats architecture of Imp9 and the propensity for conformational changes likely changed the kinetics of Imp9-histone binding .",
"Although histones can be deposited by RanGTP•Imp9•H2A-H2B onto DNA or the tetrasome , it remains unclear how H2A-H2B is released from Imp9 in cells .",
"Assembling nucleosomes may release H2A-H2B from RanGTP•Imp9•H2A-H2B or the histones may be passed to another histone chaperone or nucleosome assembly factor as part of a chaperone hand-off cascade in the nucleus .",
"These questions and the one regarding potential additional roles for Imp9 in the cytoplasm are topics for future studies ."
],
[
"Wild-type human Imp9 and Imp9 mutants ( Imp9ΔH8loop , residues 371–396 replaced with SGSTGGSGS linker; Imp9ΔH18-19loop , residues 890–906 replaced with GSGTGSGSS; Imp9ΔH19loop , residues 941–996 ( GGS ) 12 ) were cloned into the pGEX-4T3 vector ( GE Healthcare , USA ) or the pmalE vector ( New England BioLabs , Ipswich , MA ) modified to contain a TEV cleavage site ( Chook and Blobel , 1999; Chook et al . , 2002 ) and express His6-MBP instead of MBP ( pHis6-Mal-TEV ) .",
"Plasmids expressing the X . laevis histones H2A and H2B were a gift from Bing Li , UT Southwestern Medical Center .",
"The construct for mutant H2BΔ ( 1-35 ) was PCR-amplified from the wildtype H2B construct and cloned into pET-3A vector ( Novagen , USA ) .",
"Imp9 and Imp9 mutants were expressed in BL21 ( DE3 ) E . coli cells ( induced with 0 . 5 mM isopropyl-β-d-1-thiogalactoside ( IPTG ) for 12 hr at 20°C ) .",
"Cells were harvested by centrifugation , resuspended in lysis buffer ( 50 mM Tris-HCl ( pH 7 . 5 ) , 0 . 1 mM NaCl , 1 mM EDTA , 2 mM DTT , 20% glycerol and complete protease inhibitors ( Roche Applied Science , Mannheim , Germany ) ) and then lysed with the EmulsiFlex-C5 cell homogenizer ( Avestin , Ottawa , Canada ) .",
"GST-Imp9 was purified using Glutathione Sepharose 4B ( GSH; GE Healthcare ) and the GST tag was cleaved using Tev protease on the GSH column .",
"Imp9 was further purified by anion exchange chromatography followed by size-exclusion chromatography ( Superdex200 , GE Healthcare; final buffer – 20 mM HEPES ( pH 7 . 3 ) , 110 mM potassium acetate , 2 mM magnesium acetate , 2 mM DTT , 15% glycerol ) .",
"MBP-Imp9 was purified using amylose resin ( NEB ) affinity chromatography .",
"The MBP tag was left intact on MBP fusion proteins , which were used for in vitro pull-down binding assays and analysis by size exclusion chromatography .",
"Wild type and mutant Xenopus histones H2A , H2B proteins were expressed individually in E . coli BL21 DE3 plysS cells , which were lysed by sonication .",
"The lysate centrifuged at 16000 rpm and the washed pellet was resuspended in unfolding buffer ( 7 M guanidinium HCl , 20 mM Tris HCl , pH 7 . 5 , 10 mM DTT ) and dialyzed overnight in SAU-200 buffer ( 7 M urea , 20 mM sodium acetate , pH 5 . 2 , 200 mM NaCl , 1 mM EDTA , 5 mM β-mercaptoethanol ) .",
"The unfolded histone protein samples were further purified with cation exchange chromatography in SAU buffer ( 200–600 mM NaCl ) followed by dialysis overnight in cold water .",
"Mutant H2BΔ ( 1-35 ) was purified as described above .",
"Mutant proteins H2AΔTail ( contains residues 14–119 of H2A ) and H2BΔTail ( contains residues 25–123 of H2B ) used for Isothermal titration calorimetry were obtained from The Histone Source ( Colorado , United States ) .",
"H2A-H2B was reconstituted by mixing equimolar concentrations of H2A and H2B in unfolding buffer followed by overnight dialysis into refolding buffer ( 2 M NaCl , 10 mM Tris HCl , 1 mM EDTA , 5 mM β-mercaptoethanol ) .",
"The dialyzed sample was concentrated and purified using size-exclusion chromatography in refolding buffer ( Luger et al . , 1997 ) .",
"Mutant histone dimers ( H2A- H2AΔTail-H2B , H2A-H2BΔ ( 1-35 ) and H2AΔTail-H2BΔTail ) were reconstituted and purified as described above for full-length wild type H2A-H2B ( Luger et al . , 1997 ) .",
"His-tagged full-length S . cerevisiae Nap1 ( C200A/C249A/C272A ) in pHAT4 vector was expressed in BL21 ( DE3 ) E . coli cells .",
"Nap1 was purified by affinity chromatography using a GE HisTrap SP FF column followed by ion-exchange chromatography using GE Mono Q 10/100 GL column and gel filtration chromatography using GE Superdex-200 16/600 column ( 20 mM Tris pH 7 . 5 , 300 mM NaCl , 0 . 5 mM TCEP ) .",
"Ran ( Gsp1 ( 1–179 , Q71L ) ) and MBP-Ran were expressed in E . coli BL21 ( DE3 ) cells as His6 –tag proteins ( induced with 0 . 5 mM IPTG for 12 hr at 20°C ) .",
"Harvested cells were lysed with the EmulsiFlex-C5 cell homogenizer ( Avestin , Ottawa , Canada ) and the proteins purified by affinity chromatography on Ni-NTA column .",
"Eluted proteins were loaded with GTP , and RanGTP and MBP-RanGTP were further purified by cation exchange chromatography followed by exchanging into buffer containing 20 mM HEPES ( pH 7 . 5 ) , 100 mM NaCl , 4 mM magnesium acetate , 1 mM DTT , 10% glycerol ( Chook and Blobel , 1999; Fung et al . , 2015 ) .",
"Purified Imp9 was mixed with 10-fold molar excess of H2A-H2B in gel filtration buffer ( 20 mM HEPES ( pH 7 . 3 ) , 110 mM potassium acetate , 2 mM magnesium acetate , 2 mM DTT , 15% glycerol ) .",
"Imp9•H2A-H2B was separated from excess histones by size-exclusion chromatography and concentrated to 18 mg/ml for crystallization .",
"Selenomethionyl-labeled Imp9 was expressed as described previously ( Doublié , 1997 ) and purified as for Imp9 .",
"Selenomethionyl-Imp9•H2A-H2B complex was assembled as for the native complex .",
"Initial crystals were obtained by the sitting drop vapor diffusion method from commercial screens ( reservoir solution - 40 mM MES pH 6 . 5 , 3 M potassium formate , and 10% glycerol ) and were further optimized by the hanging drop vapor diffusion method .",
"Crystals were cryoprotected in reservoir solution that was supplemented with 15% glycerol , and flash frozen in liquid nitrogen .",
"Selenomethionyl-Imp9•H2A-H2B crystals were obtained in the same conditions as native crystals and were prepared similarly for crystallographic data collection .",
"Imp9•H2A-H2B native crystals diffracted to a minimum Bragg spacing ( dmin ) of 2 . 70 Å and exhibited the symmetry of space group P21212 with cell dimensions of a = 127 . 4 Å , b = 223 . 3 Å , c = 131 . 8 Å and contained two heterotrimers per asymmetric unit .",
"All diffraction data were collected at beamline 19-ID ( SBC-CAT ) at the Advanced Photon Source ( Argonne National Laboratory , Argonne , Illinois , USA ) and processed in the program HKL-3000 ( Minor et al . , 2006 ) with applied corrections for effects resulting from absorption in a crystal and for radiation damage ( Borek et al . , 2003; Otwinowski et al . , 2003 ) , the calculation of an optimal error model , and corrections to compensate the phasing signal for a radiation-induced increase of non-isomorphism within the crystal ( Borek et al . , 2010; Borek et al . , 2013 ) .",
"These corrections were crucial for successful phasing and stable model refinement .",
"Crystals of Imp9•H2A-H2B displayed mildly anisotropic diffraction .",
"To minimize radiation damage and maximize redundancy , native data was collected in two separate scans of 125 degrees for a total of 250 degrees by translating a single crystal in the X-ray beam .",
"Analysis of the self-Patterson function calculated with the native data revealed a significant off-origin peak at approximately ( 1/2 , 1/2 , 1/2 ) and 27% the height of the origin peak , indicating translational pseudosymmetry .",
"Phases were obtained from a single wavelength anomalous dispersion ( SAD ) experiment using the selenomethionyl-Imp9•H2A-H2B protein with data to 2 . 65 Å .",
"Fifty-four selenium sites were located , phases improved and an initial model containing over 50% of all Imp9•H2A-H2B residues was automatically generated in the AutoBuild routine of the Phenix ( Adams et al . , 2010 ) program suite .",
"Completion of this model was performed by manual rebuilding in the program Coot ( Emsley et al . , 2010 ) .",
"Positional and isotropic atomic displacement parameter ( ADP ) as well as TLS ADP refinement of native Imp9•H2A-H2B with NCS restraints was performed to a resolution of 2 . 70 Å using the Phenix program suite with a random 2 . 1% of all data set aside for an Rfree calculation .",
"The final model for Imp9•H2A-H2B ( Rwork = 20 . 9% , Rfree = 24 . 0% ) contained 2275 residues and 356 waters .",
"The relatively high Rwork and Rfree values are likely due to the presence of translational pseudosymmetry .",
"A Ramachandran plot generated with the program MolProbity ( Chen et al . , 2010 ) indicated that 97 . 1% of all protein residues are in the most favored regions and 0 . 1% in disallowed regions .",
"Illustrations were prepared with PyMOL ( Schrodinger LLC , 2015 ) .",
"Data collection and structure refinement statistics are summarized in Figure 1—source data 1 .",
"Imp9 and mutant Imp9 proteins were expressed and purified as described above .",
"The wild type full-length H2A , H2B and mutant H2BΔ ( 1-35 ) proteins were purified as described above .",
"Mutant H2AΔTail and H2BΔTail proteins were obtained from Histone Source ( Colorado , USA ) .",
"H2A-H2B , H2AΔTail -H2B , H2A-H2BΔ ( 1-35 ) and H2AΔTail-H2BΔTail heterodimers were reconstituted and purified as described above .",
"Imp9 or mutant Imp9 proteins and H2A-H2B or H2A-H2B mutant dimers were dialyzed in ITC buffer containing 20 mM Tris-HCl ( pH 7 . 5 ) , 150 mM NaCl , 5 mM TCEP and 5% glycerol .",
"ITC experiments were carried out using ITC-200 calorimeter ( Microcal , LLC , Northampton , MA , USA ) at 20°C with 0 . 035 mM of Imp9 or mutant Imp9 protein in the sample cell and 0 . 35 mM H2A-H2B or mutant H2A-H2B protein in the syringe .",
"All samples were thoroughly degassed and then centrifuged at 16000 g for 10 min to remove precipitates .",
"21 injections each of 1 . 9 μl except for the first ( 0 . 5 μl ) were sequentially made in each experiment .",
"The injections were mixed at 300 rpm and consecutive injections were separated by 300 s to allow the peak to return to baseline .",
"All experiments were carried out in triplicates .",
"Data were integrated and baseline corrected using NITPIC ( Keller et al . , 2012 ) .",
"The baseline corrected and integrated data were globally analyzed in SEDPHAT ( Houtman et al . , 2007; Zhao et al . , 2015 ) using a model considering a single class of binding sites .",
"SVD-reconstructed thermogram provided by NITPIC , the fit-isotherms and the residuals from SEDPHAT were all plotted using GUSSI ( Brautigam , 2015 ) .",
"Individual experiments in the triplicate sets are differently color-coded in Figure 1—figure supplement 1A .",
"For error reporting , we used F-statistics and error-surface projection method to calculate the 68 . 3% confidence intervals of the fitted data ( Bevington ) .",
"The KD ( nM ) , ΔH ( kCal/mol ) , ΔS ( Cal/mol . K ) , ΔG ( kCal/mol ) and the Imp9 local concentration correction factors for each set of triplicate experiments are reported in the Table 1 .",
"Pull-down binding assays were performed by immobilizing purified MBP-Imp9 or MBP-RanGTP ( S .",
"cerevisiae Gsp1 ( 1–179/Q71L ) on amylose resin ( New England BioLabs , Ipswich , MA ) .",
"40 μl of 100 μM MBP-Imp9 or MBP-RanGTP was immobilized on 200 μl of amylose resin ( 50% slurry ) in binding assay ( BA ) buffer containing 20 mM HEPES pH 7 . 3 , 110 mM potassium acetate , 2 mM magnesium acetate , 2 mM DTT and 15% glycerol .",
"100 μl of ~20 μM of immobilized MBP-Imp9 resin was incubated with 100 μl of 400 μM of purified H2A-H2B in a total reaction volume of 400 μl for 30 min at 4°C , followed by five washes each with 400 μl BA buffer .",
"100 μl of ~20 μM of MBP–RanGTP resin were incubated with 100 μl of 50 μM of purified Imp9•H2A-H2B in a total volume of 400 μl for 30 min at 4°C , followed by five washes each with 400 μl BA buffer .",
"For RanGTP dissociations assays , a gradient of 10 μl , 20 μl , 40 μl or 60 μl of approximately 500 μM purified RanGTP was added to 50 μl of ~20 μM of immobilized MBP-Imp9 that were pre-bound with H2A-H2B , in a total reaction volume of 400 μl .",
"These binding reactions contain 12 . 5 μM , 25 μM , 50 μM or 75 μM of RanGTP added to 2 . 5 μM MBP-Imp9•H2A-H2B .",
"Binding was followed by five washes each with 400 μl of the BA buffer .",
"From each of the reactions , 30 μl of beads after final wash was suspended in 30 μl of BA buffer .",
"10 μl of the bead slurry sample was analyzed on 12% SDS-PAGE gels and stained with Coomassie Blue for visualization .",
"A control experiment involving immobilized GST-Kapβ2 , MBP-PY-NLS ( PY-NLS of hnRNP A1 ) , and a gradient of 12 . 5 μM , 25 μM , 50 μM and 75 μM of RanGTP ( prepared as described above for the MBP-Imp9•H2A-H2B experiments ) was carried out similarly to show that RanGTP dissociates PY-NLS bound to Kapβ2 .",
"2% of the input Ran-GTP added in each of the binding reactions and approximately 2% of flow-through is also shown in the Coomassie-stained SDS-PAGE gels .",
"Pull-down binding assay to probe Ran binding to Imp9 versus Imp9Δ1–144 were performed by immobilizing GST-Imp9 or GST-Imp9Δ1–144 on Glutathione Sepharose 4B resin ( GE Healthcare Life Sciences ) .",
"12 . 5 ml of lysate from 500 ml cell culture ( OD600 = 1 ) pellet of E . coli expressing GST-Imp9 or GST-Imp9Δ1–144 ( containing ~8 mg/ml of GST-Imp9 protein ) were incubated on 1 ml of 50% Glutathione Sepharose 4B slurry in BA buffer .",
"The GST-Imp9 or GST-Imp9Δ1–144 bound resin was washed five times , each with 6 ml BA buffer , before the binding assay .",
"200 μl of 50% slurry GST-Imp9 or GST-Imp9Δ1–144 resin ( ~12 μM proteins ) was incubated with 10 μl of ~500 μM RanGTP in a total reaction volume of 400 μl for 30 min at 4°C , followed by five washes ( each with 400 μl BA buffer ) .",
"After washing , 30 μl of 50% beads slurry was suspended in 30 μl BA buffer .",
"10 μl of the resulting bead slurry sample was analyzed by Coomassie-stained SDS-PAGE .",
"A control experiment using empty GSH sepharose beads and RanGTP was performed as described above .",
"The interaction between RanGTP and Imp9•H2A-H2B complex was probed by size exclusion chromatography ( SEC ) .",
"Imp9 , RanGTP , H2A-H2B were purified as described above .",
"First , a series of SEC experiments titrating RanGTP was performed .",
"SEC of Imp9 alone ( 20 μM ) , RanGTP alone ( 60 μM ) , H2A-H2B alone ( 20 μM ) , Imp9 +H2A-H2B 1:1 molar ratio ( 20 μM ) with no RanGTP , 0 . 5 , 1 , 2 and 3 molar equivalents of RanGTP were performed in buffer containing 20 mM HEPES pH 7 . 4 , 200 mM sodium chloride , 2 mM magnesium acetate , 2 mM TCEP and 8% ( v/v ) glycerol .",
"The experiments were performed using a Superdex 200 Increase 10/300 GL column .",
"A second series of SEC experiments using 1:1 Imp9 +H2A-H2B ( 70 μM ) and 1:1:1 Imp9 , H2A-H2B , and Ran ( 70 μM ) in the same column and the same buffer were performed with higher concentrations of proteins for visualization of proteins in the SEC fractions by Coomassie-stained SDS-PAGE .",
"A third SEC series involves the mutant MBP-Imp9Δ1–144 that does not bind RanGTP and using a different Superdex 200 Increase10/300 GL column with buffer containing 20 mM HEPES pH 7 . 4 , 200 mM sodium chloride , 2 mM magnesium acetate , 2 mM DTT and 10% glycerol .",
"The sedimentation coefficients of individual proteins and protein complexes in the mixture were estimated by monitoring their sedimentation properties in a sedimentation velocity experiment carried out in Beckman-Coulter Optima XL-1 Analytical Ultracentrifuge ( AUC ) .",
"The individual proteins and mixtures of proteins were analyzed in AUC buffer containing 20 mM HEPES pH 7 . 3 , 200 mM sodium chloride , 2 mM magnesium chloride , 2 mM TCEP and 8% glycerol ( details below ) .",
"Protein samples ( 450 µl ) and AUC buffer ( 450 µl ) were loaded into a double sector centerpiece and centrifuged in an eight-hole An-50Ti rotor to 50000 rpm at 20°C .",
"The double sectors were monitored for absorbance at 280 nm ( A280 ) .",
"A total of 140 scans were collected and the first 80 scans were analyzed .",
"Buffer density , viscosity of the buffer and partial specific volume of the protein was estimated using SEDNTERP ( http://www . rasmb . bbri . org/software/windows/sednterp-philo/ ) .",
"Sedimentation coefficient distributions c",
"( s ) ( normalized for absorption differences ) were calculated by least squares boundary modeling of sedimentation velocity data using SEDFIT program ( Schuck , 2000 ) .",
"Sedimentation coefficients sw ( weighted-average obtained from the integration of c",
"( s ) distribution ) and frictional ratios f/f0 were obtained by refining the fit data in SEDFIT ( Schuck , 2000 ) .",
"For error reporting , F-statistics and Monte-Carlo for integrated weight-average s values were used ( Bevington ) .",
"Data were plotted using GUSSI ( Brautigam , 2015 ) .",
"Individual proteins , Imp9 , RanGTP and H2A-H2B , were purified as described above and dialyzed into the AUC buffer before mixing the samples to the final volume of 450 μL for the AUC experiments .",
"Samples for the AUC experiments contain: 1 ) 450 μL Imp9 alone ( 3 μM ) , 2 ) 450 μL RanGTP alone ( 10 μM ) , 3 ) 450 μL H2A-H2B ( 10 μM ) , 4 ) 3 μM Imp9 + 3 μM RanGTP in a total volume of 450 μL , 5 ) 3 μM Imp9 +3 μM H2A-H2B in a total volume of 450 μL , 6 ) 3 μM Imp9 + 3 μM H2A-H2B + 10 μM RanGTP in a total volume of 450 μL .",
"The proteins were mixed overnight before loading into the AUC cell .",
"SAXS experiments examining Imp9 , Imp9•H2A-H2B , Imp9•RanGTP , and RanGTP•Imp9•H2A-H2B samples were carried out at Beamline 4–2 of the Stanford Synchrotron Radiation Lightsource ( SSRL ) in the SLAC National Accelerator Laboratory .",
"At SSRL , the beam energy and current were 11 keV and 500 mA , respectively .",
"A silver behenate sample was used to calibrate the q-range and detector distance .",
"Data collection was controlled with Blu-Ice ( McPhillips et al . , 2002 ) .",
"We used an automatic sample delivery system equipped with a 1 . 5 mm-diameter thin-wall quartz capillary within which a sample aliquot was oscillated in the X-ray beam to minimize radiation damage ( Martel et al . , 2012 ) .",
"The sample was placed at 1 . 7 m from a MX225-HE ( Rayonix , USA ) CCD detector with a binned pixel size of 292 by 292 μm ( Figure 4—source data 1 ) .",
"All protein samples for SAXS were expressed and purified as described above .",
"Purified Imp9 was exchanged into SAXS buffer ( 20 mM HEPES pH 7 . 3 , 110 mM potassium acetate , 2 mM magnesium acetate , 2 mM DTT , and 10% glycerol ) by SEC and concentrated to 5 mg/ml ( 43 µM of Imp9 ) for SAXS analysis .",
"The Imp9•H2A-H2B was purified as described above and then exchanged into SAXS buffer by SEC and concentrated to 5 mg/ml ( 35 µM of Imp9•H2A-H2B ) for SAXS analysis .",
"To prepare the Imp9•RanGTP complex , previously purified Imp9 was first mixed with 5-fold molar excess of RanGTP for SEC to separate the Imp9•RanGTP complex from excess Ran .",
"This Imp9•RanGTP complex was then buffer exchanged into SAXS buffer in another round of SEC and concentrated to 5 mg/ml ( 37 µM of Imp9•RanGTP ) for SAXS .",
"To prepare the RanGTP•Imp9•H2A-H2B complex , previously purified Imp9•H2AH2B was mixed with 5-fold molar excess of RanGTP in SAXS buffer for SEC to separate RanGTP•Imp9•H2A-H2B from excess RanGTP .",
"Fractions containing RanGTP•Imp9•H2A-H2B were pooled , concentrated and subjected to a second round of SEC in SAXS buffer , after which the complex was concentrated to 5 mg/ml ( 31 µM of RanGTP•Imp9•H2A-H2B ) for SAXS .",
"The 10% glycerol in the SAXS buffer protects the protein samples from radiation damage during X-ray exposure ( Kuwamoto et al . , 2004 ) and our early studies show that low glycerol concentrations ( 5–20% ) do not affect protein compaction ( Yoshizawa et al . , 2018 ) .",
"All solutions were filtered through 0 . 1 μm membranes ( Millipore ) to remove any aggregates .",
"The SAXS profiles were collected at protein concentrations ranging from 0 . 5 to 5 . 0 mg/ml .",
"20 one-second exposures were used for each sample and buffer maintained at 15°C .",
"Each of the resulting diffraction images was scaled using the transmitted beam intensity , azimuthally integrated by SASTool ( SasTool , 2013 ) and averaged to obtain fully processed data in the form of intensity versus q [q = 4πsin ( θ ) /λ , θ = one half of the scattering angle; λ = X ray wavelength] .",
"The buffer SAXS profile was subtracted from a protein SAXS profile .",
"Subsequently , the mean of the lower concentration ( 0 . 5–1 . 5 mg/ml ) profiles in the smaller scattering angle region ( q < 0 . 15 Å−1 ) and the mean of the higher concentration ( 2 . 0–5 . 0 mg/ml ) profiles in the wider scattering angle region ( q > 0 . 12 Å−1 ) were merged to obtain the final experimental SAXS profiles that are free of the concentration-dependent aggregation or polydispersity effect ( Kikhney and Svergun , 2015 ) .",
"The merged SAXS profiles were initially analyzed using the ATSAS package ( Petoukhov et al . , 2012 ) to calculate radius of gyration ( Rg ) , maximum particle size ( Dmax ) , and pair distribution function ( P",
"( r ) ) ( Figure 4—figure supplement 3 and Figure 4—source datas 1 and 2 ) .",
"The molecular weight ( MWSAXS ) of each SAXS sample was estimated using SAXS MOW ( Fischer et al . , 2010 ) with a threshold of qmax = 0 . 25–0 . 3 Å−1 ( Figure 4G and Figure 4—source datas 1 and 2 ) .",
"HeLa cells expressing H2BmCherry ( Ke et al . , 2011 ) ( gift from Prof . Hongtao Yu , UT Southwestern ) .",
"The HeLa Tet-ON cells ( Cellosaurus Accession: HeLa Tet-On ( CVCL_IY74 ) ) stably expressing H2B-mCherry were originally created ( with cell identity confirmation carried out by STR profiling ) in Dr . Hongtao Yu’s lab at University of Texas Southwestern Medical Center , Dallas , Texas USA .",
"Mycoplasma negative status of the cell line was confirmed using the LookOut Mycoplasma PCR Detection kit , Sigma MP0035-1KT .",
"The cells were grown to 80% confluency , and total-cell lysate was prepared by suspending the cells in TB buffer containing 20 mM HEPES–KOH pH 7 . 3 , 110 mM potassium acetate , 2 mM magnesium acetate , 5 mM sodium acetate , 0 . 1 mM EGTA , 1 mM DTT and protease inhibitor cocktail ( Kimura et al . , 2017 ) on ice for 15 min , sonicating three times ( 5 s pulse , 10 s rest ) , then centrifuging the lysed cells at 15 , 000 g for 20 min at 4°C .",
"Nuclear and cytoplasmic fractions were prepared using the NE-PER Nuclear and Cytoplasmic Extraction reagents ( Thermo Scientific ) as per manufacturer’s instruction .",
"Protein concentration was quantitated using the Bradford protein assay kit ( BioRad ) .",
"The RFP-Trap ( high quality Red Fluorescent Protein ( RFP ) binding protein coupled to a monovalent magnetic matrix , ChromoTek GmbH ) was incubated with the cell lysates for 2 hr at 4°C .",
"The matrix was first washed with TB buffer supplemented with 200 mM NaCl , and then once with TB buffer supplemented with 150 mM NaCl .",
"The proteins bound to the beads were dissolved in SDS sample buffer for immunoblot analysis .",
"Cell lysate and protein samples dissolved in SDS sample buffer were separated by SDS–PAGE , and blotted with the indicated antibodies: Rabbit polyclonal antibody against tagRFP ( 1:1000 dilution , Cat no . AB233 , Evrogen ) , rabbit polyclonal antibody against Imp9 ( 1:1000 dilution , Cat no . A305-474A-T , Bethyl Laboratories , Inc ) , mouse monoclonal against Ran ( 1:2000 dilution , Cat no . 610340 , BD Biosciences ) , mouse monoclonal against Nuclear Pore Complex Proteins Antibody [MAb414] ( 1:5000 dilution , Cat no . 902903 , BioLegend ) and mouse monoclonal against PCNA ( 1:2000 dilution , Cat no . 307901 , BioLegend ) .",
"Goat anti-Rabbit IgG ( H + L ) , HRP-conjugated ( 1:6000 dilution , Cat no . 31460 , Thermo Fisher Scientific ) and Goat anti-Mouse IgG ( H + L ) , HRP-conjugated ( 1:6000 dilution , Cat no . 31430 , Thermo Fisher Scientific ) were used as the secondary antibodies , and immunoblots were developed using the SuperSignal West Pico PLUS Chemiluminescent Substrate ( Cat no . 34580 , Thermo Fisher Scientific ) according to the manufacturer's protocols and followed by detection using a Gel Doc EZ System ( Bio-Rad Laboratories , Hercules , CA , USA .",
"Cells ( 5 × 104 cells per chamber ) were seeded into collagen coated culture coverslip ( BD Falcon ) The next day , cells were rinsed with ice-cold PBS and fixed with 4% paraformaldehyde for 10 min at room temperature followed by permeabilization with 0 . 1% sodium citrate plus 0 . 1% Triton X-100 .",
"The cells were subjected to immunofluorescence staining using rabbit polyclonal antibody against Imp9 ( 1:250 dilution , Cat no . A305-474A-T , Bethyl Laboratories , Inc ) and mouse monoclonal antibody against Ran ( 1:250 dilution , Cat no . 610340 , BD Biosciences ) , for 2 hr at room temperature .",
"The cells were then washed with cold PBS three times for 1 min each and incubated with Alexa 480-labeled anti-rabbit secondary antibody ( 1:800 ) ( Invitrogen ) and Alexa 405-labeled anti-mouse secondary antibody ( 1:800 ) ( Invitrogen ) at room temperature for 1 hr .",
"Subsequently cells were washed with cold PBS three times for 1 min each and mounted with ProLong Gold Antifade Mountant ( Invitrogen ) .",
"Image acquisition was performed with a spinning disk confocal microscope system ( Nikon-Andor ) with a 100 × oil lens and the MetaMorph softwar .",
"Images were acquired from randomly selected fields as a z-stack with step size of 0 . 1 μm to give a total of 196 slices .",
"For each selected field of view , three images were taken , an Alexa488 ( Imp9 ) image , and Alexa405 ( Ran ) ."
]
] | [
"We report the crystal structure of nuclear import receptor Importin-9 bound to its cargo , the histones H2A-H2B .",
"Importin-9 wraps around the core , globular region of H2A-H2B to form an extensive interface .",
"The nature of this interface coupled with quantitative analysis of deletion mutants of H2A-H2B suggests that the NLS-like sequences in the H2A-H2B tails play a minor role in import .",
"Importin-9•H2A-H2B is reminiscent of interactions between histones and histone chaperones in that it precludes H2A-H2B interactions with DNA and H3-H4 as seen in the nucleosome .",
"Like many histone chaperones , which prevent inappropriate non-nucleosomal interactions , Importin-9 also sequesters H2A-H2B from DNA .",
"Importin-9 appears to act as a storage chaperone for H2A-H2B while escorting it to the nucleus .",
"Surprisingly , RanGTP does not dissociate Importin-9•H2A-H2B but assembles into a RanGTP•Importin-9•H2A-H2B complex .",
"The presence of Ran in the complex , however , modulates Imp9-H2A-H2B interactions to facilitate its dissociation by DNA and assembly into a nucleosome ."
] | [
"Cells contain two meters of DNA which , if left to its own devices , would soon end up in a knot .",
"To keep things organized , the genetic code is wrapped around protein ‘spools’ called histones , meaning it can all fit within a part of the cell known as the nucleus .",
"The cell makes a copy of its DNA every time it divides , and this copy needs a new set of histones to keep it tidy .",
"The machinery required to construct new histones sits outside the nucleus and getting the histones into position in the nucleus can be a challenge .",
"Histones have a positive charge , which helps to keep the negatively charged DNA wound around the spool .",
"Yet without supervision , histones can stick to other charged molecules in the cell and cause blockages .",
"The proteins responsible for histone transport are called importins .",
"These proteins normally recognize their cargo by molecular patterns called “nuclear localization signals” .",
"These patterns work like a postal address , telling the importin to take the cargo into the nucleus .",
"When they arrive at their destination , another protein called Ran interacts with the importins to release the cargo .",
"Strangely , removing the predicted address pattern from histones does not stop them getting to the nucleus .",
"To find out what was going on , Padavannil et al . solved the three-dimensional structure of an importin bound to a pair of histones via a technique called X-ray crystallography .",
"This made it possible to see how the proteins fit together .",
"The structure revealed that , rather than interact with the predicted address pattern , the importin wraps around the core of the histones .",
"This blocks the positive charges , stopping the histones sticking to other molecules on their way to the nucleus .",
"The next challenge was to find out how the cell unhooks the histone cargo from the importin when it arrives in the nucleus; with the positive charges covered by the importin , the histones could not stick to the DNA .",
"Yet , something changed when the levels of Ran were high .",
"Rather than unhook the histone , Ran joined the importin-histone complex .",
"This then made it possible for the histones to attach to DNA , helping them to get into position without sticking to the wrong molecules .",
"These findings form the first step in understanding how the cell transports sticky histones without getting in a knot .",
"The next step is to find out whether these interactions , shown in test tubes , happen in the same way inside living cells ."
] | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"stem cells and regenerative medicine",
"developmental biology"
] | Distinct progenitor populations mediate regeneration in the zebrafish lateral line | elife-43736-v2 | [
[
"The regenerative potential of a given tissue is dependent on the availability of progenitor cells that are able to functionally replace lost or damaged cells within that tissue .",
"For instance , bulge cells in the hair follicle can repair the surrounding epidermis ( Rompolas and Greco , 2014; Hsu et al . , 2014 ) , new intestinal epithelial cells arise from crypt cells ( Santos et al . , 2018; Yousefi et al . , 2017 ) , and horizontal and globose basal cells can regenerate cells in the olfactory epithelium ( Choi and Goldstein , 2018; Schwob et al . , 2017 ) .",
"Depletion of these progenitors can severely diminish the regenerative capacity of the tissue , and tissues that lack a progenitor pool altogether are unable to regenerate .",
"To gain further insight into how different tissues regenerate , a greater understanding of the mechanisms that define and regulate progenitor function are needed .",
"The zebrafish lateral line system has long been recognized as an excellent model for studying regeneration .",
"The sensory organ of the lateral line , the neuromast , is comprised of mechanosensory hair cells organized on the surface of the head and body ( Thomas et al . , 2015 ) .",
"Lateral line hair cells regenerate rapidly following damage , with the system returning to quiescence after regeneration is complete ( Harris et al . , 2003; Hernández et al . , 2007; Ma et al . , 2008 ) .",
"The surrounding nonsensory support cells serve as progenitors for new hair cells .",
"This replenishment is proliferation-dependent and occurs symmetrically , with each progenitor dividing to give rise to two daughter hair cells ( Wibowo et al . , 2011; Mackenzie and Raible , 2012; López-Schier and Hudspeth , 2006; Romero-Carvajal et al . , 2015 ) .",
"Three key observations of support cell behavior during regeneration suggest that different support cell populations may be differentially regulated in response to regeneration .",
"First , the support cell proliferation that follows hair cell death occurs mainly in the dorsal and ventral compartments of the neuromast ( Romero-Carvajal et al . , 2015 ) , indicating that progenitor identity is spatially regulated .",
"The most peripheral support cells , often called mantle cells , do not proliferate in response to hair cell damage ( Ma et al . , 2008; Romero-Carvajal et al . , 2015 ) .",
"Second , the regenerative capacity of the neuromast is not diminished over multiple regenerations ( Cruz et al . , 2015; Pinto-Teixeira et al . , 2015 ) , indicating that progenitor cells must also be replaced in addition to hair cells .",
"Finally , in addition to regeneration in response to acute damage , lateral line hair cells undergo turnover and replacement under homeostatic conditions ( Cruz et al . , 2015; Williams and Holder , 2000 ) .",
"However , it remains unknown whether there are distinct support cell populations within the neuromast ( e . g . hair cell progenitors and those that replenish progenitors ) , as well as how progenitor populations are regulated .",
"In this study , we have used CRISPR to generate novel transgenic lines in which distinct , spatially segregated populations of support cells are labeled in vivo .",
"Fate mapping studies using these lines show that these populations are functionally distinct with respect to their ability to contribute new hair cells during homeostasis and to generate hair cells after damage .",
"We also show that targeted ablation of one of these populations significantly reduces hair cell regeneration .",
"Other fate mapping studies show that these support cell populations can replenish each other in the absence of hair cell damage .",
"Finally , we show that Notch signaling differentially regulates these populations .",
"These results demonstrate that there are a number of distinct progenitor populations within lateral line neuromasts that are independently regulated , providing flexibility for hair cell replacement under a variety of circumstances ."
],
[
"Previous studies have shown that the majority of support cell proliferation occurs during the first twenty-four hours following hair cell death ( Ma et al . , 2008 ) .",
"We replicated this finding by administering a pulse of F-ara-EdU ( EdU ) , which has been shown to be far less toxic than BrdU ( Neef and Luedtke , 2011 ) .",
"The EdU pulse was administered for twenty-four hours following neomycin-induced hair cell ablation at 5 days post fertilization ( five dpf ) and neuromasts were imaged at seventy-two hours post treatment ( 72 hpt ) , the time at which regeneration is nearly complete ( Figure 1A ) .",
"In neomycin-treated larvae , roughly 78% of regenerated hair cells were EdU-positive , compared to 6% in mock-treated larvae ( Figure 1B–C; p<0 . 0001 ) .",
"We noticed that at the same timepoint that 28% of EdU-positive cells remained support cells ( Figure 1E and B arrowheads ) .",
"We hypothesized that these cells may represent hair cell progenitors that had been replaced via proliferation .",
"If so , then these EdU-positive cells should have the capacity to generate a new round of hair cells after subsequent damage .",
"In order to test this , we subjected larvae to two rounds of hair cell ablation and regeneration .",
"EdU was administered for 24 hr following the first ablation , and BrdU was administered for the same duration following the second ablation ( Figure 1F ) .",
"Of the 90 neuromasts analyzed , 14 retained EdU labeling .",
"Within these 14 neuromasts , we observed hair cells after the second regeneration that were both EdU- and BrdU-positive ( Figure 1G–J , arrowheads; 1 . 50 ± 1 . 45 hair cells per neuromast; mean ±SD ) , indicating that support cells that divide after the first ablation can in fact serve as hair cell progenitors after subsequent damage .",
"However , we also observed double-positive cells that remained support cells ( Figure 1K–N , arrowheads; 1 . 07 ± 0 . 73 support cells per neuromast; mean ±SD ) , as well as support cells that were only labeled by EdU ( Figure 1G–N , asterisks ) .",
"These observations indicate that support cells that divided after the first round of damage do not always serve as hair cell progenitors ( or even as progenitors at all ) .",
"Altogether , these data provide evidence of proliferation-mediated replenishment of hair cell progenitors by support cells in the neuromast , but also suggest that new hair cells arise from a pool of progenitors that are not strictly defined by their proliferation history .",
"We next sought to determine whether hair cell progenitors could be defined via gene expression .",
"As part of a broader screen to be described elsewhere , we employed CRISPR-mediated transgenesis ( Kimura et al . , 2014; Ota et al . , 2016 ) to target expression constructs to genes that had been shown to be differentially expressed in support cells ( Jiang et al . , 2014; Steiner et al . , 2014 ) .",
"This method inserts a construct with a minimal hsp70l1 promoter driving fluorescent protein expression to a targeted break , so that expression is regulated by adjacent genomic elements .",
"During this screen , we found that targeted insertion in three genes ( sfrp1a , tnfsf10l3 , and sost ) resulted in distinct spatial expression in primary neuromast support cells: a transgene inserted in the sfrp1a locus is restricted to the most peripheral support cells ( Peripheral cells; Figure 2A ) ; insertion in tnfsf10l3 is more broadly expressed throughout the periphery but is enriched in anteroposterior support cells ( AP cells; Figure 2C ) ; and insertion in sost is limited to the dorsal and ventral support cells ( DV cells; Figure 2E ) .",
"Secondary neuromasts are oriented orthogonally to primary neuromasts ( Lopez-Schier et a . , 2004 ) ; we found that the position of the distinct support cell populations are correspondingly rotated ( Figure 2—figure supplement 1 ) .",
"We also generated GFP lines for each insertion site .",
"We did not observe GFP labeling in hair cells in stable lines ( Figure 2—figure supplement 2 ) .",
"We used these distinct expression patterns to examine whether there were any functional differences between marked support cells with respect to their ability to serve as hair cell progenitors .",
"To this end , we generated stable transgenic lines for all three loci: Tg[sfrp1a:nlsEos]w217; Tg[tnfsf10l3:nlsEos]w218; and Tg[sost:nlsEos]w215 ( hereafter known as sfrp1a:nlsEos , tnfsf10l3:nlsEos , and sost:nlsEos , respectively ) .",
"Eos is a photoconvertible protein that switches from green to red fluorescence ( shown in magenta throughout this paper ) after exposure to UV light ( Wiedenmann et al . , 2004 ) .",
"The converted protein is stable for months .",
"Its nuclear localization presumably protects it from degradative elements in the cytoplasm , allowing for a more permanent label than a standard fluorescent reporter ( Cruz et al . , 2015; McMenamin et al . , 2014 ) .",
"We could thus chase this label from support cell to hair cell if these cells serve as hair cell progenitors , as hair cells that derived from these support cells would have converted Eos in their nuclei .",
"To ensure that expression of the nlsEos transgene didn’t disrupt hair cell development or regeneration , we used the vital dye FM 1-43FX to label hair cells in transgenic fish and their non-transgenic siblings .",
"In all three lines , we observed no significant difference between transgenic fish and their siblings ( Figure 2—figure supplement 3 ) .",
"Because the uptake of FM 1-43FX requires proper mechanotransduction and can thus be used as a proxy for proper hair cell function ( Seiler and Nicolson , 1999; Gale et al . , 2001; Meyers et al . , 2003 ) , we conclude that expression of these nlsEos transgenes does not impact hair cell number or function during development or regeneration .",
"We first examined how these different support cell populations contributed to hair cell development and turnover under homeostatic conditions .",
"All three nlsEos lines were crossed to a hair cell-specific transgenic line ( Tg[Brn3c:GAP43-GFP]s356t ( Xiao et al . , 2005 ) , hereafter known as brn3c:GFP ) in order to distinguish hair cell nuclei .",
"Eos in support cells was photoconverted at 5 dpf and larvae were fixed and immunostained for GFP either immediately or at 8 dpf .",
"At 5 dpf , 19% of hair cells were labeled with Eos expressed by the Peripheral cell transgene , and this number remained the same by 8 dpf ( Figure 2B; p=0 . 7047 ) .",
"Eos from the AP cell transgene labeled about 6% of hair cells at both 5 and 8 dpf ( Figure 2D; p=0 . 9668 ) .",
"Since there is no change over the three-day span , neither of these populations are responsible for generating new hair cells under homeostatic conditions .",
"In contrast , the amount of hair cells labeled with photoconverted Eos from the DV cell transgene increased from 39% to 56% over that three-day span ( Figure 2F; p<0 . 0001 ) .",
"Thus , the DV cell population seems to be predominantly involved in ongoing hair cell generation during homeostasis .",
"We next used these transgenic lines to determine whether there was any functional difference between these support cell subpopulations regarding their ability to serve as hair cell progenitors during regeneration .",
"Each of the nlsEos lines were once again crossed to brn3c:GFP fish in order to distinguish hair cell nuclei .",
"Eos in support cells was photoconverted at 5 dpf , and larvae were subjected to neomycin-induced hair cell ablation and then fixed and immunostained for GFP at 72 hpt ( Figure 3A ) .",
"Only 4% of regenerated hair cells were derived from the Peripheral cell population , whereas the AP cell and DV cell populations contributed significantly more , generating 20% and 61% of regenerated hair cells , respectively ( Figure 3B–D , arrowheads; Figure 3E; p=0 . 003 [Peripheral vs . AP] , p<0 . 0001 [Peripheral/AP vs . DV] ) .",
"In order to ensure that this difference in Eos incorporation was not simply due to relative proportion of available Eos-positive support cells , we counted the number of Eos-positive support cells in each transgenic line at 5 dpf , prior to hair cell ablation .",
"There were about half as many Peripheral cells relative to the other two populations , but no significant difference between the number of AP cells and the number of DV cells ( Figure 3F; Peripheral = 14 . 30 ± 4 . 17; AP = 22 . 8 ± 4 . 40; DV = 23 . 86 ± 4 . 45; p<0 . 0001 [Peripheral vs . AP/DV] , p>0 . 9999 [AP vs . DV] ) .",
"Thus , the difference in regenerative capacity between these populations is not simply a reflection of the number of available cells , but rather of differences in the progenitor identity of the populations .",
"Notch-mediated lateral inhibition plays a crucial role in ensuring the proper number of hair cells are regenerated , and inhibition of Notch signaling following hair cell damage dramatically increases the number of regenerated hair cells ( Ma et al . , 2008; Wibowo et al . , 2011; Romero-Carvajal et al . , 2015 ) .",
"Thus , we examined how Notch inhibition impacted the progenitor function of our three support cell populations .",
"We crossed all of our nlsEos lines to the brn3c:GFP line , and treated double-positive larvae with 50 μM LY411575 ( LY ) , a potent γ-secretase inhibitor ( Mizutari et al . , 2013; Romero-Carvajal et al . , 2015 ) , for 24 hr immediately following neomycin treatment .",
"Fish were fixed at 72 hpt and immunostained for GFP .",
"In all three lines , after Notch inhibition ( Neo/LY ) there were roughly twice as many hair cells as control fish ( Neo ) ( Figure 4A–B , E–F , I–J; p<0 . 0001 [all lines] ) , consistent with previous studies .",
"The small number of Peripheral cell-derived hair cells was no different between LY-treated fish and non-treated fish ( Figure 4C; 0 . 62 ± 1 . 28 [Neo] vs . 1 . 15 ± 2 . 16 [Neo/LY]; p=0 . 2481 ) .",
"By contrast , the number of nlsEos-positive hair cells from both AP and DV cells increased in fish treated with LY .",
"Moreover , while the number of nlsEos-positive hair cells derived from DV cells doubled in LY-treated fish ( Figure 4K; 7 . 40 ± 2 . 13 [Neo] vs . 15 . 25 ± 6 . 36 [Neo/LY]; p<0 . 0001 ) , those derived from AP cells increased roughly five-fold ( Figure 4G; 2 . 22 ± 1 . 94 [Neo] vs . 11 . 38 ± 4 . 23 [Neo/LY]; p<0 . 0001 ) .",
"As a consequence , the percentage of hair cells derived from DV cells decreased correspondingly ( Figure 4L; 67 . 86 ± 14 . 63 [Neo] vs . 54 . 69 ± 14 . 01 [Neo/LY]; p<0 . 0001 ) , whereas those derived from AP cells doubled ( Figure 4H; 25 . 19 ± 21 . 72 [Neo] vs . 50 . 68 ± 19 . 23 [Neo/LY]; p<0 . 0001 ) .",
"These data suggest that generation of hair cells from both the AP and DV populations is regulated by Notch signaling ( with the AP population being regulated to a greater extent ) , whereas Peripheral cells are not responsive to Notch signaling .",
"Since the DV cell population generates roughly 60% of regenerated hair cells , we sought to determine whether these cells were required for hair cell regeneration .",
"To this end , we generated a transgenic line in which an enhanced-potency nitroreductase ( epNTR; Tabor et al . , 2014 ) fused to GFP was introduced into the sost locus using CRISPR ( Tg[sost:epNTR-GFP]w216 , hereafter known as sost:NTR-GFP ) .",
"Nitroreductase is a bacterial enzyme that selectively binds its prodrug Metronidazole ( Mtz ) , converting Mtz into toxic metabolites that kill the cells expressing it ( Curado et al . , 2007 ) .",
"We then compared the extent of sost:NTR-GFP expression in DV cells , as defined by the sost:nlsEos transgene .",
"At three dpf , soon after the initiation of transgene expression , we see considerable overlap between NTR-GFP and nlsEos .",
"All NTR-GFP +cells were also positive for nlsEos , while an additional subset of cells expressed nlsEos alone .",
"When we compared expression at five dpf , the size of the double-positive ( NTR-GFP+; nlsEos+ ) population did not change , whereas the number of cells expressing nlsEos alone increased significantly , occupying a more central location ( Figure 5A–B , arrowheads; Figure 5C; NTR-GFP/nlsEos: 9 . 04 ± 2 . 39 [3 dpf] vs . 8 . 47 ± 2 . 27 [5 dpf]; nlsEos only: 6 . 10 ± 2 . 27 [3 dpf] vs . 10 . 86 ± 2 . 72 ( 5 dpf ) ; p>0 . 9999 [NTR-GFP/nlsEos] , p<0 . 0001 [nlsEos only] ) .",
"These observations are consistent with the idea that both transgenes initiate expression at the same time , but that nlsEos protein is retained longer than NTR-GFP protein as cells mature and as a result , NTR-GFP is expressed in a subset of DV cells .",
"We next tested to the efficacy of DV cell ablation at 3 and 5 dpf .",
"Treatment of these fish with 10 mM Mtz for 8 hr was sufficient to ablate the majority of NTR-GFP cells .",
"Treating fish with Mtz for 8 hr at five dpf ( Mtz5 ) slightly but significantly decreased the number of support cells solely expressing nlsEos by about 13% .",
"Treating fish with Mtz for 8 hr at three dpf , followed by a second 8 hr Mtz treatment at five dpf ( Mtz3/5 ) decreased the number of solely nlsEos-positive cells even further , by about 40% ( Figure 5D–G; Mock: 11 . 18 ± 2 . 04; Mtz5: 9 . 72 ± 2 . 03; Mtz3/5: 6 . 76 ± 2 . 12; p=0 . 0288 [Mock vs . Mtz5] , p<0 . 0001 [Mock vs . Mtz3/5 , Mtz5 vs . Mtz3/5] ) .",
"We next tested the impact of DV cell ablation on hair cell regeneration .",
"We compared two groups: neomycin exposure followed by Mtz treatment at 5 dpf ( Neo/Mtz5 ) , compared to Mtz treatment at 3 dpf , then neomycin treatment at 5 dpf followed by a second Mtz treatment ( Mtz3/Neo/Mtz5; Figure 6A ) .",
"For both groups , nlsEos was photoconverted at 5 dpf , just prior to neomycin treatment , and larvae were fixed at 72 hpt and immunostained for GFP and Parvalbumin ( to label NTR-GFP+ cells and hair cells , respectively ) .",
"The Neo/Mtz5 treatment resulted in a small but significant reduction in both hair cells and nlsEos-positive hair cells per neuromast relative to normal regeneration ( Figure 6B–C , E–F; Total hair cells: 11 . 73 ± 2 . 10 [Neo] vs . 9 . 33 ± 1 . 88 [Neo/Mtz5]; p=0 . 0001; nlsEos+ hair cells: 7 . 78 ± 2 . 36 [Neo] vs . 4 . 90 ± 2 . 02 [Neo/Mtz5]; p=0 . 0003 ) .",
"The Mtz3/Neo/Mtz5-treated larvae exhibited even fewer hair cells per neuromast ( Figure 6D , E; 11 . 73 ± 2 . 10 [Neo] vs . 7 . 52 ± 1 . 74 [Mtz3/Neo/Mtz5]; p<0 . 0001 ) , with nlsEos-labeled hair cells decreased to a mere 14% of total regenerated hair cells ( Figure 6G; 65 . 81 ± 14 . 89 [Neo] vs . 14 . 29 ± 18 . 10 [Mtz3/Neo/Mtz5]; p<0 . 0001 ) .",
"Importantly , Mtz treatment of siblings without the sost:NTR-GFP transgene had no impact on hair cell regeneration ( Figure 6—figure supplement 1; Neo: 9 . 5 ± 1 . 50; Mtz3/Neo/Mtz5: 9 . 98 ± 1 . 51; p=0 . 2317 ) .",
"Transgenes also had no obvious effect on hair cell development or regeneration in the absence of Mtz either alone or in combination ( Figure 6—figure supplement 2 ) .",
"Thus , ablation of DV cells reduces the number of hair cells regenerated .",
"We then examined how Notch signaling impacted hair cell regeneration in the context of DV cell ablation .",
"We treated sost:NTR-GFP larvae with 50 μM LY for 24 hr following ablation ( Mtz3/Neo/Mtz5/LY ) and assayed hair cell number at 72 hpt ( Figure 7A ) .",
"As expected , the number of regenerated hair cells increased significantly after LY treatment in all groups ( Figure 7B–F; p<0 . 0001 ) , and DV cell ablation significantly decreased hair cell regeneration ( Figure 7B , D , F; 9 . 42 ± 1 . 85 [Neo] vs . 6 . 86 ± 1 . 76 [Mtz3/Neo/Mtz5]; p=0 . 0058 ) .",
"However , LY treatment following Mtz ablation resulted in significantly fewer regenerated hair cells than LY alone ( Figure 7C , E , F; 21 . 08 ± 4 . 42 [Neo/LY] vs . 15 . 06 ± 3 . 51 [Mtz3/Neo/Mtz5/LY]; p=0 . 0029 ) , indicating that inhibiting Notch signaling cannot fully compensate for the loss of the DV population .",
"While the DV population generates roughly 60% of hair cells after damage , the other 40% must derive from a different population .",
"Consistent with this observation , reduction of DV cells by Mtz treatment only partially blocks new hair cell formation , indicating that there must be additional progenitor populations .",
"We believed that AP cells could define this additional population , since they were capable of generating roughly 20% of regenerated hair cells ( Figure 3E ) .",
"However , there may be some overlap between the expression of tnfsf10l3:nlsEos defining the AP domain and sost:nlsEos defining the DV domain .",
"When we crossed the tnfsf10l3:nlsEos and sost:nlsEos lines together , we found that roughly 88% of regenerated hair cells were nlsEos positive when larvae expressed both transgenes , compared to 65% from sost:nlsEos alone and 28% from tnfsf10l3:nlsEos alone ( Figure 8A–E; p<0 . 0001 ) .",
"Thus , while not completely additive , these data suggest that the AP population is distinct from the DV population in terms of its progenitor function .",
"We next examined how the AP population would respond to the ablation of the DV population .",
"We crossed the tnfsf10l3:nlsEos line to the sost:NTR-GFP line , sorted out double-positive larvae , and compared normal regeneration to that after Mtz treatment ( Mtz3/Neo/Mtz5 , since this had served to be the best treatment paradigm ) .",
"As above , nlsEos was photoconverted at 5 dpf , immediately prior to neomycin treatment , and larvae were fixed at 72 hpt and immunostained for GFP and Parvalbumin .",
"Mtz-ablated larvae had significantly fewer hair cells than non-ablated larvae , as in previous experiments ( Figure 9C; 10 . 36 ± 1 . 60 [Neo] vs . 7 . 98 ± 1 . 74 [Mtz3/Neo/Mtz5]; p<0 . 0001 ) , but the number of nlsEos-positive hair cells was no different between the two groups ( Figure 9A–B , arrowheads; Figure 9D; 2 . 88 ± 1 . 83 [Neo] vs . 3 . 14 ± 1 . 43 [Mtz3/Neo/Mtz5]; p=0 . 3855 ) .",
"Thus , the AP population’s progenitor function remains unchanged following DV ablation , providing further support that it is a separate progenitor population from the DV population .",
"When examining hair cell regeneration following DV cell ablation , we consistently noticed that there was an increase in NTR-GFP +cells at 72 hpt .",
"This led us to hypothesize that DV cells were capable of regeneration even in the absence of hair cell damage .",
"To test this , we first administered a 48 hr pulse of EdU ( changing into fresh EdU solution after the first 24 hr ) immediately following Mtz ablation at 5 dpf and fixed immediately after EdU washout .",
"At 48 hr post ablation , we observed slightly more than half the number of the NTR-GFP +cells relative to unablated larvae ( Figure 10C; 8 . 94 ± 1 . 62 [Mock] vs . 5 . 34 ± 2 . 14 [Mtz]; p<0 . 0001 ) .",
"However , 58% of NTR-GFP +cells were EdU-positive in fish treated with Mtz , compared to just 15% in unablated larvae ( Figure 10A–B , arrowheads; Figure 10D; p<0 . 0001 ) .",
"These results indicate that new DV cells arise from proliferation .",
"To determine the source of new DV cells , we crossed sost:NTR-GFP fish to our three different nlsEos lines .",
"Double-transgenic fish were photoconverted at 5 dpf , Mtz-ablated , and then fixed at 72 hpt and immunostained for GFP .",
"Following ablation , 56% of NTR-GFP+ cells expressed photoconverted nlsEos when DV cells were labeled , compared to 97% in unablated controls ( Figure 11A–B , arrowheads; Figure 11C; p<0 . 0001 ) .",
"31% of NTR-GFP+ cells expressed photoconverted nlsEos when Peripheral cells were labeled , compared to 6% in controls ( Figure 11D–E , arrowheads; Figure 11F; p<0 . 0001 ) and 21% of NTR-GFP+ cells expressed photoconverted nlsEos when AP cells were labeled , compared to 7% in controls ( Figure 11G–H , arrowheads; Figure 11I; p=0 . 0004 ) .",
"Thus , DV cells are capable of being replenished after Mtz ablation by other DV cells as well as by both AP and Peripheral cells ."
],
[
"The data shown above indicate that there are at least three functionally distinct progenitor populations within the neuromast: ( 1 ) a highly regenerative , dorsoventral ( DV ) population , marked by sost:nlsEos , which generates the majority of regenerated hair cells; ( 2 ) an anteroposterior ( AP ) population , marked by tnfsf10l3:nlsEos , which also contributes to hair cell regeneration albeit to a far lesser extent than sost; and ( 3 ) a peripheral population ( Peripheral ) , marked by sfrp1a:nlsEos , that does not contain hair cell progenitors ( Figure 12 ) .",
"This model of high regenerative capacity in the dorsoventral region and low regenerative capacity in the anteroposterior region is consistent with the label-retaining studies performed by Cruz et al . ( 2015 ) as well as the BrdU-localization studies of Romero-Carvajal et al . ( 2015 ) .",
"However , an examination of the overlap in expression between sost:nlsEos and sost:NTR-GFP reveals distinctions even amongst this DV progenitor population .",
"We hypothesize that cells that express only nlsEos have matured from those that express both NTR-GFP and nlsEos .",
"We posit that these more mature nlsEos cells serve as hair cell progenitors .",
"Consistent with this idea , Mtz treatment at five dpf that spares nlsEos cells not expressing NTR-GFP has only a small effect on hair cell regeneration while Mtz treatment at both 3 and 5 dpf results in substantial reduction in hair cell regeneration .",
"While we have identified distinct hair cell progenitor populations ( DV and AP ) , these populations do not account for all of the hair cell progenitors in the neuromast .",
"The combination of these cells generated 88% of new hair cells , meaning that the remaining 12% were derived from other sources .",
"Furthermore , the AP population only accounted for 40% of new hair cells generated after neomycin treatment following DV cell ablation .",
"Thus , there must be some other population ( or populations ) of support cells that are serving as hair cell progenitors that we have not labeled with our transgenic techniques .",
"The best candidates for this role are centrally located support cells found ventral to hair cells , although the identity of these cells remains to be determined .",
"It is worth noting that Peripheral and DV lines ( both nlsEos and NTR-GFP ) were generated by targeted integration of the transgene into exons ( see Materials and methods for details ) .",
"Although CRISPR-mediated exonic integration has been shown to generate loss-of-function mutations ( Ota et al . , 2016 ) , proper hair cell development and regeneration were unaffected in lines heterozygous for transgene insertion ( Figure 2—figure supplement 3 ) .",
"Zebrafish expressing both nlsEos and NTR-GFP in DV cells ( Figures 5 , 6 and 11A–C ) would theoretically be null for sost function , yet these double positive larvae have the same number of hair cells during development ( five dpf ) and after hair cell regeneration as their non-transgenic and heterozygotic siblings ( Figure 6—figure supplement 2 ) .",
"This would suggest that sost function is dispensable for hair cell development and regeneration , in spite of the contribution DV cells make to both processes .",
"However , we did not formally test whether sost function was actually disrupted by transgene insertion , so it is possible that these double-positive larvae are not indicative of true sost loss-of-function or that there are mechanisms to compensate for the loss of sost .",
"It is possible that our transgenic lines do not reflect the corresponding gene expression of the locus of insertion , although this CRISPR-mediated transgenesis method has been shown to do so for other genes ( Kimura et al . , 2014; Ota et al . , 2016 ) .",
"While we have not verified that the expression patterns of our transgenes correspond to those of the endogenous loci by in situ hybridization , a recent comprehensive analysis of gene expression in neuromast support cells ( Lush et al . , 2019 ) confirms that the endogenous transcripts of sfrp1a , sost , and tnfsf10l3 have similar patterns to those of the transgenic insertions reported here .",
"We stress that the purpose of this study is not to correlate progenitor function to specific gene function , but to examine the functional differences between populations of support cells marked by transgene insertion .",
"While our study may not definitively link the action of underlying loci with progenitor identity , our experiments demonstrate that these genetically labeled support cells have distinct progenitor functions , and can serve as important tools in future studies determining the precise mechanisms underlying regeneration in the lateral line .",
"Neuromasts located on the trunk develop at different times from different migrating primordia .",
"Within a given neuromast , hair cells are arranged such that their apical stereocilia respond to directional deflection in one of two directions along the body axis .",
"Hair cells derived from the first primordium ( primI ) respond along the anteroposterior axis , and hair cells derived from the second primordium ( primII ) respond along the dorsoventral axis ( López-Schier et al . , 2004; López-Schier and Hudspeth , 2006 ) .",
"Spatial restriction of support cell proliferation is orthogonal to hair cell planar polarity , with proliferation occurring dorsoventrally in primI-derived neuromasts and anteroposteriorly in primII-derived neuromasts ( Romero-Carvajal et al . , 2015 ) .",
"This 90-degree switch between prim1- and primII-derived neuromasts is reflected in the distribution of labeled cell populations as well: tnfsf10l3:nlsEos and sost:nlsEos retain their asymmetric localization in primII-derived neuromasts , but tnfsf10l3:nlsEos is predominantly expressed in the dorsoventral region and sost:nlsEos is restricted to the anteroposterior region ( Figure 2—figure supplement 1 ) .",
"Thus , within a given neuromast along the trunk , expression of sost:nlsEos is orthogonal to hair cell planar polarity , and that of tnfsf10l3:nlsEos is parallel to hair cell planar polarity .",
"The relationship between asymmetric progenitor localization and hair cell planar polarity remains unknown .",
"Planar cell polarity ( PCP ) signaling often drives asymmetry in other tissues and has been implicated in the planar polarity of lateral line hair cells .",
"Zebrafish deficient in Vangl2 , a critical component of the PCP pathway , still develop neuromasts , but their hair cells are oriented randomly toward one another and do not respond along a single axis ( López-Schier and Hudspeth , 2006 ) .",
"Furthermore , this random orientation stems from misaligned divisions of hair cell progenitors ( Mirkovic et al . , 2012 ) .",
"The transcription factor Emx2 has also been recently implicated in determining hair cell planar polarity ( Jiang et al . , 2017 ) .",
"Whether these genes or other components of PCP signaling mediate the asymmetric expression of sost:nlsEos and tnfsf10l3:nlsEos remains to be determined .",
"It would also be interesting to examine whether hair cell planar polarity is influenced by the asymmetric localization of these progenitor populations , or vice versa .",
"Since zebrafish are able to properly regenerate their hair cells after multiple successive insults ( Cruz et al . , 2015; Pinto-Teixeira et al . , 2015 ) , and both daughters of progenitors give rise to hair cells , there must be a means of replenishing hair cell progenitors .",
"Our EdU/BrdU double labeling experiment qualitatively demonstrated that hair cell progenitors could be replenished via proliferation of other support cells .",
"It was thus unsurprising that DV cells could themselves regenerate .",
"It is notable , however , that DV cells could be replenished even in the absence of hair cell damage , which means that hair cell death is not the sole signal that triggers support cell proliferation .",
"Support cell regeneration in the absence of hair cell death has been observed in mammals , as certain types of cochlear support cells ( inner border cells and inner phalangeal cells ) are capable of regeneration following selective ablation , a process that occurs via transdifferentiation ( Mellado Lagarde et al . , 2014 ) .",
"In contrast , zebrafish DV cell regeneration primarily occurs via proliferation , as a majority of new DV cells were EdU-positive following Mtz-induced ablation .",
"DV cells that were not EdU-positive could have arisen after the EdU pulse , been retained due to incomplete ablation , or potentially resulted from transdifferentiation from another source .",
"All three labeled support cell populations were able to replenish DV cells following Mtz-induced ablation .",
"DV cells themselves contributed nearly 60% of new DV cells , although we cannot rule out that this number is inflated due to incomplete ablation .",
"This result suggests that DV cells choose to either generate new hair cells or replenish lost DV cells , undergoing a form of self-renewal that does not require asymmetric division .",
"The AP population is also capable of replenishing DV cells .",
"That both of these populations can generate new DV cells is consistent with recent findings from Viader-Llargués et al . ( 2018 ) .",
"This study defined support cells as a peripheral mantle population and a central sustentacular population .",
"Following laser ablation of large portions of the neuromast , they found that sustentacular cells were able to regenerate mantle cells and other sustentacular cells , as well as hair cells , and could thus be considered tripotent progenitors .",
"The transgenic lines they used to label sustentacular cells were broadly expressed , and thus should encompass both AP and DV populations .",
"We were unfortunately unable to drive functional expression of NTR in Peripheral or AP cells , and were thus unable to test how ablation of these support cell populations would affect regeneration .",
"However , we can conclude that the DV and AP populations are at least bipotent , since they can both generate new hair cells and new DV cells .",
"We found that the Peripheral population could also generate new DV cells following Mtz-induced ablation .",
"Furthermore , it contributes more to DV regeneration than does the AP population .",
"This was especially surprising since proliferation has rarely been observed in peripheral cells , at least during normal hair cell regeneration ( Ma et al . , 2008; Romero-Carvajal et al . , 2015 ) .",
"However , the loss of a progenitor population could be considered to be a case of extreme damage to the neuromast , thus prompting the Peripheral cell population to respond .",
"That Peripheral cells can serve as progenitors only in extreme circumstances is consistent with the findings of Romero-Carvajal et al . ( 2015 ) and Viader-Llargués et al . ( 2018 ) .",
"Both studies suggested that mantle cells are capable of regenerating other cell types in the neuromast following extreme damage .",
"However , the latter study found that mantle cells could only generate other mantle cells .",
"Whether the Peripheral cell population in particular is capable of doing the same remains to be tested .",
"Mantle cells have also been shown to proliferate following tail amputation , forming a migratory placode that forms new neuromasts along the regenerated tail ( Jones and Corwin , 1993; Dufourcq et al . , 2006 ) .",
"Given the differences across studies , we have hesitated to designate the sfrp1a:nlsEos labeled cells as mantle cells and have instead adopted the ‘Peripheral’ label .",
"Since Peripheral cells can generate hair cell progenitors , we have characterized them as ‘upstream progenitors’ ( Figure 12 ) .",
"The zebrafish lateral line system continues to grow through larval and adult stages ( Nuñez et al . , 2009; Ledent , 2002; Sapède et al . , 2002 ) , with new neuromasts formed from budding from extant neuromasts ( Nuñez et al . , 2009; Wada et al . , 2013a; Wada et al . , 2013b ) and generated anew from interneuromast cells , latent precursors deposited between neuromasts by the migrating primordium ( Nuñez et al . , 2009; Grant et al . , 2005 ) .",
"These interneuromast cells can also serve as progenitors in the event of whole neuromast ablation ( Sánchez et al . , 2016 ) .",
"We note that the sfrp1a:nlsEos transgene is expressed in interneuromast cells as well as Peripheral neuromast cells .",
"Whether these cells share similar properties to generate new neuromasts remains to be tested .",
"Our model of neuromast progenitor identity does bear some similarities with other regenerative tissues .",
"Both the hair follicle and intestinal epithelium contain a niche of stem cells ( bulge cells and crypt cells , respectively ) which generate transit-amplifying cells that are able to generate other cell types ( Ito et al . , 2005; Taylor et al . , 2000; Barker et al . , 2007 ) .",
"Due to their high rate of proliferation and multipotency , the DV cells in the neuromast could be likened to these transit-amplifying cells .",
"However , progenitors in the neuromast may bear the most similarity to those of the olfactory epithelium , which contains two distinct progenitor populations: globose basal cells ( GBCs ) , which are transit-amplifying cells that can restore lost olfactory neurons; and horizontal basal cells ( HBCs ) , a quiescent population that can generate multiple cell types , including GBCs , in instances of extreme damage ( Iwai et al . , 2008; Leung et al . , 2007 ) .",
"In our model , the DV and Peripheral cells are comparable to the GBCs and HBCs , respectively .",
"However , we cannot make the claim that the Peripheral population is a resident stem cell population ( like bulge cells , crypt cells , and HBCs ) , as we do not yet know if it is capable of self-renewal or of generating every cell type within the neuromast .",
"Inhibition of Notch signaling during hair cell regeneration significantly increased the number of hair cells derived from both DV and AP cells , which was not unexpected given that both are hair cell progenitor populations .",
"However , Notch inhibition had a greater impact on the AP population than on the DV population , suggesting that it may be more strongly regulated by Notch signaling .",
"The receptor notch3 , in particular , is most strongly expressed in the anteroposterior portions of the neuromast ( Wibowo et al . , 2011; Romero-Carvajal et al . , 2015 ) .",
"Furthermore , a transgenic reporter of Notch activity is also expressed in the anteroposterior region ( Romero-Carvajal et al . , 2015; Wibowo et al . , 2011 ) .",
"It is thus likely that asymmetrically-localized Notch signaling maintains quiescence among AP cells during homeostasis and is responsible for suppressing the contribution of the AP population to hair cell regeneration ( compared to DV contribution ) .",
"Since Notch signaling is not as strong in the dorsoventral regions of the neuromast , the DV population is less affected and could already be more ‘primed’ to serve as hair cell progenitors than the AP population .",
"In addition , Notch signaling regulates Wnt signaling in the neuromast , particularly through activation of the Wnt inhibitor dkk2 ( Romero-Carvajal et al . , 2015 ) .",
"It is possible that Notch signaling may repress Wnt signaling in AP cells , further contributing to their low regenerative capacity .",
"More work needs to be done regarding the role of Wnt signaling in these support cell populations .",
"Notch inhibition did not have any impact on the Peripheral population’s contribution to hair cell regeneration , indicating that Notch signaling does not suppress hair cell production by Peripheral cells .",
"While it is difficult to tell from in situ expression , it seems that the Notch reporter is not active in the peripheral mantle cells ( Wibowo et al . , 2011; Romero-Carvajal et al . , 2015 ) .",
"Thus , there must be some other mechanism , either intrinsic or extrinsic , that maintains relative quiescence among the Peripheral population .",
"It is not clear why these distinct populations of progenitors exist , as there is no clear difference in the types of hair cells they produce .",
"Hair cells polarized in opposing direction are daughters of the final division of the hair cell progenitor ( López-Schier and Hudspeth , 2006 ) .",
"Heterogeneity has also been recently described in the synaptic responses of lateral line hair cells ( Zhang et al . , 2018 ) , but these differences appear lineage-independent .",
"Instead , the allocation of distinct progenitors may serve an advantage with respect to their differential regulation .",
"For example , our data suggest that DV cells contribute more to homeostatic addition of new hair cells to the neuromast in the absence of damage , while both AP and DV cells are engaged after hair cell damage .",
"AP cells were unable to overcome the loss of the DV population , suggesting that feedback mechanisms regulating both hair cell production and progenitor replacement operate independently .",
"While both AP and DV populations are regulated by Notch signaling , this regulation appears to operate independently as Notch inhibition does not compensate for DV ablation .",
"Independent progenitors may offer the flexibility to add new hair cells under a variety of conditions for both ongoing hair cell turnover and in the face of catastrophic hair cell loss ."
],
[
"Experiments were conducted on 5–8 dpf larval zebrafish ( except for the double hair cell ablation experiment , which was conducted on 15–18 dpf fish ) .",
"Larvae were raised in E3 embryo medium ( 14 . 97 mM NaCl , 500 μM KCL , 42 μM Na2HPO4 , 150 μM KH2PO4 , 1 mM CaCl2 dihydrate , 1 mM MgSO4 , 0 . 714 mM NaHCO3 , pH 7 . 2 ) at 28 . 5°C .",
"All wildtype animals were of the AB strain .",
"Zebrafish experiments and husbandry followed standard protocols in accordance with University of Washington Institutional Animal Care and Use Committee guidelines .",
"The myo6b:mKate2 construct was generated via the Gateway Tol2 system ( Invitrogen ) .",
"A pME-mKate2 ( the mKate2 sequence being cloned from pMTB-Multibow-mfR , Addgene #60991 ) construct was generated via BP Recombination , and then a pDEST-myo6b:mKate2 construct was generated via LR recombination of p5E-myo6b , pME-mKate2 , p3E-pA , and pDEST-iTol2-pA2 vectors .",
"The mbait-GFP construct was a gift from Shin-Ichi Higashijima’s lab .",
"The mbait-nlsEos construct was also generated via Gateway LR recombination of p5E-mbait/HSP70l , pME-nlsEos , p3E-pA , and pDEST-iTol2-pA2 vectors .",
"The mbait-epNTR-GFP construct was generated via Gibson assembly ( Gibson et al . , 2009 ) , inserting the coding sequence of epNTR ( cloned from pCS2-epNTR obtained from Harold Burgess’ lab ) plus a small linker sequence in front of the GFP in the original pBSK mbait-GFP vector .",
"All plasmids were maxi prepped ( Qiagen ) prior to injection .",
"Gene-specific guide RNA ( gRNA ) sequences were as follows: All gRNAs were synthesized according to the protocol outlined in Shah et al . ( 2015 ) , but were purified using a Zymo RNA Clean and Concentrator kit .",
"Upon purification , gRNAs were diluted to 1 μg/μL , aliquoted into 4 μL aliquots , and stored at −80°C .",
"Sfrp1a and tnfsf10l3 gRNA sequences were designed via http://crispr . mit . edu , and the sost gRNA sequences were designed via http://crisprscan . org .",
"The tnfsf10l3 gRNA was targeted 388 base pairs upstream of the gene’s start ATG codon , whereas the sfrp1a and sost guides were targeted to exons ( the sfrp1a guide was targeted to exon 1 , and the sost guides were targeted to exon2 and exon 1 , respectively , in the order listed above ) .",
"The Tg[myosin6b:mKate2]w232 ( hereafter called myo6:mKate2 ) line was generated via Tol2 transgenesis .",
"1–2 nL of a 5 μL injection mix consisting of 20 ng/μL myo6b:mKate2 plasmid , 40 ng/μL transposase mRNA , and 0 . 2% phenol red were injected into single cell wildtype embryos .",
"Larvae were screened for expression at three dpf and transgenic F0 larvae were grown to adulthood .",
"F0 adults were outcrossed to wildtype fish , transgenic offspring were once again grown to adulthood , and the resulting adults were used to maintain a stable line .",
"All support cell transgenic lines were generated via CRISPR-mediated transgenesis , first described by Kimura et al . ( 2014 ) .",
"This technique utilizes the targeted double strand breaks of the CRISPR/Cas9 system to insert a reporter construct into a gene of interest .",
"In addition to a gene-specific gRNA and Cas9 protein , single cell-embryos are injected with a plasmid that contains a reporter ( such as GFP or nlsEos ) downstream of a minimal promoter ( in this case a heat-shock promoter ) and a ‘bait’ sequence that is not present in the zebrafish genome ( we used the mbait sequence ) in addition to another gRNA that targets the bait sequence .",
"Following injection , Cas9 will recognize both the gene-specific gRNA as well as the bait gRNA , cleaving both the genomic DNA and linearizing the plasmid .",
"This reporter plasmid can then be integrated into the genome via non-homologous end joining .",
"These reporters can be targeted just upstream of the start codon of a gene of interest ( about 200–600 base pairs ) in order to co-opt the gene’s cis-regulatory elements ( the strategy we used for tnfsf10l3 insertions ) or can also be targeted directly to exons ( as we did for sfrp1a and for sost insertions ) .",
"Exonic insertions can thus be used to visualize potential loss of function mutations ( Ota et al . , 2016 ) .",
"For most injections , a 5 μL injection mix was made consisting of 200 ng/μL gene-specific gRNA , 200 ng/μL mbait gRNA , 800 ng/μL Cas9 protein ( PNA Bio #CP02 ) , 20 ng/μL mbait-reporter plasmid , and 0 . 24% phenol red .",
"The gRNAs and Cas9 protein were mixed together first , then heated at 37°C for 10 min , after which the other components were added .",
"In the case of sost , in which two gRNAs were co-injected , each gRNA was added to the mix at a final concentration of 100 ng/μL ( so 200 ng/μL of total guide-specific gRNA ) .",
"When reconstituting the Cas9 protein , DTT was added to a final concentration of 1 mM DTT ( per manufacturer’s instructions ) .",
"This is highly recommended to reduce needle clogging during the injection process .",
"1–2 nL of these injection mixes were injected into single cell wildtype embryos .",
"Larvae were screened for expression at three dpf and transgenic F0 larvae were grown to adulthood .",
"F0 adults were outcrossed to wildtype fish , transgenic offspring were once again grown to adulthood , and the resulting adults were used to maintain a stable line .",
"In order to photoconvert multiple nlsEos fish at once , larvae were transferred to a 60 × 15 mm petri dish and placed in a freezer box lined with aluminum foil .",
"Then , an iLumen 8 UV flashlight ( procured from Amazon ) was placed over the dish and turned on for 15 min .",
"Following the UV pulse , larvae were returned to standard petri dishes to await experimentation .",
"For all drug treatments , zebrafish larvae were placed in baskets in six well plates to facilitate transfer of larvae between media .",
"Larvae were treated at five dpf unless otherwise noted .",
"All wells contained 10 mL of drug , E3 embryo medium with the same effective % DMSO as the drug ( for mock treatments ) , or plain E3 embryo medium for washout .",
"Following treatment , the fish were washed twice into fresh E3 embryo medium by moving the baskets into adjacent wells in the row , then washed a third time by transferring them into a 100 mm petri dish with fresh E3 medium .",
"All drugs were diluted in E3 embryo medium .",
"The drug treatment paradigms were as follows: for hair cell ablation , 400 μM neomycin ( Sigma ) for 30 min; for sost:NTR ablation , 10 mM metronidazole ( Mtz; Sigma ) with 1% DMSO; for Notch inhibition , 50 μM LY411575 ( LY; Sigma; effective DMSO concentration of 0 . 5% ) for 24 hr; for the sost ablation/Notch inhibition experiment ( Figure 7 ) : 10 mM Mtz/50 μM LY for 8 hr , then 50 μM LY for 16 hr .",
"For double hair cell ablation studies , larvae that were treated with neomycin were raised on a nursery in the UW fish facility beginning at seven dpf and then treated with 400 μM neomycin again at 15 dpf in standard petri dishes ( 10 days following the first neomycin treatment ) .",
"These juvenile fish were washed into fresh system water multiple times before being returned to the nursery and were then fixed three days later ( 18 dpf ) .",
"Following hair cell ablation with neomycin , larvae were incubated in 500 μM F-ara-EdU ( Sigma #T511293 ) for 24 hr .",
"Following sost:NTR ablation with Mtz , larvae were incubated in the same concentration of EdU for 48 hr .",
"Larvae were placed into fresh EdU after the first 24 hr .",
"F-ara-EdU was originally reconstituted in 50% H2O and 50% DMSO to 50 mM , so the working concentration of DMSO of 500 μM was 0 . 5% .",
"In the case of the double ablation studies , juvenile fish were incubated in 10 mM BrdU ( Sigma ) with 1% DMSO in system water ( used in the UW fish facility ) following the second neomycin treatment for 24 hr .",
"Following treatment , larvae were washed in fresh system water several times .",
"Zebrafish larvae were fixed in 4% paraformaldehyde in PBS containing 4% sucrose for either 2 hr at room temperature or overnight at 4°C .",
"Larvae were then washed three times ( 20 min each ) in PBS containing 0 . 1% Tween20 ( PBT ) , incubated for 30 min in distilled water , then incubated in antibody block ( 5% heat-inactivated goat serum in PBS containing 0 . 2% Triton , 1% DMSO , 0 . 02% sodium azide , and 0 . 2% BSA ) for at least one hour at room temperature .",
"Larvae were then incubated in mouse anti-parvalbumin or rabbit anti-GFP ( or sometimes both simultaneously ) diluted 1:500 in antibody block overnight at 4°C .",
"The next day , larvae were once again washed three times ( 20 min each ) in PBT , then incubated in a fluorescently-conjugated secondary antibody ( Invitrogen , Alexa Fluor 488 , 568 , and/or 647 ) diluted 1:1000 in antibody block for 4–5 hr at room temperature .",
"From this point onward , larvae were protected from light .",
"Larvae were then rinsed three times ( 10 min each ) in PBT and then stored in antibody block at 4°C until imaging .",
"For BrdU immunohistochemistry , juvenile fish were rinsed once in 1N HCl , then incubated in 1N HCl following washout of Click-iT reaction mix ( see below ) .",
"IHC proceeded as above , except that the antibody block contained 10% goat serum and the fish were incubated in mouse anti-BrdU at a dilution of 1:100 .",
"All wash and incubation steps occurred with rocking .",
"Cells that had incorporated F-ara-EdU were visualized via a Click-iT reaction .",
"In the case of the double hair cell ablation experiment , Click-iT was performed before immunohistochemistry .",
"Following fixation , fish were washed three times ( 10 min each ) in PBT , then permeabilized in PBS containing 0 . 5% Triton-X for 30 min , then washed another three times ( 10 min each ) in PBS .",
"Next , fish were incubated for 1 hr at room temperature in a Click-iT reaction mix consisting of 2 mM CuSO4 , 10 μM Alexa Fluor 555 azide , and 20 mM sodium ascorbate in PBS ( made fresh ) .",
"Fish were protected from light from this point onwards .",
"Afterwards , the standard IHC protocol listed above was performed ( beginning with the 3 20 min washes in PBT ) .",
"For the sost:NTR regeneration experiment , the Click-iT reaction was performed after IHC .",
"Following incubation in secondary antibody , larvae were washed three times ( 10 min each ) in PBS , then incubated in 700 μL of the Click-iT reaction mix ( again , made immediately prior to incubation ) for 1 hr at room temperature .",
"Larvae were then washed six times ( 20 min each ) in PBT to ensure proper clearing of background labeling and stored in antibody block at 4°C until imaging .",
"With the exception of imaging requiring a far-red laser ( Figure 1F–N , Figure 3C–I , Figure 5 ) and Figure 2—figure supplement 2A and D , all imaging was performed using an inverted Marianas spinning disk system ( Intelligent Imaging Innovations , 3i ) with an Evolve 10 MHz EMCCD camera ( Photometrics ) and a Zeiss C-Apochromat 63x/1 . 2W numerical aperture water objective .",
"For Figure 2—figure supplement 2A and D , larvae were imaged with a Zeiss Plan-Apochromat 20x/0 . 8 objective .",
"All imaging experiments were conducted with fixed larvae ages 5–8 dpf .",
"Fish were placed in a chambered borosilicate coverglass ( Lab-Tek ) containing 2 . 5–2 . 5 mL E3 embryo medium and oriented on their sides with a slice anchor harp ( Harvard Instruments ) .",
"Imaging was performed at ambient temperature , generally 25°C .",
"Fish were positioned on their sides against the cover glass in order to image the first five primary neuromasts of the posterior lateral line ( P1-P5 ) .",
"All imaging was performed with camera intensification of 650 , gain of 3 .",
"Exposure time between 25–1500 ms was determined empirically to maintain imaging within the camera’s linear range; imaging parameters were held constant across all groups within an experiment .",
"Step size was 1 μm .",
"All 3i Slidebook images were exported as .",
"tif files to Fiji .",
"In cases when a far-red laser was required , imaging was performed on a Zeiss LSM 880 microscope with a Zeiss C-Apochromat 40x/1 . 2W numerical water objective .",
"Fish were immersed in a solution of 50%glycerol/50% PBS , and then mounted on a plain microscope slide ( Richard-Allen ) beneath a triple wholemount coverslip .",
"Imaging was performed at ambient temperature , generally 25°C .",
"Fish were positioned on their sides against the cover glass in order to image the first five primary neuromasts of the posterior lateral line ( P1-P5 ) .",
"For the double hair cell ablation experiment , following fixation the tails of fish were cut off and mounted underneath a single wholemount coverslip .",
"The 3 neuromasts of the terminal cluster were imaged per tail .",
"All imaging was performed at 4-5x digital zoom with master gain between 500–800 ( as set in controlling software ) for 488 , 561 , and 647 lasers , and a step size of 1 μm .",
"All images were captured through the Zen Black software and opened in Fiji as .",
"czi files .",
"All statistical analyses were done with GraphPad Prism 6 . 0 .",
"The Mann Whitney U test was used for comparisons between two groups , whereas the Kruskal-Wallis test , with a Dunn’s post-test , was used for comparisons between three or more groups .",
"Statistical significance was set at p=0 . 05 .",
"All data presented in this paper are from individual , non-pooled experiments .",
"However , the following data are representative of multiple experimental trials ( number of trials in parentheses ) : Figure 1B–D ( 3 ) ; Figure 2 ( 2 ) ; Figure 3A–E ( 3 ) ; Figure 6 ( 2 ) ; Figure 7 ( 2 ) ; Figure 9 ( 2 ) ; Figure 10 ( 2 ) ; Figure 11D–I ( 2 ) ."
]
] | [
"Mechanosensory hair cells of the zebrafish lateral line regenerate rapidly following damage .",
"These renewed hair cells arise from the proliferation of surrounding support cells , which undergo symmetric division to produce two hair cell daughters .",
"Given the continued regenerative capacity of the lateral line , support cells presumably have the ability to replenish themselves .",
"Utilizing novel transgenic lines , we identified support cell populations with distinct progenitor identities .",
"These populations show differences in their ability to generate new hair cells during homeostasis and regeneration .",
"Targeted ablation of support cells reduced the number of regenerated hair cells .",
"Furthermore , progenitors regenerated after targeted support cell ablation in the absence of hair cell damage .",
"We also determined that distinct support cell populations are independently regulated by Notch signaling .",
"The existence of independent progenitor populations could provide flexibility for the continued generation of new hair cells under a variety of conditions throughout the life of the animal ."
] | [
"Deep inside our ears , tiny specialized cells called hair cells constantly detect and relay sound and spatial information to our brain .",
"Without them , we lose our sense of hearing and balance .",
"Unfortunately , the number of hair cells drops with age or after exposure to loud noises , and there is no way for our body to replace them .",
"This can lead to permanent hearing and balance problems .",
"Zebrafish rely on similar hair cells to sense their environment .",
"In particular , clusters of hair cells make up the lateral line system , an organ that helps fish perceive vibration and pressure in the water .",
"However , unlike us , zebrafish can quickly and completely regenerate their hair cells .",
"When these get damaged , surrounding ‘support cells’ divide to form new hair cells , but the details of this process were still vague .",
"For example , it was unclear whether all support cells could create new hair cells , or only a certain population .",
"There was also little evidence to show that support cells could regenerate themselves .",
"To investigate , Thomas and Raible used a precise genetic tool called CRISPR to label subsets of support cells that differed in their gene expression .",
"Then , these cells were followed over time to see what they would become .",
"This highlighted three distinct populations that played separate roles when hair cells were regenerated .",
"The support cells located at the top and bottom of the lateral line organs ( dorsal and ventral cells ) made most of the new hair cells .",
"The cells located at the front and back ( anterior and posterior cells ) made a few; and the cells around the edges ( peripheral cells ) did not make any .",
"Further experiments then showed that all three types of support cell could transform to replenish the stock of dorsal and ventral support cells that make new hair cells .",
"By being able to label and track precise groups of support cells , researchers will be able to dissect exactly how hair cells are regenerated in fish .",
"Armed with this knowledge , it may become possible to explore ways to encourage human support cells to replace damaged hair cells in our ears ."
] | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"developmental biology"
] | The Brm-HDAC3-Erm repressor complex suppresses dedifferentiation in Drosophila type II neuroblast lineages | elife-01906-v1 | [
[
"The mechanism by which self-renewal and differentiation are balanced is a crucial issue in stem cell and cancer biology .",
"The neural stem cells , or neuroblasts , of the Drosophila larval brain have emerged as a new model for studying stem cell self-renewal and tumorigenesis .",
"In Drosophila larval central brains , there are at least two classes of neuroblast lineages ( Bello et al . , 2008; Boone and Doe , 2008; Bowman et al . , 2008 ) .",
"A type I neuroblast that expresses both Deadpan ( Dpn ) and Asense ( Ase ) divides asymmetrically to generate a self-renewing neuroblast and a ganglion mother cell ( GMC ) , which is committed to a differentiation pathway .",
"In contrast , a type II neuroblast that expresses Dpn , but not Ase , divides asymmetrically to generate a neuroblast and a transient amplifying cell known as an intermediate neural progenitor ( INP ) ( Bello et al . , 2008; Boone and Doe , 2008; Bowman et al . , 2008 ) .",
"Following maturation , the INP undergoes a limited number of asymmetric divisions to self-renew and to produce multiple GMCs ( Weng et al . , 2010 ) .",
"In both types of lineages , asymmetric division is dependent on apically localized proteins , including atypical protein kinase C ( aPKC ) ; basally localized proteins , such as Miranda and Numb; as well as several cell cycle regulators ( Chang et al . , 2012; Gonzalez , 2013 ) .",
"The failure of asymmetric division in either type of neuroblast can result in the hyperproliferation of these cells and the induction of brain tumors ( Caussinus and Gonzalez , 2005; Wang et al . , 2006 , 2007 , 2009 , 2011; Lee et al . , 2006a , 2006b; Cabernard and Doe , 2009; Chabu and Doe , 2009 , 2011; Chang et al . , 2010 ) .",
"The type II neuroblast lineage is highly analogous to the mammalian neural stem cell lineages , because both involve transient amplifying cells that are used to expand the progenitor cell population .",
"It is prone to impaired neuroblast homeostasis , if the limited self-renewing potential of INPs is unrestrained .",
"Brain tumor ( Brat ) and the Notch antagonist Numb function cooperatively to ensure that immature INPs undergo maturation and commit to the INP fate ( Boone and Doe , 2008; Bowman et al . , 2008 ) .",
"Notch signaling maintains neuroblast identity and its overactivation leads to dedifferentiation of INPs to ectopic neuroblasts ( Wang et al . , 2006; Bowman et al . , 2008; Weng et al . , 2010 ) .",
"A small number of transcription factors have been implicated in the control of INP identity and proliferative potential ( Carney et al . , 2012 ) .",
"Specifically expressed in INPs , a Zinc-finger transcription factor Earmuff ( Erm ) plays a critical role in maintaining the restricted developmental potential of the INPs ( Weng et al . , 2010 ) .",
"The Ets transcription factor Pointed ( PntP1 ) is specifically expressed in type II neuroblasts and INPs and is both necessary and sufficient for the suppression of Ase in type II neuroblasts and the generation of INPs ( Zhu et al . , 2011 ) .",
"Prospero that is basally localized in mitotic type I neuroblast , but absent from type II neuroblasts , triggers cell cycle exit and GMC differentiation ( Bello et al . , 2006; Betschinger et al . , 2006; Choksi et al . , 2006; Lee et al . , 2006c ) .",
"However , the underlying mechanism by which Erm prevents dedifferentiation is poorly understood .",
"ATP-dependent chromatin-remodeling factors are critical for the expression of the eukaryotic genome .",
"Four major classes of ATP-dependent chromatin remodeling complexes have been identified , including the extensively studied SWI/SNF complexes ( Narlikar et al . , 2002; Reisman et al . , 2009 ) .",
"The mammalian SWI/SNF complex termed the Brahma ( Brm or Brg1 ) complex regulates critical cellular processes such as differentiation and cell cycle arrest ( Klochendler-Yeivin et al . , 2002 ) .",
"Drosophila Brm complex acts similarly to control cell proliferation ( Brumby et al . , 2002 ) and differentiation ( Marenda et al . , 2003 ) .",
"A genome-wide RNAi study in Drosophila neuroblasts showed that the knockdown of genes encoding several core subunits of the SWI/SNF Brahma ( Brm ) remodeling complex may lead to neuroblast overproliferation ( Neumuller et al . , 2011 ) .",
"However , the precise role of the Brm remodeling complex during neuroblast self-renewal and the mechanism that underlying underlies this effect mechanism remain to be elucidated .",
"Besides ATP-dependent chromatin remodeling complexes , the other major class of chromatin remodelers is histone modifiers .",
"Histone deacetylases ( HDACs ) remove acetyl groups from the tails of core histones in the nucleosome and are often associated with transcriptional co-repressors ( Dokmanovic et al . , 2007 ) .",
"However , despite the critical role for histone modifiers in transcriptional regulation , it is unknown whether histone modifications play any role in Drosophila larval brain neuroblasts .",
"In this study , we report the critical role of a central chromatin remodeler , the Brm complex in preventing the formation of ectopic neuroblasts in type II lineages .",
"We show that another chromatin remodeling factor , HDAC3 functions cooperatively with the Brm complex to suppress the formation of ectopic type II neuroblasts .",
"Interestingly , multiple components of the Brm complex and HDAC3 physically associate with Erm .",
"brm and hdac3 interact genetically with erm to prevent type II neuroblast overgrowth .",
"Thus , the Brm-HDAC3-Erm complex is a novel repressor complex that suppresses dedifferentiation of INPs back into type II neuroblasts ."
],
[
"We independently identified brm from a RNA interference ( RNAi ) screen in which brm RNAi knockdown in larval brains resulted in an increase of Miranda-positive neuroblast-like cells in larval central brains ( Figure 1—figure supplement 1A ) , showing a phenotype similar to one previously reported ( Neumuller et al . , 2011 ) .",
"Brm is a DNA-dependent ATPase and a major component of a multi-protein SWI/SNF chromatin remodeling complex , which controls gene expression by altering chromatin structure ( Klochendler-Yeivin et al . , 2002 ) .",
"The number of cells expressing the proto-oncogene dMyc was significantly increased upon brm RNAi knockdown ( Figure 1—figure supplement 1B ) , consistent with the neuroblast overgrowth phenotype .",
"To determine the function of Brm in different neuroblast lineages , we generated MARCM clones in two brm loss-of-function alleles .",
"Type I wild-type ( wt ) clones always contained one neuroblast that is positive for both Dpn and Ase ( data not shown ) .",
"Similarly , only one neuroblast was present in both amorphic brm2 and hypomorphic brmT362 type I clones ( data not shown ) , indicating that Brm has no significant effect on type I neuroblast numbers .",
"Each wt type II MARCM clone also possessed only one neuroblast that was positive for Dpn , but negative for Ase ( Figure 1A; n = 25 ) .",
"Unlike the wt control , 6 . 4 ± 3 . 3 and 4 . 5 ± 2 . 6 ectopic neuroblasts were observed in brm2 ( Figure 1B , B′ , D; 88 . 6% , n = 34 ) brmT362 ( Figure 1C , C′ , D; 75 . 9% , n = 58 ) type II clones , respectively .",
"These phenotypes in brm alleles could be rescued by expressing a wild-type brm transgene ( Figure 1—figure supplement 1C ) .",
"Consistent with phenotypes in brm clones , knockdown of brm by RNAi using a type II neuroblast-specific neuroblast driver worniu ( wor ) -Gal4 , ase-Gal80 ( henceforth referred to as ‘type II driver’; ‘Materials and methods’ ) was sufficient to produce ectopic neuroblasts in 97 . 4% of type II neuroblast lineages ( Figure 1F , K; 7 ± 3 . 4 neuroblasts/lineage , n = 38 ) , while each control clone always has one neuroblast ( Figure 1E , K; n = 80 ) .",
"We therefore conclude that Brm suppresses the formation of ectopic neuroblasts in type II neuroblast lineages . 10 . 7554/eLife . 01906 . 003Figure 1 . The Brm complex suppresses the formation of ectopic type II neuroblasts .",
"( A–C )",
"Type II MARCM clones of control ( the MARCM driver; D ) , brm2 ( B , B′ ) and brmT362 ( C , C′ ) were labeled with Dpn ( blue ) , Ase ( red ) and CD8::GFP ( green ) .",
"( D ) Quantification of neuroblast number per type II MARCM clone for A–C .",
"( E–H )",
"Type II neuroblast lineage from control ( ‘the type II driver’: wor-Gal4 ase-Gal80; E ) , brm knockdown ( F ) , snr1 knockdown ( 108599 KK; G ) , and bap60 knockdown ( H ) were labeled with Dpn ( blue ) , Ase ( red ) and CD8 ( green ) .",
"( I–J′ ) type II MARCM clone of control ( I ) and bap55LL05955 ( J , J′ ) were labeled with Dpn ( blue ) , Ase ( red ) and CD8 ( green ) .",
"( K ) Quantification of neuroblast number per type II lineage for E–I .",
"Arrows indicate neuroblasts .",
"Clones are marked by CD8::GFP and indicated by white dotted line .",
"Scale bars , 10 µm .",
"*** indicates p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 01906 . 00310 . 7554/eLife . 01906 . 004Figure 1—figure supplement 1 . Analysis of chromatin remodelers in larval brains .",
"( A and B )",
"Larval brains of control ( elav-Gal4 driver ) and brm knockdown under the control of elav-Gal4 driver were labeled with Insc and Mira ( A ) and dMyc ( B ) .",
"Central brain is to the left of white dotted line .",
"( C ) Type II MARCM clones of brmT362 , brm2 with or without the expression of UAS-Brm were labeled with Dpn , Ase and CD8 .",
"( D ) Type II driver control , snr1 TRiP RNAi ( BDRC#32372 ) , and snr1 VDRC RNAi ( 12645GD ) under the type II driver were labeled with Dpn , Ase and CD8 .",
"( E ) Control ( MARCM driver ) and snr1R3 type II MARCM clones were labeled with Dpn , Ase and CD8 .",
"( F ) Control ( MARCM driver ) and bap55LL5955 type I MARCM clones were labeled with Dpn , Ase and CD8 .",
"( G ) Type II neuroblast lineages of control ( type II driver ) , iswi knockdown , nurf301 knockdown and acf1 knockdown were labeled with Dpn , Ase and CD8 .",
"Arrows , neuroblasts .",
"Clone outline is indicated by white dotted line ( C–G ) .",
"Scale bars , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01906 . 004 Concomitantly with the formation of supernumerary neuroblasts , the number of mature INPs that are positive for Dpn and Ase were dramatically reduced in both brm2 ( Figure 2B , B′ , D; 5 . 3 ± 4 . 3/clone , n = 39 ) and brmT362 ( Figure 2C , C′ , D; 9 . 6 ± 4 . 6/clone , n = 56 ) , compared with the control type II clones ( Figure 2A , A′ , D; 18 . 9 ± 3 . 7 , n = 22 ) .",
"The number of Dpn− PntP1+ immature INPs and early mature INPs appeared to be normal or slightly increased in brm2 ( Figure 2F; 8 . 4 ± 2 . 5 , n = 26 ) and brmT362 ( Figure 2G; 7 . 1 ± 2 . 1 , n = 33 ) clones compared with the control ( Figure 2E; 5 . 9 ± 1 . 1 , n = 26 ) .",
"These results suggested that ectopic neuroblasts in brm− clones likely originate from INPs that fail to undergo maturation .",
"To further determine whether INPs undergo dedifferentiation back into neuroblast , we used INP-specific RNAi to knock down brm in INPs by erm-Gal4 , an INP-specific driver .",
"In 42 . 5% of INP clones with brm knockdown , ectopic Dpn+ Ase− neuroblasts were observed ( Figure 2I , I′; 1 . 2 ± 1 . 6 neuroblasts /INP clone , n = 40 ) .",
"In contrast , none of the INP clones from the driver control contained any neuroblasts ( Figure 2H; 0 neuroblast/INP clone , n = 53 ) .",
"The relatively weak phenotype is likely due to incomplete knockdown of Brm , as shown by the reduced Brm staining in the INP clones ( Figure 2—figure supplement 1 ) .",
"Thus , our data suggest that Brm functions in INPs to prevent INP dedifferentiation back into neuroblasts . 10 . 7554/eLife . 01906 . 005Figure 2 . The Brm complex suppresses INP dedifferentiation into type II neuroblasts .",
"( A–C′ )",
"Type II MARCM clones of control ( the MARCM driver; A , A′ ) , brm2 ( B , B′ ) and brmT362 ( C , C′ ) were labeled with Dpn ( blue ) , Ase ( red ) and CD8::GFP ( green ) .",
"( D ) Quantifications of INP number per type II clone for A–C′ .",
"*** indicates p<0 . 001 .",
"( E–G )",
"Type II MARCM clones of control ( E ) , brm2 ( F ) and brmT362 ( G ) were labeled with Dpn ( blue ) , PntP1 ( red ) and CD8::GFP ( green ) .",
"( H–I′ )",
"INP clones of a control ( driver: erm-Gal4 [II]; erm-Gal4 [III]; ( H ) and brm RNAi under erm-Gal4 ( II ) ; erm-Gal4 ( III ) with UAS-Dcr2 UAS-CD8-GFP ( I , I′ ) were labeled with Dpn ( blue ) , Ase ( red ) and CD8 ( green ) .",
"White arrows indicate neuroblasts , yellow arrows indicate Dpn+ Ase+ mature INPs and yellow arrowheads indicate Dpn− PntP1+ INPs .",
"Clones are marked by CD8::GFP and indicated by white dotted line .",
"Scale bars , 10 µm ( A–G ) and 5 µm ( H–I′ ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01906 . 00510 . 7554/eLife . 01906 . 006Figure 2—figure supplement 1 . Partial knock down of brm in INP clones . Left panels , INP clones of a control ( driver: erm-Gal4 [II]; erm-Gal4 [III] ) and brm RNAi under erm-Gal4 ( II ) ; erm-Gal4 ( III ) with UAS-Dcr2 UAS-CD8-GFP were labeled with Brm ( red ) and CD8 ( green ) .",
"Right panels , Brm is absent in type II neuroblast clones under the type II driver .",
"Clones are indicated by white dotted line .",
"Scale bars , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 01906 . 006 To assess whether Brm was affecting apico-basal cell polarity , we examined the localization of aPKC , Numb and Brat , which are asymmetrically localized in wild-type neuroblasts in prometaphase/metaphase .",
"brm2 MARCM clones did not show any defects in the localization of these markers ( Figure 3—figure supplement 1A–C ) , suggesting that Brm is not important for the apical-basal polarity regulation in neuroblasts .",
"We then assessed the function of other core subunits of the Brm remodeling complex using RNAi knockdown in larval brains .",
"Knock down either of the three core components snr1 ( Figure 1G , K , 9 . 5 ± 5 . 5 neuroblasts/lineage , 64 . 5% , n = 34 ) , bap60 ( Figure 1H , K , 5 . 1 ± 2 . 8 neuroblasts/lineage , 87 . 0% , n = 52 ) , and moira ( data not shown; also reported in Neumuller et al . ( 2011 ) ) resulted in prominent phenotypes with ectopic Dpn+ Ase− neuroblasts in type II neuroblast lineages .",
"Two additional snr1 RNAi lines under the control of the type II driver also displayed excess type II neuroblasts ( Figure 1—figure supplement 1D ) .",
"Furthermore , bap55LL5955 MARCM clones also showed ectopic neuroblasts in type II ( Figure 1J , J′ , 2 . 3 ± 1 . 7 neuroblasts/clone; 30 . 5% , n = 57 ) , but not type I neuroblast lineages ( Figure 1—figure supplement 1F; n = 20 ) .",
"The number of Dpn+ Ase+ mature INPs in bap55LL5955 was significantly reduced ( 13 . 8 ± 4 . 1; n = 49 ) compared with the control ( 18 . 9 ± 3 . 7 , n = 22 ) , while the number of Dpn− PntP1+ immature and early mature INPs ( 4 . 2 ± 1 . 9 , n = 26 ) was similar to the control ( 5 . 9 ± 1 . 1 , n = 26 ) .",
"In various mutants and RNAi lines described above , we also observed an increased number of Dpn+ PntP1+ cells ( data not shown ) , which serves as an independent set of marker for type II neuroblasts .",
"This data further supports that loss-of-function of the Brm complex caused the phenotype of ectopic type II neuroblasts .",
"We conclude that core components of the Brm remodeling complex are required to suppress ectopic neuroblast formation in type II neuroblast lineages .",
"Next , we ascertained whether other chromatin remodeling complexes such as Nucleosome remodeling factor ( NURF ) and ACF complex ( ATP-utilizing chromatin assembly and remodeling factor ) , play any role during neuroblast self-renewal .",
"RNAi knockdown of nurf301 or ACF complex components iswi and acf1 ( Figure 1—figure supplement 1G ) in the type II neuroblast lineages did not result in any obvious neuroblast overgrowth .",
"These data suggest that they may not be important for type II neuroblast lineages or their RNAi targeting was insufficient to induce an effect .",
"To assess the involvement of histone modifications in type II neuroblast lineages , we screened a collection of 43 histone modifiers ( Table 1; Kirilly et al . , 2011 ) by RNAi under the control of a type II-specific driver but failed to identify any RNAi lines with ectopic neuroblasts .",
"We reasoned that histone modifiers may act cooperatively with the Brm complex in type II neuroblast lineages and the phenotype may be masked due to the presence of the functional Brm complex .",
"We therefore re-screened the same collection of potential histone modifiers in a brm RNAi background and showed that the simultaneous knockdown of both brm and hdac3 under the control of the type II driver resulted in a more severe phenotype of ectopic neuroblasts ( Figure 3D , E; 22 . 1 ± 7 . 4 neuroblasts/clone , n = 21 ) compared with brm RNAi ( Figure 3B , E; 10 . 3 ± 5 . 6 neuroblasts/clone , n = 30 ) or hdac3 RNAi knockdown alone ( Figure 3C , E; 1 . 1 ± 0 . 5 neuroblasts/clone , n = 85 ) .",
"This finding suggests that HDAC3 functions cooperatively with Brm to regulate type II neuroblast lineages .",
"Next , we took advantage of an existing deletion mutant snr16c hdac36c , which removes the entire snr1 coding region and the C-terminal region of hdac3 .",
"Type II neuroblast MARCM clones from snr16c hdac36c homozygotes possessed a large number of ectopic neuroblasts ( Figure 3G , H , 22 ± 11 . 5 neuroblasts/clone , 83 . 0% , n = 22 ) .",
"The number of mature Dpn+ Ase+ INPs in each snr16c hdac36c type II neuroblast clone was modestly reduced to 15 . 6 ± 4 . 4 ( n = 22 ) compared with 20 . 3 ± 3/clone ( n = 20 ) in control , while the number of Dpn− PntP1+ immature and early mature INPs ( 6 . 3 ± 3/clone , n = 21 ) are slightly greater compared with the control clones ( 4 . 1 ± 0 . 9 , n = 21 ) .",
"In contrast , type I neuroblast clones of this double mutant appeared normal , as there was only one neuroblast per clone ( Figure 3—figure supplement 1D , n = 17 ) .",
"Similar to hdac3 RNAi ( Figure 3C ) , neither type I nor type II neuroblast mutant clones of a loss-of-function hdac3N allele had ectopic neuroblasts ( Figure 3—figure supplement 1E and data not shown ) .",
"Despite that ectopic neuroblasts were observed in multiple snr1 RNAi lines ( Figure 1—figure supplement 1D ) , snr1R3 MARCM clones did not show obvious ectopic type II neuroblasts ( Figure 1—figure supplement 1E ) .",
"Because Snr1 protein was speculated to have extended perdurance in somatic clones of its null allele ( Marenda et al . , 2004 ) , the lack of phenotype in snr1R3 is likely due to protein perdurance in the neuroblast clones .",
"Our data suggest that HDAC3 acts cooperatively with the Brm complex to suppress the formation of ectopic type II neuroblasts .",
"To ascertain whether snr16c hdac36c causes tumorigenesis , larval brain tissues carrying snr16c hdac36c MARCM clones were transplanted into the abdomen of wild-type hosts .",
"A significant portion of the mutant tissue ( Figure 3J; 21% , n = 14 ) proliferated massively and formed malignant tumors , whilst control clones did not proliferate after the implantation ( Figure 3I; n = 25 ) .",
"In subsequent rounds of transplantation , 70% ( T1 , n = 10 ) and 80% ( T2 , n = 5 ) of the snr16c hdac36c mutant brain tissues developed tumors , suggesting that snr16c hdac36c can induce malignant tumor-like growth after allograft culture . 10 . 7554/eLife . 01906 . 007Table 1 . Histone modifiers and their RNAi linesDOI: http://dx . doi . org/10 . 7554/eLife . 01906 . 007S/No . Gene nameFull nameCG #Main functionVDRC RNAi lines1enokEnoki mmushroomCG11290HATKK108400 , GD375272nejNejire/CBPCG15319HATKK1051153CG1894CG1894HATGD41575 , GD415744CG2051CG2051HATGD334585MofMales absent on the firstCG3025HATKK1053706Rpb4Rpb4CG33520HATGD21985 , GD233087PcafGcnCG4107HATKK108943 , GD217868YL-1YL-1CG4621HATGD219039ChmChameauCG5229HATKK10554210DikDisketteCG7098HATGD4632011lidLittle imaginal discsCG9088HATGD42203 , KK10383012Ada2bCG9638HATGD2407613Sirt7CG11305HDACGD18043 , GD1804514HDAC4CG1770HDACGD2052215HDAC3CG2128HDACKK10707316HDACXCG31119HDACKK10809817Sirt4CG3187HDACGD40295 , KK11063918Sirt2CG5085HDACKK10379019Sir2CG5216HDACGD23199 , KK108241 , KK10550220Bin1Bicoid interacting proteinCG6046HDACKK105352 , GD1571021Sirt6CG6284HDACGD2248322GugGrungeCG6964HDACGD1368723Rpd3HDAC1CG7471HDACGD46929 , GD30600 , GD4692924Sin3aCG8815HDACKK10585225Rtf1CG10955Methyl transferaseKK11039226Vig2CG11844Methyl transferaseKK107081 , GD1724527eggegglessCG12196Methyl transferaseKK101677 , GD3373028escextra sexcombsCG14941Methyl transferaseGD5690 , GD569229set2CG1716Methyl transferaseGD3070730g9aCG2995Methyl transferaseGD2547431pr-set7CG3307Methyl transferaseKK10542232trrtrithorax-relatedCG3848Methyl transferaseGD10749 , KK11027633CG40351CG40351Methyl transferaseGD40683 , GD10833 , GD4526734CG4565CG4565Methyl transferaseGD566535mes-4CG4976Methyl transferaseGD1083636Art4Arginine methyl transferase 4CG5358Methyl transferaseKK10700937Su ( var ) 3–9auppressor of variegation 3–9CG6476Methyl transferaseGD3937738Art1Arginine methyl transferase 11CG6554Methyl transferaseGD40388 , KK11039139ash2absent , small or homeotic discs 2CG6677Methyl transferaseKK10071840LKRLysine ketoglutarate reductaseCG7144Methyl transferaseGD5134641Su ( z ) 12Suppressor of Zeste 205CG8013Methyl transferaseGD42422 , GD4242342Su ( var ) 205Suppressor of variegation 205CG8409Methyl transferaseKK10747743Ash1Absent , small or homeotic discs 1CG8887Methyl transferaseGD2892810 . 7554/eLife . 01906 . 008Figure 3 . HDAC3 acts cooperatively with the Brm complex to suppress the formation of ectopic type II neuroblasts .",
"( A–D )",
"The driver control ( A ) , brm RNAi ( B ) , hdac3 RNAi ( C ) , brm hdac3 double knockdown ( D ) under the type II driver were labeled with Dpn , Ase , and CD8 .",
"( E ) Quantification of neuroblast number per type II MARCM clone in A–D .",
"( F–G )",
"Type II MARCM clones from the driver control ( F ) and snr16c hdac36c ( G ) homozygous MARCM clones were labeled with Dpn , Ase and CD8 .",
"Arrows indicate neuroblasts .",
"( H ) Quantification of neuroblast number per type II MARCM clone in F–G .",
"*** indicates p<0 . 001 .",
"( I–J )",
"Clones are marked by CD8::GFP and indicated by white dotted line .",
"Larval brain tissues from the wild-type MARCM clones ( I ) and snr16c hdac36c MARCM clones ( J ) were implanted into the abdomen of wild-type hosts .",
"Scale bar , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 01906 . 00810 . 7554/eLife . 01906 . 009Figure 3—figure supplement 1 . Brm is not important for the apical-basal polarity of neuroblasts .",
"( A–C )",
"Neuroblast of control MARCM clones and brm2 MARCM clones were co-labeled with aPKC ( white ) , CD8 ( green ) and Phospho-Histone H3 ( PH3; green ) ( A ) or Numb , GFP and DNA ( B ) or Brat , GFP and DNA ( C ) .",
"Lower panels are enlarged images of the boxed region .",
"( D ) Type I MARCM clones from control ( MARCM driver ) and snr16c hdac36c were labeled with Dpn , Ase and CD8 .",
"( E ) Type II MARCM clones from control ( driver ) and hdac3N were labeled with Dpn , Ase and CD8 .",
"Arrows , neuroblasts .",
"Scale bars , 5 µm ( A–C ) , 10 μm ( D–E ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01906 . 009 Given that Brm is ubiquitously expressed in various cell types of larval brains , including neuroblasts , INPs , GMCs and neurons ( Figure 2—figure supplement 1 and data not shown ) , it is conceivable that the Brm complex may associate with regulatory protein ( s ) or co-factor ( s ) that is/are specifically expressed in type II neuroblast lineages to suppress the formation of ectopic neuroblasts .",
"We therefore assessed whether Brm could associate with two such type II-specific transcription factors , Erm and PntP1 .",
"Flag-tagged Brm was co-transfected with Myc-tagged Erm in S2 cells .",
"Following immunoprecipitation ( IP ) of Flag-Brm , Erm can be specifically detected in the immune complex ( Figure 4A ) .",
"Consistently , Flag-Brm was detected in the immune complex following the IP of Myc-Erm ( Figure 4A ) .",
"In contrast , Myc-Brm did not associate with Flag-PntP1 in similar co-IP experiments ( Figure 4—figure supplement 1A ) , suggesting that Brm specifically associates in a protein complex with Erm , but not with PntP1 .",
"Since full-length Erm had very low expression levels in S2 cells , we expressed two truncated proteins , Erm N-terminal 1-441aa ( Erm-N containing N-terminal region and four of six zinc-finger domains ) and Erm C-terminal 332-611aa ( Erm-C containing the last four zinc-finger domains and its C-terminus ) , and used them for the subsequent co-IP analysis ( Figure 4B ) .",
"In co-IP experiments , Myc-Brm associated strongly with Flag-Erm-N and weakly with Flag-Erm-C ( Figure 4C ) .",
"Furthermore , we ascertained whether Brm could associate with Erm in a protein pull-down assay .",
"Full-length Erm could not be efficiently expressed when fused with Maltose-Binding Protein ( MBP ) in bacteria; we therefore expressed truncated MBP-Erm-N or MBP-Erm-C .",
"These fusion proteins were then bound to amylose resin , and subsequently incubated with protein extracts from S2 cells transfected with Myc-Brm .",
"Following the pull-down of amylose resin , Myc-Brm associated intensely with MBP-Erm-N and weakly with Erm-C , but not with the MBP control ( Figure 4H ) .",
"These data suggest that Brm physically associates with Erm and Erm N-terminus appears to be more important for this association . 10 . 7554/eLife . 01906 . 010Figure 4 . The Brm remodeling complex physically associates with Erm and HDAC3 . ( A ) Co-immunoprecipitation ( Co-IP ) between Flag-Brm and Myc-Erm .",
"( B ) An illustration of Erm domains and truncated constructs .",
"( C ) Co-IP between Myc-Brm and Flag-Erm-N or Flag-Erm-C .",
"( D ) Co-IP between Flag-Bap60 and Myc-Erm-N or Myc-Erm-C .",
"( E ) Co-IP between Myc-Snr1 and Flag-Erm-N or Flag-Erm-C .",
"( F ) Co-IP was Flag-HDAC3 and Myc-Brm .",
"( G ) Co-IP between Flag-HDAC3 and Myc-Erm-N or Myc-Erm-C .",
"IP was performed using anti-Flag or anti-Myc antibodies .",
"Western blot was performed using anti-Flag and anti-Myc antibodies .",
"( H ) Protein pull-down assay .",
"MBP , MBP-Erm-N and MBP-ErmC bound beads were incubated with protein extracts from S2 cells expressing Myc-Brm , Myc-Snr1 or Myc-HDAC3 .",
"Western blot was performed using an anti-Myc antibody .",
"Coomassie blue ( CB ) staining showed 10% input of various purified MBP or MBP fusion proteins . DOI: http://dx . doi . org/10 . 7554/eLife . 01906 . 01010 . 7554/eLife . 01906 . 011Figure 4—figure supplement 1 . Brm and Erm may regulate gene expression of some common downstream targets .",
"( A ) Co-IP was performed using S2 cells expressing Flag-PntP1 and Myc-Brm .",
"IP was performed using anti-Flag or anti-Myc antibodies .",
"Western blot was performed using anti-Flag and anti-Myc antibodies .",
"( B ) The DNA binding preferences of the first zinc-finger ‘GTAG’ and the fourth zinc-finger ‘RAAA’ .",
"They are observed to be enriched in 270 Brm binding sites .",
"( C ) The distant distribution between the ChIP–chip peak and the occurrences of the motif in ( D ) .",
"( D ) The de novo Erm-binding motif learned by SEME based on the 270 Brm binding sites . DOI: http://dx . doi . org/10 . 7554/eLife . 01906 . 011 We next ascertained whether Erm associates with other components of the Brm remodeling complex , such as BAP60 and Snr1 .",
"In co-IP experiments , Flag-BAP60 was detected in the immune complex following IP of Myc-Erm-N but not Myc-Erm-C ( Figure 4D ) .",
"Likewise , Myc-Erm-N was detected in the immune complex when IP was performed using Flag-BAP60 ( Figure 4D ) .",
"Moreover , Myc-Snr1 associated with Flag-Erm-N or Flag-Erm-C in co-IP experiments ( Figure 4E ) .",
"Consistently , Myc-Snr1 associated with MBP-Erm-N and weakly with MBP-Erm-C in protein pull-down assays ( Figure 4H ) .",
"Therefore , we conclude that the several components of the Brm remodeling complex specifically associates with Erm .",
"Given that HDAC3 functions cooperatively with the Brm complex to suppress the generation of ectopic neuroblasts , we ascertained whether HDAC3 can physically associate with both Brm and Erm .",
"Flag-HDAC3 and Myc-Brm were co-transfected in S2 cells .",
"Following immunoprecipitation of Flag-HDAC3 , Myc-Brm was clearly detected in the immune complex ( Figure 4F ) .",
"Similarly , Flag-HDAC3 was detected in the immune complex after IP of Myc-Brm ( Figure 4F ) .",
"Interestingly , HDAC3 also physically associates with Erm-N , but not Erm-C , in both co-IP ( Figure 4G ) and protein pull-down assay ( Figure 4H ) .",
"Taken together , these results show that Brm physically associates with Erm and HDAC3 in the protein complex .",
"Given that Brm and Erm associate in a protein complex , and both of them suppress the formation of ectopic neuroblasts , we assessed whether brm genetically interacts with erm to prevent dedifferentiation of INPs to neuroblasts .",
"First , we ascertained whether erm knockdown can exacerbate the brm RNAi phenotype in type II neuroblast lineages .",
"The simultaneous knockdown of brm and erm resulted in a much more severe phenotype with a large number of ectopic neuroblasts in each lineage ( Figure 5D , E; 37 . 1 ± 7 . 6 neuroblasts/lineage , n = 20 ) , in contrast to brm knockdown alone ( Figure 5B , E; 9 . 5 ± 2 . 8 , n = 32 ) .",
"It was reported that in erm- mutants , dedifferentiated neuroblasts can establish ectopic type II neuroblast lineages and form ectopic glial chambers ( Weng et al . , 2010 ) .",
"Presumably due to incomplete knockdown of erm that only led to weak ectopic type II neuroblasts phenotypes , erm RNAi under the type II driver resulted in ectopic type II lineages with each lineage containing one type II neuroblast ( Figure 5C , E; 1 neuroblast/lineage , n = 60 ) .",
"This dramatic enhancement suggests that brm and erm genetically interact to prevent the dedifferentiation of INPs back to neuroblasts .",
"Furthermore , knock down of erm by RNAi in the brmT362 MARCM clones ( Figure 5—figure supplement 1A , B; 9 . 9 ± 5 . 5 neuroblasts/clone , n = 32 ) also significantly enhanced neuroblast overgrowth compared with brmT362 clones ( Figure 5—figure supplement 1A , B; 4 . 1 ± 2 . 4 neuroblasts/clone , n = 30 ) .",
"However , the size of the brmT362 clones with erm knockdown remained smaller than the control clones , probably due to the reduced number of INPs that are required to expand the clonal size .",
"Erm overexpression has previously been shown to result in premature differentiation of type II neuroblasts ( Weng et al . , 2010 ) .",
"Similarly , we found that overexpression of Erm in type II MARCM clones caused 100% of the neuroblasts to undergo premature differentiation; 29 . 3% of the clones contained a neuroblast that gained Ase expression and 41 . 5% of the clones contained a neuroblast that had gained Ase expression with strongly reduced Dpn expression , while the rest of the clones showed no obvious neuroblasts ( Figure 5G and data not shown; n = 41 ) .",
"To assess whether this effect is dependent on its association with Brm , we overexpressed Erm in brm2 type II neuroblast clones .",
"The premature differentiation of type II neuroblasts was dramatically suppressed in a brm loss-of-function mutant background , as there were still 37 . 8% of clones contained ectopic neuroblasts ( Figure 5I ) , while 62 . 2% of type II neuroblasts underwent premature differentiation ( Figure 5H; n = 37 ) .",
"Similarly , the simultaneous knockdown of both erm and snr1 resulted in a more severe phenotype of ectopic neuroblasts ( Figure 5M–N; 862 ± 106 . 8 neuroblasts/brain lobe , n = 20 ) compared with either erm knockdown ( Figure 5K , N; 76 . 6 ± 14 . 2 neuroblasts/brain lobe , n = 20 ) or snr1 knockdown ( Figure 5L , N; 219 . 5 ± 52 . 2 neuroblasts/brain lobe , n = 20 ) .",
"Thus , we conclude that brm and snr1 genetically interact with erm to prevent dedifferentiation of INPs to neuroblasts .",
"Furthermore , the simultaneous knockdown of hdac3 and erm under the control of type II driver also resulted in a more dramatic increase of ectopic type II neuroblasts ( Figure 5Q , R; 565 . 4 ± 68 . 1 type II neuroblasts/brain lobe , n = 20 ) compared with either erm knockdown ( Figure 5P , R; 76 . 0 ± 7 . 7 type II neuroblasts/brain lobe , n = 20 ) or hdac3 knockdown ( Figure 5R; 8 type II neuroblasts/brain lobe , n = 20 ) , suggesting that hdac3 and erm genetically interact in type II neuroblast lineages .",
"Taken together , these results indicate that Brm , HDAC3 , and Erm function as a repressor complex to prevent INP dedifferentiation into type II neuroblasts . 10 . 7554/eLife . 01906 . 012Figure 5 . Brm genetically interacts with Erm to prevent dedifferentiation of INPs to neuroblasts .",
"( A–D )",
"Type II clones of control ( the type II driver; A ) , brm knockdown ( B ) , erm knockdown ( C ) and brm erm double knockdown ( D ) were labeled with Dpn ( blue ) , Ase ( red ) and CD8 ( green ) .",
"( E ) Quantifications of neuroblast number per type II neuroblast lineage for A–D .",
"( F–I )",
"Type II MARCM clones of brm2 ( F , F′ ) , UAS-Erm ( G ) and UAS-Erm , brm2 ( H–I ) were labeled with Dpn ( blue ) , Ase ( red ) and CD8 ( green ) .",
"( J–M )",
"Larval brains of control ( J , elav-Gal4 driver ) , erm knockdown ( K ) , snr1 knockdown ( L ) and erm snr1 double knockdown ( M ) were labeled with Dpn ( blue ) , Ase ( red ) and Mira ( green ) .",
"( N ) Quantifications of the number of type II neuroblasts per brain hemisphere in various genotypes in J–M .",
"Control ( elav-Gal4 ) , 7 ± 0; erm RNAi , 76 . 6 ± 14 . 2; snr1 RNAi , 219 . 5 ± 52 . 2; erm snr1 double knockdown ( KD ) , 862 . 0 ± 106 . 7 .",
"( O–Q )",
"Larval brains of control ( driver; O ) , erm knockdown ( P ) and erm hdac3 double knockdown ( Q ) under the type II driver were labeled with Dpn ( blue ) , Ase ( red ) and CD8 ( green ) .",
"( R ) Quantifications of neuroblast number per brain hemisphere in O–Q .",
"Central brain is to the left of white dotted lines .",
"Arrows indicate neuroblasts .",
"Clones were indicated by white dotted lines .",
"Scale bars , 10 µm .",
"*** indicates p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 01906 . 01210 . 7554/eLife . 01906 . 013Figure 5—figure supplement 1 . Knocking down of erm enhanced the neuroblast overgrowth observed in brm mutants .",
"( A ) Type II MARCM clones of brmT362 and UAS-Dcr2; erm RNAi , brmT362 were labeled with Dpn , Ase and CD8 .",
"Arrows , neuroblasts .",
"Clone outline is indicated by white dotted line .",
"Scale bar , 10 µm .",
"( B ) Quantification of neuroblast number per type II MARCM clone .",
"MARCM Control , 1 . 0 ± 0; brmT362 , 4 . 1 ± 2 . 4; erm RNAi , brmT362 , 9 . 9 ± 5 . 5 .",
"( C ) Simultaneous knockdown of brm and notch in type II neuroblast lineages partially suppressed the ectopic neuroblast phenotype , compared with brm knockdown alone .",
"Type II neuroblast lineages are labeled with Dpn , Ase and CD8 .",
"( D ) Quantification of genotypes in C . brm notch double knockdown , 6 . 0 ± 4 . 7 neuroblasts/lineage , n = 76; brm knockdown , 10 . 8 ± 4 neuroblasts/lineage , n = 39 .",
"*** indicates p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 01906 . 013"
],
[
"Here , we report a critical function of the Drosophila Brm remodeling complex in suppressing the formation of ectopic type II neuroblasts in larval brains .",
"Mutants of major components of the Brm complex , including Brm and Bap55 , and RNAi targeting of several Brm components formed ectopic type II neuroblasts .",
"Therefore , the Drosophila Brm remodeling complex displays a tumor suppressor-like function in larval brains .",
"Multiple subunits of the SWI/SNF complex are associated with various cancers .",
"BAP47 ( homologous to Snr1 ) is a bona fide tumor suppressor and the gene is deleted in pediatric rhabdoid tumors ( Reisman et al . , 2009 ) .",
"Mutations in epigenetic regulators are found in approximately half of hepatocellular carcinoma and bladder cancers , and represent a significant portion of mutated genes in medulloblastoma ( Gui et al . , 2011; Fujimoto et al . , 2012; Pugh et al . , 2012 ) .",
"Drosophila Brm complex is essential for intestinal stem cell proliferation and commitment in the adult intestine ( Jin et al . , 2013; Zeng et al . , 2013 ) .",
"Two other chromatin remodeling factors , Iswi and Domino control germline stem cell and somatic stem cell self-renewal in the ovary ( Xi and Xie , 2005 ) .",
"We have demonstrated that Brm physically associates with Erm , a type II-specific transcription factor that prevents the dedifferentiation of INPs back into neuroblasts .",
"Furthermore , Bap60 and Snr1 , two other components of the Brm complex , also physically associate with Erm in a protein complex .",
"Therefore , we have provided the first molecular link during the regulation of type II neuroblast lineages .",
"We speculate that the association with Erm may provide functional specificity of the Brm remodeling complex in type II neuroblast lineages .",
"We have also shown that brm genetically interacts with the type II-specific transcription factor erm .",
"Ectopic neuroblast phenotype resulting from brm knockdown was dramatically enhanced by simultaneous knockdown of erm .",
"Furthermore , brm knockdown , similar to erm− ( Weng et al . , 2010 ) , can be partially suppressed by loss of notch ( Figure 5—figure supplement 1C , D ) .",
"These functional data suggest that Erm is a co-factor of the Brm remodeling complex in type II neuroblast lineages .",
"However , it is uncertain how the Brm–Erm protein complex functions to prevent dedifferentiation in type II neuroblast lineages .",
"Our bioinformatic analysis has identified a 14 bp-long motif as the de novo Erm DNA-binding motif ( Figure 4—figure supplement 1B–D; and Supplementary methods ) and 202 sites out of the 270 known genomic loci harboring Brm ( Negre et al . , 2011 ) also contain the de novo Erm DNA-binding motif ( Table 2 , Gene list ) .",
"As there are many genes that are potentially co-occupied by Brm and Erm , it is possible that Brm–Erm complex results in a unique configuration of the chromatin ‘landscape’ in INPs to prevent INP dedifferentiation into neuroblasts .",
"Therefore , disruption of chromatin remodelers may cause widespread changes to the transcriptome , thus amplifying the effect of the single genetic mutation . 10 . 7554/eLife . 01906 . 014Table 2 . Predicted common target genes of Brm and ErmDOI: http://dx . doi . org/10 . 7554/eLife . 01906 . 014S/No . CG nameGene name1CG10033for2CG10117ttv3CG10137CG101374CG10159BEAF-325CG10388Ubx6CG10610ECSIT7CG1071E2f28CG10844RyR9CG1100Rpn510CG11228hpo11CG11309CG1130912CG11589VhaM9 . 7-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-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 ( 2 ) k01209113CG4996CG4996114CG5229chm115CG5393apt116CG5505scny117CG5548CG5548118CG5588Mtl119CG5599CG5599120CG5611CG5611121CG5613CG5613122CG5824l ( 3 ) 07882123CG5836SF1124CG6022Cchl125CG6202Surf4126CG6218CG6218127CG6235tws128CG6241CG6241129CG6272CG6272130CG6322U4-U6-60K131CG6343ND42132CG6401CG6401133CG6511CG6511134CG6556cnk135CG6565CG6565136CG6604H15137CG6634mid138CG6829Ark139CG6948Clc140CG6951CG6951141CG6983CG6983142CG7082papi143CG7085l ( 2 ) s5379144CG7186SAK145CG7191CG7191146CG7372CG7372147CG7379CG7379148CG7564CG7564149CG7597Cdk12150CG7632CG7632151CG7685CG7685152CG7734shn153CG7771sim154CG7828APP-BP1155CG7845CG7845156CG7849CG7849157CG7957MED17158CG7961alphaCop159CG8067CG8067160CG8241pea161CG8287Rab8162CG8360CG8360163CG8372CG8372164CG8396Ssb-c31a165CG8409Su ( var ) 205166CG8481CG8481167CG8790Dic1168CG8798Lon169CG8817lilli170CG9042Gpdh171CG9054Ddx1172CG9063Rich173CG9065CG9065174CG9243CG43345175CG9243CG43346176CG9244Acon177CG9249CG9249178CG9250Mpp6179CG9305CG9305180CG9376CG9376181CG9473MED6182CG9596CG9596183CG9635RhoGEF2184CG9641CG9641185CG9730mRpL21186CG9750rept187CG9829poly188CG9865CG9865 Most class I HDACs are recruited into large multi-subunit co-repressor complexes for maximal activity ( Wen et al . , 2000 ) .",
"HDAC1 and 2 are found in multiple co-repressor complexes , while to date HDAC3 appears to be uniquely recruited to the Silencing mediator of retinoic and thyroid receptors ( SMRT ) /Nuclear receptor co-repressor ( N-CoR ) complex ( Guenther et al . , 2000; Li et al . , 2000 ) .",
"Here , we report that Drosophila HDAC3 is recruited to a novel multi-subunit complex containing Brm and Erm and that this co–repressor complex prevents dedifferentiation of INPs into type II neuroblasts .",
"The SMRT complex appears not to be important for type II neuroblasts , as knockdown of smrter that encodes a core component of the SMRT complex ( Heck et al . , 2012 ) neither resulted in any ectopic type II neuroblasts nor enhanced the phenotype of ectopic neuroblasts by brm knockdown ( data not shown ) .",
"We also showed that HDAC3 dramatically enhanced the phenotype of ectopic neuroblast upon loss of brm or snr1 , two core components of the Brm complex .",
"By identifying this novel repressor complex , we have provided a mechanistic link between transcriptional repression and histone deacetylation during the suppression of dedifferentiation .",
"HDACs are typically recruited by oncogenic protein complexes in lymphoma and leukemia and HDAC3 inhibitors are synergistic or additive with anticancer agents for therapeutics ( Dokmanovic et al . , 2007 ) .",
"Our finding that HDAC3 functions cooperatively with the Brm complex in suppressing suppressing dedifferentiation of INPs into neuroblasts and induces tumors in the allograph transplantation revealed an unexpected potential involvement of HDAC3 in tumor suppression in brain tissue .",
"It will be of interest to determine whether this effect is conserved in the mammalian central nervous system and whether it occurs in tissues other than the brain ."
],
[
"The following flies were used in this study: brmT362 is from J Treisman; erm1 , erm2 , UAS-ErmCTHA , UAS-Brm ( AK Dingwall ) , 9D11-Gal4 ( erm-Gal4; GM Rubin ) .",
"brm2 , bap55LL05905 , Erm RNAi ( #26778; BDSC ) are from Bloomington Drosophila stock center .",
"VDRC RNAi lines used: Brm ( GD37720 and 37721GD ) , Bap60 ( KK103634 ) , Snr1 ( KK108599 , GD12645 , and BDRC#32372 ) , Bap55 ( GD24704 ) , Moira ( GD6969 ) , Bap180 ( KK108618 ) , dMi-2 ( KK107204 ) , nurf301 ( GD46645 ) , Acf1 ( GD33446 ) and ISWI ( GD24505 ) .",
"The type II neuroblast driver: w; UAS-Dicer 2 , wor-Gal4 , ase-Gal80/CyO; UAS-mCD8-GFP/TM3 , Ser ( Neumuller et al . , 2011 ) .",
"The primary antibodies used were: guinea-pig anti-Dpn ( 1:1000 , J Skeath ) , anti-Insc ( 1:1000 ) ; rabbit anti-aPKCζ C20 ( 1:100; Santa Cruz Biotechnologies , Dallas , TX ) ; guinea-pig anti-Numb ( 1:1000; J Skeath ) ; mouse anti-Mira ( 1:50; F Matsuzaki ) ; rat anti-CD8 ( 1:250; Caltag laboratories , United Kingdom ) ; rabbit anti-GFP ( 1:500; Molecular Probes , Eugene , OR ) ; rabbit anti-Asense ( 1:1000; YN Jan ) ; rabbit anti-PntP1 ( 1:100; J Skeath ) ; rabbit anti-Brm ( 1:100; L Zhang ) ; rat anti-phospho-Histone H3 ( 1:1000; Cell Signaling , Danvers , MA ) ; rabbit anti-phospho-Histone H3 ( 1:200; Sigma , St Louis , MO ) ; mouse anti-dMyc ( 1:5; B Edgar ) .",
"Antibodies for western blotting used were: mouse anti-Myc ( 1:2000; Abcam , United Kingdom ) and mouse anti-Flag ( 1:1000; Sigma ) .",
"Third instar larval brains were dissected and fixed with 3 . 7% formaldehyde in PBS .",
"Fixed brains were blocked with 3% BSA for one hour and then incubated with primary antibody in 3% BSA ( in 0 . 3% PBS-T ) over night at 4°C .",
"Following three times washing ( 10 min each ) , larval brains were incubated with secondary antibody diluted in 0 . 3% PBS-T for 1 . 5 hr .",
"After two times washing ( 10 min each ) , DNA was labeled by ToPro-3 ( 1:5000; Invitrogen , Carlsbad , CA ) in 0 . 3% PBS-T for 20 min .",
"Larval brains were mounted in vector shield ( Vector Laboratory , Burlingame , CA ) for confocal microscopy .",
"Images were obtained using a Zeiss LSM 700 confocal microscope and processed with Adobe Photoshop CS5 . 1 .",
"MARCM clones were generated as previously described ( Lee and Luo , 1999 ) .",
"Briefly , larvae were heat shocked at 37°C for 90 min at 24 hr ALH and at 10–16 hr after the first heat shock .",
"Larvae were further aged for 3 days at 25°C , and larval brains were dissected and processed for immunohistochemistry .",
"To generate type II neuroblast clones , UAS lines were crossed to the type II driver at 25°C and shifted to 29°C at 24 hr ALH .",
"Wandering third instar larvae were dissected after incubation for 3 or 4 days at 29°C .",
"Drosophila S2 cells were cultured in Shields and Sang M3 insect medium ( Sigma-Aldich ) , and supplemented with 10% fetal bovine serum ( FBS; Hyclone , Logan , UT ) .",
"Flag-Erm or Myc-Brm generated by Gateway cloning was transfected into S2 cells using Effectene Transfection Reagent ( QIAGEN , The Netherlands ) .",
"S2 cells were collected 48 hr after transfection for protein homogenization .",
"80 μg S2 cells are homogenized with lysis buffer ( 25 mM Tris pH8/27 . 5 mM NaCl/20 mM KCl/25 mM sucrose/10 mM EDTA/10 Mm EGTA/1 mM DTT/ 10% ( vol/vol ) glycerol/0 . 5% Nonidet P40 ) with Proteases inhibitors ( Complete , Boeringher; PMSF 10 μg/ml , Sodium orthovanadate 10 μg/ml ) .",
"The supernatants were used for immunoprecipitation with anti-Myc or anti-Flag for overnight at 4°C , followed by incubation with protein A/G beads for two hours ( Pierces , Rockford , IL ) .",
"Protein A/G beads were washed with cold PBS for three times .",
"Bound proteins were separated by SDS-PAGE and analyzed by western blotting .",
"MBP or MBP fusion proteins were expressed in BL21 cells and bound on amylose resin ( Cart# E8021L; NEW ENGLAND Biolabs Inc . , United Kingdom ) .",
"50 μg of purified MBP fusion proteins bound on amylose resin were incubated for 3 hr at 4°C with protein extracts from 100 μg S2 cells that were homogenized in lysis buffer with proteases inhibitors .",
"After washing amylose resin three times for 7 min each with 1 ml lysis buffer , bound proteins were separated by SDS-PAGE and analyzed by western blotting .",
"Allograft culture of larval brain tissue was carried out as previously described ( Castellanos et al . , 2008 ) .",
"Third instar larval brains are dissected and the tissue is cut into pieces .",
"A piece of tissue is collected with the tip of a glass needle and injected in the mid-ventral abdomen of a young female fly .",
"Plasmid constructs were generated using either pENTR Directional TOPO Cloning Kit ( Invitrogen ) or In-Fusion HD Cloning Kit ( Clontech , Mountain View , CA ) .",
"ESTs used in this study were GH14092 ( Erm ) , LD36356 ( Brm ) , LD09078 ( Bap60 ) , GH08712 ( Snr1 ) ( Drosophila Genomics Resource Centre [DGRC] , Bloomington , IN ) .",
"Briefly , coding region of genes were amplified by PCR , inserted into the pENTR/D-TOPO vector ( Invitrogen ) and destination vectors ( pAMW or PAFW ) were generated by LR recombination .",
"From the previously reported ChIP–chip data , we obtained a list of 270 Brm binding sites .",
"To determine if Erm also binds to these binding sites , we first analyzed the DNA binding domains of Erm , which contain 6 zinc fingers .",
"Each zinc-finger domain was assigned a DNA binding preference ( position weighted matrix ) based on published methods ( Kaplan et al . , 2005 ) .",
"We then scanned these six DNA binding preferences of approximately +/−200 bp around the 270 Brm binding sites and found the DNA binding preferences of the 1st zinc-finger ‘GTAG’ and the 4th zinc-finger ‘RAAA’ are enriched in the 270 Brm binding sites .",
"The sites enriched with these two binding preferences were subjected to further analysis using the de novo motif-finding program SEME ( Zhang et al . , 2013 ) , and a 14 bp-long motif was identified as the de novo Erm DNA-binding motif .",
"We scanned +/−200 bp around the 270 Brm binding sites with the de novo Erm DNA-binding motif .",
"Among them , 202 sites ( FDR<0 . 0001 ) were identified as putative Erm-binding sites with an AUC score of 0 . 73 , which is significantly higher than the AUC score computed for random motifs ( 0 . 5 ) .",
"As a negative control , the same approach was also applied to predict the motif of Zinc-finger protein ( Zif ) , which regulates asymmetric division of neuroblasts and therefore is unlikely to be a co-factor with Brm .",
"The predicted Zif DNA-binding motif differs dramatically with the predicted Erm DNA-binding motif in sequence .",
"Furthermore , it was not significantly enriched in the 270 Brm binding sites and had an AUC score of 0 . 54 , similar to the AUC score ( 0 . 5 ) for random motifs .",
"Thus , our data suggests that Brm and Erm can potentially regulate a set of common downstream targets ."
]
] | [
"The control of self-renewal and differentiation of neural stem and progenitor cells is a crucial issue in stem cell and cancer biology .",
"Drosophila type II neuroblast lineages are prone to developing impaired neuroblast homeostasis if the limited self-renewing potential of intermediate neural progenitors ( INPs ) is unrestrained .",
"Here , we demonstrate that Drosophila SWI/SNF chromatin remodeling Brahma ( Brm ) complex functions cooperatively with another chromatin remodeling factor , Histone deacetylase 3 ( HDAC3 ) to suppress the formation of ectopic type II neuroblasts .",
"We show that multiple components of the Brm complex and HDAC3 physically associate with Earmuff ( Erm ) , a type II-specific transcription factor that prevents dedifferentiation of INPs into neuroblasts .",
"Consistently , the predicted Erm-binding motif is present in most of known binding loci of Brm .",
"Furthermore , brm and hdac3 genetically interact with erm to prevent type II neuroblast overgrowth .",
"Thus , the Brm-HDAC3-Erm repressor complex suppresses dedifferentiation of INPs back into type II neuroblasts ."
] | [
"Stem cells show great promise for repairing damaged tissue , and maybe even generating new organs , but stem cell therapies will only be successful if researchers can understand and control the behaviour of stem cells in the lab .",
"Neural stem cells or ‘neuroblasts’ from the brains of larval fruit flies have become a popular model for studying these processes , and one type of neuroblast—known as a ‘type II’ neuroblast—is similar to mammalian neural stem cells in many ways .",
"When type II neuroblasts divide , they generate another neuroblast and a second cell called an intermediate neural progenitor ( INP ) cell .",
"This progenitor cell then matures and undergoes a limited number of divisions to generate more INP cells and cells called ganglion mother cells .",
"The process by which stem cells and INP cells become specific types of cells is known as differentiation .",
"However , under certain circumstances , the INP cells can undergo the opposite process , which is called dedifferentiation , and become ‘ectopic neuroblasts’ .",
"This can give rise to tumors , so cells must employ a mechanism to prevent dedifferentiation .",
"Researchers have known that a protein specifically expressed in INP cells called Earmuff is involved in this process , but many of the details have remained hidden .",
"Now , Koe et al . have discovered that a multi-protein complex containing Earmuff and a number of other proteins—Brahma and HDAC3—have important roles in preventing dedifferentiation .",
"All three proteins are involved in different aspects of gene expression: Earmuff is a transcription factor that controls the process by which the genes in DNA are transcribed to make molecules of messenger RNA; Brahma and HDAC3 are both involved in a process called chromatin remodeling .",
"The DNA inside cells is packaged into a compact structure known as chromatin , and chromatin remodeling involves partially unpacking this structure so that transcription factors and other proteins can have access to the DNA .",
"Koe et al . also showed that Earmuff , Brahma and HDAC3 combine to form a complex that prevents dedifferentiation .",
"An immediate priority is to identify those genes whose expression is regulated by this complex in order to prevent dedifferentiation ."
] | 2014 |
[
"Introduction",
"Results",
"Discussion"
] | [
"evolutionary biology",
"epidemiology and global health"
] | Using evolution to generate sustainable malaria control with spatial repellents | elife-15416-v2 | [
[
"If deflecting mosquitoes from accessing humans indoors is in itself an effective means of reducing transmission , as proposed by Achee et al . ( 2012 ) , then selection for mosquitoes which are repelled from treated houses could serve to generate new public health tools .",
"Critically , selection would depend on the relative fitness of mosquitoes that are deflected away from buildings compared to those entering buildings .",
"This in turn would depend on the proportion of properties treated with insecticides , the susceptibility or resistance ( physiological ) of mosquitoes contacting indoor insecticides and the fitness costs associated with being deflected away from the sleeping indoor hosts which vector species have evolved to exploit .",
"However , the fitness costs of being deflected from human dwellings are difficult to determine directly .",
"Following a well-established history of mathematical modeling to explore issues relating to the evolution of resistance in malaria vector populations ( for example [Rosenheim and Tabashnik , 1990; Le Menach et al . , 2007; Mandal et al . , 2011; Georghiou and Taylor , 1977; White et al . , 2014] ) , we have therefore developed an analysis to explore the possible outcome across a range of fitness scenarios , using a feeding-cycle based , two-locus , bi-allelic population genetics model , capturing the mosquito life-history characteristics of one initial mating and pre-adult development period as well as the Plasmodium development period in infected mosquitoes before transmission is possible .",
"As well as the spread of resistance and deflection alleles over time , our model also tracks adult population size and the expected number of infectious bites given by the population during each modeled time unit .",
"Each modeled time unit corresponds to the length and reproductive outcomes of a single feeding cycle , an approach used in previous models of vector population genetics ( Lynch et al . , 2012; Read et al . , 2009 ) .",
"For details of the model see Appendix",
"1 . To minimize the sensitivity of the model results to specific parameter values , we frame our key disease control results in terms of the proportionate difference between the model’s calculated infectious bite values for a given set of intervention assumptions and those assuming no intervention , minimising the impact of parameter values that are unaffected by the intervention .",
"The impact of deflection on malaria prevalence is determined by the proportion of mosquitoes deflected by a repellent and the probability ( compared to non-deflected mosquitoes ) that they will then acquire and transmit a Plasmodium infection .",
"The effect on Plasmodium transmission of deflecting vectors to outdoor biting has not been definitively measured in the field , we therefore consider a wide range of per-feed probabilities of Plasmodium transmission to deflected mosquitoes compared to the 4% probability assumed for indoor feeds .",
"Reductions in this parameter are intended to represent the effects of all potential sources of reduced transmission to the vector , including deflection to non-human and therefore non-infectious hosts .",
"In terms of model results , proportionate reductions in this parameter will have the same effect as proportionate changes in the probability that an infectious mosquito which survives to feed will give an infectious bite to a human host .",
"As such , the reductions explored can be interpreted as the product of the proportionate reductions in transmission to and from outdoor-feeding vectors .",
"Our key model assumptions .",
"( 1 ) Physiological insecticide resistance is controlled by a single locus bi-allelic autosomal gene , with the resistance allele being completely dominant to the susceptibility allele .",
"( 2 ) Deflection by a given spatial repellent is controlled by a single locus bi-allelic autosomal gene , with the deflection allele being completely dominant to the non-deflection allele .",
"( 3 ) Deflected vectors are assumed not to come into contact with the insecticide used in association with the ESR; therefore , mosquitoes that have phenotypes which combine deflection and resistance will not experience any of the fitness benefits associated with resistance if the ESR and insecticide are always present together .",
"( 4 ) The resistance and deflection loci are not linked and re-assort randomly .",
"( 5 ) The genotypes determining adult resistance and deflection phenotypes do not affect the probability of juvenile survival from egg to adult .",
"( 6 ) Mating is random and females mate once , as newly emerged adults , with males in their cohort .",
"( 7 ) Juvenile density dependence means that variation in the absolute number of eggs produced by the adult population does not materially change the rate at which new adults join the population .",
"Whilst we cannot predict the form that the genetic determinants of resistance , behavioural or otherwise , may take , there are examples of single-locus insecticide-resistance genes , including the knockdown resistance ( kdr ) alleles that provide resistance to DDT and pyrethroids ( Ndiath et al . , 2012; Chandre et al . , 2000; Jones et al . , 2012; Dabiré et al . , 2012 ) .",
"Whilst the genetic basis of deflection behaviour is unknown and may often be more complex than that of insecticide resistance , it is parsimonious to model this process initially by assuming simple single genes that determine the likelihood of such responses .",
"The impact on the predictions of continuous traits , or , should suitable data become available , of specific more complex genetic assumptions , can be incorporated into future work .",
"The model considers four possible phenotypes , as shown in Table 1 , with associated genotypes ( resistance alleles represented by R , and deflection alleles by D ) . 10 . 7554/eLife . 15416 . 003Table 1 . Phenotype definitions and characteristics . DOI: http://dx . doi . org/10 . 7554/eLife . 15416 . 003PhenotypeFitnessResistantDeflectedGenotypesSusceptibleFSNoNorr\\ddResistantFRYesNoRr\\dd RR\\ddDeflected and not resistantFDNoYesrr\\Dd rr\\DDDeflected and resistantFRDYesYesRr\\Dd Rr\\DD RR\\Dd RR\\DD The average fitness of offspring into which deflection alleles are inherited , F¯D , is ( Equation 1 ) F¯D=FS+[Dr][−R]+[DR][D−] ( FRD−FS ) +[Dr][−r][D−] ( FD−FS ) With [dr] , [dR] , [Dr] , [DR] , [d−] and [D−] representing , in the zygote genotypes for the population at a given time point , the proportion of alleles at the deflection locus which are non-deflection alleles paired with susceptible alleles , non-deflection alleles paired with resistant alleles , deflection alleles paired with susceptible alleles , deflection alleles paired with resistant alleles , non-deflection alleles paired with any resistance allele , and deflection alleles paired with any resistance allele , respectively , assuming the same proportions in gametes of mating males and newly emerged females .",
"The average fitness of offspring into which non-deflection alleles are inherited , F¯d , is ( Equation 2 ) F¯d=FS+[dR] ( 1+[dr][d−] ) ( FR−FS ) +[dr][Dr][d−] ( FD−FS ) + ( [DR]+[dR][Dr][d−] ) ( FRD−FS ) In order for the proportion of deflection alleles in the population to increase , we need the average fitness of the offspring into which deflection alleles are inherited to be greater than the average fitness of the offspring into which non-deflection alleles are inherited .",
"This is true when the following inequality applies: ( Expression 1 ) F¯D>F¯d↔[dr]2[d−] ( FD−FS ) >[dR] ( 1+[dr][d−] ) ( FR−FD ) + ( [DR] ( 1−1[D−] ) +[Dr] ( [dR][d−]−[−R][D−] ) ) ( FRD−FD ) The equivalent expression for spread of the resistance allele is: ( Expression 2 ) F¯R>F¯r↔[rd] ( FR−FS ) +[rD] ( FR−FD ) + ( [rD]+[RD][−d][R−]−[Rd][rD][r−] ) ( FRD−FR ) >[rd][rD][r−] ( FD−FS ) See Appendix 2 for derivation of Equations 1 and 2 and Expressions 1 and",
"2 . We assume that ESR is only relevant where the fitness cost of being susceptible to an IRS insecticide is greater than the fitness cost of being deflected by an ESR , so FD>FS , requiring that the mortality associated with a mosquito that has a susceptible phenotype entering an insecticide-treated property is greater than that associated with a mosquito being deflected from a property .",
"From Expression 1 , it can be seen that the spread of the deflection allele in the vector population will be favoured by maximising the fitness difference between susceptible and deflected phenotypes ( increasing the value of the left-hand side of the expression ) , and by maximising the fitness of deflected relative to resistant phenotypes ( reducing the value of the right-hand side of the expression ) .",
"Avoiding the use of ESR in properties without insecticide , minimising Y3 ( Table 2 ) , and using ESR in all insecticide-treated properties , minimising Y2 , improves the survival of deflected phenotypes ( given B<I ) without affecting the survival probability of susceptible or resistant mosquitoes , enhancing the desired fitness relationships and hence favouring the spread of deflection alleles in the vector population and the initial establishment of a new evolved spatial repellent . 10 . 7554/eLife . 15416 . 004Table 2 . Feeding related survival probabilities . DOI: http://dx . doi . org/10 . 7554/eLife . 15416 . 004Feeding-related survival probabilitiesProportion of propertiesY1Y2Y3Y4PhenotypeBaseline fitness adjustmentUntreated propertyInsecticide onlyESR onlyInsecticide and ESRAverage survivalSusceptibleU U−I UU−IU−I ( Y2+Y4 ) ResistantCOR1UUUUUResistant and deflectedCOR2UUU−BU−BU−B ( Y3+Y4 ) DeflectedUU−IU−BU−BU−IY2−B ( Y3+Y4 ) U=no-treatment survival , I=survival reduction caused by insecticide in susceptible mosquitoes , B=survival reduction caused by deflection from protected building , Yi=proportion of properties in each treatment category , COR1=fitness cost of resistance experienced by resistant non-deflected phenotypes , COR2=fitness cost of resistance experienced by resistant deflected phenotypes .",
"From Expression 1 , it can be seen that the spread of deflection alleles is dependent not only upon the relative fitness values of the different phenotypes but also upon there being sufficiently low initial levels of resistance alleles in the population .",
"Since the genotype proportions will change over time , this is a dynamic relationship .",
"In order for the D allele to spread at all , initial allele proportions and fitness relationships must comply with the inequality in Expression 1 , but the spread of the resistance allele over time may eventually reverse the relationship , so that the proportion of non-deflection alleles will begin to increase instead .",
"The rate of spread of the resistance allele will also determine whether the deflection allele will spread and be sustained in the population .",
"From Expression 2 , which shows the conditions necessary for the resistance allele to spread , it can be seen that the fitness differential between resistant and susceptible phenotypes and that between resistant and deflected-resistant phenotypes , by helping to determine whether the resistance alleles spread , are also determinants of whether the deflection allele , and hence deflected phenotypes , will spread and be maintained in the population .",
"The spread of the deflection allele when resistance alleles are present in the population is also critically determined by the initial proportion of deflection alleles at the deflection locus and by the proportions of deflection and non-deflection alleles that are paired with resistance alleles .",
"These interactions are explored in Figures 1–4 . 10 . 7554/eLife . 15416 . 005Figure 1 . Spread of deflection in a population over time .",
"( i ) Phenotype and",
"( ii ) genotype proportions over time for a population subject to insecticide interventions applied in combination with an ESR .",
"Illustrating",
"( a ) long-term establishment of ESR ,",
"( b ) transient establishment of ESR , and",
"( c ) failure to establish ESR .",
"The parameter values used to generate the plots in panels",
"( a ) are: 20% per cycle survival of susceptible phenotypes , 60% per cycle survival of resistant phenotypes 45% per cycle survival of deflected phenotypes , 0 . 5% initial proportion of resistance alleles and 25% initial proportion of deflection alleles .",
"For the panels in",
"( b ) and",
"( c ) , the per cycle survival of deflected phenotypes is reduced to 40% .",
"For panel",
"( c ) , other parameters are also amended to 30% per cycle survival for susceptible phenotypes , 5% initial prevalence of resistance alleles and 10% initial prevalence of deflection alleles . DOI: http://dx . doi . org/10 . 7554/eLife . 15416 . 005 In the absence of resistance , Expression 1 reduces to F¯D>F¯d↔[d−] ( FD−FS ) >0 , so deflection would be expected to spread to fixation provided that deflected phenotypes have greater fitness than non-deflected phenotypes .",
"If deflection reaches fixation , then Expression 2 reduces to F¯R>F¯r↔[rD] ( FRD−FD ) >0 , and resistance will only spread if resistant-deflected phenotypes are fitter than deflected phenotypes .",
"In this instance , the strategy of using ESR in all insecticide-treated properties would prevent further spread of resistance because deflected mosquitoes would not enter any insecticide-treated properties and would experience no benefits from resistance .",
"hence the fitness of resistant deflected phenotypes will be the same as or lower than that of non-resistant deflected phenotypes ( depending upon cost of resistance ) , giving FRD≤FD ."
],
[
"We carried out a numerical analysis using the model to explore the establishment over time of a new ESR and the associated population-level changes in infectious bite rate .",
"Assumed baseline parameter values are:",
"( i ) time from egg laying to adult emergence equivalent to the length of three gonotrophic cycles ,",
"( ii ) probability per feed that a non-deflected mosquito will acquire a Plasmodium infection is 4% ,",
"( iii ) probability per feed that an infectious mosquito gives an infectious bite on a human host is 80% ,",
"( iv ) time to infectiousness of Plasmodium infection in the vector is approximately equivalent to the length of three gonotrophic cycles .",
"Where not stated otherwise , we use 20% survival of susceptible phenotypes per cycle , 60% survival of resistant phenotypes per cycle , 45% survival of deflected phenotypes per cycle , 0 . 5% initial proportion of resistance alleles and 25% initial proportion of deflection alleles .",
"The first question considered is whether , and under what circumstances , selection could generate an effective spatial repellent from a substance that initially repelled only a part of the population .",
"Consistent with Expression 1 , we found that the spread of a deflection allele through the population depended on the initial proportions of deflection and resistance alleles in the population and the fitness differentials between susceptible , resistant , deflected and resistant deflected phenotypes .",
"As illustrated in Figure 1 , for some combinations of fitness values and initial allele proportions , the deflection allele spreads rapidly to near-fixation , and remains consistently at that level for at least 300 cycles ( panels labelled ‘a’ ) .",
"In other cases , the deflection allele spreads initially , but falls away within 300 cycles as the resistance allele spreads ( panels labelled ‘b’ ) , and in some cases the deflection allele shows only minimal spread before being lost as resistance spreads ( panels labelled ‘c’ ) .",
"For cases like that in ‘b’ , where deflection spreads initially but then falls away , we considered the outcome if the insecticide used is swapped for an alternative , for which resistance alleles are still relatively rare , whilst the deflection allele is close to its peak prevalence .",
"In some cases this allows a ‘ratchet’ effect , whereby the deflection allele is able to spread and reach sustained high levels .",
"There is presently little or no direct information available about the fitness costs of a switch to outdoor biting .",
"Furthermore , this would be expected to vary with mosquito species , the degree of anthropophilly , the type and accessibility of outdoor hosts , and various other factors .",
"The initial proportion of deflection and resistance alleles in the population will depend upon the choice of insecticide and ESR , but will also be expected to vary between specific populations .",
"We therefore carried out analyses for a range of parameter values , the results of which are summarized in Figure 2 . 10 . 7554/eLife . 15416 . 006Figure 2 . Combinations of per cycle survival values for deflected and non-deflected resistant phenotypes , which support the spread and maintenance of deflected phenotypes in the population . Grid plots indicating which combinations of resistant ( x-axis ) and deflected ( y-axis ) phenotype per-cycle survival values ( in 1% increments ) give rise to a population comprising at least 80% deflected phenotypes after 300 modeled time periods .",
"When this is achieved directly , the applicable square is bright green .",
"Dark green squares indicate combinations for which the required outcome can be achieved via a ‘ratchet’ where the initial paired insecticide is swapped once for a new insecticide , with allele proportions at the time of the swap assumed to be 0 . 5% resistance alleles and the maximum percentage of deflection alleles achieved whilst using the first insecticide .",
"Results are calculated for 1% increments in each survival value .",
"Gridlines and diagonals are to aid visual location of results on the grid .",
"The baseline parameters ( panel",
"i ) are: 20% per cyclesurvival of susceptible phenotypes; resistant deflected phenotypes have the same survival probability as non-resistant deflected phenotypes; 0 . 5% initial prevalence of resistance alleles; and 25% initial prevalence of deflection alleles .",
"Parameter values for panels",
"( ii ) to",
"( iv ) differ from the baseline values as follows: panel",
"( ii ) 10% initial prevalence of deflection alleles; panel",
"( iii ) 2% initial prevalence of resistance alleles; and panel",
"( iv ) 60% per cycle survival of susceptibles .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 15416 . 00610 . 7554/eLife . 15416 . 007Figure 2—figure supplement 1 . Effect of incomplete deflection on fitness combinations which support the spread and maintenance of deflection . Grid plot indicating when the tested combinations of fitness values give rise to a population comprising at least 80% deflected phenotypes after 300 modeled time periods ( corresponding to the length of 300 gonotrophic cycles ) .",
"The baseline parameters are: 20% survival of susceptible phenotypes per cycle; resistant+deflected phenotypes have a fitness equal to 75% that of deflected phenotypes plus 25% that of resistant phenotypes; 0 . 5% initial prevalence of resistance alleles; and 25% initial prevalence of deflection alleles .",
"The assumptions used are consistent with a situation in which deflected phenotypes are deflected 75% of the time and ESR is always and only applied with insecticide , or where 75% of insecticide-treated properties are treated with ESR , and deflected phenotypes are always deflected by the ESR .",
"Comparison of this plot with the light-green elements of panel",
"( i ) of Figure 2 confirms that the establishment and maintenance of an ESR is possible when deflected+resistant phenotypes have greater fitness than deflected+susceptible phenotypes , but for a more limited range of combinations of deflected and resistant survival values . DOI: http://dx . doi . org/10 . 7554/eLife . 15416 . 007 A comparison of panels",
"( i ) to",
"( iv ) in Figure 2 shows that the fitness values for all phenotypes , and the initial prevalence of deflection and resistance alleles all affect the potential for deflection alleles to spread and be maintained in the population , consistent with the relationships shown in Expression 1 .",
"For example , with the baseline parameter values , in a context in which insecticide-resistant phenotypes have average per-cycle survival of 60% and deflected phenotypes have per-cycle survival of 45% , an ESR introduced at a time when the prevalence of resistance and deflection alleles are 0 . 5% and 25% will become established , with the deflection allele spreading and deflected phenotypes comprising more than 80% of the population 300 cycles after introduction ( panel",
"i ) , as shown by the light green square for 45% deflected survival with 60% resistant survival .",
"However , if the initial prevalence of deflection alleles is only 10% , then with the same per-cycle survival rates , deflection alleles spread but are not sustained .",
"Deflection can , however , be established by replacement of the initial insecticide whilst deflection alleles are at their maximum prevalence ( panel",
"ii ) , as shown by the dark green square for 45% deflected survival with 60% resistant survival .",
"From comparison of Figure 2 panel",
"( i ) and Figure 3 , it can be seen that if a cost of resistance affects resistant deflected phenotypes this serves to increase the range of deflection and resistance fitness combinations for which deflection alleles can spread and be maintained . 10 . 7554/eLife . 15416 . 008Figure 3 . Effect of cost of resistance on resistant and deflected per-cycle survival combinations which support the spread and maintenance of deflected phenotypes in the population . Combinations of phenotype survival values which can result in more than 80% of the population having deflected phenotypes after 300 cycles , assuming various costs of resistance .",
"Colours indicate the lowest cost of resistance ( COR ) incurred by deflected resistant phenotypes which achieves the threshold 80% deflection phenotypes in the population after 300 cycles ( without assuming any ratchet ) .",
"COR here represents the reduction in per cycle survival of deflected resistant phenotypes arising as result of having a resistant phenotype , so the resistant deflected phenotype has per-cycle survival equal to that for the deflected phenotype ( y-axis ) less the applicable COR .",
"The survival values shown for the resistant phenotype ( x-axis ) are those after taking account of any cost of resistance that affects non-deflected resistant phenotypes .",
"The baseline parameters are: 20% per cycle survival of susceptible phenotypes; 0 . 5% initial prevalence of resistance alleles; and 25% initial prevalence of deflection alleles . DOI: http://dx . doi . org/10 . 7554/eLife . 15416 . 008 Once the ESR is established , the impact of the ESR–insecticide combination treatment on the transmission of Plasmodium will critically depend on the reduced vectorial capacity arising from the exclusion of mosquitoes from treated properties .",
"This will have two components .",
"Reduced survival of deflected mosquitoes will reduce the population of adult mosquitoes , the probability of infected mosquitoes surviving to give an infectious bite , and the number of bites that an infectious mosquito will survive to give .",
"In addition , anything which reduces the probability of transmission from human host to a feeding mosquito , or from a feeding mosquito to a human host , will enhance the reduction in transmission arising from deflection away from human dwellings .",
"The level of transmission of Plasmodium to or from mosquitoes that have transitioned to outdoor feeding is not yet well-explored .",
"In calculating the levels of infectious bites that correspond to given levels of deflected mosquitoes in the population , we therefore represent all the possible sources of reduced transmission by deflected vectors as a range of possible parameter values for their probability per feed of acquiring a Plasmodium infection , assuming probabilities of 4% , 2% , 1% and 0% ( 100% , 50% , 25% or 0% of the value assumed in the absence of deflection ) .",
"From Figure 4 , it can be seen that for a context in which transmission is as efficient for deflected outdoor-feeding vectors as for vectors exposed to no intervention , the initial reduction in infectious bites achieved when using an ESR ( blue line with squares ) is comparable with , but not quite equal to , the reduction in infectious bites achievable using an insecticide alone ( pink line with crosses ) .",
"However , the reduction in bites achieved using the ESR is maintained at a high level in the long term , while resistance rapidly eliminates the effectiveness of the unpaired insecticide .",
"When assuming some reduction in transmission for deflected mosquitoes ( lines with slash , circle and triangular markers ) , this trade-off between immediate and long-term benefits is reduced , with the ESR offering an initial reduction in infectious bites very similar to that achieved initially with insecticide alone , with the benefit again maintained or improved over the long term . 10 . 7554/eLife . 15416 . 009Figure 4 . Effect of evolved spatial repellent on infectious bites from a vector population over time . The plots represent the infectious bites from the vector population per unit of time as a proportion of that with no intervention , assuming use of insecticide with and without ESR and probabilities per feed that ESR-deflected mosquitoes will become infected with Plasmodium of 4% , 2% , 1% or 0% .",
"Plots otherwise use baseline parameter values .",
"Probability per feed that non-deflected mosquito acquires Plasmodium infection is assumed to be 4% .",
"If the size of the human population is assumed to be the same for all treatments and time periods , this equates to the entomological inoculation rate ( EIR ) as a percentage of the EIR with no intervention . DOI: http://dx . doi . org/10 . 7554/eLife . 15416 . 009 Although the results summarized in Figures 1 , 2 and 4 assume that resistant deflected phenotypes have the same fitness as susceptible deflected phenotypes , consistent with a context in which deflected phenotypes never enter a property treated with insecticide , and hence never experience any fitness benefit from resistance , note that this is not a requirement for successful establishment of an ESR , as illustrated in Figure 2—figure supplement 1 ."
],
[
"The use of DDT for indoor residual spray ( IRS ) programs proved an outstanding success in the history of public health campaigns against malaria .",
"Its withdrawal in the light of environmental concerns reversed successes that approached elimination in some regions ( Roberts , 2010; Curtis , 2002; Roberts et al . , 1997 ) .",
"By the time of its withdrawal , resistance to the toxic effects of DDT was already widely observed , but there is empirical evidence to suggest that it may nonetheless have maintained efficacy as a transmission-reduction agent through its action as an effective spatial repellent ( Roberts et al . , 2000; Roberts and Alecrim , 1991 ) .",
"In part inspired by this , the deflection of malaria-vector mosquitoes from indoor feeding at night to outdoor feeding is being actively investigated as a means to reduce malaria transmission ( Achee et al . , 2012; University of Notre Dame , 2016 ) .",
"Here , we show that combining a spatial repellent which initially repels only a small proportion of a target vector population with indoor residual spraying of a high-toxicity insecticide can serve both to create a highly effective spatial repellent and to protect the companion insecticide from the rapid evolution of direct resistance to its toxic effects .",
"Our analysis provides the initial theoretical framework to spur empirical testing of this concept .",
"There is , however , already a body of empirical evidence consistent with our proposal that selection can act to increase the efficacy of a repellent paired with a toxic insecticide .",
"Our work predicts that , given heritable behavioural traits in the exposed population , toxic substances encountered with sufficient frequency and with sufficient volatility to be detectable at a distance would be observed to show repellent action against exposed populations .",
"In fact , many substances investigated because of their repellent properties are also toxic on contact with target species .",
"These include DEET , probably the most effective and well-known of the available repellents , as well as many of the naturally repellent plant-based volatiles currently in use or under investigation for personal protection ( Xue et al . , 2003; Licciardi et al . , 2006 ) .",
"In fact , experiments that have combined repellents with insecticides on bed nets have often found that the benefits arise from the additive or synergistic toxicity of the repellents ( Pennetier et al . , 2007; Faulde et al . , 2010; N'guessan et al . , 2008 ) to an equivalent or greater degree than through their localized repellent effects .",
"A comparison of the behavioural and insecticidal effects of three public-health insecticides provides more direct evidence of toxicity giving rise to repellency .",
"The most toxic of the three , carbosulfan , also demonstrated high spatial repellence in a population of susceptible mosquitoes but not in a resistant population ( Malima et al . , 2009 ) , an observation wholly consistent with our theoretical predictions .",
"The two elements defining the public-health benefits of an ESR used to deflect vectors away from human dwellings are: ( 1 ) the proportion of the mosquito population affected by the ESR; and ( 2 ) the reduction in infectious bites resulting from deflection .",
"The former is dependent on the spread of the deflection allele in the population .",
"This is determined by the relative fitness of susceptible , resistant , deflected and resistant-plus-deflected phenotypes , and by the initial prevalence of deflection and resistance alleles when the combined ESR-insecticide intervention is introduced .",
"Choices about how the ESR and its associated insecticide are selected and deployed can influence or determine these values .",
"The potential for establishing an ESR is maximized by lower fitness values for susceptibles and by higher fitness values for deflected phenotypes , indicating that control programs should target high coverage with high-efficacy insecticides to minimize the fitness of non-deflected susceptibles .",
"Critically , all treated properties should ideally be treated with both an insecticide and ESR , rather than with either alone , in order to maximize selection for deflection and to minimize selection for physiological resistance .",
"Minimising the entry of deflected phenotypes to insecticide-treated properties avoids exposing deflected phenotypes to insecticide and hence reducing the average fitness of deflected phenotypes relative to that of resistant phenotypes .",
"Minimising the use of ESR on properties without insecticide coverage , particularly during the establishment stage of an ESR , avoids generating a fitness cost for deflected mosquitoes but not for non-deflected susceptible mosquitoes which can enter and feed .",
"The use of a well-established ESR in a small number of properties for which insecticide use is for some reason impossible may , however , comprise one of ESR’s potential benefits .",
"A high initial proportion of deflection and a low proportion of resistance alleles in the treated population also supports the spread of deflection alleles and the establishment of an ESR .",
"Although these values cannot be directly controlled , desirable values may be targeted through careful choice of insecticide and ESR for each vector population .",
"The candidate ESR can be chosen to target an initial threshold level of deflection genotype in the population .",
"The initial companion insecticide can be chosen to target low initial levels of physiological resistance in the target population , and could exploit high-toxicity insecticides that would normally show a very rapid loss to resistance , serving both to maximize selection for deflected phenotypes and to give maximum immediate transmission reduction benefits .",
"An ESR needs to be partnered with a suitable IRS insecticide , which should have low contact repellence to maximize the mortality produced in non-deflected mosquitoes .",
"Existing and new chemical insecticides with low contact repellence and high toxicity , such as bendiocarb ( Evans , 1993 ) , are therefore suitable as potential partner insecticides for an ESR program , potentially transforming them from short-term solutions to sustainable tools .",
"Furthermore fungal biopesticides being developed for vector control potentially offer an ideal partnership with a spatial repellency treatment , whether DDT or a novel chemical , as they appear to have no contact repellency and have inherent resistance management benefits ( Thomas and Read , 2007; Mnyone et al . , 2010 ) .",
"These novel biopesticides have previously been proposed as ‘evolution proof’ late-life-acting insecticides ( Read et al . , 2009; Thomas and Read , 2007 ) , offering respite from the treadmill of insecticide loss to resistance .",
"For late-life-acting insecticides , relatively low-virulence fungal strains are ideal ( Lynch et al . , 2012 ) , but much work on these organisms has focussed on generating high-virulence strains ( Fang et al . , 2012 ) .",
"Given their low or absent contact repellence , such high-virulence strains would provide ideal candidates for combination with an evolved spatial repellent , provided action can be taken before resistance becomes established in the target mosquito populations .",
"Our focus is on repellence that prevents indoor-feeding malaria vectors from entering properties , rather than on more localized repellence away from bed nets once vectors have entered properties .",
"The excito-repellent properties of some of the pyrethroid insecticides currently used on long-lasting insecticide-treated nets ( LLINs ) reduce the mortality they generate by pushing vectors away before they acquire a lethal dose ( Grieco et al . , 2007; Tananchai et al . , 2012 ) .",
"Such reduced mortality would diminish their likely efficacy as partner insecticides for ESRs .",
"They could nonetheless enhance the establishment of an ESR when deployed in combination with suitable IRS , by reducing the relative fitness both of non-deflected susceptible mosquitoes and of mosquitoes resistant to the IRS insecticide .",
"Such insecticides would also benefit from the resistance protection provided by an established ESR .",
"As one of the key public-health tools currently deployed , and one already showing signs of succumbing to resistance ( N'Guessan et al . , 2007; Ochomo et al . , 2014; Chandre et al . , 1999 ) , protecting the efficacy of these compounds could be hugely beneficial .",
"Further , in the search for replacements for pyrethroids on bednets , alternative actives with low contact repellence could make excellent partner insecticides for an ESR and again benefit from protection against a rapid loss to resistance .",
"If the partner insecticide is deployed via bednets then , as for IRS , the ESR can be deployed separately , allowing it to be applied , refreshed , removed or replaced without requiring any changes to the manufacture or maintenance of the LLINs .",
"The fitness cost of deflection away from indoor human hosts will vary according to vector species and degree of anthropophilly .",
"Clearly for species that are readily zoophagous , such as Anopheles arabiensis , the fitness cost of diverting to a livestock host would be relatively low , provided that the alternative host is present and accessible .",
"For more anthropophilic species , such as Anopheles gambiae , the fitness cost of deflection must be higher , and may be expected to vary between local populations depending on the feeding alternatives adopted .",
"In assessing where ESRs offer high potential to contribute to public health campaigns , therefore , consideration should be given to the detail of the species mix in the local population , and the availability of alternative hosts .",
"Immigration into the population will slow the spread of deflection , and so the size of a treated area is also a likely determinant of success since a large enough treatment area can minimize immigration by effectively including a whole breeding population within the treatment area .",
"Equally , an isolated settlement may provide a closed vector population over a relatively small treatment area .",
"The separation of repellence and toxicity provides additional benefits .",
"The companion insecticide can be changed whilst maintaining use of an established ESR .",
"In contexts where an ESR can only be established on a transient basis , for instance because resistance alleles are already relatively common in the population when the ESR is introduced or because the population is subject to sustained immigration of non-deflected phenotypes , the ESR may still be established using a ‘ratchet’ approach in which the insecticide used in combination with the ESR can be changed when deflection phenotypes reach their peak , so that alleles for resistance to the new product will only offer a fitness benefit if paired with the relatively small proportion of non-deflection genotypes remaining in the population , allowing the deflection allele to spread .",
"Where an ESR can become well established in populations in which the fitness of resistant phenotypes is higher than that of deflected phenotypes , unless the deflection allele reaches fixation , insecticide resistance will still spread eventually and deflection will eventually disappear .",
"However , replacement of the partner insecticide at a suitable time will preserve the benefits of the established ESR , and the spread of resistance to the new partner insecticide may be wholly suppressed .",
"This might open public-health opportunities such as the short-term use of a more expensive insecticide to generate ESR protection for the long-term use of a cheaper alternative .",
"When the conditions for establishment of an ESR are met , then differential transmission for outdoor/early biting vs indoor biting is potentially very important for the outcome in terms of reducing infectious bites .",
"Because it is easiest to establish an ESR where deflection has little impact on fitness , if the only mechanism by which deflection to outdoor biting has an effect on infectious bites is through the incremental mortality associated with outdoor biting , then ESRs are most useful in the circumstances where they are hardest to establish .",
"However , many other factors may reduce the probability per feed that a vector will acquire Plasmodium , or that , once infectious , it will transmit the parasite to a human host .",
"There is empirical evidence of reduced Plasmodium infection in at least one vector population which has transferred from indoor to outdoor biting ( Ndiath et al . , 2014 ) .",
"Ndiath et al . ( Ndiath et al . , 2014 ) found no sporozooites in a population of insecticide-susceptible An .",
"gambiae pushed to outdoor feeding by the deployment of LLINs in Dielmo , whilst transmission continued at a high level in an indoor-feeding resistant population ( Ndiath et al . , 2014 ) .",
"Whilst this specific example cannot be unambiguously attributed to reduced transmission to/from outdoor feeding vectors rather than , for example , to differential mortality rates , it is reasonable to consider other potential causes of reduced outdoor transmission rates .",
"These might include , for example , increased probabilities of taking feeds from non-human hosts ( Lefèvre et al . , 2009 ) , differential availability of infectious human hosts indoors and outdoors , reduced effectiveness of transmission outside normal biting times ( Gautret and Motard , 1999; Mideo et al . , 2013; O'Donnell et al . , 2011 ) , and increased active host response to early/outdoor feeding attempts .",
"Deflection to outdoor biting could therefore have dramatic effects on transmission that are independent of direct vector mortality effects , and there is potential for easily established ESRs which are as effective as conventional insecticides in reducing transmission , but on a much more sustainable basis .",
"There is still some controversy about the desirability of deflecting mosquitoes away from indoor biting and the well-established control methods which exploit this behaviour .",
"The spatial repellent concept ( whether evolved or conventional ) is predicated on the idea that forcing vectors and Plasmodium into behavioural options that offer them lower fitness outcomes will inherently provide a new and potentially sustainable means of reducing transmission ( Achee et al . , 2012 ) .",
"However , outdoor biting is commonly viewed as a route to increased vector activity in a context where personal protection and anti-vector measures are hard to action ( Bradley et al . , 2012 ) .",
"In some contexts , for some compounds , the ESR concept may help to generate and sustain repellents that are able to protect against outdoor biting in the vicinity of treated properties , but in some locations it may nonetheless be the case that producing a move to outdoor biting simply does not generate overall transmission reductions equivalent to those achieved by control measures applied directly to indoor-feeding vectors .",
"Transmission reductions may also change over time; for example , if outdoor biting initially generates low transmission because the accessible outdoor human hosts are all adults who have partial immunity , successful protection of children would eventually generate increasing numbers of susceptible adults who will in turn be exposed to outdoor feeding vectors , eliminating this benefit .",
"It might also be argued that , whether beneficial or detrimental , outdoor biting will inevitably evolve as a behavioural resistance mechanism , and therefore that the use of an ESR is irrelevant .",
"However , we would argue that ESRs offer benefits even in contexts where a transition to outdoor biting is ultimately found to be detrimental .",
"For vectors that preferentially feed indoors , there is an expected fitness cost to a switch to outdoor feeding .",
"Selection should therefore favour the use of a ‘cue’ to determine host choice , allowing the fitness benefits of indoor feeding to be enjoyed wherever safe , whilst avoiding the costs of entering insecticide-treated properties .",
"Where ESRs are used in such contexts , either they will have no effect at all ( i . e . they will do no harm nor good ) or at least part of the vector population selected to respond to a ‘cue’ will be ‘cued’ by the ESR .",
"This means that , should outdoor feeding prove undesirable at some point , the transition to outdoor feeding will be at least partly reversible since , unlike direct responses to an insecticide or to entering buildings , the response to an ESR will cease to affect vector behaviour if the ESR is withdrawn .",
"As well as being reversible in its effects , unlike many resistance-management strategies , ESR does not require the withdrawal or reduction of existing control measures in order to be effective .",
"All novel interventions carry potential for harm , however , if they divert limited resources away from more effective control measures .",
"In order to minimize potential harm as well as maximising potential benefits therefore , candidate ESRs should be cheap to obtain , and cheap and easy to deploy in the field .",
"Unlike IRS , there is no imperative to cover interior surfaces with an ESR as they are intended to act at a distance , deployment as a single-spot event should therefore be possible .",
"If this is formatted as a simple physical item , for example a small disc of impregnated paper , it can be deployed without the need for any special equipment .",
"For ease of acceptance , candidate ESRs should have no detectable unpleasant odour for humans .",
"To minimize unintended fitness costs for deflection , candidate substances should not be common in the natural environment of the target vector population .",
"Any carrying material used for ESR distribution should be cheap , readily available , not amenable to other practical use , resistant to degradation and inedible for animals with indoor access .",
"The evolution of behavioural change in response to pesticides is also a recognized issue for agricultural insecticide use ( Castillo-Chavez et al . , 1988; Kennedy et al . , 1987; Jongsma et al . , 2010 ) and it is interesting to note that in the early 1980s Gould ( 1984 ) suggested that some insect populations might be expected to evolve behavioural avoidance of agricultural insecticides which were not originally repellent to them , and that in many situations , physiological resistance would evolve more slowly to insecticide formulations with high repellency than to non-repellent formulations .",
"Whereas use in agriculture is commonly highly detrimental to the sustained utility of public-health tools , the parallel potential for ESRs in human health and agriculture may in some contexts allow agricultural use to enhance their public health role .",
"In areas where vectors rest in insecticide-treated crops , using a vector-control spatial repellent on crops in combination with agricultural insecticides may serve to enhance selection for deflection alleles without generating any selection pressure for physiological resistance to public health insecticides ( whilst incidentally providing some resistance-management for agricultural pesticides ) .",
"This might also be a means to apply selection to nectar-feeding males , or to enhance selection for deflection in contexts where there is some existing resistance to all available public health insecticides but not to the agricultural insecticides being used locally .",
"Work carried out since the widescale use of DDT was terminated has clarified the role of spatial repellence in its contribution to malaria control ( Roberts et al . , 2000 ) .",
"Given its highly effective spatial repellence combined with high toxicity and the ubiquity of its use , it is perhaps interesting to consider whether DDT was always an effective repellent , or whether it actually provides the first empirical example of the ESR concept in action ?",
"Our models suggest that there is clear potential to use evolution to create better control through evolved spatial repellence , and we hope to stimulate empirical work to test both our assumptions and the application of this novel approach .",
"More generally , we demonstrate the importance of taking into account and modeling the evolutionary implications of different methods of insect control and medical interventions more broadly .",
"We provide an example in which the inevitable evolution of the target insects can be used to improve rather than to reduce the effectiveness of the intervention .",
"When evaluating a new intervention detailed mathematical modeling of the evolutionary outcomes alongside the more commonly considered epidemiological outcomes has considerable potential to improve infectious disease control .",
"We recommend bringing together theoretical and empirical work to explore fully the potential of the ESR concept ."
]
] | [
"Evolution persistently undermines vector control programs through insecticide resistance .",
"Here we propose a novel strategy which instead exploits evolution to generate and sustain new control tools .",
"Effective spatial repellents are needed to keep vectors out of houses .",
"Our approach generates such new repellents by combining a high-toxicity insecticide with a candidate repellent initially effective against only part of the vector population .",
"By killing mosquitoes that enter treated properties the insecticide selects for vector phenotypes deflected by the repellent , increasing efficacy of the repellent against the target vector population and in turn protecting the insecticide against the spread of insecticide resistance .",
"Using such evolved spatial repellents offers an evolutionarily sustainable , ‘double-dip’ system of disease control combining mortality and repellence .",
"We formalize this idea using models which explore vector population genetics and disease transmission probabilities and show that using evolved spatial repellents is theoretically achievable , effective and sustainable ."
] | [
"Many of the mosquito species that transmit malaria have evolved to bite humans indoors at night , and therefore health programs target them using insecticides sprayed on surfaces inside people’s homes .",
"This strategy , however , stops working when mosquito populations evolve to resist the insecticide used , either because they are immune to its poisonous effects or because they change their behaviour to avoid it .",
"Consequently , there is now a need to develop alternative strategies to control mosquitoes that are more sustainable in the face of evolution .",
"One possibility is repellents that keep mosquitoes out of homes .",
"Lynch and Boots have now asked whether evolution could be used to create effective repellents from substances that initially repel only part of the mosquito population by pairing them with lethal insecticides sprayed inside people’s homes .",
"Mathematical models showed that , before insecticide resistance becomes widespread , this “evolved repellence” approach could reduce the spread of malaria by a similar amount to using insecticides alone .",
"This was particularly true if the models considered that , as well as surviving to give fewer infectious bites , repelled infectious mosquitoes may be less likely to transmit malaria with each feed , for example if they feed more on livestock rather than humans .",
"The models of Lynch and Boots also show that that the success of the evolved repellence concept in a given location depends on a number of factors .",
"The proportion of the starting mosquito population that is repelled or resistant can have a large effect .",
"Similarly , success will also depend on how likely normal , repelled and insecticide-resistant mosquitoes are to reproduce successfully .",
"These values can be influenced by the choice of insecticide and repellent and how the chemicals are applied .",
"Lynch and Boots show that swapping insecticides can allow an evolved repellent to be established where it would otherwise not succeed .",
"Also , the spread of resistance to the paired insecticide is slowed or prevented when the mosquito population evolves to be repelled .",
"Practical laboratory and field- work is now needed to build on this theoretical groundwork and to determine suitable locations and application strategies to exploit this concept as a way to sustainably reduce the spread of malaria in the future ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"cell biology",
"tools and resources"
] | A transcriptomics resource reveals a transcriptional transition during ordered sarcomere morphogenesis in flight muscle | elife-34058-v2 | [
[
"Sarcomeres are the stereotyped force producing mini-machines present in all striated muscles of bilaterians .",
"They are built of three filament types arrayed in a pseudo-crystalline order: actin filaments are cross-linked with their plus ends at the sarcomeric Z-disc and face with their minus ends towards the sarcomere center .",
"In the center , symmetric bipolar muscle myosin filaments , anchored at the M-line , can interact with the actin filaments .",
"Myosin movement towards the actin plus ends thus produces force during sarcomere shortening .",
"Both filament types are permanently linked by a third filament type , the connecting filaments , formed of titin molecules ( Gautel and Djinović-Carugo , 2016; Lange et al . , 2006 ) .",
"A remarkable feature of sarcomeres is their stereotyped size , ranging from 3 . 0 to 3 . 4 µm in relaxed human skeletal muscle fibers ( Ehler and Gautel , 2008; Llewellyn et al . , 2008; Regev et al . , 2011 ) .",
"Even more remarkable , the length of each bipolar myosin filament is 1 . 6 µm in all mature sarcomeres of vertebrate muscles , requiring about 300 myosin hexamers to assemble per filament ( Gokhin and Fowler , 2013; Tskhovrebova and Trinick , 2003 ) .",
"Human muscle fibers can be several centimetres in length and both ends of each fiber need to be stably connected to tendons to achieve body movements .",
"As sarcomeres are only a few micrometres in length , many hundreds need to assemble into long linear myofibrils that span from one muscle end to the other and thus enable force transmission from the sarcomeric series to the skeleton ( Lemke and Schnorrer , 2017 ) .",
"Thus far , we have a very limited understanding of how sarcomeres initially assemble into long immature myofibrils during muscle development to exactly match the length of the mature muscle fiber ( Sparrow and Schöck , 2009 ) .",
"In particular , we would like to understand how such sarcomeres mature to the very precise stereotyped machines present in mature muscle fibers .",
"Across evolution , both the pseudo-crystalline regularity of sarcomeres as well as their molecular components are well conserved ( Ehler and Gautel , 2008; Vigoreaux , 2006 ) .",
"Thus , Drosophila is a valid model to investigate the biogenesis of sarcomeres as well as their maturation .",
"In particular , the large indirect flight muscles ( IFMs ) that span the entire fly thorax are an ideal model system to investigate mechanisms of myofibrillogenesis .",
"They contain thousands of myofibrils consisting of 3 . 2 µm long sarcomeres ( Schönbauer et al . , 2011; Spletter et al . , 2015 ) .",
"Like all Drosophila adult muscles , IFMs are formed during pupal development from a pool of undifferentiated myoblasts called adult muscle precursors ( AMPs ) ( Bate et al . , 1991 ) .",
"From 8 hr after puparium formation ( APF ) , these AMPs either fuse with themselves ( for the dorso-ventral flight muscles , DVMs ) or with remodelled larval template muscles ( for the dorso-longitudinal flight muscles , DLMs ) to form myotubes ( Dutta et al . , 2004; Fernandes et al . , 1991 ) .",
"These myotubes develop dynamic leading edges at both ends and initiate attachment to their respective tendon cells at 12 to 16 hr APF ( Weitkunat et al . , 2014 ) .",
"These attachments mature and mechanical tension is built up in the myotubes , followed by the formation of the first immature periodic myofibrils at 30 hr APF when the muscle fibers are about 150 µm in length .",
"These immature myofibrils contain the earliest sarcomeres , which are about 1 . 8 µm in length ( Weitkunat et al . , 2014 ) .",
"During the remaining 3 days of pupal development , the muscle fibers grow to about 1 mm to fill the entire thorax and sarcomere length increases to a final length of about 3 . 2 µm in adult flies ( Orfanos et al . , 2015; Reedy and Beall , 1993 ) .",
"After myoblasts have fused to myotubes , the flight muscle specific selector gene spalt major ( spalt , salm ) is turned on in the developing flight muscle myotubes .",
"Spalt major is responsible for the correct fate determination and development of the flight muscles , which includes the fibrillar flight muscle morphology and the stretch-activated muscle contraction mode ( Schönbauer et al . , 2011; Syme and Josephson , 2002 ) .",
"It does so by controlling the expression of more than 700 flight muscle specific genes or gene isoforms during development ( Spletter and Schnorrer , 2014; Spletter et al . , 2015 ) .",
"However , how the interplay between all these isoforms instructs the formation of highly regular , pseudo-crystalline sarcomeres in the flight muscle is not understood .",
"Here , we studied the transcriptional dynamics of flight muscle development in detail .",
"We performed a systematic mRNA-Seq time-course of isolated muscle tissue at eight time points from the myoblast stage until the mature adult muscle stage , generating a transcriptomics resource of developing flight muscle .",
"Bioinformatic analysis of expression dynamics identified two gene clusters that are strongly enriched for sarcomeric genes .",
"The temporal dynamics of these clusters identified a transcriptional transition that is required for sarcomere morphogenesis .",
"We define sarcomere morphogenesis in three sequential although overlapping phases .",
"First , immature myofibrils assemble simultaneously; second , short sarcomeres are added to each myofibril; and third , all sarcomeres mature to their final length and diameter and acquire stretch-sensitivity .",
"Interestingly , the number of myofibrils remains constant , suggesting that every sarcomere progresses through sarcomere maturation .",
"We show that the flight muscle selector gene spalt major contributes to the observed transcriptional transition , suggesting that muscle fiber type-specific transcription is continuously required during sarcomere formation and maturation .",
"Together , these findings indicate that precise transcriptional control of the sarcomeric components enables their ordered assembly into sarcomeres and their maturation to pseudo-crystalline regularity ."
],
[
"To better understand muscle morphogenesis in general and myofibrillogenesis in particular , we focused on the Drosophila indirect flight muscles ( IFMs ) .",
"We hypothesised that major morphological transitions during IFM development may be induced by transcriptional changes , thus we aimed to generate a detailed developmental mRNA-Seq dataset from IFMs .",
"IFMs are built from AMPs that adhere to the hinge region of the wing disc epithelium and are labelled with GFP-Gma under Him control ( Figure 1A ) ( Soler and Taylor , 2009 ) .",
"At 16 hr APF , many of these myoblasts have fused to larval template muscles to build the dorsal-longitudinal flight muscle ( DLM ) myotubes , which initiate attachment to their tendons .",
"At this stage , the DLM myotubes of fibers 3 and 4 have a length of about 300 µm ( Figure 1B ) .",
"Fusion ceases at about 24 hr APF ( Figure 1C ) and attachment matures until 32 hr APF , coinciding with the strong recruitment of βPS-Integrin and the spectraplakin homolog Shortstop ( Shot ) to the attachment sites .",
"At this stage the myofibers have built up mechanical tension and compacted to a length of about 150 µm , coinciding with the appearance of long Shot-positive tendon extensions that anchor the muscles within the thorax .",
"This important developmental transition is highlighted by the assembly of immature myofibrils visualised by strong F-actin staining throughout the entire muscle fiber ( Figure 1D ) ( Weitkunat et al . , 2014 ) .",
"After 32 hr APF , the myofibers undergo another developmental transition and begin to grow dramatically .",
"They elongate 3-fold to reach a length of about 480 µm by 48 hr APF ( Figure 1E ) and about 590 µm by 56 hr APF ( Figure 1F ) .",
"Concomitantly , the tendon extensions shrink with the myofibers being directly connected to the basal side of the tendon cell epithelium by 72 hr APF ( Figure 1G ) .",
"At the end of pupal development ( 90 hr APF ) , wavy muscle fibers with a length of about 780 µm containing mature myofibrils ( Figure 1H ) are present within the thorax .",
"To quantify transcriptional dynamics across the entire developmental time-course , we focused on the major developmental transitions and isolated mRNA from dissociated myoblasts of dissected or mass-isolated third instar wing discs and from hand-dissected IFMs at 16 , 24 , 30 , 48 , 72 and 90 hr APF pupae , and adult flies 1 day after eclosion ( Figure 1I ) .",
"We performed mRNA-Seq using at least two biological replicates for each time point ( see Materials and methods ) .",
"To identify genes with similar temporal expression profiles , we used Mfuzz ( Kumar and Futschik , 2007 ) to cluster standard normalized read counts from all genes expressed above background ( 12 , 495 of 13 , 322 genes ) .",
"This allowed us to confidently identify 40 distinct genome-wide clusters ( Figure 2—figure supplement 1 ) , each of which contains a unique gene set ranging from 155 to 703 members ( Supplementary file 1 ) .",
"These clusters represent various temporal expression dynamics , with high expression at early ( myoblast proliferation and fusion ) , mid ( myotube attachment and myofibril assembly ) or late ( myofiber growth ) myogenesis stages or a combination thereof ( Figure 1I , Figure 2—figure supplement 1 ) .",
"These distinct patterns suggest a precise temporal transcriptional regulation corresponding to observed morphological transition points .",
"To verify the mRNA-Seq and cluster analysis , we selected a number of ‘indicator’ genes with available antibodies or GFP fusion proteins whose expression correlates with important developmental transitions .",
"Twist ( Twi ) is a myoblast nuclear marker at larval stages and its expression needs to be down-regulated after myoblast fusion in pupae ( Anant et al . , 1998 ) .",
"We find twi mRNA in Mfuzz cluster 27 , with high expression in myoblasts until 16 hr APF and a significant down-regulation from 24 hr APF , which we were able to verify with antibody stainings ( Figure 2A–C ) .",
"The flight muscle fate selector gene spalt major ( salm ) ( Schönbauer et al . , 2011 ) and its target , the IFM splicing regulator arrest ( aret , bruno ) ( Spletter et al . , 2015 ) are members of cluster 26 and 14 , respectively .",
"Expression of both clusters is up-regulated after myoblast fusion at 16 hr APF , which we were able to verify with antibody stainings ( Figure 2D–F , Figure 2—figure supplement 2A–C ) .",
"The apparent salm mRNA peak at 72 hr APF , which does not appear to cause a further protein increase , represents a mere 1 . 3 fold increase in expression and would need further confirmation as Salm , like many transcription factors , is expressed at low levels .",
"For the initiation of muscle attachment , we selected Kon-tiki ( Kon ) ( Schnorrer et al . , 2007; Weitkunat et al . , 2014 ) , member of cluster 15 , which is transiently up-regulated after myoblast fusion , before it is down-regulated again after 30 hr APF .",
"Consistently , we found Kon-GFP present at muscle attachment sites at 30 hr APF but not at 72 hr APF ( Figure 2G–I ) .",
"A similar expression peak shifted to slightly later time points is found in cluster 34 , which contains β-tubulin 60D ( βTub60D ) ( Leiss et al . , 1988 ) .",
"Consistently , we find β-Tub60D-GFP ( Sarov et al . , 2016 ) expression in IFMs at 30 and 48 hr but not 72 hr APF ( Figure 2J–L ) .",
"After attachment is initiated , the attachments need to mature and be maintained .",
"As expected , we found the essential attachment components βPS-Integrin ( mys ) and Talin ( rhea ) in clusters that are up-regulated after myoblast fusion until adulthood ( clusters 7 and 25 , respectively ) .",
"This is consistent with continuous high protein expression of βPS-Integrin-GFP and Talin-GFP at muscle attachment sites ( Figure 2M–O , Figure 2—figure supplement 2D–F ) .",
"Taken together , these semi-quantitative protein localisation data nicely validate the temporal mRNA dynamics found in the mRNA-Seq data , confirming our methodology .",
"Hierarchical clustering of the core expression profiles from the 40 identified Mfuzz clusters defines eight temporally ordered groups ( Figure",
"3 ) that show progressive expression dynamics as muscle development proceeds .",
"A time-dependent shift in gene ontology ( GO ) term enrichments is apparent between the eight groups , reflecting the different stages of IFM development .",
"GO-Elite analysis ( Zambon et al . , 2012 ) for gene set enrichment identified GO terms related to cell proliferation and development as enriched in the early clusters ( such as the twi cluster 27 or the kon cluster 15 ) , whereas terms related to actin filament dynamics are more enriched in the middle clusters ( such as the βPS-Integrin cluster seven and the Talin cluster 25 ) , reassuring that the clustering approach is valid ( Figure 3 , Supplementary file 2 ) .",
"Strikingly , the only two clusters that display a strong enrichment for genes important for sarcomere organisation are clusters 13 and 22 , both of which are late up-regulated clusters ( Figure 3 ) .",
"Members of both clusters just become detectable at 30 hr APF ( Unc-89/Obscurin-GFP , Act88F-GFP , Mhc-GFP ) or even later at 48 hr APF ( Strn-Mlck-GFP , Mf-GFP ) .",
"In all cases , we could confirm the strong up-regulation from 30 hr to 72 hr APF in the mRNA-Seq data at the protein level using GFP fusion proteins under endogenous control ( Figure 2P–U , Figure 2—figure supplement 2J–P ) ( Sarov et al . , 2016 ) .",
"We additionally verified the late up-regulation of Flightin ( Fln ) , a member of cluster 3 , which is detectable at 72 hr but not at 30 hr APF ( Figure 2—figure supplement 2G–I ) .",
"At late stages of flight muscle development , mitochondrial density strongly increases ( Clark et al . , 2006 ) .",
"Using GO-Elite , we found a strong enrichment for mitochondrial related pathways in four late up-regulated clusters , namely 3 , 28 , 39 as well as the sarcomere cluster 22 ( Figure 3 ) .",
"By comparing the clusters to systematic functional data acquired at all stages of Drosophila muscle development ( Schnorrer et al . , 2010 ) , we find enrichments in clusters throughout the time-course .",
"Interestingly , genes highly expressed in flight muscle compared to other muscle types , identified as ‘salm-core genes’ ( Spletter et al . , 2015 ) , are also enriched in the late clusters , including the mitochondrial enriched clusters 3 , 28 , 39 and the sarcomere enriched cluster 22 ( Figure 3 ) .",
"These data highlight the changes in biological process enrichments that parallel expression dynamics , with a particular transition happening during later stages of muscle development after 30 hr APF .",
"This corresponds to a time period after immature myofibrils have been assembled , which thus far remained largely unexplored .",
"To examine the temporal expression dynamics in more detail , we performed a principle component analysis ( PCA ) of the mRNA-Seq time points and Mfuzz clusters and found that the major variance is developmental time , with a notable change after 30 hr APF ( Figure 4A , Figure 4—figure supplement 1A ) .",
"There are a large number of genes being up-regulated as well as down-regulated between 30 and 48 hr and between 48 and 72 hr APF , with major differences between the sets of genes expressed at early ( 16–30 hr APF ) versus late ( 72–90 hr APF ) stages of development ( Figure 4B , Figure 4—figure supplement 1B ) .",
"Thus , we focused our attention on the transcriptional transition between 30 and 72 hr APF , which correlates with major growth of the flight muscle fibers ( Figure 1 ) .",
"A large number of genes are significantly up- or down-regulated from 30 hr to 72 hr APF , as visualized on a volcano plot displaying log2fold changes ( FC ) ( Figure 4C ) , suggesting a major change in gene expression .",
"In particular , many sarcomeric genes are strongly up-regulated .",
"To identify fine details in expression dynamics , we took all genes significantly up-regulated between 30 and 72 hr APF and performed hierarchical clustering of their DESeq2 normalized counts values ( Figure 4D ) .",
"We noted that many genes are turned on from 30 hr to 72 hr APF , whereas others are already expressed at 30 hr and strongly increase their expression until 72 hr ( Figure 4D ) , suggesting a transcriptional transition after 30 hr APF .",
"Consistently , we found GO-terms of cell proliferation , cell cycle and Notch signalling down-regulated , whereas actin cytoskeleton , sarcomere , muscle function and mitochondrial related gene sets are strongly up-regulated from 30 hr to 72 hr APF ( Figure 4E ) .",
"Finally , the genes up-regulated from 30 hr to 72 hr APF are enriched for sarcomeric proteins and the ‘salm core genes’ ( Figure 4—figure supplement 1C , D ) , and the Mfuzz gene clusters with the most up-regulated members are the mitochondrial and both sarcomeric gene containing clusters 13 and 22 ( Figure 4F ) .",
"Together , these data suggest that in particular expression of the sarcomeric and mitochondrial genes is strongly induced after 30 hr APF .",
"The strong up-regulation of sarcomeric gene expression after immature myofibrils have been assembled ( Figure",
"1 ) ( Weitkunat et al . , 2014 ) caught our interest and prompted us to more closely investigate the later stages of myofibrillogenesis during which myofibers grow dramatically ( Figure 1 ) .",
"We stained the myofibers with phalloidin to reveal myofibril morphology and with the titin isoform Kettin ( an isoform of the sallimus gene ) to label the developing Z-discs and systematically quantified sarcomere length and myofibril width ( see Materials and methods ) ( Figure 5A , Supplementary file 3 ) .",
"By measuring the total muscle fiber length , we calculated the total number of sarcomeres per myofibril at a given stage .",
"We found that the sarcomere length and width remain relatively constant at about 2 . 0 and 0 . 5 µm , respectively until 48 hr APF ( Figure 5B , C , Supplementary file 3 ) .",
"However , the number of sarcomeres per myofibril dramatically increases from about 100 at 34 hr to about 230 at 48 hr APF .",
"After 48 hr only a few more sarcomeres are added , resulting in about 270 sarcomeres per myofibril at 60 hr APF .",
"This number remains constant until the fly ecloses ( Figure 5D , Supplementary file 3 ) .",
"Moreover , by analysing fiber cross-sections we found that the growth of the individual myofibril diameter correlates with growth of the entire muscle fiber .",
"Both fiber diameter and myofibril diameter remain constant from 30 hr to 48 hr APF .",
"After 48 hr the myofibril diameter grows nearly 3-fold from 0 . 46 µm to 1 . 43 µm in adult flies ( Figure 5E–G ) , while fiber cross-sectional area grows nearly 4-fold from 1 , 759 µm2 to 6 , 970 µm2 .",
"Strikingly , during the entire time period from 30 hr APF to adults , the total number of myofibrils per muscle fiber remains constant ( about 2000 per muscle fiber , Figure 5H ) .",
"Taken together , these quantitative data lead us to propose ordered but somewhat overlapping phases of sarcomere morphogenesis: ( 1 ) During the sarcomere assembly phase , about 100 immature sarcomeres self-assemble within each immature myofibril .",
"( 2 ) Many short sarcomeres are added to each myofibril increasing its length .",
"( 3 ) The short sarcomeres grow in length and thickness to reach the mature pseudo-crystalline pattern .",
"No new myofibrils are built after the initial myofibril assembly phase .",
"We gained additional evidence to support this ordered myofibrillogenesis model on both the molecular and functional levels .",
"First , the initial assembly versus the later sarcomere maturation complement the transition in gene expression we observe from 30 hr to 72 hr APF .",
"Using members of the late induced Mfuzz clusters that contain sarcomeric components , we found that indeed a subset of sarcomeric proteins , such as Unc-89/Obscurin and Mhc , are already detectable in a periodic pattern on immature myofibrils at 30 hr .",
"By contrast , other important components , including Fln , Myofilin ( Mf ) and Strn-Mlck , are only incorporated into myofibrils from 48 hr APF , showing high levels by 72 hr APF ( Figure 5—figure supplement 1 ) .",
"Second , to investigate muscle function , we used Talin-YPet as a muscle attachment marker and quantified the number of spontaneous muscle contractions in intact pupae ( see Materials and methods ) .",
"Interestingly , we found that immature myofibrils already start to spontaneously contract at 30 hr APF .",
"These spontaneous contractions increase in strength and frequency until 48 hr APF , but then cease , producing no detectable spontaneous contractions at 72 hr APF ( Figure 5I , J , Figure 5—video 1 ) .",
"This demonstrates that during the sarcomere assembly phase , immature contractile myofibrils are generated , which then likely acquire stretch-sensitivity as the immature myofibrils mature and thus cease contracting .",
"How does the transcriptional transition of the various sarcomeric components instruct myofibrillogenesis ?",
"As the identified sarcomeric clusters 13 and 22 are enriched for ‘salm-core genes’ ( Spletter et al . , 2015 ) ( Figure 3 ) , we chose to investigate gene expression in developing spalt-major knock-down ( salmIR ) flight muscles compared to wild type ( Supplementary file 4 ) .",
"salmIR IFM shows a strong down-regulation in gene expression , notably of mRNAs coding for sarcomeric and mitochondrial components at 24 and 30 hr APF , and in particular at 72 hr APF ( Figure 6A–C , Figure 6—figure supplement 1A–D ) .",
"The genes down-regulated in salmIR IFM are enriched for GO terms associated with sarcomere assembly , flight behaviour and mitochondrial genes , as well as for the mitochondrial Mfuzz clusters 3 , 28 , 39 and the sarcomeric Mfuzz clusters 13 and 22 ( Figure 6—figure supplement 1E–G ) .",
"Interestingly , members of cluster 22 , which is strongly enriched for sarcomeric and mitochondrial genes , are not only down-regulated at 72 hr APF in salmIR ( Figure 6B ) but are also less strongly induced from 30 hr to 72 hr APF in salmIR muscle compared to wild type ( Figure 6D ) , suggesting that salm in addition to other factors is indeed required for the strong induction of sarcomeric protein expression after 30 hr APF .",
"Salm is expressed shortly after myoblast fusion and constitutive knock-down of salm with Mef2-GAL4 results in a major shift of muscle fiber fate ( Schönbauer et al . , 2011 ) , which may indirectly influence transcription after 30 hr APF .",
"Hence , we aimed to reduce Salm levels only later in development , to directly address its role during sarcomere maturation .",
"To this end , we knocked-down salm with the flight muscle specific driver Act88F-GAL4 , which is expressed from about 18 hr APF and requires salm activity for its expression ( Bryantsev et al . , 2012; Spletter et al . , 2015 ) .",
"This strategy enabled us to reduce Salm protein levels at 24 hr APF resulting in undetectable Salm levels at 72 hr APF ( Figure 6—figure supplement 2 ) .",
"To test if Salm contributes to the transcriptional boost of sarcomeric components after 30 hr APF , we performed quantitative imaging using unfixed living flight muscles expressing GFP fusion proteins under endogenous control .",
"We used green fluorescent beads to normalise the GFP intensity between different samples , and could verify the induction of Mhc , Unc-89 , Fln and Strn-Mlck on the protein level from 30 hr to 72 hr APF ( Figure 6—figure supplement 3 ) .",
"While overall sarcomere morphology is not strongly affected in Act88F >> salmIR muscles , we found that the levels of Strn-Mlck , Fln and Mhc proteins are strongly reduced at 90 hr as compared to wild-type controls ( Figure 6D–H ) .",
"This suggests that salm indeed contributes to the transcriptional transition that boosts the expression of a number of sarcomeric proteins after 30 hr APF .",
"To investigate the consequences of late salm knock-down , we quantified the myofibril and sarcomere morphology from 48 hr onwards .",
"The myofibrils display a fibrillar morphology , confirming that the early function of Salm to determine IFM fate was unaffected by our late knock-down .",
"At 72 hr APF and more prominently at 90 hr APF and in adults , Act88F >> salmIR myofibrils showed actin accumulations at broadened Z-discs ( Figure 7A–H ) , which are often a landmark of nemaline myopathies ( Sevdali et al . , 2013; Wallgren-Pettersson et al . , 2011 ) .",
"The myofibril width was not significantly different in these myofibrils ( Figure 7I ) .",
"However , the sarcomeres of Act88F >> salmIR muscles displayed a strong defect in sarcomere length growth after 48 hr APF ( Figure 7J , Supplemental File 3 ) , with sarcomeres only obtaining a length of 2 . 8 µm in adult flies , demonstrating that Salm in addition to other factors is required for normal sarcomere maturation .",
"Given the defects in sarcomere length and sarcomere gene expression in Act88F >> salmIR muscles , we explored the function of these abnormal muscle fibers .",
"As expected , Act88F >> salmIR flies are flightless ( Figure 7—figure supplement 1A ) and we observed rupturing of the adult muscle fibers within 1d after eclosion ( Figure 7—figure supplement 1B–G ) , demonstrating the importance of proper sarcomere maturation to prevent muscle atrophy .",
"Based on our finding that spontaneous flight muscle contractions stop by 72 hr APF , we hypothesized that if Salm truly regulates sarcomere maturation , we may see spontaneous contraction defects during development .",
"At 48 hr APF , Act88F >> salmIR fibers twitch , but less often than and without the double twitches observed in control fibers ( Figure 7K , L , O; Figure 7—video 1 ) .",
"Strikingly , at 72 hr APF salmIR fibers fail to stop contracting and moreover show frequent and uncoordinated spontaneous contractions in which different myofibril bundles of the same fiber twitch at different times ( Figure 7M–O , Figure 7—video 2 ) , demonstrating that sarcomere maturation is indeed disrupted , with the likely consequence that myofibrils fail to acquire normal stretch-activation sensitivity .",
"To directly test the function of a sarcomeric component during the sarcomere maturation phase , we investigated the role of the prominently induced Salm target Strn-Mlck .",
"Strn-Mlck is only expressed after 30 hr APF and is largely incorporated during sarcomere maturation ( Figure 5—figure supplement 1E , Figure 6—figure supplement 3E ) , and thus is also a bone-fide example of a gene regulated during the transcriptional transition .",
"In Strn-Mlck mutants , sarcomere and myofibril morphology , including myofibril width , is initially normal .",
"However , at 80 hr APF the sarcomeres overgrow , consistently reaching lengths of more than 3 . 5 µm and resulting in slightly longer muscle fibers at 80 hr APF ( Figure 8 ) .",
"After overgrowing , sarcomeres appear to hyper-contract resulting in short , thick sarcomeres in 1-day-old adults ( Figure 8E , J , K , L ) .",
"Like Act88F >> salmIR flies , Strn-Mlck mutant adults are flightless ( Figure 7—figure supplement 1A ) and display ruptured fibers during the first days of life ( Figure 7—figure supplement 1K–M ) ( Spletter et al . , 2015 ) .",
"Together , these data demonstrate that sarcomere maturation must be precisely controlled at the transcriptional level to enable the precise growth of sarcomeres to their final mature size .",
"This ensures the lifelong function of the contractile apparatus of muscle fibers ."
],
[
"In this study , we generated a systematic developmental transcriptomics resource from Drosophila flight muscle .",
"The resource quantifies the transcriptional dynamics across all the major stages of muscle development over five days , starting with stem cell-like myoblasts and attaching myotubes to fully differentiated , stretch-activatable muscle fibers .",
"We have specifically focused on the transcriptional regulation of sarcomere and myofibril morphogenesis; however , the data we provide cover all other expected dynamics , such as mitochondrial biogenesis , T-tubule morphogenesis , neuromuscular junction formation , tracheal invagination , etc .",
"Thus , together with the available systematic functional data of Drosophila muscle development ( Schnorrer et al . , 2010 ) , our data should be a versatile resource for the muscle community .",
"It nicely complements existing systematic data from vertebrate muscle , which thus far are largely restricted to postnatal stages ( Brinegar et al . , 2017; Drexler et al . , 2012; Lang et al . , 2017; Zheng et al . , 2009 ) .",
"Furthermore , Drosophila flight muscle contains a single muscle fiber type , in contrast to the mixed fiber types found in mammals ( Schiaffino and Reggiani , 2011; Spletter and Schnorrer , 2014 ) .",
"Hence , in this model the transcriptional dynamics of a single fiber type muscle can be followed with unprecedented precision .",
"Earlier work has shown that the flight muscle myotubes first attach to tendon cells and then build-up mechanical tension .",
"This tension triggers the simultaneous assembly of immature myofibrils , converting the myotube to an early myofiber ( Weitkunat et al . , 2014 ) .",
"This suggested a tension-driven self-organisation mechanism of myofibrillogenesis ( Lemke and Schnorrer , 2017 ) .",
"Here we discovered that myofibrillogenesis is not only regulated mechanically , but to a large extent also transcriptionally .",
"This enabled us to extend our model for ordered myofibrillogenesis also to later developmental stages and to define three sequential although somewhat overlapping phases ( Figure 9 ) .",
"During the sarcomere self-assembly phase at about 30 hr APF , a large number of genes coding for sarcomeric proteins , including Mhc , Act88F and Unc-89/Obscurin , become up-regulated to enable the self-organization of short , immature sarcomeres within thin , immature myofibrils .",
"Strikingly , all of the about 2000 myofibrils assemble during this phase .",
"This is followed by a sarcomere addition phase during which a transcriptional transition is initiated and the expression of the sarcomeric proteins increases .",
"Concomitantly , the muscle fibers grow in length by addition of new sarcomeres to all the immature myofibrils , increasing the sarcomere number from about 80 to 230 per fibril at 48 hr APF .",
"These sarcomeres are contractile , but remain short and thin ( Figure 9 ) .",
"After the transcriptional transition , myofibrillogenesis enters the final sarcomere maturation phase .",
"Proteins present in immature myofibrils like Mhc , Act88F and Unc-89/Obscurin are expressed to even higher levels , and additional , often flight-muscle specific proteins like Mf , Fln and the titin-related isoform Strn-Mlck , begin to be expressed at high levels and are incorporated into the maturing sarcomeres .",
"This facilitates a dramatic growth of all immature sarcomeres in length and particularly in diameter with all 2000 myofibrils reaching a pseudo-crystalline regularity within about two days of development ( Figure 9 ) .",
"Importantly , these matured sarcomeres no longer contract spontaneously , likely because they acquired the stretch-activated mechanism of contraction described for mature Drosophila flight muscles ( Bullard and Pastore , 2011; Josephson , 2006 ) .",
"Our ordered sarcomere morphogenesis model is strongly supported by the observation that the number of myofibrils remains largely constant during the entire sarcomere morphogenesis period , suggesting that in flight muscles no new myofibrils are added after the initial assembly of immature myofibrils at about 30 hr APF .",
"As all sarcomeres are present shortly after 48 hr APF , they all need to mature simultaneously to achieve their final pseudo-crystalline regularity .",
"Our model is further supported by previous studies .",
"We and others found that immature myofibrils have a width of about 0 . 5 µm ( Weitkunat et al . , 2014 ) , which corresponds to about four thick filaments across each myofibril at the EM level at 42 hr APF ( at 22°C ) ( Reedy and Beall , 1993 ) .",
"This ‘core’ myofibril structure built until 48 hr APF , when most sarcomeres have formed , is expanded dramatically after 48 hr APF , reaching a mature width of 1 . 5 µm , corresponding to about 35 thick filaments across each myofibril at the EM-level ( Reedy and Beall , 1993 ) .",
"Recent data showed that the ‘core’ myofibril structure built until 48 hr APF contains already highly ordered actin filaments , which gain even higher order to reach their pseudo-crystalline regularity at 90 hr APF ( Loison et al . , 2018 ) .",
"In total , each adult myofibril contains around 800 thick filaments per sarcomere ( Gajewski and Schulz , 2010 ) .",
"The ‘core’ myofibril structure was also revealed by the preferential recruitment of over-expressed actin isoforms ( Röper et al . , 2005 ) and more importantly , by selective incorporation of a particular Mhc isoform that is only expressed at mid-stages of flight muscle development ( Orfanos and Sparrow , 2013 ) .",
"This Mhc isoform expression switch coincides with the global transition in sarcomeric gene expression between the sarcomere assembly and the sarcomere maturation phases that we defined here .",
"It also fits with the recent discovery that the formin family member Fhos is selectively required for actin filament elongation and recruitment of new actin filaments and thus myofibril diameter growth after 48 hr APF ( Shwartz et al . , 2016 ) .",
"Expression of Fhos is also induced after 30 hr APF .",
"Fhos is a member of Mfuzz Cluster 28 , another strongly induced cluster , underscoring the general relevance of the transcriptional transition for ordered sarcomerogenesis .",
"Mature indirect flight muscles employ a stretch-activated mechanism of muscle contraction , thus Ca2+ is not sufficient to trigger muscle contractions without additional mechanical stretch ( Bullard and Pastore , 2011; Josephson , 2006 ) .",
"This is different to cross-striated body muscles of flies or mammals that contract synchronously with Ca2+ influx .",
"Hence , it is intriguing that immature flight muscle myofibrils do in fact contract spontaneously , with the contraction frequencies and intensities increasing until 48 hr APF .",
"It was recently proposed in Drosophila cross-striated abdominal muscles and in developing cross-striated zebrafish muscles that spontaneous contractions are important for the proper formation of the cross-striated pattern ( Mazelet et al . , 2016; Weitkunat et al . , 2017 ) .",
"A similar role for contractions was found in C2C12 cells by stimulating the contractions optogenetically ( Asano et al . , 2015 ) .",
"This shows that spontaneous contractions are a necessary general feature for the assembly of cross-striated muscle fibers across species .",
"However , flight muscles are not cross-striated in the classical sense , but have a fibrillar organisation in which each myofibril remains isolated and is not aligned with its neighbouring myofibrils ( Figure 5 ) ( Josephson , 2006; Schönbauer et al . , 2011 ) .",
"We can only speculate about the mechanism that prevents alignment of the myofibrils in the flight muscles , but it is likely related to their stretch-activated contraction mechanism .",
"This mechanism prevents spontaneous twitching due to increased Ca2+ levels , because it additionally requires mechanical activation that can only occur during flight in the adult .",
"Thus , flight muscle sarcomeres not only grow and mature their sarcomere structure , they also gain their stretch-activatability .",
"We identified an important transition in gene expression between the early sarcomere assembly and the late sarcomere maturation phases .",
"Similar large scale transcriptome changes have also been observed during postnatal stages of mouse ( Brinegar et al . , 2017 ) , chicken ( Zheng et al . , 2009 ) and pig ( Zhao et al . , 2015 ) skeletal muscle development or during regeneration after injury in fish ( Montfort et al . , 2016 ) and mouse muscles ( Warren et al . , 2007 ) , indicating that muscle maturation generally correlates with large scale transcriptional changes .",
"It is well established that general myogenic transcription factors , in particular Mef2 , are continuously required in muscles for their normal differentiation ( Sandmann et al . , 2006; Soler et al . , 2012 ) .",
"Mef2 regulates a suit of sarcomeric proteins in fly , fish and mouse muscle important for correct sarcomere assembly and maturation ( Hinits and Hughes , 2007; Kelly et al . , 2002; Potthoff et al . , 2007; Stronach et al . , 1999 ) .",
"In Drosophila , Mef2 cooperates with tissue-specific factors , such as CF2 , to induce and fine-tune expression of structural genes ( Gajewski and Schulz , 2010; García-Zaragoza et al . , 2008; Tanaka et al . , 2008 ) .",
"General transcriptional regulators , such as E2F , further contribute to high levels of muscle gene expression during myofibrillogenesis , in part through regulation of Mef2 itself ( Zappia and Frolov , 2016 ) .",
"However , it is less clear if muscle type-specific identity genes are continuously required to execute muscle type-specific fate .",
"Spalt major ( Salm ) is expressed after myoblast fusion in flight muscle myotubes and is required for all flight muscle type-specific gene expression: in its absence the fibrillar flight muscle is converted to tubular cross-striated muscle ( Schönbauer et al . , 2011; Spletter et al . , 2015 ) .",
"Here we demonstrated that Salm is continuously required for correct sarcomere morphogenesis , as late salm knock-down leads to defects in sarcomere growth during the late sarcomere maturation phase , causing severe muscle atrophy in adults .",
"It might do so by modifying the cooperation between Mef2 and E2F or by changing chromatin states , as vertebrate spalt homologs are recently discussed as epigenetic regulators ( Yang , 2018; Zhang et al . , 2015 ) .",
"However , Salm cannot be solely responsible for the transcriptional transition after 30 hr APF , as the transition still partially occurs in its absence .",
"Here we defined ordered phases of sarcomere morphogenesis in Drosophila flight muscles .",
"Is this a general concept for sarcomere morphogenesis ?",
"Reviewing the literature , one finds that in other Drosophila muscle types which display a tubular cross-striated myofibril organisation , such as the fly abdominal muscles , the striated sarcomeres also first assemble and then grow in length ( PerezPérez-Moreno et al . , 2014; Weitkunat et al . , 2017 ) , suggesting a conserved mechanism .",
"In developing zebrafish skeletal muscles , young myofibers present in younger somites show a short sarcomere length of about 1 . 2 µm , which increases to about 2 . 3 µm when somites and muscle fibers mature ( Sanger et al . , 2017; Sanger et al . , 2009 ) .",
"Interestingly , sarcomere length as well as thick filament length increase simultaneously during fish muscle maturation , indicating that as in flight muscles , the length of all sarcomeres in one large muscle fiber is homogenous at a given time ( Sanger et al . , 2009 ) .",
"Similar results were obtained in mouse cardiomyocytes measuring myosin filament length at young ( two somite ) and older ( 13 somite ) stages ( Du et al . , 2008 ) and even in human cardiomyocytes , in which myofibrils increase nearly threefold in width and become notably more organized and contractile from 52 to 127 days of gestation ( Racca et al . , 2016 ) .",
"These data suggest that sarcomeres generally may go through a series of ordered but overlapping developmental phases .",
"Interestingly , these changes in sarcomere morphology correlate with a switch in myosin heavy chain isoform expression changing from embryonic to neonatal to adult during skeletal muscle development ( Schiaffino et al . , 2015 ) .",
"Importantly , mutations in embryonic myosin ( MYH3 in humans ) result in severe congenital disorders characterised by multiple facial and limb contractures ( Toydemir et al . , 2006 ) .",
"As a similar ordered expression of myosin isoforms is also found during muscle regeneration after injury in adults ( Ciciliot and Schiaffino , 2010; Schiaffino et al . , 2015 ) , we hypothesize that our sequential sarcomere morphogenesis model may also be applicable to vertebrate skeletal and possibly heart muscles .",
"It will be a future challenge to identify possible feedback mechanisms that indicate the successful end of the sarcomere assembly phase or a possible re-entry into the sarcomere assembly phase during muscle regeneration or exercise induced muscle fiber growth .",
"It is enticing to speculate that the assembling cytoskeleton itself would measure its assembly status and provide a mechanical feedback signal to modify the activity of muscle-specific transcription factors ."
],
[
"Fly stocks were maintained using standard culture conditions .",
"Characterization of normal IFM sarcomere and fiber growth was performed in w1118 grown at 27°C .",
"salm RNAi was performed with previously characterized GD3029 ( referred to as salmIR ) and KK181052 ( Schönbauer et al . , 2011 ) from VDRC ( http://stockcenter . vdrc . at ) ( Dietzl et al . , 2007 ) at 25°C using Act88F-GAL4 to induce knock-down after 24 hr APF .",
"Act88F-GAL4 x w1118 served as control .",
"The Strn-Mlck-MiMIC insertion MI02893 into IFM-specific IsoR ( Bloomington stock 37038 ) and TRiP hairpin JF02170 were obtained from Bloomington ( Ni et al . , 2011 ) .",
"The salm-EGFP line was used to sort wing discs ( Marty et al . , 2014 ) .",
"Tagged genomic fosmid reporter fly lines include strn4 ( Strn-Mlck-GFP , Isoform R ) ( Spletter et al . , 2015 ) , fTRG500 ( Mhc-GFP , Isoforms K , L , M ) , fTRG501 ( Mf-GFP , Isoforms A , G , N ) , fTRG587 ( Rhea-GFP , Isoforms B , E , F , G ) , fTRG876 ( Fln-GFP ) , fTRG932 ( mys-GFP ) , fTRG958 ( βTub60D-GFP ) , fTRG1046 ( unc-89-GFP ) , and fTRG10028 ( Act88F-GFP ) ( Sarov et al . , 2016 ) .",
"To label myoblasts , we utilized the enhancer for Holes-in-muscle ( Him ) , which is expressed in dividing myoblasts and promotes the progenitor fate .",
"Him-nuc-eGFP flies were a gift of M . Taylor ( Soler and Taylor , 2009 ) .",
"Him-GAL4 flies were created by cloning an EcoRI to SacII fragment of the Him enhancer ( Liotta et al . , 2007 ) upstream of GAL4 into pStinger .",
"UAS-BBM ( UAS-palmCherry ) ( Förster and Luschnig , 2012 ) was driven with Him-GAL4 to label myoblasts .",
"Him-GFP-Gma flies were created by PCR amplifying GFP-Gma with AscI and PacI overhangs and then cloning downstream of the Him enhancer in pStinger to generate a gypsy insulator-Himenh-Gma-GFP-SV40-gypsy insulator cassette .",
"The rhea-YPet line used to label muscle ends for live imaging of twitch events was generated by CRISPR-mediated gene editing at the endogenous locus ( S . B . L and F . S . , details will be published elsewhere ) .",
"The kon-GFP line was generated by inserting GFP into the kon locus after its transmembrane domain using the genomic fosmid FlyFos021621 , which was integrated using Φ-C31 into VK00033 ( I . Ferreira and F . S . , details will be published elsewhere ) .",
"Flight tests were performed as previously described ( Schnorrer et al . , 2010 ) .",
"Act88F-GAL4 crosses were kept at 25°C , as higher temperatures negatively impacted flight ability , because of the very high GAL4 expression levels in this strain .",
"Adult males were collected on CO2 and recovered at least 24 hr at 25°C before testing .",
"Flies were introduced into the top of a 1 m long cylinder divided into five zones .",
"Those that landed in the top two zones were considered ‘normal fliers’ , those in the next two zones ‘weak fliers’ and those that fell to the bottom of the cylinder ‘flightless’ .",
"Wing-discs were dissected from 3rd instar wandering larvae in 1x PBS and fixed in 4% PFA in PBS-T .",
"Discs were stained as described below for anti-GFP .",
"Adult and pupal flight muscles were dissected and stained as previously described ( Weitkunat and Schnorrer , 2014 ) .",
"Briefly , early pupae ( 16–60 hr APF ) were freed from the pupal case , fixed for 20 min . in 4% PFA in relaxing solution and washed in 0 . 5% PBS-Triton-X100 ( PBS-T ) .",
"72 hr APF and older samples were cut sagittally with a microtome blade .",
"All samples were blocked for at least 1 hr at RT in 5% normal goat serum in PBS-T and stained with primary antibodies overnight at 4°C .",
"Primary antibodies include: guinea pig anti-Shot 1:500 ( gift of T . Volk ) , rat anti-Kettin 1:50 ( MAC155/Klg16 , Babraham Institute ) , rabbit anti-GFP 1:1000 ( ab290 , Abcam ) , rat anti-Bruno 1:500 ( Filardo and Ephrussi , 2003 ) , rabbit anti-Salm 1:50 ( Kühnlein et al . , 1994 ) , mouse anti-βPS-integrin 1:500 ( CF . 6G11 , DSHB ) , rabbit anti-Twi 1:1000 ( gift of Siegfried Roth ) and rabbit anti-Fln 1:50 ( Reedy et al . , 2000 ) ( gift of Jim Vigoreaux ) .",
"Samples were washed three times in 0 . 5% PBS-T and incubated overnight at 4°C with secondary conjugated antibodies ( 1:500 ) from Invitrogen ( Molecular Probes ) including: Alexa488 goat anti-guinea pig IgG , Alexa488 donkey anti-rat IgG , Alexa488 goat anti-mouse IgG , Alexa488 goat anti-rabbit IgG , rhodamine-phalloidin , Alexa568 goat anti-rabbit IgG and Alexa633 goat anti-mouse IgG .",
"Samples were washed three times in 0 . 5% PBS-T and mounted in Vectashield containing DAPI .",
"Head , wings and abdomen were removed from one day old w1118 flies and thoraxes were fixed overnight at 4°C in 4% PFA .",
"For 30–90 hr APF samples , pupae were freed from the pupal case , poked 3–5 times with an insect pin in the abdomen and fixed overnight at 4°C in 4% PFA .",
"Thoraxes or pupae were then sunk in 30% sucrose in 0 . 5% PBS-T overnight at 4°C on a nutator .",
"Thoraxes or pupae were embedded in Tissue-Tek O . C . T . ( Sakura Finetek ) in plastic moulds ( #4566 , Sakura Finetek ) and frozen on dry ice .",
"Blocks were sectioned at 30 µm on a cryostat ( Microm vacutome ) .",
"Sections were collected on glass slides coated with 1% gelatin +0 . 44 μM chromium potassium sulfate dodecahydrate to facilitate tissue adherence .",
"Slides were post-fixed for 1 min . in 4% PFA in 0 . 5% PBS-T at RT , washed in 0 . 5% PBS-T , incubated with rhodamine-phalloidin for 2 hr at RT , washed three times in 0 . 5% PBS-T and mounted in Fluoroshield with DAPI ( #F6057 , Sigma ) .",
"Images were acquired with a Zeiss LSM 780 confocal microscope equipped with an α Plan-APOCHROMAT 100x oil immersion objective lens ( NA 1 . 46 ) .",
"To compare if indicator protein expression replicates the mRNA-Seq expression dynamics , we imaged three time points from each expression profile with the same confocal settings .",
"Laser gain and pinhole settings were set on the brightest sample and reused on remaining time points in the same imaging session .",
"All samples were additionally stained with the same antibody mix on the same day and if possible in the same tube .",
"Images were processed with Fiji ( Schindelin et al . , 2012 ) and Photoshop , and displayed using the ‘Fire’ look-up table .",
"Fiber length and fiber cross-sectional area were measured with freehand drawing tools in Fiji based on rhodamine-phalloidin staining .",
"Sarcomere length , myofibril width , and myofibril diameter were measured automatically using a custom Fiji plug-in , MyofibrilJ , available from https://imagej . net/MyofibrilJ and code from https://github . com/giocard/MyofibrilJ ( Cardone , 2018; copy archived at https://github . com/elifesciences-publications/MyofibrilJ ) .",
"All measurements are based on rhodamine-phalloidin staining , except 34 hr APF sarcomere lengths , which are based on both rhodamine-phalloidin and Unc-89-GFP staining .",
"‘Sarcomeres per fibril’ was calculated as average individual fiber length divided by sarcomere length for fiber 3 or 4 .",
"‘Fibrils per fiber’ was calculated as average number of fibrils per unit area multiplied by individual fiber cross sectional area .",
"For determining myofibril diameter , samples were imaged using a 3x optical zoom ( 50 nm pixel size ) .",
"At least 20 cross-section images from different fibers for >10 flies were acquired for each time point .",
"The number of fibrils per section and fibril diameter were determined with the tool ‘analyze myofibrils crosswise’ from MyofibrilJ ( https://imagej . net/MyofibrilJ ) .",
"In this tool , an initial estimate of the diameter is obtained by finding the first minimum in the radial average profile of the autocorrelation ( Goodman , 1996 ) of the image .",
"This estimate is used to calibrate the optimal crop area around all the cross-sections in the image , their position previously detected by finding the local intensity peaks .",
"All of the detected cross-sections are then combined to obtain a noise-free average representation of the fibril section .",
"Finally , the diameter is calculated by examining the radial profile of the average and measuring the full width where the intensity is 26% of the maximum range .",
"For determining sarcomere length and myofibril width , for each experiment between 10 and 25 images were acquired from more than 10 individual flies .",
"From each image , nine non-overlapping regions of interest were selected , which were rotated to orient fibrils horizontally , when necessary .",
"The tool ‘analyze myofibrils lengthwise’ from MyofibrilJ reports the sarcomere length ( indicated as repeat ) and myofibril width ( indicated as thickness ) .",
"Because of the periodic nature of sarcomere organization , their length is estimated by means of Fourier analysis , identifying the position of the peaks on the horizontal axis of the Fourier transformed imaged .",
"Quality of the estimate was evaluated by visual inspection of the Fourier transformed image , overlaid with the peaks detected , as generated by the plug-in .",
"Myofibrils width is estimated from the position of the first minimum in the vertical intensity profile of the autocorrelation of the image .",
"Live imaging of developmental spontaneous contractions was performed on a Leica SP5 confocal microscope .",
"Prior to imaging , a window was cut in the pupal case , and pupae were mounted in slotted slides as previously described ( Lemke and Schnorrer , 2018; Weitkunat and Schnorrer , 2014 ) .",
"At the specified developmental time point , IFMs were recorded every 0 . 65 s for 5 min .",
"General movement within the thorax was distinguished from IFM-specific contraction , and each sample was scored for the number of single or double contractions observed per 5 min time window .",
"Data were recorded in Excel and ANOVA was performed in GraphPad Prism to determine significant differences .",
"Movies were assembled in Fiji ( Image J ) , cropped and edited for length to highlight a selected twitch event .",
"Quantitative imaging of fosmid reporter intensity was performed at 30 hr APF , 48 hr APF , 72 hr APF , 90 hr APF and in 1 day adult in live IFM by normalizing to fluorescent beads ( ThermoFisher ( Molecular Probes ) , InSpeck Green Kit I-7219 ) .",
"IFMs were dissected from five flies , mounted with fluorescent microspheres ( 0 . 3% or 1% relative intensity , depending on the reporter intensity ) in the supplied mounting medium and immediately imaged ( within 20 min ) .",
"Intensity measurements were obtained at 40x for at least 10 flies in regions where both IFM and at least three beads were visible .",
"Control Act88F-GAL4;; fosmid-GFP x w1118 and Act88F-GAL4;; fosmid-GFP x salmIR ( fosmids used included Strn-Mlck-GFP , Mhc-GFP , Fln-GFP , and Unc-89-GFP ) were imaged in the same imaging session .",
"Relative fluorescence fiber to beads was calculated for each image in Fiji by averaging intensity for three fiber ROIs and three bead ROIs .",
"Data were recorded in Excel and Student’s t-test for significance and plotting were performed in GraphPad Prism .",
"We previously published mRNA-Seq analysis of dissected IFMs from Mef2-GAL4 , UAS-GFP-Gma x w1118 at 30 hr APF , 72 hr APF and 1d adult , and Mef2-GAL4 , UAS-GFP-Gma x salmIR in 1d adult ( Spletter et al . , 2015 ) .",
"We expanded this analysis in the present study to include myoblasts from third instar larval wing discs ( see below ) and dissected IFMs from Mef2-GAL4 , UAS-GFP-Gma x w1118 at 16 , 24 , 30 , 48 , 72 , 90 hr APF and from 1 day adults as well as IFMs from Mef2-GAL4 , UAS-GFP-Gma x salmIR flies at 24 , 30 , 72 hr APF and from 1 day adults .",
"IFMs were dissected from groups of 15 flies in 30 min to minimize changes to the transcriptome , spun down in PBS for 5 min at 7500 rpm and immediately frozen in 100 µl TriPure reagent ( #11667157001 , Roche ) on dry ice .",
"RNA was isolated after combining IFMs from 150 to 200 flies , with biological duplicates or triplicates for each time point .",
"Poly ( A ) +mRNA was purified using Dynabeads ( #610 . 06 , Invitrogen ) and integrity was verified on a Bioanalyzer .",
"mRNA was then fragmented by heating to 94°C for 210 s in fragmentation buffer ( 40 mM TrisOAc , 100 mM KOAc , 30 mM MgOAc2 ) .",
"First-strand cDNA synthesis was performed with the Superscript III First-Strand Synthesis System ( #18080–051 , Invitrogen ) using random hexamers .",
"The second strand was synthesized with dUTP and submitted to the Vienna Biocenter Core Facilities ( VBCF , http://www . vbcf . ac . at ) for stranded library preparation according to standard Illumina protocols and sequenced as SR100 on an Illumina HiSeq2500 .",
"Libraries were multiplexed two to four per lane using TrueSeq adaptors .",
"To perform mRNA-Seq on fusion competent myoblasts that will form the IFMs , we first dissected wing discs from wandering third instar larvae and manually cut the hinge away from the wing pouch .",
"mRNA was isolated in TriPure reagent and sequenced as described above .",
"We estimate this sample ( Myo1 ) is ~50% myoblast , as the myoblasts form a nearly uniform layer over the underlying epithelial monolayer .",
"To obtain a purer myoblast sample , we performed large-scale imaginal disc sorting followed by dissociation .",
"We used particle sorting to isolate imaginal discs from Him-GAL4 , UAS-BBM ( UAS-palmCherry ) ; salm-EGFP flies based on the green fluorescent signal .",
"10–12 ml of larvae in PBS were disrupted using a GentleMACS mixer ( Miltenyl Biotec ) and discs were collected through a mesh sieve ( #0278 in , 25 opening , 710 µm ) .",
"Fat was removed by centrifugation for 10 min . at 1000 rpm at 4°C , discs were rinsed in PBS and then re-suspended in HBSS .",
"Discs were further purified on a Ficoll gradient ( 25%:16% ) .",
"Discs were then sorted on a Large Particle Flow Cytometer ( BioSorter ( FOCA1000 ) , Union Biometrica , Inc .",
") , obtaining 600–1000 discs per sample .",
"Discs were spun for 5 min at 600 rcf in a Teflon Eppendorf tube and then re-suspended in the dissociation mixture ( 200 µl of 10x Trypsin , 200 µl HBSS , 50 µl collagenase ( 10 mg/mL ) , 50 µl dispase ( 10 mg/ml ) ) .",
"The tube was incubated for 10 min . at RT and then transferred to a thermal shaker for 30 min . at 25°C at 650 rpm .",
"Myoblasts were filtered through a 35 µm tube-cap filter and spun at 600 rcf for 5 min . to pellet the cells .",
"Cells were re-suspended in HBSS for evaluation or frozen in TriPure reagent for RNA extraction .",
"We obtained samples with ~90% purity based on counting the number of red fluorescent cells/non fluorescent+green fluorescent cells in three slide regions .",
"mRNA was isolated in TriPure reagent and sequenced as described above , generating the Myo2 and Myo3 samples .",
"FASTA files were de-multiplexed and base called using Illumina software .",
"Reads were trimmed using the FASTX-toolkit .",
"Sequences were mapped using STAR ( Dobin et al . , 2013 ) to the Drosophila genome ( BDGP6 . 80 from ENSEMBL ) .",
"Mapped reads were sorted and indexed using SAMtools ( Li et al . , 2009 ) , and then bam files were converted to bigwig files .",
"Libraries were normalized based on library size and read-counts uploaded to the UCSC Browser for visualization ( http://genome . ucsc . edu/cgi-bin/hgTracks ? hgS_doOtherUser=submit&hgS_otherUserName=Ayeroslaviz&hgS_otherUserSessionName=IFMTP . leg . TCpaperHub1; http://genome . ucsc . edu/cgi-bin/hgTracks ? hgS_doOtherUser=submit&hgS_otherUserName=Ayeroslaviz&hgS_otherUserSessionName=AretSalmIFMTP . TCpaperHub2 ) .",
"Mapped sequences were run through featureCounts ( Liao et al . , 2014 ) and differential expression analysis was performed on the raw counts using DESeq2 ( Love et al . , 2014 ) .",
"We performed all pairwise comparisons across the time-course as well as between wild-type and salmIR samples ( Supplementary file 1 and 4 ) .",
"All original and processed data can be found as supplemental data or in the Gene Expression Omnibus submission ( accession number GSE107247 ) .",
"R packages employed in the analysis include ComplexHeatmap ( Gu et al . , 2016 ) , CorrPlot ( https://github . com/taiyun/corrplot ) , VennDiagram ( Chen and Boutros , 2011 ) , plyr ( Wickham , 2011 ) , reshape2 ( Wickham , 2007 ) , ggplot2 ( Wickham , 2009 ) and RColorBrewer ( Neuwirth , 2015 ) .",
"Genome-wide soft clustering was performed in R with Mfuzz ( Futschik and Carlisle , 2005 ) , using the DESeq2 normalized count values .",
"We filtered the dataset to include all genes expressed at one time point or more , defining expression as >100 counts after normalization .",
"We then set all count values < 100 to 0 , to remove noise below the expression threshold .",
"DESeq2 normalized data was standardized in Mfuzz to have a mean value of zero and a standard deviation of one , to remove the influence of expression magnitude and focus on the expression dynamics .",
"We tested ‘k’ ranging from 10 to 256 .",
"We then performed consecutive rounds of clustering to obtain three independent replicates with similar numbers of iterations , ultimately selecting a final k = 40 clusters with iterations equal to 975 , 1064 and 1118 .",
"We calculated a ‘stability score’ for each cluster by calculating how many genes are found in the same cluster in each run ( Supplementary file 1 ) .",
"Figures are from the 1064 iterations dataset .",
"Mfuzz cluster core expression profiles were calculated as the average standard-normal expression of all genes with a membership value greater than or equal to 0 . 8 , and then core profiles were clustered in R using Euclidean distance and complete linkage .",
"Enrichment analysis was performed with GO-Elite ( Zambon et al . , 2012 ) using available Gene Ontology terms for Drosophila .",
"We additionally defined user provided gene lists for transcription factors , RNA binding proteins , microtubule associated proteins , sarcomeric proteins , genes with an RNAi phenotype in muscle ( Schnorrer et al . , 2010 ) , mitochondrial genes ( http://mitoXplorer . biochem . mpg . de ) and salm core fibrillar genes ( Spletter et al . , 2015 ) .",
"Full results and gene lists are available in Supplementary file 2 .",
"These user-supplied lists allowed us to define more complete gene sets relevant to a particular process or with a specific localization than available in existing GO terms .",
"Analysis was performed with 5000 iterations to generate reliable significance values .",
"Processed data from DESeq2 , Mfuzz and GO-Elite are available in Supplementary file 1 , 2 , 4 .",
"mRNA-Seq data are publicly available from NCBI’s Gene Expression Omnibus ( GEO ) under accession number GSE107247 .",
"mRNA-Seq read counts are further publicly accessible as track hubs in the UCSC Genome Browser at the following links: [1] ( wild-type IFM time course ) http://genome . ucsc . edu/cgi-bin/hgTracks ?",
"hgS_doOtherUser=submit&hgS_otherUserName=Ayeroslaviz&hgS_otherUserSessionName=IFMTP . leg . TCpaperHub1 and [2] ( salm timecourse ) http://genome . ucsc . edu/cgi-bin/hgTracks ?",
"hgS_doOtherUser=submit&hgS_otherUserName=Ayeroslaviz&hgS_otherUserSessionName=AretSalmIFMTP . TCpaperHub2 .",
"Fiji scripts for analysis of sarcomere length , myofibril width and myofibril diameter are available from https://imagej . net/MyofibrilJ .",
"Raw data used to generate all plots presented in figure panels are available in the source data files for Figures 1 , 5 , 6 , 7 and 8 .",
"Data on statistical test results are presented in Supplementary file 5 ."
]
] | [
"Muscles organise pseudo-crystalline arrays of actin , myosin and titin filaments to build force-producing sarcomeres .",
"To study sarcomerogenesis , we have generated a transcriptomics resource of developing Drosophila flight muscles and identified 40 distinct expression profile clusters .",
"Strikingly , most sarcomeric components group in two clusters , which are strongly induced after all myofibrils have been assembled , indicating a transcriptional transition during myofibrillogenesis .",
"Following myofibril assembly , many short sarcomeres are added to each myofibril .",
"Subsequently , all sarcomeres mature , reaching 1 . 5 µm diameter and 3 . 2 µm length and acquiring stretch-sensitivity .",
"The efficient induction of the transcriptional transition during myofibrillogenesis , including the transcriptional boost of sarcomeric components , requires in part the transcriptional regulator Spalt major .",
"As a consequence of Spalt knock-down , sarcomere maturation is defective and fibers fail to gain stretch-sensitivity .",
"Together , this defines an ordered sarcomere morphogenesis process under precise transcriptional control – a concept that may also apply to vertebrate muscle or heart development ."
] | [
"Animals may have different types of muscles but they all have one thing in common: molecular machines called sarcomeres that produce a pulling force .",
"Conserved from fruit flies to humans , these structures line up end-to-end inside muscle cells , forming long cables called myofibrils .",
"Some of the myofibrils in a human can reach several centimetres in length , which is much longer than those in a fruit fly .",
"However , individual sarcomeres are the same length in both humans and flies .",
"To build the parts of the sarcomere , an animal cell first copies the relevant genes into intermediate molecules known as mRNAs , which are then translated to build new sarcomere proteins .",
"Developing muscle cells later tune their sarcomeres to make them sensitive to stretching .",
"This tweaks the power and force of the mature muscle , but the details of this developmental process are not fully understood .",
"Now , Spletter et al . have counted all the mRNAs in the developing flight muscles of fruit flies , with the aim of generating a resource that catalogues the changes in gene activity , or expression , that occur as muscles develop .",
"This revealed that sarcomeres form in three phases .",
"First , the cells assembled all their myofibrils .",
"Then , they added short sarcomeres to the ends of their myofibrils .",
"Finally , the sarcomeres matured to their full length and diameter , and became sensitive to stretching .",
"Fruit fly muscles had 40 patterns of gene expression , with most of the sarcomere components having one of two specific patterns .",
"The expression of these genes dramatically rose after the young muscle cells had finished assembling all their myofibrils , suggesting muscles express different genes when their sarcomeres mature .",
"A protein called spalt-major helped the cell to know when to make the transition , allowing the sarcomeres to grow in length and width .",
"Losing spalt-major late in muscle development stopped sarcomere growth and prevented the tuning process .",
"The sarcomeres failed to become sensitive to stretching , a crucial feature of mature muscle .",
"Muscles without spalt-major contracted too much and without coordination , like a muscle spasm .",
"The similarities between fruit fly and human sarcomeres suggest this developmental sequence may also occur in human muscles too .",
"Understanding these steps may help to improve repair after injury or muscle growth during exercise .",
"The next step is to test whether regenerating or growing muscles develop in the same way ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"microbiology and infectious disease",
"immunology and inflammation"
] | A sustained type I IFN-neutrophil-IL-18 axis drives pathology during mucosal viral infection | elife-65762-v1 | [
[
"Neutrophils are a critical component of the innate immune system .",
"In humans , they are the most abundant leukocytes in circulation and are often among the first wave of immune cells responding to pathogen invasion .",
"In the context of bacterial or fungal infections , including those that are sexually transmitted , neutrophils are largely protective and can help eliminate pathogens through a variety of effector functions , including phagocytosis , production of reactive oxygen species ( ROS ) , neutrophil extracellular traps ( NET ) and protease release , and cytokine and chemokine secretion ( Mayadas et al . , 2014; Pham , 2006; Tecchio et al . , 2014 ) .",
"In contrast , the role of neutrophils during viral infection is less clear ( Galani and Andreakos , 2015 ) .",
"While neutrophils have been reported to neutralize several viruses and display protective qualities in vivo ( Akk et al . , 2016; Jenne et al . , 2013; Saitoh et al . , 2012; Tate et al . , 2009; Tate et al . , 2011 ) , they have also been associated with tissue damage , loss of viral control , and increased mortality ( Bai et al . , 2010; Brandes et al . , 2013; Kulkarni et al . , 2019; Narasaraju et al . , 2011; Vidy et al . , 2016 ) .",
"Type I interferons ( IFNs ) are potent regulators of neutrophil activity in a multitude of contexts .",
"Type I IFNs can enhance recruitment of neutrophils to sites of infection , regulate neutrophil function , and drive immunopathology after infection by different classes of pathogens , including Plasmodium spp .",
", Candida spp .",
", and Pseudomonas spp .",
"( Majer et al . , 2012; Pylaeva et al . , 2019; Rocha et al . , 2015 ) .",
"However , type I IFNs can also inhibit neutrophil recruitment to the ganglia by suppressing chemokine expression after herpes simplex virus ( HSV ) infection ( Stock et al . , 2014 ) , suggesting that the interplay of IFNs and neutrophil activity may be dependent on tissue type and the pathogen .",
"The relationship between neutrophil-intrinsic type I IFN signaling and infection outcomes is less clear .",
"Type I IFNs can promote expression of interferon-stimulated genes ( ISGs ) and pro-inflammatory cytokines in neutrophils , suggesting a potential role for them in driving immunopathology ( Galani et al . , 2017 ) .",
"During Leishmania infection , however , IFNAR signaling appears to suppress neutrophil-dependent killing of parasites ( Xin et al . , 2010 ) , which emphasizes the complexity of IFN-mediated neutrophil responses .",
"Genital herpes is a chronic , sexually transmitted infection that affects over 400 million people worldwide ( World Health Organization , 2007 ) and can be caused by two members of the alphaherpesvirus family , HSV-2 or the related HSV-1 .",
"Genital herpes is characterized by recurrent episodes of inflammation and ulceration , and the factors that drive disease are unclear .",
"In humans , ulcer formation is associated with suboptimal viral control and spread during episodes of reactivation ( Roychoudhury et al . , 2020; Schiffer and Corey , 2013; Schiffer et al . , 2013 ) , while in mouse models , severity of disease often correlates with susceptibility to infection and the level of viral replication in the genital mucosa ( Gopinath et al . , 2018 ) .",
"Neutrophil infiltration into sites of active HSV-2 ulcers has also been reported in humans ( Boddingius et al . , 1987 ) , but whether these cells are helpful or harmful during HSV infection is unknown .",
"While neutrophils have been associated with tissue damage after multiple routes of HSV-1 infection ( Divito and Hendricks , 2008; Khoury-Hanold et al . , 2016; Rao and Suvas , 2019; Thomas et al . , 1997 ) , a protective role for neutrophils after genital HSV-2 infection has also been reported ( Milligan , 1999; Milligan et al . , 2001 ) , although the use of non-specific depletion antibodies has muddled the respective contribution of neutrophils and other innate immune cells such as monocytes , which are known to be antiviral ( Iijima et al . , 2011 ) .",
"Furthermore , increased neutrophil recruitment to the HSV-2-infected vaginal epithelial barrier resulted in greater epithelial cell death , suggesting that neutrophil responses may indeed be pathogenic ( Krzyzowska et al . , 2014 ) .",
"However , the factors that distinguish pathogenic vs . non-pathogenic neutrophil responses during viral infection , including HSV-2 infection , remain ill-defined .",
"To address this , we evaluated the impact of neutrophils on genital disease severity using two models of HSV infection that result in low levels ( HSV-1 ) or high levels of inflammation ( HSV-2 ) ( Lee et al . , 2020 ) .",
"Between these two states , heightened expression of type I IFN during the resolution phase of acute infection and sustained expression of ISGs in neutrophils were detected after HSV-2 infection but not HSV-1 .",
"Therapeutic antibody-mediated blockade of IFNα/β receptor 1 ( IFNAR1 ) as well as neutrophil-specific deletion of IFNAR1 reduced both genital inflammation as well as vaginal IL-18 levels during the resolution phase of acute HSV-2 infection .",
"Accordingly , therapeutic neutralization of IL-18 also ameliorated genital disease after HSV-2 infection .",
"Together , our data demonstrates that sustained type I IFN signaling is a key determinant of pathogenic neutrophil responses during viral infection , and identifies neutrophil- and type I IFN-dependent IL-18 production as a novel driver of inflammation during genital HSV-2 infection ."
],
[
"To determine the role of neutrophils in our model of vaginal HSV-2 infection , wild-type ( WT ) female C57BL/6 mice were treated with Depo-Provera ( depot medroxyprogestrone , DMPA ) to hold mice at the diestrus phase of the estrus cycle and normalize susceptibility to infection ( Kaushic et al . , 2003 ) .",
"Neutrophils were depleted in DMPA-treated mice by intraperitoneal ( i . p . ) injection of an antibody against Ly6G , a neutrophil-specific marker , or an isotype control .",
"One day later , mice were inoculated intravaginally with 5000 plaque forming units ( PFU ) of WT HSV-2 strain 186 syn+ ( WT HSV-2 ) .",
"Neutrophils were effectively reduced up to 6 days post-infection ( d . p . i . ) in the vagina ( Figure 1A ) and the blood ( Figure 1—figure supplement 1 ) .",
"In order to focus on genital inflammation , mice were monitored for 1 week after infection , as progression of disease within the second week of our infection model is largely indicative of viral dissemination into the central nervous system .",
"In both cohorts , mild genital inflammation was apparent starting at 4 d . p . i . in a small fraction of mice ( Figure 1B ) .",
"Over time , progression of disease in the neutrophil-depleted mice was significantly slower compared to the controls .",
"Remarkably , as late as 7 d . p . i . , a proportion of the neutrophil-depleted group remained uninflamed , in contrast to the isotype control group in which all mice displayed signs of inflammation ( Figure 1B ) .",
"To confirm the disparity in disease severity , we examined the vagina and genital skin by histology .",
"At 6 d . p . i . , epithelial denuding and damage was apparent in the isotype control-treated mice ( Figure 1C ) .",
"In contrast , only a limited amount of epithelial destruction was observed in neutrophil-depleted mice , with less cellular infiltrates at sites of damage and in the lumen ( Figure 1C ) .",
"Furthermore , the epithelial layer proximal to areas of damage was morphologically distinct in isotype control-treated animals compared to neutrophil-depleted animals , suggesting diverse epithelial responses after infection in the presence or absence of neutrophils ( Figure 1C ) .",
"Similarly , destruction of the epidermis and separation of the epidermis from the dermis were widespread in the genital skin of isotype control-treated mice but not in neutrophil-depleted mice ( Figure 1C ) .",
"Unexpectedly , differences in genital inflammation and mucosal damage were largely independent of changes in viral control in the absence of neutrophils , as viral shedding into the vaginal lumen ( Figure 1D ) and viral control in the tissue parenchyma ( Figure 1E ) were similar between the two groups .",
"Indeed , disease severity was decreased in neutrophil-depleted mice despite a slight delay in the resolution of viral replication at 5 d . p . i . ( Figure 1D ) .",
"We next evaluated whether the decreased inflammation after neutrophil depletion was due to changes in the cellular response against HSV-2 infection .",
"We examined the recruitment of Ly6C+ monocytes , NK cells , and CD4 and CD8 T cells ( Figure 1—figure supplement 2A ) , all of which have been implicated in either the control of HSV or modulation of disease severity ( Lee and Ashkar , 2012; Shin and Iwasaki , 2013; Truong et al . , 2019 ) .",
"To remove intravascular cells and to limit our analysis to cells within the vagina , tissues were thoroughly perfused prior to collection ( Scott et al . , 2018 ) .",
"Unexpectedly , there was no significant difference in the number of Ly6C + CD11b + cells ( Figure 1—figure supplement 2B ) , NK cells ( Figure 1—figure supplement 2C ) , total CD4 ( Figure 1—figure supplement 2D ) , or CD8 T cells ( Figure 1—figure supplement 2E ) that were recruited to the vagina over the first 6 days after infection regardless of whether neutrophils were present or not .",
"Thus , our data demonstrate that neutrophils do not play a significant antiviral role in our model of vaginal HSV-2 infection , and rather promote genital inflammation with minimal impact viral burden and recruitment of other immune cells to the vagina .",
"We next wanted to determine whether neutrophil-specific effector functions were promoting disease after HSV-2 infection .",
"NETs have been associated with tissue damage in the context of both infectious ( Jenne and Kubes , 2015 ) and non-infectious disease ( Granger et al . , 2019 ) .",
"To test whether NETs play a role in genital disease after HSV-2 infection , we first examined the ability of neutrophils to form NETs when exposed to HSV-2 .",
"In vitro stimulation of neutrophils with HSV-2 resulted in the enlargement of cell nuclei and the characteristic expulsion of DNA coated in citrullinated histones , which is a key characteristic of NETs ( Figure 1—figure supplement 3A ) .",
"The formation of NETs requires input from multiple pathways , including histone citrullination by enzymes such as PAD4 , which leads to chromatin de-condensation and the eventual release of DNA ( Li et al . , 2010 ) .",
"To generate animals that were specifically lacking PAD4 in neutrophils , we bred Padi4fl/fl x S100a8-Cre mice ( PAD4 CKO ) .",
"HSV-2 infection of these mice and their littermate controls demonstrated minimal impact on genital inflammation ( Figure 1—figure supplement 3B ) or viral replication ( Figure 1—figure supplement 3C ) in the genital mucosa .",
"Thus , our data show that PAD4 expression in neutrophils and likely NET formation are not the mechanisms by which these cells mediate disease after HSV-2 infection .",
"We next tested whether ROS production by neutrophils mediated inflammation after HSV-2 infection .",
"While production of ROS in neutrophils supports antimicrobial activity against a variety of pathogens ( Dinauer , 2019 ) , excessive oxidative stress can be associated with tissue injury ( Mittal et al . , 2014 ) .",
"We found that in vitro stimulation of neutrophils with HSV-2 led to an increase in ROS production compared to unstimulated cells ( Figure 1—figure supplement 4A ) .",
"To determine whether respiratory burst in neutrophils promoted genital inflammation after HSV-2 infection in vivo , we infected mice with germline deficiency in Ncf2 ( Ncf2 KO ) , which encodes p67phox , a key component of the NADPH oxidase complex ( Jacob et al . , 2017 ) .",
"HSV-2 infection of Ncf2 KO and heterozygous controls resulted in similar progression of disease ( Figure 1—figure supplement 4B ) and did not alter viral titer ( Figure 1—figure supplement 4C ) .",
"To confirm that tested neutrophil effector functions , including ROS production , had little impact on genital inflammation , we infected mice in which the calcium-sensing molecules STIM1 and STIM2 were deleted from neutrophils , as these calcium-sensing molecules cooperatively regulate neutrophil activation and select effector functions ( Clemens et al . , 2017 ) .",
"Stim1fl/fl x Stim2fl/fl x S100a8-Cre ( STIM1/2 DKO ) mice were infected with HSV-2 and monitored for disease .",
"As expected , there was little difference in genital inflammation severity between the STIM1/2 DKO and Cre- controls ( Figure 1—figure supplement 4D ) or viral titers ( Figure 1—figure supplement 4E ) .",
"Together , our data show that ROS production from neutrophils and other cell types play little role in driving genital inflammation after HSV-2 infection .",
"To identify the factors that drove pathogenic neutrophil responses after HSV-2 infection , we turned to a complementary model of HSV-1 genital infection that we had previously described ( Lee et al . , 2020 ) .",
"Inoculation with the same dose of HSV-1 and HSV-2 led to profound differences in genital inflammation ( Figure 2A ) despite comparable levels of mucosal viral shedding throughout most days after infection ( Figure 2—figure supplement 1A; Lee et al . , 2020 ) , although resolution of HSV-2 infection at 6 and 7 d . p . i . was delayed ( Figure 2—figure supplement 1A , B ) .",
"Importantly , magnitude of the neutrophil response in the vagina was similar between HSV-1- and HSV-2-infected mice during the course of acute mucosal infection ( Figure 2B ) , and neutrophils could be found infiltrating sites of both HSV-1- and HSV-2-infected epithelium ( Figure 2C ) .",
"In contrast to HSV-2 infection , antibody-mediated depletion of neutrophils with anti-Ly6G antibody prior to inoculation with HSV-1 did not reduce the development of genital inflammation during the first 7 days after infection ( Figure 2D ) and had minimal impact on acute viral control ( Figure 2D - Figure 2—figure supplement 1C ) .",
"Together , our data suggests that the regulation of the neutrophil response after HSV-1 or HSV-2 infection was distinct , which may contribute to the differences in disease outcomes between these infections .",
"To better understand the differences between pathogenic neutrophil responses after HSV-2 infection and the non-pathogenic neutrophil responses after HSV-1 , we performed single-cell RNA sequencing ( scRNA-seq ) on sorted live vaginal cells from a mock-infected mouse or mice infected with HSV-1 or HSV-2 using the 10x Genomics platform ( Zheng et al . , 2017 ) .",
"Each sample was composed of cells from a single animal to better delineate potential subsets within cell populations , particularly neutrophils .",
"Analysis across 21 , 633 cells in all samples revealed 17 unique clusters in the vagina during HSV infection after filtering , including myeloid cells , lymphocytes , and epithelial cells ( Figure 3A ) .",
"Neutrophils were identified by expression of known cell markers such as S100a8 and Csf3r ( Figure 3B ) .",
"In mock-infected animals , the vaginal neutrophil population was dominated by cluster 0 , and upon infection , at least two additional neutrophil subsets , cluster 2 and cluster 5 , were clearly present ( Figure 3C ) .",
"While HSV-1-infected mice retained all three subpopulations of neutrophils in the vagina at 5 d . p . i . , in HSV-2-infected mice , the presence of cluster 0 was greatly reduced , and the bulk of the neutrophils was composed of cluster 2 and 5 ( Figure 3C ) .",
"One major distinguishing characteristic between ‘homeostatic’ cluster 0 and ‘infection’ clusters 2 and 5 was the extent of ISG expression , in which cluster 0 expressed low levels of genes associated with a type I IFN response , even in infected animals , while clusters 2 and 5 expressed high levels of these genes ( Figure 3D; Liberzon et al . , 2015 ) .",
"Furthermore , the gene expression profile of clusters 2 and 5 was different between HSV-2 and HSV-1 infection at 5 d . p . i . ( Figure 3—figure supplement 1 ) , including the expression of ISGs ( Figure 3C , Figure 3—figure supplement 1 ) .",
"Differential expression of select ISGs was confirmed by quantitative reverse transcription PCR ( qRT-PCR ) analysis in the vagina at 5 days after HSV-1 or HSV-2 infection ( Figure 3—figure supplement 2 ) .",
"qRT-PCR shows that expression of CXCL10 ( Figure 3—figure supplement 2A , B ) and Gbp2 ( Figure 3—figure supplement 2C , D; Glennie et al . , 2015 ) is increased in HSV-2-infected vaginas compared to HSV-1 , while IL-15 is not ( Figure 3—figure supplement 2E , F ) , which supports the accompanying scRNA-seq analysis .",
"While type I IFN was robustly produced early during acute infection after both HSV-1 and HSV-2 infection ( Figure 3—figure supplement 3 ) , IFNβ levels were higher in the vaginal lumen after HSV-2 infection compared to HSV-1 at time points corresponding to the onset of genital inflammation ( Figure 3E ) despite similar viral burden between the two infection models ( Figure 2—figure supplement 1A ) .",
"IFNβ was undetectable in both the vaginal lumen and the parenchyma by 7 days after both HSV-1 and HSV-2 infection ( Figure 3—figure supplement 3B , C ) .",
"Thus , during viral infection , distinct neutrophil subsets can be classified by transcriptional profiling , and expression of ISGs suggests that a key difference between a pathogenic and non-pathogenic neutrophil response during viral infection may be sustained IFN production and signaling .",
"We next wanted to test whether type I IFN signaling promoted immunopathology during genital HSV-2 infection .",
"IFNAR1-deficient mice are highly susceptible to HSV , regardless of the route of inoculation ( Gill et al . , 2006; Iversen et al . , 2010; Iversen et al . , 2016; Reinert et al . , 2012; Royer et al . , 2019; Svensson et al . , 2007; Wilcox et al . , 2016 ) , and rapidly succumb to infection , mainly due to a loss of viral control .",
"To investigate the temporal effects of type I IFNs in HSV-2 genital disease , we used an antibody against IFNAR1 to block the receptor at different time points after infection ( Scott et al . , 2018 ) .",
"When mice were injected i . p . with anti-IFNAR1 antibody on the day of HSV-2 inoculation , disease progression was more rapid compared to isotype control-treated animals ( Figure 4—figure supplement 1A ) , and the mice succumbed to infection at a faster rate ( Figure 4—figure supplement 1B ) , in a manner similar to IFNAR1-deficient mice ( Iversen et al . , 2010; Iversen et al . , 2016; Lee et al . , 2017; Reinert et al . , 2012; Wang et al . , 2012 ) .",
"Inflammation and rapid disease progression were likely due to significantly elevated viral burden in the anti-IFNAR1 antibody-treated mice compared to isotype controls ( Figure 4—figure supplement 1C ) , as HSV is a highly lytic virus that is capable of independently inducing epithelial tissue damage ( Horbul et al . , 2011 ) .",
"To focus on the effects of persistent IFN signaling in the vagina after HSV-2 infection , we also treated mice with a single injection of anti-IFNAR1 antibody or an isotype control at 4 d . p . i . In stark contrast to early anti-IFNAR1 antibody treatment , one treatment with therapeutic IFNAR1 blockade led to a significant reduction in the severity of inflammation compared to isotype controls ( Figure 4A ) .",
"Histology of vaginal tissues from isotype-treated controls at 6 d . p . i . showed widespread epithelial denuding and immune cell infiltrates within the epithelial layer of the vagina ( Figure 4B ) .",
"In contrast , damage to the vaginal epithelium in anti-IFNAR1 antibody-treated mice appeared to be localized ( Figure 4B ) , similar to neutrophil-depleted mice ( Figure 1C ) .",
"Similarly , the genital skin of isotype control-treated mice displayed signs of severe inflammation and destruction of the epidermis , while the skin structure of anti-IFNAR1 antibody-treated mice was largely intact ( Figure 4B ) .",
"Furthermore , IFNAR1 blockade at 4 d . p . i . had little impact on mucosal viral shedding ( Figure 4C ) .",
"Collectively , these data show that the protective effect of type I IFN on control of genital HSV infection is limited to the early stages of acute infection , and that sustained IFN signaling in the later stages of acute HSV-2 genital infection drives inflammation with minimal effect on viral replication .",
"Single-cell transcriptional profiling data suggested that type I IFN signaling was robust in vaginal neutrophils after HSV-2 infection ( Figure 3D ) .",
"To determine whether intrinsic IFN signaling in neutrophils promoted immunopathology , we deleted IFNAR1 from granulocytes by breeding Ifnar1fl/fl x S100a8-Cre mice ( IFNAR1 CKO ) .",
"After confirming that IFNAR1 ablation was limited to the neutrophil population ( Figure 5A ) , IFNAR1 CKO mice and littermate Cre- controls were vaginally infected with HSV-2 .",
"Despite differences in IFNAR1 expression , the number of neutrophils recovered from the vaginal lumen was similar between the IFNAR1 CKO mice and their Cre- control littermates ( Figure 5B ) .",
"Strikingly , although the magnitude of the vaginal neutrophil response was similar , we found that the severity of genital inflammation presented by the IFNAR1 CKO mice was significantly reduced compared to the Cre- controls ( Figure 5C ) .",
"As observed after neutrophil depletion , a subset of the IFNAR1 CKO cohort did not develop any signs of inflammation as late as 7 d . p . i . ( Figure 5C ) .",
"Similar to our observations with therapeutic IFNAR1 blockade , IFNAR1 CKO mice exhibited less pathology in both the vagina and genital skin compared to Cre- controls ( Figure 5D ) .",
"Distinct disease outcomes between the Cre- controls and IFNAR1 CKO mice occurred independently of viral control , as viral loads in the mucosa were similar between the two groups ( Figure 5E ) .",
"Together , our data demonstrates that tissue inflammation during HSV-2 infection is largely driven by prolonged type I IFN production , which acts directly upon neutrophils to drive disease .",
"Type I IFN stimulation of neutrophils can upregulate ISGs as well as several pro-inflammatory cytokines ( Galani et al . , 2017 ) .",
"To determine whether type I IFN was driving disease by shaping the cytokine milieu within the vagina , we first measured several pro-inflammatory cytokines in the vagina at 5 d . p . i . , in the presence or absence of neutrophils .",
"The production of inflammatory cytokines such as IL-6 ( Figure 6—figure supplement 1A ) , IL-1β ( Figure 6—figure supplement 1B ) , or TNF ( Figure 6—figure supplement 1C ) , all of which have been associated with genital inflammation and HSV-2 infection in humans ( Gosmann et al . , 2017; Masson et al . , 2014; Murphy and Mitchell , 2016 ) , was similar between both neutrophil-depleted and control groups .",
"Production of IFNγ ( Figure 6—figure supplement 1D ) as well as IL-12p70 ( Figure 6—figure supplement 1E ) , both cytokines associated with a type I immune response and important for HSV control , was similar between the neutrophil-depleted and control groups .",
"However , when we measured IL-18 , an IL-1 family cytokine that is primarily known for mediating innate defense ( Harandi et al . , 2001 ) and for promoting IFNγ production from NK cells during genital HSV-2 infection ( Lee et al . , 2017 ) , we detected a notable difference between neutrophil-depleted and control mice ( Figure 6A ) , suggesting an unexpected role for this cytokine in driving disease during HSV-2 infection .",
"To determine whether type I IFN signaling regulated IL-18 production in the vagina , we assessed IL-18 levels in the vaginal lumen after therapeutic antibody-mediated IFNAR1 blockade .",
"At 5 d . p . i . , similarly to neutrophil-depleted mice , we found that IL-18 levels were markedly reduced ( Figure 6B ) .",
"Importantly , measurement of IL-18 in the vagina of IFNAR1 CKO at 5 d . p . i . also revealed a significant decrease in cytokine levels compared to littermate controls ( Figure 6C ) .",
"To determine whether IL-18 was playing a key role in driving immunopathology during genital HSV-2 infection , we therapeutically administered an IL-18-neutralizing antibody directly at the site of infection to HSV-2-infected animals starting at 3 d . p . i . in order to promote sufficient antibody concentration and activity in the relevant tissue .",
"Remarkably , neutralization of IL-18 led to a considerable reduction in disease severity ( Figure 6D ) , without any impact on viral control ( Figure 6E ) .",
"To determine the source of pathogenic IL-18 in the vagina , we probed vaginal tissues for the neutrophil marker S100A8 and IL-18 at 6 d . p . i . ( Figure 6F ) .",
"Detection of IL-18 and S100A8 around a single nucleus demonstrated that neutrophils could be a source of IL-18 during vaginal HSV-2 infection ( Figure 6F ) .",
"However , we also identified IL-18-reactive cells that were negative for S100A8 but in close proximity to neutrophils ( Figure 6F ) , suggesting the potential for multiple cellular sources of IL-18 .",
"Thus , our data demonstrate that sustained type I IFN signaling in neutrophils leads to the production of vaginal IL-18 and reveal IL-18 to be a novel regulator of disease after HSV-2 infection ."
],
[
"In this study , we evaluated drivers of a pathogenic neutrophil response using a mouse model for an important human infection .",
"We found that neutrophils promote genital inflammation without affecting antiviral activity after genital HSV-2 infection , suggesting that the neutrophil response is primarily immunopathogenic .",
"Depletion of neutrophils led to a significant decrease in disease severity without altering recruitment of other immune cells or the production of common pro-inflammatory cytokines , and deficiency in genes controlling neutrophil effector functions such as ROS production and NET formation had little impact on progression of disease .",
"Comparative analysis of single-cell transcriptional profiles revealed a strong type I IFN signature that was sustained in neutrophils responding to a highly inflammatory genital HSV-2 infection but not a less inflammatory HSV-1 infection , suggesting that host responses to these two related viruses established distinct inflammatory milieus with divergent effects on responding neutrophils .",
"In contrast to antibody-mediated blockade of IFNAR1 at the time of infection , which led to significantly worse disease outcomes , IFNAR1 blockade just prior to the resolution phase of acute mucosal infection significantly delayed the progression of genital inflammation .",
"Importantly , neutrophil-specific deficiency of IFNAR1 markedly reduced the severity of genital disease after HSV-2 infection , suggesting that persistent IFN signaling drove disease primarily by acting on neutrophils .",
"Ultimately , this sustained type I IFN signaling in neutrophils promoted the production of pro-inflammatory IL-18 , and therapeutic neutralization of this cytokine also ameliorated disease .",
"Together , our results suggest an axis of type I IFNs , neutrophils , and IL-18 as the key driver of genital disease in a mouse model of HSV-2 infection , and that sustained type I IFN signaling is a key factor in distinguishing between pathogenic and non-pathogenic neutrophil responses during mucosal viral infection .",
"Type I IFNs are a frontline of defense against viral infection , but models of chronic viral infection , including lymphocytic choriomeningitis virus ( LCMV ) ( Teijaro et al . , 2013; Wilson et al . , 2013 ) , human immunodeficiency virus ( HIV ) ( Meier et al . , 2009; Rotger et al . , 2010; Sedaghat et al . , 2008; Taleb et al . , 2017 ) , and simian immunodeficiency virus ( SIV ) ( Harris et al . , 2010; Jacquelin et al . , 2009 ) , reveal the detrimental effect of overexuberant or sustained type I IFN signaling .",
"Notably , prolonged IFN signaling during chronic viral infection can promote immunosuppression through multiple cellular and molecular mechanisms , and deletion or blockade of IFNAR1 during chronic LCMV infection can alleviate immunosuppression and enhance long-term viral control ( Cheng et al . , 2017; Taleb et al . , 2017; Teijaro et al . , 2013; Wilson et al . , 2013 ) .",
"However , unlike the LCMV model , early blockade of type I IFN signaling led to more severe disease and a complete loss of viral control after HSV-2 infection , similar to infections performed on an IFNAR1-deficient genetic background ( Iversen et al . , 2010; Iversen et al . , 2016; Lee et al . , 2017; Leib et al . , 1999; Reinert et al . , 2012; Wang et al . , 2012 ) , indicating an early antiviral role ( Lee et al . , 2017; Luker et al . , 2003 ) .",
"Rather , only therapeutic inhibition of sustained IFN signaling diminished disease without disrupting viral control , thus revealing a heretofore unappreciated , temporal division of the antiviral and immunopathological effects of type I IFN signaling during HSV-2 infection .",
"The source of sustained type I IFN production that promotes immunopathology after genital HSV-2 infection is currently unknown .",
"It is also unclear as to why type I IFN production is sustained during HSV-2 , but not after genital HSV-1 infection .",
"Our data indicates a slight delay in the control of HSV-2 infection compared to HSV-1 at 6 and 7 d . p . i . , but this did not correlate with differences in IFNβ levels between HSV-1 and HSV-2 infection .",
"Differences in viral dissemination to the nervous system ( Lee et al . , 2020 ) or function of viral proteins may account for disparities in type I IFN production and ultimately , disease severity .",
"HSV encodes numerous proteins that can suppress type I IFN production and regulate the signaling pathways ( Christensen et al . , 2016; Lin and Zheng , 2019; Melroe et al . , 2004 ) , suggesting that production of type I IFN likely occurs from a cell type that is not directly infected .",
"While plasmacytoid dendritic cells ( pDC ) are known as robust producers of type I IFN , they appear to have a limited role during genital HSV-2 infection ( Swiecki et al . , 2013 ) , indicating an alternative source of type I IFN , such as conventional DCs ( Wilson et al . , 2013 ) .",
"In humans , type I IFN can be detected at active lesion sites during recurrent episodes ( Peng et al . , 2009; Roychoudhury et al . , 2020 ) , although levels do not correlate with restriction of viral replication ( Roychoudhury et al . , 2020 ) .",
"This raises the possibility that type I IFN induction may not be antiviral and could contribute to ulcer formation , although this hypothesis is yet to be tested .",
"Human neutrophils from females are also reported to be hyper-responsive to type I IFNs ( Gupta et al . , 2020 ) .",
"Although clinical disease recurrence rates between men and women with genital herpes are similar ( Wald et al . , 2002 ) , differences in neutrophil sensitivity to type I IFN may have implications for sex-dependent mechanisms of ulcer development .",
"Synergistic effects of cytokine signaling have been reported to be important for maximizing cellular responses to infection through the upregulation of cooperative or independent molecular programs ( Bartee and McFadden , 2013 ) or through the cross-regulation of receptor signaling pathways ( Ivashkiv and Donlin , 2014 ) .",
"Upon infection , the activity of neutrophils can be modulated strongly by multiple IFNs , in a variety of tissues .",
"Type I ( Ank et al . , 2008 ) , type II ( Iijima et al . , 2008; Lee et al . , 2020 ) , and type III IFNs ( Ank et al . , 2008; Lee et al . , 2020 ) are all robustly produced during HSV-2 infection .",
"While type I and type II IFNs are crucial for the control of HSV-2 replication , endogenous type III IFNs do not appear to affect either disease severity or viral control , although exogenous application of type III IFNs can reduce viral burden ( Ank et al . , 2008; Ank et al . , 2006 ) .",
"Expression of the type III IFN receptor , IFNLR , is limited to very few cell types , including neutrophils and epithelial cells ( Blazek et al . , 2015; Mahlakõiv et al . , 2015; Sommereyns et al . , 2008 ) .",
"As epithelial cells are a major target for HSV-2 replication , dissecting the action of type III IFNs within the neutrophil and epithelial cell compartments may reveal a more detailed picture of the role type III IFNs play .",
"The impact of simultaneous type I , II , and III IFN signaling on neutrophil function is currently unclear , and due to the importance of these molecules in controlling infection , cell-specific modifications of receptor expression will be required to better understand their impact on neutrophil function .",
"In vitro stimulation of neutrophils with type I IFN leads to the upregulation of many common ISGs as well as inflammatory genes , including IL-18 ( Galani et al . , 2017 ) .",
"Importantly , type I IFN may differentially regulate expression of IL-18 and IL-1β , another IL-1 family cytokine that depends on caspase-mediated cleavage for activation ( Zhu and Kanneganti , 2017 ) .",
"It is unclear whether neutrophils are directly producing this cytokine in our model of infection and whether IL-18 production is dependent on inflammasome activation .",
"As HSV also encodes proteins that can inhibit inflammasome activity ( Maruzuru et al . , 2018 ) , one possibility is that IL-18 is produced by a cell type that is not productively infected with HSV , such as neutrophils .",
"Alternatively , neutrophil proteases released in the extracellular space have been reported to cleave and activate proIL-1 cytokines that are secreted by other cells in a caspase-1-independent manner ( Clancy et al . , 2018; Robertson et al . , 2006; Sugawara et al . , 2001 ) , suggesting a mechanism by which neutrophils may modulate IL-18 levels without directly secreting the cytokine themselves .",
"Our data show that along with neutrophils , IL-18 was present in the epithelium in cells that are in close proximity to infiltrating neutrophils .",
"Although we have not yet confirmed whether this detected IL-18 is bioactive , our data allude to multiple sources and mechanisms by which pathogenic IL-18 is produced during HSV-2 infection .",
"During HSV-2 vaginal infection , IL-18 stimulates NK cells to rapidly produce antiviral IFNγ ( Lee et al . , 2017 ) , and is thought to be important for orchestrating a protective innate immune response .",
"Accordingly , IL-18-deficient mice are more susceptible to HSV-2 infection ( Harandi et al . , 2001 ) and HSV-1 infection ( Fujioka et al . , 1999; Reading et al . , 2007 ) , presumably due to dysregulation of innate IFNγ production and loss of viral control .",
"Our study reveals a novel aspect of IL-18 biology during HSV-2 infection , and that like type I IFN signaling , there may be a temporal component to the effects of IL-18 during HSV-2 infection .",
"Currently , the mechanism by which IL-18 promotes disease during genital HSV-2 infection is unknown .",
"In the gut , the role of IL-18 is balanced between protection and pathology ( Jarret et al . , 2020; Nowarski et al . , 2015 ) .",
"The role of IL-18 during HSV-2 infection appears to be similarly complex , and further study will be required to identify the compartment on which IL-18 acts and the downstream effects of IL-18 signaling .",
"Additionally , while our results demonstrate an important role for IL-18 , the reduction in disease severity was not as profound as therapeutic IFNAR blockade in our HSV-2 model of infection .",
"Considering the complex response elicited by type I IFN , our data suggest that other IL-18-independent , IFN-dependent mechanisms that promote genital inflammation are yet to be elucidated .",
"Nevertheless , therapeutic neutralization of IL-18 reduced disease without altering viral titers in our model , suggesting that IL-18 does not have an impact on T-cell-dependent IFNγ production ( Milligan and Bernstein , 1997; Nakanishi et al . , 2009 ) or direct antiviral activity .",
"As previous studies have shown that IL-18 is also dispensable for stimulating IFNγ from adaptive memory immune responses ( Harandi et al . , 2001 ) , IL-18 , along with type I IFN , may present attractive targets for therapeutics aiming to reduce inflammation during genital herpes ."
],
[
"Six-week-old female C57BL/6J mice were purchased from Jackson Laboratories and rested for at least 1 week and infected at a minimum of 7 weeks of age .",
"Ncf2 KO mice and controls were provided by M . C . Dinauer ( Washington University , St Louis ) and generated as previously described ( Jacob et al . , 2017 ) .",
"Stim1fl/fl x Stim2fl/fl x S100a8-Cre mice were provided by G . A . Clemens ( Washington University , St Louis ) and were generated as previously described ( Clemens et al . , 2017 ) .",
"Ifnar1fl/fl mice ( Ifnar1tm1Uka ) were a gift from H . W . Virgin ( Kamphuis et al . , 2006; Nice et al . , 2016 ) .",
"Padi4fl/fl mice ( B6 ( Cg ) -Padi4tm1 . 2Kmow/J ) and S100a8-Cre ( B6 . Cg-Tg ( S100a8-cre , -EGFP ) 1Ilw/J ) were obtained from Jackson Laboratories and bred at Washington University School of Medicine .",
"Cre- littermates generated from breeding pairs were used as controls .",
"All mice were maintained on a 12 hr light/dark cycle with unlimited access to food and water .",
"This study was carried out in accordance with the recommendations in the Guide for the Car and Use of Laboratory Animals of the National Institutes of Health .",
"Vero Cells ( African green monkey kidney epithelial cells , ATCC ) were cultured in Dulbeco’s Modified Eagle Medium ( Gibco ) containing 1% fetal bovine serum ( FBS , Corning ) and maintained at 37°C with 5% CO2 .",
"Cells were regularly tested for mycoplasma contamination , and all cells used for this study were mycoplasma-free .",
"Primary neutrophils were isolated from the bone marrow ( BM ) of naive female C57BL/6J mice .",
"A Histopaque gradient was used to isolate primary neutrophils for ROS assays , while a Percoll gradient was used for NET assays .",
"For Histopaque isolation: 3 ml of Histopaque 1119 ( Sigma-Aldrich ) was overlaid with 3 ml of Histopaque 1077 ( Sigma-Aldrich ) .",
"A single-cell suspension of isolated BM cells in 1 ml of PBS was layered over the Histopaque gradient .",
"Cells were centrifuged for 30 min at room temperature ( RT ) , and neutrophils were collected from the bottom interface .",
"For Percoll isolation: BM cells were resuspended in HBSS ( Gibco ) with 20 mM HEPES ( Gibco ) and layered over 6 ml of 62% Percoll solution ( GE Healthcare ) .",
"Cells were centrifuged for 30 min at RT , and neutrophils were collected from the bottom of the tube .",
"All tissue culture experiments were performed under BSL2 containment .",
"WT HSV-2 186 syn+ ( Spang et al . , 1983 ) and HSV-1 McKrae ( Williams et al . , 1965 ) were propagated and titered on Vero cells as previously described ( Lee et al . , 2020 ) .",
"Briefly , for propagation of virus stocks , Vero cells were plated in T150 tissue culture flasks , inoculated at 0 . 01 MOI at 80% confluence , and incubated at 37°C .",
"Infected cells were harvested 2–3 days after infection , resuspended in equal volumes of virus supernatant and twice-autoclaved milk , and sonicated .",
"Lysed cells were aliquoted and used as viral stock .",
"To titer , Vero cells were plated in six-well plates and inoculated with 10-fold serial dilutions of stock virus .",
"After inoculation , overlay media with 20 μg/ml human IgG was added to each well and plates were incubated at 37°C for 2–3 days .",
"To count , Vero cells were stained with 0 . 1% crystal violet .",
"All tissue culture experiments were performed under BSL2 containment .",
"For titration of virus in the vaginal lumen , 50 ul washes with sterile PBS were collected using a pipette and a sterile calginate swab , and diluted in 950 ul of ABC buffer ( 0 . 5 mM CaCl2 , 0 . 5 mM MgCl2 , 1% glucose , and 1% FBS in sterile PBS ) .",
"For titration of virus from tissue , vaginas were harvested into pre-weighed tubes and flash frozen on dry ice .",
"ABC buffer was added to weighed tissues , which were bead-homogenized and clarified by centrifugation .",
"10-fold serial dilutions of vaginal washes or tissue homgenate were titered by plaque assays on Vero cells ( Lee et al . , 2020 ) .",
"All mice were injected subcutaneously in the neck ruff once with 2 mg of DMPA ( Depo-Provera , Pfizer ) 5–7 days prior to virus inoculation .",
"For experiments in which neutrophils were depleted , mice were i . p . injected once with 500 μg of anti-Ly6G ( clone 1A8 ) or rat IgG2a isotype control ( anti-trinitrophenol +KLH ) ( Leinco Technologies ) diluted in sterile PBS ( Sigma-Aldrich ) 1 day prior to inoculation .",
"For experiments in which IFNAR blockade was conducted , mice were i . p . injected once with 1 mg of anti-IFNAR1 ( clone MAR1-5A3 ) or mouse IgG1 isotype control ( clone HKSP ) ( Leinco Technologies ) on either the day of inoculation ( ‘early’ ) or at 4 d . p . i . ( ‘late’ ) .",
"For ‘late’ treatments , only mice without overt signs of genital inflammation were chosen for antibody injection in both anti-IFNAR and isotype control groups to avoid biasing of results .",
"For experiments in which IL-18 was neutralized , mice were treated intravaginally with 100 μg of anti-IL-18 antibody ( clone YIGIF74-1G7 ) or rat IgG2a isotype control ( clone 2A3 ) ( BioXCell ) on days 3–5 after infection .",
"Selection of mice for isotype control or experimental antibody treatment was random .",
"For intravaginal inoculation , a sterile calginate swab ( McKesson ) moistened with sterile PBS was used to gently disrupt mucous from the vaginal cavity .",
"Stock virus was diluted in sterile PBS and either 5000 PFU or 104 PFU virus was delivered into the vaginal cavity via pipette tip in a 10 μl volume .",
"Mice were weighed and monitored for signs of disease for 1 week following infection in an unblinded manner and monitored for survival for 2 weeks .",
"Genital inflammation was scored as follows: 0 – no inflammation , 1 – mild redness and swelling around the vaginal opening , 2 – fur loss and visible ulceration , 3 – severe ulceration and mild signs of sickness behavior ( lack of grooming ) , 4 –hindlimb paralysis , and 5 – moribund .",
"All tissues were harvested from animals sedated with ketamine and xylazine and thoroughly perfused with a minimum of 15 ml of PBS .",
"Vaginas were processed as follows: tissue was cut into pieces and digested for 15 min in a shaking water bath held at 37°C in a 0 . 5 mg/ml solution of Dispase II ( Roche ) in PBS .",
"Tissues were then transferred to a solution of 0 . 5 mg/ml Collagenase D ( Roche ) and 15 μg/ml DNase I ( Roche ) in RPMI media ( Gibco ) supplemented with 10% FBS ( Corning ) and 1% pen/strep ( Gibco ) and digested for 25 min in a shaking water bath held at 37°C .",
"50 μl of sterile EDTA was added to each sample and incubated at 37°C for another 5 min .",
"Tissues were then mechanically disrupted through a 70 um cell strainer into a single-cell suspension using a 3 ml syringe plunger .",
"Tissues were washed with RPMI media with 1% FBS , centrifuged , and resuspended in 200 μl RPRM with 1% FBS and 1% pen/strep .",
"Single-cell suspensions from vaginal tissues , or luminal cells collected in vaginal washes were plated in 96-well plates and incubated with Live/Dead Fixable Aqua Dead Cell Stain kit ( Molecular Probes ) for 15 min at room temperature ( RT ) in the dark .",
"Cells were then incubated with Fc block ( anti-CD16/32 , Biolegend ) for 15 min at RT in the dark .",
"Surface staining was performed in FACS buffer ( 1% FBS and 0 . 02% sodium azide in PBS ) on ice and in the dark using the following antibodies: CD3 ( clone 145–2 C11 ) , CD4 ( clone GK1 . 5 ) , CD8a ( clone 53–6 . 7 ) , CD11b ( clone M1/70 ) , CD45 ( clone 30-F11 ) , Gr-1 ( clone RB6-8C5 ) , Ly6C ( clone HK1 . 4 ) , Ly6G ( clone 1A8 ) , and NK1 . 1 ( clone PK136 ) .",
"All antibodies were purchased from Biolegend .",
"For surface staining of IFNAR1 , cells were incubated with an anti-IFNAR1 antibody or a mouse IgG1 isotype control ( Leinco Technologies ) for 20 min at 37°C .",
"Cells were washed and then surface staining of other markers proceeded as described above .",
"Cell counts were performed by adding Precision Count Beads ( Biolegend ) to samples prior to flow cytometric acquisition .",
"Samples were acquired on an LSR Fortessa ( BD Biosciences ) and analyzed by FlowJo ( Treestar ) .",
"All tissues were harvested from animals sedated with ketamine and xylazine and thoroughly perfused with a minimum of 15 ml of PBS , followed by 15 ml of PLP fixative ( 0 . 01 M NaIO4 , 0 . 075 M lysine , 0 . 0375 M sodium phosphate , and 2% paraformaldehyde [PFA] ) for immunofluorescent ( IF ) staining or 4% PFA for immunohistochemistry ( IHC ) .",
"Tissues were cryoprotected in 30% sucrose , frozen in OCT medium ( Sakura ) , and cut into 7 um sections .",
"Cryosections were blocked 5% bovine serum albumin ( BSA ) , 5% goat serum ( Jackson Immunoresearch ) , and 0 . 1% Triton-X in PBS for 1 hr at RT .",
"HSV antigens were detected with a rabbit anti-HSV primary antibody ( Dako ) , incubated overnight at 4°C , washed in PBS , and incubated for 1 hr at RT with a goat anti-rabbit IgG conjugated to AlexaFluor 488 ( Life Technologies ) .",
"S100A8 was detected with a rat anti-mouse S100A8 primary antibody ( clone 63N13G5 , Novus Biologicals ) and a goat anti-rat IgG conjugated to AlexaFluor 568 ( Life Technologies ) in a similar manner .",
"IL-18 was detected using a biotinylated rat anti-mouse IL-18 primary antibody ( clone 93–10C , MBL International ) .",
"Cryosections were blocked as described above and then treated with the Avidin/Biotin Blocking Kit ( Vector Laboratories ) according to manufacturer's protocol .",
"Endogenous peroxidases were quenched with a 2% hydrogen peroxide solution .",
"Anti-mouse IL-18 or a rat IgG1 isotype control was incubated overnight at 4°C .",
"The AlexaFluor 647 Tyramide Signal Amplification kit ( Invitrogen ) was used to visualize IL-18 and used according to manufacturer's protocol .",
"DNA was visualized with 4′ , 6-diamidino-2-phenylindole ( DAPI ) ( Life Technologies ) .",
"Sections were imaged with a Zeiss Cell Observer inverted microscope using a 40x objective , acquired with Zen software , and image brightness was adjusted using Photoshop ( Adobe ) .",
"For IHC , sections were probed with an anti-HSV antibody incubated overnight at 4°C ( Dako ) , a donkey anti-rabbit IgG-HRP antibody ( Jackson Immunoresearch ) for 1 hr at RT and then enzymatically visualized by 3 , 3’-diaminobenzidine ( DAB ) enzyme reaction ( Sigma-Aldrich ) .",
"Sections were counterstained with hematoxylin and eosin , and images were captured using Zeiss ZEN software on a Zeiss Cell Observer inverted microscope witβh an Axiocam dual B/W and color camera with a 20x objective .",
"Image brightness was adjusted using Photoshop ( Adobe ) and merged with Image J64 ( NIH ) .",
"Harvested tissues were homogenized in RLT buffer ( RNeasy Kit , Qiagen ) with approximately 100 μl of sterile 1 . 0 mm zirconia/silica beads ( Biospec Products ) in a bead beater .",
"Homogenized tissue samples were processed according to manufacturer's protocol using the RNeasy Mini Kit ( Qiagen ) and RNA quality and quantity was assessed on a Nanodrop ( ThermoFisher ) .",
"qRT-PCR was performed in 10 μl reactions using the iTaq Universal SYBR Green One-Step kit ( Biorad ) according to manufacturer's protocol .",
"Single-cell suspensions from digested vaginas were stained with Live/Dead Fixable Aqua Dead Cell Stain kit ( Molecular Probes ) for 15 min at RT in the dark .",
"Live cells were sorted on BD FacsAria II housed in a BSL2 biosafety cabinet .",
"A minimum of 16 , 000 cells were resuspended in PBS with 2% FBS and 0 . 2 U/μl RNase inhibitor at a concentration of 800–1400 cells/μl , submitted to McDonnell Genome Institute , and prepared for droplet-based 3' end scRNA-seq using the Chromium 3' v3 single-cell reagent kit as per manufacturer's protocol ( 10x Genomics ) .",
"Library sequencing was performed on a NovaSeq S4 ( Illumina ) .",
"For cytokine analysis by Bio-Plex Pro Mouse Cytokine 23-Plex Immunoassay ( Bio-rad ) : 2 × 50 μl washes with sterile PBS were collected from the vaginal lumen using a pipette .",
"Samples were centrifuged for 3 min at 13000*g to remove mucous and cells , and supernatants were added to 200 μl of ABC buffer .",
"The assay was performed according to manufacturer instructions , and plates were read on a Luminex Bioplex 100 system ( Biorad ) .",
"For measurement of IL-18 or IFNβ , 2 × 50 μl washes with sterile PBS were collected from the vaginal lumen and centrifuged to remove mucous and cells .",
"IL-18 was measured using the mouse IL-18 ELISA kit ( MBL International ) according to manufacturer's instructions at half-volumes , while IFNβ was measured using the LEGEND MAX Mouse IFNβ ELISA kit ( Biolegend ) according to manufacturer's instructions .",
"For the measurement of IFNβ in tissue homogenates , vaginal tissues were collected in pre-weighed tubes and flash frozen on dry ice .",
"NP40 lysis buffer ( 150 mM NaCl , 50 mM Tris , pH 8 . 0 , 1% NP40 alternative ) with protease inhibitor cocktail ( Sigma Aldrich ) was added to weighed tissue and bead-homogenized .",
"Lysates were clarified by centrifugation and supernatants used for ELISA .",
"To measure ROS production , isolated neutrophils were stimulated with heat-killed HSV-2 ( 56°C for 30 min ) at an MOI of 5 for 16 hr at 37°C .",
"ROS levels were quantified using DCFDA Cellular ROS Detection Assay kit ( Abcam ) according to manufacturer's protocol .",
"Fluorescence levels were measured by flow cytometry .",
"To induce NET formation , neutrophils were stimulated with heat-killed HSV-2 at an MOI of 1 for 4 hr at 37°C .",
"Cells were fixed with 8% PFA overnight and probed with a polyclonal rabbit antibody against mouse citrullinated histone H3 ( Abcam ) for 1 hr at RT in 1% BSA and 0 . 1% Triton-X for 1 hr at RT , a goat anti-rabbit antibody conjugated to AlexaFluor 488 ( Life Technologies ) for 1 hr at RT and DAPI diluted in PBS for 6 min at RT .",
"Cells were imaged with a Zeiss Cell Observer inverted microscope using a 63x objective and image brightness was adjusted using Photoshop ( Adobe ) .",
"All numerical data analyses except for scRNA-seq data analysis were performed on Graphpad Prism8 software .",
"Values were log-transformed to normalize distribution and variances where necessary .",
"Immune cell numbers and cytokine measurement were analyzed by two-way ANOVA with Bonferroni's multiple comparisons test .",
"Log-transformed viral titers were analyzed by repeated measures two-way ANOVA with Bonferroni's multiple comparisons test .",
"Inflammation scores were analyzed by repeated-measures two-way ANOVA or mixed-effects analysis with Geisser-Greenhouse correction and Bonferroni's multiple comparisons test .",
"The Geisser-Greenhouse correction was used for inflammation scores to correct any violations of sphericity and to provide a more restrictive , stringent calculation of p values .",
"ROS MFI was measured by unpaired two-tailed Student's t-test .",
"qPCR results were analyzed by one-way ANOVA with Tukey's multiple comparisons test .",
"A p<0 . 05 was considered statistically significant .",
"No experimental data points were excluded from statistical analysis , including potential outliers .",
"Mouse and sample numbers per group and experimental repeat information is provided in the figure legends .",
"All data points represent individual biological replicates , and the 'n' for each group refers to biological replicates .",
"No power calculations were performed to determine sample size; rather sample sizes were determined based on historical data ."
]
] | [
"Neutrophil responses against pathogens must be balanced between protection and immunopathology .",
"Factors that determine these outcomes are not well-understood .",
"In a mouse model of genital herpes simplex virus-2 ( HSV-2 ) infection , which results in severe genital inflammation , antibody-mediated neutrophil depletion reduced disease .",
"Comparative single-cell RNA-sequencing analysis of vaginal cells against a model of genital HSV-1 infection , which results in mild inflammation , demonstrated sustained expression of interferon-stimulated genes ( ISGs ) only after HSV-2 infection primarily within the neutrophil population .",
"Both therapeutic blockade of IFNα/β receptor 1 ( IFNAR1 ) and genetic deletion of IFNAR1 in neutrophils concomitantly decreased HSV-2 genital disease severity and vaginal IL-18 levels .",
"Therapeutic neutralization of IL-18 also diminished genital inflammation , indicating an important role for this cytokine in promoting neutrophil-dependent immunopathology .",
"Our study reveals that sustained type I interferon ( IFN ) signaling is a driver of pathogenic neutrophil responses and identifies IL-18 as a novel component of disease during genital HSV-2 infection ."
] | [
"Herpes simplex virus ( HSV ) is a human pathogen that causes genital herpes , an incurable disease that results in recurrent sores and inflammation .",
"Infection with HSV induces a strong antiviral immune response , which results in large numbers of immune cells arriving at these lesions .",
"But while some of these cells help to control viral replication , others might contribute to the inflammation that drives the disease .",
"One of the first immune cells to respond to infection are neutrophils .",
"Although neutrophils are generally protective , especially against bacteria and fungi , they have also been implicated in tissue damage and severe inflammation during viral infections .",
"But what determines whether a neutrophil will help to fight off an infection or increase disease severity is still an open question .",
"To investigate this , Lebratti , Lim et al . studied mice that had been infected with the genital herpes virus HSV-2 , which is known to cause significant amounts of inflammation in mice .",
"The experiments revealed that a signaling molecule called type I interferon , which is thought to be antiviral , causes neutrophils at the site of the infection to produce proteins , such as IL-18 , which trigger an inflammatory reaction .",
"Lebratti , Lim et al . found that type I interferon and IL-18 had shifting roles during the course of infection .",
"In the early stages , both molecules had a protective effect , confirming results from previous studies .",
"However , as the infection progressed , sustained levels of type I interferon signaling in neutrophils led to excess amounts of IL-18 .",
"Lebratti , Lim et al . discovered that blocking interferon signaling or decreasing the levels of IL-18 later during infection unexpectedly reduced the severity of the disease and resulted in less genital tissue damage .",
"Further experiments also showed that mice infected with another genital herpes virus called HSV-1 did not experience sustained levels of type I interferon .",
"This may explain why this virus causes less severe disease in mice .",
"Understanding how the immune system reacts to viruses could reveal new targets for treatments of genital herpes .",
"At the moment , there is little information about IL-18 production during genital herpes in humans .",
"So , the next step is to see whether neutrophils behave in the same way and whether IL-18 can be detected during human disease .",
"It is possible that the same immune components could promote disease in other infections too .",
"If so , this work may help uncover new drug targets for other viral diseases ."
] | 2021 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"genetics and genomics"
] | Loss of centromere function drives karyotype evolution in closely related Malassezia species | elife-53944-v2 | [
[
"Centromeres are the genomic loci on which the kinetochore , a multi-subunit complex , assembles to facilitate high-fidelity chromosome segregation .",
"The centromere-specific histone H3 variant CENP-A is the epigenetic hallmark of centromeres , as it replaces canonical histone H3 in the nucleosomes to make specialized centromeric chromatin that acts as the foundation to recruit other kinetochore proteins .",
"A remarkable diversity in the organization of centromere DNA sequences has been observed to accomplish this conserved role ( Roy and Sanyal , 2011; Yadav et al . , 2018b ) .",
"The smallest known centromeres are the point centromeres present in budding yeasts of the family Saccharomycetaceae that span <200 bp in length ( Clarke and Carbon , 1980; Gordon et al . , 2011; Kobayashi et al . , 2015 ) .",
"These centromeres are organized into conserved DNA elements I , II , and III that are recognized by a cognate kinetochore protein complex called the CBF3 complex , making them genetically defined centromeres .",
"Small regional centromeres , identified in several Candida species , form the second category ( Sanyal et al . , 2004; Padmanabhan et al . , 2008; Kapoor et al . , 2015; Chatterjee et al . , 2016 ) and have a 2–5-kb region enriched by kinetochore proteins .",
"These centromeres can either have unique DNA sequences or a homogenized core that is flanked by inverted repeats .",
"The third type of centromere structure is the large regional centromere , which is often repetitive in sequence and spans more than 15 kb .",
"Large regional centromeres can be transposon-enriched , as in Cryptococcus species , or organized into repeat structures around a central core , as in Schizosaccharomyces pombe ( Chikashige et al . , 1989; Clarke and Baum , 1990; Sun et al . , 2017; Yadav et al . , 2018b ) .",
"Although the organization of DNA elements is variable , a majority of known centromeres share AT-richness as a common feature .",
"Examples include the CDEII of point centromeres , central core sequences in S . pombe , and centromeres of Neurospora crassa , Magnaporthe oryzae , Plasmodium falciparum , and diatoms ( Fitzgerald-Hayes et al . , 1982; Iwanaga et al . , 2010; Rhind et al . , 2011; Kapoor et al . , 2015; Diner et al . , 2017; Yadav et al . , 2019 ) .",
"Even the recently described mosaic centromere structure observed in Mucor circinelloides that has lost CENP-A comprises an AT-rich kinetochore-bound core region ( Navarro-Mendoza et al . , 2019 ) .",
"Although suppression of recombination around centromeres has been correlated with reduced GC content ( Lynch et al . , 2010 ) , the genetic underpinning that determines how an AT-rich DNA region favors kinetochore assembly remains unclear .",
"Ironically , AT-rich sequences have been shown to be fragile sites within a chromosome ( Zhang and Freudenreich , 2007 ) .",
"Several lines of evidence suggest that centromeres are species-specific and are among the most rapidly evolving genomic regions , showing variation even between closely related species ( Bensasson et al . , 2008; Padmanabhan et al . , 2008; Rhind et al . , 2011; Roy and Sanyal , 2011 ) .",
"This evolution is accompanied by the concomitant evolution of CENP-A and the associated kinetochore proteins ( Talbert et al . , 2004 ) .",
"Functional incompatibilities between centromeres result in uniparental genome elimination in interspecies hybrids ( Ravi and Chan , 2010; Sanei et al . , 2011 ) .",
"The divergent nature of centromeres is proposed to be a driving force for speciation ( Henikoff et al . , 2001; Malik and Henikoff , 2009 ) .",
"Asexual organisms , by virtue of inter- and intra-chromosomal rearrangements , diversify into species clusters that are distinct in genotype and morphology ( Barraclough et al . , 2003 ) .",
"These genotypic differences include changes in both chromosomal organization and number .",
"Centromere function is directly related to karyotype stabilization following a change in chromosome number .",
"Rearrangements in the form of telomere–telomere fusions and nested chromosome insertions ( NCIs ) , wherein an entire donor chromosome is ‘inserted’ into or near the centromere of a non-homologous recipient chromosome , are among the major sources of chromosome number reduction ( Lysak , 2014 ) .",
"Such events often result in the formation of dicentric chromosomes that are subsequently stabilized by breakage-fusion-bridge cycles ( McClintock , 1941 ) or via inactivation of one centromere through different mechanisms ( Han et al . , 2009; Sato et al . , 2012 ) .",
"Well-known examples of telomere–telomere fusions include the formation of extant human chromosome 2 by fusion of two ancestral chromosomes ( Yunis and Prakash , 1982; IJdo et al . , 1991 ) , the reduction in karyotype observed within members of the Saccharomycotina such as Candida glabrata , Vanderwaltozyma polyspora , Kluyveromyces lactis , and Zygosaccharomyces rouxii ( Gordon et al . , 2011 ) , and the exchange of chromosomal arms seen in plants and fungi ( Schubert and Lysak , 2011; Sun et al . , 2017 ) .",
"NCIs have predominantly shaped karyotype evolution in grasses ( Murat et al . , 2010 ) .",
"Reduction of chromosome number by centromere loss has also been reported ( Gordon et al . , 2011 ) .",
"To investigate whether centromere breakage can be a natural source of karyotype diversity in closely related species , we sought to identify centromeres in a group of Malassezia yeast species that exhibit variation in chromosome number .",
"Malassezia species are lipid-dependent basidiomycetous fungi that are naturally found as part of the animal skin microbiome ( Theelen et al . , 2018 ) .",
"At present , the Malassezia genus includes 18 species divided into three clades: A , B , and C . These species also have unusually compact genomes of less than 9 Mb , organized into six to nine chromosomes as revealed by electrophoretic karyotyping of some of these species ( Boekhout and Bosboom , 1994; Boekhout et al . , 1998; Wu et al . , 2015 ) .",
"Fungemia-associated species such as Malassezia furfur belong to Clade A . Clade B includes common inhabitants of human skin that are phylogenetically clustered into two subgroups , namely Clade B1 that contains Malassezia globosa and Malassezia restricta and Clade B2 that contains Malassezia sympodialis and related species .",
"Clade C includes Malassezia slooffiae and Malassezia cuniculi , which diverged earlier from a Malassezia common ancestor ( Wu et al . , 2015; Lorch et al . , 2018 ) .",
"Besides humans , Malassezia species have been detected on the skin of other animals .",
"For example , M . slooffiae was isolated from cows and goats , M . equina from horses , M . brasiliensis and M . psittaci from parrots , and the cold-tolerant species M . vespertilionis from bats ( Lorch et al . , 2018; Theelen et al . , 2018 ) .",
"In addition , culture-independent studies of fungi from environmental samples showed that Malassezia species that are closely related to those found on human skin were also detected in diverse niches , such as deep-sea vents , soil invertebrates , hydrothermal vents , corals , and Antarctic soils ( Amend , 2014 ) .",
"More than ten Malassezia species have been detected as a part of the human skin microbiome ( Findley and Grice , 2014 ) .",
"The human skin commensals such as M . globosa , M . restricta , and M . sympodialis have been associated with dermatological conditions such as dandruff/seborrheic dermatitis , atopic dermatitis , and folliculitis ( Theelen et al . , 2018 ) .",
"Recent reports implicate M . restricta in conditions such as Crohn’s disease and M . globosa in the progression of pancreatic cancer ( pancreatic ductal adenocarcinoma ) ( Aykut et al . , 2019; Limon et al . , 2019 ) .",
"Elevated levels of Malassezia species and the resulting inflammatory host response have been implicated in both of these disease states .",
"The nature of genomic rearrangements in each species may influence its ability to adapt and cause disease in a specific host niche .",
"Thus , studying the mechanisms of karyotype evolution is an important step towards understanding the evolution of the Malassezia species complex .",
"Kinetochore proteins serve as useful tools in the identification of centromeres because of their centromere-exclusive localization .",
"CENP-A replaces histone H3 in the centromeric nucleosomes .",
"This has been shown as a reduction in histone H3 levels at the centromeres in Candida lusitaniae ( Kapoor et al . , 2015 ) and in a human neocentromere ( Lo et al . , 2001 ) .",
"These CENP-A nucleosomes act as a foundation to recruit CENP-C , the KMN ( KNL1C-MIS12C-NDC80C ) network , and other kinetochore proteins ( Musacchio and Desai , 2017 ) .",
"In this study , we experimentally validated all of the eight centromeres of M . sympodialis using the Mtw1 protein ( Mis12 in S . pombe ) , a subunit of the KMN complex , as the kinetochore marker .",
"The Mis12 complex proteins are evolutionarily conserved outer kinetochore proteins that link the chromatin-associated inner kinetochore proteins to the microtubule-associated outer kinetochore proteins .",
"Members of the Mis12 complex localize to centromeres in other organisms ( Goshima et al . , 1999; Goshima et al . , 2003; Westermann et al . , 2003; Roy et al . , 2011 ) .",
"Recent studies suggest that the protein domains associated with the Mis12 complex members are exclusive to kinetochore proteins and are not detected in any other proteins , making them attractive tools for identifying centromere sequences ( Tromer et al . , 2019 ) .",
"Using the features of centromeres of M . sympodialis and newly generated chromosome-level genome assemblies , we predicted centromeres in related Malassezia species carrying seven , eight , or nine chromosomes , and experimentally validated the centromere identity in representative species of each karyotype , each belonging to a different Malassezia clade .",
"We employed gene synteny conservation across these centromeres to understand their transitions from an inferred ancestral state of nine chromosomes .",
"On the basis of our results , we propose that centromere loss by two distinct mechanisms drives karyotype diversity ."
],
[
"Previous reports that are based on pulsed-field gel electrophoresis ( PFGE ) have suggested that chromosome number varies within the Malassezia species complex .",
"The early diverged species M . slooffiae of Clade C was reported to have nine chromosomes ( Boekhout et al . , 1998 ) .",
"Clade B Malassezia species are reported to have nine ( M . globosa and M . restricta ) , eight ( M . sympodialis ) , or six chromosomes ( M . pachydermatis ) .",
"Among the Clade A species , M . obtusa and M . furfur CBS14141 were both reported to have seven chromosomes ( Boekhout and Bosboom , 1994; Zhu et al . , 2017 ) .",
"A high-quality reference genome is a prerequisite to understanding the rearrangements associated with chromosome number variation .",
"In addition , such a reference genome will also assist in resolving ambiguities in PFGE-based estimates of chromosome number when similar-sized chromosomes are present .",
"Complete genome assemblies were not available for many of the species with reported numbers of chromosomes .",
"To obtain better-assembled reference genomes , we sequenced the genomes of M . slooffiae and M . globosa as representatives of the nine-chromosome state , and of M . furfur as a representative of the seven-chromosome state , using PacBio SMRT sequencing technology ( Figure 1 , Figure 1—figure supplement 1 ) .",
"The M . globosa genome was completely assembled into nine contigs with telomeres on both ends ( BioSample accession SAMN10720087 ) .",
"We validated these contigs by matching each band on the pulsed-field gel with the contig sizes from the genome assembly , and further confirmed these by chromoblot analysis following PFGE .",
"This analysis shows that chromosome 5 contains the rDNA locus and migrates further than the expected size of 902 kb , as a diffuse ensemble of different sizes along with chromosome 3 ( Figure 1A ) .",
"The assembled genome of M . slooffiae has 14 contigs of which nine contigs have telomeres on both ends , indicative of nine chromosomes ( BioSample accession SAMN10720088 ) .",
"Each of the nine contigs could be assigned to the bands observed in the pulsed-field gel ( Figure 1B ) .",
"For M . furfur , the final genome assembly consisted of seven contigs with telomeres on both ends and matched the expected chromosome sizes obtained from an earlier PFGE analysis of CBS14141 ( Figure 1C ) .",
"The complete genome assembly of M . sympodialis reported earlier is distributed into eight chromosomes with telomere repeats on both ends ( Figure 1D ) , and serves as a representative of an eight-chromosome state in this study .",
"Changes in chromosome number are always associated with the birth or loss of centromeres , which stabilizes the karyotype in organisms with monocentric chromosomes .",
"To understand the transitions between these different karyotypic states observed in the Malassezia species complex , we sought to validate centromeres in species representative of each karyotype experimentally .",
"Organisms that have point centromeres possess Ndc10 , Cep3 , and Ctf13 of the CBF3 complex , a cognate protein complex that is specific to point centromeres .",
"None of these point-centromere-specific proteins could be detected in M . sympodialis .",
"However , we could detect homologs of CENP-A , CENP-C , and most of the outer kinetochore proteins in the genome of M . sympodialis ( Figure 2A and Figure 2—figure supplement 1 ) .",
"We functionally expressed an N-terminally GFP-tagged Mtw1 protein ( Protein ID: SHO76526 . 1 ) from its native promoter , and the expression of the fusion protein was confirmed by western blotting ( Figure 2B ) .",
"Upon staining with anti-GFP antibodies and DAPI ( 4′ , 6-diamidino-2-phenylindole ) , we were able to detect punctate localization of Mtw1 at the nuclear periphery ( Figure 2C ) , consistent with the clustered kinetochore localization observed in other yeasts ( Goshima et al . , 1999; Euskirchen , 2002; Roy et al . , 2011 ) .",
"Live-cell images of MSY001 ( GFP-MTW1 ) cells revealed that the kinetochores ( GFP-Mtw1 ) remained clustered throughout the cell cycle , starting from unbudded G1 cells in interphase to large-budded cells in mitosis ( Figure 2D ) .",
"Having identified Mtw1 as an authentic kinetochore protein , we performed ChIP-sequencing using the GFP-Mtw1 expressing strain of M . sympodialis ( MSY001 ) .",
"Mapping the reads to the reference genome of M . sympodialis strain ATCC42132 ( Zhu et al . , 2017 ) identified one significantly enriched locus on each of the eight chromosomes ( Figure 3A ) .",
"The lengths of the Mtw1-enriched centromere regions identified from the ChIP-seq analysis ranged from 3167 bp to 5143 bp with an average length of 4165 bp ( Table 1 ) .",
"However , the region of maximum Mtw1 enrichment on each chromosome ( based on the number of sequenced reads aligned ) mapped to the intergenic region harboring the GC trough ( approximately 1 kb long ) , which was previously predicted to be the centromeres of M . sympodialis ( Figure 3B ) ( Zhu et al . , 2017 ) .",
"The regions of Mtw1 enrichment span beyond the core centromeres and include active genes located proximal to these troughs ( Figure 3B , Figure 3—figure supplement 1A ) .",
"However , these open reading frames ( ORFs ) do not show consensus features such as the orientation of transcription or functional classification .",
"We validated this enrichment by ChIP-qPCR analysis with primers homologous to the predicted centromeres compared to those homologous to a control region distant from the centromere ( Figure 3—figure supplement 1B ) .",
"The presence of CENP-A nucleosomes at the kinetochore should result in reduced histone H3 enrichment at the centromeres when compared to a non-centromeric locus .",
"To test this , we performed ChIP with anti-histone H3 antibodies and analyzed the immunoprecipitated ( IP ) DNA by qPCR .",
"As compared to a control ORF region that is unlinked to the centromere ( 190 kb away from CEN1 ) , the pericentric regions flanking the core centromere showed a marginal reduction in histone H3 enrichment , which was further reduced at the core , that maps to the GC trough with the highest enrichment of the kinetochore protein Mtw1 .",
"That the core centromere region showing the maximum depletion of histone H3 coincided with the regions most enriched with Mtw1 further supports that histone H3 , in these regions , is possibly replaced by its centromere-specific variant CENP-A ( Figure 3C ) .",
"To understand the features of M . sympodialis centromeres , we analyzed the centromere DNA sequences for the presence of consensus motifs or structures such as inverted repeats .",
"PhyloGibbs-MP ( Siddharthan et al . , 2005; Siddharthan , 2008 ) predicted a 12-bp-long AT-rich motif common to all of the centromere sequences of M . sympodialis ( Figure 3D ) .",
"We swept the PWM from the PhyloGibbs-MP output across each chromosome of M . sympodialis and counted the number of motif predictions in a sliding 500-bp window , sliding by 100 bp at a time .",
"Sites with log-likelihood-ratio ( LLR ) of >7 . 5 were counted as motif predictions .",
"The LLR is the natural logarithm of the ratio of the likelihood of the12-bp substring arising as a sample from the PWM to the likelihood of it being generic ‘background’ .",
"In each case , the global peak coincides with the centromere ( Figure 3—figure supplement 2A ) .",
"In each chromosome , the centromere region shows between 7 and 13 motif matches , whereas no other 500-bp window shows more than three matches .",
"This suggests that the AT-rich motif is more enriched at the centromeres than at any other region in the M . sympodialis genome ( Figure 3—figure supplement 2A ) .",
"To ensure that this is not an artifact of the GC-poor nature of the centromere , we repeated the analysis with a synthetic shuffled PWM , created by scrambling the order of the columns of the original PWM ( that is , scrambling the positions in the motif while keeping the corresponding weight vectors the same ) .",
"This shuffled motif showed more matches in the centromeres than are seen in the non-centromeric genomic sequence , but significantly fewer than are seen in the authentic centromeric sequences of most chromosomes ( Figure 3—figure supplement 2B ) .",
"A dot-plot analysis was performed to detect the presence of any direct or inverted-repeat structure associated with the centromeres in M . sympodialis .",
"Analysis of all of the centromere sequences and 5-kb flanking sequences using SyMap confirmed the lack of direct/inverted repeat structures ( Figure 3—figure supplement 2C ) .",
"In the absence of any centromere-exclusive DNA sequence , the unique and distinguishing features of centromere regions in M . sympodialis are an AT-rich core region of <1 kb ( average AT content of 78% as compared to the genome average of 41 . 5% ) that is enriched with the 12-bp motif ( Figure 3—figure supplement 3 ) within a kinetochore protein-bound region of 3–5 kb .",
"As expected , the kinetochore-bound region contains a reduced level of histone H3 .",
"Using the unique centromere features identified in M . sympodialis , we predicted one centromere locus on each of the seven M . furfur chromosomes , and these all map to chromosomal GC troughs ( Figure 4—figure supplement 1A , Table 2 ) .",
"We also predicted the centromeres in M . slooffiae , M . globosa , and M . restricta , each of which contains nine chromosomes ( Figure 4—figure supplement 1B–D , Table 2 ) .",
"Each of the predicted centromere regions is enriched with the 12-bp AT-rich motif identified in M . sympodialis centromere sequences as compared to other regions in the genomes ( Figure 4—figure supplement 2 ) .",
"To validate the centromeric loci in M . furfur experimentally , we functionally expressed the centromeric histone H3 variant CENP-A with a 3xFLAG tag at the C-terminus ( Figure 4A ) .",
"We performed ChIP in strain MF001 ( CENP-A-3xFLAG ) and analyzed immunoprecipitated DNA by qPCR using primers specific to each of the seven predicted centromeres and to a centromere-unlinked control locus 1 . 3 Mb away from CEN1 .",
"Enrichment of CENP-A at all seven centromeres over the control locus confirmed that the predicted regions are indeed centromeres in M . furfur CBS14141 ( Figure 4B , C ) .",
"Given the lack of genetic manipulation methods for M . slooffiae and M . globosa , we tested the enrichment of histone H3 at the predicted centromeres in these two species .",
"All of the nine centromeric loci in these two species contained a reduced histone H3 level when compared to a control locus that was unlinked to centromeres ( Figure 4D , E ) .",
"Furthermore , upon analyzing the enrichment profile at one centromere ( CEN1 ) in M . slooffiae , we observed a reduction in the enrichment levels of histone H3 at the GC troughs as compared to the flanking regions ( Figure 4F ) .",
"In the case of M . globosa , the regions spanning a centromere ( CEN2 ) also depicted a similar reduction in the histone H3 levels ( Figure 4G ) .",
"Taken together , the significant reduction in the histone H3 levels at the predicted centromeres , indicative of the presence of CENP-A , suggests that these putative centromere regions are indeed bona fide centromeres in these species .",
"Synteny of genes across centromeres is largely conserved in closely related species ( Byrne and Wolfe , 2005; Padmanabhan et al . , 2008; Yadav et al . , 2018b ) .",
"To understand the transition between different chromosome number states , we analyzed the conservation of gene synteny across centromeres in these species .",
"By mapping gene synteny at the centromeres of M . globosa and M . slooffiae ( each with nine chromosomes ) , compared with that of M . sympodialis ( containing eight chromosomes ) , we found complete gene synteny conservation in eight of the nine centromeres ( Figure 5A , B ) .",
"Thus , syntenic regions of all eight M . sympodialis centromeres are present in the genomes of M . globosa and M . slooffiae .",
"In the case of M . restricta , seven putative centromeres are completely syntenic with M . sympodialis centromeres and one centromere retained partial gene synteny ( Table 3 ) .",
"However , no gene synteny conservation was observed at the centromeres of Chr2 in M . globosa , Chr5 in M . slooffiae , or Chr8 in M . restricta ( Table 3 ) , indicating the loss of a centromere during the transition from the nine-chromosome state to the eight-chromosome state .",
"The GC trough corresponding to MgCEN2/MslCEN5 is flanked by genes that map to MsyChr2 on one arm and MsyChr4 on the other ( Figure 5C , D ) .",
"The centromere region in each of MgChr2 and MslChr5 marks a synteny breakpoint showing no homologous region in the M . sympodialis genome , indicating a loss of this centromere DNA sequence .",
"We also observed that the genes flanking the breakpoint are conserved in M . sympodialis , suggesting that the specific intergenic region was involved ( Figure 5E , F ) .",
"Evidence for the internalization of telomere-adjacent ORFs or for the presence of interstitial telomere repeats that are indicative of telomere–telomere fusions was not detected in the M . sympodialis genome .",
"These observations strongly support our hypothesis that breakage of MgCEN2/MslCEN5 ( or the orthologous ancestral CEN ) and fusion of the two acentric arms to other chromosomes resulted in the reduction in chromosome number observed when comparing these species .",
"To understand the basis of the change in chromosome number from nine to seven in Malassezia species , we compared the synteny of ORFs flanking the M . slooffiae or M . globosa centromeres with that of M . furfur .",
"Of the nine centromeres in M . slooffiae , three centromeres belonged to conserved gene synteny blocks and four others retained partial gene synteny conservation in M . furfur ( Figure 6A , Table 3 ) .",
"A similar pattern of gene synteny conservation was observed between M . globosa and M . furfur ( Figure 6B , Table 3 ) .",
"The genes flanking the remaining two centromeres ( CEN8 and CEN9 ) in M . slooffiae were present in conserved gene synteny blocks in the two arms of MfChr3 ( Figure 6C ) .",
"However , the regions corresponding to CEN8 and CEN9 in M . slooffiae appear to have evolved to decreased AT-richness in M . furfur .",
"A similar centromere inactivation mechanism was observed when CEN8 and CEN9 of M . globosa were compared to the corresponding syntenic regions in M . furfur ( Figure 6D ) .",
"These results suggest centromere inactivation by changes in the centromeric DNA sequence in this species ( Figure 6—figure supplement 1 ) .",
"To trace the ancestral karyotype in Malassezia , we predicted centromeres and inferred chromosome numbers in other species of clades A and B on the basis of GC troughs and gene synteny .",
"We identified putative centromeres in M . dermatis and M . nana in Clade B because of their relatively better-assembled genomes distributed in 18 and 13 scaffolds , respectively .",
"Of these , we could predict eight centromeric regions marked by GC troughs that were also enriched with the 12-bp motif in each species ( Figure 7—figure supplement 1A–B , Figure 7—figure supplement 2A–B and Table 4 ) .",
"Furthermore , in both of these species , the eight putative centromeres shared complete gene synteny with the regions spanning M . sympodialis centromeres , indicating that their common ancestor had eight chromosomes ( green circle in Figure 7A , Figure 7—figure supplement 3 and Table 3 ) .",
"To map the common ancestor in Clade B Malassezia species , we analyzed regions flanking centromeres of Chr2 of M . globosa and Chr8 of M . restricta , both of which mapped to the gene synteny breakpoint of the genome of the Malassezia species that have eight chromosomes suggesting that their common ancestor , named as Ancestor B ( Anc . B ) , also had nine chromosomes ( Figure 7A , Figure 7—figure supplement 3 ) .",
"On the basis of our centromere predictions in Clade B species and synteny analysis , we propose that centromere breakage would have occurred in the common ancestor of M . sympodialis , M . nana , and M . dermatis after divergence from the common ancestor of M . globosa and M . restricta , which retained a nine-chromosome configuration ( Figure 7A , B ) .",
"As mentioned earlier , M . furfur and M . obtusa of Clade A contain seven chromosomes each ( Boekhout and Bosboom , 1994; Boekhout et al . , 1998 ) .",
"To further understand the karyotype variations within this clade , we predicted the chromosome number in Malassezia vespertilionis and Malassezia japonica because their genomes are relatively well assembled ( Sugita et al . , 2003; Lorch et al . , 2018 ) .",
"In both of these species , we were able to predict nine GC troughs , indicative of the centromeres of nine chromosomes ( Figure 7—figure supplement 1C–D , Table 4 ) .",
"In the case of M . vespertilionis , all of the predicted centromeres showed enrichment of the 12-bp motif ( Figure 7—figure supplement 2C ) .",
"However , the 12-bp motif was found to be enriched in all of the predicted centromeres except the centromere of scaffold 7 of M . japonica ( Figure 7—figure supplement 2D ) .",
"The presence of species with nine chromosomes in Clade A suggests that the ancestral state in this clade , Anc .",
"A , also contained nine chromosomes ( Figure 7A ) .",
"We identified nine centromeres in M . slooffiae , the only species in Clade C with a well-assembled genome .",
"The presence of species with nine chromosomes in each of the three clades of Malassezia species , the conservation of gene synteny across orthologous centromeres , and the similar centromere features shared by all nine species analyzed in this study suggest that Malassezia species diverged from a common ancestor that had nine chromosomes with short regional centromeres enriched with the 12-bp AT-rich DNA sequence motif ."
],
[
"In this study , we experimentally validated the chromosome number in M . slooffiae and M . globosa by PFGE analysis .",
"We sequenced and assembled the genomes of M . slooffiae , M . globosa , and M . furfur and compared each one with the genome of M . sympodialis in order to understand the karyotype differences observed in members of the Malassezia species complex .",
"These species represent each of the three major clades of Malassezia species with chromosome numbers ranging from seven to nine .",
"Because centromere loss or gain directly influences the chromosome number of a given species , we experimentally identified the centromeres of these representative species to understand the mechanisms of karyotype diversity .",
"Kinetochore proteins are useful tools in identifying the centromeres of an organism .",
"The localization of the evolutionarily conserved kinetochore protein Mtw1 suggested that kinetochores are clustered throughout the cell cycle in M . sympodialis .",
"ChIP-sequencing analysis identified short regional ( <5 kb long ) centromeres in M . sympodialis that are depleted of histone H3 , but enriched with an AT-rich sequence motif .",
"The identification of centromeres in M . slooffiae , M . globosa , and M . furfur further suggested that centromere properties are conserved across these Malassezia species .",
"By predicting putative centromeres in five other species and by considering four species with experimentally mapped centromeres described above across three clades of Malassezia , we concluded that an AT-rich centromere core of <1 kb in length enriched with the 12-bp sequence motif is a potential signature of centromeres in the nine Malassezia species analyzed in this study .",
"Comparative genomics analysis revealed two major mechanisms of centromere inactivation resulting in karyotype change .",
"The presence of a nine-chromosome state in each of the three clades , along with conserved centromere features and conserved gene synteny , helped us infer that the ancestral Malassezia species had nine chromosomes that had short regional centromeres with an AT-rich core enriched with the 12-bp sequence motif .",
"Centromeres in the Malassezia species complex represent the first example of short regional centromeres in basidiomycetes .",
"Centromeres reported in other basidiomycetes , such as those in Cryptococcus and Ustilago species , are of the large regional type ( Yadav et al . , 2018b ) .",
"The Malassezia species analyzed in this study have a significantly smaller genome ( <9 Mb ) than other basidiomycetes and lack RNAi machinery .",
"The occurrence of short regional centromeres in RNAi-deficient Malassezia species is in line with a previous finding showing a reduction in centromere size in RNAi-deficient basidiomycete species as compared to their RNAi-proficient relatives ( Yadav et al . , 2018b ) .",
"With the presence of clustered kinetochores across cell cycle stages , and the absence of key genes encoding the RNAi machinery , these Malassezia species resemble ascomycetes such as many of the CTG clade species with short regional centromeres ( Sanyal et al . , 2004; Nakayashiki et al . , 2006; Padmanabhan et al . , 2008; Kapoor et al . , 2015; Chatterjee et al . , 2016; Yadav et al . , 2018a ) , rather than the basidiomycetes with large regional centromeres .",
"By combining these features , we conclude that the genome size and the presence of complete RNAi machinery could determine the centromere type of a species , irrespective of the phylum to which it belongs .",
"Based on the binding patterns of the kinetochore protein across the M . sympodialis genome , the 3–5-kb long region can be divided into two domains:",
"( a ) an AT-rich CEN core that maps to the intergenic region containing the GC trough , which shows maximum kinetochore binding ( <1 kb ) ; and",
"( b ) the regions flanking the core , which show basal levels of kinetochore protein binding .",
"We observed conservation of the 12-bp AT-rich motif in the centromere core across the nine Malassezia species .",
"It should also be noted that the 12-bp motif is significantly enriched at the centromeres but is not exclusive to the centromeres as it is detected across the chromosomes .",
"We did not observe any orientation bias for these motifs .",
"The functional significance of the frequent occurrence of this motif at centromeres is unknown .",
"It will be intriguing to test the roles played by this motif , the core , and the flanking sequences in centromere function .",
"Testing these domains for centromere function in vivo by making centromeric plasmids in various Malassezia species is challenging at present because of technical limitations .",
"Other than M . sympodialis , M . pachydermatis , and M . furfur , no other Malassezia species have been successfully transformed ( Ianiri et al . , 2016; Celis et al . , 2017; Ianiri et al . , 2017a ) .",
"Moreover , in all of these cases , the genetic manipulations are performed by Agrobacterium-mediated transconjugation , which cannot be used for the introduction of circular plasmids .",
"Hence , the functional significance of the 12-bp motif remains unknown and remains an important question to be addressed in the future to provide an understanding of centromere function in these species .",
"The centromeres in M . sympodialis contain transcribed ORFs , which have also been documented in the centromeres of rice , maize , and Zymoseptoria tritici ( Nagaki et al . , 2004; Wang et al . , 2014 ) .",
"In contrast to these cases , our read count analysis did not reveal any significant difference in the transcription ( RPKM values ) of centromere-associated ORFs and ORFs elsewhere in the genome of M . sympodialis .",
"We posit that the 12-bp AT-rich motif sequences could facilitate the transcription of these genes by recruiting transcription factors that have a possible role in kinetochore assembly .",
"Binding of Cbf1 at CDEI in S . cerevisiae centromeres and of Ams2 at the central core sequences of S . pombe centromeres are classic examples of transcription factors facilitating kinetochore stability in fungal systems ( Hemmerich et al . , 2000; Chen et al . , 2003 ) .",
"A fine regulation of transcription by Cbf1 , Ste12 , and Htz1 , and the resulting low-level cenRNAs , have been implicated in proper chromosome segregation in budding yeast ( Ohkuni and Kitagawa , 2011; Ling and Yuen , 2019 ) .",
"These studies reinforce the role of transcription in centromere function irrespective of the centromere structure .",
"In this study , we report three high-quality chromosome-level genome assemblies and identified centromeres in nine Malassezia species , representing all of the three Malassezia clades with differing numbers of chromosomes .",
"This will serve as a rich resource for comparative genomics in the context of niche adaptation and speciation .",
"Analysis of gene synteny conservation across centromeres using these genomes revealed breakage at the centromere as one of the mechanisms that results in a karyotype change between closely related species — those with nine chromosomes , such as M . slooffiae and M . globosa , and those with eight chromosomes , such as M . sympodialis .",
"Gene synteny breakpoints adjacent to the centromeres have been reported in C . tropicalis , which has seven chromosomes , one less than C . albicans ( Chatterjee et al . , 2016 ) .",
"Centromere loss by breakage was proposed to have reduced the Ashbya gossypii karyotype by one when compared to the pre-whole genome duplication ancestor ( Gordon et al . , 2011 ) .",
"Breakpoints of conserved gene synteny between mammalian and chicken chromosomes were also mapped to the centromeres ( International Chicken Genome Sequencing Consortium , 2004 ) .",
"Similar consequences in the karyotype have been reported in cases where centromeres were experimentally excised .",
"Besides neocentromere formation , survival by fusion of acentric chromosome arms has been shown in S . pombe ( Ishii et al . , 2008 ) .",
"Such fusions are detected upon deletion of centromeres in another basidiomycete , C . deuterogattii ( Schotanus and Heitman , 2019 ) .",
"By comparing the ancestral state ( M . slooffiae ) and other Malassezia species with either the same number or fewer chromosomes , we observed gene synteny breaks that were adjacent to centromeres ( indicated by partial synteny conservation ) , in addition to the break observed at MgCEN2 or MslCEN5 .",
"Does this suggest that Malassezia centromeres are fragile in nature ?",
"We advance the following hypothesis to explain the observed breaks at centromeres .",
"Studies of the common fragile sites in the human genome suggest different forms of replication stress as a major source of instability and subsequent breakage at these sites ( Helmrich et al . , 2011; Letessier et al . , 2011; Ozeri-Galai et al . , 2011 ) .",
"The resolution of the resulting replication fork stall has been shown to be critical for the stability of these fragile sites ( Schwartz et al . , 2005 ) .",
"Studies of the human fragile site FRA16D show that the AT-rich DNA ( Flex1 ) results in fork stalling as a consequence of cruciform or secondary structure formation ( Zhang and Freudenreich , 2007 ) .",
"Centromeres are natural replication fork stall sites in the genome ( Greenfeder and Newlon , 1992; Smith et al . , 1995; Mitra et al . , 2014 ) .",
"The AT-rich core centromere sequence in M . globosa is also predicted to form secondary structures ( Figure 7—figure supplement 4 ) , which can be facilitated by the inherent replication fork stall at the centromeres .",
"Whenever these secondary structures are unresolved and the fork restart fails , double strand breaks ( DSBs ) can occur at the centromeres .",
"Chromosomal breakage and aneuploidy resulting from such defects are known to occur in cancers ( Kops et al . , 2005 ) .",
"In mammals , centromeric DSBs are repaired efficiently compared to regions elsewhere in the genome , largely because of the presence of several homology tracts in the form of repetitive DNA sequences and the stiffness provided by the inherent heterochromatic state , which facilitates ligation ( Rief and Löbrich , 2002 ) .",
"Malassezia species are haploid in nature and lack typical pericentric heterochromatin marks .",
"Although the efficiency of centromeric DSB repair in the absence of long tracts of homologous sequences is not known in this species complex , we propose that the AT-rich core sequences , by virtue of secondary structure formation during DNA replication , could occasionally undergo DNA breakage at the centromere in Malassezia species .",
"The second mechanism of chromosome number reduction suggested by our analyses comparing the M . furfur genome with the genomes of M . slooffiae or M . globosa involves the inactivation of centromeres in the process of transition from a nine-chromosome state to a seven-chromosome state .",
"Centromere inactivation occurs in cases involving the fusion of centric chromosomal fragments , stabilizing the fusion product and generating a functionally monocentric chromosome , as seen during the origin of human Chr2 from the ancestor shared with the great apes ( Yunis and Prakash , 1982; IJdo et al . , 1991 ) .",
"A larger proportion of known centromere inactivation events were shown to be mediated by epigenetic modifications , in which inactivated centromeres are enriched with marks such as H3K9me2/3 , H3K27me2/3 , or DNA methylation , emphasizing the role of heterochromatin in this process ( Zhang et al . , 2010; Koo et al . , 2011; Sato et al . , 2012 ) .",
"Deletion of the centromere sequence corresponding to kinetochore binding has also been reported as an alternate mechanism , albeit a less frequent mechanism , in both humans and in yeast ( Stimpson et al . , 2010; Gordon et al . , 2011; Sato et al . , 2012 ) .",
"In difference to what might be expected according to the two modes described above , we observed divergence in the sequences corresponding to the inactivated centromeres ( CEN8 and CEN9 of both M . slooffiae and M . globosa ) in the arms of M . furfur Chr3 , resulting in the loss of AT-richness of these centromere core regions ( Figure 7C ) .",
"This is also suggestive of a functional role for AT-rich DNA in centromere function in these species .",
"A change in chromosome number between two closely related species such as C . albicans and C . tropicalis is associated with a change in centromere structure: centromeres in C . albicans are unique short regions that are epigenetically regulated , whereas those in C . tropicalis are associated with genetically defined homogenized inverted repeats ( Chatterjee et al . , 2016 ) .",
"Strikingly , in both the transitions described above for Malassezia species , we did not observe any change in the centromere structure .",
"The emergence of evolutionarily new centromeres , as seen in primate evolution , was not detected in Malassezia species ( Rocchi et al . , 2009; Kalitsis and Choo , 2012 ) .",
"This is particularly striking in the absence of conservation of any specific centromere-exclusive DNA sequence .",
"This suggests that a strong driving force helps to maintain the highly conserved centromere properties in closely related Malassezia species that descended from a common ancestor , even after extensive chromosomal rearrangements involving centromeres that might have driven speciation .",
"Furthermore , centromere inactivation/loss of centromere function seems to be a conserved theme mediating variation in chromosome number from unicellular yeast species to metazoans , including primates ."
],
[
"Malassezia strains were grown on modified Dixon's media ( malt extract 36 g/L , desiccated ox-bile 20 g/L , Tween40 10 mL/L , peptone 6 g/L , glycerol 2 mL/L , oleic acid 2 . 89 mL/L ) .",
"M . sympodialis , M . furfur strains were grown at 30°C .",
"Cultures of M . globosa and M . slooffiae were grown at 32°C .",
"The allele for N-terminal tagging of Mtw1 with GFP was prepared by gap repair in the Saccharomyces cerevisiae BY4741 strain ( Eckert-Boulet et al . , 2012 ) .",
"Briefly , a 1 . 6-kb fragment consisting of the upstream and promoter sequence of the MTW1 gene and a 1 . 6-kb fragment having the MTW1 ORF ( Protein ID: SHO76526 ) along with the downstream sequence were amplified from M . sympodialis genomic DNA .",
"The GFP ORF ( without the stop codon ) and NAT were amplified from plasmids pVY7 and pAIM1 , respectively .",
"S . cerevisiae was transformed with all four fragments and the linearized plasmid pGI3 ( digested with KpnI and BamHI ) and the epitope-tagged allele were assembled in an ordered way by gap repair .",
"Total DNA was isolated from S . cerevisiae and the E . coli DH5α strain was transformed .",
"The pGFP-Mtw1 construct was screened by restriction digestion and further confirmed by sequencing .",
"The pGFP-Mtw1 construct was used to transform M . sympodialis strain ATCC42132 by Agrobacterium tumefaciens-mediated transconjugation ( Ianiri et al . , 2016; Ianiri et al . , 2017a ) .",
"The allele for C-terminal tagging of CENP-A with a 3xFLAG epitope tag was prepared by gap repair in the Saccharomyces cerevisiae BY4741 strain ( Eckert-Boulet et al . , 2012 ) .",
"Briefly , a 1-kb fragment consisting of the upstream and promoter sequence of the CENP-A gene of M . furfur including the ORF ( CENP-A ORF coordinates in Chr1: 1 , 453 , 468–1 , 453 , 921 ) , as well as a 1-kb fragment containing the sequence downstream of CENP-A ORF , were amplified from M . furfur genomic DNA .",
"The 3xFLAG tag was introduced in the reverse primer annealing to the CENP-A ORF .",
"The NAT marker was amplified from plasmid pAIM1 as above .",
"S . cerevisiae was transformed with all three fragments and plasmid pGI3 ( digested with KpnI and BamHI ) and the epitope-tagged allele were assembled in an ordered way by gap repair .",
"Total DNA was isolated from S . cerevisiae and the E . coli DH5α strain was transformed .",
"The resulting pMF1 construct was screened by restriction digestion and further confirmed by sequencing .",
"The pMF1 construct was used to transform M . furfur strain CBS14141 by Agrobacterium tumefaciens-mediated transconjugation ( Ianiri et al . , 2016; Ianiri et al . , 2017a ) to obtain the epitope-tagged strain MF001 .",
"The GFP-Mtw1 strain was inoculated to 1% v/v from a saturated starter culture grown in mDixon medium .",
"After growth for 6 hr at 30 °C/ 150 rpm , these cells were pelleted at 4 , 000 rpm and washed three times with 1x phosphate-buffered saline ( PBS ) , and the cell suspension was placed on a clean glass slide .",
"A coverslip was placed on the spot and sealed prior to imaging .",
"The images were acquired at room temperature using a laser scanning inverted confocal microscope LSM 880-Airyscan ( ZEISS , Plan Apochromat 63x , NA oil 1 . 4 ) equipped with highly sensitive photo-detectors .",
"The filters used were GFP/FITC 488 excitation and GFP/FITC 500/550 band pass , long pass for emission .",
"Z-stack images were taken every 0 . 3 μm and processed using ZEISS Zen software or ImageJ .",
"All of the images were processed post-acquisition with minimal adjustments to levels and linear contrast until the signals were highlighted .",
"Cells were grown on mDixon’s medium and washed with water by centrifugation at 4000 rpm for 5 min .",
"Cells were resuspended in 10 mL of 5% ( v/v ) 2-mercaptoethanol solution in water and incubated at 30 °C/ 150 rpm for 45 min .",
"The cells were pelleted , washed , and resuspended in 3 mL spheroplasting buffer ( 40 mM citric acid , 120 mM Na2HPO4 and 1 . 2 M sorbitol ) for every 1 . 5 × 109 cells .",
"Cell clumps were dissociated by mild sonication for 30 s using the medium-intensity setting in a Bioruptor ( Diagenode ) .",
"Lysing enzymes from Trichoderma harzianum ( Sigma ) , chitosanase ( Sigma ) , and zymolyase-20T ( MP Biomedicals ) were added at 20 mg/mL , 0 . 2 µg/mL , and 100 µg/mL , respectively .",
"The spheroplasting suspension was incubated at 30°C/65 rpm for 6–8 hr .",
"The suspension was examined under a microscope to estimate the proportion of spheroplasts .",
"Spheroplasts were washed with ice-cold 1xPBS and used as per the experimental design ( adapted from Boekhout , 2003 ) .",
"The GFP-Mtw1 strain was inoculated to 1% ( v/v ) from a saturated starter culture grown in mDixon medium .",
"After growth for 6 hr , the cells were fixed by the addition of formaldehyde to a final concentration of 3 . 7% for 1 hr .",
"Post-fixing , the cells were washed with water and taken for preparation of spheroplasts ( as described above ) .",
"Spheroplasts were washed with ice-cold 1xPBS and resuspended in ice-cold 1xPBS to a cell density suitable for microscopy .",
"Slides for microscopy were washed with water and coated with poly L-lysine ( 15 µL of 10 mg/mL solution per well ) for 5 min at room temperature .",
"The solution was aspirated and washed once with water .",
"The cell suspension was added to each well ( 15–20 µL ) and allowed to stand at room temperature for 5 min .",
"The cell suspension was aspirated and the slides were washed once with water to remove unbound cells .",
"The slides were fixed in ice-cold methanol for 6 min followed by treatment with ice-cold acetone for 30 s .",
"Post fixing , blocking solution ( 2% non-fat skim milk in 1xPBS ) was added to each well , and slides were incubated at room temperature for 30 min .",
"After this , the blocking solution was aspirated and primary antibodies were added ( mouse anti-GFP antibodies [Sigma] at 1:100 dilution ) .",
"After incubation for 1 hr at room temperature , each slide was washed eight times with 1xPBS giving a 2 min incubation for every wash .",
"Secondary antibody solution ( goat anti-mouse-AlexaFluor488 [Invitrogen] at 1:500 dilution ) was added to each well and incubated for 1 hr in the dark at room temperature .",
"Post-incubation , slides were washed as described above .",
"Mounting medium ( DAPI at 100 ng/mL in 70% glycerol ) was added , incubated for 5 min and aspirated out .",
"Slides were sealed with a clean coverslip before imaging .",
"The images were acquired at room temperature using an inverted fluorescence microscope ( ZEISS Axio Observer , Plan Apochromat 100x , NA oil 1 . 4 ) .",
"Z- stack images were taken every 0 . 3 μm and processed using ZEISS Zen software/ImageJ .",
"The ChIP protocol was adapted from that implemented for C . neoformans ( Yadav et al . , 2018b ) .",
"Logarithmically grown cells were fixed with formaldehyde at a final concentration of 1% for 30 min for Mtw1 ChIP and 15 min for CENP-A , histone H3 , and histone H4 ChIP .",
"The reaction was quenched by the addition of glycine to a final concentration of 0 . 135 M . Cells were pelleted and processed for spheroplasting as described above .",
"Spheroplasts were washed once sequentially using 10 mL of the following ice-cold buffers: 1xPBS , Buffer-I ( 0 . 25% Triton X-100 , 10 mM EDTA , 0 . 5 mM EGTA , 10 mM Na-HEPES [pH 6 . 5] ) , and Buffer-II ( 200 mM NaCl , 1 mM EDTA , 0 . 5 mM EGTA , 10 mM Na-HEPES [pH 6 . 5] ) .",
"The pellet after the final wash was resuspended in 1 mL lysis buffer ( 50 mM HEPES [pH 7 . 4] , 1% Triton X-100 , 140 mM NaCl , 0 . 1% Na-deoxycholate , 1 mM EDTA ) for every 1 . 5 × 109 cells .",
"Protease inhibitor cocktail was added to 1x final concentration .",
"The resuspended spheroplasts were sonicated with a Bioruptor ( Diagenode ) using a 30 s ON/OFF pulse at high-intensity mode with intermittent incubation on ice to obtain chromatin fragments in the size range of 100–300 bp .",
"The lysate was cleared after sonication by centrifugation at 13 , 000 rpm for 10 min at 4°C .",
"The input DNA fraction was separated at this step ( 1/10th volume of lysate ) and processed for de-crosslinking by the addition of 400 µL elution buffer ( 0 . 1 M NaHCO3 , 1% SDS ) per 100 µL lysate ( processing for de-crosslinking is mentioned below ) .",
"The remaining lysate was split equally and processed as IP and control samples .",
"For GFP-Mtw1 ChIP , 20 µL GFP-trap beads and blocked agarose beads , respectively , were used for IP and control .",
"For CENP-A-3xFLAG ChIP , 20 µL anti-FLAG affinity gel and blocked agarose beads , respectively , were used for IP and control .",
"In the case of histone H3 or histone H4 ChIP , 5 µL of antibodies were used per IP fraction along with 20 µL Protein-A sepharose beads .",
"Samples were rotated for 6 hr at 4°C .",
"Post incubation , samples were sequentially washed as follows: twice with 1 mL low salt wash buffer ( 0 . 1% SDS , 1% Triton X-100 , 2 mM EDTA , 20 mM Tris [pH 8 . 0] , 150 mM NaCl ) , twice with 1 mL high salt wash buffer ( 0 . 1% SDS , 1% Triton X-100 , 2 mM EDTA , 20 mM Tris [pH 8 . 0] , 500 mM NaCl ) , once with 1 mL LiCl wash buffer ( 0 . 25 M LiCl , 1% NP-40 , 1% Na-deoxycholate , 1 mM EDTA , 10 mM Tris [pH 8 . 0] ) and twice with 1 mL 1xTE ( 10 mM Tris [pH 8 . 0] , 1 mM EDTA ) .",
"Samples were rotated in a rotaspin for 5 min at room temperature for every wash ( 15 min in case of histone H3 or histone H4 ChIP ) .",
"After washing , DNA was eluted from the beads twice using 250 µL elution buffer .",
"The samples for elution were incubated at 65°C for 5 min , rotated for 15 min and then collected by centrifugation .",
"Samples were decrosslinked by the addition of 20 µL 5 M NaCl and incubation at 65°C for 6 hr .",
"Following this , samples were deproteinized by the addition of 10 µL 0 . 5 M EDTA , 20 µL 1 M Tris [pH6 . 8] , 2 µL Proteinase K ( 20 mg/L ) and incubation at 45°C for 2 hr .",
"After incubation , samples were treated with an equal volume of phenol-chloroform-isoamyl alcohol ( 25:24:1 ) mix , and the aqueous phase was extracted by centrifugation .",
"DNA was precipitated by the addition of 1/10th volume of 3 M Na-acetate , 1 µL glycogen ( 20 mg/mL ) , 1 mL absolute ethanol and incubation at −20°C for at least 12 hr .",
"Finally , the samples were harvested by centrifugation at 13 , 000 rpm for 45 min at 4°C followed by washing the pellet once with ice-cold 70% ethanol .",
"Air-dried pellets were then resuspended in 20 µL sterile MilliQ water with 10 µg/mL RNAse .",
"Samples were either processed for library preparation for ChIP-sequencing or analyzed by qPCR with the primers listed in Supplementary file 1 .",
"GFP-Mtw1 ChIP sequencing was performed at the Clevergene Biocorp .",
"Pvt .",
"Ltd . , Bengaluru , India .",
"A total of 46 , 704 , 720 and 63 , 524 , 912 150-bp paired-end reads were obtained for IP and Input samples , respectively .",
"The reads were mapped to the M . sympodialis ATCC42132 genome using Geneious 9 . 0 ( http://www . geneious . com/ ) with default conditions .",
"Each read was allowed to map only once randomly anywhere in the genome .",
"The alignments were exported to BAM files , sorted , and visualized using the Integrative Genomics Viewer ( IGV , Broad Institute ) .",
"The images from IGV were imported into Adobe Photoshop ( Adobe ) and scaled for representation purposes .",
"RNA-sequencing data ( E-MTAB-4589 ) from a previous study was downloaded from the ArrayExpress website , sorted , and visualized using IGV .",
"GC-content was calculated using Geneious 9 . 0 with a sliding window size of 250 bp .",
"The data were exported as wig files and further visualized using IGV .",
"Protein lysates for western blot were prepared by the TCA method .",
"Overnight grown cultures of 1 mL were harvested , washed , and resuspended in 400 μL of 12 . 5% ice-cold TCA solution .",
"The suspension was vortexed briefly and stored at – 20°C for 4–6 hr .",
"The suspension was thawed on ice , pelleted at 14 , 000 rpm for 10 min , and washed twice with 350 μL of 80% acetone ( ice-cold ) .",
"The washed pellets were air-dried completely and resuspended in the desired volume of lysis buffer ( 0 . 1 N NaOH+1% SDS ) .",
"Samples were separated in 12% polyacrylamide gels and transferred onto nitrocellulose membranes .",
"For probing , mouse anti-GFP antibody ( Roche ) and the HRP conjugated goat anti-mouse secondary antibody ( Abcam ) were used at 1:3000 and 1:5000 dilution , respectively , in 2 . 5% skim milk powder in 1xPBS .",
"The blots were developed using Chemiluminescence Ultra substrate ( BioRad ) and imaged using the VersaDoc imaging system ( BioRad ) .",
"For CHEF analysis of M . globosa ( CBS7966 ) and M . slooffiae ( CBS7956 ) , the cells were grown on solid mDixon medium and then collected and resuspended in 1xPBS .",
"CHEF plugs were prepared as described in previous studies ( Sun et al . , 2014; Sun et al . , 2017 ) .",
"Chromosomes were separated in 1% Megabase certified agarose gels made with 0 . 5 × TBE , using a BioRad CHEF-DR II System running at 3 . 2 V/cm with linear ramping switching time from 90 to 360 s for 120 hr in 0 . 5xTBE at 14°C .",
"The gel was stained with EtBr and visualized under UV .",
"For the chromoblot analyses of M . globosa ( CBS7966 ) , the gel from the CHEF analysis was first transferred to a membrane , and the resulting chromoblots were then hybridized sequentially with four probes from chromosomes 3 , 4 , 5 , and 6 of the CBS7966 genome assembly , respectively ( see the Supplementary file 1 for the primer information ) , as described in previous studies ( Findley et al . , 2012; Yadav et al . , 2018b ) .",
"Sequence reads were assembled with HGAP3 included in SMRTPortal v2 . 3 ( PacBio , Menlo Park , CA , USA ) and default parameters , except for the genome size set to 9 Mb .",
"Assembly completeness was evaluated by checking for telomeric repeats .",
"Non-telomeric contig-ends were aligned to other contigs using BLAST and unique overlaps were used to build complete chromosomes .",
"Short telomere ends were extended using uniquely mapped reads longer than 10 kb and repolishing of the assembly using the resequencing pipeline in SMRTPortal v2 . 3 .",
"The assembly resulted in 19 contigs , with a total length of 9 . 2 Mb .",
"17 long and one short telomere could be identified .",
"Six contigs had telomeres on both the 5'- and the 3'-end , and thus represent full-length chromosomes ( Chr1 , 2 , 3 , 6 , 7 , and 8 ) .",
"Six contigs had only one telomere and seven contigs had no telomeric sequence .",
"Two contigs without telomeres were from the mitochondrion and two were from the ribosomal repeats .",
"Chromosome 5 was constructed from two contigs ending in ribosomal rDNA repeats .",
"The assembly contains six copies of the repeat , but read coverage suggests a length of 30–40 repeat units that cannot be resolved with the available read-length .",
"The remaining contigs were used to build chromosomes 4 and 9 , which share highly similar 5'-ends .",
"The two ends can be distinguished by two microsatellite expansions .",
"Chromosome 4 had a very short 3'-telomere from the default assembly , but the raw data contained a uniquely mapping read that extended several repeat units past the assembly end .",
"After polishing the reference , all nine chromosomes had clear 5′- and 3′- telomeres .",
"Sequence reads were assembled using HGAP3 included in SMRTPortal v2 . 3 ( PacBio , Menlo Park , CA , USA ) with default parameters .",
"This resulted in an assembly with 14 scaffolds with telomeric repeats at both ends in nine contigs .",
"Of the remaining five contigs , three of them could be assigned to mitochondrial DNA on the basis of BLAST analysis with M . globosa .",
"The remaining two contigs , of size 5 . 8 kb and 2 . 3 kb , did not show BLAST hits against the M . globosa or M . sympodialis genomes .",
"Sequence reads generated from CBS14141 were assembled using HGAP3 included in SMRTPortal v2 . 3 ( PacBio , Menlo Park , CA , USA ) with default parameters .",
"This resulted in an assembly with eight scaffolds , of which the nuclear genome was organized in seven scaffolds that had telomere repeats at both ends .",
"Analysis of gene synteny conservation across the centromeres was performed by BLAST as follows .",
"The genomes for M . restricta , M . nana , and M . dermatis were downloaded from the NCBI genomes portal .",
"The PacBio assembled genomes of M . globosa and M . slooffiae were used for synteny analysis .",
"Synteny analysis was done in the context of ORFs flanking the centromeres of M . sympodialis .",
"The protein sequences for each of these ORFs served as the query in BLAST analysis against the genome of other species .",
"The local database for each genome was set up in the Geneious software for this analysis .",
"The percentage identity values for each of the ORFs are mentioned in the boxes in Figure 7—figure supplement 3 .",
"In addition , synteny analyses between M . globosa and M . sympodialis were conducted with megablast ( word size: 28 ) and plotted together with GC content ( calculated as the deviation from the genomic mean , in non-overlapping 1-kb windows ) , using Circos ( v0 . 69–6 ) ( Krzywinski et al . , 2009 ) .",
"Additional whole-genome alignments were conducted with Satsuma ( Grabherr et al . , 2010 ) , with default parameters .",
"The linear synteny comparisons shown in Figures 5 and 6 were generated with the Python application EasyFig ( Sullivan et al . , 2011 ) .",
"To reconstruct the phylogenetic relationship among the nine Malassezia species selected , orthologs were identified using the bidirectional best-hit ( BDBH ) , COGtriangles ( v2 . 1 ) , and OrthoMCL ( v1 . 4 ) algorithms implemented in the GET_HOMOLOGUES software package ( Contreras-Moreira and Vinuesa , 2013 ) .",
"The proteome of M . sympodialis ATCC42132 was used as a reference .",
"A phylogeny was inferred from a set of 738 protein sequences as follows .",
"Individual protein sequences were aligned using MAFFT v7 . 310 ( L-INS-i strategy ) , and poorly aligned regions were trimmed with TrimAl ( -gappyout ) .",
"The resulting alignments were concatenated to obtain a final supermatrix consisting of a total of 441 , 200 amino acid sites ( 159 , 270 parsimony-informative ) .",
"This sequence was input to IQ-TREE v1 . 6 . 5 ( Nguyen et al . , 2015 ) and a maximum likelihood phylogeny was estimated using the LG+F+R4 amino acid model of substitution .",
"Branch support values were obtained from 10 , 000 replicates of both ultrafast bootstrap approximation ( UFBoot ) and the nonparametric variant of the approximate likelihood ratio test ( SH-aLRT ) implemented in IQ-TREE .",
"The best likelihood tree was graphically visualized with iTOL v4 . 3 . 3 ( Letunic and Bork , 2019 ) ."
]
] | [
"Genomic rearrangements associated with speciation often result in variation in chromosome number among closely related species .",
"Malassezia species show variable karyotypes ranging between six and nine chromosomes .",
"Here , we experimentally identified all eight centromeres in M . sympodialis as 3–5-kb long kinetochore-bound regions that span an AT-rich core and are depleted of the canonical histone H3 .",
"Centromeres of similar sequence features were identified as CENP-A-rich regions in Malassezia furfur , which has seven chromosomes , and histone H3 depleted regions in Malassezia slooffiae and Malassezia globosa with nine chromosomes each .",
"Analysis of synteny conservation across centromeres with newly generated chromosome-level genome assemblies suggests two distinct mechanisms of chromosome number reduction from an inferred nine-chromosome ancestral state:",
"( a ) chromosome breakage followed by loss of centromere DNA and",
"( b ) centromere inactivation accompanied by changes in DNA sequence following chromosome–chromosome fusion .",
"We propose that AT-rich centromeres drive karyotype diversity in the Malassezia species complex through breakage and inactivation ."
] | [
"Millions of yeast , bacteria and other microbes live in or on the human body .",
"A type of yeast known as Malassezia is one of the most abundantmicrobes living on our skin .",
"Generally , Malassezia do not cause symptoms in humans but are associated with dandruff , dermatitis and other skin conditions in susceptible individuals .",
"They have also been found in the human gut , where they exacerbate Crohn’s disease and pancreatic cancer .",
"There are 18 closely related species of Malassezia and all have an unusually small amount of genetic material compared with other types of yeast .",
"In yeast , like in humans , the genetic material is divided among several chromosomes .",
"The number of chromosomes in different Malassezia species varies between six and nine .",
"A region of each chromosome known as the centromere is responsible for ensuring that the equal numbers of chromosomes are passed on to their offspring .",
"This means that any defects in centromeres can lead to the daughter yeast cells inheriting unequal numbers of chromosomes .",
"Changes in chromosome number can drive the evolution of new species , but it remains unclear if and how centromere loss may have contributed to the evolution of Malassezia species .",
"Sankaranarayanan et al . have now used biochemical , molecular genetic , and comparative genomic approaches to study the chromosomes of Malassezia species .",
"The experiments revealed that nine Malassezia species had centromeres that shared common features such as being rich in adenine and thymine nucleotides , two of the building blocks of DNA .",
"Sankaranarayanan et al . propose that these adenines and thymines make the centromeres more fragile leading to occasional breaks .",
"This may have contributed to the loss of centromeres in some Malassezia cells and helped new species to evolve with fewer chromosomes .",
"A better understanding of how Malassezia organize their genetic material should enable in-depth studies of how these yeasts interact with their human hosts and how they contribute to skin disease , cancer , Crohn’s disease and other health conditions .",
"More broadly , these findings may help scientists to better understand how changes in chromosomes cause new species to evolve ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"computational and systems biology",
"cancer biology"
] | Non-coding cancer driver candidates identified with a sample- and position-specific model of the somatic mutation rate | elife-21778-v3 | [
[
"Cancer is caused by somatically acquired changes in the DNA sequence of genomes ( Stratton et al . , 2009 ) .",
"Recently , large-scale sequencing of cancer-genomes coordinated by the International Cancer Genome Consortium ( ICGC ) , The Cancer Genome Atlas ( TCGA ) , and others has catalogued the molecular changes across hundreds of cancer samples ( Hudson et al . , 2010; Weinstein et al . , 2013 ) .",
"The quest is now to analyze and understand the role of these changes in cancer development .",
"The aberrations in non-coding regions are of particular interest as they have only become evident with the advent of whole cancer-genomes .",
"Here , we develop the method ncdDetect for non-coding cancer driver detection .",
"The method captures the heterogeneities of the mutational processes in cancer and aggregates signals of mutational burden as well as functional impact in the significance evaluation of a candidate driver element .",
"We apply the method to 505 TCGA whole-genomes ( Fredriksson et al . , 2014 ) .",
"Cancer arises by an evolutionary process where natural selection operates on genetic variation stemming from randomly occurring somatic mutations .",
"Thousands of somatic mutations distinguish tumor tissue from healthy tissue , as a result of the mutational processes that cancer cells go through during the lifetime of a cancer patient .",
"The somatic mutations are identified through Next-Generation Sequencing ( NGS ) and commonly labeled according to their effect on cancer development: Driver mutations are subject to positive selection during the evolutionary process of cancer , as they offer the cell a growth advantage and contribute to the expansion of tumors .",
"By definition , driver genes contain one or more driver mutations .",
"Passenger mutations , on the other hand , have a neutral , or perhaps slightly negative , fitness contribution to the cell , and accumulate as passive passengers during the evolutionary process of cancer ( Stratton et al . , 2009; Pon and Marra , 2015 ) .",
"Many more passenger than driver mutations exist in cancer cells , and distinguishing the two is challenging ( Marx , 2014 ) .",
"Typically , the strategy is to search for signs of recurrent positive selection across a set of cancer genomes .",
"Signs of recurrent positive selection across cancer genomes can be detected by comparing the somatic mutation frequency to an estimated background mutation rate ( Pon and Marra , 2015 ) .",
"However , modeling the background mutation rate is complicated as it varies along the genome with a large degree of heterogeneity ( Lawrence et al . , 2013; Polak et al . , 2015; Alexandrov et al . , 2013 ) .",
"Not only does the mutation rate in cancer exhibit high variation between different cancer types; this is also the case between different samples of the same cancer type .",
"Furthermore , the mutational processes are affected by various genomic features , primarily replication timing , expression , and the position-specific sequence context ( Lawrence et al . , 2013; Bertl et al . , 2017 ) .",
"It is thus crucial to take these features into account when estimating the background mutation rate in cancer .",
"Another strategy for detecting signs of positive selection in cancer is to rank mutations according to their impact on protein function ( Marx , 2014 ) .",
"In particular , point mutations might introduce alterations in the amino acid sequence of a protein , and thereby change its function ( Reva et al . , 2011 ) .",
"Systematic mutational screens of cancer exomes have expanded the set of known cancer driver genes over the past decade ( Forbes et al . , 2015 ) .",
"Many tools exist for the identification of such genes ( Dees et al . , 2012; Lawrence et al . , 2013; Gonzalez-Perez and Lopez-Bigas , 2012; Tamborero et al . , 2013; Reimand and Bader , 2013 ) , and at present , 616 cancer driver genes are catalogued as causally implicated in cancer ( Cosmic , 2016 ) .",
"However , less than 2% of the genome codes for protein .",
"While it is established that non-coding elements play diverse roles in regulating the expression of protein-coding genes , only few studies systematically explored the role of non-coding somatic mutations in cancer development ( Weinhold et al . , 2014; Lochovsky et al . , 2015; Melton et al . , 2015; Fredriksson et al . , 2014; Mularoni et al . , 2016; Lanzós et al . , 2017 ) .",
"The first identified non-coding driver element was the TERT promoter with highly recurrent mutations across several cancer types ( Huang et al . , 2013; Horn et al . , 2013 ) .",
"In general , the functional understanding of non-coding regions is poor compared to protein-coding regions , challenging the interpretation of non-coding mutations ( Khurana et al . , 2016 ) .",
"We develop the method ncdDetect for detection of non-coding cancer driver elements .",
"With this method , we consider the frequency of mutations alongside their functional impact to reveal signs of recurrent positive selection across cancer genomes .",
"In particular , the observed mutation frequency is compared to a sample- and position-specific background mutation rate , which is estimated based on various genomic annotations .",
"A scoring scheme ( e . g . position-specific evolutionary-conservation scores ) is applied to further account for functional impact in the significance evaluation of a candidate cancer driver element .",
"To strengthen our conclusions regarding the driver potential of candidate elements , we draw on additional data sources .",
"Non-coding mutations may perturb gene expression patterns , and we thus correlate their presence with expression levels in an independent data set ( Ding et al . , 2015 ) .",
"Likewise , we correlate mutation status with survival information for these candidates .",
"What sets ncdDetect aside from other non-coding driver detection methods is the position-specificity , and the derived ability to include genomic annotations of varying resolution down to the level of individual positions .",
"In one existing non-coding driver detection method , the position- and sample-specific probabilities of mutation are derived , much like in ncdDetect but are then aggregated across a candidate element during significance evaluation ( Melton et al . , 2015 ) .",
"This means that knowledge about the exact position and probability of a mutation is not fully utilized .",
"In another method , the genome is divided into bins according to the average value of replication timing ( Lochovsky et al . , 2015 ) , and in a recent method , the significance evaluation is performed by locally conditioning on the number of observed mutations within a candidate element ( Mularoni et al . , 2016 ) .",
"To our knowledge , no existing non-coding driver detection method derive and apply position- and sample-specific probabilities of mutation in the significance evaluation of a candidate driver element , and allows the use of position-specific scores and accurate evaluation of their expectation across a candidate element .",
"This unique feature of ncdDetect means that candidate elements of arbitrary size and location can be analyzed , and that the potential large variation of mutational probabilities within a candidate element is handled .",
"With ncdDetect , we model the different levels of heterogeneity in the somatic mutation rate known to be at play in cancer and evaluate the relative merit of different position-specific scoring-schemes .",
"The result is a driver detection method tailored for the non-coding part of the genome , and with it we aim to contribute to the understanding of non-coding cancer driver elements ."
],
[
"A key feature of ncdDetect is the application of position- and sample-specific probabilities of mutation .",
"These are obtained by a statistical null model , inferred from somatic mutation calls of a collection of cancer samples ( Material and methods: Statistical null model ) ( Bertl et al . , 2017 ) .",
"The model predicts the mutation rate from a set of explanatory variables , that is genomic annotations ( Figure 1A ) .",
"In the present paper , the null model is trained on 505 whole genomes distributed across 14 different cancer types generated by TCGA ( Fredriksson et al . , 2014 ) ( Figure 1B ) . 10 . 7554/eLife . 21778 . 003Figure 1 . Variation in mutation rate at different scales and various explanatory variables .",
"( A ) The flowchart illustrates the input to the model fit that predicts the position- and sample-specific mutational probabilities .",
"( B ) The number of mutations observed per sample divided into the 14 different cancer types .",
"( C ) The set of genomic annotations used as explanatory variables are illustrated on a 300 kb region of chromosome 1 for the colorectal cancer sample CRC_TCGA-A6-6141-01A .",
"For illustrative purposes , the nucleotide sequence is shown on a 30 bp section of chromosome 1 and trinucleotides likewise on a 5 bp section . DOI: http://dx . doi . org/10 . 7554/eLife . 21778 . 00310 . 7554/eLife . 21778 . 004Figure 1—source data 1 . The number of mutations observed for each of the 505 samples . This data set relates to Figure 1B . DOI: http://dx . doi . org/10 . 7554/eLife . 21778 . 00410 . 7554/eLife . 21778 . 005Figure 1—figure supplement 1 . The average number of mutations observed per sample per bp for each of the considered element types , as well as for intergenic regions . DOI: http://dx . doi . org/10 . 7554/eLife . 21778 . 00510 . 7554/eLife . 21778 . 006Figure 1—figure supplement 1—source data 1 . The number of mutations per sample per bp for the defined element types . This data set relates to Figure 1—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 21778 . 006 As explanatory variables , the model includes genomic annotations known to correlate with the mutation rate in cancer , as well as annotations we have found to improve the model fit .",
"It is well known that the mutation rate varies between samples ( Lawrence et al . , 2013; Alexandrov et al . , 2013 ) .",
"Indeed , the mean and median number of mutations per sample is approximately 32 × 103 and 8 × 103 , respectively , with a large degree of variation between and within cancer types ( Figure 1B ) .",
"For example , the average number of mutations per sample is 73 times higher for colorectal cancer than for thyroid cancer , and within melanoma cancer , the least mutated sample has 224 times fewer mutations compared to the highest mutated sample .",
"The mutation rate depends on the position-specific sequence context ( Alexandrov et al . , 2013 ) and correlates with replication timing and gene expression level ( Lawrence et al . , 2013 ) .",
"The mutation rate also varies between different types of genomic regions ( Weinhold et al . , 2014 ) .",
"Finally , we find that the local mutation rate in a window around a given genomic position helps to capture unaccounted mutation rate variation and increases the goodness of fit .",
"The complete model specification , including the definition of the local mutation rate , is given in Material and methods: Statistical null model .",
"Consequently , genomic annotations considered as explanatory variables in the null model for each sample are replication timing , tissue-specific gene expression level , trinucleotides ( the nucleotide under consideration and its left and right flanking bases , thus taking into account the sample-specific mutational signature ) , genomic segment ( 3’ and 5’ untranslated regions ( UTRs ) , splice sites , promoter elements and protein-coding genes ) and local mutation rate ( Figure 1C ) .",
"Given these explanatory variables for a specific genomic position , the model predicts the particular probability of observing a mutation of a given type for a specific sample at this particular position .",
"Although ncdDetect is designed for the analysis of non-coding elements , it can also be applied on protein-coding genes .",
"We initially evaluate the performance of different versions of ncdDetect null models and different scoring schemes on protein-coding genes .",
"We then use it to detect driver candidates among promoter elements , splice sites , 5' UTRs and 3' UTRs ( Table 1 , Material and methods: Candidate elements ) .",
"While all the analyses presented here are pan cancer , individual cancer types can be analyzed separately . 10 . 7554/eLife . 21778 . 008Table 1 . Overview of elements analyzed with ncdDetect .",
"Regions located on chromosome X and Y are excluded from the analyses ( Material and methods: Candidate elements ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 21778 . 008Element typeNumber of elementsMedian element length ( bps ) Percentage of genome coveredprotein-coding genes20 , 15312961 . 19promoter elements20 , 0528480 . 69splice sites18 , 682300 . 033’ UTRs19 , 34610071 . 065’ UTRs19 , 0782590 . 25"
],
[
"Non-coding somatic mutations play part in tumour initiation and progression .",
"With the advent of whole genome sequencing , the systematic screening of such mutations is possible .",
"We have developed the method ncdDetect with the goal of detecting non-coding cancer driver elements and thereby gain an understanding of the underlying mechanisms of tumorigenesis .",
"With ncdDetect , we model the heterogeneous neutral background mutation-rate , taking genomic annotations known to correlate with the mutation rate into account .",
"We consider the mutational burden and functional impact to reveal signs of recurrent positive selection across cancer genomes .",
"The position- and sample-specific approach behind ncdDetect sets the stage for a number of distinct types of analyses .",
"The analysis of one contiguous region is a straight-forward application of ncdDetect , as is the combined analysis of disjoint regions , potentially with vastly different background mutation-rates .",
"The flexible setup conveniently ensures that no constraints are necessary when defining the size and location of a particular region of interest .",
"Furthermore , the method can be used to evaluate more complex functional hypotheses than those presented here .",
"For instance , different sets of regions in different samples can be jointly evaluated , and sample- or tissue-specific scoring schemes can be applied directly .",
"Not all the significant non-coding elements can be regarded as true cancer drivers .",
"ncdDetect might falsely identify driver elements ( ‘false positives’ ) for both technical and biological reasons .",
"The false positives stemming from predicting too low a mutation rate in certain regions can be reduced by adding relevant genomic annotations as explanatory variables to the null model .",
"In general , failing to include an explanatory variable that explains variation in the mutation rate will cause too little variation in the predicted mutational probabilities .",
"We handle such overdispersion of the mutation rate by adjusting each sample- and position-specific probability of mutation with an overdispersion-based correction factor .",
"This improves the model fit , although some inflation appears to remain for long elements .",
"We acknowledge that the false-positive rate among long genes is not properly controlled and likely higher than the applied FDR threshold .",
"We therefore continue to strive to improve our model of the site-specific mutational process , which will also improve power .",
"The observed mutation rate also varies for technical reasons .",
"For instance , the power to call mutations and the rate with which mutations are missed , will vary with genomic complexity , including repeats , pseudogenes , etc .",
"The predicted mutational probabilities can thus be further improved by including genomic annotations that correlate with the rate of either false-negative or false-positive mutation calls as explanatory variables .",
"Likewise , ncdDetect might miss true driver elements ( ‘false negatives’ ) .",
"Especially , with the size of the current whole cancer-genome data sets , we lack statistical power to detect infrequently mutated driver elements , or driver elements that may operate within an individual cancer type .",
"This issue will be remedied as larger sets of sequenced whole genomes become available .",
"In the near future , more than 2500 whole genomes will be available from the Pan-Cancer Analysis of Whole Genomes ( PCAWG ) project .",
"However , it is becoming evident that some instances of detected potential driver regions may be explained by local mutational mechanisms rather than recurrent selection ( Sabarinathan et al . , 2016 ) .",
"This emphasizes the importance of critical scrutinization and eventually independent validation of driver candidates emerging from ncdDetect .",
"With ncdDetect , we screen for non-coding cancer drivers and highlight cases of special interest .",
"To gain further evidence for the identified candidates , we correlate the presence of non-coding mutations with gene expression as well as patient survival: We find that mutations in the promoter and in the coding region of a gene in the Base Excision Repair pathway , SMUG1 , correlate with an increase of C→T mutations .",
"We hypothesize that SMUG1 mutations might perturb uracil-DNA glycosylase function and cause a specific mutational phenotype .",
"Although our study is limited to correlative observations between the expected mutational signature for uracil-DNA glycosylase deficiency and mutational presence as well as gene expression , perturbation experiments in cell lines support the hypothesis ( An et al . , 2005 ) .",
"We also identify non-coding regulatory regions that associate with patient survival , including the potential clinically important 5’ UTR of CD1A , the promoter and 3’UTR of PRSS3 , and the 3’UTR of SEC14L1 .",
"Finally , we identify lung cancer mutations in the splice sites of STK11 as potential driver events .",
"By extending the analysis to the larger TCGA data set , we show that these correlate significantly with expression .",
"The patients also show a strong tendency for poorer survival .",
"In this work , we have addressed the challenges associated with distinguishing driver and passenger non-coding mutations .",
"We evaluated three different scoring schemes and found that a conservation-based scheme performed better than mutation counts and log-likelihoods in our setting .",
"For selected candidate cases , we found a significant effect on expression levels and a significant decrease in survival for mutated samples .",
"The combined analyses of mutational impact on expression and survival across cancer types allowed us aggregate evidence and gain power .",
"The screen identified candidates of potential clinical relevance .",
"However , sample sizes remain small and further studies in large independent cohorts are necessary to establish their potential as prognostic biomarkers or therapeutic targets .",
"As we continue to gain larger cancer genomics data sets for driver screens , accurate modelling of the mutational heterogeneity will become increasingly important .",
"This will help control the false-positive rate as the power of the data increases .",
"As our understanding of the general differences in mutational mechanisms between cancer types improves further , this knowledge should be incorporated in ncdDetect ."
],
[
"The statistical null model that enables us to predict position- and sample-specific probabilities of mutation is a multinomial logistic regression model ( Agresti , 2013 ) .",
"The model is described in detail in Bertl et al . , 2017 .",
"Logistic regression has been used to model the background mutation rate in cancer in previous non-coding driver detection studies ( Melton et al . , 2015 ) .",
"The model considers four possible outcomes; transitions ( TS{A→G , G→A} ) , two types of transversions ( TV{A-→T , G→T} and TV{A→C , G→C} ) as well as the reference class of no mutation .",
"The use of logistic regression ensures that the predicted probabilities are restricted to lie in the interval between zero and one .",
"The reference sequence used in the model is the GRCh37 assembly ( hg19 ) for the human genome .",
"The explanatory variables of the model are listed below .",
"The multinomial logistic regression model fit is based on a so-called count-table .",
"For the purpose of creating this data structure , the three numeric variables , replication timing , local mutation rate , and expression level are each discretized into five bins .",
"For each combination of explanatory variable levels ( 505 × 5 × 2 × 16 × 6 × 5 × 5 = 12 , 120 , 000 combinations ) , the number of genomic positions as well as the number of mutations of each type are counted .",
"Before constructing the count-table , all COSMIC genes were excluded from the data set .",
"For fast and memory-efficient estimation , the multinomial logistic regression model is split up into three binomial logistic models ( Begg and Gray , 1984 ) .",
"Estimation is conducted in R ( R Development Core Team , 2008 ) ( RRID:SCR_001905 ) using the function glm4 from the contributed package MatrixModels ( Bates and Maechler , 2015 ) , which provides efficient estimation for GLMs with sparse design matrices .",
"Three-fold multiple imputation is used to handle missing values in the variable replication timing ( Schafer , 1997 ) .",
"The imputed values are randomly drawn from the marginal distribution of observed replication timing values .",
"In the implementation of ncdDetect , the scores must be discrete values .",
"For speed efficiency , integer values are recommended .",
"The conservation scoring scheme is based on the phyloP scores .",
"To ensure that all scores are positive , the phyloP values are shifted by adding 20 to all values .",
"For computational reasons , the values are rounded downwards to the first decimal point , and multiplied by a factor of 10 to create integers .",
"No mutation is associated with a score value of zero .",
"The log-likelihood scoring scheme defines the scores as minus the natural logarithm of the sample- and position-specific neutral somatic mutation probabilities predicted by the null model .",
"The scores are converted into integer values by the same procedure used for the conservation scores .",
"Effectively , this means that positions with no mutations will be given a score of zero .",
"The candidate elements are defined based on the protein-coding transcript annotations of GENCODE version 19 basic annotation set ( GENCODE , 2016 ) .",
"Regions are divided into five categories; protein-coding genes , promoter elements , splice sites , 3' UTRs , and 5' UTRs .",
"The different regions are defined per transcript and collapsed per gene .",
"Splice site regions are defined as the two intronic bases on either side of all internal exons .",
"Promoter elements are defined as 500 bps in either direction from transcriptional start sites .",
"A hierarchy for the categories is defined as protein-coding genes > splice sites > 3' UTRs > 5' UTRs > promoter elements .",
"Bps included in two or more categories are retained only for the category higher in the hierarchy .",
"Region definitions are available in Supplementary file 4 .",
"Elements located on chromosome X and Y are not considered in the analyses conducted here .",
"The number of analyzed elements thus differ from the total number of elements defined ( Table 1 ) .",
"We analyze 19 , 256 protein-coding regions , 19 , 157 promoter elements , 17 , 867 splice sites , 18 , 481 3’ UTRs and 18 , 220 5’ UTRs .",
"The length distributions of the element types are depicted in Figure 5—figure supplement 3 .",
"To correct for overdispersion of the mutation rate , we adjust each sample- and position-specific mutational probability by an overdispersion-based mutation rate correction factor .",
"The correction factor is modeled with a beta binomial model .",
"Details are given in Appendix , section 1 .",
"To support our findings from the 505 whole genome TCGA samples , we obtain mutation calls , expression values and survival data based on the larger set of TCGA exomes ( Weinstein et al . , 2013; Supplementary file 7 ) .",
"These data are applied in the expression analyses , in the analyses of enrichment of G→A mutations in SMUG1 mutated samples , as well as in the survival analyses performed .",
"We obtain mutation calls for 5802 samples .",
"Of these , we remove 348 samples , which are also present in the original 505-sample data set .",
"The final mutation call set thus consists of 5454 TCGA exome samples .",
"We obtain expression data for 8471 samples , and after removing samples present in the 505-sample set , a total of 4295 samples have both mutation calls and expression data available .",
"A total of 5336 TCGA exome samples have both mutation calls and clinical survival data available , after subtracting samples that are also present in the 505 whole genome TCGA sample set .",
"For a given candidate , we perform a two-sided Wilcoxon rank sum test .",
"With this , we test the hypothesis that there is no difference in gene expression levels between samples that are mutated and samples that are not mutated in a given candidate element .",
"Such a test is performed for each individual cancer type , and the p-values are combined across cancer types using Fisher’s method .",
"These analyses are performed for both the 505 whole genome TCGA samples and the 4295 TCGA exome samples with the necessary data available .",
"Data overview and results are available in Supplementary file",
"5 . In order to test if the proportion of G→A ( including C→T ) mutations outside CpG sites are greater for samples harboring a SMUG1 mutation , compared to samples not carrying such a mutation , we first count the 192 ( =4 × 4 × 4 × 3 ) different mutation types ( including left and right flanking bases for a given mutated position ) for each of the samples .",
"For each sample , we count the number of G→A mutations , excluding those in CpG sites and , for melanoma samples , those part of the CC→TT UV induced mutational signature ( Alexandrov and Stratton , 2014 ) .",
"The counts are normalized by the total number of mutations for the sample .",
"These proportions are referred to as the uracil-DNA glycosylase deficiency signature .",
"For a given cancer type , we divide the samples into two groups; those that carry a SMUG1 mutation , and those that do not .",
"A one-sided Wilcoxon rank sum test is performed to test the null hypothesis that SMUG1-mutated samples do not have higher values of the uracil-DNA glycosylase deficiency signature statistic , compared to samples without SMUG1 mutations .",
"Fisher’s method is used to combine p-values across cancer types .",
"This type of analysis is performed for the 505 whole genome TCGA sample set .",
"We further analyze whether there is a correlation between the uracil-DNA glycosylase deficiency signature and SMUG1 , UNG , or SMUG1 × UNG gene expression .",
"This type of analysis is performed for both the 505 whole genome TCGA samples and the 4295 TCGA exome samples with the necessary data available .",
"The uracil-DNA glycosylase deficiency signature statistic calculated on the basis of the TCGA exome data set is based only on captured CDS regions .",
"Results and data overview are available in Supplementary file",
"5 . We investigate the correlation between mutation status and survival data in the following manner: We download clinical data from the TCGA data portal ( TCGA Data Portal , 2016 ) ( running date 01/11/2015 ) using the RTCGAToolbox R library ( Samur , 2014 ) .",
"For a candidate driver element , the difference in survival between mutated and non-mutated samples is tested per cancer type using a one-sided Log-rank test on the Kaplan-Meier estimated survival curves ( Kaplan and Meier , 1958 ) .",
"The tests are only performed when at least four mutations are observed within a given cancer type .",
"Evidence is combined across cancer types with Fisher’s method .",
"The survival analysis is performed for each of the top-50 ranked ncdDetect candidates of each non-coding element type ( promoters , splice sites , 3’ UTRs and 5’ UTRs ) , or all significant elements of a given type .",
"The analyses are performed for both the 505 whole genome TCGA sample set , and the 5336 TCGA exome sample set with mutation calls and clinical survival data available .",
"Results and data overview are available in Supplementary file",
"6 . The use of mathematical convolution on the fine grained sample- and position-specific scores and probabilities makes ncdDetect computationally intensive ( Figure 3—figure supplement 1 ) .",
"Convolution is the procedure of calculating the distribution function of the sum of independent discrete random variables .",
"The algorithm is implemented using dynamic programming ( Touzet and Varré , 2007 ) and can be thought of as filling out a matrix from the bottom left corner to the upper right corner .",
"Convoluting each cell in the matrix has time complexity O ( 1 ) , and the running time is thus determined by the size of the matrix .",
"The time complexity of the algorithm is O ( m × k × smax ) , where m is the element size , k is the number of samples and smax is the maximum score .",
"ncdDetect is implemented in the software environment R ( R Development Core Team , 2008 ) ( RRID:SCR_001905 ) , using the Rcpp and RcppArmadillo packages ( Eddelbuettel , 2016 ) for speed optimization .",
"The core ncdDetect functions to perform convolution are collected in the R package ncdDetectTools available at github . com .",
"The package can be installed using the devtools package ( Tools to Make Developing R Packages Easier , 2016 ) : install_github ( ‘MaleneJuul/ncdDetectTools’ ) .",
"A few examples of the package functionalities are given in the package vignette , also available in the github repository .",
"The null model estimates used for the current application is provided at http://moma . ki . au . dk/ncddetect/ along with a tutorial on how to obtain p-values from these estimates using ncdDetect .",
"Mutational , expression and clinical data for the TCGA samples are administered by dbGaP ( https://dbgap . ncbi . nlm . nih . gov ) ( RRID:SCR_002709 ) .",
"The additional datasets supporting the conclusions of this article are included within the article and its supplementary files ."
]
] | [
"Non-coding mutations may drive cancer development .",
"Statistical detection of non-coding driver regions is challenged by a varying mutation rate and uncertainty of functional impact .",
"Here , we develop a statistically founded non-coding driver-detection method , ncdDetect , which includes sample-specific mutational signatures , long-range mutation rate variation , and position-specific impact measures .",
"Using ncdDetect , we screened non-coding regulatory regions of protein-coding genes across a pan-cancer set of whole-genomes ( n = 505 ) , which top-ranked known drivers and identified new candidates .",
"For individual candidates , presence of non-coding mutations associates with altered expression or decreased patient survival across an independent pan-cancer sample set ( n = 5454 ) .",
"This includes an antigen-presenting gene ( CD1A ) , where 5’UTR mutations correlate significantly with decreased survival in melanoma .",
"Additionally , mutations in a base-excision-repair gene ( SMUG1 ) correlate with a C-to-T mutational-signature .",
"Overall , we find that a rich model of mutational heterogeneity facilitates non-coding driver identification and integrative analysis points to candidates of potential clinical relevance ."
] | [
"Cancers are diseases caused by changes in DNA sequences .",
"Some changes occur in the protein-coding part of the DNA sequence , in other words , in the stretches of DNA that include the instructions to make a given protein .",
"Other changes occur in the remaining parts of the DNA that do not code for proteins , which accounts for about 98% of the human genome .",
"Modern technologies allow us to identify these DNA changes , but , up until recently , this has only been possible for the protein-coding part of the DNA .",
"Many studies have thus analyzed DNA changes in the protein-coding part of the human genome , while the larger , non-coding part remains rather unexplored .",
"Advances in technology means that large datasets are becoming available where changes in DNA sequences are identified across the entire genomes of a collection of cancer patients .",
"However , it is not clear which of these DNA changes play a role in the development of cancer and which are neutral with no effect on cancer .",
"Now , Juul et al . have developed a new method , named 'ncdDetect' , to search the human genome and identify stretches of DNA that when changed give cancer cells an advantage and allow them to grow .",
"Juul et al . refer to these DNA stretches as 'driver elements' , and , after analyzing the genomes from 505 patients with cancer , they identified some known driver elements and some potentially new ones .",
"For example , possible driver elements were found in non-coding parts of the DNA that regulate genes called SMUG1 and CD1A .",
"Both of these genes encode proteins that had been linked to cancer in the past , but driver elements had not previously been described in the nearby non-coding regions .",
"Juul et al . also found a number of possible driver elements that might be important to consider in the treatment of cancers .",
"Importantly , not all the candidate driver elements identified with ncdDetect are true drivers .",
"The changes in DNA vary greatly between different types of cancer and even between different cases of a single type of cancer .",
"Understanding and describing this variation continues to be a challenge in identifying driver elements , and so Juul et al . plan to keep improving the method to make sure that the driver elements it identifies are all trustworthy ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"ecology"
] | Spatial self-organization favors heterotypic cooperation over cheating | elife-00960-v1 | [
[
"Cooperation , providing a benefit available to others at a cost to self , has been postulated to drive major transitions in evolution ( Maynard Smith and Szathmary , 1998 ) .",
"Cooperation may take place between similar individuals contributing and sharing identical benefits ( homotypic cooperation ) or between two populations exchanging distinct benefits such as in some forms of mutualism ( heterotypic cooperation ) .",
"Both homotypic and heterotypic cooperation are vulnerable to cheaters ( Turner and Chao , 1999; Strassmann et al . , 2000; Bronstein , 2001; Rainey and Rainey , 2003; Travisano and Velicer , 2004 ) .",
"Cheaters exploit cooperative benefits without contributing their fair share and are therefore competitively superior to their cooperating counterparts .",
"How might cooperation avoid being taken over by cheaters ?",
"The answer lies in ‘positive assortment’ ( Fletcher and Doebeli , 2009 ) , in which benefit-supplying individuals interact more with other benefit-supplying individuals than with cheaters .",
"In homotypic cooperation that involves genetic relatives , positive assortment can be realized through ‘kin discrimination’ , which is based on the active recognition and preferential treatment of more closely related individuals over distantly related ones ( Sachs et al . , 2004 ) .",
"Positive assortment can also be realized through ‘kin fidelity’ ( Sachs et al . , 2004 ) .",
"For example , restricted migration in a spatial environment causes homotypic cooperators and cheaters to cluster with their respective progeny .",
"This clustering allows cooperators to preferentially interact with each other ( Figure 1A , top ) .",
"Both mechanisms of positive assortment can favor cooperation ( Hamilton , 1964a; Hamilton , 1964b; Maynard Smith , 1964; Chao and Levin , 1981; Nowak and May , 1992; Fletcher and Doebeli , 2006; Kerr et al . , 2006; MacLean and Gudelj , 2006; West et al . , 2006; Lion and Baalen , 2008; Wild et al . , 2009; West and Gardner , 2010 ) .",
"A spatial environment may also impede homotypic cooperation by intensifying competition among cooperators ( Taylor , 1992; Wilson et al . , 1992; West et al . , 2002 ) and in certain cases , by potentially encouraging cheating strategies ( Hauert and Doebeli , 2004 ) . 10 . 7554/eLife . 00960 . 003Figure 1 . A spatial environment favors heterotypic cooperation over cheating .",
"( A ) Top: clustering with self-type can favor homotypic cooperators ( yellow ) over cheaters ( black ) .",
"Bottom: clustering with self-type should not favor heterotypic cooperation since cooperator clusters ( red ) and competitively superior cheater clusters ( blue ) should have equivalent access to the heterotypic cooperative partner ( green ) .",
"( B ) We engineered three yeast strains: a red-fluorescent R→A←L strain requiring lysine and releasing adenine; a green-fluorescent G→L←A strain requiring adenine and releasing lysine; and a cyan-fluorescent C←L strain requiring lysine and not releasing adenine .",
"The three strains purely competed ( ‘Comp’ ) or additionally cooperated and cheated ( ‘Co&Ch’ ) , depending on whether the medium contained or lacked adenine ( ‘Ade’ ) and lysine ( ‘Lys’ ) , respectively .",
"( C ) In competitive communities , R→A←L:C←L dropped below the initial value of 1 ( dotted line ) during community growth due to the fitness advantage of C←L over R→A←L .",
"In contrast , R→A←L:C←L rose above 1 when the strains engaged in cooperation and cheating .",
"Population ratios in experiments were measured using flow cytometry .",
"All communities started from an initial density of 3000 total cells/mm2 with the three strains at a 1:1:1 ratio on top of an agarose column ( Figure 1—figure supplement 1A ) and were analyzed after approximately six to eight generations ( Figure 1—figure supplement 2 ) .",
"p Values are from the Wilcoxon signed rank test , comparing the median with 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 00310 . 7554/eLife . 00960 . 004Figure 1—figure supplement 1 . Community setup .",
"( A ) Unless otherwise stated ( see B ) , experiments and simulations were initiated by randomly distributing the three cell populations on top of a filter placed above an agarose column of equal size .",
"The agarose column was 6 mm in diameter and 11 mm in height .",
"( B ) Alternatively , a 2 mm-diameter spot of cell mixture was deposited at the center of a membrane filter on top of an agarose pad ( 24 mm × 24 mm × 4 mm ) .",
"In this configuration , the initial inoculum spot had ∼8 × 104 total cells/mm2 .",
"Unless otherwise stated , all communities were initiated from equal proportions of R→A←L , G→L←A , and C←L populations . DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 00410 . 7554/eLife . 00960 . 005Figure 1—figure supplement 2 . In experimental spatial communities , cooperators R→A←L are favored over cheaters C←L .",
"( A–D )",
"In the communities of heterotypic cooperation and cheating ( Figure 1—figure supplement 1A ) , the R→A←L:C←L ratio increased over time before leveling off .",
"The initial surface cell density was 3000 cells/mm2 in ( A and B ) and 104 cells/mm2 in ( C and D ) .",
"The ratios did not change considerably in late stages of community growth ( approximately six to eight generations in B and D ) and were therefore used for reporting results ( e . g . , in Figure 1C ) .",
"Since sampling was destructive , all data points were from independent communities grown to various times and generations .",
"The consistency in the data trend highlights small variations among independent spatial communities .",
"The leveling off of the R→A←L:C←L ratio in later generations likely reflects the fact that by that time , not much open space is available for self-organization ( Figure 7 ) .",
"As a reference , 104/mm2 is two generations away from confluence .",
"( E and F )",
"Unlike in ( A–D ) where the growth area was limited to the original inoculation domain ( Figure 1—figure supplement 1A ) , the R→A←L:C←L ratio continued to increase during later generations when the growth medium extended beyond the original inoculation domain ( Figure 1—figure supplement 1B ) .",
"The initial decrease in R→A←L:C←L suggests that C←L was more starvation-tolerant and therefore fitter than R→A←L in this lysine-limited environment before sufficient cell growth and spatial organization occurred .",
"Exp: experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 005 Heterotypic cooperation can occur between populations that are genetically related but phenotypically differentiated , such as between different cell types in a multicellular organism .",
"Alternatively , heterotypic cooperation can involve two genetically unrelated populations ( e . g . , species ) exchanging distinct benefits that are costly to produce .",
"For example , legume plants supply organic carbon and other essential nutrients to rhizobia , and rhizobia reciprocate with fixed nitrogen ( Udvardi and Poole , 2013 ) .",
"Positive assortment in heterotypic cooperation can be achieved through ‘partner choice’ and ‘partner fidelity feedback’ ( Bull and Rice , 1991; Sachs et al . , 2004; Foster and Wenseleers , 2006; Weyl et al . , 2010 ) .",
"In partner choice , active mechanisms ( disruptable through for example mutational or pharmacological means ) enable an individual to differentially reward cooperative instead of non-cooperative partners based on a signal .",
"Thus , discrimination mediated by partner choice can occur in advance of exploitation ( Bull and Rice , 1991 ) .",
"For instance , in the mutualism between client fish and cleaner fish in which cleaner fish obtain food from removing client parasites , client fish recognize and avoid cheating cleaners that also bite healthy tissues ( Bshary , 2002 ) .",
"In partner fidelity feedback , fitness-feedback from repeated interactions ensures that aiding the partner helps self .",
"Examples of partner fidelity feedback can be found in mutualism between hosts and their vertically transmitted symbionts , for example , between eukaryotes and their endosymbioic mitochondria and chloroplasts ( Sachs et al . , 2011 ) .",
"Natural heterotypic cooperative systems often benefit from a combination of partner choice and partner fidelity feedback .",
"Often , for a particular system , it is challenging to determine which mechanism is mainly responsible for fending off cheaters .",
"For instance , the mutualism between fig and fig wasp and between legume and rhizobia have been thought to employ partner choice by some investigators ( Kiers et al . , 2003; Sachs et al . , 2004; Foster and Wenseleers , 2006; Jandér and Herre , 2010 ) and partner fidelity feedback by others ( Weyl et al . , 2010 ) .",
"In this study , using engineered yeast strains and mathematical models devoid of possibilities for partner choice , we examined how through partner fidelity feedback heterotypic cooperation between microbes may be protected against cheaters .",
"Spatial environment , which facilitates repeated interactions between neighboring individuals , has been shown to promote heterotypic cooperation ( Boucher et al . , 1982; Doebeli and Knowlton , 1998; Yamamura et al . , 2004; West et al . , 2007; Harcombe , 2010; Mitri et al . , 2011 ) .",
"However , the mechanism for how partner fidelity feedback unfolds in a spatial environment is not well understood .",
"Specifically , how might spatial correlation in the tendency to contribute arise between genetically unrelated heterotypic cooperators when such correlation was initially absent ( Frank , 1994 ) ?",
"If population viscosity was the sole driving force , then clusters of cooperators and clusters of competitively superior cheaters would be expected to have equivalent access to clusters of heterotypic cooperative partners .",
"This would seem to favor cheaters ( Figure 1A , bottom ) .",
"Instead , we show that in a spatial environment , asymmetric fitness effects of cooperators and cheaters on partners during cell growth into open space drives assortment .",
"This emergence of non-random patterns from initially random spatial distributions , known as self-organization , automatically grants cooperators instead of cheaters more access to heterotypic cooperative partners , disfavoring cheaters .",
"Thus , partner fidelity feedback through self-organization excludes cheaters without evolving recognition mechanisms ."
],
[
"To examine how partner fidelity feedback unfolds in a spatial environment , we started with an engineered experimental system incapable of partner recognition ( Shou et al . , 2007; Waite and Shou , 2012 ) .",
"This system consisted of three reproductively isolated Saccharomyces cerevisiae strains: a green-fluorescent strain requiring adenine and releasing lysine ( G→L←A ) , a red-fluorescent strain requiring lysine and releasing adenine ( R→A←L ) , and a cyan-fluorescent strain requiring lysine and not releasing adenine ( C←L ) .",
"Release of lysine or adenine was caused by metabolite overproduction due to a mutation that made the first enzyme of the biosynthetic pathway ( Lys21 and Ade4 , respectively ) insensitive to end-product inhibition ( Armitt and Woods , 1970; Feller et al . , 1999 ) .",
"C←L still produced adenine for itself at the wild-type level , but without the overproduction mutation , the adenine produced by C←L was not sufficient to support the growth of G→L←A ( Figure supplement 6 in Shou et al . , 2007 ) .",
"These strains engaged in different types of interactions depending on the environment .",
"In minimal medium supplemented with abundant adenine and lysine , they competed for nutrients required by all three strains ( e . g . , glucose and nitrogen ) and limited space ( Figure 1B , ‘Comp’ ) .",
"In minimal medium without supplements , in addition to competition , G→L←A and R→A←L exchanged essential metabolites lysine and adenine ( Shou et al . , 2007 ) , while C←L consumed lysine without releasing adenine .",
"Not overproducing adenine , C←L showed a ∼2% growth rate advantage over R→A←L when lysine was abundant ( competition assay in Figure 1 of Waite and Shou , 2012 ) .",
"In the absence of lysine , there was no significant difference in the death rates of R→A←L and C←L ( Figure S1 in Waite and Shou , 2012 ) .",
"Finally , in media lacking lysine and adenine , binary cocultures of R→A←L and G→L←A could grow from low to high cell densities ( Figure 1 in Shou et al . , 2007 ) , whereas cocultures of C←L and G→L←A failed to grow ( Figure supplement 6 in Shou et al . , 2007 ) .",
"These results collectively suggest that C←L acts as a cheater variant of R→A←L ( Waite and Shou , 2012 ) .",
"In other words , in the absence of supplements , cooperator R→A←L and the competitively superior cheater C←L competed for the lysine supplied by the heterotypic cooperative partner ( partner ) G→L←A ( Figure 1B , ‘Co&Ch’ ) .",
"Cooperator R→A←L ‘reciprocated’ by releasing adenine , which is essential for partner G→L←A , but cheater C←L did not release adenine .",
"We first verified that a spatial environment could stabilize heterotypic cooperation against a competitively superior cheater in our system .",
"We initiated experimental communities from randomly distributed equal proportions of the three cell populations on agarose pads ( Figure 1—figure supplement 1A ) .",
"During pure competition in the presence of supplemented adenine and lysine , the R→A←L:C←L ratio dropped below the original value of 1 after approximately six to eight generations ( Figure 1C ) , consistent with the known 2% growth advantage of C←L over R→A←L ( Waite and Shou , 2012 ) .",
"In contrast , during cooperation and cheating in the absence of adenine and lysine supplements , cooperating R→A←L was favored over cheating C←L ( Figure 1C , time course in Figure 1—figure supplement 2 ) .",
"If the spatial aspect of the environment is disrupted , either by periodically mixing a community or by growing it as a well-mixed liquid coculture , partner fidelity feedback should not operate and cheaters are expected to be favored over cooperators .",
"However , the ratio of cooperators R→A←L to cheaters C←L in periodically mixed replicate communities varied dramatically ( Figure 2—figure supplement 1 ) .",
"This was because the lysine-limited environment strongly selected for adaptive mutants in C←L and R→A←L ( Waite and Shou , 2012 ) .",
"Thus , C←L and R→A←L engaged in an ‘adaptive race’ ( Waite and Shou , 2012 ) akin to clonal interference: if C←L had the best mutation to grow under lysine limitation , then the coculture was quickly destroyed by cheaters; if R→A←L had the best adaptive mutation , then the coculture quickly purged cheaters .",
"As a result , C←L outcompeted R→A←L or R→A←L outcompeted C←L depending on which population had a better mutation , not because of social interactions .",
"This kind of phenomenon has also been observed for non-engineered cooperating and cheating microbes ( Morgan et al . , 2012 ) .",
"We chose two evolving cocultures in which the C←L populations were increasing in frequency , When we grew these two cocultures in well-mixed and in periodically perturbed spatial environments , C←L was favored ( Figure 2—figure supplement 2; ‘Materials and methods’ ) .",
"However , in a spatial environment , R→A←L was favored ( Figure 2—figure supplement 2 ) , consistent with previous experiments on a different microbial system ( Harcombe , 2010 ) .",
"Our experiments involved non-clonal and non-isogenic populations .",
"In nature , cheaters can be of different species ( such as the non-pollinating wasps of fig ) and therefore not isogenic with their cooperating counterpart .",
"Additionally , upon environmental stresses , originally isogenic cooperators and cheaters can quickly acquire different mutations and become nonisogenic ( Morgan et al . , 2012; Waite and Shou , 2012 ) .",
"Regardless , we resorted to mathematical models to eliminate the confounding influence of adaptive mutations .",
"We extended a three-dimensional individual-based model of community growth ( previously described as the ‘diffusion model’ in Momeni et al . , 2013 ) to include cooperators , cheaters , and heterotypic partners .",
"The main assumptions of this model were: ( 1 ) that the growth of individual cells depended on consumption of the limiting metabolite , and the consumption rate in turn depended on the local concentration of the limiting metabolite according to Michaelis–Menten kinetics; ( 2 ) that spatial distribution of metabolites was governed by release , diffusion , and consumption; and ( 3 ) that cells rearranged when necessary to accommodate new cells as per experimental observations ( ‘Materials and methods’ ) .",
"Parameters of this model ( such as the rates of growth , death , and metabolite consumption and release ) were experimentally determined ( Momeni et al . , 2013 ) , mostly through characterizing properties of monocultures ( Figure 2—source data 1 ) .",
"C←L was assumed to have a constant intrinsic fitness advantage over R→A←L at all lysine concentrations , as modeled by a higher maximum uptake rate ( vm , C>vm , R in ‘The diffusion model’ in ‘Materials and methods’ ) .",
"In the absence of adaptations , simulation results confirmed that a spatial environment favored cooperators over cheaters and that disrupting the spatial aspect of the environment gave cheaters an advantage over cooperators ( Figure 2 ) . 10 . 7554/eLife . 00960 . 006Figure 2 . In simulated communities , a spatial environment is required to promote heterotypic cooperation . The R→A←L:C←L ratios of simulated ( ‘Sim’ ) spatial cooperating and cheating communities were grown either unperturbed ( purple circles ) or periodically mixed ( orange squares ) .",
"To simulate periodic mixing , the arrangement of cells was completely randomized every 12 hr .",
"In each mixing event , the concentration of adenine and lysine throughout the community was assigned to be the average value over the entire community .",
"Error bars show the standard deviation of ratios in six independent communities .",
"The solid black line shows the ratio in a simulated well-mixed liquid coculture using the same parameters as simulated communities on agarose ( Figure 2—source data 1 ) .",
"The fitness advantage of cheater over cooperator was either 2% ( top panel ) or 8% ( bottom panel ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 00610 . 7554/eLife . 00960 . 007Figure 2—source data 1 . Parameter values used in the diffusion model simulations . DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 00710 . 7554/eLife . 00960 . 008Figure 2—figure supplement 1 . Stochastic cheater outcomes in periodically mixed communities of ancestral cooperators , cheaters , and partners . The R→A←L:C←L ratios diverged in six replicate experimental communities periodically mixed during growth .",
"Cell migration during mixing allowed the mutant type most adapted to the low-lysine environment to sweep through the entire community .",
"The most adaptive mutation could occur in either R→A←L or C←L , which led to stochastic outcomes similar to those observed in well-mixed liquid cocultures ( Waite and Shou , 2012 ) .",
"We started these communities from a spot at a density of ∼8 × 104 total cells/mm2 ( Figure 1—figure supplement 1B ) and physically mixed them daily ( approximately every two generations ) using a glass rod .",
"The cells attached to the glass rod ( estimated to be ∼10% of the community population ) were suspended in water and analyzed using flow cytometry .",
"Exp: experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 00810 . 7554/eLife . 00960 . 009Figure 2—figure supplement 2 . A spatial environment is required to favor heterotypic cooperation over cheating .",
"( A ) To preadapt cooperators and cheaters in the lysine-limited coculture environment so that no new mutations of large fitness benefits could quickly arise , we started eight well-mixed replicates ( marked by different symbols ) consisting of rsp5 R→A←L , rsp5 C←L , and the ancestral G→L←A ( ‘Materials and methods’ ) .",
"The rsp5 mutation was previously found to be highly adaptive for the lysine-requiring cells in a lysine-limited environment ( Waite and Shou , 2012 ) .",
"The initial stochastic phase was indicative of additional rounds of adaptive races ( Waite and Shou , 2012 ) between rsp5 R→A←Land rsp5 C←L .",
"After 250 hr , the R→A←L:C←L ratios showed steady trends , suggesting no additional rapid adaptive races .",
"Two of these cultures ( brown ) that had R→A←L:C←L ratios close to 1:1 were revived from frozen stocks .",
"( B ) In the two revived cocultures where the evolved populations were denoted with a “ ʹ ” , R′→A←L:C′←L continued to decline steadily .",
"The broken axis indicates the period of time elapsed during which a small revived inoculum ( 30 μl frozen stock into 200 μl minimal medium and then expanded to 2 ml minimal medium ) grew to detectable densities .",
"( C ) When preadapted communities were grown unperturbed on agarose pads , cooperators were favored as communities grew ( purple circles ) .",
"In contrast , cheaters were favored when the spatial aspect of the environment was either disrupted ( daily mixing after day 5 , orange squares ) or absent ( well-mixed liquid cocultures , black diamonds ) .",
"We started all spatial communities at ∼8 × 104 total cells/mm2 in a ∼2 mm-diameter spot ( Figure 1—figure supplement 1B ) and liquid communities at 5 × 105 total cells/ml in 3 ml SD .",
"Exp: experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 009 During spatial growth of the yeast community , ancestral R→A←L and C←L should also engage in an adaptive race to adapt to the lysine-limited environment .",
"However , unlike in the liquid environment , mutants in R→A←L and C←L were spatially restricted to the neighborhood of their origins , and thus these mutants could not sweep through the entire community .",
"Consequently , population dynamics from different replicates were highly reproducible ( Figure 1—figure supplement 2 ) .",
"Thus , we used ancestral R→A←L and C←L for all other experiments .",
"How might a spatial environment promote heterotypic cooperation ?",
"The relative positioning of cells in a community , the ‘spatial pattern’ of a community , can develop differently based on the fitness effects of cell–cell interactions ( Momeni et al . , 2013 ) .",
"Due to the inability of confocal or two-photon microscopy to yield three-dimensional patterns of cells in yeast communities , we only had access to the top-views ( xy ) and vertical cross-sections ( z ) , with the latter being obtained through cryosectioning ( Momeni and Shou , 2012 ) .",
"In vertical cross-sections , we have previously shown that in the absence of adenine and lysine supplements , R→A←L and G→L←A as a strongly cooperative pair ( i . e . , there is a large fitness gain from interacting with the other population ) are expected to spatially mix ( Momeni et al . , 2013 ) .",
"In contrast , R→A←L and C←L , a competing pair , should form segregated columns , with each column consisting primarily of a single cell type ( Momeni et al . , 2013 ) .",
"Finally , for the commensal pair G→L←A and C←L in which each G→L←A cell can support the local growth of multiple C←L cells , C←L is expected to grow over G→L←A ( Momeni et al . , 2013 ) .",
"However , it is unclear what patterns would form when the three strains grow together in a community and how the patterns might impact cooperators and cheaters differently .",
"To examine how cooperation and cheating might affect community patterns , we compared communities grown in a spatial environment under conditions of competition ( ‘Comp’ ) or cooperation and cheating ( ‘Co&Ch’ ) ( Figure 1B ) .",
"Specifically , equal proportions of R→A←L , G→L←A , and C←L cells were randomly distributed on top of an agarose surface and allowed to grow .",
"Top-views of experimental ( ‘Exp’ ) communities grown in the presence of adenine and lysine supplements revealed that R→A←L , G→L←A , and C←L , when engaged in pure competition , were evenly distributed in the horizontal xy plane ( Figure 3A , top panel ) .",
"This pattern was caused by the initial cells growing into microcolonies which , after running into each other , were forced to grow upward ( Figure 3—figure supplement 1A ) ( Momeni et al . , 2013 ) .",
"Indeed , vertical cross-sections exhibited patterns consistent with our expectations that competitive populations should form segregated columns ( Momeni et al . , 2013 ) ( Figure 3A , bottom panel ) . 10 . 7554/eLife . 00960 . 010Figure 3 . Growing cells self-organize to exclude cheaters from heterotypic cooperators .",
"( A and B )",
"Experimentally , as the initially randomly distributed cells grew , different patterns emerged depending on whether the medium contained or lacked adenine and lysine supplements and consequently whether the dominant cell-cell interaction was respectively competition ( ‘Comp’ ) or cooperation and cheating ( ‘Co&Ch’ ) .",
"Top-views: ‘xy’; vertical sections: ‘z’ .",
"( C ) Compared to C←L , R→A←L had a higher level of association with G→L←A during cooperation and cheating ( ARG/CG>",
"1 ) but not during competition .",
"In ( C ) , the communities were analyzed after approximately six to eight generations .",
"( D , E and F )",
"We observed similar results in the simulated communities .",
"In simulated top-views , higher color intensity indicates a greater number of cells of the corresponding fluorescent color stacked at that position .",
"In simulated vertical cross-sections , low and high color intensity represent dead and live cells , respectively .",
"Scale bar: 100 μm .",
"In C and F , grey: competition; magenta: cooperation and cheating .",
"p Values are from the Mann–Whitney U-test .",
"All communities started from an initial density of 3000 total cells/mm2 ( Figure 1—figure supplement 1A ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 01010 . 7554/eLife . 00960 . 011Figure 3—figure supplement 1 . Cooperation-cheating and pure competition led to distinct community patterns in top-views .",
"( A ) On media supplemented with adenine and lysine , R→A←L , G→L←A , and C←L competed for shared resources .",
"Individual cells initially grew into microcolonies ( hour 5 ) and expanded until they reached other microcolonies ( hours 9–13 ) .",
"Subsequent growth was mainly in the vertical direction due to physical constraints ( hour 46 ) , and top-view patterns remained static ( hours 46–120 ) .",
"( B ) On media not supplemented with adenine or lysine , R→A←L and G→L←A were the heterotypic cooperative pair and C←L was the cheater .",
"After small microcolonies formed ( hour 81 ) , the positive feedback between R→A←L and G→L←A led to the formation of pods consisting of mixed heterotypic cooperators ( red and green ) surrounded by isolated patches of cheaters ( blue ) ( hours 101–171 ) .",
"All parameters in simulations are similar to those in Figure 3 .",
"‘Sim w/delay’ incorporated a 60 hr delay in the death of G→L←A and in the consequent release of lysine according to experimental observations ( Momeni et al . , 2013 ) .",
"This simulation generated results qualitatively similar to simulations without incorporating the delay ( ‘Sim’ ) .",
"For generality , we opted to use simulations without such a delay throughout the paper .",
"Scale bar: 100 μm .",
"Co&Ch: cooperating and cheating; Comp: competing; Exp: experiment; Sim: simulation . DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 01110 . 7554/eLife . 00960 . 012Figure 3—figure supplement 2 . The partner association indexes in simulated communities showed more association between R→A←L and G→L←A than between C←L and G→L←A during cooperation and cheating but not during competition .",
"( A ) Quantifying the partner association index in top-views ( ‘xy’ ) and vertical cross-sections ( ‘z’ ) of simulated communities shows trends similar to the experimental results in Figure 3C .",
"( B ) When quantified using a three-dimensional neighborhood , the partner association indexes showed similar results as in ( A ) .",
"The partner association indexes are less variable when averaged across each three-dimensional community using a three-dimensional neighborhood instead of across each two-dimensional slice of a community using a two-dimensional neighborhood ( compare B with A ) .",
"Co&Ch: cooperating and cheating; Comp: competing; Sim: simulation . DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 012 In the absence of adenine and lysine supplements , the three populations engaged in heterotypic cooperation and cheating in addition to competition for shared resources .",
"Top-views of these experimental communities showed patterns distinct from those of purely competitive communities .",
"Regions dominated by a mixture of the cooperating pair R→A←L and G→L←A appeared isolated from regions dominated by the cheater C←L ( Figure 3B , top panel and Figure 3—figure supplement 1B ) .",
"Vertical cross-sections of these communities revealed that cooperating R→A←L and G→L←A intermixed and formed tall ‘pods’ .",
"In contrast , cheating C←L was relegated to the periphery of these pods and grew relatively poorly ( Figure 3B , bottom panel ) .",
"To quantify differential partner association , the result of partner fidelity feedback , we define ‘partner association index’ ARG/CG: for those R→A←L and C←L cells bordering at least one other cell type ( ‘Materials and methods’ ) , ARG/CG is the ratio of the average number of G→L←A in the immediate neighborhood of R→A←L to the average number of G→L←A in the immediate neighborhood of C←L .",
"A partner association index ARG/CG> 1 indicates more G→L←A neighbors surrounding R→A←L than surrounding C←L .",
"Using a two-dimensional neighborhood to quantify ARG/CG in top-views and vertical cross-sections , we found that ARG/CG significantly exceeded 1 during cooperation and cheating but not during competition ( Figure 3C ) .",
"This self-organization—the formation of non-random patterns from initially randomly distributed individuals purely driven by internal local interactions ( Camazine et al . , 2003; Solé and Bascompte , 2006 ) —automatically makes G→L←A partner more accessible to cooperating R→A←L than to cheating C←L .",
"Similar to the experiments , top-views and vertical cross-sections in the communities simulated through the diffusion model ( ‘Sim’ ) also showed that cooperating R→A←L and G→L←A preferentially associated with each other and formed tall pods , while cheating C←L was isolated ( Figure 3E ) .",
"Such self-organization was absent in pure competition ( Figure 3D ) .",
"We quantified the partner association index of simulated communities using a three-dimensional neighborhood averaged across the entire community ( ARG/CG3D ) , which turned out to be much less variable than analyzing two-dimensional slices ( Figure 3—figure supplement 2 ) .",
"During cooperation and cheating , ARG/CG3D increased from an initial value of 1 to a steady level greater than 1 ( Figure 3F , top ) .",
"This greater-than-1 ARG/CG3D favored cooperating R→A←L over cheating C←L , as the ratio R→A←L:C←L continued to increase even after ARG/CG3D had leveled off ( Figure 3F , bottom ) .",
"In contrast , during pure competition , ARG/CG3D was close to 1 and C←L was favored over R→A←L ( Figure 3F ) .",
"In addition to generating information difficult to obtain from experimental communities ( such as the detailed time course of ARG/CG3D described above ) , simulations enabled us to explore a broader class of cooperator–cheater communities .",
"Simulations showed that self-organization also allowed discrimination among cooperators of varying quality ( Figure 4 ) .",
"Specifically , we initiated diffusion-model simulations using three populations: G→L←A , R→A←L , and R→A , d←L .",
"The adenine release rate of R→A , d←L was a fraction d ( <",
"1 ) of that of R→A←L , and like C←L , R→A , d←L had a constant growth rate advantage over R→A←L at all lysine concentrations .",
"When grown in the absence of supplements , R→A , d←L were isolated and outcompeted by R→A←L .",
"This effect was quantitative , as a lower d value resulted in a larger ARG/RdG ( i . e . , the less R→A , d←L released , the more it was excluded; Figure 4A , B ) , and a greater advantage of R→A←L over R→A , d←L ( Figure 4C , D ) .",
"In contrast , ARG/RdG was not highly sensitive to the intrinsic growth rate advantage of R→A , d←L over R→A←L ( Figure 4—figure supplement 1 ) .",
"R→A , d←L was still strongly disfavored even when it had relatively large ( 50% ) intrinsic growth rate advantage over R→A←L ( Figure 4—figure supplement 1 ) .",
"Taken together , self-organization favors cooperators that supply the most benefits . 10 . 7554/eLife . 00960 . 013Figure 4 . Self-organization leads the most giving cooperator to associate most with the heterotypic cooperative partner , allowing discrimination of cooperators of varying quality . In diffusion model simulations , R→A , d←L produced adenine at a rate d-fold ( 0 ≤ d < 1 ) of the release rate of R→A←L .",
"R→A , d←L had a 5% fitness advantage over R→A←L at all lysine concentrations .",
"R→A , d←L that released less ( smaller values of d ) were isolated more in spatial patterns ( A , steady-state values summarized in B ) and disfavored more as the community grew ( C and D ) .",
"In ( D ) , the fitness advantage of R→A←L over R→A , d←L was calculated from the rate of changes in the ratio R→A←L:R→A , d←L between generations 2 and 6 in ( C ) .",
"In ( B ) and ( D ) , data from six replicates were plotted .",
"The communities were initiated at 3000 total cells/mm2 .",
"Sim: simulation . DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 01310 . 7554/eLife . 00960 . 014Figure 4—figure supplement 1 . Spatial self-organization allowed R→A←L to increase in frequency even when R→A , d=0←L was much fitter . In diffusion model simulations , the percent growth rate advantage of R→A , d←L over R→A←L , marked in the insets , was assumed to be fixed at all concentrations of lysine .",
"Here , d = 0 .",
"The partner association index ( ARG/RdG3D ) did not show strong dependence on the intrinsic advantage of R→A , d←L over R→A←L ( A and B ) .",
"The overall advantage of R→A←L over R→A , d←L in the self-organized communities was reduced when the intrinsic growth rate advantage of R→A , d←L over R→A←L increased ( C ) .",
"However , R→A , d←L were still disfavored compared to R→A←L , and a larger intrinsic advantage of R→A , d←L over R→A←L did not translate proportionally into better overall survival of R→A , d←L in the community ( D ) .",
"The partner association indexes in ( B ) corresponded to communities after seven generations of growth .",
"The R→A←L advantage over R→A , d←L in ( D ) was calculated from changes in the ratio R→A←L :R→A , d←L between generations 2 and 6 in ( C ) .",
"The communities were initiated at 3000 total cells/mm2 .",
"Sim: simulation . DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 01410 . 7554/eLife . 00960 . 015Figure 4—figure supplement 2 . Cheaters with a much higher affinity for cooperative benefits than cooperators can destroy heterotypic cooperation even in a spatial environment . The evolved cheater C←L–LYP1 ( with a mutation in LYP1 ( see CT10 in Table 1 of Waite and Shou . , 2012 ) is much more efficient in taking up lysine at low lysine concentrations than the ancestral cooperator R→A←L ( Waite and Shou , 2012 ) .",
"Thus , the evolved cheater consumed lysine and grew while keeping the lysine concentration at very low levels where the ancestral R→A←L could hardly grow .",
"In a spatial environment with no supplements , starting from equal proportions of R→A←L:G→L←A:C←L–LYP1 , cheaters were favored ( purple circles ) .",
"When supplied with abundant adenine and lysine ( gray triangles ) , the high affinity of C←L–LYP1 for low concentrations of lysine was no longer advantageous; the R→A←L:C←L–LYP1 ratio slowly changed in favor of cheaters in this case , similar to the ‘Comp’ case in Figure 1C .",
"Exp: experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 01510 . 7554/eLife . 00960 . 016Figure 4—figure supplement 3 . In obligatory byproduct mutualism ( benefit production incurs no cost and non-producers have no fitness advantage over producers ) , high levels of non-producers can still destroy byproduct mutualism in a spatial environment . In simulations , we examined the growth of communities of R→A←L , G→L←A , and C←L at different initial ratios in well-mixed ( A and B ) and spatial ( C and D ) environments lacking supplements .",
"The initial population size of G→L←A was always one third of the initial total population size , which was kept constant .",
"R→A←L:C←L varied among 1:10 , 1:1 , and 10:1 ( dashed , solid , and dotted lines , respectively ) .",
"Even though non-producer C←L was assumed to have no fitness advantage over producer R→A←L , high levels of non-producers still destroyed mutualism ( R→A←L:C←L = 1:10 ) in liquid ( B ) and spatial ( D ) environments ( cross indicates the extinction of the community ) .",
"Error bars in ( D ) are standard deviations calculated using six independent replicates .",
"Note that non-producers are disfavored in spatial , but not well-mixed , environment .",
"Sim: simulation . DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 016 One might hypothesize that among the initially randomly distributed cell populations , the occasional fortuitous proximity of a cooperator cell to a partner cell is necessary for self-organization .",
"To examine this hypothesis , we simulated communities starting from a periodic and symmetric initial distribution of cells ( Figure 5A ) .",
"In the absence of initial spatial asymmetry , other stochastic effects such as the initial metabolite-storage state of the cell , cell rearrangement , and death events broke the symmetry and self-organization still emerged ( Figure 5B ) .",
"Even though the final partner association index from random initial distribution was greater than that from periodic distribution , the latter was still significantly greater than 1 ( Figure 5C ) .",
"Consequently , cooperators still outperformed cheaters in periodic initial distribution , even though the degree of outperformance was greater in random initial distribution ( Figure 5D ) .",
"These simulation results suggest that the heterogeneity in the initial spatial distribution of cells promoted but was not required for self-organization . 10 . 7554/eLife . 00960 . 017Figure 5 . Heterogeneity in the initial spatial distribution of cells facilitates but is not required for cheater isolation . Starting from a symmetric and periodic distribution in which all cooperators R→A←L and cheaters C←L had an equal access to the partner G→L←A ( A ) , heterotypic cooperators self-organized ( B and C ) and were favored ( D ) .",
"( B–D ) corresponded to generation 6 .",
"Break of symmetry from the initial symmetric spatial distribution can be due to stochastic effects such as differences in the initial amounts of metabolites cells possessed , death of cells , or the random direction of cell division .",
"Compared to a random initial distribution , communities with a periodic initial distribution showed smaller mean ARG/CG; nonetheless , ARG/CG significantly exceeded 1 ( Wilcoxon signed rank test ) .",
"In these simulations , the growth rate advantage of C←L over R→A←L was assumed to be 10% at all concentrations of lysine .",
"The communities were initiated at 4400 total cells/mm2 .",
"Scale bar: 100 μm .",
"In simulated top-views , higher color intensity indicates a greater number of cells of the corresponding color stacked at that position .",
"Sim: simulation . DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 017 What drives differential partner association during self-organization ?",
"Since self-organization was only observed during cooperation and cheating but not during competition , the very acts of cooperation and cheating are required .",
"When the fitness effects of interactions are the major driving force of patterning , interacting populations are expected to intermix if both supply spatially localized large fitness benefit to the other ( Momeni et al . , 2013 ) .",
"In contrast , lack of benefit to either population causes population segregation ( Momeni et al . , 2013 ) .",
"Thus , we reason that the asymmetry between cooperators and cheaters in their capacity to reciprocate partner’s benefits drives self-organization .",
"To examine how this asymmetry leads to self-organization , we simulated a community in which a center stripe of partners was initially bordered by a stripe of cooperators on one side and cheaters on the other ( Figure 6 ) .",
"This simulation configuration allowed us to examine the process of self-organization from the simplest form of initial symmetry .",
"Near the cooperator side , partners intermixed with cooperators due to the spatially localized large benefits to both ( Momeni et al . , 2013; red and green in Figure 6 ) .",
"In contrast , near the cheater side , the lack of benefits to partners caused minimal intermixing between cheaters and partners ( Momeni et al . , 2013; blue and green in Figure 6 ) .",
"This isolation of cheaters allowed cooperators to increase in frequency despite the intrinsic fitness advantage of cheaters over cooperators . 10 . 7554/eLife . 00960 . 018Figure 6 . Asymmetric fitness effects of cooperators and cheaters on partners drive self-organization . Time progression of self-organization in a simulated community as observed in top-views ( top ) and vertical cross-sections ( bottom ) .",
"Heterotypic cooperative partners ( green ) supply large benefits to both cooperators ( red ) and cheaters ( blue ) .",
"Since the benefit is spatially localized , only cooperators and cheaters that are close to partners will grow .",
"Given that cells dividing toward partners will on average have more access to benefits than those dividing away from partners , both cooperators and cheaters pile over partners .",
"Cooperators reciprocate by supplying a large , but different , localized benefit to the partner , while cheaters do not .",
"Thus , partners grow and pile over cooperators but not cheaters .",
"Consequently , further growth of cooperators is facilitated , while cheaters become isolated and disfavored .",
"In this simulation , cheaters have an 8% fitness advantage over cooperators .",
"To mimic the top-view from microscopy , the top-view in this simulation represents the top-most layer of cells instead of the total intensity integrated over z at each pixel . DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 01810 . 7554/eLife . 00960 . 019Figure 6—figure supplement 1 . Localization of cooperative benefits is required for self-organization . The partner association indexes and the R→A←L:C←L ratios of simulated cooperating and cheating spatial communities were compared at different levels of localization of benefits .",
"‘Co&Ch’ communities had parameters similar to those in Figure 3—figure supplement 2B ( purple circles ) and therefore had normal levels of spatial localization of benefits .",
"In ‘Exc . release’ communities ( brown squares ) , cooperators and partners released excessive amounts of adenine and lysine , respectively ( 200-fold higher compared to the original communities ) .",
"Since the neighboring cells could not consume the released metabolites fast enough , the benefits no longer remained localized to the vicinity of the releasing cell .",
"In ‘Inst . distr . ’ cases ( black diamonds ) , the diffusion coefficient in the community was assumed to be very large , such that any released metabolite was instantly distributed among all cells .",
"When benefits were delocalized either because of excessive metabolite release or rapid distribution of metabolite throughout the community , self-organization was diminished ( A ) and cooperators were disfavored compared to cheaters ( B ) .",
"In all cases , cheaters had an 8% intrinsic fitness advantage over cooperators , and communities were not disturbed ( i . e . , cells were not repositioned ) .",
"Error bars show the standard deviation of ratios in six independent communities .",
"Sim: simulation . DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 019 If the cell densities in the initial inoculum were so high that all cell types were forced to be each other’s immediate neighbor , then the degree of self-organization might be limited .",
"To test whether access to open space is required for self-organization , we initiated communities at high initial cell densities such that two cell layers covered the inoculation spot .",
"We then allowed communities to grow unperturbed in a spatial environment ( Figure 1—figure supplement 1B ) .",
"Compared to the center , the expanding front where open space offered opportunities for self-organization showed a significantly higher partner association index ( Figure 7 , Figure 7—figure supplement 1 ) .",
"In the community center , cooperators were not favored over cheaters .",
"In contrast , R→A←L:C←L increased progressively above the initial value of 1 as the community grew into open space away from the inoculum ( Figure 7 ) .",
"Thus , similar to what has been observed for homotypic cooperation ( Datta et al . , 2013; Van Dyken et al . , 2013 ) , growth into open space during range expansion also favors heterotypic cooperation over cheating . 10 . 7554/eLife . 00960 . 020Figure 7 . Cell growth into open space is required for self-organization and cheater isolation . In an unperturbed spatial environment , a community starting from a high-density ( ∼105 total cells/mm2 ) confluent inoculum expanded to new territories ( purple ) .",
"Compared to ‘Center’ , self-organization was significantly more in ‘Expanding front’ .",
"Consequently , the initially 1:1 R→A←L:C←L ratio changed in favor of cooperators as the community expanded outward but not in ‘Center’ .",
"Sections were collected from three independent communities .",
"Using the Mann–Whitney U-test , the association indexes of the ‘Center’ and of the ‘Expanding front’ were significantly different ( p = 4 × 10−4 ) .",
"Exp: experiment . DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 02010 . 7554/eLife . 00960 . 021Figure 7—figure supplement 1 . Vertical cross-sections of communities started from a high-density inoculum . A schematic diagram ( A ) shows positions ( gray solid lines ) of cross-sections ( B–E ) taken from a community initiated from a high-density inoculum ( dashed circle ) .",
"Vertical cross-sections in ( B ) and ( C ) were 100 μm and 180 μm from the edge of the community , respectively .",
"( D ) and ( E ) show respectively the center portion and the expanding front portion of a vertical cross-section through the initial inoculum region .",
"Cheater patches became smaller or disappeared as the community front expanded out .",
"Scale bar: 200 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 00960 . 021"
],
[
"Using an engineered yeast community and a mathematical model devoid of partner recognition , we examined how partner-fidelity feedback unfolds in a spatial environment at the individual cell level .",
"In our system , partner cells released essential metabolites for cooperators and cheaters , and cooperators reciprocated with a different essential metabolite while cheaters did not .",
"We found that despite an initially random or periodic spatial distribution , cells ‘self-organized’ into a non-random and non-symmetric pattern: cooperators had more partner neighbors than cheaters did .",
"The level of differential partner association , as quantified by the partner association index , is correlated with how much cooperators outperform cheaters despite the intrinsic fitness advantage of cheaters over cooperators .",
"What is required for self-organization ?",
"Self-organization is driven by the asymmetry between cooperators and cheaters in the amount of spatially localized benefits they supply to the heterotypic partner during cell growth into open space .",
"Our previous work has shown that if two distinct populations receive spatially localized large fitness benefits from each other , then the two populations are expected to intermix ( Momeni et al . , 2013 ) .",
"In contrast , when the fitness benefit to at least one population is small , then cell types are not expected to intermix ( Momeni et al . , 2013 ) .",
"Consequently , partners intermix with cooperators but not cheaters ( Figures 3–7 ) .",
"This difference in the tendency to intermix causes differential partner association , which facilitates the growth of cooperators and isolates and disfavors cheaters .",
"Indeed in simulations , if intermixing was prevented through delocalizing benefits ( Momeni et al . , 2013; Allen et al . , 2013 ) , or if intermixing was imposed on all cells through high initial cell densities , self-organization and cooperator advantage over cheaters diminished ( Figure 6—figure supplement 1 , Figure 7 ) .",
"Similar trends are observed in recent works which mathematically examined diffusion of public good in homotypic cooperation ( Allen et al . , 2013; Borenstein et al . , 2013 ) .",
"Simulations showed that spatial self-organization could discriminate among cooperators that supply different levels of benefits ( Figure 4 ) .",
"When cooperative benefits were limiting , the most ‘helpful’ cooperator ended up with the highest number of partners and was most favored .",
"In our mathematical model , we could be sure that this was not due to partner recognition .",
"Thus , in theory , self-organization through partner fidelity feedback is capable of achieving finer levels of discrimination without requiring sophisticated cognition and memory .",
"Without knowing the molecular mechanisms of interactions , one could easily confuse partner fidelity feedback with partner recognition , because both are capable of discriminating partners of varying cooperative qualities .",
"Our work suggests that any argument on partner recognition in a system that is intrinsically spatial ( such as legume and rhizobia and fig and colonizing fig wasp ) will require identifying variants that fail to distinguish cooperators from cheaters .",
"Otherwise , interpreting cheater discrimination as ‘partner choice’ can be misleading .",
"Spatial self-organization can fend off cheaters with large fitness advantages over cooperators ( Figure 4D ) .",
"However , if ( non-isogenic ) cheaters are much better than cooperators at taking up low concentrations of benefits , then cheaters will destroy heterotypic cooperation even in a spatial environment ( experimental results in Figure 4—figure supplement 2 ) .",
"In byproduct mutualism ( benefit production incurs no cost and thus non-producers have no fitness advantage over producers ) , non-producers are excluded and disfavored in a spatial environment ( Figure 4—figure supplement 3 ) .",
"Furthermore , high levels of non-producers can still destroy byproduct mutualism in a spatial environment ( simulation results in Figure 4—figure supplement 3 ) .",
"This is because non-producers compete with producers for benefits from the partner .",
"If the initial level of non-producers is too high , producers receive few benefits and suffer , which in turn negatively impacts the partner .",
"Conceptually , spatial self-organization favoring heterotypic cooperation can be considered as an example of niche construction or environmental feedback ( Lehmann , 2007; Pepper , 2007 ) .",
"Cooperators construct favorable niches and cheaters construct unfavorable niches for partners .",
"Through differential partner association , partners construct better niches for cooperators than for cheaters .",
"Niche construction not only affects the growth of current cells , but also that of the future progeny .",
"This reciprocal niche construction favors heterotypic cooperation over cheating .",
"In summary , spatial self-organization is the mechanism for partner fidelity feedback .",
"Not requiring the evolution of partner recognition ( Travisano and Velicer , 2004; Foster and Wenseleers , 2006 ) , spatial self-organization offers a simple and fundamental mechanism solely driven by acts of strong cooperation and cheating during cell growth into open space .",
"Even in natural heterotypic cooperative systems with long evolutionary histories , self-organization may act either alone or in synergy with potential recognition mechanisms ( Weyl et al . , 2010 ) to exclude cheaters ."
],
[
"R→A←L , G→L←A , and C←L were respectively WS950 ( MATa ste3::kanMX4 lys2Δ0 ade4::ADE4 ( PUR6 ) ADHp-DsRed . T4 ) , WS954 ( MATa ste3::kanMX4 ade8Δ0 lys21::LYS21 ( fbr ) ADHp-venus-YFP ) , and WS962 ( MATa ste3::kanMX4 lys2Δ0 ADHp-CFP ) .",
"To grow yeast communities , agarose columns were prepared by pouring 2× concentrated SD minimal medium ( Guthrie and Fink , 1991 ) with 2% low melting temperature agarose either into flat-bottom 96-well plates ( Figure 1—figure supplement 1A ) or into rectangular petri dishes ( Figure 1—figure supplement 1B ) .",
"Agarose in the rectangular petri dishes was subsequently cut into 24 mm × 24 mm × 4 mm pads .",
"For competition experiments , 2× SD was supplemented with lysine and adenine ( 650 μM and 430 μM final concentrations , respectively ) .",
"The communities on agarose were inoculated either from a uniform distribution of all cells ( Figure 1—figure supplement 1A at 3000 or 10 , 000 total cells/mm2 ) or a high-density inoculum of ∼2 mm diameter ( Figure 1—figure supplement 1B at ∼8 × 104 total cells/mm2 ) , as specified .",
"We grew well-mixed liquid cocultures in 3 ml of 1× SD without supplementing adenine or lysine , with initially 5 × 105 total cells/ml .",
"In all cases , cultures were initiated from equal proportions of R→A←L , G→L←A , and C←L populations .",
"Flow cytometry was used to measure population sizes of different types in a community ( Momeni et al . , 2013 ) .",
"Fluorescent imaging equipment and procedures are described in Momeni et al . ( 2013 ) .",
"Cryosectioning to obtain vertical cross-sections of yeast communities followed Momeni and Shou ( 2012 ) .",
"We quantified the relative association of cooperators and cheaters with the heterotypic partner by dividing the average number of immediate G→L←A neighbors per focal R→A←L cell by the average number of immediate G→L←A neighbors per focal C←L cell when a focal cell neighbored at least one different population ( ARG/CG , partner association index ) .",
"We chose to count the immediate G→L←A neighbors because nearest neighbors presumably had the greatest impact on the growth of the focal cell due to spatially localized benefits .",
"We chose to analyze focal cells neighboring at least one different population because cells surrounded by their own types do not contribute as much to population growth as cells surrounded by partners .",
"In simulations , a three-dimensional neighborhood around each cell was used to quantify the association index ( ARG/CG3D ) , whereas in experiments , a two-dimensional neighborhood was used in two-dimensional top-views or cross-sections of the community .",
"Based on fluorescence intensities in the DsRed , YFP , and CFP channels , cell types were assigned to each pixel in fluorescent images .",
"Pixels having fluorescence intensities less than 30% above the background in all fluorescence channels were defined as ‘no signal’ .",
"Otherwise , fluorescence intensities in each channel were normalized to their respective image-wide 90th percentile values and pixel identity was assigned to be the same as the fluorescence channel with the highest normalized intensity .",
"For cross-sections taken from the center of communities in Figure 7 , the top crown of cross-sections appeared very bright , whereas the middle and lower regions appeared dim ( Figure 7—figure supplement 1 ) .",
"This large dynamic range caused artifacts in cell identification .",
"The intensity thresholds in these sections were therefore manually adjusted to increase the accuracy of cell identification .",
"Eliminating manual adjustments did not alter the conclusions .",
"To simulate the growth of three-dimensional yeast communities , we used the agent-based diffusion model ( Momeni et al . , 2013 ) .",
"In this model , metabolites are released by cooperators and partners , diffuse throughout the community and agarose , and are consumed by cells that need the metabolite .",
"Most parameters were measured experimentally ( Figure 2—source data 1 ) .",
"We provide a summary of the most relevant features of the model below , without repeating the implementation details that can be found in Momeni et al . ( 2013 ) .",
"Cells take up their required metabolites depending on the local concentration of the metabolite according to the Michaelis–Menten equation:vi ( Si ) =vm , iSiSi+Ki , where , i = R→A←L , G→L←A , or C←L corresponding to each cell type , Si is the concentration of the required metabolite for each cell type ( Si = SL , lysine concentration for R→A←L and C←L , and Si = SA , adenine concentration for G→L←A ) , vm , i is the maximum uptake rate when metabolites are abundant , and Ki is assumed to be equal to the Monod constant ( the concentration of limiting nutrient at which half maximal growth rate is achieved ) for each cell type .",
"R→A←L and C←L cells require αL fmole of lysine and G→L←A cells require αA fmole of adenine to produce a new daughter cell .",
"The distribution of metabolites is modeled using the diffusion equation , with uptake and release as sinks and sources .",
"Using simplified notations of SA=SA ( t ) and SL=SL ( t ) at time t , the metabolite distributions after a time step tu are calculated asSA ( t+tu ) =SA+tu[∇⋅ ( D∇SA ) −vm , GSASA+KAnG+γAnR] , andSL ( t+tu ) =SL+tu[∇⋅ ( D∇SL ) −vm , RSLSL+KMM , RnR−vm , CSLSL+KMM , CnC+βLtunG′] .",
"Here , nR , nC , and nG are , respectively , the densities of live R→A←L , C←L , and G→L←A cells in a focal community grid .",
"∇=∂∂xx^+∂∂yy^+∂∂zz^ is the vector differential operator .",
"nG′ is the density of G→L←A cells that die ( which is distributed as binomial ( nG , p ) where p = tu·dG , with dG being the death rate of G→L←A ) and release lysine in the time step tu .",
"D is a spatially varying function representing the diffusion coefficient in the environment ( the value of D was 360 μm2/s inside agarose , 20 μm2/s inside yeast communities , and 0 μm2/s in the surrounding air , according to experimental measurements ) .",
"Considering D as a spatially varying function simplifies the numerical calculations by automatically incorporating the boundary conditions at the community–air interface .",
"vm , i , the maximum uptake rate of the limiting nutrient per cell , relates to the maximum growth rate rm , i through vm , i=αirm , i/ln2 , where αi is the amount of limiting nutrient required to produce a new cell ( αR=αC=αL and αG=αA ) .",
"C←L has a fixed intrinsic fitness advantage over R→A←L at all lysine concentrations .",
"This advantage was modeled as a higher uptake rate for the limiting nutrient .",
"For example , a 2% fitness advantage of cheaters means vm , C=1 . 02 vm , R or equivalently rm , C=1 . 02 rm , R .",
"βL is the amount of lysine released upon the death of a G→L←A cell , and γA is the release rate of adenine per R→A←L cell .",
"C←L cheaters do not release any adenine .",
"For other types of cheaters that release adenine with a lower rate compared to cooperators ( e . g . , in Figure 4 ) , a corresponding release term is included in the equation .",
"To solve this diffusion equation , we follow the above finite difference time-domain equations over time .",
"The diffusion equation is solved over two separate spatial domains ( Momeni et al . , 2013 ) , one containing the agarose ( with a 60 μm grid size ) , and the other containing the community and the air above it ( with a 15 μm grid size ) .",
"These grid sizes accommodated different diffusion coefficients in agarose and in community and represented the average distance nutrient molecules diffuse in 3 . 5 s .",
"When we used the same diffusion coefficient ( 360 μm2/s and one grid size of 50 μm ) for agarose and community , similar results were obtained .",
"No-flow ( ∂Si/∂z=0 ) boundary conditions are applied to the top and bottom surfaces of the simulation domain and periodic boundary conditions are applied to the four vertical sides of the domain .",
"To incorporate the effects of competition for other shared resources , the growth of all cells also depended on , in addition to adenine or lysine , a shared resource ( for instance , glucose ) that was initially supplied in the medium ( Figure 2—source data 1 ) .",
"In such simulations , diffusion and uptake of glucose were also simulated in a way similar to the above equations .",
"Each cell divided only after acquiring enough glucose , in addition to adenine or lysine .",
"Once a cell had accumulated one metabolite sufficient for one cell division , it stopped consuming that metabolite and continued to acquire the second metabolite until a sufficient amount had been acquired to trigger the birth of a daughter cell .",
"In simulations , R→A←L , C←L , and G→L←A cells are initially randomly distributed on the surface of solid medium .",
"The cells start from random initial storage of their required metabolites .",
"In each tu time step , each live cell takes up its required metabolites according to the Michaelis–Menten equation shown above .",
"Each cell type is assumed to require its limiting metabolite and a shared metabolite ( αL lysine for R→A←L and C←L , αA adenine for G→L←A , and αG for the shared glucose for all cell types , all listed in Figure 2—source data",
"1 ) to divide .",
"The state of cells is examined at every τ time interval ( τ = 6 min , which contains several diffusion tu time steps , but is still much shorter than the minimum cell doubling time of ∼2 hr ) .",
"The cells that have acquired the required amount of limiting metabolites divide .",
"Cell divisions in a three-dimensional community often required cell rearrangement .",
"Assumptions concerning cell rearrangement were derived from experimental observations ( Momeni et al . , 2013 ) .",
"Time-lapse images of the growth of a single fluorescent cell into a microcolony showed that the center of the microcolony became brighter due to multiple cell layers when the microcolony grew to larger than a five-cell radius ( Momeni et al . , 2013 ) .",
"Thus , we assumed that each cell initially budded in the horizontal plane and pushed others in its immediate neighborhood to the side along the shortest path to empty space .",
"Once a cell was completely surrounded on each side by roughly five cells , it either budded directly upward with a probability of 70% or randomly budded to one of the sides at the same level , pushing up the displaced cell and all the cells above .",
"The probability of 0 . 7 ( instead of",
"1 ) of dividing directly upward is estimated from experimental observations: when individual green-fluorescent cells were surrounded by many equally fit competing red-fluorescent cells , vertical cross-sections showed that as z increased , the progeny of the green-fluorescent cell ‘diffused’ laterally ( instead of remaining a vertical line ) ( Figure 3—figure supplement 1F in Momeni et al . , 2013 ) .",
"Since we cannot experimentally track all possible outcomes of cell rearrangement , this assumption is a simplification of reality .",
"This simplification could have contributed to the discrepancy between experimental and simulation patterns , although our conclusions from experiments and simulations are similar .",
"The cells also die stochastically corresponding to their fixed death rates ( dR , dG , or dC as listed in Figure 2—source data 1 ) .",
"After each cell state update ( τ ) , the diffusion coefficient is updated: the diffusion coefficient in each community diffusion grid ( 15 μm × 15 μm × 15 μm , maximally containing 3 × 3 × 3 = 27 cells of size 5 μm × 5 μm × 5 μm ) is assumed to be proportional to the occupancy of that grid , changing from 0 to 20 μm2/s .",
"In this three-dimensional agent-based model of community growth , the initial conditions of cells , cell death , random direction of growth , and cell rearrangement are the only sources of stochasticity; metabolite uptake and diffusion of metabolites in the environment are modeled as deterministic phenomena .",
"Most of the parameters used for the simulations ( Figure 2—source data",
"1 ) are measured experimentally .",
"More details of the implementation and assumptions can be found in Momeni et al . ( 2013 ) .",
"An example of the implementation of this model as a MATLAB code is included in additional files ( Source code 1 ) .",
"When the ancestral R→A←L , G→L←A , and C←L were periodically mixed on an agarose pad lacking adenine and lysine , the final R→A←L:C←L ratios were stochastic , either in favor of cheaters or cooperators ( Figure 2—figure supplement 1 ) .",
"This is consistent with previous experiments in liquid cocultures ( Waite and Shou , 2012 ) that also yielded stochastic cheater outcomes due to an adaptive race between R→A←L and C←L .",
"During the adaptive race , both R→A←L and C←L sampled from the same set of mutations that enhanced cell fitness in the lysine-limited cooperative environment .",
"The population with the fittest mutant rapidly dominated the coculture ( Figure 2—figure supplement 1; Waite and Shou , 2012 ) .",
"To mitigate the confounding effect of adaptive race , we used R→A←L and C←L populations preadapted to the cooperative environment .",
"First , R→A←L containing a mutation in RSP5 ( CT8 , see Table 1 in Waite and Shou , 2012 ) , known to significantly improve the fitness of lysine-requiring cells under lysine-limitation ( Waite and Shou , 2012 ) , was crossed to the ancestral C←L to produce a diploid ( WS1421 ) .",
"Sporulation of the diploid yielded a cyan-fluorescent cooperator ( WS1447 ) and a red-fluorescent cheater ( WS1448 ) , both harboring the rsp5 mutation .",
"The temperature sensitivity of this rsp5 allele allowed its easy selection at 37°C .",
"To avoid confusion , we indicate these cooperators and cheaters as rsp5 R→A←L and rsp5 C←L , respectively .",
"Several well-mixed cocultures consisting of rsp5 R→A←L , rsp5 C←L , and the ancestral partner G→L←A ( WS954 ) were initiated at a ratio of 1:1:1 .",
"The initial stochastic phase in population dynamics was indicative of additional rounds of adaptive races ( Waite and Shou , 2012 ) between rsp5 R→A←L and rsp5 C←L ( Figure 2—figure supplement 2A ) .",
"After 250 hr , the R→A←L:C←L ratios showed steady trends , suggesting the absence of further rapid adaptive races ( Figure 2—figure supplement 2A ) .",
"After ∼500 hr , two of the lines ( brown ) that displayed a steady change of the rsp5 R→A←L:rsp5 C←L ratio in favor of cheaters and a final ratio close to 1:1 were frozen down .",
"After reviving these preadapted cocultures ( hereafter marked as “ ’ ” ) , R′→A←L:C′←L continued to decline steadily ( Figure 2—figure supplement 2B ) .",
"Propagating well-mixed liquid cocultures from these lines for an additional 400 hr , we observed that the R′→A←L:C′←L ratio exhibited a steady decline ( Figure 2—figure supplement 2C , ‘In liquid , well-mixed’ ) , suggesting a cheater C′←L fitness advantage of around 8% ( 7 . 5 ± 0 . 8% SD ) over R′→A←L .",
"It should be noted that since R′→A←L and C′←L are not isogenic , the fitness advantage of C′←L over R′→A←L is likely not solely due to the lack of adenine overproduction by C′←L .",
"Nevertheless , this case models an advantage for the cheating type over the cooperating type , similar to what might be observed in nature if cheaters and cooperators are of different species ( Côté and Cheney , 2005; Jandér and Herre , 2010 ) ."
]
] | [
"Heterotypic cooperation—two populations exchanging distinct benefits that are costly to produce—is widespread .",
"Cheaters , exploiting benefits while evading contribution , can undermine cooperation .",
"Two mechanisms can stabilize heterotypic cooperation .",
"In ‘partner choice’ , cooperators recognize and choose cooperating over cheating partners; in ‘partner fidelity feedback’ , fitness-feedback from repeated interactions ensures that aiding your partner helps yourself .",
"How might a spatial environment , which facilitates repeated interactions , promote fitness-feedback ?",
"We examined this process through mathematical models and engineered Saccharomyces cerevisiae strains incapable of recognition .",
"Here , cooperators and their heterotypic cooperative partners ( partners ) exchanged distinct essential metabolites .",
"Cheaters exploited partner-produced metabolites without reciprocating , and were competitively superior to cooperators .",
"Despite initially random spatial distributions , cooperators gained more partner neighbors than cheaters did .",
"The less a cheater contributed , the more it was excluded and disfavored .",
"This self-organization , driven by asymmetric fitness effects of cooperators and cheaters on partners during cell growth into open space , achieves assortment ."
] | [
"Cooperation between individuals of the same species , and also between different species , is known to be important in evolution .",
"Large fish , for example , rely on small cleaner fish to remove parasites , while the small fish benefit from the nutrients in these parasites .",
"However , cooperation can be undermined by other individuals or species who “cheat” by taking advantage of those who cooperate , without providing any benefits in return .",
"For example , some cleaner fish cheat by biting off healthy tissue from their host , in addition to parasites .",
"Genetically-related individuals who cooperate by sharing identical benefits can combat cheaters by giving preferential treatment to their relatives ( a process known as kin discrimination ) or by staying close to the relatives to form clusters ( kin fidelity ) .",
"However , two genetically-unrelated populations that mutually cooperate by sharing different benefits cannot employ these methods to overcome cheaters .",
"Instead they rely on either partner choice or partner fidelity feedback .",
"Partner choice – the approach adopted by cleaner fish and their hosts – relies on one population recognizing a signal from the other population and responding accordingly: for example , large fish observe cleaner fish and approach those that cooperate with their current host and avoid those that cheat .",
"Partner fidelity feedback , on the other hand , relies on repeated interactions between the two populations providing an advantage in terms of evolutionary fitness to both: for example , organelles called mitochondria and chloroplasts live inside cells , helping the cells to harvest energy and providing energy for themselves and the host cells in the process .",
"In some cases – such as the cooperation between figs and fig wasps , or between certain plants and the bacteria that fix nitrogen in their roots – researchers cannot agree if the populations are relying on partner choice or partner fidelity feedback .",
"Now Momeni et al . have used a combination of experiments on yeast and mathematical modeling to explore partner fidelity feedback in greater detail .",
"They started by using genetic engineering techniques to produce two species of yeast that mutually cooperate , each providing a metabolite that is essential to the other , but are not able to recognize each other: this means that these populations cannot rely on partner choice to combat cheaters .",
"Momeni et al . then observed how these two species interacted with each other and a third species of yeast that cheated by consuming one of the metabolites without releasing any metabolite of its own .",
"Momeni et al . found that as long as there was space for the yeast cells to grow into , the two species that cooperated self-organized into mixed clusters , with the cheating species being excluded from these clusters .",
"The self-organization was driven by a positive feedback loop involving the two species that cooperated , with each species helping to increase the fitness of the other .",
"The results of Momeni et al . demonstrate that it is possible for two genetically unrelated populations to cooperate and combat cheaters without the use of partner choice ."
] | 2013 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"cell biology"
] | A functional link between the co-translational protein translocation pathway and the UPR | elife-07426-v2 | [
[
"Secretory and transmembrane proteins are essential for linking intracellular trafficking and extracellular environments and , in metazoans , play broad roles in all aspects of intracellular communication .",
"These proteins contain either a signal sequence or transmembrane domain ( TMD ) that is co-translationally captured by the SRP and targeted to the endoplasmic reticulum ( ER ) membrane ( Akopian et al . , 2013 ) .",
"At the ER membrane , the Sec61 translocon facilitates insertion or translocation of these polypeptides into the ER membrane ( Park and Rapoport , 2012 ) .",
"The ER contains a network of chaperones and enzymes to assist in folding proteins into their native conformations ( Braakman and Bulleid , 2011 ) .",
"When the influx of nascent polypeptides exceeds the ER protein folding capacity , misfolded proteins accumulate in the ER and lead to ER stress .",
"Under ER stress , signaling pathways , collectively termed the Unfolded Protein Response ( UPR ) , are activated to restore ER homeostasis ( Walter and Ron , 2011 ) .",
"The activation of the UPR leads to various cellular processes that include: transcriptional upregulation of UPR target genes , attenuation of translation , activation of ER associated degradation , and ER expansion .",
"However , if ER homeostasis cannot be restored , the UPR induces cell death pathways to eliminate non-functional cells ( Walter and Ron , 2011 ) .",
"The significance of the UPR is underscored by the fact that aberrations in UPR signaling can lead to a multitude of diseased states including neurological disorders , diabetes , and inflammatory disorders ( Wang and Kaufman , 2012 ) .",
"In solid tumors , the UPR is constitutively activated as an adaptive response pathway for survival under adverse conditions , such as hypoxia ( Wang and Kaufman , 2012 ) .",
"Several ER transmembrane proteins act as ER stress sensors ( Walter and Ron , 2011 ) .",
"The most ancient member of these , inositol-requiring enzyme 1 ( Ire1 ) , is conserved from yeast to mammals ( Cox et al . , 1993; Mori et al . , 1993 ) .",
"Ire1 contains an ER luminal domain involved in sensing misfolded proteins and cytoplasmic kinase/RNase domains , which are involved in the activation of downstream pathways .",
"Mammals have two Ire1 paralogs , Ire1α and Ire1β .",
"While Ire1α is a ubiquitously expressed gene , Ire1β expression is restricted to the gastrointestinal tract ( Tirasophon et al . , 1998; Wang et al . , 1998 ) .",
"Upon ER stress , the Ire1α RNase domain is activated by self-oligomerization and subsequently excises a 26 base intron from the cytosolic unspliced form of XBP1u mRNA ( Yoshida et al . , 2001; Calfon et al . , 2002 ) .",
"The resulting mRNA fragments are ligated in the cytosol by RtcB ligase generating the spliced form of XBP1 mRNA ( Jurkin et al . , 2014; Kosmaczewski et al . , 2014; Lu et al . , 2014 ) .",
"The Ire1α mediated splicing step is critical for mounting the UPR , as only the spliced XBP1 mRNA produces an active transcription factor that induces hundreds of genes responsible for increasing ER abundance to accommodate the demand for protein production ( Shaffer et al . , 2004; Sriburi et al . , 2004; Acosta-Alvear et al . , 2007 ) .",
"In addition to XBP1 mRNA splicing , Ire1α also reduces the load of incoming proteins by cleaving ER-localized mRNAs in a process termed regulated Ire1-dependent decay ( RIDD ) ( Hollien and Weissman , 2006; Han et al . , 2009; Hollien et al . , 2009 ) .",
"While considerable attention has been paid to the mechanism of Ire1α activation , little is known about how the low abundant Ire1α efficiently finds and cleaves its mRNA substrates during ER stress .",
"Recent studies have shown that XBP1u mRNA is recruited to the ER membrane through its nascent chain , but the components involved in the specific recruitment of XBP1u mRNA to the Ire1α cleavage site in the ER membrane remain unidentified .",
"In this study , we have discovered a complex between Ire1α and the Sec61 translocon channel in the ER membrane .",
"We show that this interaction is specific by identifying key residues in Ire1α , and that it is stable even during ER stress conditions .",
"Surprisingly , we find that a hydrophobic region in the XBP1u protein mimics a TMD and is co-translationally captured by the signal recognition particle ( SRP ) .",
"The SRP bound XBP1u-ribosome nascent chain ( RNC ) is then delivered to the Sec61 translocon where its mRNA engages with Ire1α .",
"Despite its interaction with the Sec61 translocon , the XBP1u nascent chain inefficiently inserts into the ER membrane due to its weak hydrophobic region .",
"Furthermore , siRNA mediated depletion of SRP , the SRP receptor or the Sec61 translocon in human cells impairs the Ire1α-mediated splicing of XBP1u mRNA .",
"Mutations in Ire1α that disrupts its association with the Sec61 translocon lead to reduced Ire1α-mediated cleavage of ER-targeted mRNAs .",
"Over all , our studies establish an important link between the UPR and the co-translational protein translocation pathway , which ensures efficient cleavage of ER-targeted mRNAs during ER stress conditions ."
],
[
"To investigate how the low abundant Ire1α efficiently finds and cleaves its mRNA substrates , we searched for Ire1α associated proteins that could facilitate interactions with its mRNA substrates .",
"To this end , we performed immunoaffinity purification from detergent solubilized microsomes derived from HEK 293 cells expressing hemagglutinin ( HA ) -tagged Ire1α .",
"The affinity-purified material was subjected to SDS-polyacrylamide gel electrophoresis and bands not present in the control were subjected to analysis by mass spectrometry .",
"Remarkably , in addition to a known Ire1α interacting protein , BiP ( Bertolotti et al . , 2000 ) , we identified all three subunits of the Sec61 translocon ( Sec61α , Sec61β , and Sec61γ ) and Sec63 ( Figure 1A and Figure 1—figure supplement 1 ) .",
"We were intrigued by the interaction between the Sec61 translocon and Ire1α since we reasoned that it could facilitate Ire1α access to RIDD mRNA substrates ( Hollien and Weissman , 2006 ) that are targeted to the Sec61 translocon via their nascent chains .",
"To determine if this interaction occurs at endogenous levels of Ire1α and the Sec61 translocon , we co-immunoprecipitated the endogenous Sec61 translocon from a detergent cell extract of non-transfected HEK 293 cells using Sec61β antibodies .",
"We could detect a significant amount of endogenous Ire1α precipitating with Sec61β and Sec61α by immunoblotting ( IB ) ( Figure 1B ) .",
"Furthermore , immunodepletion of the Sec61 translocon nearly quantitatively depleted the endogenous Ire1α , indicating that almost all Ire1α is in a complex with the Sec61 translocon in cells ( Figure 1—figure supplement 2 ) .",
"Interestingly , this interaction remained stable even after treatment of cells with the ER stress inducer DTT , which impairs protein folding by preventing disulfide bond formation in the ER lumen ( Figure 1B ) .",
"These results implied that Ire1α interaction with the Sec61 translocon might be functionally important during the conditions of ER stress .",
"We further verified that the Sec61 translocon selectively associated with Ire1α , but not with the Ire1α paralogue Ire1β or with the other ER stress sensors PERK ( Harding et al . , 1999; Sood et al . , 2000 ) and ATF6α ( Haze et al . , 1999 ) ( Figure 1C ) .",
"To determine whether the Ire1α interaction with the Sec61 translocon is direct , we treated HA-tagged Ire1α expressing cells with a lysine-reactive reversible crosslinker , DSP .",
"After quenching the crosslinker , the complex was denatured in urea and SDS , which dissociates noncovalently bound proteins , and immunoprecipitated ( IP ) with HA antibodies .",
"The resulting IP was treated with DTT to reverse the crosslinking and analyzed by IB .",
"The α subunit of the Sec61 translocon could be crosslinked with Ire1α , as judged by the increase in Sec61α signal with increasing concentration of crosslinker ( Figure 1D ) , thus supporting a direct interaction between Ire1α and the Sec61 translocon .",
"Consistent with the result from Sec61β immunoprecipitation ( Figure 1C ) , the crosslinked adduct was visible even when cells were treated with DTT before the crosslinker reaction , supporting a model where Ire1α associates with the Sec61 translocon even under ER stress conditions .",
"( Figure 1D ) . 10 . 7554/eLife . 07426 . 003Figure 1 . Identification of a complex between Ire1α and the Sec61 translocon .",
"( A ) The detergent extracts of either microsomes derived from HEK 293 cells ( control ) or cells expressing hemagglutinin ( HA ) -tagged Ire1α were bound to anti-HA resin and eluted with a low pH glycine buffer .",
"The eluted proteins were analyzed by SDS-PAGE and stained with coommassie blue .",
"( B ) The cell lysates from non-transfected HEK 293 cells treated with or without DTT were immunoprecipitated ( IP ) with anti-GFP antibodies as a control or anti-Sec61β antibodies .",
"The bound material was eluted with sample buffer and analyzed along with starting lysates ( input , 2% loading ) by immunoblotting ( IB ) using antibodies against the indicated antigens .",
"Calnexin , an abundant endoplasmic reticulum ( ER ) trans membrane protein was probed as a control .",
"( C ) Cell extracts from HEK 293 cells transfected with the indicated FLAG tagged constructs were subject to IP with FLAG antibody .",
"The resulting samples were analyzed by IB with indicated antibodies .",
"( D ) HEK 293 cells stably expressing HA-tagged Ire1α were either treated with 10 mM DTT or left untreated for 2 hr .",
"Cells were then semipermeabilized with 0 . 015% digitonin and treated with the indicated concentration of DSP crosslinker for 30 min at room temperature .",
"Samples were denatured and IP with anti-HA antibodies .",
"The resulting IP was analyzed by IB .",
"Control denotes non-transfected HEK293 cells . DOI: http://dx . doi . org/10 . 7554/eLife . 07426 . 00310 . 7554/eLife . 07426 . 004Figure 1—figure supplement 1 . Peptides of Sec61α , Sec61β , and Sec61γ identified by mass spectrometry sequences of Sec61α , Sec61β , and Sec61γ annotated to indicate the peptides ( yellow ) identified by mass spectrometry . DOI: http://dx . doi . org/10 . 7554/eLife . 07426 . 00410 . 7554/eLife . 07426 . 005Figure 1—figure supplement 2 . Ire1α is codepleted with the Sec61α translocon .",
"( A ) The co-depletion of Ire1α with the Sec61 translocon was compared between endogenous Ire1α and recombinant Ire1α-HA .",
"The cell lysates either from non-transfected HEK 293 cells or HEK 293 cells stably expressing various amount of Ire1-HA were passed through an anti-Sec61β resin to deplete the Sec61 translocon .",
"The flow through fractions was analyzed along with starting lysates ( input ) by IB .",
"( B ) The co-depletion of Ire1α with the Sec61 translocon was quantified by ImageJ64 from the immunoblot shown in A . Note that nearly all endogenous Ire1α in HEK 293 cells was codepleted with the Sec61 translocon , whereas overexpression of Ire1α leads to freeunbound Ire1α , presumably due to a saturation of the Sec61 translocon binding sites . DOI: http://dx . doi . org/10 . 7554/eLife . 07426 . 005 To exclude the possibility that this interaction was captured during Ire1α synthesis at the Sec61 translocon , we set out to identify specific residues in Ire1α that are required for the interaction with the Sec61 translocon .",
"We therefore performed co-immunoprecipitation with HA antibodies using detergent extracts of cells expressing mutant versions of Ire1α-HA .",
"While deletion of the luminal domain of Ire1α ( amino acids 30 to 408 ) had no effect on the interaction with the Sec61 translocon , deletion of an evolutionarily conserved 10 amino acid region ( amino acids 434 to 443 ) in the luminal portion of Ire1α adjacent to its TMD nearly abolished the interaction with the Sec61 translocon ( Figure 2A , C ) .",
"Mutagenesis of single residues within this region further revealed that Val437 , Asp438 , Met440 , Leu441 and Asp443 are crucial for the interaction since replacing any of these amino acids with alanine significantly reduced the interaction with the translocon ( Figure 2B , C ) .",
"We next asked whether the Ire1α interaction with the Sec61 translocon is important for XBP1u mRNA cleavage during ER stress .",
"To address this , we transiently depleted the Sec61α subunit , which forms the translocon channel , in cells by siRNA-mediated knock down .",
"Indeed , Ire1α mediated splicing of XBP1u mRNA was substantially reduced in Sec61α depleted cells during ER stress ( Figure 2D ) . 10 . 7554/eLife . 07426 . 006Figure 2 . Key residues in Ire1α important for the interaction with the Sec61 translocon .",
"( A ) The cell lysates of the indicated versions of HA-tagged Ire1α were IP with anti-HA antibodies , eluted with sample buffer and analyzed by IB .",
"Ire1α–venus served as a control .",
"The mutation K907 to A907 impairs the RNase activity of Ire1α ( Tirasophon et al . , 2000 ) .",
"Deletion of the Ire1α cytosolic domain from amino acid 477 to 977 or luminal domain from amino acid 30 to 408 is labeled as ΔCD or ΔLD ( Volmer et al . , 2013 ) , respectively .",
"Ire1α Δ34 carry a deletion from amino acid 409 to 443 and Ire1α Δ10 lacks amino acids 434 to 443 .",
"( B ) The indicated Ire1α mutants were analyzed as described in panel A . ( C ) Comparison of the sequences of the 10 amino acid region of Ire1α in vertebrates .",
"Triangle depicts amino acid residues of Ire1α in which alanine scanning mutations disrupt binding to the Sec61 translocon .",
"( D ) HeLa cells were transfected with control siRNA or siRNA targeting Sec61α .",
"After 48 hr of transfection , cells were transfected again with siRNA which was followed by transfection with FLAG-tagged XBP1u .",
"96 hr after the first transfection , cells were treated with 10 mM DTT for the indicated time periods .",
"Total proteins and RNA were isolated from Trizol harvested cells and analyzed by IB against the indicated antigens and by an RT-PCR reaction to monitor splicing of XBP1u mRNA ( Calfon et al . , 2002 ) , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 07426 . 006 Our findings of Ire1α in a complex with the Sec61 translocon raised the important question of how XBP1u mRNA would be recruited to this complex in order to be cleaved by Ire1α during ER stress .",
"Although XBP1u mRNA encodes a soluble protein and shuttles between the cytosol and nucleus ( Yoshida et al . , 2006 ) , recent studies have shown that XBP1u is co-translationally targeted to the ER membrane for efficient splicing of its mRNA by Ire1α ( Yanagitani et al . , 2009 ) .",
"This targeting reaction also depends on a C-terminal hydrophobic region 2 ( HR2 ) as well as a translational pausing sequence located in the extreme C-terminus of XBP1u ( Yanagitani et al . , 2009 , 2011 ) .",
"Combined , HR2 and the pausing sequence are speculated to facilitate direct interaction of RNCs of XBP1u with the ER lipid bilayer ( Yanagitani et al . , 2009 ) .",
"We reasoned that the interaction between XBP1u and lipids might not selectively direct its mRNA-RNCs to the ER membrane within the cell .",
"We therefore hypothesized that XBP1u-RNCs may directly interact with the Sec61 translocon for its specific recruitment to the ER membrane as well as to engage with Ire1α .",
"To first determine if the recruitment of XBP1u-RNCs requires any ER membrane factor ( s ) , we reconstituted XBP1u recruitment to the ER membrane in vitro .",
"XBP1u transcripts lacking a stop codon were translated using a rabbit reticulocyte lysate containing 35S-labelled methionine and ER-derived rough microsomes ( RM ) .",
"As expected , the truncated XBP1u transcripts produced XBP1u-RNCs that were recruited to RM , as indicated by XBP1u peptides in the pellet fraction ( Figure 3A , B ) .",
"However , a mild trypsin digestion of RM rendered them inactive for XBP1u recruitment .",
"XBP1u recruitment to the ER membrane was dependent on the presence of HR2 since its deletion ( XBP1ΔHR2 ) abolished the membrane recruitment , while replacing HR2 with a stronger hydrophobic TMD from the transferrin receptor ( XBP1u-TR ) restored XBP1u recruitment to RM ( Figure 3A , B and Figure 3—figure supplement 1 ) .",
"We obtained similar results when we analyzed full-length versions of XBP1u , thus arguing against membrane recruitment due to artificial stalling of XBP1u at the ribosome ( Figure 3B , bottom ) .",
"These results supported our hypothesis that XBP1u-RNCs can be recruited to the ER membrane via an interaction with an ER membrane factor . 10 . 7554/eLife . 07426 . 007Figure 3 . XBP1u utilizes the signal recognition particle ( SRP ) pathway for targeting its mRNA to the ER membrane .",
"( A ) Diagram of constructs derived from XBP1u .",
"Blue box denotes the previously described hydrophobic region 2 ( HR2 ) of XBP1u ( Yanagitani et al . , 2009 ) .",
"Dark blue indicates the transmembrane domain ( TMD ) from the transferrin receptor in lieu of HR2 .",
"( B ) The indicated versions of XBP1u transcripts lacking a termination codon were translated in rabbit reticulocyte lysate in the presence of rough microsomes ( RM ) or trypsin digested RM ( tRM ) .",
"The reactions were separated by centrifugation to analyze pellet ( P ) and soluble fractions ( S ) by SDS-PAGE and autoradiography .",
"( C ) Affinity purified ribosome associated nascent chains ( RNCs ) of the indicated versions of FLAG-tagged XBP1u were analyzed by IB for the indicated antigens .",
"L13a is a ribosomal protein .",
"TRC40 is a control protein .",
"Autoradiography of the blot revealed equal recovery of translated substrates .",
"( D ) XBP1u transcripts lacking a termination codon were translated in the wheat germ translation system including purified SRP , puromycin/potassium acetate treated RM ( PK-RM ) or both .",
"RM alone was included as a control .",
"An aliquot of the total translation reaction was analyzed by IB for SRP54 and Sec61β , which indicate the presence of SRP and PK-RM , respectively .",
"An autoradiograph of the blot revealed equal translation of substrate XBP1u in all reactions .",
"The reactions were separated and analyzed as in panel B . DOI: http://dx . doi . org/10 . 7554/eLife . 07426 . 00710 . 7554/eLife . 07426 . 008Figure 3—figure supplement 1 . Sequence and hydrophobicity of XBP1u constructs . The last 80 residues of the indicated XBP1u constructs used in this study were analyzed for hydrophobicity using the Kyte-Doolittle scale .",
"( A ) The red color denotes HR2 in XBP1u .",
"( B ) The HR2 deleted position is indicated as an open arrow .",
"( C ) The green color denotes the replacement of HR2 with the TMD from the transferrin receptor . DOI: http://dx . doi . org/10 . 7554/eLife . 07426 . 008 Based on the above observation and the presence of a hydrophobic region ( HR2 ) ( Yanagitani et al . , 2009 ) in the XBP1u protein , we hypothesized that XBP1u mRNA-RNC targeting to the ER membrane may be mediated by the SRP pathway .",
"To test this , we affinity-purified RNCs of XBP1u from in vitro translation reactions via an N-terminal FLAG-tag and analyzed for SRP recruitment by IB .",
"Indeed , SRP was enriched in RNCs of XBP1u but not in RNCs of XBP1ΔHR2 ( Figure 3C ) .",
"As expected , XBP1u-TR exhibited slightly increased binding to SRP since it contains a genuine TMD ( Figure 3C and Figure 3—figure supplement 1 ) .",
"We next wondered whether the binding of SRP to HR2 of XBP1u-RNCs is essential for targeting to the ER membrane .",
"To address this , we translated XBP1u mRNA in vitro using a wheat germ extract that lacks SRP that is compatible with the mammalian SRP receptor ( Walter and Blobel , 1981 ) .",
"When RM stripped of ribosomes/SRP by puromycin and high salt ( PK-RM ) was added to this reaction , XBP1u nascent peptides were localized in the soluble fraction ( Figure 3D ) .",
"In contrast , adding back purified SRP to the original level found in RM shifted XBP1u localization to the pellet fraction , mirroring the membrane recruitment of XBP1u .",
"To examine whether Ire1α mediated splicing of XBP1u mRNA in cells also depends on the SRP mediated targeting of its RNCs to the ER membrane , we depleted SRP or the SRP receptor in cells by siRNA-mediated knock down .",
"Cells depleted of the SRP subunits SRP14 or SRP54 showed sharply reduced XBP1u mRNA splicing upon treatment with the ER stress inducer DTT ( Figure 4A ) .",
"Similarly , depletion of the α subunit of the SRP receptor ( SRα ) nearly abolished splicing of XBP1u mRNA ( Figure 4B ) .",
"Importantly , these effects were not due to a defect in the biosynthesis of Ire1α since its level was unchanged by transient depletion of either SRP or its receptor ( Figure 4A , B ) .",
"To rule out the possibility that the reduction in XBP1u mRNA splicing observed in SRP knockdown experiments was not due to diminished ER substrate burden and Ire1α activation , we examined the activation of ER stress sensors under knockdown conditions ( Figure 4A , C ) .",
"Auto-phosphorylation of PERK and Ire1α in response to DTT treatment was identical under both control and SRP54/14 knockdown conditions ( Figure 4A ) .",
"In addition , depletion of SRP14 , SRα or Sec61α had little effect on the amount of PERK phosphorylation and ATF6 cleavage in response to ER stress ( Figure 4C ) .",
"These data suggest that the reduced XBP1u mRNA splicing in the SRP pathway knockdown experiments is not an indirect effect but a result of reduced XBP1u mRNA targeting to the ER membrane .",
"Together these results suggest that SRP binds to HR2 of XBP1u-RNC and recruits it to the ER membrane for Ire1α mediated splicing of XBP1u mRNA under ER stress conditions . 10 . 7554/eLife . 07426 . 009Figure 4 . SRP mediated targeting to the ER ensures efficient spicing of XBP1u mRNA .",
"( A ) HEK 293 cells were transfected with shRNAs against luciferase ( control ) , SRP14 , or SRP54 .",
"5 days after transfection , the cells were replated for transfection with XBP1u and treated with 10 mM DTT for the indicated time periods .",
"The Trizol harvested cells were analyzed as in Figure 2D .",
"A phos-tag gel was used for the Ire1α immunoblot .",
"p-Ire1α and p-PERK indicate the phosphorylated forms of Ire1α and PERK , respectively .",
"* denotes a background band .",
"( B ) HeLa cells were transfected with control siRNA or siRNA targeting the α subunit of the SRP receptor ( SRα ) and analyzed as described in Figure 2D .",
"( C ) HeLa cells were transfected with the indicated siRNA oligos and treated with 10 mM DTT for 2 hr after 96 hr post-transfection .",
"Cells were harvested in SDS sample buffer and analyzed for IB with indicated antibodies .",
"Upon DTT treatment , the ATF6α band disappears due to the cleavage of its N-terminal cytosolic domain .",
"Our ATF6α antibodies were not suitable for detecting the cleaved N-terminal cytosolic domain ( not shown ) .",
"Note that depletion of Sec61α caused significant reduction of the transmembrane proteins PERK and ATF6α .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07426 . 009 Since the XBP1u protein does not possess a typical TMD , we wondered whether the SRP bound XBP1u-RNC is actually delivered to the Sec61 translocon in the ER membrane .",
"To examine this , we isolated the ER membrane-targeted RNCs of XBP1u from in vitro translation reactions and treated them with a cysteine reactive chemical crosslinker .",
"We observed a weak crosslinking between XBP1u and the Sec61α subunit ( Figure 5A , lanes 2 and 4 ) .",
"By contrast , XBP1u strongly crosslinked to the β subunit of the translocon , presumably via a single cysteine residue localized in its cytosolic domain ( Figure 5A , lanes 2 and 5 ) .",
"This result implied that the moderate hydrophobicity of HR2 of XBP1u might prevent full engagement of the translocon .",
"This was further supported by the improved crosslinking between XBP1u and Sec61α observed when we used XBP1u-TR , which contains a genuine TMD ( Figure 5A , compare 4 and 7 ) .",
"It has been previously shown that the Sec61 translocon can discriminate between functional and non-functional or weak signal sequences ( Jungnickel and Rapoport , 1995 ) .",
"Accordingly , the functional signal sequence is able to form a protease resistant tight junction with the Sec61 translocon .",
"To directly test if RNCs of XBP1u form a weak or tight junction with the Sec61 translocon , we performed a proteinase K ( PK ) accessibility assay with the ER membrane targeted RNCs of XBP1u .",
"We noticed efficient protection of the XBP1u signal sequence HR2 under physiological salt concentration but it became partially PK sensitive under high salt conditions , suggesting that XBP1u HR2 forms a weak junction with the translocon ( Figure 5B compare band 1 in lane 2 and 3 ) .",
"By contrast , we observed a tight complex between XBP1u-TR and the translocon as judged by an increased protection of XBP1-TR relative to XBP1u under high salt conditions ( Figure 5B compare band 1 in lane 3 and 8 ) .",
"Interestingly , upon high salt and puromycin treatment , which releases nascent chains from ribosomes , we detected membrane-protected fragments for XBP1-TR that disappeared after treating with a detergent ( Figure 5B lane 9 , 10 ) , demonstrating that insertion of XBP1u-TR occurs after its release from the ribosome .",
"However , we failed to detect membrane-protected fragments for XBP1u , suggesting that XBP1u HR2 is rejected by the Sec61 translocon after its release from the ribosome .",
"Interestingly , we noticed ribosome protected fragments even after puromycin treatment , indicating that the interaction between the translational pausing sequence of XBP1u and the ribosome exit tunnel remains stable ( Figure 5B band 2 in lanes 5 and 10 ) . 10 . 7554/eLife . 07426 . 010Figure 5 . XBP1u nascent chains interact with the Sec61 translocon , but inefficiently insert into the ER membrane .",
"( A ) The membrane-targeted RNCs of XBP1u or XBP1u-TR were isolated by centrifugation and treated with BMH crosslinker .",
"An aliquot was directly analyzed ( input , 4% loading ) , while the remainder was IP with the indicated antibodies .",
"Anti-GFP antibodies were used as a control .",
"The XBP1u crosslinked adducts are indicated by ‘XBP x’ .",
"* indicates an unidentified crosslinked product .",
"( B ) The ER membrane targeted RNCs of XBP1u variants were subjected to a proteinase K ( PK ) accessibility assay in the presence of the indicated salt concentrations and salt plus puromycin ( pur . ) .",
"* indicates the inclusion of a detergent in the reaction .",
"FL indicates full-length versions of XBP1u .",
"Band 1 indicates protease-protected fragments of either ribosome translocon protected fragments or protected fragments after insertion into the membrane ( lane 9 ) .",
"Band 2 indicates fragments protected by ribosomes .",
"( C ) The indicated versions of XBP1u transcripts containing a glycan acceptor site at the C-terminus were translated in vitro in the presence of RM .",
"The reactions were stopped at the indicated time points by directly mixing with the sample buffer and analyzed by autoradiography .",
"gXBP1u denotes the glycosylated form .",
"( D ) Cell lysates from HEK 293 cells expressing the indicated FLAG tagged XBP1u versions containing a glycan acceptor site were treated with endoglycosidase H ( EndoH ) or peptide-N-glycosidase F ( PNGase ) and analyzed by IB with FLAG . DOI: http://dx . doi . org/10 . 7554/eLife . 07426 . 01010 . 7554/eLife . 07426 . 011Figure 5—figure supplement 1 . Insertion assays with XBP1u and its variants . For the co-translational protein insertion , versions of XBP1 transcripts containing a glycan acceptor site were translated in vitro in the presence of liposomes or RM .",
"The reactions were stopped after 30 min by directly mixing with the endoglycosidase H ( EndoH ) denaturation buffer and digested with Endo H before analyzing by autoradiography .",
"gXBP1u denotes the glycosylated form of XBP1u .",
"For the post-translational protein insertion , the indicated XBP1u transcripts were initially translated in the absence of RM and the nascent chains were released by the puromycin treatment .",
"The post ribosomal supernatant was used for the insertion assay . DOI: http://dx . doi . org/10 . 7554/eLife . 07426 . 011 Since we observed an interaction between XBP1u and the translocon , we wondered whether this interaction facilitates integration of XBP1u into the ER membrane .",
"To address this , we introduced an N-glycan acceptor site prior to the stop codon of XBP1u constructs and translated them in the presence of RM .",
"Glycosylation , a post-translocational event diagnostic of successful insertion , was readily detected for XBP1u , as determined by endoglycosidase H ( Endo H ) deglycosylation ( Figure 5—figure supplement 1 ) .",
"As expected , no glycosylation was detected for XBP1ΔHR2 , whereas XBP1-TR showed increased glycosylation .",
"Importantly , the co-translational protein insertion pathway was solely responsible for these insertion activities since almost no glycosylation was detected during a post-translational protein insertion assay ( Figure 5—figure supplement 1 ) .",
"Furthermore , a time course experiment revealed a decreased rate of glycosylation for XBP1u relative to its counterpart XBP1u-TR , suggesting that its weak hydrophobic region HR2 impedes efficient insertion into the membrane ( Figure 5C ) .",
"Corroborating the in vitro results , in cells expressing XBP1u constructs we detected the HR2 dependent glycosylation of XBP1u , which was sensitive to the Endo H or peptide-N-glycosidase ( PNGase ) treatment ( Figure 5D ) .",
"As expected , the XBP1u glycosylation was significantly less efficient relative to that of XBP1u-TR ( Figure 5D ) .",
"These findings are consistent with recent observations that a fraction of XBP1u could be inserted into the ER membrane in cells ( Chen et al . , 2014 ) .",
"Collectively , these results suggest that the sequence of XBP1u HR2 has evolved in a way that it manages to follow the co-translational protein translocation pathway , but avoids efficient insertion into the ER membrane .",
"We next examined whether disrupting the interaction between Ire1α and Sec61 translocon impairs Ire1α mediated cleavage of XBP1u mRNA .",
"To this end , we complemented Ire1α or the translocon interaction defective Ire1α mutants into either HEK 293 Ire1α−/− cells generated by the CRISPR/Cas9 system or mouse embryonic fibroblast ( MEF ) Ire1α−/− cells ( Lee et al . , 2002 ) .",
"The complementation of Ire1α into Ire1α−/− cells led to restoration of XBP1u mRNA splicing in an ER stress dependent manner , whereas the complementation of Ire1α mutants either ∆10 or D443A showed sharply reduced XBP1u mRNA splicing ( Figure 6A , B ) .",
"In addition , the Ire1α mutant D443A also exhibited a significant deficiency in downregulation of the RIDD mRNA substrates Blos1 and Scara3 ( Hollien et al . , 2009 ) ( Figure 6C ) .",
"These effects were not due to a defect in activation of Ire1α mutants under ER stress conditions since we observed similar Ire1α auto-phosphorylation in both wild type and ∆10 Ire1α expressing cells ( Figure 6D ) .",
"These results support the model that the Sec61 translocon bridges Ire1α and its mRNA substrates ( Figure 7 ) . 10 . 7554/eLife . 07426 . 012Figure 6 . The Ire1α interaction with the Sec61 translocon ensures efficient cleavage of ER-targeted mRNAs .",
"( A ) HEK 293 Ire1α−/− cells generated by CRISPR/Cas9 were stably complemented with Ire1α-HA or its mutant ( Δ10 ) .",
"The expression of these constructs was controlled by doxycycline , but the cells were not induced with doxycyline in order to achieve low expression levels of Ire1α .",
"Cells were harvested in Trizol after either treatment with tunicamycin ( TM: 5 μg/ml ) , thapsigargin ( Tg: 2 . 5 μg/ml ) or DTT ( 10 mM ) for the indicated time periods and analyzed by XBP1u mRNA splicing assay and IB with the indicated antibodies .",
"( B ) Mouse embryonic fibroblast ( MEF ) Ire1α−/− cells complemented with Ire1α-HA or its mutant ( D443A ) were harvested after either treatment with TM ( 5 μg/ml ) for 5 hr or DTT ( 10 mM ) for 2 hr and analyzed by XBP1u mRNA splicing assay and IB as described in Figure 2D .",
"( C ) The MEF Ire1α−/− cells complemented with indicated Ire1α variants were treated with TM ( 5 μg/ml ) for 6 hr and analyzed by qPCR to measure Blos1 and Scara3 mRNA abundance .",
"We normalized all mRNA abundance measurements to the housekeeping control Rpl19 mRNA .",
"( D ) HEK 293 Ire1α−/− cells stably expressing Ire1α-HA or its mutant ( Δ10 ) were treated with DTT for 2 hr , TM for 5 hr , Tg for 5 hr and analyzed for phosphorylated Ire1α .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07426 . 01210 . 7554/eLife . 07426 . 013Figure 7 . Model for Ire1α-mediated cleavage of ER-localized mRNAs . Ire1α forms a complex with the Sec61 translocon , to which XBP1u mRNA is recruited by its ribosome nascent chains ( RNCs ) through the SRP pathway .",
"Despite interacting with the Sec61 translocon , the XBP1u nascent chain is inefficiently inserted into the ER membrane due to its weak hydrophobic region .",
"Upon ER stress , Ire1α is activated through self-oligomerization and cleaves XBP1u mRNA to yield an active transcription factor , XBP1s , as well as to cleave ER-localized mRNAs through regulated Ire1-dependent decay ( RIDD ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07426 . 013"
],
[
"In the present study , we have addressed how the low abundant Ire1α effectively finds and cleaves its substrate mRNAs that are associated with ribosomes in the ER membrane .",
"Our results have established a direct link between the co-translational translocation pathway and the UPR that facilitates efficient cleavage of ER-targeted mRNAs by Ire1α during ER stress ( Figure 7 ) .",
"Specifically , we have identified a complex comprising Ire1α and the Sec61 translocon , which is stable even during ER stress conditions .",
"We have shown that this interaction is specific and is not captured while Ire1α is being synthesized in the Sec61 translocon since the other ER stress sensors , Ire1β , PERK or ATF6 , fail to interact with the Sec61 translocon .",
"Moreover , our domain mapping studies identified a conserved region in the luminal domain of Ire1α required for this interaction .",
"Several observations suggest that Ire1α may directly interact or at least be in close proximity to the Sec61 translocon .",
"First , our Ire1α pull down experiment identified the Sec61 translocon as one of the major interacting proteins in addition to Sec63 and BiP ( Figure 1A ) .",
"We can exclude the possibility that Ire1α associates with the Sec61 translocon through BiP since the interaction is stable even during ER stress conditions , whereas BiP dissociates from Ire1α ( Bertolotti et al . , 2000 ) .",
"Second , our chemical crosslinking studies captured a crosslinked adduct between Ire1α and the α subunit of the Sec61 translocon ( Figure 1D ) .",
"However , interaction studies with purified Ire1α and the Sec61 translocon are required to demonstrate if these proteins directly interact with each other .",
"Interestingly , the Sec61 translocon interaction region is conserved only in vertebrates ( Figure 2C ) , which suggests that the Ire1α interaction with the Sec61 translocon may not be absolutely essential for cleavage of its mRNA substrates but would increase the fidelity and efficiency of Ire1α .",
"We hypothesized that XBP1u mRNA might be targeted and localized in proximity to the Ire1α-Sec61 translocon complex in order to be efficiently cleaved during ER stress to yield an active transcription factor for UPR target genes .",
"Initial biochemical fractionation studies have shown that XBP1u mRNA is localized in the ER membrane although its encoded protein is soluble and localized both in the cytosol and nucleus ( Stephens et al . , 2005; Yoshida et al . , 2006 ) .",
"Elegant studies from the Kohno group revealed that XBP1u mRNA is co-translationally targeted to the ER membrane as RNCs ( Yanagitani et al . , 2009 ) .",
"They further showed that this targeting reaction also depends on a hydrophobic region present in the C-terminus of XBP1u as well as a translational pausing sequence ( Yanagitani et al . , 2011 ) .",
"Based on the observation that the HR2 of XBP1u associates with protein-free liposomes , they concluded that XBP1u RNCs could directly bind with the ER lipid bilayer .",
"Although these studies have established the importance of co-translational targeting of mRNA-XBP1 RNCs to the ER membrane , it was not clear how these nascent chains are specifically recruited to the ER membrane in the presence of other membrane compartments in the cell .",
"In an attempt to address this issue , we reproduced the ER membrane recruitment assay with XBP1u RNCs in vitro .",
"Consistent with the previous findings , XBP1u RNCs were robustly recruited to the ER membrane in an HR2 dependent manner ( Figure 3B ) .",
"However , XBP1u RNCs could not be efficiently recruited to ER membranes treated with trypsin , which suggested that an ER membrane factor might be required for binding with XBP1u RNCs ( Figure 3B ) .",
"The hydrophobic sequence HR2 dependent targeting of XBP1u RNCs to the ER membrane led us to propose that the SRP pathway might be involved in this targeting reaction .",
"Our biochemical reconstitution studies have revealed that SRP can capture HR2 of XBP1u RNCs , which are then associated with the ER membrane by an interaction with the Sec61 translocon ( Figure 3C , D and Figure 5A ) .",
"It is possible that the weak hydrophobic sequence of HR2 of XBP1u RNCs can be captured in vitro but may not effectively compete for the SRP binding in cells due to the presence of numerous substrates of SRP , which are likely of stronger hydrophobicity than HR2 of XBP1u .",
"However , our results argue against this idea since siRNA-mediated depletion of co-translational protein translocation components showed significantly reduced Ire1α-mediated cleavage of XBP1u mRNA in cells ( Figure 4A , B ) .",
"We speculate that the weaker signal sequence of HR2 might be strengthened by a translational pausing sequence in XBP1u , thus providing an increased time window for SRP binding .",
"This is supported by the fact that co-translational pausing of XBP1u nascent chains are crucial for targeting to the ER membrane in vitro and in cells ( Yanagitani et al . , 2011 ) .",
"We have shown that the SRP bound XBP1 RNCs are efficiently delivered to the Sec61 translocon , but do not form a tight complex with the Sec61 translocon ( Figure 5A , B ) .",
"Thus most are released into the cytosol and less than 10% are inserted into the ER membrane ( Figure 5C , D ) .",
"Future studies are required to precisely determine whether XBP1u nascent chains are released at the Sec61 translocon or after insertion into the ER lipid bilayer .",
"Our results suggest that the moderate hydrophobicity of XBP1u HR2 impedes efficient insertion into the ER membrane , since replacing it with a stronger TMD from the transferrin receptor significantly improved insertion into the ER membrane ( Figure 5C , D ) .",
"It is puzzling why XBP1u evolved with a combination of a weak C-terminus HR2 and a translational pausing sequence to utilize the SRP pathway rather than a stronger TMD or a signal sequence .",
"In plants , Ire1 catalyzes the cytoplasmic splicing of bZIP60 mRNA to produce an active transcription factor ( Mishiba et al . , 2013 ) .",
"Interestingly , bZIP60 mRNA is likely targeted to the ER membrane by the SRP pathway since it encodes a typical transmembrane protein .",
"Therefore , the specific advantage of this unique targeting mechanism utilized by XBP1u mRNA in vertebrates is unclear .",
"It may be that the presence of a soluble form of XBP1u is necessary for a fully switchable UPR .",
"This hypothesis is supported by earlier studies showing that the XBP1u protein binds to the active transcription factor XBP1s and routes it for proteasomal degradation ( Lee et al . , 2003; Yoshida et al . , 2006 ) .",
"This feed back loop has been shown to be important for accurately turning off UPR genes by XBP1s when the ER stress is restored .",
"Recent studies have shown that numerous cytosolic mRNAs are localized in the ER membrane through poorly understood mechanisms ( Reid and Nicchitta , 2015 ) .",
"Our findings of the promiscuous substrate selectivity exhibited by SRP in binding moderate hydrophobic region in XBP1u suggests that other cytosolic proteins encoding mRNAs could be targeted to the ER membrane through the SRP pathway .",
"We hypothesize that one of the reasons Ire1α has evolved a specific interaction with the Sec61 translocon is to overcome the limitations imposed by its low abundance in the ER membrane relative to Sec61 , where its substrate mRNAs are recruited ( Ghaemmaghami et al . , 2003 ) .",
"Indeed , our complementation experiments show that the Sec61 interaction defective Ire1α mutants were not able to efficiently mediate splicing of XBP1u mRNA during ER stress as well as cleavage of the RIDD mRNA substrates ( Figure 6A–C ) .",
"However , we found that overexpressing Ire1α mutants restores the inefficient cleavage of mRNAs ( data not shown ) , suggesting that the Sec61 translocon interaction is important to bridge the low abundant Ire1α and its mRNA substrates .",
"It is currently unclear how Ire1α is localized to the specific translocon where XBP1u mRNA may be found , though it is feasible that Ire1α may dynamically monitor the Sec61 translocon population and thus increase the likelihood of contact with translocon-localized XBP1u mRNA .",
"Another intriguing hypothesis is that Ire1α may interact with a subset of the translocon population where XBP1u mRNA could be preferentially localized .",
"In any case , it is likely that another layer of complexity may exist in order to facilitate efficient co-localization of Ire1α and XBP1u mRNA .",
"In addition , since Ire1α interacts with the Sec61 translocon , both under normal and ER stress conditions ( Figure 1C , D ) , it is unclear what prevents Ire1α from spuriously cleaving mRNAs associated with the Sec61 translocon under normal conditions .",
"Most likely , accumulation of misfolded proteins triggers self-oligmerization and activation of the translocon-associated Ire1α only during ER stress conditions .",
"How Ire1α is specifically arranged with the Sec61 translocon to access its substrate mRNAs and how it is coordinated with several other translocon-associated proteins remain important questions for future studies ."
],
[
"The in vitro expression Sp64 vector ( Promega , Madison , WI ) based construct encoding N-terminus FLAG-tagged XBP1u was generated from human XBP1u cDNA ( Sino Biological , Inc . China ) using standard molecular biology methods .",
"XBP1u∆HR2 was created by deleting the amino acid coding sequence 186–208 using both 5′ phosphorylated oligos and the Phusion site directed mutagenesis protocol .",
"XBP1u-TR was constructed by replacing HR2 ( 186–208 ) with the oligonucleotides encoding the TMD of human transferrin receptor ( IAVIVFFLIGFMIGYLGY ) by an overlap extension PCR method .",
"The TMD of transferrin receptor serves as a signal sequence for recognition by SRP as previously described ( Mariappan et al . , 2010 ) .",
"We appended a 3F4-tag sequence ( GTNMKHMAGAAA ) to the C-terminus of XBP1u constructs , which end with asparagine amino acid ( N ) , thus yielding an N-glycosylation motif ( NGT ) .",
"For the preparation of RNCs , the open reading frames were PCR amplified using a forward 5′ primer annealing to SP6 ( Sharma et al . , 2010 ) , and a reverse primer lacking a stop codon .",
"For mammalian cell expression , we generated FLAG-tagged XBP1u with its 3′ UTR by following the previously described procedure ( Yanagitani et al . , 2009 ) and cloned into pcDNA5/FRT/TO ( Invitrogen , Carlsbad , CA ) .",
"By overlap extension PCR , a partial 3F4 tag sequence ( GTNMKHM ) was added prior to the stop codon of FLAG-XBP1u 3′ UTR in pcDNA5/FRT/TO , resulting in an NGT .",
"The coding region of human Ire1α was amplified from Ire1α-pcDNA3 . EGFP ( Addgene plasmid #13009 , kindly provided by Dr Fumihiko Urano ) and cloned into pcDNA5/FRT/TO carrying a C-terminal TEV protease cleavage site followed by either the HA- or the FLAG-tag .",
"Similarly , the coding region of either mouse Ire1β or mouse PERK ( Addgene plasmid #21880 , #21814 , kindly provided by Dr David Ron ) was cloned into pcDNA5/FRT/TO carrying a C-terminal TEV protease cut site followed by the FLAG-tag .",
"The human ATF6α was amplified including the 3xFLAG tag from p3xFLAG-ATF6 ( Addgene plasmid #11975 , kindly provided by Dr Ron Prywes ) and cloned into pcDNA5/FRT/TO carrying a C-terminal TEV protease cut site followed by the HA-tag .",
"The Ire1α ( K907A ) RNase mutant ( Tirasophon et al . , 2000 ) construct was made by site directed mutagenesis .",
"Deletion of the Ire1α cytosolic domain ( ΔCD ) lacking amino acids 477–977 , luminal domain ( ΔLD ) lacking amino acids 30–408 ( Volmer et al . , 2013 ) , Δ34 lacking amino acids 409 to 443 and Δ10 lacking amino acids 434 to 443 were constructed using the Phusion site-directed mutagenesis protocol with the use of 5′ phosphorylated primers .",
"Alanine scanning mutagenesis was performed using an efficient one step site directed mutagenesis protocol ( Zheng et al . , 2004 ) .",
"All PCR reactions were performed with Phusion high fidelity DNA polymerase ( New England Biolabs , Ipswich , MA ) , except for site directed mutagenesis , which used Pfu-Ultra polymerase ( Agilent Technologies , Santa Clara , CA ) .",
"3% DMSO was included in all PCR reactions to enhance amplification .",
"The coding regions of all constructs were sequenced to preclude any sequence error .",
"Antibodies were from the following sources: anti- L13a ( Santa Cruz Biotech , Dallas , TX ) , anti-FLAG M2 antibody and anti-FLAG M2 affinity gel ( Sigma–Aldrich , St . Louis , MO ) , anti-Ire1α ( Cat . No . #3294 , Cell Signalling , Beverly , MA ) , anti-PERK ( Cat . No . #3192 , Cell Signalling ) , anti-HA agarose ( Cat . No . #11815016001 , Roche , Switzerland ) and complete protease inhibitor cocktail tablets ( Roche ) , anti-Tubulin ( Cat . No . #ab11312 , Abcam , UK ) , anti-SRP54 ( BD Biosciences , Franklin Lakes , NJ ) , anti-SRP14 ( Cat . No . #PA5-27554 , Fisher Scientific ) , Anti-HA ( 16B12 , Cat . No . #MMS-101P-200 , Covance , Princeton , NJ ) , anti-ATF6 ( Cat . No . #sc-22799 , Santa Cruz , Dallas , TX ) , Calnexin ( Cat . No . #SPA-865 , Enzo life sciences ) , Anti-mouse Goat HRP ( Cat . No . #11-035-003 , Jackson Immunoreserach , West Grove , PA ) , Anti-rabbit Goat HRP ( Cat . No . #111-035-003 , Jackson Immunoreserach ) and Antibodies against TRC40 , SRα , Sec61α , Sec61β , and GFP were previously described ( Snapp et al . , 2004 ) .",
"The purified SRP and the wheat germ translation system were purchased from tRNA probes , Texas .",
"Reagents were from the following sources: Digitonin ( EMD Millipore , Billerica , MA ) , DTT ( American Bioanalytical , Natick , MA ) , Dithiobis ( succinimidyl propionate ) ( DSP , Thermo Scientific , Waltham , MA ) Bismaleimidohexane ( BMH , Thermo Scientific ) and protein-A agarose ( RepliGen , Waltham , MA ) .",
"siRNA oligos were purchased from Integrated DNA Technologies ( San Jose , CA ) .",
"Sec61α 3′ UTR siRNA ( 5′-CACUGAAAUGUCUACGUUUtt-3′ ) was previously described ( Lang et al . , 2012 ) .",
"SRα siRNA ( 5′-UAUAAACUGGACAACCAGUtt-3′ ) sequence was previously described , but it was used as an shRNA plasmid ( Lakkaraju et al . , 2007 ) .",
"shRNA plasmids of luciferase , SRP14 , and SRP54 have been previously described ( Lakkaraju et al . , 2007 ) .",
"HeLa , HEK 293-Flp-In T-Rex ( Invitrogen ) , and MEF Ire1α−/− FRT cell lines ( Hollien et al . , 2009 ) were cultured in high glucose DMEM containing 10% FBS at 5% CO2 .",
"Transfections with either plasmids or siRNA oligos were performed with Lipofectamine 2000 ( Invitrogen ) according to the manufacture's protocol .",
"For the siRNA mediated gene silencing , HeLa cells were transfected with 40 pmol of siRNA oligos per well in a 12 well plate using Lipofectamine 2000 .",
"At 24 hr after transfection , cells were replated for the second round of transfection with siRNA oligos at 48 hr . 6 hr later cells were transfected with 200 ng of pcDNA5-FLAG-XBP1u-3′ UTR .",
"96 hr after the first siRNA transfection , cells were harvested using the Trizol reagent ( Invitrogen ) for isolation of total proteins and RNA and analysed by IB and RT-PCR of XBP1-mRNA , respectively .",
"shRNA mediated gene silencing in HEK 293 cells was performed by following the previously established protocol ( Lakkaraju et al . , 2007 ) except that at fifth day of transfection , cells were replated for transfection with FLAG-XBP1u-3′ UTR and harvested in Trizol after treatment with DTT at sixth day of initial transfection for analysis by both IB and RT-PCR of XBP1u mRNA .",
"To establish stable cell lines , HEK 293-Flp-In T-Rex or MEF Ire1α−/− FRT cells were transfected with 1 μg of pOG44 vector ( Invitrogen ) and 0 . 1 μg of FRT vectors containing Ire1α or its mutants using Lipofectamine 2000 .",
"MEF Ire1α−/− FRT cells were plated in hygromycin ( 50 μg/ml ) 24 hr after transfection , while HEK 293-Flp-In T-Rex cells were plated in hygromycin ( 100 μg/ml ) plus blasticidin ( 10 μg/ml ) .",
"The medium was replaced every 3 days until colonies appeared .",
"To regulate the expression of Ire1α or its mutants in complemented MEF Ire1α−/− cells , we transfected with pCDNA/tetR ( Invitrogen , a kind gift from Dr Dhasakumar Navaratnam ) and selected with blasticidin ( 10 μg/ml ) until colonies appeared .",
"The colonies were picked and the expression of recombinant Ire1α or its mutants was compared with control MEF cells .",
"For purification of Ire1α associated proteins , HEK293 cells or HEK293 stable cells expressing Ire1α -HA were induced with 250 ng/ml of Doxycycline for 48 hr .",
"Cells were lysed in Buffer A ( 10 mM Hepes pH 7 . 4 , 250 mM Sucrose , 2 mM MgCl2 , 1× protease inhibitor cocktail ) by repeated passage through a 23-gauge syringe needle .",
"ER microsomes were isolated from low speed supernatants ( 2823×g for 30 min ) by centrifugation for 1 hr at 75 , 000×g .",
"Microsomes were resuspended in Buffer B ( 10 mM Hepes pH 7 . 4 , 250 mM Sucrose , 2 mM MgCl2 , 0 . 5 mM DTT ) and solubilized in lysis buffer ( 50 mM Hepes , 150 mM NaCl , 5 mM MgAc , 1 mM DTT , 1× protease inhibitor cocktail , 1% digitonin ) for 30 min at 4°C .",
"The supernatant was collected by centrifugation at 20 , 000×g for 15 min and incubated with rat anti-HA-agarose ( Roche ) .",
"The beads were extensively washed with lysis buffer , but containing 0 . 2% digitonin .",
"The bound material was eluted from the column using 0 . 1 M glycine , pH 2 . 3 and 0 . 5% Triton-X100 .",
"The elutions were TCA precipitated and analysed by SDS-PAGE , followed by Coomassie blue stain .",
"Bands of interest were identified by mass spectrometry at Keck MS and Proteomics Resource , Yale School of Medicine .",
"For co-immunoprecipitation of endogenous Ire1α with the Sec61 translocon , HEK 293 cells were treated either with or without 10 mM DTT for 2 hr .",
"Cells were lysed in Buffer A ( 50 mM Tris-HCl , pH 8 . 0 , 150 mM NaCl , 1% digitonin ) by rotating 30 min at 4°C .",
"The supernatant was collected by centrifugation at 20 , 000×g for 15 min and incubated with anti-GFP or anti-Sec61β antibodies conjugated to protein-A agarose .",
"The beads were extensively washed with Buffer A , but containing 0 . 2% digitonin .",
"The bound material was eluted from the beads by directly boiling in SDS sample buffer .",
"The clean blot IP system ( Thermo Scientific ) was used for secondary antibodies to minimize background from the primary antibodies .",
"For co-immunoprecipitation of recombinant Ire1α and the endogenous Sec61 translocon , HEK 293 cells were transiently transfected with HA-tagged Ire1α versions .",
"After 36 to 48 hr of transfection , cells were harvested in 1xPBS and lysed as above .",
"The resulting digitonin cell extract was bound to anti-HA-agarose ( Roche ) , washed , eluted SDS sample buffer and analysed by IB .",
"RM derived from canine pancreas have been described ( Walter and Blobel , 1983 ) RM was treated with 20 μg/ml of trypsin ( Sigma ) for 1 hr on ice .",
"The reaction was stopped by adding 2 mM PMSF with continued incubation for 15 min on ice .",
"The trypsin digested RM was sedimented through 0 . 5 M sucrose in a physiological salt buffer ( PSB: 50 mM Hepes pH 7 . 4 , 100 mM KAc , 2 mM MgAc ) for 12 min at 70 , 000 rpm/TLA100 . 3 ( Beckman , Brea , CA ) to remove trypsin .",
"The resulting pellet was resuspended in membrane buffer ( 50 mM Hespes pH 7 . 4 , 250 mM Sucrose , 100 mM KAc , 2 mM MgAc , and 1 mM DTT ) .",
"As a control , RM were treated similarly in parallel but without trypsin as a control .",
"This was done as described previously ( Yanagitani et al . , 2011 ) with the following modifications .",
"Transcripts encoding versions of XBP1u lacking or containing a stop codon were translated in a rabbit reticulocyte lysate translation system ( Sharma et al . , 2010 ) supplemented with 35S-methionine in the presence or absence of membranes for 20 min at 32°C .",
"The translation reaction was layered on 1 M sucrose prepared in PSB .",
"After sedimentation for 15 min at 20 , 000×g , the supernatants and the pellets were analysed by SDS-PAGE and autoradiography .",
"RNCs of XBP1u versions were affinity purified using anti-FLAG agarose , similarly to previous methods ( Mariappan et al . , 2010 ) .",
"In brief , 300 μl reactions were translated for 20 min and immediately chilled on ice .",
"The samples were adjusted to 2 mM cycloheximide , diluted to 1 ml with PSB , and incubated with 20 μl suspension of anti-FLAG affinity resin recognizing the N-terminal FLAG-tag for 1 . 5 hr .",
"After being washed extensively with PSB , the bound RNCs were eluted in SDS sample buffer , and analysed by both IB and autoradiography .",
"XBP1u transcripts lacking a termination codon were translated in a wheat germ extract supplemented with 35S-methionine , 32 nM purified SRP ( tRNA probes ) and/or puromycin/KAc treated RM ( PK-RM ) .",
"After incubation at 25°C for 45 min , the reactions were sedimented through 0 . 25 M sucrose in PSB for 15 min at 20 , 000×g .",
"The supernatants and the pellets were analysed by SDS-PAGE and autoradiography .",
"Transcripts encoding versions of XBP1u lacking a termination codon were translated in the presence of RM for 25 min at 32°C .",
"The membrane targeted RNCs were isolated by centrifugation through a 0 . 5 M sucrose cushion for 12 min at 70 , 000 rpm/TLA100 . 3 , and the resulting pellet was resuspended in PSB .",
"Crosslinking was performed with 400 μM BMH ( a homo-bifunctional cysteine-reactive crosslinker ) for 6 min at 25°C and quenched with 25 mM 2-mercaptoethanol .",
"The resulting products were denatured with 1% SDS , 100 mM Tris-HCl pH 8 . 0 for 30 min at 55°C and diluted 10-fold IP buffer .",
"The respective antibodies were added and incubated for 1 . 5 hr at 4°C , followed by incubation with protein-A agarose ( Repligen ) for 1 . 5 hr at 4°C .",
"The beads were washed at least three times with IP buffer , eluted with SDS sample buffer and analysed after heating to 95°C , but 55°C/30 min for the Sec61α sample .",
"Samples were treated with RNase A ( 100 μg/ml ) for 10 min at 37°C before analyzing by SDS-PAGE and autoradiography .",
"This was done as described previously with the following modifications ( Oyadomari et al . , 2006 ) .",
"HEK 293 cells stably expressing HA-tagged Ire1α were semipermeabilized with 0 . 015% digitonin containing buffer ( 20 mM Hepes pH 7 . 4 , 110 mM KAc , 2 mM MgAc ) and treated with various concentrations of DSP crosslinker for 30 min at room temperature .",
"Samples were collected in quenching/denaturing buffer containing 2% SDS , 6 M urea , 100 mM Tris-HCl pH 8 . 0 , incubated for 30 min at 37°C , diluted 20-fold IP buffer and IP with anti-HA antibodies .",
"The resulting IP was treated with DTT containing SDS sample buffer for 30 min at 37°C to reverse the crosslinking and analyzed by IB .",
"For all RNA analyses , the total RNA was isolated from treated or non-treated cells using Trizol reagent ( Invitrogen ) , treated with RQ1 RNase-Free DNase ( Promega , Madison , WI ) to remove residual DNA , and cDNA was synthesized using 2 μg of total RNA as a template , random hexamers , and M-MuLV reverse transcriptase ( NEB ) .",
"We measured relative mRNA abundance by real time quantitative PCR ( BioRad , Hercules , CA ) with SYBR green as the fluorescent dye .",
"Each sample was measured in triplicate and normalized to Rpl19 mRNA levels .",
"The primers for mouse Rpl19 , Blos1 and Scar3 were previously described ( Hollien et al . , 2009 ) .",
"A 6% polyacrylamide gel was made containing 25 μM Phos-tag ( Wako , Japan ) and 50 μM MnCl2 .",
"SDS-PAGE was conducted at 100 V for 3 hr , followed by Mn chelation with 1 mM EDTA .",
"The gel was transferred to nitrocellulose and probed with an anti-Ire1 antibody ( Cell Signalling ) .",
"The Ire1α targeting sequence ( 5′ GATGGCAGCCTGTATACGCTTGG 3′ ) was cloned into the gRNA expression vector ( Mali et al . , 2013 ) in order to direct Cas9 nuclease activity toward the fourth coding exon of Ire1α .",
"HEK 293-Flp-In T-Rex cells were plated in a six-well plate and transfected at 70% confluence with 250 ng of the gRNA expression vector and 250 ng of the pSpCas9 ( BB ) -2A-Puro ( Ran et al . , 2013 ) expression plasmid with Lipofectamine 2000 .",
"Expression of Cas9 was selected for by puromycin treatment ( 2 . 5 μg/ml ) for 48 hr , after which cells were returned to non-selecting media for 72 hr .",
"Cells were then plated at 0 . 5 cells/well in 96 well plates and expanded for 3 weeks .",
"Individual clones were examined for Ire1α knock out by IB against endogenous Ire1α ."
]
] | [
"Upon endoplasmic reticulum ( ER ) stress , the transmembrane endoribonuclease Ire1α performs mRNA cleavage reactions to increase the ER folding capacity .",
"It is unclear how the low abundant Ire1α efficiently finds and cleaves the majority of mRNAs at the ER membrane .",
"Here , we reveal that Ire1α forms a complex with the Sec61 translocon to cleave its mRNA substrates .",
"We show that Ire1α's key substrate , XBP1u mRNA , is recruited to the Ire1α-Sec61 translocon complex through its nascent chain , which contains a pseudo-transmembrane domain to utilize the signal recognition particle ( SRP ) -mediated pathway .",
"Depletion of SRP , the SRP receptor or the Sec61 translocon in cells leads to reduced Ire1α-mediated splicing of XBP1u mRNA .",
"Furthermore , mutations in Ire1α that disrupt the Ire1α-Sec61 complex causes reduced Ire1α-mediated cleavage of ER-targeted mRNAs .",
"Thus , our data suggest that the Unfolded Protein Response is coupled with the co-translational protein translocation pathway to maintain protein homeostasis in the ER during stress conditions ."
] | [
"Proteins are made up of long chains of smaller building blocks called amino acids .",
"To build this chain , a molecule called mRNA is ‘translated’ into the sequence of amino acids by a molecular machine called a ribosome .",
"In order to work , the protein chain must then be folded into a complex shape .",
"For many proteins , this happens inside a cell compartment called the endoplasmic reticulum .",
"Newly made proteins are guided to the endoplasmic reticulum by ‘signal recognition particles’ , and then enter the endoplasmic reticulum through a channel protein called Sec61 .",
"If too many protein chains arrive at once , or they are folded too slowly , the accumulation of unfolded proteins can stress the endoplasmic reticulum .",
"To fix this , cells trigger a process called the unfolded protein response .",
"In mammals , an enzyme called Ire1α detects when the endoplasmic reticulum is becoming stressed and responds by cleaving mRNA molecules .",
"One particular target of Ire1α is the mRNA molecule that encodes a protein called XBP1 , which can activate hundreds of genes to increase the size—and hence reduce the stress—of the endoplasmic reticulum .",
"This protein is only made if a section of the mRNA molecule is removed from it; thus , by cleaving the mRNA , Ire1α enables the protein to be made .",
"It remains unknown , however , how Ire1α finds and cleaves its mRNA targets .",
"Plumb , Zhang et al . identified the proteins that bind to Ire1α in human cells , and found that the Sec61 channel is one such protein .",
"This interaction localizes Ire1α to the Sec61 channel in the endoplasmic reticulum membrane .",
"The XBP1 protein is then brought to this channel by a signal recognition particle while it is still being translated—that is , when it is still attached to the ribosome and its mRNA molecule .",
"Ire1α can then cleave the XBP1 mRNA .",
"In cells that lack the signal recognition particle or the Sec61 channel protein , Ire1α cannot efficiently cleave the XBP1 mRNA molecule .",
"In addition , if Ire1α is unable to interact with the channel protein , it does not efficiently cleave mRNA molecules at the endoplasmic reticulum membrane .",
"This work establishes a new link between the unfolded protein response and the pathway that brings new proteins to the endoplasmic reticulum membrane .",
"It provides a basis for future studies examining the details of Ire1α signaling in mammals and , in particular , work investigating the mechanism of insulin mRNA cleavage by Ire1α , which has been implicated in type 2 diabetes ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"cell biology"
] | Sister kinetochore splitting and precocious disintegration of bivalents could explain the maternal age effect | elife-11389-v3 | [
[
"Once during every menstrual cycle , an oocyte segregates half of its chromosomes into a small cell , the polar body , to prepare for fertilization ( Petronczki et al . , 2003 , Clift and Schuh , 2013 ) .",
"Segregating the chromosomes accurately is vital for the development of healthy human embryos upon fertilization .",
"Surprisingly , oocytes segregate their chromosomes much more inaccurately than do human mitotic cells ( Nagaoka et al . , 2012 , Knouse et al . , 2014 ) , human spermatocytes ( Pacchierotti et al . , 2007 , Templado et al . , 2011 ) or mouse oocytes ( Pacchierotti et al . , 2007 , Danylevska et al . , 2014 , Hassold and Hunt , 2001 ) .",
"The accuracy of chromosome segregation drops even further with increasing age ( Nagaoka et al . , 2012 , Chiang et al . , 2010 , Fragouli et al . , 2011 , Kuliev et al . , 2011 ) .",
"This may be one of the reasons behind an increase in miscarriages , trisomic pregnancies and infertility with advanced maternal age .",
"Notably , eggs from young women are also often aneuploid ( Pacchierotti et al . , 2007 , Obradors et al . , 2011 ) .",
"Why chromosomes missegregate so frequently , even in oocytes from young women , is still poorly understood .",
"We showed previously that human oocytes assemble a spindle by an unusually long and error-prone mechanism that is likely to sensitize human oocytes of all ages to abnormal kinetochore-microtubule attachments and chromosome segregation errors ( Holubcova et al . , 2015 ) .",
"However , it is still unclear why chromosome segregation errors in oocytes become more frequent as women get older .",
"Research in mouse oocytes suggests that changes in chromosome architecture contribute to this age-dependent increase in aneuploidy ( Lister et al . , 2010 , Chiang et al . , 2011 , Yun et al . , 2014 , Sakakibara et al . , 2015 ) .",
"The architecture of chromosomes differs in meiosis and mitosis .",
"During the first meiotic division , the oocyte segregates entire chromosomes instead of sister chromatids as is the case in mitosis .",
"To facilitate this , the chromosome pairs are linked with each other through meiotic recombination ( Nagaoka et al . , 2012 , Kong et al . , 2004 , Baudat et al . , 2013 ) .",
"The sister kinetochores of each chromosome become unified so that they function as a single kinetochore ( Watanabe , 2012 ) .",
"This prevents the undesirable bi-orientation of sister kinetochores during meiosis I ( Watanabe , 2012 , Yokobayashi and Watanabe , 2005 , Kim et al . , 2015 ) .",
"The resulting unit of two linked chromosomes with two functional kinetochores is called a bivalent chromosome .",
"The cohesion complex has a crucial role in maintaining the integrity of the bivalents: it holds the homologous chromosomes together and promotes the close association of the sister kinetochores ( Petronczki et al . , 2003 , Watanabe , 2012 ) .",
"Recent work in mouse oocytes suggests that the cohesin complex is gradually lost from oocyte chromosomes as mice get older ( Lister et al . , 2010 , Tachibana-Konwalski et al . , 2010 , Chiang et al . , 2010 ) .",
"This loss of cohesin has been suggested to lead to an increase in sister kinetochore distances and the precocious dissociation of bivalents into individual chromosomes ( Yun et al . , 2014 , Sakakibara et al . , 2015 ) , often referred to as univalents in meiosis .",
"Whether such a loss of cohesin is also relevant in human oocytes is still unclear ( Garcia-Cruz et al . , 2010 , Tsutsumi et al . , 2014 , Handyside et al . , 2012 ) .",
"The aim of this study was to investigate whether age-related changes in chromosome architecture also contribute to the maternal age effect in human oocytes .",
"To this end , we analyzed the organization of chromosomes and how they interact with the spindle in over 200 live and fixed human oocytes from donors aged between 23 and 46 years ."
],
[
"We first investigated the chromosome organization in oocytes from young women ( ≤30 years ) by recording high resolution z-stacks ( 300 nm in x and y , 650 nm in z ) of 1 , 051 chromosomes and their kinetochores in 23 oocytes .",
"Unexpectedly , 60% of sister kinetochores appeared as two discrete spots when observed by light microscopy ( Figure 1A , B ) .",
"This is in contrast to oocytes from young mice , whose sister kinetochores generally appear as a single spot ( Chiang et al . , 2010 , Lister et al . , 2010 , Shomper et al . , 2014 ) .",
"The sister kinetochores were spaced up to 1 . 7 µm apart ( Figure 2C ) and sometimes separated by prominent gaps ( Figure 1A , B ) .",
"Such bivalents with separated kinetochores were a frequent phenomenon , observed throughout the entire cohort of oocyte donors ( Figure 1C; Figure 1—figure supplement 1 , 2 ) , including very young donors ( Video 1 ) . 10 . 7554/eLife . 11389 . 003Figure 1 . Most sister kinetochores are not unified and attach to two discrete k-fibers during meiosis I in human oocytes .",
"( A ) Maximum intensity z-projection immunofluorescence images of kinetochores and chromosomes in meiosis I oocytes from young donors ( ≤30 years old ) .",
"Representative examples from 5 different oocytes .",
"Scale bar represents 1 µm .",
"( B ) Categories of sister kinetochore configurations shown in ( A ) and their frequency in oocytes from young donors ( ≤30 years old ) .",
"1 , 051 kinetochore pairs from 23 oocytes were analyzed .",
"( C ) Examples of kinetochore configurations from 26 human oocytes across all age groups in which sister kinetochores are separated ( marked with white arrows ) .",
"Scale bar represents 1 µm .",
"( D ) Maximum intensity z-projection immunofluorescence images of microtubules and distinct sister kinetochore pairs in cold-treated meiotic spindles from human oocytes across all age groups .",
"Drawings illustrate the different modes of microtubule attachments in the immunofluorescence images .",
"Scale bar represents 5 µm in overview and 1 µm in insets .",
"( E ) Quantification of the number of individual k-fibers attaching to sister kinetochore pairs .",
"949 attachments from 25 cold-treated human oocytes from donors across all age groups were evaluated .",
"( F ) Frequency of the different modes of kinetochore-microtubule attachments shown in ( D ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 00310 . 7554/eLife . 11389 . 004Figure 1—figure supplement 1 . The majority of sister kinetochores are split during meiosis I in human oocytes . Representative images of kinetochores from 5 human oocytes co-labelled with CREST ( magenta ) and Hec1 ( green ) .",
"Schemes on top of the panel show the different kinetochore configurations as determined from Hec1 labelling .",
"Arrows of the same colour highlight sister kinetochores .",
"Scale bar represents 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 00410 . 7554/eLife . 11389 . 005Figure 1—figure supplement 2 . Sister kinetochore separation is already evident in the initial stages of spindle assembly . Representative bivalents from 3 different human oocytes fixed before completion of bipolar spindle assembly are shown .",
"Arrows of the same colour highlight sister kinetochores .",
"Scale bar represents 5 µm in overview and 1 µm in insets . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 00510 . 7554/eLife . 11389 . 006Figure 1—figure supplement 3 . Sister kinetochores can interact with k-fibers that originate from different regions of the meiotic spindle .",
"( A ) Illustrations and representative images of k-fiber attachment types to unified sister kinetochore pairs in cold-treated human oocyte spindles .",
"Arrows indicate attachment types defined in ( 1D ) .",
"Scale bar represents 1 µm .",
"( B ) Illustrations and representative images of k-fiber attachment types to distinct kinetochore pairs in cold-treated human oocyte spindles .",
"Arrows indicate attachment types defined in ( 1D ) .",
"Scale bar represents 1 µm .",
"( C ) Illustrations and representative images of k-fiber attachment types to separated kinetochore pairs in cold-treated human oocyte spindles .",
"Arrows indicate attachment types defined in ( 1D ) .",
"Scale bar represents 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 00610 . 7554/eLife . 11389 . 007Figure 1—figure supplement 4 . Individual kinetochores can attach to multiple k-fibers during meiosis I in human oocytes . Illustrations and representative images of kinetochore-microtubule interactions in cold-treated human oocyte spindles showing multiple k-fiber attachments ( indicated by arrows ) to individual kinetochore pairs .",
"Scale bar represents 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 00710 . 7554/eLife . 11389 . 008Figure 2 . Age-dependent increase in sister kinetochore separation correlates with abnormal kinetochore-microtubule attachments .",
"( A , B )",
"Overview of a cold-treated meiotic spindle and examples of its bivalents in an oocyte from ( A ) a young ( 24 years old ) and ( B ) an older ( 40 years old ) donor .",
"Scale bars represent 5 µm in overviews and 1 µm in examples .",
"( C ) Relative frequency of individual sister kinetochore pair distances across different age groups .",
"2 , 588 measurements were obtained from 57 oocytes .",
"Distances between unresolvable sister kinetochore configurations of the indistinguishable and overlapping categories ( as shown in ( 1B ) ) are presented as 0 µm .",
"( D ) Age-related increase in the incidence of separated sister kinetochores in 58 human oocytes .",
"****p≤0 . 0001 .",
"( E ) Occurrence of kinetochore configurations defined in ( 1B ) across different age groups .",
"*p≤0 . 05 , **p≤0 . 01 , ***p≤0 . 001 , ****p≤0 . 0001 .",
"Significance analyses were performed for changes relative to <30 years old age group .",
"( F ) Quantification of the proportion of microtubule attachments ( shown in H ) in each category of sister kinetochore configurations .",
"*p≤0 . 05 , **p≤0 . 01 , ***p≤0 . 001 ( Fisher’s exact test ) .",
"Significance analyses were performed for changes in the frequency of merotelic ( *** ) and lateral ( * ) attachment modes relative to the unified kinetochore group .",
"( G ) The proportion of kinetochore-microtubule attachments ( shown in ( H ) ) in cold-treated spindles of oocytes from each age group .",
"N=25 oocytes .",
"*p≤0 . 05 , **p≤0 . 01 , ***p≤0 . 001 , ****p≤0 . 0001 .",
"Significance analyses were performed for changes in the frequency of merotelic attachment mode relative to <30 years old age group .",
"( H ) Representative maximum intensity z-projections of immunofluorescence images of cold-treated spindles showing different microtubule attachment types across the three categories of sister kinetochore configurations defined in ( 1B ) .",
"Arrowheads indicate kinetochore pairs with the specified microtubule attachment type .",
"Scale bar represents 5 µm in overview , 1 µm in insets . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 00810 . 7554/eLife . 11389 . 009Figure 2—figure supplement 1 . Mean sister kinetochore distance in meiosis I increases with donor’s age in a linear manner . Correlation analysis between mean sister kinetochore distance and donor’s age .",
"57 oocytes from donors aged 24–40 years were analyzed .",
"98% of the sister pairs were resolved and included in the analysis . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 00910 . 7554/eLife . 11389 . 010Figure 2—figure supplement 2 . Age-dependent increase in separation of sister kinetochores in human oocytes is conserved from meiosis I to meiosis II .",
"( A ) Overview of a cold-treated MII spindle and examples of its chromosomes in an oocyte from a young ( 23 years old ) donor .",
"Scale bars represent 5 µm in overviews and 1 µm in examples .",
"( B ) Overview of a cold-treated meiotic spindle and examples of its bivalents in an oocyte from an older ( 40 years old ) donor .",
"Scale bars represent 5 µm in overviews and 1 µm in examples .",
"( C ) Correlation analysis between mean sister kinetochore distance in meiosis II and oocyte donor age .",
"46 oocytes from donors aged 23–46 years were analyzed . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 01010 . 7554/eLife . 11389 . 011Figure 2—figure supplement 3 . Frequency of merotelic attachments are correlated with kinetochore configuration and donor’s age . Frequency of merotelic kinetochore-microtubule attachments as in Figure 2G stratified by kinetochore configuration and donor’s age . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 01110 . 7554/eLife . 11389 . 012Video 1 . A projection through confocal sections of a meiosis I spindle in an oocyte from a young donor ( 24 years old ) stained for microtubules ( green ) , chromosomes ( grey ) and kinetochores ( magenta ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 012 We then analyzed how the split kinetochores interact with the spindle .",
"Around 90% of split kinetochores were attached to two separate kinetochore fibers ( Figure 1D , E ) .",
"The two kinetochore fibers did not always run in parallel , but often linked the sister kinetochores to distant positions at the spindle poles ( Figure 1D , F ) .",
"Thus , consistent with the high degree of separation , the spindle recognizes most split sister kinetochores in human oocytes as independent units ( Figure 1—figure supplement 3 , 4 ) .",
"The fraction of split kinetochores in human oocytes increased with maternal age .",
"Around 75% of all kinetochores were split in oocytes from women between 30 and 35 .",
"This increased to 87% in women over 35 ( Figure 2A–E; Videos 1 , 2 ) .",
"The fraction of sister kinetochores that was separated from each other by a prominent gap also increased dramatically with advanced maternal age ( Figure 2D ) .",
"Sister kinetochores also sometimes become distinguishable in oocytes from very old mice ( Chiang et al . , 2010 , Lister et al . , 2010 ) .",
"But in contrast to human oocytes , their sister kinetochores are not separated by a prominent gap .",
"Consistent with the remarkably large distances between sister kinetochores during meiosis I ( Figure 2C , Figure 2—figure supplement 1 ) and meiosis II ( Duncan et al . , 2012 ) in human oocytes , sister kinetochores were also separated by very large gaps during meiosis II ( Figure 2—figure supplement 2; Videos 3 , 4 ) . 10 . 7554/eLife . 11389 . 013Video 2 . A projection through confocal sections of a meiosis I spindle in an oocyte from an older donor ( 40 years old ) stained for microtubules ( green ) , chromosomes ( grey ) and kinetochores ( magenta ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 01310 . 7554/eLife . 11389 . 014Video 3 . A projection through confocal sections of a meiosis II spindle in an oocyte from a young donor ( 23 years old ) stained for microtubules ( green ) , chromosomes ( grey ) and kinetochores ( magenta ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 01410 . 7554/eLife . 11389 . 015Video 4 . A projection through confocal sections of a meiosis II spindle in an oocyte from an older donor ( 40 years old ) stained for microtubules ( green ) , chromosomes ( grey ) and kinetochores ( magenta ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 015 Together , these data show that the majority of bivalent chromosomes in human oocytes feature three or four functionally distinct kinetochores .",
"Each of these kinetochores is able to establish its own kinetochore fiber .",
"Thus , human oocytes question the paradigm that sister kinetochores are unified during meiosis I .",
"We then went on to analyze whether the split nature of sister kinetochores in human oocytes could affect chromosome segregation .",
"First , we assessed if having three or four functionally distinct kinetochores per bivalent increased the probability of abnormal kinetochore-microtubule attachments .",
"During meiosis I , the sister kinetochores of each chromosome should be attached to a single spindle pole only ( amphitelic attachment ) .",
"In this way , they can be segregated accurately after anaphase onset .",
"If a pair of sister kinetochores is attached to opposite spindle poles instead ( merotelic attachment ) , the chromosome will be pulled from both sides of the spindle upon anaphase onset .",
"Such pulling will cause a chromosome to lag behind during anaphase ( Cimini et al . , 2001 , Lane et al . , 2012 ) .",
"This can lead to aneuploidy when the ingressing cytokinetic furrow partitions chromosomes inaccurately between the egg and the polar body .",
"To assess how the bivalent chromosomes were attached to the spindle , we briefly placed the oocytes on ice before fixation .",
"This selectively preserves the more stable microtubules that interact with kinetochores ( Zhai et al . , 1995 ) .",
"We then stained the oocytes for microtubules , chromosomes and kinetochores .",
"Separated sister kinetochores were more likely to be merotelically attached to the spindle than unified sister kinetochores .",
"Interestingly , we observed that the degree of separation correlated with the probability of a pair being merotelically attached: the fraction of merotelic attachments increased from 12% in unified kinetochore pairs to 17% when the sister kinetochores were distinct but still linked , and up to 28% in completely separated kinetochore pairs ( Figure 2F , H ) .",
"This could potentially be due to improved accessibility from the opposite spindle pole , when two tightly joined sister kinetochores are compared with two separated sister kinetochores .",
"The number of merotelic kinetochore-microtubule attachments also increased with maternal age , from around 7% in women under 30 to around 21% in women over 35 ( Figure 2G ) .",
"This is consistent with the increased fraction of separated sister kinetochores with advanced maternal age .",
"Separated kinetochores were more likely to be merotelically attached in oocytes from older women than in oocytes from young women ( Figure 2—figure supplement 3 ) .",
"This could be explained by the increase in the degree of separation between sister kinetochores as women get older ( Figure 2C ) , as well as additional age-related defects that could contribute to aberrant attachments .",
"The remarkably large distances between some sister kinetochores raised an unexpected problem: a stable arrangement of bivalent chromosomes on the spindle could theoretically also be achieved if the split sister kinetochores are facing towards opposite poles instead of the same spindle pole ( Figure 3A scheme , middle row; fully-rotated ) .",
"Such a chromosome would be rotated on the spindle by 90 degrees and could not segregate accurately during anaphase . 10 . 7554/eLife . 11389 . 016Figure 3 . Sister kinetochore separation allows bivalents to rotate and twist on the meiotic spindle .",
"( A ) Schematic representation of the possible orientations of bivalents on the meiosis I spindle .",
"( B ) Representative images of the different orientations that bivalents can adopt relative to the axis of the metaphase I spindle .",
"Arrows of the same colour highlight sister kinetochores .",
"Scale bar represents 5 µm , 1 µm in insets .",
"( C ) Representative images of Shugoshin-1 staining in bivalents rotated relative to the axis of the metaphase I spindle .",
"Arrows of the same colour highlight sister kinetochores .",
"Scale bar represents 1 µm .",
"( D ) Occurrence of one or more inverted bivalents in fully assembled metaphase I spindles .",
"Half-inverted bivalents were scored only in oocytes subjected to cold treatment which selectively preserves kinetochore fibers and hence allows for detection of bioriented kinetochore pairs .",
"Fully-inverted bivalents were scored both in cold-treated and non-treated meiosis I spindles .",
"( E ) Proportion of bivalents that are fully- or half-inverted on late metaphase I spindles .",
"( F ) Occurrence of fully inverted bivalents in oocytes from donors across all age groups . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 01610 . 7554/eLife . 11389 . 017Figure 3—figure supplement 1 . Shugoshin-1 is a reliable marker of sister kinetochores in meiosis I human oocytes and can be used to identify rotated bivalents .",
"( A ) Representative images of Shugoshin-1 localization in bivalents from 10 oocytes .",
"Scale bar represents 1 µm .",
"( B ) Distribution of Shugoshin-1 labelling relative to inter-kinetochore distance within bivalents from 20 meiosis-I oocytes .",
"( C ) Percentage of oocytes containing at least one fully-inverted bivalent .",
"Inversion was scored based on inter-kinetochore distance and localization of Shugoshin-1 labelling .",
"The majority of oocytes analysed were from donors older than 30 years .",
"( D ) Representative images and schematic diagrams of Shugoshin-1 labelling in inverted bivalents .",
"For each inverted bivalent , an additional in-axis bivalent from the same spindle is shown for comparison .",
"Arrows of the same colour highlight sister kinetochores .",
"Scale bar represents 1 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 01710 . 7554/eLife . 11389 . 018Figure 3—figure supplement 2 . Despite their non-conventional geometry , rotated bivalents can form stable k-fiber attachments .",
"( A ) Representative immunofluorescence images of in-axis and rotated bivalents in cold-treated human oocyte meiotic spindles .",
"Chromosomes labelled with Hoechst .",
"Arrows of the same colour highlight sister kinetochores .",
"Scale bars represent 5 µm in overviews and 1 µm in insets .",
"( B ) Proportion of kinetochore configurations in fully-inverted bivalents in human oocytes .",
"( C ) Microtubule attachment states of each of the four kinetochores in rotated bivalents in cold-treated human oocyte spindles .",
"( D ) Configuration of kinetochore pairs in half-inverted bivalent chromosomes . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 018 To our surprise , a large fraction of oocytes had one or more rotated bivalent chromosomes , in which the sister kinetochores of both pairs faced opposite spindle poles ( Figure 3A–C , middle row; Figure 3D; Figure 3—figure supplement 1; Videos 5 , 6 ) .",
"Given that the orientation of sister kinetochores in these rotated bivalents is inverted , we called these bivalents ‘inverted bivalents’ .",
"We also often observed half-inverted bivalents , in which only one pair of the sister kinetochores was oriented towards opposite spindle poles ( Figure 3A–C bottom row , half-inverted; Figure 3—figure supplement 1D , right panel ) , while the other pair was correctly oriented , with both sister kinetochores facing a single spindle pole ( Figure 3D ) .",
"Bivalent rotation was also evident from the distribution of Shugoshin-1 , which marked the pairs of sister kinetochores ( Figure 3C , Figure 3—figure supplement 1; Videos 5 , 6 ) .",
"Interestingly , Shugoshin-1 associated with a wider domain than in meiosis I mouse oocytes ( Lee et al . , 2008 ) .",
"It will be interesting to investigate how meiotic cohesion is achieved in human oocytes and whether the unexpected kinetochore configurations that we observed could be linked to species-specific differences in this process . 10 . 7554/eLife . 11389 . 019Video 5 . A projection through confocal sections of a meiosis I spindle in an oocyte stained for Shugoshin-1 ( green ) , chromosomes ( grey ) , kinetochores ( magenta ) , and in which all bivalents are oriented in axis . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 01910 . 7554/eLife . 11389 . 020Video 6 . A projection through confocal sections of a meiosis I spindle in an oocyte stained for Shugoshin-1 ( green ) , chromosomes ( grey ) , kinetochores ( magenta ) , and in which two bivalents are fully-inverted as indicated by Shugoshin-1 staining ( marked with white arrows ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 020 In total , more than 2% of all bivalents were half or fully inverted ( Figure 3E ) .",
"Importantly , kinetochores of inverted bivalents were linked to the spindle through cold-resistant and thus stable microtubules ( Figure 3—figure supplement 2A , C ) .",
"This indicates that the altered orientation of kinetochores in inverted bivalents does not completely compromise their ability to capture microtubules .",
"Moreover , the incidence of inverted bivalents increased with maternal age , from 12 . 5% in oocytes from women under 30 to 40% in oocytes from women over 35 years old ( Figure 3F; Figure 3—figure supplement 1C ) .",
"This is consistent with the increased separation of sister kinetochores with maternal age , which is expected to promote the rotation ( Figure 3—figure supplement 2B , D ) .",
"Having four instead of only two independent microtubule attachment sites also allowed bivalents to be twisted along their axis ( Figure 4A ) .",
"In around 20% of bivalents with split sister kinetochores , the chromosomes were twisted , as judged by the observation that their sister kinetochore pairs were oriented perpendicular instead of parallel to each other ( Figure 4B ) .",
"Such twisting is likely to exert additional forces on arm cohesion , which is possibly already weakened in oocytes at the time of meiotic resumption ( Duncan et al . , 2012 , Chiang et al . , 2010 ) .",
"Arm cohesion was indeed reduced in a large fraction of chromosome bivalents ( Figure 5A , C , D ) .",
"In total , 10% of all bivalents were weakly associated as evident from a gap in the bivalents’ center .",
"In some cases , the chromosomes were separated by a gap of a few 100 nm only .",
"In other cases , gaps of up to 3 . 2 µm between two chromosomes were visible ( Figure 5A ) .",
"Despite the gap , the chromosome pairs were under tension , with each pair of sister kinetochores facing the correct spindle pole .",
"This suggests that chromosomes with reduced arm cohesion still behave as functional bivalents . 10 . 7554/eLife . 11389 . 021Figure 4 . Kinetochore separation allows twisting of bivalents .",
"( A ) Representative three-dimensional images and schematic illustrations of undistorted and twisted bivalent geometries .",
"Arrows of the same colour highlight sister kinetochores .",
"Scale bar represents 1 µm .",
"( B ) Geometric categories of bivalents displaying a split sister kinetochore configuration ( defined in ( 1B ) ) .",
"Undistorted and twisted geometries were defined as bivalents with kinetochore pairs that are parallel and perpendicular to each other , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 02110 . 7554/eLife . 11389 . 022Figure 5 . Precocious dissociation of bivalents into univalent contributes to aneuploidy .",
"( A ) Illustrations and representative images of homologs showing gaps between weakly-associated bivalents ( indicated by arrowheads ) .",
"Scale bar represents 5 µm in overviews and 1 µm in insets .",
"( B ) Representative immunofluorescence images and illustrations showing complete disintegration of bivalents into two proximally or distally positioned univalents .",
"Lower panel insets and cartoons show univalents ( dark grey , numbered in magenta ) and bivalents ( light grey , numbered in black ) that were determined by manual tracing and identification of homologs within the spindle .",
"Scale bar represents 5 µm in overviews and 1µm in insets .",
"( C ) Quantification of weakened chromosome arm cohesion and bivalent disintegration in 50 human oocytes .",
"( D ) Proportion of intact , weakly-associated ( with gaps ) or disintegrated bivalents amongst 1 , 137 chromosomes across different age groups .",
"* p≤0 . 05 ( Fisher’s exact test ) .",
"Significance analyses were performed for changes relative to <30 years old age group .",
"( E ) Proportion of univalents that achieved bi-orientation on the meiosis I spindle .",
"( F ) Proportion of human oocytes that contained at least one univalent pair across different age groups . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 022 Insufficient arm cohesion was also evident from the complete disintegration of 1 . 2% of bivalents into univalents ( Figure 5B–F ) .",
"95% of these univalents aligned in the center of the spindle , with their sister kinetochores facing opposite spindle poles , suggesting that they could segregate equally into two sister chromatids upon anaphase onset ( Figure 5E ) .",
"The fraction of bivalents affected by loss of arm cohesion increased significantly with maternal age .",
"In women under 30 , 5% of all chromosome pairs were separated by gaps or completely disintegrated into univalents .",
"This increased to 11% of chromosome pairs in women over 35 years old ( Figure 5D ) .",
"A recent study also reported the presence of univalents in oocytes from older women ( Sakakibara et al . , 2015 ) .",
"Our data suggests that the premature dissociation of bivalents into univalents also contributes to aneuploidy in oocytes from young women ( Figure 5F ) .",
"It also provides further evidence from a larger number of oocytes that an increase in univalents is a likely contributor to the maternal age effect , because more than 40% of oocytes from women over 35 featured univalents ( Figure 5F ) .",
"Having more than two functional kinetochores on each bivalent chromosome is expected to hinder the correct attachment of chromosomes to the spindle .",
"Live imaging in human oocytes showed that around 16 . 3 ± 2 . 3 hr were needed to align the chromosomes in the center of the spindle upon nuclear envelope breakdown ( Holubcova et al . , 2015 ) .",
"The period of chromosome congression and alignment coincided in time with spindle instability and reorganization ( Figure 6—figure supplement 1 ) .",
"It is conceivable that the reorganization of the spindle is required to attach the bivalent chromosomes correctly to the spindle , consistent with a previous study in mouse oocytes ( Kitajima et al . , 2011 ) .",
"Difficulties in correctly attaching bivalents to the spindle would only lead to aneuploidy if oocytes are allowed to progress into anaphase in the presence of abnormal kinetochore-microtubule attachments .",
"Thus , a decisive question in understanding how aneuploidy arises is whether human oocytes have effective control mechanisms that monitor progression into anaphase .",
"In mitosis , the spindle assembly checkpoint delays progression into anaphase until all chromosomes are correctly attached to the spindle ( Musacchio and Salmon , 2007 ) .",
"Whether human oocytes have similar mechanisms is unclear .",
"Live imaging revealed that all oocytes without chromosome alignment defects progressed into anaphase .",
"Similarly , all oocytes with mild alignment defects and 92% of oocytes with severe misalignment progressed into anaphase ( Figure 6A , B; Videos 7 , 8 ) .",
"Thus , the presence of chromosome alignment defects did not preclude progression into anaphase .",
"In addition , the onset of anaphase was not severely delayed when misaligned or lagging chromosomes were present ( Figure 6C , D ) .",
"Less prominent delays might however be masked by some variability in anaphase timing between oocytes .",
"Collectively , these results suggest that human oocytes progress into anaphase with normal efficiency and without major delays if chromosomes are misaligned . 10 . 7554/eLife . 11389 . 023Figure 6 . Efficiency and timing of anaphase progression are unaffected by chromosome alignment and segregation defects in human oocytes .",
"( A ) Frames from time lapse movies of live human oocytes progressing through anaphase without alignment defects ( row 1 ) and with misaligned chromosomes ( row 2 and 3 ) .",
"Blue arrows indicate misaligned bivalents while white arrows point to bivalents that are located at spindle poles at anaphase onset .",
"Time represented in 00 h: 00 min format .",
"Scale bar represents 10 µm .",
"( B ) Efficiency of progression through anaphase relative to the presence and severity of chromosome alignment defects .",
"( C ) Timing of anaphase onset relative to severity of chromosome misalignment in live human oocytes .",
"NEBD stands for Nuclear Envelope Breakdown .",
"( D ) Timing of anaphase onset relative to incidence of lagging chromosomes in live human oocytes . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 02310 . 7554/eLife . 11389 . 024Figure 6—figure supplement 1 . Spindle remodeling in human oocytes persists after chromosome congression and decreases following chromosome alignment . States of chromosomes during meiotic progression in human oocytes and their relationship to spindle remodeling .",
"Each of the colors in the coded time plots represents the state of chromosomes in a single oocyte progressing through meiosis I . Only oocytes that went through anaphase with no misaligned chromosomes were included in the analysis . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 02410 . 7554/eLife . 11389 . 025Video 7 . Time-lapse movie of a live human oocyte progressing through anaphase I with a misaligned chromosome at the spindle pole ( white arrow ) .",
"Microtubules and chromosomes are labelled in green and magenta respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 02510 . 7554/eLife . 11389 . 026Video 8 . Time-lapse movie of a live human oocyte progressing through anaphase I with misaligned chromosomes ( white and yellow arrows ) .",
"Microtubules and chromosomes are labelled in green and magenta respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 026"
],
[
"Our findings suggest possible answers to the two key questions related to human oocyte meiosis .",
"Firstly , why is chromosome segregation in human oocytes in general more error-prone than in mitosis , even in oocytes from young women ?",
"Our previous work established that the spindles in human oocytes form over a lengthy and error-prone process , during which the spindle undergoes extensive reorganization .",
"Chromosomes frequently remain merotellically attached to the spindle close to anaphase onset .",
"These merotelic attachments could be favoured by the high instability and morphology of the spindle in human oocytes ( Holubcova et al . , 2015 ) .",
"The data presented in this study suggest that merotelic attachments are also promoted by the split nature of sister kinetochores in human oocytes .",
"It is conceivable that the specialized architecture of bivalents in human oocytes and spindle reorganization are directly linked: the fact that the four sister kinetochores of a bivalent chromosome can interact with microtubules independently may make it more challenging for the oocyte to correctly attach the bivalents to the spindle .",
"Thus , more extensive spindle reorganization may be required to correct attachment errors that arise during spindle assembly .",
"Conversely , repeated rounds of microtubule attachment and detachment during spindle reorganization may further exacerbate splitting of sister kinetochores and cause strain on chromosome arm cohesion , promoting the twisting of bivalents and the precocious dissociation of bivalents into univalents .",
"In this way , spindle reorganization could contribute to chromosome segregation defects in human oocytes .",
"Secondly , what causes the maternal age effect ?",
"We show that bivalents disintegrate precociously into univalents as women get older .",
"Univalents were observed in more than 40% of oocytes from women over 35 , suggesting that they are a major contributor to the maternal age effect , consistent with a recent study ( Sakakibara et al . , 2015 ) .",
"We also demonstrate that the degree of sister kinetochore separation increases with advanced maternal age .",
"Sister kinetochore separation correlated with merotelic kinetochore-microtubule attachment .",
"In addition , it allowed bivalents to rotate on the spindle with each of their sister-kinetochores facing towards opposite spindle poles .",
"This is unexpected , because it is generally believed that mechanisms exist that prevent the biorientation of sister kinetochores in meiosis I .",
"We observed half- or fully-inverted bivalents in a large fraction of oocytes .",
"It is possible that some of these abnormal attachments are corrected by the time of anaphase onset .",
"However , it is unclear whether mechanisms exist in the oocyte that could detect these inverted bivalents and correct their attachment .",
"Our observation that human oocytes progress into anaphase efficiently even if misaligned chromosomes are present suggests that oocytes could in principle also progress into anaphase with inverted bivalents ( Figure 7 ) .",
"Such inverted bivalents could lag behind during anaphase if centromeric cohesion is still intact in one or both of the two pairs of sister kinetochores .",
"If centromeric cohesion is compromised in one of the two sister kinetochores , the bivalent could disintegrate into one univalent and two sister chromatids .",
"If centromeric cohesion is compromised in both pairs of sister kinetochores , the bivalent could disintegrate into four individual sister chromatids upon anaphase onset , and two non-sister chromatids of different primary parental origin would remain in the egg ( Figure 7 ) .",
"Interestingly , a recent genetic study has reported that precisely this segregation outcome , which was called reverse segregation , seems to be a major cause of chromosome segregation errors during meiosis I in human oocytes ( Ottolini et al . , 2015 ) .",
"The mechanisms behind this reverse segregation pattern have remained unclear .",
"Our study indicates that inverted bivalents are likely to contribute to this segregation pattern . 10 . 7554/eLife . 11389 . 027Figure 7 . Schematic representation of bivalents with varying degree of rotation and their expected segregation outcomes following anaphase I . DOI: http://dx . doi . org/10 . 7554/eLife . 11389 . 027 In addition , the precocious separation of bivalents into univalents is likely to contribute to this pattern in oocytes from both young and older women .",
"The vast majority of univalents bioriented on the MI spindle .",
"If the sister chromatids segregated equally to the two spindle poles upon anaphase onset , this would also result in the reverse segregation pattern that was previously observed .",
"Together , these data establish a mechanistic framework whereby the separated nature of sister kinetochores in human oocytes and the precocious dissociation of bivalents into univalents , combined with spindle instability ( Holubcova et al . , 2015 ) and unrestrained progression into anaphase , prime oocytes from women of all ages for aneuploidy ."
],
[
"The use of immature unfertilized human oocytes in this study has been approved by the UK’s National Research Ethics Service under the REC reference 11/EE/0346; IRAS Project ID 84952 .",
"Immature unfertilized oocytes were donated by women receiving assisted reproduction treatment at Bourn Hall Clinic ( Cambridge , UK ) between September 2012 and November 2015 .",
"94 oocytes ( from 61 donors ) were used for live imaging; 87 MI oocytes ( from 53 donors ) and 46 MII eggs ( from 29 donors ) for fixations .",
"The donors were aged between 23 and 46 years and underwent ovarian stimulation ( Faddy et al . , 2011 ) for intracytoplasmic sperm injection ( ICSI ) .",
"Couples were referred for ICSI treatment because of male factor associated infertility ( 69 . 4% ) , absence of a male partner ( 6 . 0% ) , polycystic ovary/anovulatory syndrome ( 5 . 2% ) , tubal damage ( 2 . 3% ) , endometriosis ( 0 . 7% ) , idiopathic infertility ( 11 . 9% ) or a combination of the above/other factors ( 3 . 5% ) .",
"Only oocytes that were immature at the time of the ICSI procedure and thus could not be used for in vitro fertilization were used in this study .",
"All patients participating in this study gave informed consent for these surplus oocytes to be used in this study and for the results of this study to be published .",
"Oocytes were collected and cultured as previously described ( Holubcova et al . , 2015 ) .",
"In brief , immature live oocytes were collected within 5 hr after retrieval from ovaries and transported from Bourn Hall Clinic to the MRC .",
"The oocytes were microinjected with 10–15 µl ( 1–2% of human oocyte volume ) of 1–2 µg/µl mRNA encoding fluorescently labelled proteins using mercury-filled needles based on previously published methods ( Jaffe and Terasaki , 2004 , Schuh and Ellenberg , 2007 ) .",
"Oocyte culture , micromanipulation and live imaging were performed in G-MOPS medium supplemented with 10% FBS under mineral oil ( Paraffin , Merck ) at 37°C .",
"Only oocytes that were morphologically normal and underwent NEBD within 24 hr of retrieval from ovaries were used in this study .",
"None of the oocytes used in this study had been freeze-thawed .",
"For in vitro mRNA synthesis , pGEMHE-H2B-mRFP1 ( Schuh and Ellenberg , 2007 ) and pGEMHE-EGFP-MAP4 ( Schuh and Ellenberg , 2007 ) were linearized with AscI .",
"Capped mRNA was synthesized with T7 RNA polymerase ( mMessage mMachine kit , Ambion ) and dissolved in 11 µl water .",
"mRNA concentrations were determined on ethidium bromide agarose gels by comparison with an RNA standard ( Ambion ) .",
"Time-lapse images were acquired at 37ºC using a Zeiss LSM710 confocal microscope equipped with an environmental chamber and a 40x C-Apochromat 1 . 2 NA water immersion objective ( Carl Zeiss Limited , Cambridge UK ) .",
"Images were typically acquired at a temporal resolution of 5–10 min and a spatial resolution of 4 µm confocal sections covering 70 µm .",
"We either recorded a single oocyte or multiple oocytes in parallel using Zeiss’ MultiTime Series Macro .",
"Care was taken not to expose cells to laser intensities that perturb oocyte maturation .",
"To determine k-fiber stability , non-kinetochore-bound microtubules were depolymerized by placing oocytes at 4°C for 6 min .",
"Cells were immediately fixed following cold treatment and processed for immunofluorescence microscopy as described below .",
"K-fiber attachments were quantified from three-dimensional volume reconstructions of spindles using Imaris ( Bitplane ) .",
"Oocytes were fixed for 60 min at 37°C in 100 mM HEPES ( pH 7 ) ( titrated with KOH ) , 50 mM EGTA ( pH 7 ) ( titrated with KOH ) , 10 mM MgSO4 , 2% formaldehyde ( MeOH free ) and 0 . 2% Triton X-100 , based on previously published methods ( Strickland et al . , 2004 ) .",
"To analyze the morphology and orientation of bivalents close to anaphase onset , non-injected oocytes were fixed ≥15 hr after NEBD .",
"In cases where NEBD was not evident , microinjected oocytes were assessed periodically and briefly ( ≤1 min ) for maturation and fixed once the spindle migrated to the cell surface and acquired a size corresponding to about 15 h after NEBD , as defined by our previous study ( Holubcova et al . , 2015 ) .",
"None of the oocytes used for fixed cell analysis were subjected to long-term live imaging before fixation .",
"After fixation , oocytes were extracted in PBS , 0 . 1% Triton X-100 overnight at 4°C .",
"All antibody incubations were performed in PBS , 3% BSA and 0 . 1% Triton X-100 , either overnight at 4°C ( for primary antibodies ) or for 3 hr at room temperature ( for secondary antibodies ) .",
"The primary antibodies used were rat anti-α-tubulin ( MCA78G , Serotec; 1:3000 ) , mouse anti-Hec1 ( ab3613 , Abcam; 1:100 ) , mouse anti-SgoL1 ( H00151648-M01 , Abnova 1:100 ) and human ACA centromere CREST autoantibody ( FZ90C-CS1058 , Europa Bioproducts; 1:1000 ) .",
"As secondary antibodies , Alexa-Fluor-488/564/647 labelled anti-rat/anti-human/anti-mouse ( all Molecular Probes; 1:400 ) were used .",
"DNA was stained with 0 . 5 µg/ml Hoechst 33342 ( Molecular Probes ) .",
"Immunostained oocytes were imaged on a Zeiss LSM710 confocal microscope equipped with a 63x C Apochromat 1 . 2 NA water immersion objective at a spatial resolution of 0 . 3 µm optical sections ( xy pixel 1024x1024 ) .",
"For analysis , images were deconvolved using Huygens Professional ( Scientific Volume Imaging ) ."
]
] | [
"Aneuploidy in human eggs is the leading cause of pregnancy loss and Down’s syndrome .",
"Aneuploid eggs result from chromosome segregation errors when an egg develops from a progenitor cell , called an oocyte .",
"The mechanisms that lead to an increase in aneuploidy with advanced maternal age are largely unclear .",
"Here , we show that many sister kinetochores in human oocytes are separated and do not behave as a single functional unit during the first meiotic division .",
"Having separated sister kinetochores allowed bivalents to rotate by 90 degrees on the spindle and increased the risk of merotelic kinetochore-microtubule attachments .",
"Advanced maternal age led to an increase in sister kinetochore separation , rotated bivalents and merotelic attachments .",
"Chromosome arm cohesion was weakened , and the fraction of bivalents that precociously dissociated into univalents was increased .",
"Together , our data reveal multiple age-related changes in chromosome architecture that could explain why oocyte aneuploidy increases with advanced maternal age ."
] | [
"Older women are more likely to experience a miscarriage or give birth to a child who has a developmental disorder .",
"This occurs because age increases the chances that a woman’s egg cells will have the wrong number of chromosomes .",
"If a sperm fertilizes an egg with too many or too few copies of a chromosome , the resulting embryo will have the wrong number of copies for many genes .",
"Many of these embryos fail to develop and die , but some are born with developmental conditions like Down's syndrome and Turner syndrome .",
"New egg cells develop from immature egg cells that are present in a woman from birth .",
"In an immature egg cell , chromosomes that came from the woman’s father are paired up with the matching chromosomes from the woman’s mother and the handle-like structures on each chromosome ( called the kinetochores ) are fused .",
"Just before the immature egg cell divides , a molecular machine called ‘the spindle’ attaches to the chromosome handles .",
"The spindle then separates these pairs of chromosomes such that each new cell receives only one copy of each chromosome .",
"However , while it is known that this process sometimes goes wrong , it is not clear why mistakes happen more often in older women .",
"Now , Zielinska et al . used powerful microscopes to observe cell division in over 200 preserved or living immature egg cells donated by women between the ages of 23 and 46 .",
"First , the experiments examined over 1 , 000 chromosomes in preserved immature egg cells that were about to divide .",
"This revealed that the chromosome handles that were supposed to be fused had often disconnected in women over 35 years old .",
"Chromosome pairs without correctly fused handles were also prone to rotating during the division process , and sometimes the pairs simply fell apart too soon .",
"Further experiments with living immature egg cells then revealed that the spindle struggled to grip and separate the chromosomes correctly , possibly because the chromosome handles were not properly fused .",
"These events increased the likelihood of a new egg cell receiving too many or too few chromosomes .",
"Finally , Zielinska et al . found that immature egg cells lack a robust control mechanism that can detect when these problems occur .",
"Together these findings help to explain why miscarriages and chromosome abnormalities are more common in the children of older women .",
"Research building on these findings may in the future help women in their late 30s and early 40s to increase their chances of having a family ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Material and methods"
] | [
"structural biology and molecular biophysics",
"microbiology and infectious disease"
] | Mechanistic insight into the conserved allosteric regulation of periplasmic proteolysis by the signaling molecule cyclic-di-GMP | elife-03650-v1 | [
[
"Biofilms are complex agglomerations of sessile , microbial cells surrounded in a self-secreted extracellular matrix that is composed primarily of exopolysaccharides , proteins , and nucleic acids ( Hall-Stoodley et al . , 2004 ) .",
"These communities are prevalent in natural as well as industrial and hospital settings and can form on a wide range of biotic and abiotic surfaces .",
"Biofilm-forming pathogenic bacteria have been associated with numerous persistent and nosocomial infections in humans , such as infection of the ear or urinary tract or colonizing the lungs of patients suffering from cystic fibriosis ( Parsek and Singh , 2003 ) .",
"Because bacteria in biofilms can withstand antibiotic treatment , many clinically relevant antibiotics are ineffective in the treatment of biofilm-related bacterial infections ( Mah and O'Toole , 2001 ) .",
"Such antibiotic tolerance is a matter of concern especially in the context of the increasing number of multi-drug resistant strains and a rather slow rate of discovery of new antimicrobial agents ( Spellberg et al . , 2004 ) .",
"Hence , it is crucial to understand the molecular basis of biofilm formation , maintenance , and dispersal in order to identify novel targets that could potentially be used for disrupting these bacterial aggregates .",
"The decision to transition between a planktonic and a biofilm life-style is orchestrated by the near ubiquitous bacterial second messenger cyclic-di-GMP ( c-di-GMP ) , a dinucleotide known to modulate many different aspects of bacterial physiology ( Ross et al . , 1987; Hengge , 2009 ) .",
"The dinucleotide is synthesized from two molecules of GTP by diguanylate cyclases ( DGCs ) containing a GGDEF domain , and hydrolyzed by phosphodiesterases ( PDEs ) with either an EAL or a HD-GYP domain ( Tal et al . , 1998; Simm et al . , 2004; Ryan et al . , 2006; Schirmer and Jenal , 2009; Krasteva et al . , 2012 ) .",
"Often these juxtaposing domains appear together in the same polypeptide chain , and in a particular subset , as part of enzymatically inactive multi-domain proteins providing them with the ability to sense and respond to changing levels of intracellular c-di-GMP .",
"In Pseudomonas fluorescens , biofilm formation is dependent on such a signaling protein , referred to as LapD ( Hinsa and O'Toole , 2006; Newell et al . , 2009 ) .",
"This transmembrane receptor contains a cytoplasmic GGDEF–EAL domain module that lacks enzymatic activity , though the EAL domain has retained its capacity to bind c-di-GMP .",
"Cytosolic c-di-GMP levels in P . fluorescens are regulated in response to nutrient availability , specifically the amount of inorganic phosphate ( Figure 1A; Monds et al . , 2007 ) .",
"Limiting phosphate increases the expression of a PDE , which lowers intracellular c-di-GMP levels , ultimately resulting in the dispersion of P . fluorescens biofilms .",
"Conversely , when environmental phosphate levels are high , multiple DGCs contribute to high intracellular c-di-GMP levels , resulting in stable cell attachment and subsequently biofilm formation ( Newell et al . , 2011b ) .",
"LapD is a c-di-GMP receptor that translates changes in the concentration of this cytosolic second messenger into cell-surface events via an inside-out signaling mechanism that ultimately controls cell adhesion ( Newell et al . , 2009 ) .",
"Specifically , c-di-GMP-bound LapD engages the periplasmic protease LapG through its Per-Arnt-Sim ( PAS ) -like domain , preventing it from cleaving the cell surface-bound large adhesin LapA , a protein mediating cell adhesion to multiple substrates ( Navarro et al . , 2011; Newell et al . , 2011a; El-Kirat-Chatel et al . , 2014 ) .",
"In contrast , c-di-GMP-unbound LapD adopts an autoinhibited conformation and loses its affinity for LapG .",
"LapG in turn accesses and cleaves LapA's N-terminus , releasing the bulk of the adhesin from the cell surface and destabilizing cell attachment ( Boyd et al . , 2012; Chatterjee et al . , 2012; Boyd et al . , 2014 ) .",
"Thus , LapD acts as the central hub in P . fluorescens that converts environmental cues into the formation of or dispersal from biofilms . 10 . 7554/eLife . 03650 . 003Figure 1 . The LapD signaling system and structures of the LapD periplasmic domain .",
"( A ) Overview of LapD-mediated signaling .",
"The primary structure and domain organization of the LapD receptor is shown ( top panel ) .",
"The cartoon ( bottom panel ) summarizes the current model of LapD-mediated regulation of biofilm formation in P . fluorescens by the differential recruitment of the periplasmic protease LapG in response to exogenous inorganic phosphate ( Pi ) availability ( reviewed in Boyd and O'Toole , 2012 ) .",
"Low bioavailability of Pi induces the expression of phosphodiesterases ( PDEs ) via the Pho system , which results in low intra-cellular concentrations of c-di-GMP and LapD adopting an autoinhibited state .",
"Auto-inhibited LapD cannot sequester the periplasmic protease LapG , allowing the LapG enzyme to cleave the cell-surface adhesin LapA , releasing it from the cell surface .",
"Conversely , when environmental Pi is present in sufficient concentration , a subset of diguanylate cyclases ( DGCs ) produce intra-cellular c-di-GMP to levels sufficient for binding to the EAL domain of LapD , thus activating this receptor .",
"Activated LapD binds to and sequesters LapG in the periplasm , which promotes biofilm formation by preventing cleavage of LapA .",
"( B ) Crystal structures of the periplasmic output domain from P . fluorescens LapD and L . pneumophila CdgS9 .",
"Two orthogonal views of each output domain structure are shown in ribbon representation , with the two protomers colored pink and gray , respectively ( P . fluorescens LapDOutput , PDB 3PJV; Navarro et al . , 2011 ) .",
"Distances between the tryptophan residues in the conserved GWxQ motif of each protomer are indicated .",
"The relative position of the inner cell membrane ( gray bar ) and connection to the flanking transmembrane ( TM ) helices are indicated .",
"( C ) Surface conservation of the output domain .",
"The sequences of 18 orthologs of LapD were aligned and the sequence conservation was mapped on to the solvent-accessible surface of CdgS9Output .",
"The surface is colored according to the degree of conservation . DOI: http://dx . doi . org/10 . 7554/eLife . 03650 . 003 Given that orthologs to LapD and LapG are found in a variety of bacteria , including several pathogens , understanding the molecular mechanism by which c-di-GMP binding to LapD's cytosolic EAL domain alters the ability of its periplasmic domain to bind LapG is of great interest .",
"Previous work from our groups has begun to establish some of these intrinsic regulatory principles ( Navarro et al . , 2011; Newell et al . , 2011a ) .",
"The PAS-like periplasmic domain of LapD that engages LapG is flanked by two putative transmembrane helices , the second of which connects to an intracellular , juxatamembrane HAMP domain followed by the aforementioned GGDEF-EAL domain module ( Figure 1A ) .",
"HAMP domains have been extensively studied in several other systems , as this domain is a crucial signal relay module in a large number of diverse proteins , not only in bacteria but also in archaea and eukaryotes ( Galperin et al . , 2001 ) .",
"These domains are known to be a homodimeric assembly adopting a parallel four-helix bundle ( Hulko et al . , 2006; Ferris et al . , 2011 ) , although the exact mechanism by which conformational changes are propagated through a signaling protein via HAMP domains remains a topic of great interest ( Swain and Falke , 2007; Zhou et al . , 2009; Ames et al . , 2014 ) .",
"In LapD , the HAMP domain couples to the GGDEF-EAL domain module via a common extension of HAMP domains , the so-called signaling or S helix ( Anantharaman et al . , 2006 ) .",
"Our previous structure-function analyses revealed that the S helix docks onto the lateral surface of the EAL domain and positions the GGDEF domain above the c-di-GMP binding site at the EAL domain , preventing dinucleotide binding , and hence stabilizing the autoinhibited state .",
"Conversely , c-di-GMP binding to the EAL domain is mutually exclusive with this autoinhibited conformation , disrupting inhibitory interactions , leading to an activation of LapD ( Navarro et al . , 2011 ) .",
"Through a poorly understood process , these conformational changes in the GGDEF-EAL domain module are sensed by the HAMP domain , which then propagates the signal to the periplasmic domain of LapD , ultimately resulting in a change in affinity for LapG ( Newell et al . , 2009; Newell et al . , 2011a ) .",
"The elucidation of a complete functional circuit from nutritional input to the output machinery , and many of the underlying molecular principles , makes the Lap system one of the best-understood c-di-GMP signaling systems to date , and a potent model system to study mechanistic questions .",
"In addition , considering the prevalence of the intracellular HAMP-GGDEF-EAL module ( currently 1296 annotated genes in Pfam ) and extracellular PAS-like output domain in LapD-like c-di-GMP receptors , as well as in active enzymes for environmental sensing , the mechanism underlying LapD function will be broadly relevant for understanding bacterial transmembrane signaling .",
"Our previous work elucidated the mechanistic underpinnings and regulation of the cytoplasmic portion of LapD ( Navarro et al . , 2011 ) .",
"Here , we focus on the periplasmic events and how they are coupled to the cytoplasmic control elements in the context of the global , c-di-GMP-dependent switching mechanism of the full-length , transmembrane receptor .",
"In particular , we present an activation model for inside-out signaling via LapD based on structures of an unliganded and LapG-bound output domain , and further informed by protein and cell-based switching assays .",
"The results shed light on the control of bacterial proteolysis in the periplasm via conserved signal transduction modules ."
],
[
"We previously identified conserved LapD/LapG-containing operons in many , diverse bacterial species ( Navarro et al . , 2011; Newell et al . , 2011a ) .",
"In addition , we reported the crystal structures of the isolated periplasmic output domain of P . fluorescens LapD and the LapG protease ortholog from Legionella pneumophila ( Navarro et al . , 2011; Chatterjee et al . , 2012 ) .",
"The former structure revealed an unusual domain-swapped , V-shaped dimer resembling PAS domains ( Figure 1B ) , yet several questions remained unanswered .",
"While we identified a surface-exposed loop that contains a strictly conserved GWxQ motif important for LapG binding ( Navarro et al . , 2011 ) , how access of LapG to the loop is controlled was not apparent .",
"Also , the structure of LapG revealed a transglutaminase-like fold with functionally important calcium ions bound near its active site ( Chatterjee et al . , 2012 ) , but did not explain how this protease interacts with LapD .",
"To address these shortcomings , we report here two structures , that of the output domain of a LapD ortholog from L . pneumophila , also known and referred to here as CdgS9 ( Levi et al . , 2011 ) , and that of LapG in complex with the same output domain .",
"The comparison of the apo- and LapG-bound states elucidates the periplasmic switching mechanism .",
"The structure of L . pneumophila CdgS9Output ( residues of 22–152 ) was determined from data collected on a crystal grown from selenomethionine-derivatized protein by using single anomalous dispersion ( SAD ) phasing ( see ‘Material and methods’ , and Table 1 for details ) .",
"The asymmetric unit contains one molecule of CdgS9Output , which adopts a PAS-like domain fold with the highest structural homology to CitA ( Reinelt et al . , 2003 ) .",
"A potential biologically relevant dimer involves a crystallographic symmetry mate , burying 3020 Å2 surface area at the twofold symmetry axis via favorable interactions ( estimated ΔGint = −20 . 2 kcal/mol ) ( Krissinel and Henrick , 2007 ) ( Figure 1B , bottom right panel ) .",
"The residues at the CdgS9Output homo-dimer interface tend to be more conserved than residues outside of this region , based on an alignment of 18 putative LapD orthologs from different bacterial species ( Figure 1C; Navarro et al . , 2011 ) . 10 . 7554/eLife . 03650 . 004Table 1 . Data collection and refinement statisticsDOI: http://dx . doi . org/10 . 7554/eLife . 03650 . 004CdgS9Output ( SeMet ) LapG/CdgS9Output ( SeMet ) LapG/CdgS9Output ( native ) Data collectiona Space groupP6122P21P21 Unit cell axes a , b , c ( Å ) 61 . 58 , 61 . 58 , 147 . 5359 . 08 , 74 . 69 , 62 . 8875 . 85 , 73 . 67 , 88 . 22 α , β , γ ( ο ) 90 . 0 , 90 . 0 , 120 . 090 . 0 , 101 . 8 , 90 . 090 . 0 , 92 . 9 , 90 . 0 Resolution Limits ( Å ) 30–2 . 14 ( 2 . 22–2 . 14 ) 30–0 . 29 ( 2 . 37–2 . 29 ) 44–2 . 10 ( 2 . 17–2 . 10 ) Unique Observations9611 ( 924 ) 22 , 884 ( 2231 ) 55 , 783 ( 4959 ) Completeness98 . 6 ( 97 . 6 ) 93 . 8 ( 92 . 2 ) 97 . 7 ( 87 . 3 ) Multiplicity21 . 9 ( 18 . 6 ) 7 . 2 ( 5 . 4 ) 4 . 3 ( 3 . 2 ) Average I/σ35 . 4 ( 10 . 5 ) 20 . 1 ( 2 . 3 ) 12 . 4 ( 2 . 8 ) Rsym ( % ) 7 . 7 ( 30 . 9 ) 8 . 9 ( 67 . 1 ) 8 . 2 ( 38 . 7 ) Refinement Rcryst/Rfree ( % ) 21 . 9/24 . 317 . 0/21 . 1 No .",
"protein molecules16 No .",
"protein residues131886 No .",
"water molecules90677 Total number atoms11587826 rmsd bond angles ( o ) 1 . 021 . 05 rmsd bond lengths ( Å ) 0 . 0040 . 004 <B> protein ( Å2 ) 41 . 234 . 0 <B> water ( Å2 ) 59 . 138 . 8 <B> calcium ( Å2 ) N/A35 . 3 Ramachandran Plot ( % ) Favored9898 Outliers00PDB code4U644U65aNumbers in parentheses correspond to values in the highest resolution .",
"The protomers form a parallel dimer , with the termini lining up at one end of the complex , consistent with their connectivity to the transmembrane helices that flank the periplasmic domain .",
"There is only a minor entanglement of the terminal short β-strands , which connect to the transmembrane helices ( Figure 1B ) .",
"Such an arrangement is in stark contrast to our previously determined structure of P . fluorescens LapDOutput ( Figure 1B , left panel ) , in which the PAS domain dimer originated from an unusually extensive domain-swap .",
"Although the folds of the dimer half-sites between the P . fluorescens LapDOutput and CdgS9Output are almost identical ( rmsd of 1 . 9 Å ) and the conserved GWxQ motifs , which are important for LapG binding , are positioned in identical , surface-exposed loops within each half-site , the dimer topology of the two output domain ortholog structures is substantially different ( Figure 1B ) .",
"In addition , one notable difference between the crystallographic models for the output domains of LapD and CdgS9 is the relative position of the conserved GWxQ motifs .",
"CdgS9Output adopts a ‘parallel’ conformation as opposed to the open , V-shaped form that we observed in case of LapDOutput .",
"As a consequence , the tryptophan residues ( W125 in P . fluorescens LapD; W126 in L . pneumophila CdgS9 ) that form the distal tip of the PAS fold and appear to be a main anchor point for LapG binding ( Navarro et al . , 2011 ) are farther apart in the P . fluorescens dimer than the L . pneumophila LapD ortholog ( 50 Å vs 35 Å between their respective Cβ positions ) ( Figure 1B ) .",
"Considering the discrepancy in the two output domain structures , we developed a structure-guided assay , which allows us to distinguish between the two topologies in the context of the intact , full-length LapD protein from P . fluorescens ( Figure 2 ) .",
"The approach relies on the site-specific incorporation of a non-natural amino acid with altered functionality , in this case para-azido-phenylalanine ( pAzF ) for target cross-linking ( Figure 2A; Chin et al . , 2002; Mehl et al . , 2003; Peeler and Mehl , 2012 ) .",
"Upon UV irradiation , pAzF reacts efficiently with N-H and C-H bonds in its vicinity , forming covalent adducts within a radius of approximately 3 Å ( Keana and Cai , 1990 ) .",
"Specifically , we introduced pAzF via an amber codon-suppression system at sites that we predict would yield cross-links either in only one of the two topologies ( A50 , T54 , A115 , based on V-shaped P . fluorescens model; A48 , S52 , L66 , based on a homology model using the parallel L . pneumophila structure as the target ) , in both ( H41 , Y75 ) or in none ( F71 , V91 , R93 ) ( Figure 2B ) .",
"Choosing several sites in each category increases confidence in the results .",
"LapD variants containing pAzF incorporated at these positions were expressed in a construct wherein the protein was fused to super-folder green fluorescent protein ( sfGFP ) ( Pedelacq et al . , 2006 ) at its C-terminus ( Figure 2A ) , which enables the use of in-gel fluorescence to detect monomeric and cross-linked species in SDS-PAGE with high specificity and sensitivity using crude lysates or partially purified proteins .",
"In this particular case , we expect to observe a second band corresponding to a covalent LapD-sfGFP dimer only if the pAzF residue was incorporated at or very close to the dimerization interface . 10 . 7554/eLife . 03650 . 005Figure 2 . Distinguishing between the ‘open’ domain-swapped and ‘closed’ parallel conformations of the output domain of LapD .",
"( A ) Non-natural amino acid incorporation into protein .",
"The UV-photo-activatable non-natural amino acid para-azido-phenylalanine ( pAzF ) was site-specifically incorporated into the output domain of full-length P . fluorescens LapD , which was fused C-terminally to superfolder GFP ( sfGFP ) , using a two-plasmid amber-suppression expression system .",
"( B ) Cross-linking strategy based on structural models .",
"Two different models of the output domain of LapD are shown with the eleven sites of pAzF incorporation shown as sticks and color-coded according to structure-based cross-linking predictions .",
"The left panel shows the domain-swapped , open conformation determined previously ( PDB 3PJV; Navarro et al . , 2011 ) , while the right panel shows a homology model of the ‘closed’ , parallel conformation based on the crystal structure of CdgS9Output .",
"( C ) Crosslinking patterns support a ‘closed’ , parallel model .",
"Cells expressing full-length LapD fused to sfGFP with pAzF incorporated at the indicated positions were harvested by centrifugation and lysed by sonication .",
"Membranes were isolated by ultracentriguation and resuspended in buffer lacking detergent supplemented with and without c-di-GMP .",
"After equilibration , half of each sample was exposed to short-wave UV light .",
"These membrane suspensions were immediately solubilized in 2% SDS and subjected to SDS-PAGE analysis .",
"Gels were imaged by fluorescence .",
"As indicated , cross-links appear as band-shifts with a higher molecular weight compared to LapD-sfGFP . DOI: http://dx . doi . org/10 . 7554/eLife . 03650 . 005 We expressed each full-length P . fluorescens LapD variant carrying a site-specific pAzF residue in Escherichia coli , isolated the membrane fraction , and exposed half of the respective samples to UV , the other half was processed identically but without excitation of pAzF .",
"Proteins were separated in SDS-PAGE , and fluorescent bands were detected using a gel imager .",
"In the absence of UV light , no cross-linked products were observed .",
"Also , no cross-links occurred in the wild-type protein lacking pAzF .",
"Notably , no cross-linked LapD dimers were observed when pAzF was introduced at positions that would support dimerization in the crystallographic domain-swapped , V-shaped LapDOutput model , either in the absence or presence of c-di-GMP .",
"The only cross-linked LapD dimers were seen with proteins that contained pAzF at interfacial positions that were consistent with both models ( H41 , Y75 ) or identified in the parallel , unswapped model ( e . g . , A48 , S52 , L66 ) based on the CdgS9Output structure .",
"Taken together , the cross-linking data clearly indicate that the topology observed in the CdgS9Output structure represents the biologically relevant model , which is conserved in the two LapD orthologs , LapD and CdgS9 , despite only moderate overall sequence conservation ( 31% identity/52% similarity ) .",
"We suspect our previously published domain-swapped model of the P . fluorescens output domain was an artifact of expression and/or crystallization .",
"A deeper understanding of the regulation of periplasmic proteolysis via LapD requires a detailed structural model of a LapG–LapDOutput complex .",
"We previously showed that the isolated output domain of P . fluorescens or L . pneumophila LapD orthologs binds its corresponding LapG protease counterparts readily ( Navarro et al . , 2011; Chatterjee et al . , 2012 ) .",
"In addition , we demonstrated that the mode of interaction is conserved since we also could generate mixed complexes between the output domain from one strain and the protease of the other , and these interactions relied on the presence of the surface-exposed tryptophan residue ( W125 in P . fluorescens LapD , W126 in L . pneumophila CdgS9 ) in the conserved GWxQ loop of LapD ( Chatterjee et al . , 2012 ) .",
"While the homogeneous complexes failed to crystallize thus far despite the best of our efforts , we were successful in determining the structure of L . pneumophila CdgS9Output bound to P . fluorescens LapG ( see details in ‘Materials and methods’ ) ( Figure 3 ) .",
"The use of orthologous proteins as surrogates is common practice in obtaining sought-after crystal structures , and our validation experiments ( see below ) attest to the conservation of key features and the functional relevance of these structure-based models . 10 . 7554/eLife . 03650 . 006Figure 3 . Crystal structure of a P . fluorescens LapG and L . pneumophila CdgS9Output complex .",
"( A ) Overview of the complex structure .",
"The LapG-CdgS9Output complex is shown as a ribbon representation , with the two protomer chains of CdgS9Output colored in pink and gray , and the N- and C-terminal lobes of LapG shown in slate and cyan , respectively .",
"The conserved catalytic triad ( cysteine-histidine-aspartate; C-H-D ) is shown as sticks with the carbon atoms in orange .",
"The highly conserved tryptophan residue ( W126 ) in each protomer of CdgS9Output is presented as sticks , calcium ions of LapG are shown as green spheres .",
"The relative position of the inner cell membrane ( gray bar ) is indicated .",
"Two orthogonal views are shown .",
"( B ) Interaction interface between CdgS9Output and LapG .",
"The interface footprints on LapG and the CdgS9 half-sides ( pink , purple and black ) were highlighted on the accessible surface area of the individual proteins ( left panel ) .",
"The binding pocket for CdgS9 W126 in LapG is shown as a close-up view ( right panel ) .",
"A 180° rotation was applied to LapG from the complex structure to view the interface .",
"( C ) Surface conservation of CsgS9Output and LapG interaction interface .",
"Based on the alignment of 18 sequences of LapD and LapG orthologs , the sequence conservation was mapped onto the accessible surface of each protein .",
"The surface is colored according to the degree of sequence conservation .",
"For LapG , two views , separated by a 180° rotation , are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 03650 . 006 The crystallized complex consists of two CdgS9 output domain molecules that adopt a similar parallel dimer conformation as in the apo-CdgS9Output structure ( Figures 1B and 3A ) .",
"The output domain dimer is bound to one LapG molecule at a lateral docking site ( Figure 3A ) , with an interaction surface of 1290 Å2 ( with a calculated ΔGint = −16 . 3 kcal/mol; PISA [Krissinel and Henrick , 2007] ) and involving both molecules of the CdgS9Output dimer to a similar extent ( Figure 3B ) .",
"As predicted by the biochemical analysis ( Navarro et al . , 2011; Chatterjee et al . , 2012 ) , the aforementioned conserved tryptophan residue ( W126 ) is the major anchor point on CdgS9 as it inserts into a hydrophobic pocket at the bottom of LapG ( Figure 3A , right panel ) .",
"The pocket is formed by strand β4 of LapG's C-terminal lobe and several other disjointed motifs preceding β4 .",
"While there are contributions from a limited number of hydrogen bonds and a water-mediated interaction ( Figure 3A ) , the interface appears to be dominated by hydrophobic contacts and shape-complementarity of the interacting elements .",
"Besides the binding pocket for the conserved tryptophan residue , LapG contributes surfaces surrounding the pocket and the central strand β4 that binds to the CdgS9Output dimer interface , yielding a continuous interfacial area bridging both protomers of the CdgS9Output dimer .",
"Based on an alignment of 18 distantly related LapDG ortholog pairs ( Navarro et al . , 2011 ) , we mapped sequence conservation onto the molecular surface of CdgS9Output and LapG .",
"Notably , there is a disparity regarding surface conservation between the two proteins: LapG presents a cluster of conserved residues on the side that coincides with the active site and the region that interacts with CdgS9Output , while the opposite side of the protease shows poor sequence conservation ( Figure 3C , right panel ) .",
"In contrast , CdgS9Output is less conserved overall ( 37% identical to P . fluorescens LapDOutput ) and shows only a moderate clustering of conserved residues ( Figure 3C , left panel ) .",
"Yet , the main docking site for the CdgS9–LapG interaction , the loop that contains the GW126xQ motif , is well conserved .",
"As discussed below , pronounced shape complementarity between the interacting surfaces may contribute to the stable recruitment of LapG to the periplasmic output domain of LapD orthologs .",
"Determination of the structure of apo- and LapG-bound CdgS9Output permits us to describe the conformational changes that tune the affinity of LapD-like receptors for LapG proteases .",
"Given that the core LapG-binding motif ( the GWxQ loop ) is surface-exposed in the apo-CdgS9Output structure and is potentially available for engaging LapG , it was important to address how differential LapG binding would be achieved in the two distinct states .",
"For their comparison , we superimposed the apo-CdgS9Output dimer onto the CdgS9Output domain of the complex ( Figure 4 ) .",
"The conformation of the individual output domain protomers varies only slightly in the two states ( rmsd between 1 . 1 and 1 . 7 Å ) .",
"However , from a top view a change in the output domain dimer interface is apparent ( Figure 4A , left panel ) .",
"As a result , a cradle is created , which buttresses strand β4 of LapG while providing additional anchor points via the conserved GWxQ binding loop on one dimer half-side and helix α2 on the other .",
"The CdgS9 helix butts against the β-sheet of the C-terminal LapG lobe , just underneath the protease active site . 10 . 7554/eLife . 03650 . 007Figure 4 . Conformational changes in CdgS9Output between the apo-state and in complex with LapG .",
"( A ) Overview of the superposition of apo-CdgS9Output and LapG-bound CdgS9Output .",
"Chain A of the CdgS9Output dimer was used as the reference for the superposition .",
"Chains A and B of apo-CdgS9Output are colored tan and yellow , while chains A and B of LapG-bound CdgS9Output are colored pink and gray , respectively .",
"The N- and C-terminal lobes of LapG are colored in slate and cyan , respectively .",
"Both cartoon ( left panel ) and surface ( right panel ) representations of LapG are shown .",
"( B ) Components of the conformational change .",
"The superposition in ( A ) is shown as a side-view , depicting the helical motions of α1 , α2 , and α4 .",
"Two perpendicular views are shown , with the right panel showing slices of the top view .",
"( C ) Cartoon model .",
"The structural transition from an apo-state ( yellow/tan ) to a LapG binding-competent state ( pink/gray ) of CdgS9Output based on the analysis shown in panels ( A ) and ( B ) is illustrated .",
"The top pair depicts changes at the distal tip of the output domain; the bottom pair illustrates changes in the membrane proximal region of the output domain . DOI: http://dx . doi . org/10 . 7554/eLife . 03650 . 007 This conformational change is realized through the differential packing of the central helices within the output domain dimer ( Figure 4B , C ) .",
"In the apo-state , the output domain dimer of CdgS9 contains central four-helix bundles , either via packing of helices α1 and α2 of the PAS domain fold close to the distal end , or helices α1 and α4 at the membrane-proximal region .",
"The types of interactions that facilitate the formation of the two 4-helix bundle segments are quite different .",
"Closer to the membrane , a cluster of 4 phenylalanine residues ( 2 per protomer; F33 and F34 of CdgS9 ) stack in a perpendicular fashion , as opposed to the packing near the distal tip , which involves the burying of two symmetry-related arginine residues ( R75 of CdgS9 ) ( Figure 4C ) .",
"As a consequence , the overall structure of the output domain appears bulged ( Figure 4B ) .",
"When engaged with LapG ( Figures 3A and 4B ) , this perfect symmetry between the two protomers observed in the apo-state is broken .",
"The resulting complex is asymmetric with regard to the output domain dimer , manifested by a 5 Å tilt and vertical offset of one PAS domain fold relative to the other .",
"The central helices α1 are pushed closer together—a movement permitted by smaller residues at the interface ( S48 and S52 ) , a perpendicular-to-parallel stacking transition of one of the F33–F34 residue pairs , and the relocation of R75 from the dimer interface to a more surface-exposed position .",
"The net result is a transition from a four-helix bundle with poor shape complementarity with LapG to a two-helix bundle and an overall shape that is able to accommodate LapG ( Figure 4C ) .",
"We argue that the isolated output domain is in a deregulated state and spontaneously populates the high-affinity conformation for LapG binding ( on-state ) since regulatory domains that would lock the receptor in its off-state ( the apo state ) or on-state are lacking .",
"This conclusion is consistent with the observation that the isolated output domain is an inefficient competitor for the interaction between LapG and full-length LapD ( data not shown ) .",
"Nevertheless , the conservation of the interfaces and motifs involved in LapG binding in the otherwise fairly divergent LapD family of receptors suggest that the proposed mechanism is more generally applicable .",
"Taken together , the crystal structures of apo-CdgS9Output and a CdgS9Output-LapG complex and our previous work ( Newell et al . , 2009; Navarro et al . , 2011; Newell et al . , 2011a ) yield a detailed model for the activation of LapD by c-di-GMP binding to its cytosolic module .",
"It involves the release of an autoinhibitory interaction , triggering a conformational change in the periplasmic domain through the juxtamembrane HAMP domain and transmembrane segments .",
"High-affinity LapG binding to the off-state of LapD is prevented by an apparent surface mismatch .",
"In contrast , activated LapD displays a new surface that is cryptic in the apo state , spans both protomers of the output domain dimer , and is optimized for interaction with LapG ( Video 1 ) . 10 . 7554/eLife . 03650 . 008Video 1 . Composite structural overview . The video shows several interpolations between the apo-state and the LapG-bound output domain dimer of CdgS9 , based on the crystallographic data presented here .",
"It depicts the conformational changes between the two states of the periplasmic output domain and how they lead to LapG recruitment . DOI: http://dx . doi . org/10 . 7554/eLife . 03650 . 008 Previously , we determined the crystal structure of calcium-bound L . pneumophila LapG ( Chatterjee et al . , 2012 ) , a protease with transglutaminase fold ( Ginalski et al . , 2004 ) .",
"Here , we obtained a molecular model of the CdgS9-bound ortholog from P . fluorescens .",
"In order to ascertain the degree of conservation of the two LapG protease orthologs and to detect any potential conformational changes that may occur upon binding to a LapD ortholog , the two structures were superimposed and analyzed for differences ( Figure 5A ) .",
"Despite moderate sequence conservation between the two orthologs ( 40% identity/54% similarity ) , the two structures are very similar with regard to their overall fold ( Cα rmsd of 1 . 09 Å over 136 atoms ) .",
"However , there are a few significant differences between the two structures .",
"First , we identified a rare di-calcium binding motif in the N-terminal lobe of P . fluorescens LapG , which is in contrast to the single calcium ion bound at the same site in the L . pneumophila LapG structure ( Figure 5B ) .",
"Yet , the two orthologs share the same conserved coordinating residues required for di-calcium binding ( Figure 5C ) ( Chatterjee et al . , 2012 ) .",
"Additional experiments will be necessary to ascertain the functional purpose of these alternate calcium-binding modes; however , it is tempting to suggest these differences help tune their respective substrate specificities given the proximity of the calcium atoms to the catalytic triad . 10 . 7554/eLife . 03650 . 009Figure 5 . Structure of P . fluorescens LapG in complex with CdgS9Output .",
"( A ) The structure of L . pneumophila LapG shown in gray ( PDB: 4FGO , Chatterjee et al . , 2012 ) was superimposed on that of P . fluorescens LapG , colored in slate and cyan , when in complex with CdgS9Output .",
"A close-up view of the differences observed in the C-terminal lobe of the two LapG structures is shown ( right inset ) .",
"Disordered regions not resolved in the crystal structures are shown as dots .",
"Calcium ions are represented as magenta spheres .",
"The catalytic triad residues ( C-H-D ) are shown as sticks with the carbon atoms colored in orange .",
"( B ) The Ca2+ binding sites of LapG .",
"P . fluorescens ( top panel , blue cartoon ) and L . pneumophila LapG ( bottom panel , gray cartoon ) coordinate two or one calcium ion , respectively .",
"In P . fluorescens LapG , an Fo–Fc omit map contoured at 3σ ( green mesh , inset of top panel ) shows strong evidence for a second calcium ion not seen in the L . pneumophila structure , which is made possible by local conformational differences in the so-called calcium binding loop that bring D111 of P . fluorescens into position to coordinate the additional calcium ion .",
"A LIGPLOT ( Wallace et al . , 1995 ) ( right panels ) depicts the coordination of calcium ions .",
"( C ) Conservation of the di-calcium binding site .",
"A WebLOGO plot ( Crooks et al . , 2004 ) generated from an alignment of 18 LapG ortholog sequences demonstrates the conservation of coordinating residues ( blue font ) as well as the nucleophilic cysteine ( yellow font ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 03650 . 009 The second conformational change observed between CdgS9Output-bound P . fluorescens LapG and the L . pneumophila LapG ortholog localizes to the site of interaction with LapD orthologs , which involves the peeling away of β5 upon binding to LapD , whereas β5 and part of the linker are ordered in the stand-alone LapG structures ( Figure 5A ) .",
"Notably though , even in these structures this region appears to be more flexible than the core of the protease based on an analysis of their corresponding temperature factors and the presence of largely disordered segments in its vicinity .",
"Undoubtedly there are concerns when working with a mixed complex containing isolated domains of proteins from different bacterial genera , foremost the validity of the crystallographic models and their relevance for LapD function .",
"This issue is particularly important considering that intact LapD is a transmembrane protein with a cytoplasmic module that is crucial for the regulation of signal transmission across the membrane .",
"To assess the soundness of our structure-based models , we again turned to a cross-linking assay utilizing non-natural amino acid pAzF incorporation .",
"The cross-linking assay awards several advantages over other binding experiments .",
"It provides site-specific information that can be directly correlated with crystallographic data .",
"In addition , it is very sensitive , allowing us to work at low , more physiologically relevant protein concentrations that support c-di-GMP-dependent regulation of LapD , which is lost at higher protein concentrations required in alternative binding assays .",
"Also , the assay reflects on equilibrium binding .",
"Finally , although the cross-linking efficiency is low , the results are semi-quantitative since empirically the fraction of the successfully established cross-links appears to be proportional to the number of bound molecules .",
"We followed the strategy that we employed to assess LapD homo-dimerization ( Figure 2 ) , but in contrast to previous experiments , we introduced pAzF site-specifically into sfGFP-tagged LapG and assessed UV-induced cross-linking to LapD variants , which can be detected as up-shifts of a fluorescent band in SDS-PAGE gels ( Figure 6A ) . 10 . 7554/eLife . 03650 . 010Figure 6 . Validation of the crystallographic CdgS9Output-LapG complex interface .",
"( A ) Schematic overview of the Lapd-LapG cross-linking assay .",
"The right panels show a representative experiment in which detergent-solubilized LapD-sfGFP is incubated with non-fluorescent LapG-containing pAzF incorporated at a site predicted to be at the LapD–LapG binding interface in the presence and absence of c-di-GMP .",
"Upon UV irradiation , a fluorescent band corresponding to LapD-sfGFP covalently linked to LapG is observed , which is more intense in the presence of c-di-GMP indicative of more LapD–LapG binding .",
"Other experiments were carried out with a LapG-sfGFP/LapD pairs .",
"( B ) Structure-guided cross-linking of LapD variants and LapG .",
"The complex structure ( left panel ) highlights four sites on LapG ( A169 , Y176 , V205 , and Y206 ) that span the width of the binding interface plus an additional fifth site ( Y108 ) that is not at the interface in which pAzF was incorporated .",
"Each of these five LapG-sfGFP pAzF-containing variants were purified and incubated with purified non-fluorescent CdgS9Output , LapDOutput and c-di-GMP activated , detergent-solubilized full-length P . fluorescens LapD prior to irradiation with UV light .",
"Samples were analyzed by SDS-PAGE and gels imaged by fluorescence as described in Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 03650 . 010 Specifically , we introduced pAzF at positions in P . fluorescens LapG that are close to the crystallographic LapG-CdgS9Output interface ( A169 , Y176 , V205 , or Y206 ) or at a site distal to the interfacial region ( Y108 ) ( Figure 6B ) .",
"The purified pAzF-containing , sfGFP-tagged P . fluorescens LapG variants were mixed with",
"( i ) L . pneumophila CdgS9Output to validate the crystallographic complex;",
"( ii ) P . fluorescens LapDOutput to assess the conservation of interactions in a homologous complex; and",
"( iii ) full-length , c-di-GMP-activated P . fluorescens LapD , which will enable us to extend results to the native , regulated receptor .",
"We also included a control where we do not add any LapD variant , which will allow us to distinguish between LapG–LapD interactions and possible LapG homo-dimers ( Figure 6B ) .",
"Without UV irradiation , no cross-links were observed in any of the conditions .",
"We also did not observe any band shift in the sample that only contained pAzF-derivatized LapG variants but no LapD , consistent with our previous studies that did not indicate signs of LapG oligomerization ( Chatterjee et al . , 2012 ) .",
"In addition , pAzF at position Y108 of LapG , which is distal to the LapD–LapG interface , did not support cross-linking .",
"In contrast , all the LapG variants that contained a pAzF residue at positions that , based on the structure , are predicted to be close to the interaction site yielded band shifts to variable extents ( V205pAzF > Y206pAzF/Y176pAzF/A169pAzF >> Y108pAzF ) ( Figure 6B ) .",
"It is worth noting that the isolated output domains of L . pneumophila CdgS9 and P . fluorescens LapD cross-link to LapG readily , suggesting that they are in a binding-competent state , which is in agreement with our crystallographic analysis .",
"However , cross-linking is more robust in the presence of c-di-GMP-activated full-length LapD protein ( Figure 6A ) .",
"These data are consistent with the conclusion that our structural models and assays accurately report on the complex formation between full-length LapD and LapG .",
"Next , we sought support for the asymmetric ensemble of a CdgS9Output dimer bound to one LapG protease observed in the crystal structure of the complex .",
"To assess whether the asymmetric 2:1 complex observed in the crystal structure was compatible with the binding of a second LapG molecule , we used the CdgS9Output protomer with the unliganded W126 residue ( CdgS9B; Figure 7A ) as a reference in a superposition of the output domain protomer that engages LapG involving the conserved tryptophan residue .",
"From this analysis , it is clear that the potential second binding site does not allow as tight a binding as the crystallographically determined docking site due to sub-optimal shape complementarity ( Figure 7A , note gap indicated in bottom panel; statistic shape complementarity ( Lawrence and Colman , 1993; Collaborative Computational Project N , 1994 ) of 0 . 72 for the crystallographic interface vs 0 . 31 for the modeled , second binding site ) .",
"This observation is consistent with the asymmetric distribution of conformational changes at the output domain dimer interface ( see above; Figure 4A ) . 10 . 7554/eLife . 03650 . 011Figure 7 . Stoichiometry of the LapD–LapG complex .",
"( A ) Structural model of a 2:2 CdgS9Output–LapG complex .",
"The CdgS9Output domain that engages LapG via its GW126xQ loop was superimposed onto the output domain of the complex structure , which is not bound to LapG .",
"The open arrow indicates poor shape complementarity at the putative second LapG binding site .",
"( B ) Stoichiometry of CdgS9Output-LapG complex in solution .",
"SEC-MALS was used to determine the absolute molecular weight of the CdgS9Output-P .",
"fluorescens LapG complex used for crystallization ( top panel ) , a co-expressed complex of L . pneumophila LapG bound to CdgS9Output ( middle panel ) , and the latter complex incubated with an excess L . pneumophila LapG ( bottom panel ) .",
"Red traces and dashed lines show the signal of the light scattering and UV detector , respectively .",
"Dotted , vertical lines indicate the theoretical molecular weight of the 2:1 and 2:2 output domain-protease complexes based on their primary sequence .",
"Blue points across the elution peaks refer to the calculated molecular weight as the proteins elute from the gel filtration column .",
"( C ) Assessment of LapG binding to full-length LapD by covalent cross-linking .",
"Purified , non-fluorescent LapG possessing pAzF at positions V205 ( top gel ) or Y206 ( bottom gel ) was titrated into a fixed quantity of detergent solubilized LapD-sfGFP in the absence ( left side of gel ) or presence ( right side of gel ) of c-di-GMP ( 20 μM ) and allowed to incubate for 20 min .",
"Samples were irradiated with UV light for 5 min and analyzed by SDS-PAGE .",
"Band intensities of LapD-sfGFP cross-linked to LapG from three independent experiments were measured , normalized to non-crosslinked LapD-sfGFP , averaged , and plotted as function of the LapG:LapD molar ratio ( ±SD ) , which showed maximum crosslinking at ∼0 . 3–0 . 5 LapG molecules to 1 LapD molecule ( bottom panels ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 03650 . 011 Further experimental evidence is provided by SEC-MALS experiments that report the absolute molecular weight of proteins and complexes as they elute from a size-exclusion chromatography column ( Figure 7B ) .",
"The purified protein complex that was derived from co-expression of its components and yielded the native complex crystals elutes in a single peak , with an experimentally determined average molecular weight of 56 . 9 kDa ( theoretical molecular weight for a CdgS9Output dimer bound to one LapG molecule: 52 . 3 kDa ) .",
"Investigation of the measurements across the peak reveals a trend towards slightly higher molecular weights at the beginning of the elution peak , but it never reaches values that would indicate the formation of a stable 2:2 complex ( two LapG molecules bound to a CdgS9Output dimer; theoretical molecular weight of 75 . 2 kDa ) .",
"We also purified the homogeneous complex with both LapG and CdgS9Output coming from L . pneumophila .",
"This sample also yielded a single peak with a molecular weight of 50 . 9 kDa , consistent with a 2:1 complex ( a CdgS9Output dimer bound to one LapG molecule; Figure 5B , middle panel ) .",
"The trend towards higher molecular weights was less apparent , attesting to the homogeneity of this complex .",
"More importantly , even addition of an excess L . pneumophila LapG to the purified complex failed to drive the formation of a 2:2 complex ( Figure 7B , bottom panel ) .",
"Similar experiments for the P . fluorescens proteins could not be performed due to complexity of the LapDOutput elution profiles ( data not shown ) , which indicated high polydispersity and the presence of multiple species that complicated the analysis by SEC-MALS .",
"As an alternative , we turned to the site-specific cross-linking assay introduced earlier to assess complex formation between full-length P . fluorescens LapD and LapG in a semi-quantitative manner ( Figure 7C ) .",
"Here , we titrated sfGFP-tagged full-length P . fluorescens LapD with increasing amounts of P . fluorescens LapG with pAzF replacing either V205 or Y206 , two positions that supported robust cross-linking but would tether to opposite protomers of the LapD dimer ( Figure 6C ) .",
"Based on the crystal structure of the complex , Y206pAzF is predicted to cross-link to the LapD protomer that engages LapG with its GWxQ loop , whereas V205pAzF faces helix α2 of the other protomer .",
"Both LapG variants showed poor but detectable cross-linking in the absence of c-di-GMP ( Figure 7C ) , consistent with previous pull-down experiments ( Newell et al . , 2011a ) .",
"Activation of LapD by the addition of c-di-GMP produced more robust cross-linking across the LapG concentration series .",
"Strikingly , in all cases , maximum cross-linking was achieved at sub-stoichiometric LapG concentrations , roughly corresponding to a ratio of one LapG molecule per LapD dimer .",
"While the LapG-V205pAzF variant resembled a regular saturation-binding curve , LapG-Y206pAzF cross-linking peaked between LapG:LapD molar ratios of 0 . 3–0 . 5 and then dropped at higher LapG concentrations ( Figure 7C ) .",
"No double-shift was observed at any condition , which would indicate the formation of a 2:2 complex , suggesting that this is a rather rare event .",
"These data are consistent with the formation of an asymmetric LapD–LapG complex in P . fluorescens , but they also suggest distinct modes of binding , an argument that is based on the disparity observed between LapG-V205pAzF and LapG-Y206pAzF .",
"The difference in cross-linking profiles may be rationalized by the observation that V205pAzF could form a covalent bond with the LapD output domain protomer for which binding does not require interaction with the tryptophan-containing loop ( Figures 6B and 8A ) .",
"Taken together , these experiments establish the asymmetric complex observed in the crystals as the predominant species in solution and the membrane , and this complex appears to be conserved across the two bacterial genera assessed here . 10 . 7554/eLife . 03650 . 012Figure 8 . Attenuating LapD–LapG interactions with a peptide containing the GWxQ binding motif .",
"( A ) Cross-linking assay using purified proteins .",
"The CdgS9Output-LapG crystal structure depicts the GWxQ β-hairpin motif of CdgS9Output ( yellow cartoon ) as well as the two sites of pAzF incorporation ( blue mesh ) on LapG used to assess LapD–LapG interactions ( top panel ) .",
"To assess the effects of the MDGW126MQA peptide on LapD–LapG interactions , detergent-solubilized , full-length LapD-sfGFP was incubated with purified non-fluorescent LapG containing pAzF at V205 ( inset in bottom panel ) or Y206 ( main graph in bottom panel ) , and increasing concentrations of peptide .",
"Half of the sample was subjected to UV irradiation for 5 min prior to analysis by SDS-PAGE .",
"The average intensity of the fluorescent band corresponding to LapD-sfGFP crosslinked to LapG from three independent measurements was normalized to non-crosslinked LapD-sfGFP , averaged , and plotted as a function of peptide concentration ( ±SD ) ( red line; bottom panel ) .",
"The peptide MDGE126MQA was used as a negative control ( gray line; bottom panel ) .",
"( B ) Pull-downs of LapG-HA from lysates of P . fluorescens .",
"LapD-His6 and LapG-HA were co-expressed in a P . fluorescens strain with deleted lapD and lapG genes .",
"Western blots using HA ( top panel ) and His6 ( bottom panel ) specific primary antibodies were used to assess the levels of LapD-His6 that co-purify with LapG-HA in the presence or absence of peptides and c-di-GMP . DOI: http://dx . doi . org/10 . 7554/eLife . 03650 . 012 Our studies to date have established LapDG as an efficient signaling node to control cell adhesion and biofilm formation ( Newell et al . , 2009; Navarro et al . , 2011; Newell et al . , 2011a; Newell et al . , 2011b ) .",
"The structures provide not only mechanistic insight but also a blueprint for the development of vehicles that would disrupt the LapG–LapD interaction , and hence facilitate dispersal of biofilms .",
"As proof-of-concept , we designed peptides that contain the conserved GWxQ binding motif and would be predicted to compete with the native interaction .",
"As a control , we used a peptide with the corresponding W125E mutations , which in analogy to the mutant LapD variant reported previously ( Navarro et al . , 2011; Chatterjee et al . , 2012 ) , should not bind to LapG and hence be a less effective competitor of the LapD–LapG interaction .",
"Initially , we used the cross-linking assay to assess the effect of the peptides on the P . fluorescens LapD–LapG interactions .",
"Purified sfGFP-fused LapD was mixed with LapG variant Y206pAzF or V205pAzF that had been pre-incubated with either the MDGWMQA or the MDGEMQA peptides .",
"We used both cross-linking positions since they yielded non-equivalent results previously .",
"Based on the crystal structure of the complex , Y206pAzF would be predicted to covalently attach to the LapD dimer half-side that engages with LapG via its GWxQ loop , whereas V205pAzF points towards the other protomer that uses the interaction surface distal to its GWxQ loop ( Figures 6B and 8A ) .",
"In our previous experiments , we observed stronger cross-linking with V205pAzF , while Y206pAzF showed similar trends but unique cross-linking behavior in the LapG titration experiments , which indicated that the Y206pAzF is more sensitive and may report on a more regulated molecular interaction than the V205pAzF position .",
"Here , we demonstrate that increasing amounts of the MDGWMQA peptide , but not of the mutated MDGEMQA , decrease the cross-linking efficiency of Y206pAzF ( Figure 8A ) .",
"At the highest peptide concentration , cross-linking is reduced to about 60% of the unperturbed interaction .",
"Although such robust effects require a large excess of the competitor , they are specific for the tryptophan-containing peptide .",
"Inhibition is less pronounced when we used the LapG-V205pAzF variant ( Figure 8A , inset ) .",
"Finally , the addition of the peptide with the native sequence to LapG pull-downs from P . fluorescens lysates reduces the amount of LapD coming down with LapG , whereas the mutant peptide does not compete with the native interaction ( Figure 8B ) .",
"Together , these data indicate that inhibition of the native LapG–LapD interaction presents a feasible strategy to interfere with this and similar signaling systems .",
"Previously , we elucidated intra-molecular interactions in the cytoplasmic module that stabilize an autoinhibited conformation of LapD .",
"In particular , we showed that the S helix binds to the EAL domain of the same molecule , placing the GGDEF domain on top of the c-di-GMP binding site of the EAL domain , preventing activation at low cytoplasmic c-di-GMP levels .",
"We also identified mutations ( S229D and F222E ) that confer a hyper-biofilm phenotype in P . fluorescens and fail to respond to the appropriate environmental cue , in this case phosphate availability .",
"S229D and F222E locate to the S helix and are thought to destabilize its interaction with the EAL domain ( Navarro et al . , 2011 ) .",
"Using the site-specific cross-linking assay , we interrogated the underlying molecular mechanism of these and other , structure-guided mutants in more detail by assessing directly how mutations in the signaling domains of LapD impacted the interactions between the periplasmic domain of this receptor and LapG .",
"Consistent with previous experiments , we observed basal levels of cross-linking of LapG to LapD in the absence of c-di-GMP in a UV-dependent manner , however addition of c-di-GMP stimulated complex formation .",
"The basal and stimulated binding is abrogated in the output domain mutants W125E and D72K , both located at the LapG binding site , corroborating our structural model .",
"These findings also indicate that LapG binding to apo-LapD is still specific , albeit weaker and/or more transient compared to the activated , c-di-GMP-bound state .",
"S helix mutations S229D and F222E or the HAMP domain mutation I187E , which we predict to bias the HAMP domain towards an activated conformation in analogy to orthologous studies on other HAMP domain-containing proteins ( Zhou et al . , 2011 ) , result in a higher basal level of LapG cross-linking , but also an elevated level of cross-linking when c-di-GMP is present ( Figure 9A ) . 10 . 7554/eLife . 03650 . 013Figure 9 . Mutations in the HAMP domain and S helix sensitize LapD toward c-di-GMP .",
"( A ) Effect of structure-guided point mutations on LapD function .",
"Detergent solubilized LapD-sfGFP variants ( 2 μM ) were incubated with non-fluorescent LapG ( 1 μM ) containing pAzF at positions V205 ( upper gel ) and Y206 ( lower gel ) in the presence and absence of c-di-GMP prior to exposure to UV light and SDS-PAGE analysis .",
"Fluorescently imaged gels indicated LapD mutations W125E and D72K abolish LapD–LapG interactions while HAMP domain and S helix mutations S229D , F222E , and I187E have higher levels of interaction with LapG in the absence and presence of c-di-GMP compared to wild-type .",
"( B ) C-di-GMP sensitivity of LapD .",
"C-di-GMP was titrated into a fixed amount of detergent solubilized LapD-sfGFP ( 2 μM ) variants and non-fluorescent LapG ( 1 μM ) containing pAzF at position V205 , after which crosslinking was initiated by UV irradiation and analyzed by SDS-PAGE .",
"A sharp transition in crosslinking is observed between 0 . 75 μM and 1 μM c-di-GMP for wild-type LapD-sfGFP but not for the other activating mutations , which have higher basal levels of crosslinking and a less pronounced transition . DOI: http://dx . doi . org/10 . 7554/eLife . 03650 . 013 Another subtle , but mechanistically important difference is apparent when we determined the sensitivity of the mutant alleles to c-di-GMP ( Figure 9B ) .",
"Spiking wild-type LapD with increasing amounts of c-di-GMP shows an appreciably sharp transition in LapG binding , with the switch occurring between 0 . 75 μM and 1 μM dinucleotide .",
"In contrast , the mutant variants of LapD with alterations in the S helix ( S229D and F222E ) or the HAMP domain ( I187E ) show a shallower dose–response curve , in addition to the aforementioned higher basal levels ( Figure 9B ) .",
"These experiments establish that the deregulation of LapD through destabilization of regulatory interactions in the cytoplasmic domains is a result of altered sensitivity of these mutant c-di-GMP receptors .",
"These studies also reveal a switch-like behavior of the wild-type protein , which suggests cooperativity as an important regulatory feature for LapD activation .",
"Next , we asked whether LapD regulation in vivo can be altered through mutations in the periplasmic output domain .",
"In particular , we targeted either strictly conserved residues and/or residues that we identified as important for establishing the distinct crystallographic conformations and for the proposed activation mechanism ( Figure 10A , Figure 10—figure supplement 1 ) .",
"In this analysis , we also included a panel of HAMP domain mutations , including I187E , for which switching phenotypes had been established in chemoreceptors such as TSR ( Figures 10A , Figure 10—figure supplement 2; Zhou et al . , 2011 ) .",
"As done previously ( Navarro et al . , 2011; Newell et al . , 2011a ) , the mutations were introduced in a lapD/lapG co-expression plasmid to ensure balanced expression of both genes in P . fluorescens .",
"As the read-out , the static biofilm that forms preferentially at the air-liquid interface was quantified using an established crystal violet-based assay .",
"Deletion of lapD and lapG ( ΔlapD/lapG ) results in a hyperbiofilm phenotype , since the adhesin LapA cannot be processed due to the absence of the protease LapG and thus the adhesin is retained at the cell surface ( Newell et al . , 2011a ) .",
"On the other end of the spectrum , expression of LapG alone in the double-deletion strain results in hypo-biofilm formation or maximal dispersal , since LapG will process LapA in a constitutive , unregulated fashion .",
"Simultaneous expression of wild-type P . fluorescens LapD and LapG in the double-deletion strain restores the phenotype of the parent strain with native LapDG levels , characterized by a low-to-intermediate biofilm mass ( Figure 10A , B ) .",
"This range of phenotypes allows us to assess the functionality of mutant constructs .",
"A non-functional LapD variant would phenocopy a double-deletion strain overexpressing only LapG .",
"In contrast , a constitutively active LapD variant would present itself in a hyper-biofilm phenotype , resembling the phenotype of the double-deletion strain .",
"Any phenotype in-between the two extremes would be indicative of a functional LapD protein that retained regulation via c-di-GMP and near-wild-type levels of LapG recruitment . 10 . 7554/eLife . 03650 . 014Figure 10 . Mutational analysis of LapD regulation .",
"( A ) Biofilm phenotypes of LapD point mutants .",
"A structural model of full-length LapD is shown , which includes the closed , parallel output domain that is linked to the cytoplasmic S helix-GGDEF-EAL domain module via transmembrane and HAMP domains ( left panel ) .",
"Homology models of output domain ( middle panel ) and a HAMP domain ( right panel ) of P . fluorescens LapD were used to design mutations predicted to alter the activity of LapD .",
"Biofilm phenotypes of these LapD variants were assessed using previously established in vivo assays ( lower panel ) .",
"Representative data from 8–12 wells for at least three biological replicates are shown as means ±SD .",
"( B ) Biofilm phenotypes of chimeric constructs .",
"LapD constructs were created that contained either the periplasmic or cytoplasmic domains of L . pneumophila CdgS9 replacing the respective native P . fluorescens LapD domains ( left panel ) .",
"ΔD/ΔG: P . fluorescens strain lacking lapD and lapG genes , pDG: dual expression plasmid for LapD and LapG , p: empty plasmid , pG: expression plasmid for LapG . DOI: http://dx . doi . org/10 . 7554/eLife . 03650 . 01410 . 7554/eLife . 03650 . 015Figure 10—figure supplement 1 . Pairwise alignment of LapD and CdgS9 . Strictly conserved residues are highlighted in red .",
"The positions of point mutations used in this study are marked by asterisks .",
"Arrows indicate positions of the chimeric swap points .",
"Other functional motifs are highlighted and labeled . DOI: http://dx . doi . org/10 . 7554/eLife . 03650 . 01510 . 7554/eLife . 03650 . 016Figure 10—figure supplement 2 . Multiple-sequence alignment of HAMP domains . The alignment highlights conserved hydrophobic residues ( gray ) , other residues implicated in HAMP domain switching ( red ) , and divergent substitutions ( yellow ) .",
"The hydrophobic positions in helices 1 and 2 are central elements for the conformational transitions from a complementary x-da to a knobs-into-holes supercoil packing through a 26° helical rotation within the HAMP domain's four-helix bundle , according to the gear-box model ( Hulko et al . , 2006 ) .",
"Only a few positions show non-conservative changes in LapD and CdgS9 , while the overall alignment indicates that these HAMP domain adopt a canonical fold established for Af1503 ( PDB 2L7H ) ( Ferris et al . , 2011 ) and Aer2 ( PDB 3LNR ) ( Airola et al . , 2010 ) .",
"Residues that were targeted for site-directed mutagenesis of LapD are highlighted by asterisks ( * ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 03650 . 016 Considering the group of output domain mutations tested , there is a distinct lack of penetrant phenotypes despite the fact that non-conservative mutations were introduced at pivotal positions based on the structural analysis and sequence alignments ( Figure 10A , Figure 10—figure supplement 1 ) .",
"Some of these mutations would have been expected to be activating ( i . e . , hyper-biofilm forming ) via destabilization of the apo-LapDOutput conformation , but no such phenotypes were observed .",
"The more apparent hypo-biofilm phenotypes of mutant LapD variants D72K and Y76R can be explained by disturbance of the LapG binding interface by these mutations .",
"The weaker phenotypes of the output domain mutants are in stark contrast to the effect of sequence alterations in the HAMP domain .",
"More than half of the single-point mutations made in the HAMP domain result in hyper-biofilm phenotypes indicative of a constitutive LapD–LapG interaction .",
"Of note , the positions in the helical segments of the HAMP domain , which are important for the differential packing interactions within the dimeric HAMP domain ( positions a and d in the knobs-into-holes packing; Figure 10 , Figure 10—figure supplement 2 ) , have the most pronounced effect on LapD function .",
"These positions are the key elements in the gear-box model for HAMP domain switching ( Hulko et al . , 2006; Ferris et al . , 2011 , 2012 ) , which describes two states of the HAMP four-helix bundle that are separated by 26° rotations of the helices .",
"This observation indicates that LapD's HAMP domain adopts a canonical fold and is likely subject to a similar mode of regulation as has been described for other HAMP domains .",
"Together , these results confirm the regulatory role of the HAMP domain , but also indicate a clear hierarchy of LapD's control elements , with the HAMP domain driving the main regulatory effects , which may be important to bias the system to inside-out signaling and to prevent accidental activation through basal LapD–LapG encounters .",
"Conservation of key residues and surfaces does not imply functional conservation; however , our previous results indicated that complexes between the output domain and LapG from two different bacterial strains form readily and in a physiologically relevant manner ( Chatterjee et al . , 2012 ) .",
"To take this point further , and to demonstrate conservation of mechanism and regulation in cells , we created chimeric full-length LapD constructs , in which we replaced the P . fluorescens output domain or the cytoplasmic domain with the corresponding domains of CdgS9 , the putative L . pneumophila LapD ortholog ( Figure 10B ) .",
"Constructs Lpg-Pfl C1 and C2 contain the L . pneumophila output domain with a cytoplasmic module from P . fluorescens LapD .",
"Pfl-Lpg C2 shares the swap point with Lpg-Pfl C2 , but is the reciprocal construct consisting of output domain and transmembrane helices of LapD and the entire cytoplasmic fragment of CdgS9 .",
"In constructs Pfl-Lpg C3 , C4 , and C5 , the chimeric swap was introduced at different positions in the HAMP domain ( in-between secondary structure elements ) .",
"The swap-point in C5 is at the HAMP-S helix junction , which would leave the intra-molecular S helix-GGDEF-EAL control module intact ( Navarro et al . , 2011 ) .",
"The constructs were introduced in the dual expression plasmid that also carried the P . fluorescens lapG gene for the balanced expression of both proteins , similar to the native operon .",
"Biofilm phenotypes were assessed as before ( see above; Figure 10A ) .",
"Notably , the majority of our chimeric constructs supported biofilm regulation similar to the levels observed with the native P . fluorescens proteins ( Figure 10B ) .",
"Importantly , both the output domain and the cytoplasmic HAMP-GGDEF-EAL module from L . pneumophila appeared fully functional in this context .",
"The only construct that generated a hyper-biofilm phenotype indicative of a constitutive interaction between LapG and LapD was the one in which the L . pneumophila S helix-GGDEF-EAL fragment replaced the respective P . fluorescens LapD domains with the HAMP domain coming from P . fluorescens ( construct Pfl-Lpg C5 ) .",
"This result hints at the importance of the connection between the HAMP domain and the S helix .",
"Most importantly for this study , these data demonstrate conservation of the key mechanism between moderately conserved LapD orthologs and indicate that , despite the mixed origin of the complex structure components , CdgS9 is a suitable proxy for a c-di-GMP receptor that mediates inside-out signaling to control periplasmic proteolysis .",
"Consequently , the two crystal structures of the output domain of a LapD-like protein in two distinct conformations , one representing the inactive state , the other the active , LapG-bound state , provide fundamental insights into the switching mechanism ."
],
[
"Biofilm formation in P . fluorescens as a response to nutritional cues relies on the lap operon , which contains genes encoding the signal-integrating transmembrane protein LapD , a c-di-GMP receptor that regulates periplasmic proteolysis through a sequestration of the protease LapG .",
"LapG engagement is regulated through conformational changes in LapD that originate from c-di-GMP binding to LapD's cytoplasmic domain and are transmitted through the juxtamembrane HAMP domain .",
"Our structural analyses establish that LapD's output domain adopts a PAS domain fold and forms homo-dimers with distinct differences in the LapG binding-competent , active state and its inactive state ( Video 1 ) .",
"Notably , the packing transition that the structures depict can be described as a transition from a four-helix to a two-helix bundle , and follows two principle components: in a piston motion , one half-side of the dimeric output domain shifts vertically along the second half-side , whereas scissor-like motions encompass a tilting of the half-sides along the dimer interface .",
"Considering the current model for HAMP domain-switching ( Hulko et al . , 2006; Airola et al . , 2013 ) , which involves undergoing small amplitude translations and/or rotations during signal transduction similar to the ones we describe for CdgS9's periplasmic output domain , we predict that the output domain follows the conformational changes in the HAMP domain as a direct response .",
"The cross-over of the membrane proximal elements observed in both output domain conformations ( Figure 4B ) may function as the fulcrum about which the output and HAMP domain pivot and/or tilt .",
"As for the mode of LapG interaction , the four-helix bundle of the output domain of inactive LapD presents a surface that appears suboptimal for LapG binding , while the activated LapD conformation is characterized by extensive shape complementarity and effective interaction anchors at one face of the LapD output domain dimer , foremost the conserved tryptophan in the GWxQ loop .",
"The notion of geometric compatibility , as opposed to an increase in specific per-residue interactions as a predominant driver for high-affinity LapG binding , is supported by the overall low surface conservation of LapD orthologs .",
"The asymmetry of the LapD–LapG complex further suggests mechanisms inherent to the output domain that counteract an activating signal and may ultimately contribute to anti-cooperativity , which has been described for other PAS domain-containing proteins ( Moore and Hendrickson , 2012 ) .",
"In the current model , ( transient ) occupation of the second LapG binding site on the dimeric output domain of LapD would release LapG at the first , high-affinity site .",
"While the crystal structures provide distinct snapshots of possible conformations , they contain little information regarding the dynamics of the transitions and stability of the functional states .",
"Based on our lack of success to create a constitutively active LapD through structure-guided mutations in the output domain , we offer two explanations for the apparent disconnect between the crystallographic data that are based on the isolated output domain and the functional analysis of the regulated full-length protein .",
"In the first model , the regulatory HAMP and GGDEF-EAL domains would constrain the conformation of the LapD output domain to an extent that mutations are insufficient to destabilize the output domain dimer interface and/or induce the correct high-affinity LapG-binding state .",
"Another , non-mutually exclusive model also consistent with our data is that the apo-state may be described as a dynamic ensemble , preventing efficient LapG binding .",
"It would follow that the crystallized output domain in the absence of LapG depicts only one possible conformation of the ensemble with the mutations only having minimal effect on the activity .",
"At the same time , crucial interaction motifs are exposed even in this state , consistent with a basal level of LapG binding in the absence of c-di-GMP .",
"LapD activation by c-di-GMP on the other hand increases the affinity of LapG with the output domain following conformational changes dictated by the cytoplasmic HAMP domain .",
"It is interesting to note that both principles , switching through dynamic states or between more stable conformations have also been discussed for HAMP domains ( Ferris et al . , 2011 , 2012; Zhu et al . , 2013; Ames et al . , 2014; Mechaly et al . , 2014; Stewart , 2014 ) , consistent with the idea that the output domain may mirror the behavior of the regulatory domains .",
"Future experiments investigating the dynamics of full-length LapD , including those in a lipid environment ( Wang et al . , 2014 ) , should help to distinguish between the two models .",
"The dominance of the HAMP domain over the output domain in regulating LapD may bias the system towards inside-out signaling and against tenacious conformational changes induced by LapD–LapG encounters in the periplasm .",
"At the same time , the observed basal and/or transient interactions of LapG with LapD's output domain at low c-di-GMP levels may translate into low frequency conformational changes in the cytoplasmic domains , which would yield pre-activated receptors that would lock in place only when c-di-GMP levels are high .",
"Such a mechanism would explain how c-di-GMP has access to the EAL domain in the first place , given that the autoinhibited conformation is incompatible with c-di-GMP binding .",
"The mechanism would be best described as ‘coincidence detection’ , requiring both c-di-GMP and LapG being available for LapD engagement and full activation .",
"The HAMP domain would function as an attenuator in the inactive state , yet determine the active conformation upon c-di-GMP binding , which would prevent an immediate reversion to the autoinhibited state .",
"The presence of LapD-like signaling nodes was validated in a few other biofilm-forming bacteria .",
"Unfortunately , while we have evidence that they are regulated in a similar fashion , we know very little about their physiological role .",
"Gene deletion of the L . pneumophila LapD ortholog locus cdgS9 has no obvious phenotype , yet overexpression of CdgS9 has an effect on intracellular growth of the pathogen ( Levi et al . , 2011 ) , suggesting functional relevance of this ortholog .",
"Stronger functional similarities to the P . fluorescens system are apparent in more closely related bacteria such as Pseudomonas putida ( Gjermansen et al . , 2005 , 2006 , 2010 ) .",
"Future studies will address the physiological pathways that LapD and LapG orthologs regulate .",
"Until then , proteins from these other systems remain orphan c-di-GMP receptors and proteases , respectively .",
"Our work lays the foundation and establishes several tools that will be useful to interrogate these signaling nodes in other bacteria .",
"As part of these efforts , we demonstrate that , in principle , the LapD–LapG interaction , and likely analogous binding between similar protein pairs , is therapeutically accessible , since LapD-derived peptides containing the conserved GWxQ motif specifically diminish LapG binding to LapD .",
"While effective competition relied on high peptide concentrations , one needs to consider that we used a non-constrained peptide , which competes with the native binding site presented by full-length , c-di-GMP-activated LapD that presents an interaction surface far greater than just the peptide sequence .",
"Unfortunately , the peptides are not bioavailable and likely do not cross the outer membrane of P . fluorescens , preventing us to assess whether inhibiting the LapD–LapG interaction would result in increased biofilm dispersal , as would be predicted if the peptides would release LapG from LapD in vivo .",
"Nevertheless , our data indicate that targeting the interaction site with small-molecule inhibitors could be a fruitful strategy to perturb bacterial biofilm formation and dispersal .",
"Optimized competitors may include synthetic stapled or otherwise designed peptides that mimic the loop structure in solution ( Razavi et al . , 2014 ) .",
"Based on the conservation of binding events , regulation and key sequence motifs , we predict that our model is universal for proteins with LapD-like architecture , which includes receptors that signal in the opposite direction ( ‘outside-in’ ) by translating environmental signals emerging in the periplasm into changes in DGC and/or PDE activity in their cytoplasmic GGDEF and EAL domains , respectively .",
"As an example , a LapDG-like mode of regulation , yet in the opposite signaling direction and involving an active DGC , controls the clinically important GGDEF domain-containing protein YfiN from Pseudomonas aeruginosa and E . coli ( Malone et al . , 2010; Sanchez-Torres et al . , 2011; Malone et al . , 2012; Giardina et al . , 2013 ) .",
"In this case , the periplasmic protein YfiR suppresses YfiN's DGC activity through a direct interaction with YfiN's periplasmic PAS domain ( Malone et al . , 2012 ) .",
"YfiR becomes sequestered by the outer membrane protein YfiB , which activates YfiN , likely involving conformational changes in its HAMP-GGDEF domain module ( Malone et al . , 2012; Raterman et al . , 2013 ) .",
"Recently , a similar outside-in signaling model describing a rheostat-like control mechanism has been established for an autoinducing peptide-sensitive histidine kinase reconstituted into membrane nano-discs ( Wang et al . , 2014 ) .",
"Future studies will ascertain the generality of our model and the potential variations thereof ."
],
[
"The periplasmic output domain of L . pneumophila cdgS9 ( lpg0829; residues 22–152 ) and P . fluorescens lapG lacking the signal peptide ( Pfl01_0130; residues 50–251 ) was amplified from genomic DNA by standard PCR and cloned either separately into a pET28a-based vector ( Merck Millipore , Darmstadt , Germany ) that adds an N-terminal , cleavable His6-SUMO tag , or together into a bacterial dual expression vector , pETDuet-1 ( Merck Millipore , Darmstadt , Germany ) .",
"In the latter case , L . pneumophila His6-SUMO-CdgS9Output and P . fluorescens LapG were cloned into multiple cloning site 1 and 2 , respectively .",
"P . fluorescens lapD ( Pfl01_0131; residues 1–648 ) was amplified from genomic DNA by standard PCR and ligated into the NdeI and NotI restriction sites of a pET24a vector ( Merck Millipore , Darmstadt , Germany ) .",
"To create LapD-sfGFP and LapG-sfGFP fusion proteins , lapD was amplified from the pET24a-lapD construct and ligated into the NcoI and XhoI sites of the vector pBrew , which is a pET28a-based vector possessing a 3ʹ-linked tobacco etch virus ( TEV ) protease cleavage site followed by a superfolder GFP-His6 ( sfGFP-His6 ) gene .",
"Site-directed mutagenesis was carried out using the Quikchange kit ( Agilent Technologies , Santa Clara , CA ) following the manufacturer's instructions , followed by validation through DNA sequencing .",
"Chimeric LapD constructs were made by overlap-extension PCR .",
"Native and selenomethionine-substituted proteins were over-expressed in E . coli BL21 T7 Express or T7 Express Crystal cells ( New England Biolabs , Ipswich , MA ) , respectively .",
"For the expression of native proteins , cultures were grown at 37°C in Terrific Broth ( TB ) medium supplemented with 50 μg/ml kanamycin .",
"At an OD600 of ∼1 , the temperature was reduced to 18°C , and protein expression was induced by adding 0 . 5 mM IPTG .",
"Selenomethionine-labeled proteins were expressed in cells grown at 37°C in M9 minimal medium supplemented with the desired antibiotic , vitamins ( 1 μg/ml thiamin and 1 μg/ml biotin ) , carbon source ( 0 . 4% glucose ) , trace elements , and amino acids ( 50 μg/ml of each of the 20 amino acids with selenomethionine substituting for methionine ) .",
"Protein expression was induced at an OD600 corresponding to ∼0 . 6 .",
"To site-specifically incorporate the cross-linking amino acid para-azidophenylalanine ( pAzF ) into LapD or LapG , an amber ( TAG ) stop codon was introduced at the desired location within the genes .",
"BL21 T7 express cells ( for pAzF-derivatized LapD expression ) or BL21ai cells ( Life Technologies , Grand Island , NY; for pAzF-derivatized LapG expression ) containing both the amber-disrupted plasmid and the pDule2-pCNF machinery plasmid ( Miyake-Stoner et al . , 2009 ) were grown in TB medium supplemented or auto-induction medium ( Peeler and Mehl , 2012 ) , respectively , with 50 μg/ml kanamycin and 100 μg/ml spectinomycin to an OD600 of ∼0 . 8 , at which time the temperature was dropped to 22°C , and 0 . 5 mM IPTG and 1 mM pAzF were added .",
"In all cases , protein expression was allowed to proceed for 16 hr at 18°C , after which cells were harvested by centrifugation , re-suspended in Ni-NTA buffer A ( 25 mM Tris–HCl [pH 8 . 5] , 500 mM NaCl and 20 mM imidazole; used for LapG , LapDOutput and CdgS9Output ) , Ni-NTA buffer Aʹ ( 25 mM Tris–HCl [pH 8 . 5] , 200 mM NaCl , and 20 mM imidazole used for the LapG–CdgS9Output complex ) , or Ni-NTA buffer Aʺ ( 25 mM Tris [pH 7 . 6] , 500 mM NaCl , and 10% glycerol; used for full-length LapD ) , and flash frozen in liquid nitrogen .",
"For soluble proteins , cell suspensions were thawed and lysed by sonication .",
"Cell debris was removed by centrifugation and the clear lysates were incubated with NiNTA resin ( Qiagen , Valencia , CA ) that was pre-equilibrated with Ni-NTA buffer A . The resin was washed with 20 column volumes of Ni-NTA buffer A , followed by protein elution with five column volumes of Ni-NTA buffer B ( 25 mM Tris–HCl [pH 8 . 5] , 500 mM NaCl , and 300 mM imidazole ) .",
"The eluted proteins were buffer-exchanged into gel filtration buffer ( 25 mM Tris–HCl [pH 8 . 5] and 150 mM NaCl ) on a fast desalting column ( GE Healthcare , Little Chalfont , UK ) .",
"Proteins were subjected to size exclusion chromatography on a Superdex 200 column ( GE Healthcare , Little Chalfont , UK ) pre-equilibrated with gel filtration buffer .",
"Where indicated , the His6-SUMO moiety was cleaved off by using the yeast protease Ulp-1 following the desalting step .",
"Ulp-1 , uncleaved protein and the cleaved fusion tags were removed by NiNTA affinity chromatography prior to the final gel filtration .",
"For co-purification of the CdgS9Output–LapG complex , the procedure remained identical except that all buffers contained 200 mM NaCl instead of the amount indicated above .",
"Purified proteins were concentrated on Amicon filters with an appropriate size cutoff to concentrations >30 mg/ml , flash frozen in liquid nitrogen and stored at −80°C .",
"To isolate membranes containing LapD ( or LapD-sfGFP ) , 0 . 5 mg/ml lysozyme was added to thawed cells , which were then lysed by sonication .",
"Unlysed cells were pelleted by centrifugation at 5000×g for 5 min .",
"Membranes were pelleted by centrifuging the supernatant at 180 , 000×g for 1 hr .",
"The resulting supernatant was decanted and the membrane pellet was resuspended in membrane buffer ( 25 mM Tris [pH 7 . 5] , 500 mM NaCl , and 10% glycerol ) , sonicated briefly to homogenize the membranes and frozen in liquid N2 .",
"Where specified , LapD was solubilized for subsequent purifications by incubating membranes in membrane buffer supplemented with 2% Triton X-100 ( Sigma-Aldrich , St . Louis , MO ) .",
"After 1 hr , insoluble material was pelleted by centrifugation at 180 , 000×g for 40 min .",
"The clarified supernatant was incubated with NiNTA resin for 1 hr and washed thoroughly with membrane buffer supplemented with 4 mM β-mercaptoethanol ( BME ) , 20 mM imidazole , and 1% Triton X-100 .",
"The Triton X-100 detergent was exchanged for 0 . 01% ( wt/vol ) lauryl maltose neopentyl glycol ( LMNG ) in a step-wise fashion while LapD was still bound to the NiNTA resin , followed by elution of LapD with LapD elution buffer ( 25 mM Tris [pH 7 . 6] , 500 mM NaCl , 10% glycerol , 4 mM BME , 300 mM imidazole , and 0 . 01% LMNG ) .",
"The eluted protein was buffer-exchanged into a low-salt LapD buffer ( 25 mM Tris–HCl [pH 7 . 5] , 250 mM NaCl , 5% glycerol , 4 mM BME , and 0 . 01% LMNG ) on a fast desalting column .",
"LapD was concentrated by ultra-filtration and subjected to size exclusion chromatography on a Superdex 200 column pre-equilibrated with LapD gel filtration buffer ( 25 mM Tris–HCl [pH 7 . 6] , 150 mM NaCl , 5% glycerol , 4 mM BME , and 0 . 0025% LMNG and 0 . 02% CHAPS ) .",
"The protein was stored at 4°C and used within 72 hr .",
"To prepare LapD-sfGFP for cross-linking assays , isolated membranes were solubilized in membrane buffer supplemented with 1% lauryl maltose neopentyl glycol ( LMNG ) and 0 . 2% CHAPS for 1 hr at 4°C , followed by centrifugation at 100 , 000×g for 30 min .",
"The concentration of solubilized LapD-sfGFP ( and LapG-sfGFP ) was determined by comparing the amount of fluorescence in the supernatant to a standard curve of purified sfGFP ( excitation: 488 nm , emission: 510 nm ) .",
"Proteins were used in cross-linking assays immediately after solubilization .",
"To produce non-fluorescent LapG with pAzF , purified LapG-sfGFP was incubated with TEV protease overnight at 4°C .",
"The His6-tagged sfGFP and TEV protease were subsequently removed by incubation with NiNTA resin .",
"Unbound , non-fluorescent LapG containing site-specifically incorporated pAzF was collected in the flow through , concentrated to >2 mg/ml , frozen in liquid nitrogen , and stored at −80°C .",
"Crystals were obtained by hanging drop vapor diffusion mixing equal volumes of protein ( 10–30 mg/ml ) and reservoir solution followed by incubation at 20°C .",
"For the native and selenemethionine-derivatized L . pneumophila CdgS9Output crystals , the reservoir solution contained 0 . 1 M Bis-Tris ( pH = 5 . 0 ) , 14% PEG3350 , and 4% vol/vol 2 , 2 , 2-trifluoroethanol .",
"Crystals of the native LapG–CdgS9Output complex were obtained from a reservoir solution containing 0 . 1 M Bis-Tris ( pH = 6 . 0 ) and 0 . 1 M magnesium formate .",
"Our initial efforts directed towards experimental phase determination proved intractable due to extremely low expression levels of the selenomethione-substituted LapG–CdgS9Output complex .",
"Hence phases were determined based on data sets collected on crystals grown from protein mixtures containing selenomethionine-derivatized P . fluorescens LapG and native L . pneumophila CdgS9Output , which grew at conditions identical to the native complex .",
"For cryo-protection , crystals were soaked in reservoir solution supplemented with 20–25% xylitol .",
"Cryo-preserved crystals were flash-frozen and stored in liquid nitrogen .",
"Data were collected on frozen crystals at 100 K using synchrotron radiation at the Cornell High Energy Synchrotron Source ( CHESS , Ithaca ) .",
"Data reduction was carried out with the software package HKL2000 ( Otwinowski and Minor , 1997 ) .",
"Experimental phases for the initial structure determination were obtained from Single Anomalous Diffraction ( SAD ) experiments on crystals grown from selenomethionine-derivatized proteins by using the software package PHENIX ( Adams et al . , 2002 ) .",
"Refinement in PHENIX and COOT ( Emsley and Cowtan , 2004 ) yielded the final models .",
"Data collection and refinement statistics are summarized in Table 1 .",
"Illustrations were made in Pymol ( Schrödinger ) .",
"Alignments were generated using ClustalW2 ( Larkin et al . , 2007 ) .",
"Homology models were created using the program Modeller ( Eswar et al . , 2006 ) using default inputs .",
"Crystallographic software is provided through SBGrid ( Morin et al . , 2013 ) .",
"Unless specified otherwise , LapG–LapD cross-linking was performed with 1 μM purified LapG ( either fused to sfGFP or not ) containing pAzF at the desired locations and solubilized variants of LapD at 2 μM final concentration ( either fused to sfGFP or not ) in binding buffer ( 25 mM Tris [pH 7 . 5] , 250 mM NaCl , 2 . 5 mM CaCl2 , 0 . 01% LMNG , and 0 . 2% CHAPS ) .",
"When indicated , 10 μM c-di-GMP was added to the reaction .",
"All reaction components were allowed to incubate at room temperature for 20 min , at which time crosslinking was initiated by irradiation with short wave UV light ( ∼254 nm ) for 5 min .",
"After irradiation , samples were mixed with SDS sample buffer and immediately subjected to SDS-PAGE without boiling .",
"Gels were imaged by fluorescence using a BioRad Gel Doc system .",
"Band intensities of the cross-linked adducts were measured using ImageLab software ( Biorad , Hercules , CA ) and normalized to the intensity of non-cross-linked sfGFP-labeled protein .",
"Reported values of band intensities are the average of three replicates .",
"SEC-MALS measurements ( De et al . , 2010 ) were carried out by injecting purified proteins ( 100 µM ) onto a Phenomenex gel filtration column pre-equilibrated with MALS buffer ( 25 mM Tris–HCl [pH 7 . 4] and 100 mM NaCl ) .",
"The chromatography system was coupled to an 18-angle , static light scattering detector and a refractive index detector ( DAWN HELEOS-II and Optilab T-rEX , respectively; Wyatt Technology , Santa Barbara , CA ) .",
"Data were collected at 25°C every second at a flow rate of 1 ml/min and analyzed with the software ASTRA , yielding the molecular weight and mass distribution ( polydispersity ) of the samples .",
"For data quality control and normalization of the light scattering detectors , monomeric bovine serum albumin ( Sigma-Aldrich , St . Louis , MO ) was used .",
"P . fluorescens was routinely cultured with liquid LB medium or on solidified LB with 1 . 5% agar at 30°C .",
"Gentamicin ( Gm ) was used at 30 μg/ml .",
"To induce expression of the PBAD promoter from pMQ72 , arabinose was added at 0 . 2% ( vol/vol ) .",
"For biofilm and pull down assays , P . fluorescens was grown in K10T-1 medium , previously described as a phosphate-rich medium ( Monds et al . , 2006 ) .",
"K10T-1 consists of 50 mM Tris–HCl ( pH 7 . 4 ) , 0 . 2% ( wt/vol ) Bacto tryptone , 0 . 15% ( vol/vol ) glycerol , 0 . 61 mM MgSO4 , and 1 mM K2HPO4 .",
"Static biofilm formation assays of P . fluorescens were performed as previously described ( Newell et al . , 2011b ) .",
"Polyvinyl chloride 96-well round bottom microtiter plates ( Corning , Corning , NY ) , containing 100 μl of K10T-1/well were inoculated with 1 . 5 μl of an overnight LB liquid culture of P . fluorescens .",
"Biofilm assays were statically grown at 30°C .",
"After 6 hr of growth , unattached bacteria and medium were removed and the wells were stained with 0 . 1% ( wt/vol ) solution of crystal violet in water .",
"To quantify biofilm biomass , crystal violet was solubilized in a solution containing 30% methanol and 10% acetic acid and a Spectra Max M2 microplate reader ( Molecular Devices , Sunnyvale , CA ) was used to read the absorbance at 550 nm , as previously described ( Monds et al . , 2007 ) .",
"Quantification of 8–10 wells for at least three biological replicates was obtained for each P . fluorescens strain assessed .",
"Peptide competition for the native LapD–LapG interaction was assessed using an established pull-down assay ( Newell et al . , 2011a ) .",
"P . fluorescens-clarified cell extracts co-expressing LapD-His6 and LapG-HA were prepared in pull down buffer consisting of 20 mM Tris ( pH 8 . 0 ) , 10 mM MgCl2 and in the presence of 10 μM chemically pure c-di-GMP ( GLSynthesis Inc . , Worcester , MA ) and 1 mM custom manufactured peptides ( Elim Biopharm ) .",
"Immunoprecipitations contained 450 μl of clarified cell extract , 40 μl Protein A sepharose ( Genscript , Piscataway , NJ ) , 0 . 8% thesit , and 0 . 5 μl monoclonal , mouse anti-HA antibody ( Covance , Princeton , NJ ) .",
"Immunoprecipitations were incubated at 4°C for 60 min and then washed three times at room temperature with gentle shaking in pull down buffer with the addition of c-di-GMP to a final concentration of 10 μM .",
"Immunopreciptations were analyzed by SDS-PAGE and Western blotting to detect LapD-His6 ."
]
] | [
"Stable surface adhesion of cells is one of the early pivotal steps in bacterial biofilm formation , a prevalent adaptation strategy in response to changing environments .",
"In Pseudomonas fluorescens , this process is regulated by the Lap system and the second messenger cyclic-di-GMP .",
"High cytoplasmic levels of cyclic-di-GMP activate the transmembrane receptor LapD that in turn recruits the periplasmic protease LapG , preventing it from cleaving a cell surface-bound adhesin , thereby promoting cell adhesion .",
"In this study , we elucidate the molecular basis of LapG regulation by LapD and reveal a remarkably sensitive switching mechanism that is controlled by LapD's HAMP domain .",
"LapD appears to act as a coincidence detector , whereby a weak interaction of LapG with LapD transmits a transient outside-in signal that is reinforced only when cyclic-di-GMP levels increase .",
"Given the conservation of key elements of this receptor system in many bacterial species , the results are broadly relevant for cyclic-di-GMP- and HAMP domain-regulated transmembrane signaling ."
] | [
"While bacteria often live as unicellular microorganisms , many bacteria are capable of sticking together on a surface and forming a multicellular structure called a biofilm .",
"Bacterial biofilms occur frequently in nature; for example , on the roots of plants and submerged rocks .",
"While these biofilms are generally innocuous , others pose significant health threats to humans , causing tooth decay , gum disease , and—when they occur on implanted devices such as prosthetic heart valves—potentially serious infections .",
"When in biofilms , many bacteria are tolerant to antibiotics; therefore , working out how to disrupt these films is crucial for developing new treatments .",
"The microorganism Pseudomonas fluorescens is an example of a bacterium that can be found living in a complex biofilm .",
"In response to certain environmental cues , free-swimming P . fluorescens cells adhere to a surface and produce a slime that encases them in a robust biofilm .",
"The decision to shift between a free-swimming and a biofilm life-style is orchestrated by a signaling molecule found inside the bacteria called cyclic-di-GMP .",
"In P . fluorescens , the availability of nutrients—in particular , phosphate—controls how much cyclic-di-GMP is produced inside the cell .",
"If not enough phosphate is available , the level of cyclic-di-GMP falls and the biofilm disperses .",
"Cyclic-di-GMP affects the stability of the biofilm via a group of proteins called the Lap system .",
"When levels of cyclic-di-GMP are high , cyclic-di-GMP binds to a protein called LapD , which can then in turn bind to an enzyme known as LapG .",
"When bound to LapD , LapG is unable to break apart the molecules that help P . fluorescens cells bind to a surface , and so a biofilm can form .",
"If cyclic-di-GMP levels drop , fewer LapD molecules can bind to cyclic-di-GMP .",
"As cyclic-di-GMP-unbound LapD proteins interact poorly with LapG , this leaves some LapG molecules able to destabilize the attachments between the cells and the surface , which disperses the biofilm .",
"Here , Chatterjee et al . reveal the molecular mechanism by which LapD and LapG interact in P . fluorescens .",
"When cyclic-di-GMP is bound to LapD , the shape of LapD changes to produce features that fit into the surface of LapG .",
"It is this shape compatibility , more so than an increase in the number or quality of interactions between the chemical groups that make up the proteins , that enables LapD to bind to LapG .",
"Chatterjee et al . also provide evidence that the LapD–LapG interaction can be disrupted , thereby raising the possibility that biofilm formation could be manipulated by targeting this system .",
"Given that systems similar to the P . fluorescens Lap system exist in numerous other bacterial species , including important pathogens , the findings of Chatterjee et al . could assist efforts to develop medicines and products that eradicate bacterial biofilms .",
"LapD also shares many structural elements with a large number of other signaling proteins; therefore , these findings could also improve the understanding of how other cell signaling systems work ."
] | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"tools and resources",
"neuroscience"
] | Application of optogenetic Amyloid-β distinguishes between metabolic and physical damages in neurodegeneration | elife-52589-v2 | [
[
"Alzheimer’s disease ( AD ) is a debilitating , age-associated , neurodegenerative disease which affects more than 46 . 8 million people worldwide and represents the 6th leading cause of death in the United States of America ( Ahn et al . , 2001; De-Paula et al . , 2012; Hawkes , 2016; Kumar et al . , 2015; Zhang et al . , 2011 ) .",
"Despite extensive efforts over the last 50 years , no disease-modifying therapy has been found and , most recently , several high-profile Phase III clinical trials have failed ( Anderson et al . , 2017; Lim and Mathuru , 2018; Park et al . , 2018 ) .",
"The AD clinical trial landscape has largely been dominated by the amyloid cascade hypothesis , with more than 50% of the drugs targeting amyloid beta ( Aβ ) in Phase III trials alone ( Cummings et al . , 2018 ) .",
"The amyloid cascade hypothesis , in its original form , posits the deposition of Aβ , in particular the extracellular Aβ plaque , as the main driver of AD ( Hardy and Higgins , 1992 ) .",
"However , the causative role of Aβ plaques has recently been challenged , because of the failure of numerous interventions targeting Aβ plaques in Phase III trials and the observation of Aβ plaques in brains of non-AD symptomatic individuals ( Cummings , 2018 ) .",
"Model organisms ranging from nematodes to mice have further shown that AD pathology can be modeled in the absence of obvious Aβ plaques ( Duff et al . , 1996; Fong et al . , 2016; Teo et al . , 2019 ) .",
"These observations point to a different mechanism for Aβ neurotoxicity perhaps through Aβ’s intracellular accumulation ( LaFerla et al . , 2007 ) .",
"Amyloid plaques are macroscopic extracellular protein aggregates , but intracellular Aβ must function in smaller units of peptides , oligomers or aggregates .",
"Of these , soluble Aβ oligomers appear to be the main toxic species in AD ( Ferreira and Klein , 2011 ) .",
"For example , Aβ oligomers have been shown to induce neurotoxic effects including memory loss in transgenic animal models reviewed in Mroczko et al . ( 2018 ) .",
"These studies , however , mostly rely on in vitro injection of synthetic Aβ oligomers or oligomers extracted from AD brains ( Mroczko et al . , 2018 ) .",
"There is currently a lack of tools that can directly control Aβ oligomerization in vivo , which would allow direct examination of the effects of Aβ oligomerization .",
"Here , we describe an optogenetic method to study Amyloid-β ( Aβ ) protein oligomerization in vivo in different model organisms and apply it to delineating mechanisms of disease progression .",
"Optogenetics hinges on light responsive proteins that can be fused to genes of interest , allowing for the spatial and temporal regulation of proteins in a highly precise manner .",
"Temporal-spatial control is achieved simply by the exposure of the target system to lights of a specific wavelength , without the need to introduce other external agents ( Fenno et al . , 2011; Möglich and Moffat , 2010 ) .",
"One such optogenetic protein , a modified version of the Arabidopsis thaliana cryptochrome 2 ( CRY2 ) protein , oligomerizes into photobodies quickly and reversibly in the presence of blue light at 488 nm ( Más et al . , 2000 ) .",
"In cultured cells , expression of the photolyase homology region of CRY2 fused to mCherry led to puncta within 10 s of exposure to blue light and these puncta dispersed within minutes ( Bugaj et al . , 2013 ) .",
"This basic unit of CRY2-mCherry attached to a variety of proteins have , for instance , been used to study cortical actin dynamics in cell contractility during tissue morphogenesis , the dynamics of Wnt and EGF signaling , plasma membrane composition and transcriptional regulation ( Bugaj et al . , 2018; Bugaj et al . , 2015; Guglielmi et al . , 2015; Huang et al . , 2017; Idevall-Hagren et al . , 2012; Johnson et al . , 2017; Kaur et al . , 2017; Kennedy et al . , 2010 ) ."
],
[
"We generated transgenic animals expressing the 42-amino-acid human Aβ peptide ( Aβ1–42 ) fused to Cryptochrome 2 and the fluorescent protein mCherry ( Aβ-CRY2-mCh , Figure 1a , please note the relative sizes ) .",
"Drosophila lines were under the control of the GAL4/UAS system ( Brand and Perrimon , 1993 ) and expressed either ubiquitously via AD-Gal4 driver ( Figure 1b–c ) or specifically in neurons using Elav-Gal4 driver ( Figure 1d–e , Figure 1—figure supplement 1 ) .",
"C . elegans lines were made with the heat shock promoter ( Figure 1f–h , Figure 1—figure supplement 2 ) .",
"In order to observe oligomerization and clustering of intracellular Aβ , we took a live imaging approach in Drosophila embryos using a lightsheet microscope with a 488 nm laser activating CRY2 and the 561 nm laser imaging mCh over time .",
"We observed clusters of mCh fluorescence forming in embryos exposed to 488 nm light , but fewer in embryos that were not exposed to blue light ( Figure 1—figure supplement 3 , Videos 1–2 ) .",
"The intensity of the clusters was quantified using Imaris quantification software to show the intensity of oligomer formation ( Figure 1c , note color scale for cluster intensity ) .",
"We proceeded to test the effectiveness of light induced clustering of Aβ-CRY2-mCh in C . elegans .",
"As this construct is driven by a heat shock promoter , worms were heat shocked at 35°C for 90 min followed by resting at 20°C before being immobilized and imaged using confocal microscopy ( Figure 1f–h ) .",
"We observed an increase in clusters in worms exposed to blue light over their siblings not exposed to blue light .",
"The results were quantified as fluorescence intensity and represented in ( Figure 1h ) showing the formation of Aβ clusters .",
"Induction of chaperones from heat-shock can impact the dynamics of Aβ .",
"In particular , heat-shock treatment in another transgenic C . elegans strain , CL4176 , reduced Aβ-mediated paralysis and Aβ oligomerization ( Winblad , 1989; Wu et al . , 2010 ) .",
"Overexpression of chaperone protein suppressed Aβ-toxicity in C . elegans ( Fonte et al . , 2008 ) .",
"The use of hsp-16 promoter in the transgenic C . elegans , which requires heat-shock induction , may hence be a limitation of our current approach .",
"In view of this limitation , we performed several other controls to understand the impact of heat-shock treatment on Aβ dynamics , and whether or not heat-shock treatment itself contributes to the metabolic and phenotypic detriments observed in the transgenic animals .",
"To understand how heat-shock treatment may impact the dynamics of Aβ in our transgenic strains , we compared the dynamics of Aβ between the transgenic Aβ worms with and without heat shock .",
"We found that transgenic animals in both heat-shock ( Aβ HS L ) and non-heat-shock ( Aβ -HS L ) condition expressed detectable Aβ-CRY2-mCh protein ( Figure 1—figure supplement 2 ) , however animals that had been heat-shocked had significantly higher Aβ levels compared to non-heat-shocked animals , suggesting that heat-shock , as intended , drove higher level of Aβ expression and that this was not compensated for by secondary induction of chaperones .",
"Even though the CRY2 system predominantly oligomerizes , we also observed the formation of intracellular Aβ aggregates , using a direct stain for Aβ aggregates ( Figure 1—figure supplement 4 ) .",
"Importantly , we observed that Aβ aggregation was not reversible as had previously been observed for signaling molecules ( Huang et al . , 2017; Johnson et al . , 2017; Kaur et al . , 2017 ) , rather CRY2 appeared to initiate clustering leading to Aβ bundles that did not come apart once blue light was turned off .",
"We further confirmed that turning on blue light alone without the transgene expression did not lead to any Aβ expression or cluster formation ( Figure 1—figure supplement 2b , Ab HS L vs Ab –HS L ) , suggesting that the oligomerization process was CRY2-specific and not an artifact of light .",
"We performed FRAP experiments to establish the stability of the Aβ clusters ( Figure 2 ) .",
"When compared to an unrelated CRY2 construct , we observed a very slow recovery for Aβ-CRY2-mCh compared to Arm-CRY2-mCh ( Figure 2; Kaur et al . , 2017 ) .",
"Together , our results showed that the Aβ-CRY2-mCh transgene was functional in both organisms , with a high specificity to blue light inducing irreversible oligomerization of Aβ .",
"To test the functionality of Aβ-CRY2-mCh , we next looked for phenotypes associated with light-induced intracellular Aβ oligomerization .",
"We performed lifespan studies to assess the effects of Aβ-CRY2-mCh by expressing Aβ-CRY2-mCh either ubiquitously ( AD-Gal4 ) or specifically in the nervous system ( Elav-Gal4 ) .",
"Newly eclosed adult flies were separated into two groups in each case , one of which was exposed to ambient white light and the other kept in the dark .",
"In both cases , flies exposed to light died much more quickly with half the mean and maximum lifespans ( Figure 3a–b ) compared to those reared in the dark .",
"We extended this analysis to C . elegans , though starting at Day 6 due to the heat-shock manipulation step .",
"Under light conditions , C . elegans showed markedly decreased lifespans as compared to the transgenic C . elegans kept in dark conditions ( Figure 3c ) .",
"Most significantly , we observed that the lifespan decrease was reversible , as taking animals exposed to light for 24 days and moving them into darkness showed a recovery of their lifespan , or a rescue ( Figure 3a–b ) .",
"To ensure that these reduced lifespans were physiological , we examined the fitness of the animals .",
"Fitness was severely decreased in light-exposed flies and worms as quantified by fertility and locomotive assays ( Figure 3—figure supplement 1 ) .",
"Interestingly , light-induced Aβ oligomerization caused further impairment in sensorimotor function of transgenic Aβ nematodes ( Figure 3—figure supplement 1c ) .",
"These results suggesed that light-induced oligomerization of Aβ not only affected lifespan and fitness , but also impaired complex sensorimotor function and behavior .",
"The lethality mediated by light-induced Aβ oligomerization led us to examine the morphological effects of Aβ-CRY2-mCh clustering during embryonic stages in Drosophila .",
"Embryos expressing Aβ in only the nervous system developed at a normal pace and did not show any morphogenetic defects and even hatched in the absence of blue light ( Video 1 ) .",
"In contrast , sibling embryos exposed to blue laser light arrested in late neurogenesis stages where the central nervous system stopped developing and the embryos died ( Video 2 and Figure 4 , Figure 1—figure supplement 3 and Figure 4—figure supplement 1 ) .",
"These embryos showed a variety of abnormalities in the positioning of neurons and glial cells ( Compare to complete normal development , Video 3 ) suggesting that light-induced Aβ oligomerization caused physical , structural damage that affects neuronal development .",
"The severity of the physical damage phenotype in embryos exposed to blue light ( Figure 4a–d’’ ) led us to investigate the differences in mechanism between the two conditions by exploring inflammatory response , mitochondrial health and oxidative damage .",
"Expression of Aβ alone , without light-induced Aβ oligomerization , led to a small increase in inflammatory markers ( Figure 4e; Westfall et al . , 2018 ) .",
"In embryos exposed to light , two of these markers increased to a much greater level ( Figure 4e ) .",
"We observed a significant reduction in the number of mitochondria in light-treated Aβ animals ( Figure 4f ) .",
"Markers of oxidative damage in light and dark exposed C . elegans showed differential expression of antioxidant defense genes , specifically Catalase , Trx-2 and Sod-3 , suggestive of the occurrence of oxidative stress induced by Aβ oligomerization ( Figure 5 ) .",
"These results suggested that light-induced Aβ oligomerization phenocopied many of the hallmarks of AD .",
"We proceeded to look at light-induced Aβ oligomerization in metabolic impairments in transgenic C . elegans .",
"Heat-shocked mutants exposed to light ( Aβ HS L ) had lower ATP levels compared to the non-transgenic control ( Ctrl -HS D ) ( Figure 6 ) .",
"However , heat-shocked mutants in the dark condition ( Aβ HS D ) did not show any significant differences in ATP level compared to the non-transgenic control .",
"This result suggested that presence of Aβ alone is insufficient to induce ATP deficits , and that light-induced oligomerization of Aβ is required for the defect to manifest .",
"Aβ expression alone was also insufficient to affect the nematodes’ maximum respiratory capacity , as heat-shocked mutants in the dark condition ( Aβ HS D ) did not display differences in maximum and spare respiration capacity compared to control animals ( Ctrl -HS D ) .",
"However , light-induced Aβ oligomerization significantly reduced maximum respiration capacity in C . elegans .",
"Heat-shocked mutants in the light condition ( Aβ HS L ) displayed significantly lower maximum and spare respiration capacity compared to controls ( Ctrl -HS D ) and to heat-shocked mutants in the dark condition ( Aβ HS D ) ( Figure 6b–d ) .",
"Together , these results highlighted the metabolic differences between the two conditions and showed that metabolic deficits were mediated by light-induced Aβ oligomerization .",
"To further examine whether the metabolic deficits in transgenic C . elegans were mediated by heat-shock treatment , we included a heat-shocked non-transgenic control in the metabolic and phenotypic assays .",
"We found that heat-shock treatment does not affect ATP levels in the control animals , suggesting that the ATP detriment was a result of Aβ expression , but not heat-shock treatment ( Figure 6a ) .",
"On the other hand , heat-shock treatment appears to reduce the respiratory capacity of the control animals , suggesting that the decline in respiratory capacity was mediated by both heat-shock and Aβ expression ( Figure 6c ) .",
"Comparing the heat-shocked transgenic animals in light and dark conditions , we found that light treatment , which induces oligomerization , worsened the phenotypic and metabolic detriments in the transgenic animals .",
"This suggested that regardless of the effects of heat-shock and chaperone induction , oligomerization still negatively impacted the phenotypes .",
"The drug Li+ is used for treatment of neurological conditions ( Licht , 2012 ) .",
"A high drinking water Li+ concentration correlated with lower human mortality ( Zarse et al . , 2011 ) , and Li+ increased the lifespan of C . elegans and Drosophila ( Castillo-Quan et al . , 2016; McColl et al . , 2008; Tam et al . , 2014; Teo et al . , 2020a; Teo et al . , 2020b; Zarse et al . , 2011 ) .",
"The molecular mechanisms by which Li+ functions include inhibition of inositol monophosphotase ( IMPA ) and glycogen synthase kinase-3 ( GSK-3 ) ( Kerr et al . , 2018 ) , reduction of oxidative damage ( Kasuya et al . , 2009; Kerr et al . , 2017; Khan et al . , 2015 ) and longevity via a GSK-3/NRF-2 dependent , hormetic mechanism ( Castillo-Quan et al . , 2016 ) .",
"Among the mechanisms for neuroprotection , the Wnt signaling pathway plays a role in modulating AD pathology and its progression ( De Ferrari et al . , 2003; Jin et al . , 2017; Parr et al . , 2015; Toledo and Inestrosa , 2009 ) .",
"Activation of Wnt signaling through lithium ( Li+ ) treatment attenuated Aβ aggregation and increased the lifespan of Aβ models ( Toledo and Inestrosa , 2009 ) .",
"By inhibiting the enzyme glycogen synthase kinase-3 ( GSK-3 ) , Li+ compounds drive the downstream production of intracellular β-catenin activating the Wnt–β-catenin signaling pathway ( Klein and Melton , 1996 ) .",
"Li+ treatment through Wnt activation also rescued behavioral impairment and neurodegeneration induced by Aβ fibrils ( De Ferrari et al . , 2003 ) .",
"Taken together , these studies point to a putative AD therapeutic intervention through Li+ .",
"To test the usefulness of optogenetic Aβ for drug testing , we supplemented the food for Drosophila expressing Aβ-CRY2-mCh with Li+ .",
"Drosophila exposed to light , but consuming Li+ had significantly increased lifespans , suggesting both a rescue effect induced by Li+ ( Figure 3a–b ) , and that the optogenetic model can be used for inducible expression drug testing .",
"Rapid drug testing can be also be achieved through cell culture .",
"We extended this assay to HEK293 cells transfected with Aβ-CRY2-mCh where Aβ clusters formed upon stimulation with blue light ( Figure 7a ) .",
"The number of clusters per cell was greatly reduced by the addition of Li+ or the specific GSK3 inhibitor CHIR99021 , but CHIR99021 additionally reduced the signal intensity of the clusters ( Figure 7b–c , quantified 7d ) .",
"These data demonstrated the potential use of the optogenetics system for drug testing .",
"Finally , we also extended these findings to a vertebrate system .",
"We generated a zebrafish permanent line with UAS:Aβ-CRY2-mCh in the transgenic TgBAC ( gng8:GAL4 ) c416 background ( Hong et al . , 2013 ) that limited expression to a few cells .",
"We observed a diffuse mCh fluorescence in a small subset of neurons in the olfactory epithelium , the interpeduncular nucleus and in a few neurons sparsely distributed in the forebrain at 5 dpf .",
"Upon blue light exposure ( 488 nM ) the detectable intensity of the fluorescence increased rapidly ( Figure 8—figure supplement 1 ) .",
"Several neurons among those expressing appeared to bleb and began to die within 40 to 50 min of initial exposure to the blue light ( Video 4 ) .",
"Such damage was not observable in matched controls expressing Aβ-mCh driven by a CMV promotor lacking the CRY2 protein ( Video 5 ) .",
"Our approach leads to the expression of Aβ-CRY2-mCh in only a small subset of neurons making analysis of AD hallmarks difficult , so we used transient expression of Aβ-CRY2-mCh driven by a ubiquitin promotor in 48 hr old embryos to quantify the effects of light-induced Aβ oligomerization on mitochondria health and metabolic deficits .",
"Similar to the impairments observed in C . elegans , Aβ-CRY2-mCh expressing embryos showed lower maximum and spare respiration capacity compared to un-injected control ( Figure 8 ) .",
"In addition , light exposure of Aβ-CRY2-mCh expressing embryos caused a significant reduction in ATP levels ( Figure 8a ) ."
],
[
"Aggregation of Aβ has been studied extensively , but our results represent the first study to demonstrate that induced oligomerization of intracellular Aβ can be used to separate different pathologies ( induced by Aβ oligomerization vs expression alone ) .",
"A second approach to making inducible aggregates using a chemically controlled fluorescent protein has been published , but not yet widely tested ( Miyazaki et al . , 2016 ) .",
"Other previous approaches lacked an inducible oligomerization tool and could only demonstrate Aβ oligomers’ toxicity through exogenous injection of Aβ oligomers ( Mroczko et al . , 2018 ) .",
"We use a tool to investigate the pathological effects of intracellular Aβ oligomerization in nematodes and flies , human kidney cells , and in the vertebrate model organism zebrafish ( Figure 8—figure supplement 1 , Video 4 ) .",
"Despite the reversibility of CRY2 clustering in previous findings , we find that CRY2 initiated clustering of Aβ appeared to be irreversible likely due to the structure of the aggregates ( Antzutkin et al . , 2012 ) .",
"Consistent with existing literature showing that Aβ oligomers induce oxidative stress and inflammation ( Butterfield et al . , 2013; Forloni and Balducci , 2018 ) , we show that light-induced oligomerization of Aβ , but not Aβ expression alone , is necessary for the metabolic defects , loss of mitochondria and inflammation in C . elegans and Drosophila .",
"Light-induced oligomerization also leads to physical damage of the nervous system in D . rerio , resembling the loss of brain tissue in AD ( Figure 8—figure supplement 1 ) .",
"The embryonic assay for physical damage phenocopies brain lesions only to some degree as the extent of the damage is enhanced by the forces applied to the developing neural tissue .",
"The irreversibility of Aβ aggregates once initiated as seen here , also hints at intrinsic properties that may be unique or unusual to this peptide and warrants further investigation .",
"It is also likely to prove a good model for testing anti-aggregation compounds .",
"We anticipate that our approach will serve as an attractive tool for carrying out drug screens and mechanistic studies for the treatment of AD .",
"This optogenetic strategy will also undoubtedly complement other techniques due to the high level of spatiotemporal specificity .",
"For example , it could be optimally positioned to gain insights into the unresolved mechanism of Aβ - whether oligomerization and subsequent accumulation or lack of effective clearance underpins AD pathologies .",
"The separation of two phases of AD progression also suggests that single drug treatments will not suffice , and perhaps combinatorial approaches should be tried ."
],
[
"DNA sequences corresponding to the human Amyloidβ1–42 amino acid sequence were synthesized ( IDT ) into a Gateway technology ( Invitrogen ) entry vector .",
"The human Aβ1–42 sequence was used for Drosophila and zebrafish due to close evolutionary conservation and a nematode-codon-optimized version of Aβ1–42 was used for C . elegans ( Fong et al . , 2016 ) .",
"The CRY2-mCh fragment was cloned into pDONR vector with attB5 and attB2 sites , and MultiSite Gateway Pro 2 . 0 recombination ( Invitrogen ) was used to recombine donor plasmids and pDONR-CRY2-mCh into the respective destination vectors for three model organisms:",
"a ) pUASg . attB . 3XHA for Drosophila to synthesize pUAS-Aβ-CRY2-mCh ( Bischof et al . , 2007 )",
"b ) pDEST-hsp-16–2 p , a kind gift from Hidehito Kuroyanagi ( Medical Research Institute , Tokyo Medical and Dental University ) to synthesize hsp-16–2 p-Aβ-CRY2-mCh for C . elegans ( Okazaki et al . , 2012 ) .",
"c ) pDEST-Tol2-PA2 ( Invitrogen ) and p5’E-Ubiquitin as described in Kibat et al . ( 2016 ) were used to make Ubiquitin driven pDEST-Tol2-PA2-Ubi-Aβ-CRY2-mCh and pDEST-Tol2-PA2 ( Invitrogen ) was used to make pDEST-Tol2-PA2-10xUAS-Aβ-CRY2-mCh for expression in Danio rerio .",
"The CRY2 sequence were removed from this plasmid and the 10xUAS promoter were replaced with CMV promoter to produce pDEST-Tol2-PA2-CMV- Aβ-mCh by Gibson Assembly ( NEB ) for expression in D . Rerio .",
"d ) pDEST40 ( Invitrogen ) was used to make CMV driven pDEST40-Aβ-CRY2-mCh for expression in HEK293 tissue culture cells .",
"For Drosophila , the transgenes were injected into attP2 ( Strain#8622 ) P[CaryP]attP268A4 by BestGene Inc ( California ) ( Groth et al . , 2004; Markstein et al . , 2008 ) .",
"Expression was driven by Elav-GAL4 the neuronal driver and two ubiquitous drivers armadillo-GAL4 and daughterless-GAL4 ( Brand and Perrimon , 1993 ) .",
"All additional stocks were obtained from the BloomingtonDrosophilaStock Center ( NIH P40OD018537 ) were used in this study .",
"Fly crosses performed were: For C . elegans , 50 ng/µl the construct hsp-16–2 p-Aβ-CRY2-mCh was co-injected with 25 ng/µl of pharyngeal-specific fluorescence marker myo-2-gfp into the distal gonads of wild type young adults .",
"Transgenes were maintained as extrachromosomal arrays , hence careful selection of transgenic animals with pharyngeal GFP expression is required prior to all experiments .",
"A myo-2-gfp strain C . elegans was also generated and used alongside as controls .",
"For Danio rerio , 50 ng/µl of the construct pDEST-Tol2-PA2-10xUAS-Aβ-CRY2-mCh and 60 ng/ µl Tol2RNA transposase were injected into TgBAC ( gng8:GAL4 ) c416 zebrafish embryos during the one-cell stage .",
"pDEST-Tol2-PA2-Ubi-Aβ-CRY2-mCh and pDEST-Tol2-PA2-CMV-Aβ-mCh were injected into one-cell stage mitfa -/- zebrafish embryos lacking melanin pigment separately .",
"Drosophila were maintained at standard humidity and temperature ( 25°C ) with food containing 6 g Bacto agar , 114 g glucose , 56 g cornmeal , 25 g Brewer’s yeast and 20 ml of 10% Nipagin in 1L final volume .",
"Transgenic Drosophila and controls were distributed into either dark or light condition on Day 1 .",
"Transgenic flies fed with a lithium-supplemented diet were maintained in the same food as above with the addition of lithium chloride to a final concentration of 5 mM .",
"C . elegans were maintained as previously described at 20°C ( Stiernagle , 2006 ) .",
"Age-synchronized nematodes were generated by hypochlorite bleaching .",
"250 µM of 5-fluoro-2’-deoxyuridine ( FUDR ) was added to prevent progeny production in all experiments except for fertility assays .",
"For Fertility assay , normal Nematode Growth Media ( NGM ) agar plates were used .",
"To induce expression of Aβ-CRY2-mCh , Day 4 young adult worms were heat shocked at 35°C for 90 min , and subsequently incubated at 20⁰C for a day ( Day",
"5 ) for recovery .",
"The heat-shocked transgenic Day 6 C . elegans were then separated into dark or light condition and was maintained at 20⁰C throughout the experimental period .",
"Non-heat shock controls were used for lifespan , fertility and locomotion studies .",
"Adult zebrafish were reared under standard zebrafish facility conditions with a 14 hr light/10 hr dark cycle .",
"Zebrafish embryos injected with different expression vectors were screened for fluorescence and distributed into either dark or light condition 24 hr post fertilization .",
"Embryos were dechorionated at 48 hr post fertilization and exposed to blue light ( 488 nM ) for 1–2 hr at room temperature to induce oligomerization and subsequently used for mitochondria metabolic flux assay or ATP assay .",
"Adult fly midguts were dissected in 200 µl of 1x PBS in a PYREX Spot Plates concave glass dish ( FisherScientific ) .",
"Subsequently , the guts were carefully transferred onto a small droplet of 1x PBS on a 35 mm glass bottom dish .",
"Using fine forceps , the gut was repositioned to resemble its natural orientation .",
"PBS was then removed from the area surrounding the gut , leaving a small amount of excess PBS to hold the gut in place and prevent desiccation .",
"The 3 mm glass bottom dish was then mounted onto the Zeiss LSM800 ( Carl Zeiss AG , Germany ) for imaging .",
"The super-resolution imaging function on the Zeiss LSM800 was used for the bleaching and fluorescence recovery .",
"A small square within a cell was defined as the bleaching area and this same square was used for all bleaching experiments .",
"Bleaching was performed at 4% laser power using the 488 nm laser for 1 s and the recovery was followed every 30 s for 20 minutes .",
"The fluorescence intensity of the bleaching square ( FS ) , the entire cell ( FC ) and the background ( FB ) at each time point was computed by the ZEN 3 . 1 ( Blue edition ) software ( Carl Zeiss , Germany ) .",
"For determination of percentage fluorescence recovery , the background fluorescence was subtracted from the fluorescence of the bleaching square ( FS-FB ) and the fluorescence of the entire cell ( FC-FB ) .",
"The ( FS-FB ) / ( FC-FB ) was computed at each time point and the ( FS-FB ) / ( FC-FB ) before bleaching was set to 100% .",
"This was done for all FRAP assays for both constructs and 3 separate experiments were conducted per construct .",
"Drosophila and C . elegans were counted daily for the number of dead subjects and the number of censored subjects ( excluded from the study ) .",
"Drosophila that failed to respond to taps were scored as dead and those stuck to the food were censored .",
"C . elegans that failed to respond to plate-tapping were scored as dead , those that burrowed to the side of the plate were censored .",
"Drosophila locomotion was assessed using an established geotaxis assay previous described in Rival et al . ( 2009 ) .",
"In brief , 25 Drosophila were enclosed in a plastic column ( 25 cm tall , with a 1 . 5 cm internal diameter ) and were tapped to the bottom .",
"The number of Drosophila at the top ( Ntop ) of the column , and that at the bottom ( Nbot ) were counted after 20 s .",
"Three trials with the same sample were performed within 30 s interval .",
"Performance index was defined as ( 15+Ntop-Nbot ) /30 .",
"Heat-shocked C . elegans were assessed at Day 9 , by placing worms onto individual NGM plates , pre-spotted with Escherichia coli OP50 .",
"C . elegans were placed on the side of the bacteria spot on the NGM plate using a platinum worm picker and were left undisturbed to move freely for 15 min .",
"Distance travelled was determined by imaging and measuring of worm tracks on bacteria using a stereomicroscope .",
"25 pairs of male and female Drosophila expressing MD-Aβ-Cry2-mCh were kept in a single tube under dark or light conditions .",
"The number of eggs laid were scored after 5 hr 30 min for Days 5 , 6 , 7 , 13 , 14 and 20 .",
"Day 5 heat-shocked C . elegans were transferred onto individual NGM plates spotted with E . coli .",
"Each worm was transferred to a fresh plate daily from Day 6 to Day 8 , allowing 24 hr’ egg laying period .",
"The number of new young worms were counted on each plate as the progeny of a single individual .",
"ATP assay using firefly lantern extract ( Sigma-Aldrich ) was performed as described by Tsujimoto et al . ( 1970 ) .",
"Each C . elegans sample containing 100 D2 adult worms was freeze-thawed in liquid nitrogen and sonicated in 10% Trichloroacetic acid ( TCA ) buffer while each zebrafish sample containing five 48hpf embryos was lysed in 10% TCA buffer .",
"The extracted ATP from the different conditions and ATP standards were loaded onto a 96-well plate and injected with firefly lantern extract .",
"Luminescence was measured using a Cytation 3 Imaging Reader ( Biotek ) .",
"Mitochondrial metabolic flux assay for C . elegans was performed as described by Fong et al . ( 2017 ) using XF96 Extracellular Flux Analyzer .",
"60 D2 adult worms from each condition 24 hr post light-treatment were transferred to a 96-well Seahorse plate containing M9 buffer .",
"Final concentrations of the drugs used in the OCR experiment were 10 µM FCCP and 50 mM sodium azide .",
"For zebrafish embryos , mitochondrial metabolic flux assay was performed as described by Stackley et al . ( 2011 ) with some modifications .",
"Oxygen consumption rate ( OCR ) measurements were performed using the XF96 Extracellular Flux Analyzer .",
"Each well contained one embryo ( 48hpf ) in 175 µL of E3 medium ( fish system water ) .",
"Final concentrations of the drugs used in the OCR experiment were 9 . 4 µM oligomycin , 2 . 5 µM FCCP and 20 mM sodium azide .",
"Drosophila embryos were dechorionated using bleach , rinsed twice with water and dried , and loaded into a capillary filled with 1% low-melting agarose Type VII-A in water ( Sigma ) ( Colosimo and Tolwinski , 2006 ) .",
"C . elegans and D . rerio were embedded directly in the agarose .",
"Samples were imaged with a Lightsheet Z . 1 microscope .",
"Cry2 was activated by a dual-side illumination with 10% power 488 nm laser for 29 . 95 ms for every 2 . 5 min for 500 cycles .",
"Controls were only exposed to 561 nm .",
"Images were acquired with a water immersion objective at 10x/0 . 2 Illumination Optics and W Plan-Apochromat 20x . 1 . 0 UV-VIS detection objective ( Carl Zeiss , Germany ) .",
"Image data were processed using the maximum intensity projection function of ZEN 2014 SP software ( Carl Zeiss , Germany ) , and were analyzed with ImageJ ( NIH ) and IMARIS 9 . 0 ( Bitplane AG , UK ) .",
"Human Embryonic Kidney ( HEK293T ) cells were obtained from ATCC through the local distributor Bio-REV Pte Ltd after Short Tandem Repeat profiling , confirmed mycoplasma-free and maintained in Dulbecco’s Modified Eagle’s Medium with 10% Fetal Bovine Serum and 1% Penicillin and Streptomycin ( Invitrogen ) .",
"Cells were plated at 80% confluence on 35 mm TC treated glass bottom dish 24 hr prior to transfection with 2 . 5 mg of pDEST40-Aβ-CRY2-mCh using the Effectene Transfection Reagent ( Qiagen ) .",
"A transfection master mix containing the plasmid and transfection reagent was prepared for all treatment plates during each biological replicate to ensure the transfection efficiency was the same across all plates .",
"Two hours after transfection , the control plate was topped up with 250 µl of complete media without antibiotics .",
"For cells treated with drugs , LiCl or CHIR99021 were also added at this point to achieve a final concentration of 2 mM LiCl or 3 mM CHIR99021 respectively .",
"Cells transfected with only the pDEST40-Aβ-CRY2-mCh plasmid were used as negative control .",
"Cells were imaged 24 hr after transfection using the Zeiss LSM800 confocal microscope with 63x oil immersion lens .",
"Drosophila embryos 9 hr after deposition were incubated at 25°C in either light or dark conditions for 13 hr before being dechorionated using bleach .",
"Embryos were then fixed with Heat-Methanol treatment ( Müller and Wieschaus , 1996 ) or with heptane/4% formaldehyde in phosphate buffer ( 0 . 1M NaPO4 pH 7 . 4 ) ( Tolwinski and Wieschaus , 2001 ) .",
"Staining , detection and image processing as described in Colosimo and Tolwinski ( 2006 ) .",
"Primary antibodies used were the glial cell marker anti-Repo ( mouse mAb , 8D12 ) and the neuronal cell marker anti-Elav ( rat mAb , 7EA810 ) from Developmental Studies Hybridoma Bank ( DSHB developed under the auspices of the NICHD and maintained by The University of Iowa , Department of Biological Sciences , Iowa City , IA 52242 ) .",
"Secondary antibodies used were Alexa Flour 488 anti-rat and Alexa Flour 647 anti-mouse ( Invitrogen ) .",
"For detection of Aβ aggregates , thioflavin T ( ThT ) staining were performed as previously described Iijima et al . ( 2008 ) .",
"Formaldehyde-fixed embryos were incubated in 50% EtOH containing 0 . 1% ThT ( Sigma ) overnight .",
"Embryos were destained in 50% EtOH for 10 min , followed by three washes in PBS .",
"Embryos were then mounted on microscope slides using Aquapolymount ( Polysciences , Inc ) .",
"Images were acquired on the Zeiss LSM 800 ( Carl Zeiss , Germany ) using the following settings: 1% laser power for 488 nm; 5% laser power for 561 nm; 2% laser power for 647 nm .",
"Images were processed using the ZEN 2014 SP1 software ( Carl Zeiss , Germany ) and Imaris ( Bitplane AG ) .",
"Drosophila embryos 9 hr after deposition were incubated at 25°C in either light or dark conditions for 13 hr were used .",
"Embryos were dechorionated and washed with 100% ethanol prior to RNA extraction using the ISOLATE II RNA Mini Kit’s protocol ( Bioline , UK ) .",
"The extracted RNA was quantified using Nanodrop ( Thermo Fisher Scientific ) .",
"cDNA synthesis was done according to the SensiFAST cDNA Synthesis Kit‘s protocol ( Bioline ) .",
"Primers pairs used: AttA , IMD , DptA , Def , Duox , Rel , catalase , Prdx3 , Trx1 , Trx2 , SOD-1 , SOD-2 , SOD-3 and reference gene Rpl32 .",
"Quantitative PCR was performed using SYBR Green .",
"Expression data were normalized to the dark controls .",
"For mitochondrial copy number , the above collection method was used except the following: the embryos were stored in tritonX-100 and the primers used were Mitochondria Cytochrome b ( MtCyb ) and reference gene RNAse P . Aβ aggregates were quantified in Drosophila using ImageJ ( NIH ) and in C . elegans and HEK cells with IMARIS .",
"For HEK cells , the number of clusters larger than 0 . 4 µm in diameter were determined for at least 3 cells for the control and drug treated cells to calculate the average number of clusters per cell .",
"The mean intensity per cluster across all these cells was also determined to calculate the average intensity of the clusters for the control and drug treated cells .",
"For statistical analysis of the expression of genes , student’s t-test was used for except for cases where the data showed unequal standard deviation ( F-test , p<0 . 05 ) , in which the Mann-Whitney nonparametric test was performed .",
"Statistical analysis of lifespan studies and behavioral assays were performed using OASIS 2 ( Han et al . , 2016 ) .",
"The number of samples was determined empirically .",
"All graphs were plotted using Graphpad PRISM 6 ( Graphpad Software ) .",
"New model organisms generated for this paper are available to the community by contacting the authors .",
"Stable lines and stocks will be made available through stock centers .",
"Drosophila line UAS-Aβ-Cry2-mCh inserted on Chromosome 3 site attP2:"
]
] | [
"The brains of Alzheimer’s disease patients show a decrease in brain mass and a preponderance of extracellular Amyloid-β plaques .",
"These plaques are formed by aggregation of polypeptides that are derived from the Amyloid Precursor Protein ( APP ) .",
"Amyloid-β plaques are thought to play either a direct or an indirect role in disease progression , however the exact role of aggregation and plaque formation in the aetiology of Alzheimer’s disease ( AD ) is subject to debate as the biological effects of soluble and aggregated Amyloid-β peptides are difficult to separate in vivo .",
"To investigate the consequences of formation of Amyloid-β oligomers in living tissues , we developed a fluorescently tagged , optogenetic Amyloid-β peptide that oligomerizes rapidly in the presence of blue light .",
"We applied this system to the crucial question of how intracellular Amyloid-β oligomers underlie the pathologies of A . We use Drosophila , C . elegans and D . rerio to show that , although both expression and induced oligomerization of Amyloid-β were detrimental to lifespan and healthspan , we were able to separate the metabolic and physical damage caused by light-induced Amyloid-β oligomerization from Amyloid-β expression alone .",
"The physical damage caused by Amyloid-β oligomers also recapitulated the catastrophic tissue loss that is a hallmark of late AD .",
"We show that the lifespan deficit induced by Amyloid-β oligomers was reduced with Li+ treatment .",
"Our results present the first model to separate different aspects of disease progression ."
] | [
"Alzheimer's disease is a progressive condition that damages the brain over time .",
"The cause is not clear , but a toxic molecule called Amyloid-β peptide seems to play a part .",
"It builds up in the brains of people with Alzheimer's disease , forming hard clumps called plaques .",
"Yet , though the plaques are a hallmark of the disease , experimental treatments designed to break them down do not seem to help .",
"This raises the question – do Amyloid-β plaques actually cause Alzheimer's disease ?",
"Answering this question is not easy .",
"One way to study the effect of amyloid plaques is to inject clumps of Amyloid-β peptides into model organisms .",
"This triggers Alzheimer's-like brain damage , but it is not clear why .",
"It remains difficult to tell the difference between the damage caused by the injected Amyloid-β peptides and the damage caused by the solid plaques that they form .",
"For this , researchers need a way to trigger plaque formation directly inside animal brains .",
"This would make it possible to test the effects of plaque-targeting treatments , like the drug lithium .",
"Optogenetics is a technique that uses light to control molecules in living animals .",
"Hsien , Kaur et al . have now used this approach to trigger plaque formation by fusing light-sensitive proteins to Amyloid-β peptides in worms , fruit flies and zebrafish .",
"This meant that the peptides clumped together to form plaques whenever the animals were exposed to blue light .",
"This revealed that , while both the Amyloid-β peptides and the plaques caused damage , the plaques were much more toxic .",
"They damaged cell metabolism and caused tissue loss that resembled late Alzheimer's disease in humans .",
"To find out whether it was possible to test Alzheimer's treatments in these animals , Hsien , Kaur et al . treated them with the drug , lithium .",
"This increased their lifespan , reversing some of the damage caused by the plaques .",
"Alzheimer's disease affects more than 46 . 8 million people worldwide and is the sixth leading cause of death in the USA .",
"But , despite over 50 years of research , there is no cure .",
"This new plaque-formation technique allows researchers to study the effects of amyloid plaques in living animals , providing a new way to test Alzheimer's treatments .",
"This could be of particular help in studies of experimental drugs that aim to reduce plaque formation ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Decision and navigation in mouse parietal cortex | elife-42583-v2 | [
[
"Posterior parietal cortex ( PPC ) is recognized as a key nexus of sensorimotor integration ( Milner and Goodale , 2006 ) , and the large literature concerning its functions has led to disparate views as to its role .",
"These views originate from a broad variety of experiments , some performed in primates and others in rodents .",
"A number of studies have related PPC activity to the control of body movement ( Andersen and Buneo , 2002; Andersen and Cui , 2009; Andersen and Mountcastle , 1983; Bisley and Goldberg , 2010; Cohen and Andersen , 2002; Park et al . , 2014 ) .",
"For instance , neurons in monkey PPC can be influenced by movements of eyes , head , limbs , and body ( Cohen and Andersen , 2002 ) , by the intention to execute such movements ( Andersen and Buneo , 2002 ) , or by the attention devoted to the resulting position ( Bisley and Goldberg , 2010 ) .",
"Neurons in rat PPC , moreover , can encode attributes of body posture ( Mimica et al . , 2018 ) .",
"Another set of experiments indicates a role of PPC in decision making , especially for decisions based on vision ( Andersen and Cui , 2009; Erlich et al . , 2015; Goard et al . , 2016; Gold and Shadlen , 2007; Katz et al . , 2016; Latimer et al . , 2015; Licata et al . , 2017; Platt and Glimcher , 1999; Raposo et al . , 2014; Sugrue et al . , 2004 ) .",
"Studies in rodents found decision signals to be widespread in PPC populations , where they are mixed with other signals ( Goard et al . , 2016; Pho et al . , 2018; Raposo et al . , 2014 ) .",
"One study , in particular , probed memory-based decision signals and found them to be remarkably common , with each PPC neuron firing only for a particular decision and only at a particular moment in a stereotyped sequence ( Harvey et al . , 2012 ) .",
"A third set of studies , performed in rodents , suggested an important role of PPC in spatial navigation ( McNaughton et al . , 1994; Nitz , 2006; Nitz , 2012; Save and Poucet , 2000; Save and Poucet , 2009; Whitlock et al . , 2012; Wilber et al . , 2014 ) .",
"Neurons in rat PPC encode combinations of spatial location and body movement ( McNaughton et al . , 1994; Nitz , 2006; Nitz , 2012; Whitlock et al . , 2012; Wilber et al . , 2014 ) .",
"Inactivating it , moreover , can impair navigation ( Save and Poucet , 2000; Save and Poucet , 2009 ) .",
"In principle , all of these views may be correct .",
"Neuronal populations may benefit from a strategy of ‘mixed selectivity’ , where neurons are tuned to mixtures of task-related variables ( Rigotti et al . , 2013 ) .",
"Mixed selectivity has been observed in PPC neurons of both monkeys ( Freedman and Assad , 2009; Park et al . , 2014; Rishel et al . , 2013 ) and rats ( Raposo et al . , 2014 ) To investigate how spatial navigation , body movement , and decision-making are reflected in PPC activity , we took advantage of the capabilities allowed by virtual reality ( Harvey et al . , 2009 ) .",
"We trained mice in a virtual navigation task that relies on visual decision-making , and recorded from populations of PPC neurons .",
"The results closely replicated the apparent selectivity of PPC activity on choice , including the arrangement of neurons in choice-dependent activation sequences ( Harvey et al . , 2012 ) .",
"However , we could predict the activity of PPC neurons based on simple spatial signals: the position of the mouse in the virtual environment and its heading angle .",
"Given the trajectories taken by the mouse , the preferences of the neurons for these attributes predicted the activation of individual neurons and explained their arrangement in sequences .",
"Selectivity for choice , in particular , was fully explained by preferred heading .",
"The predictions improved only slightly when we explicitly added decision as a predictor , and they worsened when we used alternative models based on vision or body movement ."
],
[
"We trained mice in a virtual navigation task driven by perceptual decisions ( Figure 1a–c ) .",
"Head-fixed mice performed a visual two-alternative forced-choice ( 2AFC ) contrast-detection task by walking on an air-suspended ball ( Dombeck et al . , 2010 ) through a virtual corridor ( Figure 1a , Video 1 ) .",
"One of the corridor’s side walls , chosen randomly , contained a vertical grating , and mice indicated that side by turning into the left or right arms at the end of the corridor ( Figure 1b ) .",
"Successful trials were followed by a water reward , and unsuccessful ones by a brief white-noise sound .",
"To control the task difficulty , we varied the grating’s contrast randomly across trials .",
"Accordingly , contrast exerted a powerful influence on performance: mice frequently chose the correct side for high-contrast stimuli , and performed at chance when contrast was low or zero ( Figure 1c ) .",
"These visual decisions strongly influenced navigation throughout the corridor , as mice typically turned towards the intended side before reaching the end ( Figure 1d–h ) .",
"To describe the navigation trajectories , we considered two variables: position along the corridor ( z ) and heading angle ( θ ) .",
"In these coordinates , the paths that ended in left and right choices progressively deviated from each other: the animal started heading towards the chosen side before reaching the end of the corridor .",
"This dependence of heading angle θ on decision was particularly clear for trials with higher contrast , which were easier ( Figure 1f ) .",
"The final choice could thus be predicted from the heading angle with increasing accuracy as the mouse reached the end of the corridor ( Figure 1g , h ) .",
"While mice performed this task , we measured PPC population activity using 2-photon calcium imaging ( Figure 1i , j ) .",
"To identify the borders of visual cortical areas we obtained retinotopic maps using widefield imaging ( Figure 1i ) , and identified PPC as a region anterior to the V1 region , along a contour of pixels that represent a retinotopic azimuth of 60–80 degrees .",
"The average stereotaxic coordinates of this region ( Figure 1—figure supplement 1 ) were close to the coordinates used in previous studies of mouse PPC ( 2 . 0 mm posterior , 1 . 7 mm lateral , Goard et al . , 2016; Harvey et al . , 2012 ) .",
"We then targeted 2-photon imaging to this region while the mouse was performing the task ( Figure 1j ) .",
"To obtain calcium traces from well-identified cell bodies we applied Suite2p , an image-processing pipeline that provides image registration , cell detection , and neuropil correction , followed by manual curation ( Pachitariu et al . , 2016 ) .",
"In agreement with previous observations made in a memory-based task ( Harvey et al . , 2012 ) , all the recorded PPC cells could be divided into two groups forming distinct , choice-dependent sequences of activation ( Figure 1k–n ) .",
"One group of cells responded primarily during trials that ended in a leftward choice ( Figure 1k , l ) , and the other during trials that ended in a rightward choice ( Figure 1m , n ) .",
"Moreover , cells could be ordered so that the responses of each group of cells could be arranged in a sequence of activations ( Harvey et al . , 2012 ) .",
"While some cells that responded during the initial part of the task tended to fire in both trials that ended with left and right choices , the rest of the cells unambiguously fired only in one or the other of those trials .",
"To investigate this apparent dependence of activity on choice , we plotted the firing of individual neurons as a function of the animal’s position and heading ( Figure 2a , b ) .",
"Consider an example neuron , which fired mostly during trajectories that ended with rightward choices .",
"Plotting the neuron’s activity on top of the individual trajectories reveals that the neuron tended to respond in precise combinations of position z ( ~60 cm into the corridor ) and heading angle θ ( ~20 deg to the right ) .",
"This combination occurred most frequently in trajectories that ended with rightward choices ( Figure 2b ) but also occasionally in trajectories that ended with leftward choices ( Figure 2a ) .",
"The neuron was active whenever a trajectory brought the mouse to the appropriate combinations of position and heading , regardless of final choice .",
"Indeed , a simple ‘position-heading field’ was sufficient to accurately predict the calcium activity of the neuron ( Figure 2c–g ) .",
"We obtained an activity map of the neuron as a function of position and heading ( Figure 2e ) , and found that it could be used effectively to predict responses as a function of time ( Figure 2f ) .",
"The model performed well across trials ( Figure 2c , d ) , capturing not only the overall preference for trajectories that ended in rightward choices , but also detailed differences in responses in individual trials ( Figure 2c , d ) .",
"For instance , the position-heading field correctly predicted the occasional trials when the neuron responded during leftward choices ( above the black bar in Figure 2c , d ) .",
"Position-heading fields provided a good account of the activity across the population ( Figure 2h–j ) .",
"High correlation between data and predictions was not associated with a particular preference for position or heading: cells whose responses were accurately predicted could have a variety of position-heading fields ( Figure 2h–j ) .",
"For all these cells , the model performed well in describing trial-by-trial activity ( Figure 2—figure supplement 1 ) .",
"The median correlation between model predictions and calcium traces was 27% ( ±16% m . a . d . , n = 7 , 646 cells ) when pooling across seven mice , and ranged from 19% to 41% in individual mice ( Figure 4—figure supplement 1 ) .",
"These values are high , given that all measures were cross-validated .",
"Position-heading fields were also largely sufficient to explain the arrangement of parietal cortex responses in choice-dependent sequences ( Figure 3 ) .",
"For each PPC neuron we predicted responses for all trials and averaged these predictions depending on whether the trials ended in leftward choices ( Figure 3a ) or rightward choices ( Figure 3b ) .",
"The resulting predictions produce orderly , choice-dependent sequences of activation , replicating the essential features of those seen in the data ( Figure 1k–n ) .",
"These choice-dependent sequences of activation emerge from a combination of two factors: the fact that mice take different trajectories in trials that end with leftward vs . rightward choices ( Figure 1d–f ) , and the fact that different cells prefer different combinations of position and heading ( Figure 3c , d ) .",
"The preferred heading of these PPC cells was thus sufficient to explain their selectivity for choice: cells with preferred leftward heading were more likely to fire when the animal headed leftward , which occurred most often when the animal ultimately chose the left arm .",
"Indeed , cells that responded preferentially in trials ending in leftward choices almost invariably preferred negative ( leftward ) heading values ( Figure 3c ) , and the same was true for rightward choices and positive ( rightward ) heading values ( Figure 3d ) .",
"Decision was a worse predictor of PPC activity than heading ( Figure 4a–c ) .",
"A model f ( z , d ) where responses depend on position and decision alone is implicit in representations where neurons are arranged in choice-selective activation sequences ( Harvey et al . , 2012 ) .",
"It could in principle explain the sequences seen in our data ( Figure 1k–n ) .",
"However , it provided only a rough approximation of the trial-by-trial activity of individual cells , missing its graded dependence on heading angle θ ( Figure 4a ) .",
"To compare the model with the position-heading model f ( z , θ ) , we calculated the correlation between trial-averaged measurements and model predictions , and we cross-validated the results .",
"Before averaging activity in each trial , we excluded positions where decision and heading angle were so highly correlated as to be indistinguishable as predictors .",
"The range of positions varied from session to session and invariably included the end of the corridor .",
"This analysis gave a clear advantage to the position-heading model , with correlations higher by 5 . 6% than for the position-decision model ( ±9 . 8% , m . a . d . , n = 7646 neurons , Figure 4b ) .",
"This advantage was visible also in individual sessions ( Figure 4c ) .",
"Furthermore , a model with all three predictors performed only slightly better than the model with position and heading alone ( Figure 4d–f ) .",
"We extended the position-heading model to obtain a model f ( z , θ , d ) that includes explicit knowledge of the mouse decisions ( d = left or right ) .",
"This extended model effectively endows the cell with two position-heading fields for trajectories that end in leftward vs . rightward choices .",
"The extended model thus predicted slightly different curves for trials ending in left vs . right choices ( Figure 4d ) .",
"Across all neurons , the extended model performed at a similar cross-validated level as the simple position-heading model , improving the correlations by only 0 . 4% ( ±2 . 9% , m . a . d . , n = 7646 neurons , Figure 4e ) .",
"Similar results were seen in individual sessions ( Figure 4f ) .",
"Conversely , adding heading angle θ to a model that already includes d considerably improved the fits , with a median increase in correlation of 6 . 1% ( ±6 . 1% m . a . d . , n = 7646 neurons ) .",
"Therefore , heading angle was a much better predictor of responses than decision .",
"Taken together , these results suggest that much of the activity of PPC neurons in our task can be explained by two spatial attributes: position and heading .",
"A possible caveat in this conclusion , however , is that in our experiment those spatial attributes may covary with visual and motor factors .",
"Position and heading determined the visual scene , and the visual scene could in turn determine the activity of PPC neurons , especially given that mouse PPC overlaps at least partially with regions of higher visual cortex ( Wang and Burkhalter , 2007; Zhuang et al . , 2017 ) .",
"Likewise , position in z-θ space is itself determined by the animal’s movement on the ball and therefore by motor factors such as linear and angular velocity , which may in turn determine PPC activity ( McNaughton et al . , 1994; Nitz , 2006; Whitlock , 2014; Whitlock et al . , 2012 ) .",
"To assess the role of visual factors , we ran a control experiment where the animal passively viewed a playback of visual scenes presented in the task on the same session .",
"In this playback condition only a minority of PPC neurons maintained their preferences for position and heading , and even in those neurons the responses were much weaker ( Figure 4—figure supplement 2a ) .",
"Moreover , for the majority of PPC cells , activity in the playback condition was not predictable from the preferences for position and heading estimated during the task .",
"This is perhaps remarkable , given that parietal areas of the mouse cortex are considered to overlap with visual areas RL , A , and AM ( Hovde et al . , 2018; Kirkcaldie , 2012 ) , which contain retinotopic visual representations ( Garrett et al . , 2014; Wang and Burkhalter , 2007 ) .",
"By comparison , responses measured in primary visual cortex ( V1 ) during the task and during playback were in better agreement ( Figure 4—figure supplement 2b ) .",
"To assess the role of motor factors , we evaluated a model where PPC activity depends on the mouse’s movement , measured by the ball’s angular and linear velocities .",
"These quantities are related to the derivatives of position z and heading θ , but they are not identical because they are in mouse-centered coordinates and are unconstrained by the boundaries of the virtual corridor .",
"The model based on motor factors performed markedly worse than the model based on position and heading in virtual reality ( Figure 4—figure supplement 3 ) .",
"We finally asked if PPC population activity and its dependence on position and heading were sufficient to decode the details of the mouse’s trajectories and choices ( Figure 5 ) .",
"Using established methods ( Oram et al . , 1998; Zhang et al . , 1998 ) we implemented a simple Bayesian decoder ( Figure 5—figure supplement 1 ) .",
"The decoder successfully predicted the position of the animal in position-heading space at individual time points ( Figure 5a ) and replicated the animal’s trajectory ( Figure 5b; Video 2 ) .",
"In predicting the final choice , in fact , the population decoder was just as good as the animal’s actual heading: both showed a similar dependence on position z ( Figure 5c ) and stimulus contrast ( Figure 5d ) .",
"This result provides further support for the view that during visually-guided navigation , populations of PPC neurons encode spatial position and heading angle ."
],
[
"We used a virtual reality task where mice use vision to decide and navigate , and we found that the activity of PPC neurons can be accurately predicted based on two simple spatial measures: position of the animal along the corridor , and heading angle .",
"Using only these attributes , we could predict a large fraction of PPC activity during a complex task involving body movement , vision , decision , and navigation .",
"These predictions are superior to those based purely on vision or on body movement .",
"These results resonate with the view that rodent PPC encodes combinations of spatial attributes ( McNaughton et al . , 1994; Nitz , 2006; Nitz , 2012; Save and Poucet , 2000; Save and Poucet , 2009; Whitlock et al . , 2012; Wilber et al . , 2014 ) , but are also fully consistent with the observation that PPC cells can be divided into groups forming distinct sequences of activations depending on upcoming choice ( Harvey et al . , 2012 ) .",
"However , in our data , this division into choice-dependent sequences , and indeed the sequences themselves , could be explained by the effect on PPC of two measurable factors: the trajectories taken by the mouse in different trials , and the preferences of different cells for different combinations of position and heading .",
"The selectivity of PPC cells for heading and position explained the cells’ apparent selectivity for the mouse’s decision .",
"Our results therefore appear to support a different view of PPC function to that proposed by Harvey et al . ( 2012 ) .",
"Perhaps this discrepancy is due to a difference between the tasks: for example in our task , unlike the main task by Harvey et al , the spatial cues indicating the appropriate decision were visible until the end of the corridor .",
"This might have caused the animals to employ different strategies , resulting in genuinely different types of PPC coding across the two tasks .",
"It is also possible , however , that the same combination of spatial factors also contributed to the results of Harvey et al . ( 2012 ) .",
"Indeed , the animals’ trajectories in that study did exhibit differences in heading angle that correlated with the final decision , but trials showing such differences were progressively excluded from analysis until the difference in mean heading angle no longer reached statistical significance .",
"It would therefore be interesting to test whether preferences for position and heading may also contribute to the apparent decision-selectivity in Harvey et al . ’s full data set .",
"In conclusion , we found that the activity of neurons in PPC during a task involving movement , vision , decision , and navigation , can be accurately predicted based on the selectivity of the neurons for two spatial variables: the position of the mouse along the corridor , and its heading angle .",
"Consideration of this selectivity , and the mouse’s trajectories through virtual space , fully accounts for the apparent formation of decision-dependent activity sequences in this task .",
"In other words , when mice use vision to guide navigation , parietal cortex encodes navigational attributes such as position and heading rather than visual signals or abstract decisions .",
"Our results , however , do not completely rule out a possible role of decision signals in shaping the responses of PPC neurons .",
"We found that adding decision as an explicit variable barely improved the fits of the position-heading model , but did not make them worse as it could have , given that the model fits were cross-validated .",
"Our task , in fact , cannot fully distinguish encoding of navigation variables and of decisions , because trajectory signals might strongly covary with an evolving decision variable .",
"Such an evolving decision variable might be seen not only in covert neural signals , but also in ongoing body movements ( Dotan et al . , 2018; Resulaj et al . , 2009; Song and Nakayama , 2009; Spivey et al . , 2005 ) .",
"In our task , the clear dependence of trajectories on stimulus contrast is consistent with a close relationship between a decision variable and the animal’s trajectories .",
"It may not be possible to distinguish the neural encoding of the two .",
"Moreover , PPC activity could be influenced by a multitude of postural attributes that we did not monitor , and some of these may correlate with the apparent decision signals that improved our fits .",
"Indeed , there is mounting evidence that postural and other motor variables act as powerful determinants of activity throughout the cortex ( Musall et al . , 2018; Stringer et al . , 2018 ) including PPC ( Mimica et al . , 2018 ) .",
"The same observation , in fact , can be made for the apparent selectivity of PPC neurons for heading: perhaps neurons in PPC are sensitive to postural variables that in turn predict heading .",
"The precise motor , postural , or cognitive correlates of heading and decision need further examination , ideally with tasks explicitly designed to distinguish these correlates .",
"In the meantime , our results indicate that the activity of large populations of PPC neurons can be explained based on simple embodied quantities such as heading and position in the environment ."
],
[
"For the initial surgery the animal was anesthetized with isoflurane ( Merial ) at 3–5% for induction , and 0 . 75–1 . 5% subsequently .",
"Carprofen ( 5 mg/kg weight , Rimadyl , Pfizer ) was administered subcutaneously for systemic analgesia , and dexamethasone ( 0 . 5 mg/kg weight , Colvasone , Norbrook ) was administered to prevent brain swelling .",
"The scalp was shaved and disinfected , and a local analgesic ( Lidocaine , 5% ointment , TEVA UK; or intradermal injection , 6 mg/kg , Hameln Pharmaceuticals Ltd ) was applied prior to the incision .",
"The eyes were covered with eye-protective gel ( Viscotears , Alcon; or Chloramphenicol , Martindale Pharmaceuticals Ltd ) .",
"The animal was positioned in a stereotaxic frame ( Lidocaine ointment was applied to the ear bars ) , the skin covering and surrounding the area of interest was removed , and the skull was cleaned of connective tissue .",
"A custom headplate was positioned above the area of interest and attached to the bone with Superbond C and B ( Sun Medical ) .",
"Then , a round craniotomy ( 3–4 mm diameter ) was made with a fine-tipped diamond drill and/or a biopsy punch ( Kai Medical ) .",
"A cranial window was inserted into the craniotomy and fixed with Vetbond ( 3M ) and Superbond C and B . The cranial window consisted of two superimposed round coverslips ( WPI , #1 thickness ) – one matching the inner diameter of the craniotomy ( 3–4 mm ) , and the other one providing mechanical support on the skull ( typically 5 mm diameter ) .",
"The two coverslips were glued together beforehand using a Norland optical UV curing adhesive ( NOA61 , ThorLabs Inc . ) .",
"After the surgery the animal was allowed to recover for at least three days before any behavioral or physiological measurements .",
"In C57Bl/6 mice , we injected the virus AAV2/1 . Syn . GCaMP6f . WPRE . SV40 at a final concentration of 2 . 3e12 GC/ml before covering the craniotomy with the window .",
"100 nl of the virus was injected 300 µm below the brain surface at each of two locations targeting PPC ( AP = −2 mm , ML = 1 . 7 mm ) and V1 ( AP = −3 . 5 mm , ML = 2 . 5 mm ) .",
"The virus was injected at a rate of 2 . 3 nl every 6 s ( Nanoject II , Drummond ) .",
"The injection pipette was kept in place for about 10 min after the injection to allow full absorption of the virus solution in the tissue .",
"To obtain maps of retinotopy we performed widefield imaging: fluorescence imaging on transgenic mice ( GCaMP6f-TTA-Emx1-Cre ) , and intrinsic imaging on wildtype ( C57bl/6 ) mice , with methods described previously ( Garrett et al . , 2014; Pisauro et al . , 2013 ) .",
"To motivate the mice to perform the task we controlled their water intake .",
"Mice obtained a drop of water ( typically 2 or 4 μl ) for every correct choice .",
"If the water obtained during the task was below the minimum daily dose ( 40 ml/kg/day ) , mice received the rest of the dose through an appropriately weighted amount of Hydrogel .",
"On rest days ( typically Saturday and/or Sunday ) the mice received all their dose through Hydrogel .",
"The mouse was head fixed with a headplate holder that did not obstruct the visual field .",
"The mouse was free to run on an air-suspended Styrofoam ball ( 20 cm in diameter ) , whose rotation was measured by two optical computer mice ( Dombeck et al . , 2010 ) and then used in a custom virtual reality engine implemented in Matlab utilizing OpenGL through the Psychophysics Toolbox ( Brainard , 1997; Pelli , 1997 ) to control the visual scene .",
"The rotation of the ball along the axis perpendicular to the animal was responsible for forward movement in virtual reality , and the rotation of the ball along the vertical axis ( at 20% gain ) was responsible for turning in virtual reality .",
"The lateral displacement of ball ( rotation along the axis parallel to the animal’s orientation ) was ignored .",
"The virtual reality scene was presented on three computer monitors ( Iiyama ProLite E1980SD , 1280 × 1024 pixels , 60 Hz ) positioned in a U-shaped configuration around the mouse spanning 270 degrees of the visual field horizontally and 75 degrees vertically .",
"We used a multiplex video card ( Matrox TripleHead2Go Digital Edition ) to present the visual stimulus on three monitors in a synchronized manner .",
"The light intensity response of the green and the blue channels of the monitors was linearized , while the red component was switched off to reduce light contamination in the fluorescence channel .",
"In addition , to compensate for the light intensity drop-off at sharp viewing angles we attached three Fresnel lenses ( f = 22 cm , BHPA220-2-6 , Wuxi Bohai Optics Apparatus Electronic Co . , Ltd , Wuxi , Jiangsu , China ) in front of the monitors .",
"The virtual reality environment consisted of a 110 cm long T-Maze with a 20 cm wide corridor .",
"Virtual position was never allowed to be less than 5 cm to any wall .",
"During a trial , a vertical grating appeared on the left or right wall of the corridor .",
"The grating was superimposed on a noise texture ( 20% visual contrast ) , which was identical on both walls and across trials .",
"The spatial frequency of the grating was 14 cycles/m , and the resulting spatial frequency in the mouse visual field ( in cycles/deg ) varied depending on the distance and angle of the wall relative to the mouse .",
"To provide additional visual flow and context , a 40% visual contrast noise pattern was applied to the floor of the corridor ( and the ceiling , in some experiments ) .",
"The sequence of grating contrasts was randomly drawn from a uniform distribution , with negative values indicating positions on the left wall , and positive values positions on the right wall .",
"However , to prevent the animal from developing behavioral biases , trials ending in a wrong choice were repeated until finished correctly , at which point the next trial in the sequence was again a random trial .",
"This ‘baiting’ strategy was applied mainly when the animals displayed a behavioral bias .",
"Correct trials were indicated by a brief beep ( 0 . 1 s , 6 . 6 kHz ) tone , while error trials were indicated by a brief ( 0 . 2",
"s ) white noise sound .",
"During the inter-trial interval ( ~2",
"s ) the screen was gray .",
"Trials not finished within 45 s were timed out and a longer ( 3",
"s ) white-noise sound was played .",
"Mice typically performed between 200 and 400 trials per session ( session duration 45–60 min ) , with typical duration of the finished trials of 6 . 0 ± 2 . 0 ( median ±m . a . d . ) seconds , varying between 4 . 7 and 11 . 6 s ( median values ) for individual animals .",
"The behavioral session was aborted when either the animal stopped performing , or stopped consuming the water reward .",
"Mice required different numbers of training sessions to reach behavioral performance sufficient to be considered for imaging ( 7 to 36 sessions , 21 on average across seven animals ) .",
"Playback trials were run immediately after the actual behavioral session , while recording the same cells .",
"The water spout was removed , and the visual stimulus was constructed by chopping the sequence of visual scenes into 0 . 5 s segments and presenting them in randomized order .",
"To reduce the flickering effect of the visual stimulus , each 0 . 5 s segment was modified by sinusoidally modulating the contrast at the frequency of 2 Hz , thus smoothing the transition between sequential segments .",
"The animal was free to run on the ball , but this had no effect on the visual stimuli presented .",
"Typically , during these measurements the animals chose to alternate bouts of running with periods of rest .",
"Two-photon imaging ( total of 33 recording sessions ) was performed using a standard resonant B-Scope microscope ( ThorLabs Inc . ) equipped with Nikon 16x , 0 . 8 NA objective , and controlled by ScanImage 4 . 2 ( Pologruto et al . , 2003 ) .",
"Frame rate was set to ~30 Hz , with the field of view of ~500×500 µm ( 512 × 512 pixels ) .",
"This frame rate was further shared between 3–5 imaging planes spanning the depth of L2/3 using a piezo focusing device ( P-725 . 4CA PIFOC , Physik Instrumente ) resulting in a 6–10 Hz effective sampling rate per cell .",
"Laser power was depth-adjusted and synchronized with piezo position using an electro-optical modulator ( M350-80LA , Conoptics Inc . ) .",
"The imaging objective and the piezo device were light shielded using a custom-made metal cone , a tube , and black cloth to prevent contamination of the fluorescent signal caused by the monitors’ light .",
"Excitation light at 970 nm was delivered by an Ultra II femtosecond laser ( Coherent , UK ) Preprocessing of the two-photon data routinely included registration , segmentation , and neuropil correction ( . The whole cell detection pipeline is explained in Pachitariu et al . , 2016 . ) .",
"To analyze playback experiments we also applied a deconvolution algorithm to extract spikes from the continuous calcium data ( Vogelstein et al . , 2010 ) .",
"To calculate ΔF/F , the baseline fluorescence F0 was taken as the 20th percentile of the overall level of fluorescence of a cell .",
"To estimate the position-heading field of each neuron we used a local likelihood approach ( Loader , 1999 ) .",
"First , we used the data to estimate the occupancy map Mocc ( z , θ ) and the accumulated fluorescence signal map Msig ( z , θ ) .",
"Then , we filtered both maps with a Gaussian filter , and we calculated the resulting position-heading field as:Fz , θ=Msigfiltz , θ+λ ⋅FmeanMoccfiltz , θ+λ Where Fmean is the mean fluorescence of the cell , and λ is a small number used for regularization , to prevent large estimation errors in location where little or no data is available in z-θ space .",
"The size of the Gaussian filter σz , σθ was optimally chosen for each cell through a 10-fold cross-validation procedure .",
"In this procedure , for each set of values σz , σθ 90% percent of the trials ( training subset ) were chosen to estimate F^ ( z , θ ) .",
"Then , performance of the model F^ ( z , θ ) was measured on the remaining 10% of the trials ( test subset ) .",
"On each fold the following error function was used to estimate the performance of the fit:ϵ ( σz , σθ ) =⟨ ( F^ ( z",
"( t ) , θ",
"( t ) ) −F ( z",
"( t ) , θ",
"( t ) ) ) 2⟩t⟨ ( F ( z",
"( t ) , θ",
"( t ) ) −Fmeantrain ) 2⟩t Where Fmeantrain is the average ΔF/F of the training ( 90% ) subset of the data .",
"The procedure was repeated 10 times for different 90/10 partitions of the data .",
"The set of filter values σz , σθ , which resulted in the best overall performance of the model was chosen as optimal and used in subsequent analyses for that neuron .",
"Models incorporating decision were estimated using a similar cross-validation approach .",
"The data for trials ending in leftward and rightward choices were treated separately .",
"For each half of the data , we estimated the optimal σz ( for the f ( z , d ) model ) or σz , σθ ( for the f ( z , θ , d ) model ) using the same cross-validation procedure as for the f ( z , θ ) model , explained previously .",
"This effectively resulted in two sub-models for each model ( f ( z , d ) or f ( z , θ , d ) ) , with two sets of optimal smoothing parameters – one for trials ending in leftward choices and one for trials ending in rightward choices .",
"The two sub-models were then taken together to predict calcium activity on the full set of trials .",
"Depending on the model , different combinations of behavioral parameters ( z-θ , z-d , or z-θ-d ) were used to predict each cell’s activity .",
"For each session the data were randomly divided into 10 groups of trials ( with balanced rightward and leftward trials in each group ) .",
"For each group of trials , the activity was predicted using the other 90% of the dataσz or σz , σθ .",
"The Pearson’s correlation coefficient between mean trial-by-trial actual and predicted responses was used to evaluate and compare performance of different models .",
"At certain positions ( z ) of the T-Maze ( typically towards the end of the corridor ) , heading ( θ ) and final choice ( d ) become strongly correlated .",
"To increase sensitivity of the comparison between the models we excluded data coming from these times ( effectively , when the mouse is at certain z positions ) when estimating quality of fits ( Figure 4 ) .",
"For each behavioral session and for each position z we have estimated the area under the ROC ( auROC ) curve for two variables – heading and choice ( which is a measure of how well one can be predicted from another ) .",
"On a session-by-session basis , data from positions z where the auROC curve was greater than 0 . 95 were excluded from the estimation of fit quality .",
"Calcium dynamics measured with GCaMP6f is slow , with decay times as long as a few hundreds of milliseconds ( Chen et al . , 2013 ) .",
"During playback presentation of 0 . 5 s visual stimuli , this slowly decaying signal will cross-contaminate responses to sequential stimuli .",
"Therefore , for the analysis of the playback experiment , where this issue is critical , we used inferred firing rate ( Vogelstein et al . , 2010 ) and not ΔF/F .",
"To compare activity between the behavior and playback conditions at the corresponding 0 . 5 s segments , we used the correlation coefficient between the inferred firing rates ( binned at 0 . 5",
"s ) .",
"To predict the distribution of locations in z-θ space visited by the animal during the session ( Prz , θ ) we used the position-heading fields ( μiz , θ estimated separately for each cell i ) , and employed a Bayesian decoding approach .",
"Below we show that in this approach , the posterior probability distribution for the animal’s position in z-θ space at time t is:logPost ( z , θ ) =−∑i ( μi ( z , θ ) −ri",
"( t ) ) 22σi2+logPr ( z , θ ) +constwhere rit is the response of cell i at time t , and σi2 is the overall variance of the response of cell i throughout the session .",
"To see this , assume that the response ( ΔF/F , or even simply F ) of cell i at each location z , θ is a Gaussian random variable:Riz , θ~ Nμiz , θ , σiz , θwhere μiz , θ is the expected fluorescence of the cell at position z , θ , that is its position-heading field , and σiz , θ is the variability of fluorescence at this location .",
"Let’s make the additional assumption that σiz , θ=σi , that is it depends on the cell i but not on location in z , θ .",
"Given these assumptions , the probability of this random variable to equal the value ri at time t is:PrRiz , θ=rit=12π⋅σi⋅exp-μiz , θ-rit22σi2and the likelihood of a measured population response is:Lz , θ=∏iPrRiz , θ=rit=∏i12π⋅σi⋅exp-μiz , θ-rit22σi2 Taking the logarithm yields:logL ( z , θ ) =−∑i ( μi ( z , θ ) −ri",
"( t ) ) 22σi2−∑ilog ( σi ) −∑ilog ( 2π ) If we want to incorporate the position prior ( occupancy map ) to get the posterior probability distribution:Postz , θ=Lz , θ⋅Prz , θlogPost ( z , θ ) =logL ( z , θ ) +logPr ( z , θ ) =−∑i ( μi ( z , θ ) −ri",
"( t ) ) 22σi2−∑ilog ( σi ) −∑ilog ( 2π ) +logPr ( z , θ ) Dropping the constants leaves only position-dependent variables , to obtain the expression at the beginning of this section ."
]
] | [
"Posterior parietal cortex ( PPC ) has been implicated in navigation , in the control of movement , and in visually-guided decisions .",
"To relate these views , we measured activity in PPC while mice performed a virtual navigation task driven by visual decisions .",
"PPC neurons were selective for specific combinations of the animal's spatial position and heading angle .",
"This selectivity closely predicted both the activity of individual PPC neurons , and the arrangement of their collective firing patterns in choice-selective sequences .",
"These sequences reflected PPC encoding of the animal’s navigation trajectory .",
"Using decision as a predictor instead of heading yielded worse fits , and using it in addition to heading only slightly improved the fits .",
"Alternative models based on visual or motor variables were inferior .",
"We conclude that when mice use vision to choose their trajectories , a large fraction of parietal cortex activity can be predicted from simple attributes such as spatial position and heading ."
] | [
"When we step out of our homes in the morning , we scan our surroundings to decide which path we should take .",
"It is still unclear whether we use different brain areas to examine the environment , decide on a route , and then set our trajectory , or if a single region can play a role in all three processes .",
"An area in the top of our brain , named the posterior parietal cortex ( PPC ) , may be an intriguing candidate: some studies find that this region is involved in vision , others highlight that it takes part in decision-making , and a third group of experiments shows that it is important for setting paths .",
"Could the PPC be integrating all three types of information , or might the activity of the neurons in this area be better explained by just one of these processes ?",
"Here , Krumin et al . trained mice to use visual clues to navigate a virtual reality maze , where they have to ‘walk’ down a corridor and then choose to ‘go down’ the right or the left arm .",
"The rodents move on a ball suspended in mid-air , which acts as a treadmill .",
"Meanwhile , the head of the animal is kept still , making it easier to image the activity of hundreds of neurons in the PPC .",
"The experiments show that when the mouse uses its vision to choose its trajectory , the PPC does not encode visual signals or abstract decisions .",
"Instead , two navigational attributes – the position of the animal along the corridor and its heading angle – activate neurons in this area .",
"Knowing these two features was enough for Krumin et al . to accurately predict the activity of the neurons in the PPC .",
"This means that we can forecast the activity of neurons deep in the brain by recording simple behavioral features .",
"The results also suggest that the PPC may be more important for setting trajectories than for processing visual images or making abstract decisions .",
"If these findings were to be confirmed in humans , where the parietal cortex is much more complex , they might help understand better the problems that arise when this area is damaged , for example after a stroke ."
] | 2018 |
[
"Introduction",
"Materials and methods"
] | [
"short report",
"microbiology and infectious disease"
] | A molecular portrait of maternal sepsis from Byzantine Troy | elife-20983-v1 | [
[
"During excavations of a Late Byzantine era cemetery at the periphery of the ancient city of Troy , Anatolia ( in present day Turkey ) ( Figure 1—figure supplement 1 ) , we discovered two calcified nodules among a woman’s remains .",
"The woman was estimated to be 30 ( ±5y ) at the time of death ( Appendix ) .",
"She was found alone in a stone-lined grave ( Figure 1A ) within the graveyard of a farming community ( Kiesewetter , 2014 ) .",
"The nodules , which are 2–3 cm in diameter and composed of concentric layers ( Figure 1B ) , were discovered at the base of the ribs .",
"Radiocarbon dating of the decedent’s ulna yielded 790-860y BP ( Supplementary file 1A ) , in agreement with the archeological assessment of the age of the cemetery ( early 13th century AD , Appendix ) . 10 . 7554/eLife . 20983 . 003Figure 1 . Calcified nodule found among the skeletal remains at Troy .",
"( A ) Burial x24 . 177 ( grave 14 , cemetery in quadrat x24 ) .",
"Photo credit Gebhard Bieg , 2005 .",
"( B ) Cross-section of nodule ( sample no x24 . 177 ) , photo credit: Pathologie Nordhessen 2009 .",
"Scale represents 1 cm .",
"( C ) Location of Troy .",
"Modern day Turkey is shaded in gray . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 00310 . 7554/eLife . 20983 . 004Figure 1—figure supplement 1 . Map of Troy showing the cemetery in Grid Square x24 and areas of excavation 1988–2012 . Areas of excavation are in gray and the cemetery is marked with a red square .",
"North is at the top of the plan . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 00410 . 7554/eLife . 20983 . 005Figure 1—figure supplement 2 . Metagenomic profiles of shotgun DNA libraries from nodules , based on BLAST analysis of all reads >35 bp length .",
"( A ) Nodule one ( Nod1_1h-UDG ) , 28 , 713 , 282 reads total ( B ) Nodule two ( Nod2-UDG ) , 6 , 038 , 994 reads total . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 00510 . 7554/eLife . 20983 . 006Figure 1—figure supplement 3 . Fragment length distributions for non-UDG treated human mitochondrial assemblies . These FLDs were generated from the Ulna enriched libraries ( A + B ) as well as the non enriched nodule ( C ) using mapDamage2 ( Jonsson et al . , 2013 ) from merged nonUDG data sets assembled to the human mitochondrial reference genome ( Andrews et al . , 1999 ) , NCBI accession NC_012920 . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 00610 . 7554/eLife . 20983 . 007Figure 1—figure supplement 4 . Ancient DNA damage assessment of human mitochondrial reads . Damage profiles of non-UDG treated ( ‘nonU’ ) as well as UDG treated merged reads assembled to the human mitochondrial rCRS reference genome ( NC_012920 ) for ( A ) Ulna_Enr1-nonU round one human mitochondrial enrichment , ( B ) Ulna_Enr2-nonU round 2 , and ( C ) a Nod1_1h-UDG reads ( which have been UDG treated ) .",
"Damage profiles were generated using mapDamage2 . ( Jonsson et al . , 2013 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 00710 . 7554/eLife . 20983 . 008Figure 1—figure supplement 5 . Ancient DNA damage assessment of reads mapped to hg38 chrX , chrY and autosomes . Damage profiles generated using mapDamage2 ( Jonsson et al . , 2013 ) of non-UDG treated ( ‘nonU’ ) reads from the NOD1_nonU and NOD2_nonU data set ( total of 1 , 468 , 381 trimmed and merged reads ) with minimum 35 bp length and map quality 30 , mapping to ( A ) hg38 chrX , and ( B ) hg38 chrY and C ) hg38 autosomes . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 00810 . 7554/eLife . 20983 . 009Figure 1—figure supplement 6 . Haplogroup U3 Bayesian Maximum Clade Credibility tree . Complete human mtDNA genomes assigned to haplogroup U3 ( n = 137 ) were collected from GenBank and aligned with the Troy consensus sequence ( highlighted in red ) .",
"Tree was generated using BEAST v 1 . 856 and TreeAnnotator . 57",
"Posterior probabilities are shown at nodes . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 00910 . 7554/eLife . 20983 . 010Figure 1—figure supplement 7 . Heatmap of most common taxa in metagenomic samples . The heatmap gives the log of the frequency of the most common taxa in each sample along the diagonal ( if the most frequent is already shown , then second most frequent is added for that sample; Nares and Ear , Nod1_1h-UDG and Nod2-UDG ) .",
"The taxa in order are - 76775 , Malassezia restricta; 76773 , Malassezia globosa; 729 , Haemophilus parainfluenzae; 60133 , Prevotella pallens; 28117 , Alistipes putredinis; 47770 , Lactobacillus crispatus; 562 , Escherichia coli; 487 , Neisseria meningitidis; 2001 , Streptosporangium roseum; 29385 , Staphylococcus saprophyticus; 2702 , Gardnerella vaginalis . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 01010 . 7554/eLife . 20983 . 011Figure 1—figure supplement 8 . PCA of Human Microbiome Project and ancient metagenomic taxa . Taxa were identified using LMAT .",
"PCA performed using prcomp function in R . Legend indicates the origin of the category and the number of samples combined into each category .",
"The first principal component axis separates the placental and ancient samples from the remaining samples .",
"The second principal component axis separates the Sediment-UDG and Ulna-UDG data sets , which likely contain soil contamination , from the remaining samples . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 01110 . 7554/eLife . 20983 . 012Figure 1—figure supplement 9 . Sketch of skeletal preservation . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 012 Nodule one ( Figure 2—figure supplement 1 , Supplementary file 1B ) is primarily composed of two phosphate phases , hydroxylapatite ( bioapatite ) and whitlockite ( as well as small amounts of calcite ) , both of which have been found in human calcified pathological concretions ( Lagier and Baud , 2003 ) .",
"Based on their size and concentric layered structure , the nodules could be urinary stones .",
"However , struvite ( magnesium ammonium phosphate ) and calcium oxalate , common constituents of urinary stones , were absent in both XRD and SEM-EDS analyses ( Supplementary file 1B-D ) .",
"SEM of the nodules ( Figure 2 , Figure 2—figure supplement 2 ) revealed aggregates of spherical structures with dimensions typical of bacterial cells , as well as extracellular polymeric substances ( EPS – a glycocalyx secreted by the cells during biofilm formation [Decho and Thiel , 2011] ) . 10 . 7554/eLife . 20983 . 013Figure 2 . SEM image of nodule at ( A ) 2000x and ( B ) 20 , 000x magnification . Bacterial cells indicated with red arrow are between ~1 µm and 2 µm ( within range expected for Staphylococcus ) .",
"Extracellular polymeric substances ( EPS ) are indicated by yellow arrows . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 01310 . 7554/eLife . 20983 . 014Figure 2—figure supplement 1 . XRD analysis of nodule . A ) Video alignment and XRD frames .",
"Left; crosshairs indicate the center of the region examined .",
"Right; the four frames collected to obtain a 2θ range of 8–103° .",
"At a low angle , air scatter from the main beam is evident .",
"B ) Background subtracted powder pattern .",
"C ) Search/match results . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 01410 . 7554/eLife . 20983 . 015Figure 2—figure supplement 2 . SEM image of nodule at 10 , 000x magnification . Possible inflammatory ( neutrophils ) cells indicated by blue arrows . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 015 We extracted DNA from both nodules and made Uracil DNA Glycosylase ( UDG ) and non-UDG treated dsDNA libraries .",
"Shotgun sequences from all libraries yielded astonishingly high proportions of endogenous human and bacterial DNA: 24–48% human , 37–66% S . saprophyticus , and 5–7% G . vaginalis ( Figure 1—figure supplements 2–5 ) .",
"From these data , we reconstructed a human mitochondrial genome at 30 . 1x unique read depth , the consensus of which belongs to haplotype U3b3 .",
"In phylogenetic analyses of the mitogenome from Troy and modern mitogenomes , the Troy sample groups most closely with those from the Caucasus and Middle East , both of which were within the eastern limits of Late Byzantine influence ( Figure 1—figure supplement 6 ) .",
"To investigate whether the nodules belonged to the associated female individual , we extracted DNA from her ulna , constructed a dsDNA library , enriched for , sequenced , and reconstructed the mitogenome to an average unique coverage depth of 30 . 8x .",
"The nodule and the ulna share the identical mitochondrial haplotype ( Supplementary file 1E ) , indicating that they stem from the same individual or a maternal relative .",
"The metagenomic profile of the nodules suggests they derive from an amalgam of human and bacterial cells , as in an abscess .",
"The high concentration of S . saprophyticus and G . vaginalis DNA suggests an origin in genitourinary tissue .",
"To exclude an exogenous environmental source of the bacterial DNA and to further investigate the tissue of origin , we performed metagenomic profiling of the nodules , ulna and sediment from the gravesite .",
"The similarity in abundance of G . vaginalis in the nodules and modern Human Microbiome Panel ( HMP ) vaginal samples ( Figure 1—figure supplement 7 ) points to a likely origin for the nodules in the female reproductive tract .",
"The metagenomic profile of the nodules ( minus their associated blanks ) is distinct from the sediment , whereas the reads from the ulna group closely with the sediment sample ( Figure 1—figure supplement 8 ) .",
"These results indicate that the nodules were less prone to leaching of environmental DNA .",
"Our SEM-EDS and XRD findings suggest that bacterial and inflammatory cells were replicated in calcium phosphate minerals ( ‘ghost cells’ ) ; it is likely that this mineralization provided a remarkable degree of protection from DNA degradation and environmental leaching as seen in the bones .",
"The ectopic , inflammation-related calcification observed here is an apparently highly effective mechanism of bacterial fossilization that rivals mineralization occurring at much slower rates in the environment .",
"Sexing analyses of the remains ( and associated blanks ) using the method of Skoglund et al . ( 2013 ) assign the nodules as female -XX ( Supplementary file 1F ) .",
"More thorough analyses of the human DNA present in the nodules yielded an intriguing finding that helps pinpoint their tissue of origin .",
"Shotgun sequencing data from the nodules contained a small number of reads ( 884 ) conservatively mapping to the Y chromosome ( Supplementary file 1G ) .",
"The length distributions of the reads overlapped with those mapping to the X chromosome and autosomes , suggesting an endogenous , ancient origin ( Supplementary file 1G , Figure 1—figure supplement 5 ) ; we searched for but did not find similar bona fide ancient Y chromosome reads in sequence data from the ulna , the sediment , or any negative control ( Supplementary file 1G ) .",
"The presence of Y chromosome reads in the nodule but not in the ulna could be explained by a placental origin of the mineralized abscesses , indicating chorioamnionitis in the decedent while pregnant with a male fetus .",
"Chorioamnionitis – inflammation and infection of the placenta and fetal membranes – involves an inflammatory response on the part of the fetus as well as the mother ( Kraus et al . , 2004 ) , which would explain a female ( maternal ) origin of the nodular tissue with a minority male ( fetal ) component .",
"Chorioamnionitis is a mixed infection in which vaginal bacteria reach the upper reproductive tract , placenta , and fetal membranes; G . vaginalis is often identified in infected tissues ( Hillier et al . , 1988 ) .",
"S . saprophyticus can be found among the genitourinary and gastrointestinal flora of healthy women ( Ringertz and Torssander , 1986; Rupp et al . , 1992; Schneider and Riley , 1996 ) .",
"It is a common cause of urinary tract infection ( UTI ) in reproductive aged women ( Kahlmeter , 2003 ) and has also been known to cause puerperal infections ( Arianpour et al . , 2009 ) .",
"To gain further insights into the pathogens associated with this historical genitourinary infection , we pooled reads from all nodule DNA libraries , mapped and reconstructed the ancient S . saprophyticus and G . vaginalis genomes and analyzed them in conjunction with existing and newly acquired genomic data from extant and historical organisms ( Supplementary file 1H , I ) .",
"We used a combination of paired-end reference guided assembly and iterative assembly to reconstruct a nearly complete genome of S . saprophyticus Troy , including >100 Kb of novel sequence compared to reference strain ATCC 15305 .",
"The genome is 2 , 471 , 881 bp long , with an average unique coverage depth of 298 . 6x ( Figure 4—figure supplement 3 ) , which represents an unprecedented , detailed and complete picture of an ancient pathogen genome from shotgun sequencing data .",
"We also reconstructed a 22 . 6 Kb plasmid , pSST1 .",
"We were unable to reconstruct a contiguous G . vaginalis genome due to high variability in coverage and lack of synteny in both ancient and modern genomic data ( Ahmed et al . , 2012 ) .",
"Instead , we used a de novo approach to reconstruct G . vaginalis Troy gene content using reads that mapped to the annotated coding regions of all available G . vaginalis genomes .",
"This enabled us to assess the gene content of our ancient genome compared to the modern strains .",
"Using this method , we recovered 1187 unique contigs ( total length 1 , 435 , 761 bp ) corresponding to 972 annotated genes and an average unique coverage depth of 57 . 0x ( Figure 3—figure supplement 3 ) .",
"Our sample of 35 isolates of G . vaginalis was grouped into four previously defined clades ( Figure 3 , Figure 3—figure supplement 4 ) , which have been proposed to represent distinct species ( Ahmed et al . , 2012 ) .",
"G . vaginalis Troy sits within Clade 1 , among vaginal and endometrial isolates collected from both healthy women and patients with bacterial vaginosis .",
"Interestingly , the 800-year-old sample from Troy ( Turkey ) falls within contemporary genetic diversity ( Supplementary file 1I ) . 10 . 7554/eLife . 20983 . 016Figure 3 . Phylogenetic analysis of Gardnerella vaginalis . A maximum likelihood tree estimated using RAxML ( Stamatakis , 2014 ) ( Figure 3—source data",
"3 ) from a core alignment of G . vaginalis genomes ( Figure 3—source data 1 , Figure 3—source data 2 ) .",
"Branches are colored based on clades originally identified in Ahmed et al . ( Ahmed et al . , 2012 ) ( green = clade 1 , blue = clade 2 , red = clade 3 , purple = clade 4 ) .",
"Tips from modern G . vaginalis isolates are labeled based on sample source ( H = healthy vagina , BV = bacterial vaginosis , HE = healthy endometrium , E = endometrium , U = unknown ) .",
"Lighter colored branches have bootstrap values less than 100 .",
"Clinical phenotypes are interspersed throughout the phylogeny , and the Troy genome is not associated with a consistently pathogenic lineage of G . vaginalis .",
"Inset: Recombinant fragments in G . vaginalis core genome identified by BratNextGen ( Figure 3—source data",
"4 ) ( Marttinen , 2012 ) .",
"Each circle represents one genome .",
"Colored blocks represent recombinant fragments , and colors correspond to the clade designations in the phylogenetic tree .",
"Plot made with Circos ( Krzywinski et al . , 2009 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 01610 . 7554/eLife . 20983 . 017Figure 3—source data 1 . Concatenated alignment of core genes in G . vaginalis . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 01710 . 7554/eLife . 20983 . 018Figure 3—source data 2 . G .",
"vaginalis core genome alignment trimmed with Gblocks . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 01810 . 7554/eLife . 20983 . 019Figure 3—source data 3 . Maximum likelihood phylogenetic analysis of trimmed G . vaginalis alignment with RAxML . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 01910 . 7554/eLife . 20983 . 020Figure 3—source data 4 . Recombinant fragments detected with BratNextGen in trimmed G . vaginalis alignment . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 02010 . 7554/eLife . 20983 . 021Figure 3—figure supplement 1 . Ancient DNA damage assessment of G . vaginalis . Damage profiles of non-UDG treated ( ‘nonU’ ) reads from a pooled NOD1_nonU and NOD2_nonU data set ( total of 1 , 565 , 548 trimmed reads >24 bp ) mapping to G . vaginalis strain ATCC 14019 .",
"Paired end reads were mapped using bwa ( Li and Durbin , 2009 ) with default settings and duplicates were removed with samtools rmdup ( Li et al . , 2009 ) .",
"Damage profiles were generated using mapDamage2 ( Jonsson et al . , 2013 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 02110 . 7554/eLife . 20983 . 022Figure 3—figure supplement 2 . Fragment length distribution ( FLD ) for G . vaginalis ATCC 14019 . All nodule shotgun libraries ( Nod1_1h-UDG , Nod1_1h-nonU , Nod2-UDG , Nod2-nonU ) were pooled , reads were restricted to a minimum length of 35 bp and mapping quality of 30 and all duplicates removed both within and between libraries .",
"The fragment length distribution of the remaining 1 , 658 , 978 reads was visualized using mapDamage2 ( Jonsson et al . , 2013 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 02210 . 7554/eLife . 20983 . 023Figure 3—figure supplement 3 . Genome coverage plots for pooled nodule shotgun libraries . G .",
"vaginalis ( NC_014644 ) , average coverage 57 . 0X .",
"All reads were restricted to minimum length of 35 bp and minimum map quality 30 with all duplicates removed .",
"Figures depict coverage of the genome in 100 bp blocks across references .",
"Concentric grey circles demarcate increments of 50X coverage in both plots . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 02310 . 7554/eLife . 20983 . 024Figure 3—figure supplement 4 . Neighbor net network of core genomes . The network created in SplitsTree v 4 ( Huson and Bryant , 2006 ) of Gardnerella vaginalis .",
"The networks recapitulate the structure of maximum likelihood tree ( Figure 3 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 024 Consistent with prior reports ( Ahmed et al . , 2012 ) , we identified extensive impacts of lateral gene transfer ( LGT ) on G . vaginalis diversity ( Figure 3 ) .",
"Even in the core genome alignment , which contains just 44% of per-isolate gene content , we estimate that 20% of sites have been affected by recombination .",
"This high rate of recombination may help to explain the remarkable preservation of genetic diversity in G . vaginalis .",
"A recent study of Helicobacter pylori , which has similarly high rates of LGT , found that genetic diversity within the species has been preserved for more than five thousand years ( Maixner et al . , 2016 ) .",
"We discovered two distinct clades of S . saprophyticus ( Figure 4 , Figure 4—figure supplement 4 ) , one of which ( Clade P ) appears to be more strongly associated with pathogenicity and includes our ancient S . saprophyticus Troy .",
"Nineteen of twenty veterinary and human clinical isolates belong to Clade P , an association that was statistically significant ( Appendix ) .",
"A second clade ( Clade E ) is made up of food and environmental isolates of S . saprophyticus , as well as a human UTI isolate from Japan . 10 . 7554/eLife . 20983 . 025Figure 4 . Phylogenetic analysis of Staphylococcus saprophyticus .",
"( A ) Maximum likelihood tree estimated using RAxML ( Stamatakis , 2014 ) ( Figure 4—source data",
"3 ) from an alignment of S . saprophyticus genomes ( Figure 4—source data 1 , Figure 4—source data 2 ) .",
"Bootstrap values less than 100 are labeled .",
"Silhouettes indicate bacterial sample source .",
"Isolates without silhouettes are from human clinical samples isolated from urine .",
"Color corresponds to country of isolation as seen on the map .",
"Full sample descriptions are in Supplementary file 1H .",
"( B ) Source countries of bacterial samples .",
"( C ) Neighbor-net network of S . saprophyticus plasmid sequences ( Figure 4—source data",
"4 ) related to pSST1 created in SplitsTree4 ( Huson and Bryant , 2006 ) .",
"The boxed inset is an enlarged version of the portion of the network from Clade P isolates .",
"Some S . saprophyticus isolates do not encode pSST1-like plasmids , and therefore , they are not included in the network .",
"Starts and stops of recombinant regions of the alignment can be found in Figure 4—source data 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 02510 . 7554/eLife . 20983 . 026Figure 4—source data 1 . S .",
"saprophyticus whole genome alignment . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 02610 . 7554/eLife . 20983 . 027Figure 4—source data 2 . S .",
"saprophyticus whole genome alignment trimmed with trimal . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 02710 . 7554/eLife . 20983 . 028Figure 4—source data 3 . Maximum likelihood phylogenetic analysis of trimmed S . saprophyticus alignment with RAxML . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 02810 . 7554/eLife . 20983 . 029Figure 4—source data 4 . S .",
"saprophyticus plasmid alignment trimmed with trimal . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 02910 . 7554/eLife . 20983 . 030Figure 4—source data 5 . Recombinant fragments detected with BratNextGen in S . saprophyticus alignment . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 03010 . 7554/eLife . 20983 . 031Figure 4—figure supplement 1 . Ancient DNA damage assessment of S . saprophyticus . Damage profiles of non-UDG treated ( ‘nonU’ ) reads from a pooled NOD1_nonU and NOD2_nonU data set ( total of 1 , 565 , 548 trimmed reads >24 bp ) mapping to S . saprophyticus strain ATCC 15305 .",
"Paired end reads were mapped using bwa ( Li and Durbin , 2009 ) with default settings and duplicates were removed with samtools rmdup ( Li et al . , 2009 ) .",
"Damage profiles were generated using mapDamage2 ( Jonsson et al . , 2013 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 03110 . 7554/eLife . 20983 . 032Figure 4—figure supplement 2 . Fragment length distribution ( FLD ) for S . saprophyticus ATCC 15305 . All nodule shotgun libraries ( Nod1_1h-UDG , Nod1_1h-nonU , Nod2-UDG , Nod2-nonU ) were pooled , reads were restricted to a minimum length of 35 bp and mapping quality of 30 and all duplicates removed both within and between libraries .",
"The fragment length distribution of the remaining 3 , 904 , 552 reads was visualized using mapDamage2 ( Jonsson et al . , 2013 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 03210 . 7554/eLife . 20983 . 033Figure 4—figure supplement 3 . Genome coverage plots for pooled nodule shotgun libraries . S .",
"saprophyticus ( NC_007350 ) , average coverage 298 . 6X .",
"All reads were restricted to minimum length of 35 bp and minimum map quality 30 with all duplicates removed .",
"Figures depict coverage of the genome in 100 bp blocks across references .",
"Concentric grey circles demarcate increments of 50X coverage in both plots . DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 03310 . 7554/eLife . 20983 . 034Figure 4—figure supplement 4 . Neighbor net network of core genomes . Networks created in SplitsTree v 4 ( Huson and Bryant , 2006 ) of S . saprophyticus .",
"The networks recapitulate the structure of maximum likelihood trees ( Figure 4 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 03410 . 7554/eLife . 20983 . 035Figure 4—figure supplement 5 . Presence of mobile genetic elements , virulence genes , and antibiotic resistance in S . saprophyticus . Novobiocin resistance is conferred by a glycine at position 85 and lysine at position 140 ( Vickers et al . , 2007 ) , which is present in all S . saprophyticus genomes examined here .",
"SSP1924 and fosB confer streptomycin and fosfomycin resistance , respectively , and are encoded in vSs15305 in the ATCC 15305 reference genome ( Kuroda et al . , 2005 ) .",
"While none of the other isolates encode the entire genomic island , fosB and SSP1924 are found in isolates from both Clade P and Clade E . The canine isolate ( K ) harbors SCCmec containing mecA conferring methicillin resistance that has been identified in human clinical isolates of S . saprophyticus ( Higashide et al . , 2008 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 03510 . 7554/eLife . 20983 . 036Figure 4—figure supplement 6 . Recombinant regions detected by BratNextGen in S . saprophyticus . Each circle in the figure represents one isolate .",
"Regions with significant evidence for recombination are shown as black or colored blocks .",
"Black ticks mark intervals of 20 kb , and positions are in reference to ATCC15305 .",
"17 . 9% of the alignment is recombinant in at least one strain .",
"After removing fragments associated with known MGEs , 15 . 0% of sites are recombinant in the core genome .",
"Isolates are colored according to clade ( purple- Clade E , green- bovine , blue- Troy , black- Clade P ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 20983 . 036 Plasmids similar to S . saprophyticus Troy pSST1 were present in isolates from both clades .",
"The relationships among plasmid sequences from S . saprophyticus Troy and other members of Clade P were distinct from those of the core genome; we also found evidence of recombination among pSST1-like plasmids ( Figure 4C , Appendix ) .",
"A long branch separates Clade P pSST1 from those of Clade E ( Figure 4C ) , recapitulating their relationship on the core genome phylogeny .",
"This was also true of pSSP2 , the only other plasmid we identified in both Clades P and E ( but not S . saprophyticus Troy; Figure 4—figure supplement 5 ) .",
"These observations suggest plasmids are more readily exchanged within Clades P and E than between them .",
"This could indicate that Clades P and E are spatially segregated , that there are mechanistic barriers to plasmid exchange between clades , or that epistatic interactions reinforce clade separation of these mobile elements .",
"The human clinical isolates in Clades P and E are nested within the phylogeny with more divergent lineages associated with other animals .",
"This suggests that the most recent common ancestor ( MRCA ) of S . saprophyticus may not have been human-associated .",
"This is in stark contrast to the major pathogen in the genus , Staphylococcus aureus , where phylogenetic studies suggest that the MRCA of human and livestock-associated lineages had a human host ( Fitzgerald , 2012; Weinert et al . , 2012; Shepheard et al . , 2013 ) .",
"S . aureus is strongly associated with its niche on the human body and is transmitted primarily from person-to-person .",
"S . saprophyticus , by contrast , appears to be a generalist that colonizes a range of environments .",
"Several lines of evidence also indicate that humans acquire S . saprophyticus infection from the environment .",
"In northern climates , there is marked seasonal variation in the incidence of S . saprophyticus UTI ( Rupp et al . , 1992; Hovelius and Mårdh , 1984; Ringertz and Torssander , 1986; Hedman et al . , 1993; Widerström et al . , 2007 ) , whereas there is no evidence of seasonality in Mediterranean climates ( Schneider and Riley , 1996 ) .",
"S . saprophyticus can be identified in environmental samples , with a strong seasonal peak that occurs just before peak rates of S . saprophyticus UTI in northern climates ( Hedman et al . , 1993; Soge et al . , 2009 ) .",
"Molecular epidemiological surveys also suggest S . saprophyticus is primarily acquired from an environmental reservoir , rather than as a result of person-to-person transmission ( Widerström et al . , 2007; Widerström et al . , 2012 ) .",
"These observations suggest that the bacteria cycle between host-associated and environmental stages , with seasonal climatic effects on their abundance in the environment .",
"The length of the branch leading to S . saprophyticus Troy is similar to those leading to the other tips ( Figure 4A ) , suggesting there is little temporal signal in the phylogeny .",
"Calibrated phylogenetic analyses ( Appendix ) confirmed the absence of temporal signal , which precludes reliable estimation of the rate of substitution or divergence times for S . saprophyticus .",
"A mixed environmental , commensal and pathogenic niche may in part explain the absence of temporal structure in our sample of S . saprophyticus .",
"Selection pressures and generation times are likely to differ between free-living and host-associated stages , which can obscure temporal signals in genetic data ( Bromham , 2009 ) .",
"In addition to producing rate variability , periods of dormancy in the environment – e . g . during the winter in northern climates , as suggested by seasonal patterns in cultivability – would be predicted to lower the overall rate at which S . saprophyticus evolves ( Bromham , 2009; Weinert et al . , 2015 ) .",
"The 800 year interval between S . saprophyticus Troy and the other tips may simply be too short relative to the overall depth of the tree to allow reliable rate inference .",
"Notably , all human-associated isolates of S . saprophyticus in Clade P form a monophyletic group , to which the bovine mastitis strain falls basally; there are no modern human pathogenic representatives of the S . saprophyticus Troy lineage .",
"This may mean that the ecology of S . saprophyticus differed in the Byzantine world , with human infections arising from a different reservoir of bacteria than they do today .",
"S . saprophyticus is readily cultured from the environment around livestock ( Hedman et al . , 1993; Cherif-Antar et al . , 2016 ) , and Byzantine era peasants in Anatolia typically shared their households with cattle ( Lefort , 2007 ) .",
"This and other historical settings are likely to have facilitated spillover events and , perhaps , the circulation of bacteria that were adapted to both livestock and humans .",
"Based on the available data , it is not possible to determine whether the human clinical isolate nested among environmental and food-associated bacteria in Clade E represents a spillover or a second emergence into humans .",
"In either event , it appears that S . saprophyticus can transition to a human pathogenic niche with relative ease .",
"We did not identify any gene content uniquely shared ( or absent ) among the pathogenic strains in our sample , which suggests that pleiotropy underlies S . saprophyticus’ flexible association with diverse niches .",
"For many bacterial genera , genetic distances between free-living organisms and pathogens are larger than observed here , and pathogen emergence is a singular event characterized by genomic decay and loss of functions required outside the pathogenic niche ( Parkhill et al . , 2001; Larsson et al . , 2009; Reuter et al . , 2014 ) .",
"More studies and wider sampling are needed to fully characterize the niche of S . saprophyticus , but our observations reinforce the notion that the adaptive paths to bacterial virulence are more diverse than has previously been appreciated .",
"Complications of pregnancy and childbirth are major causes of morbidity and mortality worldwide and new threats to maternal health continue to emerge ( WHO et al . , 2014; Mlakar et al . , 2016 ) .",
"Our analyses of the remains of a woman who died in Late Byzantine Troy connect her to this broad historical and epidemiological phenomenon .",
"Her infection was associated with exuberant calcification of the placenta , which replicated maternal , fetal , and bacterial cells in calcium phosphate minerals and preserved a high resolution molecular portrait of their contents .",
"S . saprophyticus , an organism the decedent is likely to have acquired from her environment , and G . vaginalis , a member of the native human biota , are the dominant bacterial species of the infection .",
"S . saprophyticus Troy belongs to a lineage that appears to be uncommonly associated with human disease in the modern world , whereas G . vaginalis Troy nests among modern commensal and pathogenic strains on its phylogeny .",
"This highlights the complexity of virulence as a bacterial trait and a potential role of interactions among bacterial species in shaping pathologic outcomes of infection ."
],
[
"Ethics approval for the study of the remains of the individual excavated in 2005 from grave 14 ( Troy project , University of Tübingen , bone-sample x24 . 177 ) in quadrat x24 at Troy was obtained from Hamilton Health Sciences and McMaster University ( REB# 13–146 T ) .",
"Samples of extant bacteria were provided to investigators without patient identifiers or protected health information; the members of the study team did not have access to any identifiers or protected health information associated with the bacterial isolates .",
"Sediment from the Troy site was imported to and studied at McMaster University in accordance with Canadian Food Inspection Agency guidelines , under permit number P-2012–04220 .",
"Subsamples of both nodules and the ulna bone were sent to the Keck Carbon Cycle AMS Facility ( Earth System Science Department , UC Irvine , CA ) for radiocarbon dating ( Appendix , Supplementary file 1A ) .",
"In addition to 14C dating ultrafiltered collagen from the ulna , we also attempted to measure 14C in carbonate from nodule one ( with 10–30% leaching ) and three organic fractions from nodule two: raw nodule ( including carbonate ) , residue from demineralization with room temperature 1N HCl , and residue from demineralization plus gelatinization with 60°C 0 . 01N HCl .",
"A subsample of nodule two was subjected to mineralogical analysis using XRD at the Brockhouse Institute for Materials Research ( McMaster University ) using the Bruker D8 DISCOVER with DAVINCI . DESIGN diffractometer ( Figure 2—figure supplement 1 ) .",
"Sample flakes were piled on top of a single crystal silicon wafer , and aligned to the center of the diffractometer using the laser-video alignment system .",
"The detector to sample distance was calibrated with corundum to 20 cm .",
"Four frames were collected to obtain the 2θ range of 8–103° .",
"The frames were integrated into intensity plots using DIFFRAC . EVA V . 3 . 0 ( software package from Bruker AXS ) .",
"A pattern search/match was executed using the integrated ICDD PDF-2 2011 powder database .",
"Slight mismatch in the peak positions are likely due to variation of elemental stoichiometry in the identified phase .",
"A subsample of nodule two was viewed via SEM and subjected to elemental analysis using SEM-EDS at the MAX Diffraction Facility ( McMaster University ) .",
"Sample pieces were attached to an aluminum stub with double-sided carbon tape and sputter-coated with a thin layer of Au .",
"The sample was viewed in a Tescan Vega II LSU operating at 20kV .",
"Energy dispersive spectroscopy ( EDS ) was carried out with an IMAX detector ( Oxford Instruments ) and INCA software ( Supplementary file 1B ) .",
"We made multiple DNA extractions from subsamples of two nodules , an ulna , sediment taken from the site and relevant associated blanks/controls .",
"The details of these can all be found in Supplementary file 1J .",
"As the elemental analyses of the nodules suggested a highly mineralized constituent , we extracted them in a similar fashion to bone and they are labelled as such in Supplementary file 1J hyphenated ( ‘bone’ ) .",
"‘Bone’ ( nodules ) DNA extractions , consisted of demineralization ( DM ) , removal and freezing of DM supernatant , incubation of non-demineralized tissue with a custom digestion buffer ( DB ) , removal and freezing of DB supernatant , organic extraction of one/both supernatants , and concentration via filtration .",
"They were performed as follows .",
"For Set A ( Supplementary file 1J ) , multiple consecutive rounds of DM ( with 0 . 5M EDTA ) and digestion were performed on a shaker ( 1000 rpm ) , with the supernatant ( s ) from each round subjected to organic extraction and filtration .",
"DB consisted of 20 mM Tris pH 8 . 0 , 0 . 5% sarcosyl , 250 μg/ml Proteinase K , 5 mM CaCl2 , 50 mM DTT , 1% PVP , and 2 . 5 mM PTB .",
"The breakdown of DM/DB rounds is as follows: round 1 = 1 st 0 . 5 mL overnight ( ON ) DM at room temperature ( RT ) , second 0 . 5 mL DM at RT for 24 hr , and 7 hr digestion at 55°C , rounds 2–5 = ON 1 mL DM + 7 hr digestion at 55°C , and round 6 = ON 1 mL DM only .",
"For subsequent extraction , all round one supernatants were combined and all round 2–6 supernatants were treated separately: supernatants were subjected to organic extraction using half-volume of phenol-chloroform-isopropanol ( centrifuged at 16 , 000 x g for 5m ) , the aqueous phase of which was extracted with 750 μl chloroform ( centrifuged as before ) .",
"The final aqueous phase was filtered using Amicon Ultra 0 . 5 mL 10 kDa columns ( EMD Millipore Corp . , Billerica , MA , USA ) : columns primed with 450 μl 0 . 1xTE , followed by sample filtration , washed twice with 450 μl 0 . 1xTE , and eluted in 50 μl 0 . 1xTE .",
"For Set B , nodule two , 1 mL DM was performed ON rotating at RT and the supernatant was frozen .",
"Digestion was performed for 7 hr rotating at 55°C using 0 . 5 mL of DB ( same recipe as Set A ) and the supernatant was frozen .",
"These supernatants were combined and subjected to organic extraction and filtration as in Set A . Set D , which was the ulna , followed the same protocol as Set B , except they were subjected to an additional initial 30 min demineralization with 500 μl 0 . 5M EDTA that was not extracted , and the DB did not contain DTT , PVP , or PTB and was performed ON .",
"Set C , ‘sediment’ , DNA extractions were performed using the Mo Bio PowerSoil DNA Isolation Kit ( MO BIO Laboratories , Inc . , Carlsbad , CA ) following the manufacturer’s protocol , with a final elution of 100 μl in 0 . 1xTE .",
"For each set of samples associated blanks were treated in an identical fashion .",
"Please refer to Supplementary file 1J for details for each of these four sets of extractions .",
"Ancient DNA extracts were converted to double-indexed libraries for sequencing on the Illumina platform ( list in Supplementary file 1A ) .",
"Prior to library preparation , all ulna DNA extracts ( E5-1 , 2 , 3 , 4 , 7 , 8 , 9 , and",
"10 ) were pooled to homogenize library input into multiple UDG and non-UDG libraries , as were the two set D extraction blanks ( E5-6 and 12 ) .",
"Libraries were prepared as in ( Wagner et al . , 2014 ) using either regular ( ‘non-UDG’ ) or deaminated cytosine removal ( damage repair; ‘UDG’ ) protocols , and subsequently amplified using a double-indexing protocol ( Kircher et al . , 2012; Meyer and Kircher , 2010 ) .",
"Each library set included at least one blank no-template ( water ) control reaction .",
"Extract input volumes into library preparation were 10 μl ( L25-L13 ) , 20 μl ( L01-L20 ) , or 25 μl ( L28-L38 , 1 a-1j , and 1a-blk to 1j-blk ) .",
"In non-UDG libraries L25-L38 , the blunt-ending step was modified with an extended ( 3 hr ) T4 PNK incubation prior to adding the T4 polymerase , in the same manner as the UDG protocol .",
"Double-indexing amplification was performed as in ( Wagner et al . , 2014 ) for 10 cycles each , with 20 μl non-diluted library template input ( except L25 and L13 which were used at 0 . 1x dilution ) and included at least one no-template negative control reaction .",
"All reactions were purified to 15 ul EB with the MinElute PCR Purification Kit ( Qiagen , Hilden , Germany ) .",
"Two rounds of human mitochondrion targeted enrichment were performed on the non-UDG treated ulna specimen for comparison to the nodule shotgun reads .",
"Prior to enrichment , the 7 Ulna-non-D libraries ( L31-L38 ) were pooled to homogenize input , and 9 ul of this pool was used as input into four enrichment reactions ( Ulna-D E07-E10 ) alongside the extraction blank ( E11 ) and a negative control reaction ( enrichment blank ) .",
"The enrichment reactions were performed as for the human mtDNA enrichments in ( Wagner et al . , 2014 ) , using the same parameters and custom MYbaits baitset ( MYcroarray , Ann Arbor , MI ) designed from the rCRS sequence ( http://www . ncbi . nlm . nih . gov/nucleotide/113200490 NCBI GenBank accession no . J01415 . 2 ) , but with the following modifications according to updated manufacturer recommendations: post-hybridization bead-library binding was performed rotating at high temp ( 55°C ) , Wash Buffer 1 was eliminated , and the post-washed beads were suspended in 20 μl EB and used directly in the post-enrichment amplification .",
"For the adapter-specific blocking oligos , 2 μM of four P5/P7 adapter sequence custom blocking oligos ( corresponding to one strand of each molecule ) were used for enrichment round 1 , and the manufacturer-supplied Block #3 was used for enrichment round 2 .",
"Amplification after each enrichment round was performed as in ( Wagner et al . , 2014 ) , with additional re-amplifications as required due to low output molarities ( all amplification reactions were purified over MinElute columns to 15 μl EB ) .",
"Post-round 1 amplification used 15 μl bead mixture directly as input into each 40 μl reaction ( 15 cycles ) .",
"6 . 5 μl of this purified reaction was used as input into enrichment round 2 ( E17-E21 ) , and to increase molarity prior to sequencing , 6 . 5 μl was used as a template for subsequent re-amplification reactions ( 12 cycles ) .",
"Post-round 2 amplification used 10 μl bead mixture as input into two 40 μl reactions per enrichment ( 15 cycles ) , and the supernatants from each amplification ( two per sample ) were purified together .",
"Prior to sequencing , 14 μl of this purified reaction was used as the template for a subsequent re-amplification reaction ( 16 cycles ) .",
"All relevant ancient samples ( nodules , bone and sediment ) and their associated extraction blanks were sequenced .",
"Details on the final data set names , their associated samples/libraries/enrichments , raw reads passing filter , and pre-sequencing pooling schemes can be found in Supplementary file 1K .",
"Prior to sequencing , all additional indexed libraries ( shotgun and enrichment ) were quantified via a qPCR assay targeting indexed molecules and pooled according to desired sequencing ratio .",
"All pools except Nod1_all ( pool ‘K’ ) were size-selected using an electrophoresis gel size selection procedure ( retaining molecules ~125/150/150 to 500 bp in length ) in order to exclude as much no-insert adaptimer ( and other short adapter artifacts ) as possible .",
"Pool ‘F’ also contained 10 additional samples not considered in this paper ( pooled at a ratio of ‘1’ ) .",
"Pools were sequenced across three paired-end runs on the HiSeq 1500 , all alongside other , unrelated samples: Pool ‘K’ ( 80 bp final read length ) , Pool ‘F’ ( 85 bp final read length ) , and pools ‘G'-'J’ ( 90 bp final read length ) .",
"On the last run , the enrichment round 1 and 2 samples ( pools ‘H’ and ‘J’ ) were separated on two different lanes , since they have the same index sequences .",
"For the metagenomic analyses and the ancient pathogen genome reconstructions , all data sets were trimmed of library adapter using cutadapt ( Martin , 2011 ) with settings -e 0 . 16 , -O 1 , -a AGATCGGAAGAGC ( 70 ) and reads <24 bp were removed retaining read order .",
"To obtain metagenomic profiles from our shotgun data sets we used LMAT version 1 . 2 . 3 ( Ames et al . , 2013 ) to properly identify shotgun reads from the nodules ( Nod1_1h-UDG , Nod2-UDG ) , sediment ( Sediment-UDG ) , ulna ( Ulna-UDG ) , and all metagenomic data sets available from the Human Microbiome Project ( HMP , RRID:SCR_012956 ) database , housed at Lawrence Livermore National Laboratory ( June 2015 ) .",
"Reads that could be identified at the sequence level or consistently at the species/strain level from all blank extracts were removed from final files used in the PCA analysis .",
"The PCAs were performed using prcomp ( RRID:SCR_014676 ) in ( R Core Team , 2015 ) .",
"The taxa identifications from the HMP were combined according to the origin of the sample .",
"The number of samples combined into each category is indicated in the legend to Figure 1—figure supplement 8 .",
"A small number ( 1×10−7 ) was added to those entries with zero reads assigned and then natural logs of the numbers were taken .",
"The PCA was centered and scaled .",
"Reads were initially processed as described in the previous section .",
"The S . saprophyticus Troy draft genome was reconstructed using iterative assembly to span gaps between contigs that were created from assembly to the S . saprophyticus reference genome ( NC_007350 ) .",
"Trimmed reads from Nod1_all-UDG were paired-end assembled to the S . saprophyticus reference ( NC_007350 ) using BWA ( RRID:SCR_010910 ) with default settings ( Li and Durbin , 2009 ) , and duplicates were removed using samtools ( RRID:SCR_002105 ) rmdup ( Li et al . , 2009 ) .",
"The resulting assembly was imported into Geneious ( v . 6 . 1 . 6 , Biomatters , Ltd , RRID:SCR_010519 ) and a strict ( 50% ) consensus sequence was generated .",
"From this consensus , 65 large contigs ( 880 bp – 170 , 993 bp ) that corresponded to regions of average coverage ( and that did not span rRNA/tRNA regions ) were manually extracted , which represented the non-gap regions of the assembly .",
"As gap regions could represent indel regions ( e . g . , lateral gene transfer events ) , rearrangements , or divergence , these contigs were subjected to an iterative assembly process using a set of unmapped reads in order to attempt to span gaps and connect the contigs .",
"The primary set of reads used for iterative assembly was generated by trimming 100 bp from each end of the contigs , assembling all original reads using the above settings to this set of trimmed contigs , and removing these assembled reads from consideration .",
"A subset of the unmapped reads ( 20–100% as required , depending on assembly success ) along with the full set of contigs were then subject to iterative assembly using Geneious ( v . 6 . 1 . 6 ) , seeded with the first or last 50 bp of a contig ( settings: maximum gaps per read 10% , maximum gap size 2 , word length 20 , index word length 14 , ignore words repeated >8x , maximum mismatches per read 1% , maximum ambiguity 4 , map multiple best matches randomly ) .",
"All non-rRNA-adjacent gaps were successfully spanned , except for the region that was discovered to belong on the plasmid rather than the chromosome .",
"Ancient gene sequences were reconstructed de novo , via assembly of a pool of reads that mapped to annotated G . vaginalis .",
"First , CDS annotations were extracted from 34 modern G . vaginalis assemblies ( Supplementary file 1I; except for strains 41V and 101 ) and concatenated into one reference/genome ( 100 N’s between each CDS ) .",
"Trimmed paired end reads from Nod1_1h-UDG reads were mapped to the concatenated reference .",
"All paired and unpaired reads that mapped were extracted and subjected to de novo assembly using Velvet 1 . 2 . 1 ( Zerbino et al . , 2008 ) with settings kmer 23 , insert length 51 , expected coverage 75 , minimum contig length 24 , and coverage cutoff auto ( parameters for expected coverage were chosen based on previous observation of assembly to strain ATCC 14019 ) .",
"This generated 1207 contigs that were confirmed using blastn ( default settings , RRID:SCR_004870 ) to the nr database ( April 2014 ) to detect any non-G .",
"vaginalis sequences or chimeras generated from low level bacterial species also present in the nodules .",
"Twenty contigs were excluded due to the top hit being S . saprophyticus , leaving a final set of 1187 G . vaginalis contigs ( total length 1 , 435 , 761 bp ) .",
"The final set of genomic contigs was annotated using Prokka 1 . 7 ( Seemann , 2014 ) ( RRID:SCR_014732 ) producing 972 genes .",
"Paired-end assembly of Nod1_1h-UDG reads to the final set of contigs with bowtie2 ( Langmead and Salzberg , 2012 ) and removal of duplicates with samtools rmdup ( Li et al . , 2009 ) consists of 2 , 034 , 514 readpairs .",
"Total reads mapping to G . vaginalis from paired end-assemblies are listed in Supplementary file 1L .",
"To most conservatively assess the abundance , coverage , and authenticity of our ancient reads , we ran a subset of analyses using slightly more stringent criteria .",
"CASAVA ( RRID:SCR_001802 ) processed reads were trimmed and merged with leeHom ( Renaud et al . , 2014 ) ( RRID:SCR_002710 ) using its ancient DNA parameter ( --ancientdna ) .",
"We restricted reads from the UDG-treated shotgun nodule libraries ( Nod1_1h-UDG and Nod2-UDG ) to those of minimum 35 bp length and blasted all reads against the GenBank nucleotide database retaining only the top hit .",
"For all libraries , we additionally mapped to the S . saprophyticus ATCC 15305 ( NC_007350 ) and G . vaginalis ATCC 14019 ( NC_014644 ) with a customized version of the Burrows-Wheeler Aligner ( Li and Durbin , 2009 ) obtained from https://github . com/mpieva/network-aware-bwa ) with a maximum edit distance of 0 . 01 ( -n 0 . 01 ) , allowing for no more than two gaps ( -o 2 ) and with seeding effectively disabled ( -l 16500 ) , retaining only those mapped reads which were merged or properly paired [https://github . com/grenaud/libbam/retrieveMapped_single_and_ProperlyPair . cpp] .",
"Molecules that were less than 35 bp in length , had a mapping quality score of less than 30 , or were marked as duplicates based on both 5’ and 3’ coordinates were removed [https://bitbucket . org/ustenzel/biohazard . git] .",
"We then pooled all nodule reads DNA originating from the shotgun DNA libraries ( Nod1_1h-UDG , Nod1_1h-nonU , Nod2-UDG , Nod2-nonU ) and the S . saprophyticus and G . vaginalis mappings and further removed any duplicated molecules found between libraries ( Supplementary file 1L ) .",
"For both mapping assemblies , the average coverage at each reference position was calculated using the bedtools ( Quinlan et al . , 2010 ) ( RRID:SCR_006646 ) genomecov function and then averaged over 100 bp blocks and visualized with Circos ( Krzywinski , 2009 ) ( RRID:SCR_011798 , Figure 3—figure supplement 3 , Figure 4—figure supplement 3 ) .",
"Fragment length distributions for all pooled libraries and damage plots for the non-UDG treated libraries ( Nod1_1h-nonU and Nod2-nonU ) were calculated through mapDamage2 ( Jónsson et al . , 2013 ) ( Figure 3—figure supplement 1 , Figure 4—figure supplement 1 ) .",
"CASAVA processed reads ( see directly above ) , from enriched ulna extractions ( Ulna_Enr1-nonU , Ulna_Enr2-nonU ) , shotgun reads from the UDG treated nodule extraction ( Nod1_1h-UDG ) , and corresponding extraction blanks ( EblkD_Enr1-nonU , EblkD_Enr2-nonU and EblkA-UDG ) were processed as described above , but mapped to the rCRS mitochondrial genome ( NC_012920 ) ( Andrews et al . , 1999 ) .",
"Consensus sequences were called and contamination was estimated using Schmutzi , which implements iterative probability models to infer the endogenous bases given read length and deamination patterns ( Renaud et al . , 2015 ) .",
"Contamination was estimated at 12% and 13% , respectively , for the round 1 and round 2 enriched ulna libraries .",
"These estimates are consistent with estimates from other aDNA studies ( Posth et al . , 2016 ) .",
"Contamination could not be confidently assessed from the shotgun nodule library as it had been UDG treated , which obfuscates deamination patterns and thereby lessens the differentiation between endogenous and contaminant molecules .",
"mtDNA consensus sequences were uploaded to the HaploGrep webserver [http://haplogrep . uibk . ac . at/] and haplogroups were determined in reference to Phylotree Build 16 ( Kloss-Brandstätter et al . , 2011; van Oven and Kayser , 2009 ) ( RRID:SCR_012948 ) and found to be U3b and U3b3 .",
"Haplogroup U3b was assigned to the consensus sequence generated from the first round enrichment of the ulna because there was missing data for polymorphisms diagnostic to the haplogroup U3b3 ( Supplementary file 1E ) .",
"All three consensus sequences shared an additional five private polymorphisms not diagnostic to haplogroup U3b3 .",
"137 sequences assigned to haplogroup U3 were collected from all human complete mtDNA genomes in GenBank ( 18 June 2015 ) , and aligned along with the Troy consensus sequence generated from the nodule shotgun ( Nod1_1h-UDG ) library with MUSCLE v3 . 8 ( Edgar , 2004 ) ( RRID:SCR_011812 ) .",
"It was determined that the best model of nucleotide substitute for this group of 138 sequences was HKY+I+Γ using the program jModelTest2 ( Darriba et al . , 2012 ) and the built-in Akaike Information Criterion ( Akaike , 1974 ) .",
"A Bayesian Maximum Clade Credibility tree was calculated using BEAST v1 . 8 ( Drummond et al . , 2012 ) ( RRID:SCR_010228 ) and TreeAnnotator ( Drummond et al . , 2007 ) with the nucleotide data partitioned between coding and non-coding and a strict molecular clock with evolutionary rates of 1 . 708×10−8 and 9 . 88 3×10−8 nucleotide substitutions/site/year following Soares et al . ( Soares et al . , 2009 ) ( Figure 1—figure supplement 6 ) .",
"Damage patterns and fragment length distribution of ancient DNA mapped to mitochondrial genome can be found in Figure 1—figure supplements 3–4 .",
"Reads from four shotgun libraries ( three from the nodules , Nod1_1 hr_UDG , Nod2-UDG , Nod2-nonU; one from the ulna , Ulna-UDG ) were mapped and processed as described for the mitochondrial genome above .",
"Additionally , the reads originating from the four nodule libraries were pooled together for comparison ( ‘Nodule Pooled’ ) .",
"We mapped the libraries to a hard-masked hg38 reference genome downloaded from the UCSC genome browser [http://hgdownload . soe . ucsc . edu/goldenPath/hg38/bigZips/] and recorded the number of reads mapping to chrX , chrY , mitochondrial genome and all autosomes .",
"We first filtered for mapped merged or properly paired reads with a minimum length of 35 bp and a minimum mapping quality filter of 30 .",
"Percent coverage was calculated by tallying the number of positions covered by at least one read and dividing by the total genome length with masked regions subtracted .",
"We calculated the coverage depth by summing coverage of all positions and dividing the total by this same masked genome length ( Supplementary file 1G ) .",
"Fourteen new S . saprophyticus isolates ( North America: eight human , one bovine , and one canine; Australia: two human; Japan: two human ) were sequenced for this study to provide a broader comparative genomic data set ( Supplementary file 1H ) .",
"Reads for the new modern samples were processed with reference-guided assembly via a pipeline [https://github . com/tracysmith/RGAPepPipe] .",
"For reference guided assembly , read quality was assessed and trimmed with TrimGalore ! v 0 . 4 . 0 [www . bioinformatics . babraham . ac . uk/projects/trim_galore] , a wrapper script for FastQC [www . bioinformatics . babraham . ac . uk/projects/fastqc , RRID:SCR_005539] and cutadapt ( Martin , 2011 ) ( RRID:SCR_011841 ) .",
"Reads were mapped to the ATCC 15305 reference genome using BWA-MEM v 0 . 7 . 12 ( Li , 2013 ) ( RRID:SCR_010910 ) and bam files sorted using Samtools v 1 . 2 ( Li et al . , 2009 ) ( RRID:SCR_002105 ) .",
"Read group information was edited and duplicates removed using Picard v 1 . 138 [picard . sourceforge . net , RRID:SCR_006525] .",
"Reads were locally realigned using GATK v 2 . 8 . 1 ( DePristo et al . , 2011 ) ( RRID:SCR_001876 ) .",
"Variants were called using Pilon v 1 . 16 ( Walker et al . , 2014 ) ( RRID:SCR_014731 ) with a minimum read depth of 10 , minimum mapping quality of 40 and minimum base quality of 20 .",
"Whole genome alignment of the Troy strain and de novo assemblies to ATCC 15305 was performed using Mugsy 2 . 3 ( Angiuoli et al . , 2011 ) ( RRID:SCR_001414 ) .",
"The draft genome sequences of Japanese isolates were obtained by de novo assembly using CLC genome workbench v8 . 02 ( RRID:SCR_011853 ) .",
"For North American and Australian S . saprophyticus genomes , de novo assembly was performed using the iMetAMOS pipeline ( Koren et al . , 2014; Treangen et al . , 2013 ) ( RRID:SCR_011914 ) .",
"We compared assemblies from SPAdes ( Bankevich et al . , 2012 ) , MaSurCA ( Zimin et al . , 2013 ) , and Velvet ( Zerbino et al . , 2008 ) .",
"KmerGenie ( Chikhi and Medvedev , 2014 ) was used to select kmer sizes for assembly .",
"iMetAMOS uses FastQC [www . bioinformatics . babraham . ac . uk/projects/fastqc] to check read data quality .",
"Assemblies were evaluated using QUAST ( Gurevich et al . , 2013 ) , REAPR ( Hunt et al . , 2013 ) , LAP ( Ghodsi et al . , 2013 ) , ALE ( Clark et al . , 2013 ) , FreeBayes ( Garrison and Marth , 2012 ) , and CGAL ( Rahman et al . , 2013 ) .",
"Additionally , Kraken ( Wood et al . , 2014 ) was run to detect potential contamination in sequence data .",
"The SPAdes assembly was identified as best for isolates 13 , 16 , 19 , 41 , 42 , 43 , K , M , X , and 129 .",
"The MaSurCA assembly was identified as best for isolates 14 , 15 , 18 , and 31 .",
"Genomes were annotated using Prokka 1 . 7 ( Seemann , 2014 ) ( RRID:SCR_014732 ) .",
"OrthoMCL v2 . 0 . 9 ( Li et al . , 2003 ) ( RRID:SCR_007839 ) was used to find orthologous genes in these genomes .",
"Genomes were annotated with Prokka 1 . 7 ( Seemann , 2014 ) ( RRID:SCR_014732 ) .",
"OrthoMCL v2 . 0 . 9 ( RRID:SCR_007839 ) grouped genes into orthologous groups ( Li et al . , 2003 ) .",
"Genes were filtered to include only genes present in one copy in every genome .",
"Individual genes ( n = 537 ) were aligned with TranslatorX ( RRID:SCR_014733 ) and MAFFT v7 . 130b ( Abascal et al . , 2010; Katoh and Standley , 2014 ) ( RRID:SCR_011811 ) and concatenated [https://github . com/tatumdmortimer/core-genome-alignment] .",
"Maximum likelihood phylogenetic trees were inferred using RAxML 8 . 0 . 6 ( Stamatakis , 2014 ) ( RRID:SCR_006086 ) .",
"Bootstrap replicates ( number determined by autoMR convergence criteria ) were applied to the tree with the highest likelihood of twenty using the GTRGAMMA substitution model .",
"We used SplitsTree4 ( Huson and Bryant , 2006 ) ( RRID:SCR_014734 ) to create networks of pSST1 , the chromosome in S . saprophyticus , and the core genome of G . vaginalis .",
"Recombination in a whole genome alignment of S . saprophyticus isolates and a core genome alignment of modern G . vaginalis isolates was assessed using BratNextGen ( Marttinen , 2012 ) .",
"For both S . saprophyticus and G . vaginalis analyses , one hundred permutations were performed to calculate the significance ( p<0 . 05 ) of recombinant fragments ( plots created with Circos [Krzywinski , 2009] ) .",
"Recombination was also measured for the pSST1 plasmid alignment using Phi ( Bruen et al . , 2006 ) , Max χ2 ( Smith , 1992 ) , and NSS ( Jakobsen and Easteal , 1996 ) implemented in PhiPack; results of these tests were all significant with p-values of 5×10−15 , 0 , and 0 , respectively .",
"We used SNP-sites ( Page et al . , 2016 ) to convert the alignment of S . saprophyticus isolates to a multi-sample VCF .",
"SnpEff ( Cingolani , 2012 ) ( RRID:SCR_005191 ) was used to annotate variants in this VCF as synonymous , non-synonymous , or intergenic .",
"To determine whether there was sufficient temporal structure in the S . saprophyticus phylogeny to estimate evolutionary rates , we performed a regression of root-to-tip genetic distances against year of sampling using TempEst v 1 . 4 ( Rambaut et al . , 2016 ) .",
"We also attempted to estimate evolutionary rates using BEAST v1 . 8 ( Drummond et al . , 2012 ) ( RRID:SCR_010228 ) .",
"Results of both these analyses ( Appendix ) revealed a lack of temporal structure such that rate ( and date ) estimates are unreliable .",
"We used a Bayesian tree sampling method implemented in BaTS ( vBETA2 ) ( Parker et al . , 2008 ) to determine the significance of phylogenetic clustering and population structure in our S . saprophyticus data .",
"A distribution of phylogenies was generated using BEAST v1 . 8 ( Drummond et al . , 2012 ) ( RRID:SCR_010228 ) with GTR+Γ substitution model , a strict molecular clock , and a constant population size .",
"Markov chains were run in duplicate for 10 million generations each with sampling every 1000 generations , and the first 1 million generations were discarded as burn-in ."
]
] | [
"Pregnancy complications are poorly represented in the archeological record , despite their importance in contemporary and ancient societies .",
"While excavating a Byzantine cemetery in Troy , we discovered calcified abscesses among a woman’s remains .",
"Scanning electron microscopy of the tissue revealed ‘ghost cells’ , resulting from dystrophic calcification , which preserved ancient maternal , fetal and bacterial DNA of a severe infection , likely chorioamnionitis .",
"Gardnerella vaginalis and Staphylococcus saprophyticus dominated the abscesses .",
"Phylogenomic analyses of ancient , historical , and contemporary data showed that G . vaginalis Troy fell within contemporary genetic diversity , whereas S . saprophyticus Troy belongs to a lineage that does not appear to be commonly associated with human disease today .",
"We speculate that the ecology of S . saprophyticus infection may have differed in the ancient world as a result of close contacts between humans and domesticated animals .",
"These results highlight the complex and dynamic interactions with our microbial milieu that underlie severe maternal infections ."
] | [
"Why and how have some bacteria evolved to cause illness in humans ?",
"One way to study bacterial evolution is to search for ancient samples of bacteria and use DNA sequencing technology to investigate how modern bacteria have changed from their ancestors .",
"Understanding the evolution process may help researchers to understand how some bacteria become resistant to the antibiotics designed to kill them .",
"Complications that occur during pregnancy , including bacterial infections , have long been a major cause of death for women .",
"Now , Devault , Mortimer et al . have been able to sequence the DNA of bacteria found in tissue collected from a woman buried 800 years ago in a cemetery in Troy .",
"Some of the woman’s tissues had been well preserved because they had calcified ( probably as the result of infection ) , which preserved their structure in a mineralized layer .",
"Two mineralized “nodules” in the body appear to be the remains of abscesses .",
"Some of the human DNA in the nodules came from a male , suggesting that the woman was pregnant with a boy and that the abscesses formed in placental tissue .",
"Sequencing the DNA of the bacteria in the abscess allowed Devault , Mortimer et al . to diagnose the woman’s infection , which was caused by two types of bacteria .",
"One species , called Gardnerella vaginalis , is found in modern pregnancy-related infections .",
"The DNA of the ancient samples was similar to that of modern bacteria .",
"The other bacteria species was an ancient form of Staphylococcus saprophyticus , a type of bacteria that causes urinary tract infections .",
"However , the DNA of the ancient S . saprophyticus bacteria is quite different to that of the bacteria found in modern humans .",
"Instead , their DNA sequence appears more similar to forms of the bacteria that infect currently livestock .",
"As humans lived closely with their livestock at the time the woman lived , her infection may be due to a type of bacteria that passed easily between humans and animals .",
"Overall , the results suggest that the disease-causing properties of bacteria can arise from a wide range of sources .",
"In addition , Devault , Mortimer et al . have demonstrated that certain types of tissue found in archeological remains are a potential gold mine of information about the evolution of bacteria and other microbes found in the human body ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"cell biology"
] | Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments | elife-06585-v1 | [
[
"Eukaryotic cells assemble networks of actin filaments adjacent to the plasma membrane to carry out many fundamental processes , including: maintenance of cell morphology; amoeboid locomotion; and cell–cell interaction .",
"The architecture and function of these actin networks is specified in part by the properties of membrane-associated regulatory molecules ( Bear et al . , 2002; Lacayo et al . , 2007 ) .",
"For example , new filaments are created by nucleation factors recruited to the plasma membrane by Rho-family G-proteins , while fast-growing barbed ends of actin filaments near the membrane interact with regulatory factors that alter the rate and duration of filament elongation .",
"Some factors , including formin-family proteins ( Romero et al . , 2004; Kovar et al . , 2006 ) and Enabled/Vasodilator ( Ena/VASP stimulated phosphoprotein ) proteins ( Barzik et al . , 2005; Breitsprecher et al . , 2008; Hansen and Mullins , 2010; Breitsprecher et al . , 2011 ) , accelerate filament elongation , while others , such as capping protein , terminate filament elongation ( Dinubile et al . , 1995; Kuhn and Pollard , 2007 ) .",
"Local changes in the rates of filament elongation and capping can alter the architecture and function of the actin network and thus tip the balance between membrane protrusion and retraction ( Bear et al . , 2002; Applewhite et al . , 2007 ) .",
"One important group of actin regulatory proteins is the Ena/VASP family: weakly processive actin polymerases that accelerate filament elongation and slow filament capping ( Barzik et al . , 2005; Breitsprecher et al . , 2008; Hansen and Mullins , 2010; Breitsprecher et al . , 2011 ) .",
"Recruitment of Ena/VASP proteins to the plasma membrane often promotes outgrowth of thin , finger-like filopodia and sometimes promotes rapid advance of broad lamellipodial sheets ( Lanier et al . , 1999; Rottner et al . , 1999 ) .",
"Little is known about the molecular mechanisms that determine localization and activity of Ena/VASP proteins in vivo , but previous work suggests that a combination of membrane-associated binding partners and free barbed ends work together to recruit and maintain Ena/VASP proteins at the plasma membrane ( Krause et al . , 2004 ) .",
"Stable localization of Ena/VASP to the plasma membrane requires both the Enabled/VASP homology 1 and 2 domains ( EVH1 and EVH2 ) ( Loureiro et al . , 2002; Applewhite et al . , 2007 ) .",
"The EVH1 domain is a protein–protein interaction motif that binds proline-rich target sequences ( e . g . , FPPPP ) ( Prehoda et al . , 1999 ) , and removal of the EVH1 sequence abolishes localization of Ena/VASP to focal adhesions and the plasma membrane of fibroblasts ( Loureiro et al . , 2002 ) .",
"Interaction with actin filaments is mediated by the EVH2 sequence , which contains an actin binding domain ( BD ) and a tetramerization motif required for polymerase activity .",
"When expressed by itself in cells , the EVH2 domain concentrates in lamellipodial actin networks , but fails to achieve the same tight , membrane-proximal localization pattern as the intact protein ( Bear et al . , 2002 ) .",
"Cytochalasin D , which caps actin filament barbed ends , rapidly displaces Ena/VASP proteins from the cell periphery ( Krause et al . , 2004; Lacayo et al . , 2007; Neel et al . , 2009 ) arguing strongly that free filament ends play a functional role in Ena/VASP localization .",
"With the exception of actin , the most well understood Ena/VASP binding partners interact via the EVH1 motif—a globular domain that binds short , proline-rich sequences ( Prehoda et al . , 1999 ) .",
"Ena/VASP binding partners , which include the bacterial effector ActA ( Niebuhr et al . , 1997 ) ; the focal adhesion protein , Zyxin ( Drees et al . , 2000 ) ; an organizer of dorsal stress fibers , palladin ( Boukhelifa et al . , 2004; Gateva et al . , 2014 ) ; T-cell receptor signaling proteins , Fyb/SLAP ( Krause et al . , 2000 ) ; axon guidance factors , Robo/Sax-3 ( Bashaw et al . , 2000 ) ; and the mig-10/RIAM/Lamellipodin ( MRL ) proteins ( Krause et al . , 2004; Lafuente et al . , 2004 ) , all contain tandem repeats of a high-affinity EVH1 binding sequence: D/E FPPPPXD .",
"A second group of Ena/VASP binding partners , including the formin-family protein Diaphanous ( Grosse et al . , 2003; Schirenbeck et al . , 2006; Barzik et al . , 2014; Bilancia et al . , 2014 ) and the WAVE regulatory complex ( Law et al . , 2013; Chen et al . , 2014 ) , relies on a related , but lower affinity , EVH1-binding motif: LPPPPP .",
"The MRL proteins colocalize with Ena/VASP at the plasma membrane of many different cell types in many diverse organisms , both vertebrates and invertebrates ( Krause et al . , 2004 ) .",
"In the nematode , Caenorhabditis elegans , the mig-10 protein promotes neurite outgrowth and proper axon guidance ( Manser et al . , 1997; Chang et al . , 2006 ) .",
"Similarly , in mammals the protein Lamellipodin ( Lpd ) helps determine morphology of neurons and promotes growth of lamellipodial protrusions in a variety of non-neuronal cells ( Michael et al . , 2010; Pinheiro et al . , 2011; Yoshinaga et al . , 2012; Law et al . , 2013 ) .",
"Several MRL proteins , including Lpd , contain a tandem Ras-Association and Pleckstrin Homology ( RA-PH ) domain which , together with an adjacent coiled-coil region , causes the proteins to form weak homo-dimers in solution ( Chang et al . , 2012 ) .",
"The RA-PH domain is structurally homologous to growth factor receptor-binding proteins Grb7 , Grb10 , and Grb14 ( Depetris et al . , 2009 ) but , unlike the Grb proteins , no direct interaction between small GTPases ( e . g . , Ras or Rho family ) and MRL proteins has been reported .",
"In vitro , the MRL pleckstrin homology ( PH ) domain binds phosphatidylinositol lipids: PI ( 3 , 4 ) P2 and PI ( 3 , 4 , 5 ) P2 and in vivo the PH domain is thought to target these proteins to the plasma membrane in response to extracellular ligands such as PDGF ( Krause et al . , 2004; Chang et al . , 2012 ) .",
"Although the MRL proteins have been shown to help recruit Ena/VASP proteins to the plasma membrane , their effect on Ena/VASP activity has never been characterized .",
"In addition , results from several studies indicate that the MRL proteins likely have additional , Ena/VASP-independent roles in actin network regulation ( Krause et al . , 2004; Lyulcheva et al . , 2008; Michael et al . , 2010 ) .",
"To better understand the molecular mechanisms underlying cellular control of actin assembly we characterized the interactions between human VASP , Lpd , and filamentous actin in vitro and in live cells .",
"To our surprise , Lpd binds directly to filamentous actin , both in the absence and presence of VASP , an interaction mediated by a cloud of positively charged basic residues scattered through the C-terminal region of the protein ( the Lpd Actin Binding Region ( ABR ) , residues 850–1250 ) .",
"In cells , Lpd850−1250aa , lacking the RA-PH domain localizes to leading edge membranes and undergoes retrograde flow with the actin cytoskeleton .",
"Surprisingly , the interaction between Lpd850−1250aa and the actin cytoskeleton does not require interactions with Ena/VASP proteins or SH3 containing proteins , such as Abi1 and endophilin .",
"In vitro , Lpd increases processivity of barbed end-associated VASP tetramers by tethering them to actin filaments .",
"Together these results provide mechanistic insight into how growing actin filaments feed back on the polymerases and nucleation promoting factors that regulate their assembly ."
],
[
"Lpd takes its name from the dynamic lamellipodial actin networks to which it localizes in vivo , even in the absence of Ena/VASP proteins or free actin filament barbed ends ( Krause et al . , 2004 ) .",
"This tenacious localization to leading edge actin networks suggested that Lpd might interact directly with actin filaments , so we tested this idea by simultaneously visualizing monomeric GFP-Lpd850−1250aa and individual actin filaments in vitro by Total Internal Reflection Fluorescence ( TIRF ) microscopy ( Figure 1A , B ) .",
"In buffer containing 50 mM KCl , the GFP-labeled , monomeric Lpd construct uniformly decorated actin filaments , with a measured dissociation equilibrium constant ( Kd ) of 255 ± 2 nM ( Figure 1B , C ) .",
"Consistent with a weak electrostatic interaction , Lpd binding to filamentous actin grew progressively weaker in buffers containing higher concentrations of KCl .",
"In the presence of 100 mM KCl , interaction between monomeric Lpd and single actin filaments were undetectable by TIRF-M ( Figure 1B ) .",
"In contrast to these single-filament TIRF assays , we were able to detect interactions between Lpd850−1250aa and filamentous actin by co-sedimentation in the presence of physiological salt concentrations ( Figure 1—figure supplement 1A , B ) .",
"The stronger actin filament binding observed by co-sedimentation likely results from Lpd bundling actin filaments in solution . 10 . 7554/eLife . 06585 . 003Figure 1 . Lamellipodin ( Lpd ) binds directly to single actin filaments in vitro .",
"( A ) Cartoon representation of the human Lpd850−1250aa highlighting the Enabled/Vasodilator ( Ena/VASP ) binding sites ( grey ) , Abi1/endophilin SH3 binding sites ( red ) , and basic amino acid residues comprising the actin-binding region ( blue ) .",
"( B ) Representative Total Internal Reflection Fluorescence ( TIRF ) -M images showing 500 nM monomeric GFP-Lpd850−1250aa bound to single actin filaments in the presence of TIRF buffer containing 20 mM HEPES [pH 7 . 0] , 50–100 mM KCl , 1 mg/ml BSA , and 1 mM TCEP .",
"Scale bar , 10 µm .",
"( C ) Calculation of Kd for GFP-Lpd850−1250aa actin filament binding using the average fluorescence intensity of GFP-Lpd bound to phalloidin stabilized actin filaments ( 20% Cy5 labeled ) .",
"Error bars represent standard error of the mean .",
"( D ) Mutations in all 44 lysine/arginine residues to alanine ( called Lpd44A ) abolish F-actin binding of GFP-Lpd850−1250aa .",
"Actin filament binding was visualized in the presence 500 nM GFP-Lpd850−1250aa , wild-type and 44A mutant , in the presence of 50 mM KCl containing buffer as in ( B ) .",
"Scale bar , 10 µm .",
"( E ) Purification of GFP-Lpd850−1250aa ( monomer ) , GFP-LZ-Lpd850−1250aa ( dimer ) , and his10-GFP-Lpd850−1250aa ( monomers and trimer/tetramer ) by size exclusion chromatography .",
"( F ) Cartoon representation of purified Lpd oligomers in ( E ) .",
"( G ) Oligomerization of GFP-Lpd850−1250aa enhances actin filament binding .",
"Localization of 250 nM GFP-Lpd850−1250aa ( monomer ) , GFP-LZ-Lpd850−1250aa ( dimer ) , and his10-GFP-Lpd850−1250aa ( oligomers ) bound to phalloidin stabilized actin filaments ( 20% Cy5 labeled ) in the presence of TIRF buffer containing 100 mM KCl .",
"Scale bar , 5 µm .",
"( H ) Representative SDS-PAGE showing co-sedimentation of 1 µM filamentous actin in the presence of increasing concentrations of GFP-Lpd or GFP-LZ-Lpd ( 0–10 µM monomer concentration ) .",
"Asterisks ( * ) on SDS-PAGE gel marks partially translated or proteolyzed GFP-Lpd and GFP-LZ-Lpd that could not be removed during the purification .",
"( I ) Calculation of Kd for GFP-Lpd and GFP-LZ-Lpd actin binding domains ( BDs ) by actin co-sedimentation in the presence of 100 mM KCl buffer ( ± represents error of fit; error bars are S . D . of the mean from two independent experiments ) .",
"Note that a small fraction of Lpd is non-specifically absorbed to the walls of the centrifuge tubes in the actin co-sedimentation assay .",
"As a result , the stoichiometry of Lpd bound to actin is likely over-estimated by 5–10% ( see ‘Materials and methods’ ) .",
"( J ) Kymograph showing diffusion of his10-GFP-Lpd850−1250aa oligomers along the length of a phalloidin stabilized actin filaments .",
"Vertical scale bar , 5 s .",
"( K ) Membrane bound his10GFP-Lpd850−1250aa associates with single actin filaments .",
"Localization of 50 nm extruded small unilamellar vesicles ( SUVs ) DOPC/DOGS-NTA ( Ni+2 ) ( 99:1 molar ratio ) coated with his10GFP-Lpd850−1250aa bound to Alexa568 phalloidin stabilized actin filaments .",
"25 nM his10GFP-Lpd850−1250aa from ( E ) was combined with 50 nm SUVs ( 5 µM total lipid containing 1% or 50 nM DOGS-NTA lipid ) in buffer containing 20 mM HEPES [pH7] , 100 mM KCl , 100 µg/ml BSA , 1 mM TCEP .",
"Scale bar , 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 00310 . 7554/eLife . 06585 . 004Figure 1—figure supplement 1 . Interactions between filamentous actin , GFP-Lpd ( 850–1250aa ) , and GFP-LZ-Lpd ( 850–1250aa ) measured by cosedimentation at different buffer ionic strengths .",
"( A ) Monomeric GFP-Lpd850−1250aa and dimeric GFP-LZ-Lpd850−1250aa interact with filamentous actin in the presence of 50 , 100 , 150 mM KCl .",
"SDS-PAGE from three experiments showing the cosedimentation of 2 µM filamentous actin ( +4 µM dark phalloidin ) in the presence of 1 , 2 , and 4 µM GFP-Lpd850−1250aa or GFP-LZ-Lpd850−1250aa ( monomeric protein concentration ) .",
"Buffer composition is 20 mM HEPES [pH 7] , 50–150 mM KCl , 0 . 5 mM ATP , 0 . 5 mM MgCl2 , 0 . 5 mM EGTA .",
"( B ) Average molar ratio of GFP-Lpd or GFP-LZ-Lpd bound to filamentous actin in the presence of 50 , 100 , and 150 mM KCl .",
"Error bars represent S . D . of the mean ( n = 3 experiments ) .",
"( C ) SDS-PAGE as in Figure 1H , showing the results of co-sedimentation of 1 µM filamentous actin in the presence of increasing concentrations of GFP-Lpd or GFP-LZ-Lpd ( 0–10 µM monomer concentration ) .",
"( D ) GFP-Lpd and GFP-LZ-Lpd interact with both ‘native’ and phalloidin stabilized actin filaments .",
"Actin was polymerized at a concentration of 20 µM in the absence ( termed ‘native’ ) or presence of an equal molar concentration of dark phalloidin ( indicated by ‘+’ ) .",
"After 45 min , filamentous actin was combined with an equal volume of either 2 µM GFP-Lpd850−1250aa or GFP-LZ-Lpd850−1250aa and incubated for 1 hr before ultracentrifugation ( also see ‘Materials and methods’ ) .",
"The final buffer composition was 20 mM HEPES [pH 7 . 0] , 100 mM KCl , 1 mM TCEP , 0 . 5 mM ATP , 0 . 5 mM MgCl2 , and 0 . 5 mM EGTA .",
"( C , D )",
"Asterisks ( * ) on SDS-PAGE gel marks partially translated or proteolyzed GFP-Lpd and GFP-LZ-Lpd that could not be removed during the purification . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 004 Conserved acidic residues near the amino terminus of actin create an electronegative patch on the surface of actin filaments that interacts with positively charged , basic residues in many actin binding proteins ( Fujii et al . , 2010 ) .",
"The carboxy-terminal region of human Lpd ( residues 850–1250 ) is highly basic , with an isoelectric point ( pI ) of 9 . 97 ( Figure 2A , B ) .",
"The distribution of basic residues in this region is evolutionarily conserved among Lpd homologs , but is not conserved in other Ena/VASP-binding proteins , such as Rap1-GTP-interacting adaptor molecule ( RIAM ) , zyxin , or ActA ( Figure 2B ) .",
"Mutating all 44 basic amino acid residues in the C-terminal ABR of Lpd to alanines ( to create Lpd ( 44A ) 850−1250aa ) abolished filament binding ( Figure 1D ) , but had no effect on the ability of this construct to bind to Ena/VASP proteins ( unpublished results ) .",
"Because the C-terminal region of Lpd appears to be natively unfolded , the loss of actin binding likely reflects simply a loss of electrostatic interactions rather than a change in protein structure .",
"Furthermore , bacterially expressed and purified GFP-Lpd ( 44A ) 850−1250aa showed no signs of being structurally or functionally compromised , as compared wild-type GFP-Lpd850−1250aa . 10 . 7554/eLife . 06585 . 005Figure 2 . Conservation of Lpd ( 850–1250aa ) amino acid sequence and isoelectric point ( pI ) .",
"( A ) Protein sequence alignment of human Lpd and homologs C-termini .",
"Basic amino acid residues ( arginine and lysine ) are highlighted in blue .",
"Gray boxes mark the location of the canonical Ena/VASP homology 1 ( EVH1 ) BDs ( i . e . , FPPPP or LPPPP ) , while red boxes highlight the predicted Abi1/endophilin SH3 domain binding sites ( i . e . , PxxPxR ) .",
"Secondary structure prediction algorithms suggest that the Lpd ( 850–1250aa ) lacks secondary structure ( data not shown ) .",
"( B ) Comparison of Lpd , Pico , mig-10 , RIAM , ActA , and Zyxin pIs across the canonical Ena/VASP BD containing one or more FPPPP motifs .",
"Domain boundaries for this region are termed , Lpd C-terminus ( Lpd CT ) .",
"The number of arginine and lysine residues were calculated across the region specified ‘Lpd C-terminus ( Lpd CT ) ’ .",
"The number of Ena/VASP and SH3 domain binding sites were counted across the domain boundaries defined , Lpd CT , and are shown in columns five and six , respectively .",
"The pIs for Lpd CT were calculated using EXPASY ( Wilkins et al . , 1999 ) .",
"Abbreviations are as follows: full-length ( FL ) ; binding-domain ( BD ) ; Lamellipodin ( Lpd ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 005 Structural studies indicate that full-length ( FL ) Lpd forms homo-dimers in solution , via both a coiled-coil motif and interactions in the tandem RA-PH domains ( Depetris et al . , 2009; Chang et al . , 2012 ) .",
"We , therefore , tested the effect of Lpd oligomerization on actin filament binding .",
"We created stable dimers of the C-terminal region of Lpd by fusing Lpd850−1250aa to a dimer-forming , leucine-zipper motif ( GFP-LZ-Lpd , Figure 1E , F ) .",
"Unlike the monomeric Lpd constructs , GFP-LZ-Lpd dimers bound to single actin filaments in both low- and high-salt buffers ( 100 mM KCl; Figure 1G ) .",
"Similarly , dimeric Lpd bound more strongly to filamentous actin , both ‘native’ and phalloidin stabilized , as compared to monomeric Lpd in a co-sedimentation assay ( Figure 1H , I and Figure 1—figure supplement 1 ) .",
"Based on the ratio of Lpd:Actin sedimented under saturating conditions , we estimate a stoichiometry of one GFP-Lpd850−1250aa to at least two actin protomers .",
"We also purified spontaneously formed trimeric and tetrameric oligomers of his10GFP-Lpd850−1250aa by size-exclusion chromatography and found that their affinity for filamentous actin was even further enhanced ( Figure 1E , F ) .",
"In our TIRF assay we observed oligomeric his10GFP-Lpd850−1250aa particles bind and diffuse linearly along the sides of actin filaments ( Figure 1J ) , an activity we previously observed for tetrameric Cy3-VASP constructs ( Hansen and Mullins , 2010 ) .",
"Finally , we observed enhanced filament binding when we coupled freshly gel-filtered , monomeric his10GFP-Lpd850−1250aa to small unilamellar vesicles ( SUVs ) containing DOGS-NiNTA lipids ( Figure 1K ) .",
"These Lpd-coated SUVs bound tightly to individual actin filaments in vitro , demonstrating that oligomerization and/or clustering of Lpd850−1250aa promotes stable actin filament binding , even in near physiological salt concentrations .",
"We next tested whether Lpd can interact directly with artificial lamellipodial actin networks reconstituted in vitro from purified components ( Loisel et al . , 1999; Akin and Mullins , 2008 ) .",
"We used the Arp2/3 complex , together with capping protein , to assemble dendritic actin networks on lipid-coated bead ( LCBs ) containing Ni-conjugated lipids bound to his10Cherry-SCAR .",
"In these assays we also included either membrane tethered his10GFP or his10GFP-Lpd850−1250aa ( Figure 3A , Figure 3—figure supplement 1 ) .",
"Since his10GFP-Lpd850−1250aa is tethered to the membrane via Ni-conjugated lipids , our reconstitution lacks the recruitment and dissociation dynamics that may be mediated by interactions between the Lpd PH domain and phosphatidylinositol lipids ( i . e . , PI ( 3 , 4 ) P2 ) .",
"Nonetheless , we used this assay to test whether membrane tethered Lpd modulates actin network assembly in an autonomous manner .",
"By measuring the length and density of actin comet tails at various times ( Figure 3B ) , we found that membrane-tethered Lpd significantly slowed the rate of actin network assembly ( 1 . 5 ± 0 . 25 µm/min ) compared to membrane-tethered GFP alone ( 7 . 0 ± 0 . 48 µm/min; Figure 3B , C ) . 10 . 7554/eLife . 06585 . 006Figure 3 . Membrane-tethered Lpd slows dendritic actin network assembly in vitro .",
"( A ) Cartoon illustrating his10Cherry-SCARAPWCA , his10GFP-Lpd850−1250aa , and his10GFP tethered to a lipid coated beads ( LCBs ) containing DOGS-NTA ( Ni ) lipid ( blue head groups ) .",
"Actin network assembly on the bead surface is initiated by adding monomeric actin , profilin 1 , Arp2/3 , capping protein , and buffer containing KCl .",
"( B , C )",
"Membrane tethered his10-GFP-Lpd850−1250aa slows actin network assembly on LCBs .",
"( B ) Representative actin comet tails assembled in the presence of 7 . 5 µM actin ( 5% Alexa488-Actin ) , 3 µM hProfilin 1 , 100 nM Arp2/3 , 100 nM capping protein , and buffer containing 150 mM NaCl .",
"LCBs ( 2 . 3 µm , 4% DOGS-NTA ( Ni ) : 96% DOPC ) were charged with 75 nM his10-Cherry-SCARAPWCA , plus 25 nM his10-GFP-Lpd850−1250aa or 25 nM his10-GFP ( i . e . , 75% his10-Cherry , 25% his10-GFP ) .",
"Actin network assembly and disassembly was stopped at the indicated time points by combining the bead motility assay , 1:1 , with 37 . 5 µM Latrunculin B-phalloidin mixture .",
"Scale bar , 5 µm .",
"( C ) Representative actin comet tails assembled as in ( B ) for 5 min before transitioning from actin motility mix with 7 . 5 µM actin ( 5% Alexa488 labeled , GREEN ) into an identical mix , but containing 7 . 5 µM actin ( 5% Cy3-Actin , RED ) .",
"The length of Cy3-actin incorporated into the comet tail was measured to determine the growth velocity of multiple tails ( n ≥ 50 tails ) .",
"Error ( ± ) represents the standard deviation of the mean ( p-value = 3 × 10−29; two-tailed t-test for data sets with equal variance ) .",
"Scale bar , 10 µm .",
"( D ) Homogenous distribution of his10-Cherry-SCARAPWCA and his10GFP-Lpd850−1250aa before initiating actin network assembly .",
"Scale bar , 5 µm .",
"( E , F )",
"Spatial distribution of his10Cherry-SCARAPWCA , his10GFP-Lpd850−1250aa , and his10-GFP during steady state actin tail growth and recycling ( 30 min time point ) .",
"Actin networks were assembled in the presence of 7 . 5 µM actin ( 5% Alexa488 ) , 3 µM hProfilin 1 , 100 nM Arp2/3 , 100 nM Mm capping protein , and 3 µM hCofilin .",
"( E ) his10Cherry-SCARAPWCA and his10GFP-Lpd850−1250aa concentrate on the barbed end dense side of the actin comet tail .",
"( F ) his10-Cherry-SCARAPWCA concentrates on the barbed end dense side of the actin comet tail , while his10-GFP is excluded from the barbed end attachment zone .",
"Line scans across LCBs are shown to the right .",
"Scale bar , 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 00610 . 7554/eLife . 06585 . 007Figure 3—figure supplement 1 . Actin based motility on lipid coated glass beads .",
"( A ) Montage of actin comet tails frozen at different times point with 37 . 5 µM Latrunculin B-phalloidin containing buffer .",
"Actin networks were assembled in the presence of 7 . 5 µM actin ( 5% Alexa488 ) , 50 nM Arp2/3 , 100 nM capping protein , 6 µM hPro1 , 3 µM cofilin .",
"Scale bar , 10 µm .",
"( B ) Image of actin comet tails at steady-state in the presence of cofilin dependent network recycling described in ( A ) .",
"Scale bar , 10 µm .",
"( C ) Images of asymmetric his10-Cherry-SCARAPWCA localization in the presence of actin comet tail .",
"Scale bar , 5 µm .",
"( D ) Line scan across lipid coated bead surface in ( C ) showing fluorescent intensity of asymmetric his10-Cherry-SCARAPWCA . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 007 In addition to slowing network growth , membrane-tethered Lpd also polarized in an actin-dependent manner on the surface of LCBs .",
"Similar to the polarization of N-WASP on lipid vesicles in Xenopus egg extract ( Co et al . , 2007 ) , we found that Scar/WAVE and Lpd constructs become enriched in regions of the membrane most closely associated with the growing actin network .",
"In the absence of actin , both his10Cherry-SCAR and his10GFP-Lpd850−1250aa were distributed uniformly around the LCBs ( Figure 3D ) .",
"Within 10 min of initiating actin network assembly on LCBs , however , both membrane-tethered Scar/WAVE and Lpd constructs ( his10GFP-Lpd850−1250aa ) became concentrated in the region adjacent to the actin network , a region we refer to as the ‘barbed end attachment zone’ ( Figure 3E , Figure 3—figure supplement 1B–D ) .",
"In contrast , membrane-tethered his10GFP was displaced away from the barbed end attachment zone and became concentrated on the opposite side of the microsphere ( Figure 3F ) .",
"Having found that Lpd binds actin filaments in vitro , we next worked to determine whether actin binding by Lpd plays a significant role in its cellular localization and function .",
"To address this question we visualized the localization of fluorescently tagged , FL Lpd ( GFP-Lpd1−1250aa ) in Xenopus Tissue Culture ( XTC ) cells , spread on poly-L-lysine ( PLL ) -coated coverslips ( Figure 4A ) .",
"We used XTC cells because they spread well and produce very thin peripheral actin networks , well suited for fluorescence microscopy .",
"Ectopic gene expression from a truncated cytomegalovirus ( CMV ) promoter produces very low levels of protein expression in XTC cells , ideal for single-molecule fluorescence microscopy ( Watanabe and Mitchison , 2002 ) .",
"TIRF microscopy imaging of single GFP-Lpd1−1250aa molecules revealed that FL Lpd localizes predominantly to the leading edge of ruffling membranes and cycles on and off the plasma membrane on a subsecond time scale ( Figure 4B , Figure 4—figure supplement 1 , Video 1 ) .",
"Leading edge membrane localization of GFP-Lpd1−1250aa required dynamic actin assembly and disassembly , because acute treatment of cells with a chemical inhibitor cocktail that freezes actin dynamics ( JLY drug cocktail: Jasplakinolide , Latrunculin B , and Y27632 Rock kinase inhibitor [Peng et al . , 2011] ) caused rapid loss of membrane-localized Lpd ( Figure 4C ) . 10 . 7554/eLife . 06585 . 008Figure 4 . Lpd ( 850–1250aa ) localizes to the leading edge membranes and undergoes retrograde flow with the actin cytoskeleton .",
"( A , B )",
"Plasma membrane localization of GFP-Lpd1−1250aa visualized with TIRF microscopy in Xenopus Tissue Culture ( XTC ) cells spread on poly-L-lysine ( PLL ) .",
"GFP-Lpd1−1250aa was ectopically expressed from a ( A ) cytomegalovirus ( CMV ) or ( B ) DeltaCMV promoter .",
"( B ) Maximum intensity projection of a XTC cell expressing a single molecule concentration of GFP-Lpd1−1250aa .",
"Scale bar , 10 µm .",
"Leading edge membrane marked by dashed box is enlarged below .",
"Scale bar , 5 µm .",
"( C ) Leading edge membrane localization of GFP-Lpd1−1250aa in XTC cell viewed by TIRF-M following the addition of 8 µM Jasplakinolide , 10 µM Latrunculin B , and 10 µM Y27632 ( Rock kinase inhibitor ) ( Peng et al . , 2011 ) .",
"Scale bar , 5 µm .",
"( D ) Representative image of XTC cell coexpressing GFP-Lpd850−1250aa and mCherry-Actin .",
"Scale bar , 5 µm .",
"( E ) Kymographs show retrograde flow of GFP-Lpd850−1250aa and mCherry-Actin .",
"Scale bar , 5 µm .",
"( F ) Histogram showing distribution of GFP-Lpd850−1250aa and mCherry-Actin speckle velocities .",
"Mean speckle velocities of 71 . 9 ± 17 . 5 nm/s ( n = 246 speckles ) and 73 . 5 ± 14 nm/s ( n = 373 speckles ) were calculated for GFP-Lpd850−1250aa and mCherry-Actin , respectively .",
"( G ) GFP-Lpd850−1250aa localizes to the leading edge membrane of polarized mouse B16F1 cell migrating on laminin coated glass substrate .",
"Scale bar , 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 00810 . 7554/eLife . 06585 . 009Figure 4—figure supplement 1 . Localization of GFP-Lpd ( 850–1250aa ) and GFP-LZ-Lpd ( 850–1250aa ) .",
"( A , C )",
"Representative images showing the plasma membrane localization of ( A ) GFP-Lpd1−1250aa , ( B ) GFP-Lpd850−1250aa , and ( C ) dimeric GFP-LZ-Lpd850−1250aa visualized with TIRF microscopy in XTC cells spread on PLL .",
"Image in ( A ) , marked with asterisk ( * ) , is a representative cell imaged using with wide-field epifluorescence .",
"Note the cytoplasmic localization of GFP-Lpd1−1250aa is not visible by TIRF microscopy .",
"Scale bar , 20 µm .",
"( D ) Representative images of GFP-Lpd850−1250aa localization in polarized B16F1 cell migrating on laminin coated glass and imaged with wide-field epifluorescence .",
"Scale bar , 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 00910 . 7554/eLife . 06585 . 010Figure 4—figure supplement 2 . Retrograde flow of GFP-Lpd ( 850–1250aa ) and GFP-LZ-Lpd ( 850–1250aa ) with the actin cytoskeleton .",
"( A , B )",
"Representative kymographs showing retrograde flow of ( A ) monomeric GFP-Lpd850−1250aa and ( B ) mCherry-Actin in XTC cells .",
"Images were acquired every 5 s .",
"Scale bars , 5 µm and 5 min . ( C , D ) Representative kymographs showing retrograde flow of ( C ) monomeric GFP-Lpd850−1250aa and ( D ) dimeric GFP-LZ-Lpd850−1250aa .",
"Compared to ( A , B ) , images were acquired every 2 s .",
"Scale bars , 5 µm and 1 min . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 01010 . 7554/eLife . 06585 . 011Video 1 . Localization of full length GFP-Lpd ( 1 −1250aa ) expressed at single molecule concentrations from the DeltaCMV promoter in Xenopus Tissue Culture ( XTC ) cells . Images were acquired with temporal resolution of 100 ms using Total Internal Reflection Fluorescence ( TIRF ) -M .",
"Video plays at 15 frames per second .",
"Scale bar , 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 011 To determine which Lpd binding partners—actin , Ena/VASP , Abi1/endophilin , inositol phospholipids , or Ras/Rho-family G-proteins—are required for leading edge localization , we compared the localization of various Lpd truncation mutants in XTC cells .",
"We discovered that not only does GFP-Lpd850−1250aa , which lacks the tandem RA-PH domains , localize to leading edge membranes in XTC cells ( Figure 4C ) , this construct also undergoes retrograde flow with the lamellipodial actin network .",
"Analysis of speckle velocities in XTC cells co-expressing GFP-Lpd850−1250aa and mCherry-Actin revealed that the two proteins move with the same mean velocity: 71 . 9 ± 17 . 5 nm/s for Lpd850−1250aa vs 73 . 5 ± 14 nm/s for actin ( Figure 4F , Video 2 ) .",
"In contrast to mCherry-Actin , GFP-Lpd850−1250aa speckles were observed less frequently , appeared more diffuse , and had shorter lifetimes ( Figure 4—figure supplement 2 ) .",
"We suspect that the larger GFP-Lpd850−1250aa fluorescent particles are associated with fast endophilin-mediated endocytosis ( FEME , Vehlow et al . , 2013; Boucrot et al . , 2015 ) .",
"Consistent with constitutively dimeric Lpd having a higher affinity for actin , GFP-LZ-Lpd850−1250aa displayed slightly more robust leading edge membrane localization and the intensity of presumptive endocytic particles were brighter , as compared to those observed in cells expressing GFP-Lpd850−1250aa ( Figure 4—figure supplements 1 , 2 ) . 10 . 7554/eLife . 06585 . 012Video 2 . Retrograde flow of GFP-Lpd850−1250aa and mCherry-Actin in XTC cells spread on poly-L-lysine ( PLL ) coated coverslips . Cell was imaged with a temporal resolution of 5 s using TIRF-M at 23°C .",
"GFP-Lpd850−1250aa and mCherry-Actin were ectopically expressed from either a CMV or DeltaCMV promoter , respectively .",
"Video plays at 10 frames per second .",
"Scale bar , 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 012 We also transiently expressed GFP-Lpd850−1250aa in polarized B16F1 mouse melanoma cells migrating on laminin coated glass , and observed the same leading edge membrane localization as in XTC cells ( Figure 4G , Figure 4—figure supplement 1 , Video 3 ) .",
"Importantly , this localization does not require acute receptor stimulation following serum starvation , implying that membrane recruitment of GFP-Lpd850−1250aa represents the default Lpd localization in these migrating cells . 10 . 7554/eLife . 06585 . 013Video 3 . Leading edge membrane localization of GFP-Lpd850−1250aa in polarized B16F1 mouse melanoma cell migrating on laminin coated glass . B16F1 cell was imaged with a temporal resolution of 10 s using wide-field epi fluorescence at 37°C .",
"GFP-Lpd850−1250aa was ectopically expressed from a CMV promoter .",
"Video plays at 10 frames per second .",
"Scale bar , 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 013 We next asked whether GFP-Lpd850−1250aa requires Ena/VASP binding for leading edge membrane localization in XTC cells .",
"The C-terminal region of Lpd contains six FPPPP peptide sequences , each of which can potentially bind one VASP EVH1 domain .",
"By analytical ultracentrifugation , we found that GFP-Lpd850−1250aa simultaneously binds up to four VASP EVH1 domains in solution ( Figure 5—figure supplement 1 ) .",
"We mutated all six FPPPP motifs in Lpd850−1250aa to AAPPP ( Figure 5—figure supplement",
"2 ) and expressed this mutant protein as either a his10-GFP fusion in Escherichia coli ( his10-GFP-Lpd ( AAPPPx6 ) 850−1250aa ) or a GFP fusion in XTC cells ( GFP-Lpd ( AAPPPx6 ) 850−1250aa ) .",
"Although the his10-GFP-Lpd ( AAPPPx6 ) 850−1250aa mutant failed to recruit Ena/VASP proteins to LCBs in vitro ( Figure 5A ) , the localization of the GFP-Lpd ( AAPPPx6 ) 850−1250aa mutant in XTC cells was nearly indistinguishable from GFP-Lpd850−1250aa ( Figure 5B ) .",
"The only noticeable difference was a slight decrease in the amount of protein associated with nascent focal adhesions ( Figure 5B ) . 10 . 7554/eLife . 06585 . 014Figure 5 . Interactions with Ena/VASP or Abi1/endophilin are not required for Lpd ( 850–1250aa ) membrane localization .",
"( A ) Lpd FPPPP peptide sequences are required to recruit Ena/VASP proteins to the lipid coated beads ( LCBs ) .",
"Glass microspheres were coated with SUVs containing DOPC/DOGS Ni-NTA lipids ( 96:4 molar ratio ) .",
"LCBs were then incubated with 100 nM his10-GFP-Lpd850−1250aa , ( wild-type and AAPPPx6 mutants ) for 15 min , before being mixed with 500 nM Cy3-VASP or Cy3-EVL .",
"Lpd mutant , ( AAPPP ) x6 , cannot recruit purified Cy3-VASP or Cy3-EVL to LCBs .",
"( B ) Basic residues in flanking the Ena/VASP and Abi1/endophilin binding sites are required for leading edge localization of GFP-Lpd850−1250aa in XTC cells .",
"Representative images of wild-type and mutant GFP-Lpd850−1250aa protein in XTC cells .",
"Localization of full length GFP-Lpd1−1250aa ( top panel ) is shown for comparison .",
"Scale bar , 5 µm .",
"Refer to Figure 5—figure supplement 2 for amino acid sequences of each Lpd850−1250aa mutant .",
"( C ) Cartoon schematic showing palmitoylated Lyn-GFP and Lyn-GFP-Lpd ( WT and FPPPP → AAPPPx6 mutant ) anchored in the plasma membrane .",
"The crystal structure of GFP was derived from Yang et al . ( 1996 ) ( 1GFL . pdb ) .",
"( D ) Constitutively membrane tethered Lyn-GFP-Lpd850−1250aa localizes to the leading edge .",
"Leading edge localization of Lyn-GFP-Lpd850−1250aa does not require interactions with Ena/VASP proteins or Abi1/endophilin .",
"Localization of Lyn-GFP and GFP-PLCδ ( pleckstrin homology [PH] domain that binds to PI ( 4 , 5 ) P2 ) , phenocopied the uniform membrane localization of Lyn-GFP-Lpd ( 35A and 44A ) .",
"Scale bar , 10 µm .",
"( B , D )",
"The percentage of cells with leading edge localization is indicated in the upper right-hand corner of each representative image ( n = 96–167 cells imaged for each GFP-Lpd850−1250aa construct expressed in XTC cells ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 01410 . 7554/eLife . 06585 . 015Figure 5—figure supplement 1 . Lpd-VASP binding stoichiometry determined by sedimentation equilibrium .",
"( A ) Cartoon showing domain organization of human Lpd ( 1–1250aa ) .",
"( B ) Analytical ultracentrifugation sedimentation equilibrium traces for GFP-Lpd850−1250aa in the absence ( left ) and presence of 10 , 25 , 50 , 75 , 100 µM VASP1−114aa EVH1 domain .",
"( C ) Table showing the predicted and observed molecular weight of GFP-Lpd850−1250aa in absence and presence of different VASP1−114aa EVH1 domain protein concentrations .",
"( D ) Cartoon showing GFP-Lpd850−1250aa with VASP1−114aa EVH1 domains ( grey spheres ) binding to FPPPP sites ( red triangles ) .",
"Based on the observed binding stoichiometry between GFP-Lpd850−1250aa and VASP1−114aa , we hypothesize that steric hindrance allows only a single EVH1 domain to interact with the tandem FPPPP motifs ( i . e . , SPDFPPPPPESSLVFPPPPPSPVPA and SVVEFPSPPSDSDFPPPPPETD ) .",
"The crystal structure of GFP was derived from Yang et al . ( 1996 ) ( 1GFL . pdb ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 01510 . 7554/eLife . 06585 . 016Figure 5—figure supplement 2 . Lpd ( 850–1250aa ) wild-type and mutant protein sequence alignment . Protein sequence alignment of Lpd850−1250aa wild-type and mutants highlighting the separation of function mutations targeting either the actin BD ( arg/lys; BLUE ) , Ena/VASP binding sites ( GRAY ) , or Abi1/Endophilin SH3 domain binding sites ( RED ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 01610 . 7554/eLife . 06585 . 017Figure 5—figure supplement 3 . Membrane tethered Lyn-GFP-Lpd ( 850–1250aa ) requires basic residue for leading edge localization .",
"( A ) Plasma membrane localization of Lyn-GFP-Lpd850−1250aa , wild-type and mutants , visualized using TIRF microscopy .",
"Mutations affecting the interaction with either Ena/VASP proteins ( AAPPP ) x6 or Abi1/Endophilin ( SH3* ) did not abolish the leading edge localization of Lyn-GFP-Lpd850−1250aa .",
"Mutating all lysine and arginine residues ( 44A ) or only those outside of the Abi1/Endophilin SH3 BDs ( 35A ) eliminated the leading edge localization of Lyn- GFP-Lpd850−1250aa .",
"Membrane localization of Lyn-GFP-Lpd ( 44A and 35A ) phenocopy the uniform distribution of membrane anchored Lyn-GFP .",
"Cell images highlighted with the red dashed box include representative images of cells expressing low level of Lyn- GFP-Lpd850−1250aa .",
"The intensity of these images was scaled differently than images to the left to better visualize the localization . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 017 FL human Lpd1−1250aa contains 10 potential SH3 domain binding sites ( Law et al . , 2013 ) , of which four fall within our ABR construct , Lpd850−1250aa ( Figure 2A ) .",
"To abolish the known interactions with Abi1 and endophilin , we mutated essential proline residues ( e . g . , PxxPxR → GxxGxR ) in all four SH3 domain binding sites ( Figure 5—figure supplement 2 ) .",
"This mutant , termed Lpd ( SH3* ) 850−1250aa , could still localize to the leading edge in XTC cells ( Figure 5B ) , but to a lesser extent than wild-type Lpd or the ( AAPPP ) x6 Lpd mutant which does not bind Ena/VASP EVH1 domains .",
"Combining the ( AAPPP ) x6 and SH3* mutations in the same protein further reduced the percentage of cells with leading edge membrane localization of these GFP-Lpd constructs ( Figure 5B ) .",
"In each case , mutations in the Ena/VASP or Abi1/endophilin binding sites resulted in a loss of leading edge localization that coincided with an increased cytoplasmic localization of the GFP-Lpd850−1250aa construct .",
"To test whether the cloud of basic residues in the C-terminal region of Lpd mediates interactions with the actin cytoskeleton in vivo , we created a mutant lacking lysine and arginine residues that flank the multiple Ena/VASP and Abi1/endophilin binding sites ( Figure 5—figure supplement 2 ) .",
"In vitro , we found that GFP-Lpd ( 44A ) 850−1250aa lacking all lysine and arginine residues failed to bind to filamentous actin in vitro ( Figure 1D ) .",
"Because several of these point mutations flank or fall within the SH3 binding sites , we also generated a GFP-Lpd ( 35A ) 850−1250aa actin binding mutant , which lacks only the basic residues that sit between Ena/VASP and Abi1/endophilin binding sites ( Figure 5—figure supplement 2 ) .",
"Similar to GFP-Lpd ( 44A ) 850−1250aa , the GFP-Lpd ( 35A ) 850−1250aa construct also failed to concentrate at actin-based membrane protrusion or nascent focal adhesions in XTC cells ( Figure 5B ) .",
"We attempted to increase the avidity of GFP-Lpd ( 35A ) 850−1250aa for the leading edge membrane by expressing the protein as a leucine zipper-mediated constitutive dimer ( GFP-LZ-Lpd ( 35A ) 850−1250aa ) , but this mutant remained cytoplasmic ( Figure 5B ) .",
"To determine the role of membrane association in the leading-edge localization of Lpd we attempted to rescue leading edge localization of various GFP-Lpd850−1250aa mutants by constitutively tethering each protein to the plasma membrane with a Lyn palmitolyation sequence derived from Src kinase ( Figure 5C ) ( Inoue et al . , 2005 ) .",
"Similar to what has previously been observed for membrane tethered ActA- ( FPPPP ) x4-CAAX ( Bear et al . , 2002 ) and soluble GFP-Lpd850−1250aa , described here , Lyn-GFP-Lpd850−1250aa localized to the leading edge of actin-based membrane protrusions ( Figure 5D , Video 4 ) .",
"In contrast , leading edge localization of the combined ( AAPPP ) x6 and SH3* mutant construct was observed in fewer cells than the Lyn-GFP-Lpd850−1250aa .",
"Although Lyn-GFP-Lpd ( 35A ) 850−1250aa was targeted to the plasma membrane by the palmitoyl lipid anchor , this mutant still failed to localize to the peripheral regions where Ena/VASP , Abi1 , and endophilin are located ( Figure 5D ) .",
"Localization of Lyn-GFP-Lpd ( 35A ) 850−1250aa was identical to that of a membrane-anchored Lyn-GFP construct lacking any Lpd sequences .",
"Both of these constructs were distributed uniformly across the plasma membrane ( Figure 5D ) . 10 . 7554/eLife . 06585 . 018Video 4 . Localization of membrane tethered Lyn-GFP-Lpd ( 850−1250aa ) at the leading edge membrane of XTC cells spread on PLL coated coverslips expressed at single molecule concentrations from DeltaCMV promoter . Images were acquired using TIRF microscopy with temporal resolution of 50 ms time .",
"Video plays at 15 frames per second .",
"Scale bar , 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 018 Given this loss of positive charges , it is possible that failure of Lyn-GFP-Lpd ( 35A ) to localize to the leading edge membrane reflects an inability to interact with a polarized population of anionic lipids .",
"To look for such a polarized lipid distribution we expressed a phosphatidylinositol lipid sensor , based on the pleckstrin-homology domain of phospholipase Cδ ( GFP-PLCδ PH ) .",
"We found that GFP-PLCδ PH distributes uniformly across the plasma membrane of XTC cells ( Figure 5D ) .",
"This result supports our interpretation that failure of Lyn-GFP-Lpd ( 35A ) to localize to leading edge membranes in XTC cells reflects the fact that Lpd coupling to the actin cytoskeleton is required for establishing interactions with Ena/VASP or Abi1/endophilin at actin based membrane protrusions .",
"To test whether leading edge membrane localization of GFP-Lpd850−1250aa requires interaction with free actin filament barbed ends or with the sides of actin filaments , we used pharmacological perturbations to examine how GFP-Lpd850−1250aa leading edge localization responds to changes in actin cytoskeletal dynamics .",
"First , we simply froze actin assembly and disassembly by adding a mixture of Jasplakinolide and Latrunculin B to cells , without inhibiting myosin activity .",
"Under these conditions we observed rapid retrograde movement of GFP-Lpd850−1250aa away from the leading edge , identical to the movement of mCherry-Actin ( Figure 6A , Video 5 ) .",
"The dynamics of GFP-Lpd850−1250aa translocation suggests that , in the absence of net actin assembly , myosin can pull GFP-Lpd850−1250aa off the leading edge via a tight linkage to the actin cytoskeleton .",
"In these experiments , a fraction of membrane-localized GFP-Lpd850−1250aa remained at the leading edge , presumably due to the maintenance of interaction with Ena/VASP and/or Abi1/endophilin . 10 . 7554/eLife . 06585 . 019Figure 6 . Dynamic actin filament assembly and free barbed ends are required for leading localization of GFP-Lpd ( 850–1250aa ) .",
"( A ) Dynamic actin assembly is required for maintenance of GFP-Lpd850−1250aa leading edge localization .",
"Image montage showing translocation of GFP-Lpd850−1250aa and mCherry-Actin toward the cell body , following the addition of 8 µM Jasplakinolide and 10 µM Latrunculin B . Note that a population of GFP-Lpd850−1250aa remains associated with the peripheral membrane after addition of Jasp-LatB ( yellow arrowhead ) .",
"Horizontal scale bar , 5 µm .",
"Vertical scale bar , 2 min . ( B–E ) Barbed ends are required for plasma membrane localization of ( B ) GFP-Lpd850−1250aa , ( C ) GFP-LZ-Lpd850−1250aa , ( D ) GFP-Lpd850−1250aa ( AAPPP ) x6 , and ( E ) GFP-Lpd850−1250aa ( AAPPPx6 + SH3* ) .",
"Representative kymographs showing membrane dissociation of GFP-Lpd850−1250aa , wild-type and mutants , following the addition of 100 nM Cytochalasin D ( blue arrowhead ) .",
"Horizontal scale bar , 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 01910 . 7554/eLife . 06585 . 020Video 5 . Localization of GFP-Lpd850−1250aa and mCherry-Actin in XTC cells following the addition of 8 µM Jasplakinolide and 10 µM Latrunculin B . Images were acquired with a temporal resolution of 10 s using TIRF-M at 23°C .",
"Drugs were added after 150 s of imaging .",
"Video plays at 10 frames per second .",
"Scale bar , 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 020 Next we treated cells expressing GFP-Lpd850−1250aa with Cytochalasin D , a small molecule that caps barbed ends of actin filaments .",
"Consistent with previous observations ( Krause et al . , 2004; Neel et al . , 2009 ) , addition of Cytochalasin D to XTC cells strongly inhibited the localization of GFP-VASP to leading edge membranes ( unpublished observations ) .",
"When we treated these cells with a 100 nM Cytochalasin D , we observed a large fraction of leading edge-localized GFP-Lpd850−1250aa immediately dissociate from the leading edge membrane and flow back with the actin cytoskeleton ( Figure 6B , Video 6 ) .",
"Because GFP-LZ-Lpd850−1250aa binds more strongly to filamentous actin , dissociation from the leading edge membrane was more dramatic following addition of Cytochalasin D ( Figure 6C , Video 7 ) .",
"The same membrane dissociation dynamics were observed when cells expressing GFP-Lpd850−1250aa mutants , ( AAPPP ) x6 and SH3* , were treated with Cytochalasin D ( Figure 6D , E ) .",
"Together , these results suggest that the leading edge localization of GFP-Lpd850−1250aa is regulated , in part , by a direct interaction with free actin filament barbed ends , independent of interactions with Ena/VASP or Abi1/endophilin proteins . 10 . 7554/eLife . 06585 . 021Video 6 . Localization of GFP-Lpd850−1250aa following the addition of 100 nM Cytochalasin D in a XTC cell imaged with a temporal resolution of 2 s using TIRF-M at 23°C . Cytochalasin D was added at 214 s .",
"Video plays at 50 frames per second .",
"Scale bar , 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 02110 . 7554/eLife . 06585 . 022Video 7 . Localization of GFP-LZ-Lpd850−1250aa following the addition of 100 nM Cytochalasin D in a XTC cell imaged with a temporal resolution of 2 s using TIRF-M at 23°C . Cytochalasin D was added at 102 s .",
"Video plays at 50 frames per second .",
"Scale bar , 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 022 The six VASP-binding ‘FPPPP’ motifs in the C-terminal region of Lpd do not appear to overlap the cloud of basic residues that mediate actin filament binding ( Figure 2A ) .",
"We , therefore , wondered whether Lpd could bind Ena/VASP proteins and filamentous actin simultaneously .",
"To test this idea , we first used TIRF microscopy to demonstrate that several Cy3-labeled VASP constructs—an isolated EVH1 domain and two full length , tetrameric , actin-binding mutants—failed to bind actin filaments ( Figure 7A ) .",
"Addition of GFP-Lpd850−1250aa , however , caused all of these VASP constructs to associate with the sides of actin filaments ( Figure 7B ) .",
"Interaction of the tetrameric Cy3-VASP actin-binding mutant with the monomeric Lpd construct produced higher-order VASP clusters visible on the sides of the filaments ( Figure 7B , arrowheads ) .",
"We previously observed this type of protein clustering in vitro when we combined tetrameric VASP and ActA255−392aa ( unpublished observations ) . 10 . 7554/eLife . 06585 . 023Figure 7 . Lpd can simultaneously interact with VASP and filamentous actin .",
"( A ) VASP EVH1 and FL VASP mutants cannot interact with actin filaments in vitro .",
"Images highlight the inability of 200 nM ( monomeric concentration ) wild-type Cy3-VASP1−380aa , Cy3-VASP1−114aa ( EVH1 domain ) , Cy3-VASPLIL-3A , RRRK-4A , and Cy3-VASPRRRK-4E to phalloidin stabilized actin filaments ( 20% Cy5 labeled ) .",
"Buffer contains 20 mM HEPES [pH 7] , 50 mM KCl , 1 mg/ml BSA , 1 mM TCEP .",
"Scale bar , 10 µm .",
"( B ) GFP-Lpd850−1250aa can simultaneously interact with filamentous actin and VASP EVH1 domains .",
"Colocalization of 500 nM monomeric GFP-Lpd850−1250aa phalloidin stabilized actin filaments ( 20% Cy5 labeled ) in the presence of 200 nM ( monomeric concentration ) of wild-type Cy3-VASP1−380aa , Cy3-VASP1−114aa ( EVH1 domain ) , Cy3-VASPLIL-3A , RRRK-4A ( GAB and FAB mutant ) , or Cy3-VASPRRRK-4E ( FAB mutant ) .",
"Note the formation of large clusters containing VASP and Lpd ( yellow arrowheads ) .",
"Scale bar , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 02310 . 7554/eLife . 06585 . 024Figure 7—figure supplement 1 . Lpd and VASP synergistically bundle actin filaments .",
"( A ) Montage of single actin filaments polymerizing in the presence of 2 µM actin ( 20% Cy5 labeled ) and TIRF buffer containing 100 mM KCl .",
"Compared to actin filaments elongating in the presence of 50 nM VASP ( tetrameric concentration ) or 250 nM GFP-LZ-Lpd850−1250aa ( dimer concentration ) .",
"The combined presence of VASP and GFP-LZ-Lpd850−1250aa produces large actin bundles .",
"Scale bar , 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 024 Supporting the notion that Lpd-VASP complexes can synergistically bind actin filaments , we find that the presence of both VASP and GFP-LZ-Lpd results in a large amount of actin filament bundling in the presence of 100 mM KCl ( Figure 7—figure supplement 1 ) .",
"In contrast , individual actin filaments elongating in the presence of 50 nM tetrameric VASP or 250 nM dimeric GFP-LZ-Lpd alone do not bundle actin filaments in high ionic strength buffer .",
"This means that the Lpd-VASP interaction can compensate for the weak actin filament binding observed for the individual proteins in buffer containing 100 mM KCl .",
"Because human VASP is a weakly processive actin polymerase and Lpd can interact simultaneously with both VASP tetramers and actin filaments , we hypothesized that Lpd might modulate the processivity of VASP by both clustering tetramers and tethering them to actin filaments .",
"We tested this hypothesis by visualizing actin filament elongation in the presence of either monomeric ( Lpd850−1250aa ) or dimeric ( LZ-Lpd850−1250aa ) GFP-Lpd constructs .",
"The weak interaction between monomeric GFP-Lpd850−1250aa and filamentous actin turns out to be further antagonized by the presence of monomeric actin or profilin-actin ( Figure 8A ) .",
"Also , we observed a reduction in the rate of barbed end actin filament elongation in the presence 2 µM profilin-actin and soluble GFP-Lpd850−1250aa or GFP-LZ-Lpd850−1250aa ( Figure 8B ) , consistent with Lpd binding to ( and partially sequestering ) actin monomers .",
"In contrast to monomeric Lpd , dimeric GFP-LZ-Lpd850−1250aa could transiently interact with both the sides and barbed ends of single growing actin filaments ( Figure 8D , Video 8 ) .",
"Although GFP-Lpd and GFP-LZ-Lpd associate more transiently with single actin filaments in the presence of monomeric actin , Lpd binds well enough to promote filament bundling when present at a near stoichiometric concentration relative to monomeric actin ( Figure 8—figure supplement 1 ) . 10 . 7554/eLife . 06585 . 025Figure 8 . Lpd enhances VASP barbed end processivity .",
"( A ) Monomeric actin antagonizes GFP-Lpd850−1250aa actin filament binding .",
"Visualization of 500 nM GFP-Lpd850−1250aa in the absence or presence of 4 µM monomeric actin in the presence of buffer containing 20 mM HEPES [pH 7 . 0] , 50 mM KCl , 1 mg/ml BSA , 1 mM TCEP , and 25 µM Latrunculin B . Scale bar , 10 µm .",
"( B ) GFP-Lpd850−1250aa ( 1 µM , monomer concentration ) and GFP-LZ-Lpd850−1250aa ( 0 . 25 µM , dimer concentration ) slow barbed end elongation in the presence of 2 µM profilin-Mg-ATP-actin ( 5% Cy5 labeled ) and TIRF buffer containing 50 mM KCl .",
"( C ) Single actin filament elongation rates measured as in ( B ) , but in the presence of 2 µM actin ( 20% Cy5 ) with TIRF buffer containing 75–100 mM KCl .",
"( B , C )",
"Error bars represent the standard deviation of the mean ( n ≥ 30 barbed end elongation rates measured per condition ) .",
"( D ) Dimeric GFP-LZ-Lpd850−1250aa localizes to sides and barbed ends of elongating actin filaments .",
"Kymographs showing the localization of 50 nM GFP-LZ-Lpd850−1250aa ( green ) to a single actin filament polymerized in the presence of 2 µM Mg-ATP-Actin ( 20% Cy5 , red ) .",
"Scale bars , 2 µm and 10 s .",
"( E ) Visualization of processive barbed end associated Cy3-VASP tetramers ( green ) in the absence or presence of 200 nM GFP-LZ-Lpd850−1250aa .",
"Actin filaments were polymerized in the presence of 2 µM Mg-ATP-Actin ( 20% Cy5 , red ) .",
"Scale bar , 2 µm and 10 s .",
"( F , G )",
"Calculation of Cy3-VASP barbed end dwell times in the absence ( F ) or presence of 200 nM GFP-LZ-Lpd850−1250aa ( G ) decorated actin filaments .",
"Histogram plots of Cy3-VASP barbed end associated dwell times with insets of the log10 ( 1-cumulative distribution frequency ) fit with a ( F ) single exponential curve for Cy3-VASP alone ( τ1 = 0 . 49 ± 0 . 03 s , n = 673 molecules ) or ( G ) Cy3-VASP in the presence of 200 nM GFP-LZ-Lpd850−1250aa ( τ1 = 0 . 58 ± 0 . 05 s ( 73% , fast ) , τ2 = 2 . 3 ± 0 . 4 s ( 27% , slow ) , n = 632 molecules ) .",
"Note that the dwell times for Cy3-VASP in ( F ) are shorter than previously reported ( Hansen and Mullins , 2010 ) .",
"This due to Cy5-Actin being a less favorable substrate for barbed end incorporation compared to Alexa488-Actin .",
"( H ) Clustered his10-GFP-Lpd850−1250aa increases the processivity of Cy3-VASP .",
"Image montage showing colocalization of the Cy3-VASP ( 5 nM ) and his10-GFP-Lpd850−1250aa ( 50 nM ) on actin filament barbed end elongating in the presence of 2 µM Actin ( 20% Cy5 ) and TIRF buffer contains 75 mM KCl .",
"Note the intensity of the actin filament decreases when the VASP-Lpd complex is associated with the growing actin filament barbed end , indicating that unlabeled vs Cy5-labeled actin is more favorably incorporated .",
"Scale bar , 5 µm .",
"( I ) Lpd-VASP barbed associated complexes incorporate actin monomers at a faster velocity , as compared to actin filament elongating in the presence of 50 nM tetrameric VASP .",
"Error bars represent the standard deviation of the mean ( p-value = 7 × 10−12; two-tailed t-test for data sets with unequal variance ) .",
"( J ) Calculation of the barbed end dwell times for Cy3-VASP and his10-GFP-Lpd850−1250aa complexes .",
"Plot of 1-CDF was best fit to a single exponential curve , yielding τ1 = 33 ± 2 s ( n = 87 complexes ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 02510 . 7554/eLife . 06585 . 026Figure 8—figure supplement 1 . Lpd dependent actin filament bundling .",
"( A , B )",
"Lpd bundles dynamically elongating actin filaments .",
"Montage showing single actin filaments elongating and bundling in the presence of 2 µM actin ( 5% Cy5 labeled ) and either ( A ) 1 µM GFP-Lpd850−1250aa or ( B ) 1 µM GFP-LZ-Lpd850−1250aa ( dimer concentration equals 0 . 5 µM ) in TIRF buffer containing 50 mM KCl .",
"Note that the intensity of single actin filaments is faint due to the low percentage of Cy5-labeled actin and low laser intensity used to prevent camera pixel saturation by the bright actin filament bundles .",
"Scale bar , 20 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 02610 . 7554/eLife . 06585 . 028Video 8 . Dynamic actin filament association of 200 nM dimeric GFP-LZ-Lpd ( 850−1250aa ) in the presence of 2 µM Mg-ATP ( 20% Cy5 labeled ) visualized by TIRF-M . For clarity , only GFP-LZ-Lpd ( 850−1250aa ) is shown .",
"Images were acquired with temporal resolution of 0 . 5 s using TIRF-M .",
"Video plays at 50 frames per second .",
"Scale bar , 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 028 We tried to measure the barbed end dwell time of GFP-LZ-Lpd850−1250aa , but the dissociation kinetics and diffusivity of Lpd were too rapid .",
"Using lower concentrations of GFP-LZ-Lpd850−1250aa to visualize single barbed end association events did not improve our resolution .",
"We speculate that the comet-like barbed end localization of GFP-LZ-Lpd850−1250aa ( Figure 8D ) may reflect dimers simultaneously binding to the barbed end and near the barbed end , creating a fluorescent speckle .",
"Because GFP-Lpd and GFP-LZ-Lpd reduce the rate of actin filament elongation and transiently associate with barbed ends , we tested whether VASP could alleviate the inhibitory effect of Lpd .",
"We measured barbed end elongation rates in the presence of 2 µM actin and 2 µM profilin-actin , as well as buffers of various ionic strengths ( Figure 8B , C ) .",
"Because Lpd and VASP synergistically and potently bundle actin filaments in 50 mM KCl , we tried increasing the ionic strength ( 75–100 mM KCl ) to minimize side binding , while favoring barbed end association of the Lpd-VASP complex .",
"However , for all conditions tested , we found that the rate of barbed end elongation in the presence of both Lpd and VASP was intermediate between the rates measured in the presence of either Lpd or VASP alone ( Figure 8B , C ) .",
"Interpreting these results remains challenging because multiple competing activities contribute to single filament elongation rates .",
"Because multiple , competing interactions determine the average barbed end elongation rate in the presence of Lpd and VASP , we returned to imaging single Cy3-VASP tetramers to determine whether Lpd modulates VASP's barbed-end processivity .",
"Fluorescently labeled VASP tetramers accumulate on growing actin filament barbed ends decorated with GFP-LZ-Lpd850−1250aa where they form a stable population ( τ1 = 0 . 58 ± 0 . 05 s ( 73% ) , τ2 = 2 . 3 ± 0 . 4 s ( 27% ) ; Figure 8E–G ) that dissociates much more slowly than VASP tetramers observed in the absence of GFP-LZ-Lpd ( τ1 = 0 . 49 ± 0 . 03 s; Figure 8E–G ) .",
"One challenge we faced in visualizing the colocalization of Lpd and VASP on single actin filament barbed ends was that the high solution protein concentrations required to observe complex formation .",
"Unfortunately , these high concentrations were not compatible with single molecule imaging and produced large protein complexes that were unable to associate with growing barbed ends .",
"To circumvent this problem , we used lower concentrations of his10GFP-Lpd ( 850–1250aa ) oligomers purified by size exclusion chromatography ( Figure 1E ) and characterized their effect on VASP barbed end processivity in the single actin filament TIRF assay .",
"Under these conditions we observed complexes containing both VASP and Lpd processively track growing actin filament barbed ends at a velocity of 36 ± 9 . 4 subunits/s ( Figure 8I , Video 9 ) .",
"The rate at which Lpd-VASP complexes shuttled actin monomers onto free barbed ends was faster than elongation of single actin filaments in the presence of 50 nM tetrameric VASP ( 25 ± 1 . 6 subunits/s , Figure 8I ) .",
"The dwell time for VASP-Lpd tip complexes ( τ1 = 33 ± 2 s; Figure 8J ) was also significantly longer as compared to single VASP tetramers ( τ1 = 0 . 49 ± 0 . 03 s; Figure 8F ) . 10 . 7554/eLife . 06585 . 029Video 9 . Visualization of a processive VASP-Lpd tip complex bound to the barbed end of a single actin filament . Elongation of single actin filaments were visualized in the presence of 2 µM Mg-ATP actin ( 20% Cy5 labeled ) , 5 nM Cy3-VASP , 50 nM his10-GFP-Lpd850−1250aa , and buffer containing 75 mM KCl .",
"Images were acquired with temporal resolution of 2 s using TIRF-M .",
"Video plays at 20 frames per second .",
"Scale bar , 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 029"
],
[
"We discovered a direct interaction between Lpd and filamentous actin that is mediated by a highly basic , unstructured region in the C-terminus of Lpd ( residues 850–1250 ) .",
"The cloud of positively-charged amino acid residues is conserved between Lpd homologs of different organisms .",
"These residues , however , are absent from other Ena/VASP binding proteins , such as RIAM , ActA , and Zyxin ( Figure 2B ) , suggesting that this basic region is important for cellular functions specific to Lpd .",
"The filament-BD does not overlap motifs that bind the EVH1 domains of VASP ( Figure 2A ) , enabling Lpd to simultaneously interact with both Ena/VASP proteins and filamentous actin .",
"Similar to VASP ( Hansen and Mullins , 2010 ) , monomeric Lpd molecules bind actin filaments only weakly in physiological ionic strength ( ∼150 mM KCl ) , but bind much more strongly when oligomerized or densely clustered on membranes .",
"Because of this effect , local enrichment of Lpd at the plasma membrane can exert a non-linear , cooperative effect on Ena/VASP activity ( Figure 9 ) . 10 . 7554/eLife . 06585 . 027Figure 9 . Model . Based on the canonical model ( Krause et al . , 2004 ) , Lpd is recruited to actin based membrane protrusions through interactions with phosphatidylinositol lipids ( i . e . , PI ( 3 , 4 ) P2 ) and possibly small GTPases ( i . e . , Ras or Rho family ) .",
"Similar to the Grb protein family , Lpd is predicted to form homo-dimers mediated by interactions between the coiled-coil and tandem RA-PH domain .",
"We find that the C-terminus of Lpd ( residues 850–1250 ) is sufficient for recruiting Lpd to leading edge membrane where it directly interacts with free barbed ends and/or the sides of the actin filaments .",
"Importantly , this interaction between Lamelliopodin and filamentous actin can occur independently to those mediated by Ena/VASP proteins or SH3 domains ( i . e . , Abi1/endophilin ) .",
"However , Ena/VASP proteins recruited to actin based membrane protrusion can simultaneously associate with free actin filament barbed ends and Lpd .",
"By this mechanism , we speculate that the lifetime of membrane targeted and barbed end associated Ena/VASP proteins are extended at the plasma membrane . DOI: http://dx . doi . org/10 . 7554/eLife . 06585 . 027 Several mechanisms have been proposed to modulate the activity of Ena/VASP proteins , including: post-translational modifications of the EVH2 domain ( Barzik et al . , 2005 ) ; alternative splicing of Ena/VASP transcripts ( Philippar et al . , 2008 ) ; and specificity for distinct profilin-actin isoforms ( Dugina et al . , 2009; Mouneimne et al . , 2012 ) .",
"In this study , we establish that Ena/VASP proteins are directly controlled by their membrane-targeting factors .",
"Specifically we show that Lpd-VASP complexes are more persistently localized to polymerizing ends , because the Lpd-actin interaction tethers VASP to the barbed end ( Figure 9 ) .",
"Filament tethering in combination with VASP clustering ( Breitsprecher et al . , 2008 ) greatly increases the processivity of Ena/VASP proteins .",
"In addition , Lpd-VASP complexes also enhance barbed-end polymerization more effectively compared to individual VASP tetramers , an ability likely based on Lpd's interaction with actin monomers .",
"By increasing the number of monomer binding sites near the barbed end , Lpd may directly contribute to the polymerase activity of VASP tetramers .",
"In cells both FL Lpd and its C-terminal actin BD ( Lpd850−1250aa ) localize to actin-based membrane protrusions independently of other binding proteins such as Ena/VASP or Abi1/endophilin .",
"In contrast to FL Lpd , however , we find that acute treatment of cells with cytochalasin D rapidly displaces GFP-Lpd850−1250aa from the leading edge and causes it to translocate toward the cell body with the disassembling lamellipodial actin network .",
"We observed a similar phenotype for Lpd850−1250aa mutants that cannot interact with Ena/VASP proteins or SH3 domains .",
"Taken together these results demonstrate that the C-terminal region of Lpd harbors autonomous filament binding sites that strongly influence its localization to actin rich protrusions .",
"The ability of Lpd850−1250aa to directly interact with polymeric actin extends the canonical model of Lpd membrane localization , which suggests that Lpd targets the plasma membrane by binding to PI ( 3 , 4 ) P2 and/or small GTPases ( Krause et al . , 2004 ) .",
"Future experiments will determine the hierarchical mechanisms that control membrane localization and maintenance of Lpd in response to these diverse molecular inputs .",
"In vitro , the C-terminal actin BD of Lpd reduces barbed end elongation by partially sequestering actin monomers and/or transiently capping free barbed ends .",
"This effect is more pronounced in the presence of profilin-actin , as compared to actin alone , indicating that profilin can shunt Lpd towards the filament barbed end .",
"Interestingly , Lpd contains a poly-proline domain situated upstream of the ABR characterized in this study .",
"We speculate that this poly-proline domain , similar to its function in other actin regulatory proteins such as formins and nucleation promoting factors ( Paul et al . , 2008; Campellone and Welch , 2010 ) , could bind profilin-actin and promote its interaction with the actin-binding region , influencing the partitioning of Lpd between monomeric and filamentous actin .",
"Previous work by Bae et al . ( 2010 ) indicates that the interaction between profilin and phosphatidylinositol lipids ( e . g . , PI ( 3 , 4 ) P2 ) plays a key role in regulating Lpd localization to actin-based membrane protrusions .",
"It will , therefore , be important to characterize the interplay between profilin binding to the Lpd poly-proline domain and phosphatidylinositol lipids in the future .",
"We speculate that enrichment of PI ( 3 , 4 ) P2 at the plasma membrane could liberate monomeric actin from profilin-actin complexes , thereby promoting the interaction between Lpd and actin monomers .",
"The function of Lpd at the leading edge is not simply to recruit Ena/VASP proteins to the plasma membrane: Knockdown of Lpd inhibits lamellipodium formation more potently compared to loss of Ena/VASP proteins ( Bear et al . , 2002; Krause et al . , 2004 ) and results in global reduction of lamellipodial actin density ( Lyulcheva et al . , 2008; Michael et al . , 2010; Law et al . , 2013 ) .",
"Recently both Lpd and its binding partners Ena/VASP were found to interact with the Scar/WAVE regulatory complex ( Law et al . , 2013; Chen et al . , 2014 ) , an important activator of the Arp2/3 complex at the leading edge .",
"These interactions are mediated by the SH3 domain of Abi1 , a WAVE complex component , which binds multiple PxxPxR motifs in the C-terminus of Lpd and the EVH1 domains of Ena/VASP proteins .",
"Mutations interfering with WAVE-Lpd binding impaired cell migration in wound closure assays ( Law et al . , 2013 ) , demonstrating the functional relevance of this interaction .",
"Direct binding of Lpd to filamentous actin might promote Ena/VASP and Scar/WAVE activity within the lamellipodial actin networks they construct .",
"The position of the WAVE-binding motif in Lpd suggests that Lpd could synchronously tether the WAVE regulatory complex and Ena/VASP to filamentous actin .",
"Furthermore , both Lpd ( Krause et al . , 2004; Chang et al . , 2012 ) and the WAVE regulatory complex interact with anionic lipids at the plasma membrane ( Lebensohn and Kirschner , 2009; Koronakis et al . , 2011 ) .",
"We propose that these multi-layered interactions at the leading edge guide the assembly of the actin nucleation and elongation machinery into supramolecular complexes to coordinate their activities for the efficient construction of force-generating actin networks .",
"Besides its function in regulating lamellipodial actin network assembly , Lpd has also been implicated in regulating FEME ( Vehlow et al . , 2013; Boucrot et al . , 2015 ) .",
"Similar to the interactions with the WAVE complex , the C-terminus of Lpd binds to SH3 domains in endophilin using multiple PxxPxR motifs .",
"Consistent with Lpd playing a role FEME , we observed vesicular localization of GFP-Lpd850−1250aa that translocate toward the cell body .",
"Because Lpd interacts directly with filamentous actin and multiple actin regulatory proteins , Lpd could function to orchestrate the actin nucleation machinery at the sites of FEME ."
],
[
"Lpd clones were derived from a plasmid encoding human Lpd1−1250aa ( pBSII-SK ( + ) vector ) provided by Matthias Krause ( King's College London ) .",
"his10-TEV-EGFP ( A207K ) -Lpd850−1250aa was cloned into a modified pETM-11 vector ( EMBL Protein Expression and Purification Facility; Heidelberg , Germany ) .",
"Expression vectors used for transient transfection into XTC cell , genes were cloned into the pCMV-EGFP/mCherry ( Takara Clontech Laboratories , Mountain View CA , USA ) or pDeltaCMV-EGFP vectors originally created by Naoki Watanabe ( Tohoku University , Japan ) and provided by Orion Weiner ( University of California at San Francisco , CVRI ) .",
"All of our constructs contain a monomeric EGFP variant , A207K point mutation , which significantly reduces the tendency of EGFP to dimerize .",
"Inspired by experiments described by Inoue et al . ( 2005 ) , we fused a Lyn11 palmityolation sequence ( GCIKSKGKDSA ) derived from Src family kinase to our pDeltaCMV-EGFP ( A207K ) construct to generate our Lyn-GFP and Lyn-GFP-Lpd fusions .",
"Mutations in the Lpd Ena/VASP BDs , ( AAPPP ) x6 , were introduced using Pfu Turbo DNA polymerase and standard site-directed mutagenesis techniques .",
"We made the constitutive GFP-Lpd dimer by inserting a leucine zipper derived from Saccharomyces cerevisiae Gcn4p; ( Tomishige et al . , 2002 ) in between the GFP and Lpd protein sequences [EGFP-GGGYKQLEDKVEELASKNYHLENEVARLKKLVEFGGG-Lpd] .",
"Lpd850−1250aa with all lysine and arginine residues mutated to alanine , called Lpd ( 44A ) , was synthesize by Invitrogen ( GeneArt ) and cloned into the pDeltaCMV-Lyn-EGFP ( A207K ) or his10-TEV-EGFP ( A207K ) vectors for transient cell expression and bacterial protein expression , respectively .",
"We used the Quikchange Lightning Multi site-directed mutagenesis kit ( Agilent , Cat# 210515 ) to correct lysine/arginine to alanine mutations that flanked or lied within the Abi1/endophilin SH3 domain binding sites resulting in a construct we termed Lpd ( 35A ) .",
"This kit was separately used to mutate proline residues within the four predicted SH3 domain-binding sites located in Lpd850−1250aa .",
"The pIs for Lpd and homologs were calculated with EXPASY ProtParam ( Wilkins et al . , 1999 ) and sequences were aligned using ClustalW .",
"Refer to Figure 5—figure supplement 2 for exact protein sequences of each Lpd850−1250aa mutant used in this study .",
"For a complete list of plasmid DNA used in this study , refer to Supplementary file 1 .",
"BL21 ( DE3 ) Star pRARE bacteria ( chloramphenicol resistant ) were transformed with his10-TEV-EGFP ( A207K ) -Lpd ( 850–1250aa , wild-type and mutants ) or his6-Z-tag-TEV-EGFP ( A207K ) -LZ-Lpd850−1250aa and plated on LB agar containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol .",
"The next day , 50–100 ml of TPM media ( 20 g tryptone , 15 g yeast extract , 8 g NaCl , 2 g Na2HPO4 , 1 g KH2PO4 , per liter ) containing antibiotics was inoculated with multiple bacterial colonies swiped from the agar plate .",
"This starter culture was grown for 4–5 hr at 37°C to an OD600 = 2 . 5–3 . 0 , before being diluted to OD600 = 0 . 05–0 . 1 in 6 liters of TPM media .",
"These cultures were grown to an OD600 = 0 . 9–1 . 1 at 37°C .",
"Bacteria were then induced to express Lpd with 0 . 25 mM IPTG and then shifted from 37°C to 25°C .",
"After 6 hr of expression at 25°C , bacterial cultures were harvested by centrifugation .",
"Cells were lysed into buffer containing 50 mM Na2PO4 [pH 8] , 400 mM NaCl , 0 . 4 mM BME ( Sigma , Cat# M3148-100ML ) , 1 mM phenylmethanesulfonyl fluoride ( Sigma , Cat# P7626-5G ) , and DNase ( Sigma , Cat# DN25-1G ) using a microfluidizer .",
"Lysate was clarified by centrifugation in a Beckman JA-17 rotor for 60 min , 16 , 000 rpm ( 35 , 000 rcf ) .",
"High speed supernatant was recirculated over a 5 ml HiTrap chelating column ( GE Healthcare , Cat# 17-0409-03 ) that was charged with 100 mM CoCl2 ( Sigma , Cat# 255599 ) , washed with water , and then equilibrated with lysis buffer lacking protease inhibitors and DNase .",
"Following capture of his-tagged Lpd , the HiTrap column was washed with ∼100 ml of buffer containing 50 mM Na2PO4 [pH 8 . 0] , 400 mM NaCl , 0 . 4 mM BME .",
"Proteins were gradient elution over 40 ml ( 2 ml/min ) using a buffer containing 50 mM Na2PO4 [pH 8 . 0] , 400 mM NaCl , 500 mM imidazole , 0 . 4 mM BME .",
"Using more than 0 . 4 mM BME will instantly reduce the chelated Co+2 and turn the HiTrap column brown .",
"Removal the his6-Ztag or his10 , was achieved by TEV protease digestion of the HiTrap eluate during an overnight dialysis in 4 liters of buffer containing 50 mM Na2PO4 [pH 8 . 0] , 400 mM NaCl , 0 . 4 mM BME .",
"After 20–24 hr , the dialysate containing TEV cleaved Lpd was recirculated over a HiTrap chelating column to remove uncleaved protein , his6-TEV protease , and other proteins that non-specifically bound during the first purification step .",
"EGFP-Lpd and EGFP-LZ-Lpd were then buffer exchanged using a HiPrep Desalting 26/10 G25 Sephadex column ( GE Healthcare , Cat# 17-5087-01 ) equilibrated in 20 mM HEPES [pH 7] , 75 mM NaCl , 1 mM TCEP .",
"To isolate FL EGFP-Lpd and EGFP-LZ-Lpd , these proteins were loaded onto a MonoS 5/50 GL column ( GE Healthcare , Cat# 17-5168-01 ) equilibrated with 20 mM HEPES [pH 7] , 75 mM NaCl , 1 mM TCEP .",
"Bound protein was eluted with a 75–400 mM NaCl salt gradient applied over 40 CV ( 1 CV = 1 ml ) at a flow rate of 1 ml/min .",
"EGFP-Lpd850−1250aa and EGFP-LZ-Lpd850−1250aa eluted in the presence of 170 mM NaCl and 225 mM NaCl , respectively .",
"Peak fractions were pooled , concentrated by centrifugation in a Vivispin 6 ( 30 kDa MWCO ) column .",
"EGFP-Lpd are EGFP-LZ-Lpd were then gel filtered using a 24 ml Superdex200 column equilibrated with 20 mM HEPES [pH 7 . 5] , 200 mM NaCl , 10% glycerol , and 1 mM TCEP .",
"Peak fractions were pooled , concentrated in a new Vivispin 6 ( 30 kDa MWCO ) column , and frozen with liquid nitrogen before being stored in the −80°C freezer .",
"To isolate uncleaved his10-TEV-EGFP ( A207K ) - Lpd850−1250aa , we loaded the HiTrap eluted protein onto a 323 ml Superdex200 gel filtration column equilibrated with 10 mM HEPES [pH 7 . 0] , 200 mM NaCl , 1 mM TCEP .",
"This allowed us to separate soluble aggregates from the monomeric protein .",
"Peak fractions were then combined with 10 mM HEPES [pH 7 . 5] , 1 mM TCEP ( 1:1 vol ) to effectively reduce the salt concentration to 100 mM NaCl .",
"This sample was then loaded on a MonoS column equilibrated with 10 mM HEPES [pH 7 . 0] , 100 mM NaCl , 1 mM TCEP and gradient eluted as described above .",
"Peak fractions of his10-EGFP-Lpd eluted from the MonoS column containing a small fraction of spontaneously formed oligomers .",
"Freezing in glycerol , sucrose , arginine-glutamate , or without cryo-protection also caused his10-EGFP-Lpd protein to oligomerize .",
"Consequently , his10-EGFP-Lpd was frozen after MonoS elution as 300–500 µl aliquots ( 40–50 µM ) .",
"When needed for actin bead motility assays and TIRF microscopy experiments , his10-EGFP-Lpd was thawed and gel filtered using a 24 ml Superdex200 column equilibrated with 10 mM HEPES [pH 7 . 5] , 200 mM NaCl , and 1 mM TCEP .",
"Freshly gel filtered his10-EGFP-Lpd showed no signs to proteolysis or aggregation after at least one week on ice .",
"Cytoplasmic actin was purified from Acanthamoeba castellani as described ( Gordon et al . , 1976; Hansen et al . , 2013 ) .",
"Actin was stored at 4°C in G-buffer containing 2 mM Tris [pH 8 . 0] , 0 . 5 mM TCEP , 0 . 1 mM CaCl2 , 0 . 2 mM ATP , 0 . 01% azide , and used within 6 months .",
"The quality , or degree of proteolysis , of monomeric actin was routinely monitored by SDS-PAGE .",
"Monomeric actin was labeled on Cys-374 by adding 3–7 molar excess Cy3-maleimide ( GE Healthcare , Cat# PA23031 ) or Cy5-maleimide ( GE Healthcare , Cat# PA25031 ) on ice in G-buffer for 10–15 min .",
"Reactions were quenched with 10 mM DTT and then centrifuged at 346716×g ( TLA 100 . 4 rotor , Beckman Coulter ) to remove insoluble dye and aggregated protein .",
"Labeled actin was then polymerized at room temperature by the addition of KMEI polymerization buffer ( final concentration of 10 mM imidazole [pH 7 . 0] , 50 mM KCl , 1 mM MgCl2 , 1 mM EGTA , and 1 mM ATP ) .",
"Labeled filamentous actin was then centrifuged in a TLA 100 . 4 rotor for 30 min at 195028×g .",
"The pellet was gently washed with G-buffer and then resuspended in G-buffer ( 2 mM Tris [pH 8 . 0] , 0 . 5 mM TCEP , 0 . 1 mM CaCl2 , 0 . 2 mM ATP , 0 . 01% azide ) to initiate actin filament depolymerization .",
"Following depolymerization in G-buffer for 3–5 days , actin was centrifuged at 346716×g ( TLA 100 . 4 rotor ) for 20 min and then gel filtered ( Superdex 75 ) in G-buffer .",
"We typical observed a 50–60% labeling efficiency .",
"Human VASP was expressed and purified as his6-TEV-KCK-VASP ( 1–380aa ) Cys-light ( C7S , C64S , C334A ) fusion as previously described by Hansen and Mullins ( 2010 ) .",
"Mouse EVL was expressed and purified as a his6-TEV-KCK-EVL ( 1–393aa ) Cys-light ( C7S , C177S ) fusion using the protocol described above .",
"The specific residues mutated in the VASP actin filament binding mutants described in Figure 4 , are as follows: VASPLIL-3A , RRRK-4A ( L226A , I230A , L235A , R273A , R274A , R275A , K276A ) and VASPRRRK-4E ( R273E , R274E , R275E , K276E ) .",
"Both actin binding mutants were expressed and purified in the context of the his6-TEV-KCK-VASP ( 1–380aa ) Cys-light ( C7S , C64S , C334A ) vector .",
"In all cases the his6 tag was cleaved from purified Ena/VASP protein with TEV protease before cation exchange and size exclusion chromatography ( Superdex 200 ) .",
"The Arp2/3 complex ( native , A . castellani ) , capping protein ( mouse , recombinant ) , and profilin ( human , recombinant ) were purified and handled as previously described by Akin and Mullins ( 2008 ) .",
"Purification of his10-Cherry-SCARAPWCA ( 171-559aa , human WAVE1 ) was achieved by expressing codon optimized Z-tag-TEV-his10-Cherry-SCARAPWCA in Rosetta ( DE3 ) bacteria for 20 hrs at 18°C in Terrific Broth media .",
"Cell lysate was recirculated over a 5 ml HiTrap chelating column charged with CoCl2 .",
"Eluted protein was cleaved with TEV protease overnight at 4°C and then exchanged into low ionic strength buffer using a HiPrep Desalting 26/10 G25 Sephadex column .",
"FL his10-Cherry-SCARAPWCA was then separated from partially translated fragments using anion exchange chromatography ( i . e . , MonoQ ) and then separated from protein aggregates by size exclusion chromatography ( Superdex75 column ) .",
"For sedimentation equilibrium experiments , GFP-Lpd850−1250aa and VASP1−114aa ( monomeric EVH1 domain ) were diluted into 20 mM HEPES [pH 7] , 100 mM KCl , 1 mM TCEP and then centrifuged at 346716×g ( TLA 100 . 4 rotor , Beckman Coulter ) for 20 min to remove proteins that were potentially aggregated .",
"GFP-Lpd850−1250aa ( 9–10 µM ) was then combined with 10 , 25 , 50 , 75 , or 100 µM VASP1−114aa ( 13 . 1 kDa ) and loaded into 6-well Teflon chambers with quartz windows and placed in a 4 channel Ti-60 rotor .",
"Proteins were centrifuged at 7000 , 10 , 000 , and 14 , 000 rpm in a Beckman XL-I analytical ultracentrifuge at 20°C for 14–18 hr per speed or until equilibrium was reached .",
"Continuous scans of 488 nm absorbance were acquired every 2 hr in replicates of 10 , to monitor the sedimentation of GFP-Lpd850−1250aa in the absence and presence of VASP1−114aa .",
"An extinction coefficient of 55 , 000 M−1 cm−1 was used to calculate the GFP-Lpd850−1250aa protein concentration from the absorbance measured at 488 nm for each radial position .",
"Global fitting of three equilibrium traces from all speeds ( i . e . , 7 , 10 , 14K rpm ) for each condition was performed using open source NIH Sedphit and Sedphat software ( Peter Schuck , NIH ) .",
"Equilibrium traces were globally fit using a monomer-dimer self-association model .",
"Using Sednterp , we calculated a partial specific volume of 0 . 7354 ml/g and a buffer density of 1 . 00499 g/ml .",
"Our method for fitting the equilibrium traces involved fixing the meniscus position , floating the bottom position , and varying local concentrations in the experimental parameters .",
"Using Sedphat , the apparent molecular weight of GFP-Lpd850−1250aa was calculated by fixing the dimer concentration at zero and floating the molecular weight .",
"Glass was functionalized as described by Bieling et al . ( 2010 ) with some modifications .",
"In brief , coverglass ( Corning No . 1 . 5 , 18 × 18 mm sq . ) was cleaned by sonication in 3 M NaOH , followed by Pirahna etching ( 40% hydrogen peroxide , 60% sulfuric acid ) .",
"Coverslip sandwich were then incubated with undiluted 3-glycidyloxypropyl-trimetoxysilane ( GOPTS , Sigma 440167 ) for 30 min at 75°C .",
"Glass was then washed with anhydrous acetone ( Electron Microscopy Sciences; RT 10016 ) to remove excess silane and rapidly dried with an in house manufactured coverslip spin drier .",
"Next , hydroxyl-PEG3000 Da-NH2 ( 95% ) and CH3O-Biotin-PEG3000 Da-NH2 ( 5% ) powders ( Cat# 10-3000-20 and 10-3000-25-20 respectively; Rapp Polymere ) and dissolved in anhydrous acetone .",
"Silanized coverslips were assembled into sandwiches with 75 µl of dissolved amino-PEG/amino-PEG-biotin used per coverslip sandwich .",
"The pegylation reaction was performed at 75°C in glass weigh jars ( Fisher , Cat# 03-420-5C ) for ≥2 hr .",
"Glass was then washed with copious amounts of MilliQ water before spin drying and storing at room temperature in a dust free container .",
"Counterglass slides placed in coplin jars were bath sonicated in 3 M NaOH .",
"Glass was rinsed with water , bath sonicated in 100% ethanol , and then dried .",
"To minimize protein depletion in the TIRF experiments , the counterglass was coated with PLL-PEG ( PLL-PEG ) , which was dried onto the glass surface before rinsing with water .",
"Synthesis of PLL-PEG was performed as previously described by Huang et al . ( 2001 ) .",
"PEG/biotin-PEG functionalized coverslips were then attached to PLL-PEG coated counterglass with double sided tape ( Tesa ) .",
"To reduce nonspecific binding to the PEG functionalized surface the imaging chamber was washed with a 1× PBS solution pH 7 . 2 containing 1% Pluronic F-127 and 100 µg/ml beta casein .",
"Glass was then washed with buffer containing 20 mM HEPES pH 7 , 1 mM DTT , 200 mM KCl , 100 µg/ml beta-casein ( wash buffer ) .",
"Glass was subsequently incubated for 2 min with 50 nM streptavidin , followed by 50 nM biotin-PEG11 heavy meromyosin diluted in wash buffer .",
"To minimize nonspecific binding of GFP-Lpd and actin , it is essential that the stock solution of kappa casein is made fresh in water and centrifuged for 20 min at 346716×g ( TLA 100 . 4 rotor or equivalent ) to remove aggregated protein .",
"If this is not possible , kappa casein can be left out of the 1% Pluronic F-127 or replaced with beta casein .",
"Ultimately , the assay quality will depend on your ability to block defects on the biotin-PEG functionalized glass surfaces .",
"Because Lpd850−1250aa and human VASP are highly basic proteins , poor coverslip functionalization will result in a tremendous amount of non-specific protein absorption to the glass substrates used for TIRF microscopy .",
"The single actin filament TIRF assay was performed as described by Hansen and Mullins ( 2010 ) .",
"When using phalloidin stabilized actin filaments , 2 µM actin ( 20% Cy3 or Cy5 labeled ) was copolymerized with 1 µM dark phalloidin at room temperature for 2–3 hr in buffer containing 10 mM imidazole [pH 7] , 50 mM KCl , 1 mM EGTA , and 1 mM MgCl2 .",
"For experiments involving dynamic single actin filament elongation , actin polymerization was initiated by combining 1 µl of 10× ME ( 10× ME contains 0 . 5 mM MgCl2 , 2 mM EGTA ) with 9 µl of 4 . 44 µM monomeric actin ( 5–20% Cy5 labeled and diluted in G-buffer: 2 mM Tris [pH 8 . 0] , 0 . 5 mM TCEP , 0 . 1 mM CaCl2 , 0 . 2 mM ATP , 0 . 01% azide ) .",
"Actin was then combined with 2× TIRF imaging buffer ( 20 µl volume ) and 4× protein ( i . e . , VASP , profilin , Lpd ) ( 10 µl volume ) resulting in a final buffer composition of 20 mM HEPES [pH 7] , 50–100 mM KCl , 1 mM MgCl2 , 1 mM EGTA , 0 . 2% methylcellulose cP400 , 1 mg/ml BSA , 1 mM ATP , 20 mM BME , 1 mM Trolox ( Sigma , Cat# 238813 ) , 20 mM glucose , 125 µg/ml glucose oxidase ( Serva , #22780 . 01 Aspergillus niger ) , and 20 µg/ml catalase ( Sigma , #C40-100MG Bovine Liver ) .",
"A final reaction volume of 40 µl ( 10 µl actin , 20 µl 2× TIRF buffer , 10 µl VASP/Lpd/etc ) was flowed through a PEG/biotin-PEG TIRF flow cell , sealed with VALAP ( 1:1:1 mixture of Vaseline , lanoline , and paraffin wax ) , and imaged at 23°C .",
"To calculate the dissociation rate constant ( Kd ) for GFP-Lpd850−1250aa binding to single actin filaments ( Figure 1C ) we used TIRF microscopy to image the density of GFP-Lpd850−1250aa bound to phalloidin stabilized actin filaments ( 20% Cy5 labeled ) .",
"For these experiments , increasing concentrations of monomeric GFP-Lpd850−1250aa ( 0–1 µM ) diluted in TIRF buffer containing 50 mM KCl were sequentially flowed into an imaging chamber containing immobilized actin filaments .",
"After a 5 min incubation , GFP-Lpd850−1250aa and Cy5-Actin filaments were imaged across ≥10 fields of view and ≥2 chambers .",
"The average GFP-Lpd fluorescence intensity was calculated across ≥100 µm of filamentous actin .",
"Actin polymerization was initiated by mixing 4–20 µM A . castellani monomeric actin in buffer containing a final concentration of 20 mM HEPES [pH 7 . 0] , 50 mM KCl , 1 mM MgCl2 , 1 mM EDTA , 1 mM ATP , 1 mM TCEP , in the absence ( termed ‘native’ actin ) or presence of an equal molar concentration of dark phalloidin ( Calbiochem , Cat# 516640 ) .",
"Actin was then incubated at 23°C for 45–60 min to completely polymerize .",
"In parallel , GFP-Lpd or GFP-LZ-Lpd were diluted in buffer containing a final concentration of 20 mM HEPES [pH 7 . 0] , 50–150 mM KCl ( depending on the desired final salt concentration ) , and 1 mM TCEP .",
"Diluted Lpd ( 10–20 µM ) was then subjected to high-speed ultracentrifugation for 30 min in a TLA100 rotor at 60 , 000 rpm ( 156 , 424×g ) to remove potentially aggregated protein or particulate matter .",
"Using a spectrophotometer , the Lpd protein concentration was recalculate from the measured absorbance at 280 nm ( ε280 = 41 , 370 M−1 cm−1 and 44 , 350 M−1 cm−1 for GFP-Lpd850−1250aa and GFP-LZ-Lpd850−1250aa , respectively ) .",
"Pre-centrifugation typically results in a loss of 1–2% total protein , which is comparable to the error of most spectrophotometer readings .",
"GFP-Lpd is diluted further to a desired 2× working concentration , which is then mixed 1:1 with 2× pre-polymerized filamentous actin ( final reaction volume equals 50 µl ) .",
"Lpd and filamentous actin are then allowed to equilibrate at 23°C for 60 min .",
"Actin filaments with Lpd bound are then pelleted by ultracentrifugation in a TLA100 rotor at 48 , 000 rpm ( 100 , 111×g ) for 30 min .",
"After ultracentrifugation , the supernatant is removed and pellets are resuspended in 50 µl of 1× SDS-PAGE protein sample buffer ( 50 mM Tris-HCl [pH 6 . 8] , 100 mM DTT , 2% SDS , 10% glycerol , 0 . 1% bromophenol blue ) .",
"We then load 10 µl of resuspended pellets and load samples that were not centrifuges in a pre-casted NuPAGE 4–12% Bis-Tris gradient gel ( Invitrogen , Cat# NP0323BOX ) , which are then resolved using a Tris/MES/SDS running buffer [pH 7 . 3] .",
"SDS-PAGE gels were subsequently fixed for 30 min in a solution containing methanol:water:acetic acid ( 50:43:1 ) , before being stained with Sypro Ruby ( Invitrogen , Cat# S12000 ) .",
"Gels are then scanned using a Typhoon imaging system ( 488 nm/610 nm; EX/EM ) .",
"The concentration of Lpd and actin in the load and pellet were quantifies by measuring the integrated intensity of individual protein bands in gel after applying a local average background subtraction using ImageQuant .",
"When we process samples containing GFP-Lpd or GFP-LZ-Lpd alone that were included during the co-sedimentation assays ( Figure 1—figure supplement 1D ) , we observe the equivalent of 50–100 nM ( 5–10% of a 1 µM load ) Lpd removed from the supernatant in the absence of actin .",
"Because the amount of protein lost during the pre-spin and the co-sedimentation assay are roughly the same , we conclude that protein loss likely reflects non-specific absorption to the walls of the centrifuge tubes .",
"For this reason , we are likely over-estimating the stoichiometry of Lpd bound to actin in Figure 1I and Figure 1—figure supplement 1D by 5–10% .",
"The following lipids were used to make liposomes: 18:1 ( Δ9-Cis ) PC ( DOPC ) 1 , 2-dioleoyl-sn-glycero-3-phosphocholine ( Avanti #850375C ) and 18:1 DGS-NTA ( Ni ) [1 , 2-dioleoyl-sn-glycero-3-[ ( N- ( 5-amino-1-carboxypentyl ) iminodiacetic acid ) succinyl] ( nickel salt ) ( Avanti #790404C ) .",
"All lipids were combined in clean glass vials ( National Scientific Glass tubes with PTFE screw cap lid; Fisher 03-391-7B ) under a continuous stream of Argon gas .",
"The total amount of lipids used for each liposome preparation was 4 µmoles .",
"Lipid mixtures in chloroform were dried to a film under inert gas , followed by ≥2 hr of drying in a vacuum desiccator .",
"Lipid films were then resuspended with phosphate buffer saline [pH 7 . 2] to concentration of 4 mM and freeze-thawed 10-times using liquid nitrogen and a water bath at ambient temperature .",
"SUVs were generated by micro-tip sonication ( 4 × 15 s pulses , 20% amplitude ) and then micro-centrifuged at 4°C for 30 min at 21 , 430×g .",
"The supernatant was then transferred to a new eppendorf tube and stored on ice .",
"Vesicles were used the same day .",
"Glass microspheres were washed with nitric acid by diluting a 10% wt/vol slurry of 2 . 34 µm silica beads ( Bangs Laboratories , Cat# SS05N ) to 1% wt/vol in a glass vial .",
"Glass microspheres were incubated in nitric acid for 2–3 hr at room temperature .",
"Acid-washed glass beads were pelleted by centrifugation for 5 min and then washed with four times in a glass vial with MilliQ water .",
"Beads were resuspended in water to make a 10% wt/vol slurry .",
"Lipid bilayers was assembled on acid-washed glass beads by combining 20 µl of 10% bead slurry ( vortex/sonicate before aliquoting ) with 105 µl 20 mM HEPES [pH 7] , 150 mM NaCl ( or PBS [pH 7 . 2] ) in an eppendorf tube .",
"Diluted glass beads were vortexed and then bath sonicated for 5 min .",
"Monodisperse glass beads were then combined with 25 µl of 4 mM SUVs ( 4 mM = total lipid concentration ) , vortexed briefly , and then rotated at room temperature for 30 min .",
"After assembling the lipid bilayer , 750 µl of milliQ water was added to each tube and beads were micro-centrifuged for 2 min at 200×g .",
"The supernatant was aspirated off the beads and the 750 µl water wash , followed by micro-centrifuged was repeated four times .",
"After the final spin , the supernatant was aspirated leaving ∼50 µl of water/beads .",
"Beads were then resuspended by vortexing and 150 µl of buffer containing , 20 mM HEPES [pH 7] , 200 mM KCl was added .",
"The final bead slurry was ∼1% ( wt/vol ) and contained a final KCl concentration of 150 mM .",
"To charge lipid coated glass beads with protein , we combined 5 µl of 1% bead slurry with 45 µl of 111 nM his10 tagged protein ( i . e . , his10Cherry-SCAR ) .",
"Proteins were diluted into buffer containing 20 mM HEPES [pH 7] , 150 mM KCl , 100 µg/ml BSA , 0 . 5 mM TCEP .",
"Before all experiments , we determined the quality of lipid bilayer by charging beads with a mixture of 50 nM his10GFP and 50 nM his10Cherry-SCAR .",
"If the beads are uniformly coated with GFP and Cherry , you can assume the beads have a continuous and uniform membrane .",
"However , if you observed bright cherry fluorescent patches on glass beads that do not colocalize with GFP , a non-continuous bilayer was generated .",
"We also observed that his10Cherry-SCAR , but not his10GFP bound to bare glass microspheres under these conditions .",
"The reconstituted actin bead motility mix is based on protocols developed by Akin and Mullins ( 2008 ) .",
"Our bead motility assay contained 20 mM HEPES pH 7 , 100 mM KCl , 1 mM MgCl2 , 1 mM EGTA , 1 mM ATP , 0 . 2% methylcellulose ( cP 400 ) , 2 . 5 mg/ml BSA , 20 mM beta-mercaptoethanol , 7 . 5 µM cytoplasmic actin ( A . castellani ) , 50–125 nM mouse capping protein ( recombinant ) , 100–150 nM Arp2/3 ( native , A . castellani ) , 7 . 5 µM human profilin I ( recombinant ) , 3–5 µM human cofilin ( recombinant ) , and 0 . 1% ( wt/vol ) 2 . 3 µm lipid coated glass beads charged with 100 nM his10-Cherry-SCARAPWCA for 15–20 min .",
"After reactants were mixed to initiate actin assembly , samples were with quench or immediately flowed in to glass coverslip chamber , which was then sealed with VALAP .",
"In vitro quenching of actin assembly and disassembly was accomplished by combining equal volumes of the bead motility reaction and 37 . 5 µM Latrunculin B-phalloidin ( 1:1 , therefore 5 molar excess relative to concentration of actin ) diluted in buffer containing 20 mM HEPES [pH 7] , 100 mM KCl , 100 µg/ml BSA , 0 . 5 mM TCEP .",
"Imaging chamber for the bead motility assay were assembled with glass silanized with a 2% solution of diethyldichlorosilane ( Gelest , Cat# SID 3402 . 0 ) in isopropanol ( pH 4 . 5 with acetic acid ) ( Akin and Mullins , 2008 ) .",
"Xenopus fibroblasts ( XTC cells ) were maintained at 23°C ( without CO2 ) in 70% diluted Leibovitz L-15 media ( Invitrogen/Gibco , Cat# 21083-027 no phenol red ) containing 10% heat inactivated fetal bovine serum ( Gibco ) and penicillin/streptomycin ( Watanabe and Mitchison , 2002 ) .",
"Cells that were 70% confluent in 24-well plastic dishes containing complete media were transiently transfected with 500 ng of plasmid DNA and 1 . 5 µl Lipofectamine LTX with 1 µl PLUS reagent ( Invitrogen , Cat# A12621 ) .",
"After 5 hr of transfection , media was exchanged .",
"Prior to live cell imaging , XTC cells were trypsinized and plated in 70% L-15 media lacking phenol red and FBS .",
"XTC cells were plated onto a glass bottom 96-well plate ( Matrical Bioscience , MGB096-1-2-LG ) cleaned with 3 M NaOH for 1 hr and subsequently coated with 0 . 01% ( wt/vol ) PLL ( Sigma , P-8920 ) for 20–30 min at 23–25°C .",
"Cells were imaged on a Nikon TIRF microscope at 23–25°C .",
"For experiments in which we froze actin assembly and disassembly with the JLY cocktail , cells were allowed to spread on PLL coated glass for 30 min in the presence of 10 µM Rock kinase inhibitor ( Y27632 ) .",
"Cells were then treated with 10 µM Jasplakinolide ( CalBiochem , Cat# 420107 ) , 8 µM Latrunculin B ( Enzo Life Science , Cat# T110-0001 ) , and 10 µM Y27632 Rock kinase inhibitor ( CalBiochem , Cat# 688001 ) to freeze actin assembly and disassembly in XTC cells ( Peng et al . , 2011 ) .",
"All drugs were diluted to a 2× final concentration in 70% L-15 media lacking serum and penicillin/streptomycin before being added to cells .",
"B16F1 mouse melanoma cells ( ATCC CRL-6323 ) were cultured in Dulbecco Modified Eagles High glucose containing 10% heat inactivated fetal bovine serum and penicillin/streptomycin .",
"Cells were grown in 25 cm2 flasks at 37°C in the presence of 5% CO2 and split every 3–4 days .",
"For transfection of pCMV-EGFP-Lpd ( 850–1250aa ) , cells were plated in 24-well dish to a confluency of 50–60% .",
"We added 0 . 5–1 . 0 µg DNA in complex with 2 . 5–5 . 0 µl Superfect ( Qiagen , Cat# 1006699 ) to each well containing complete media .",
"After 5–6 hr , the media was changed .",
"24–36 hr after transfection , cells were prepared for live cell imaging .",
"Circular glass coverslips ( 25 mm , Warner Instruments Cat# 64-0715 ) were cleaned with 3 M NaOH for 30 min at 23°C .",
"Coverslips were rinsed extensively with MilliQ water and coated with 50 µg/ml mouse laminin ( Sigma , Cat# 23017-015 ) diluted in PBS [pH 7 . 2] at 37°C for 2 hr .",
"Laminin coated coverslips were rinse with PBS and then assembled in stainless steel Attofluor cell imaging chamber ( Invitrogen ) .",
"Transfected cells were then trypsinized in 24-well plastic dishes , centrifuged , wash with complete media , and seeded on the laminin coated glass in the presence of filter sterilized Ham's F12 media containing 10% FBS and 50 mM HEPES [pH 7 . 2] .",
"Cells spread on laminin coated glass within 30 min .",
"Polarized cells were imaged using wide-field epifluorescence and a Nikon Plan Apo 60× TIRF ( NA 1 . 45 ) objective on a Nikon Eclipse microscope .",
"XTC cell images were acquired on an inverted Nikon Eclipse TIRF microscope using either a 60× Nikon Plan Apo 60× TIRF ( NA 1 . 45 ) or a 100× Nikon TIRF ( 1 . 49 NA ) objective at 23–25°C .",
"B16F1 cells were imaged with Nikon Plan Apo 60× TIRF ( NA 1 . 45 ) using wide-field epifluorescence illumination at 37°C in the presence of CO2 .",
"We employed the Nikon Perfect Focus instrument to maintain the focal plan throughout image acquisition .",
"GFP/Alexa488 , Cy3 , and Cy5 fluorophores were excited with 491 nm , 561 nm , and 638 nm Coherent lasers respectively .",
"Laser power was modulated with neutral density filters , an AOTF , and exposure settings such that 1–2 mW of laser power was delivered through the objective as measured with a power meter .",
"We used a single filter cube containing a multipass excitation and dichroic filter ( Chroma ) .",
"A rapid switching Sutter Instruments emission filter wheel was positioned before our camera .",
"Images of XTC cells , B16F1 cells , and single actin filament TIRF assay were collected on a cooled Andor Xion EM-CCD camera .",
"The microscope , camera , and lasers were controlled using Micromanager 1 . 4 ( Edelstein et al . , 2010 ) .",
"Image processing and data analysis was performed using ImageJ .",
"Kymographs were generated using the ImageJ plugin , MultipleKymographs .",
"Data was graphed using Kaleidagraph and Prism .",
"Figures for the manuscript were made in Adobe Illustrator CS6 ."
]
] | [
"Enabled/Vasodilator ( Ena/VASP ) proteins promote actin filament assembly at multiple locations , including: leading edge membranes , focal adhesions , and the surface of intracellular pathogens .",
"One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons .",
"To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin ( Lpd ) , actin , and VASP , both in vivo and in vitro .",
"We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity .",
"We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments .",
"This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly ."
] | [
"Actin—the most abundant protein in most eukaryotic cells—assembles into a network of filaments that spans the length and breadth of the cell .",
"Like the skeleton of an animal , this ‘actin cytoskeleton’ gives the cell its shape and strength , and enables the cell to actively move through its environment .",
"To start moving , many cells begin assembling actin filaments next to the cell membrane .",
"The growth of these filaments pushes the membrane forward and creates a two-dimensional structure called a ‘lamellipod’ , which explores the space around the cell and steers its movement .",
"The actin filaments in a lamellipod are dynamic and undergo repeated cycles of assembly and disassembly .",
"These processes are tightly regulated by a variety of other proteins .",
"Members of the Ena/VASP protein family , for example , collect the building blocks of an actin filament and rapidly stack them in place on the fast-growing end of a filament .",
"The activities of Ena/VASP proteins play an especially important role in creating lamellipodial actin networks and in driving cell movement .",
"Previous work showed that a protein called Lamellipodin binds to Ena/VASP proteins and helps recruit them to the cell membrane .",
"However , it was unclear whether Lamellipodin could affect the activity of Ena/VASP proteins or their interaction with the actin filaments .",
"Hansen and Mullins have now analyzed the interactions between Ena/VASP , Lamellipodin and actin .",
"The experiments demonstrate that Lamellipodin does not simply tether Ena/VASP proteins to the membrane but also binds directly to actin filaments , via a binding site that is distinct from the site that contacts Ena/VASP .",
"Further experiments with purified proteins revealed that Lamellipodin could interact with both actin filaments and Ena/VASP proteins at the same time .",
"Hansen and Mullins also found that purified Lamellipodin interacted with VASP proteins to form clustered protein complexes , and that together with the tethering of actin filaments to the membrane , this clustering greatly increased VASP's ability to lengthen actin filaments .",
"By visualizing Lamellipodin tagged with a green fluorescent protein in living cells , Hansen and Mullins then showed that its interaction with actin filaments was sufficient to localize Lamellipodin to the cell membrane .",
"Finally , since Lamellipodin interacts with a multitude of signaling molecules in addition to Ena/VASP proteins , the next big challenge is to understand how Lamellipodin itself is regulated .",
"Future studies could also explore how cells harness the power of the actin cytoskeleton to carry out these essential activities ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"cell biology"
] | Phospholipase D activity couples plasma membrane endocytosis with retromer dependent recycling | elife-18515-v2 | [
[
"The ability to detect photons is a fundamental property of animal photoreceptors .",
"In order to achieve this , ocular photoreceptors of animals generate an expanded region of plasma membrane that is packed with the receptor for light , rhodopsin .",
"This strategy is used regardless of the architecture of the photoreceptor .",
"For example , in ciliary photoreceptors ( e . g vertebrate rods ) , light passes along the outer segment that is stacked with membranous discs , whereas in insect photoreceptors , the plasma membrane is expanded to form actin-based microvilli; both these structures are packed with rhodopsin , and incident light is absorbed as it passes along them ( Arendt , 2003 ) .",
"The light-sensitive membranes of photoreceptors undergo stimulus-dependent turnover ( LaVail , 1976; White and Lord , 1975 ) ; such turnover will alter both membrane area and composition , thus regulating sensitivity to light [reviewed in ( Blest , 1988 ) ] .",
"The importance of this process is underscored by the human disease Best’s macular dystrophy , in which rod outer segment length and electroretinograms are altered during changes in ambient illumination , ultimately leading to macular degeneration ( Abràmoff et al . , 2013 ) .",
"Despite the importance of this process , the cellular and molecular mechanisms that regulate photosensitivemembrane turnover remains poorly understood .",
"In Drosophila photoreceptors , the apical domain is expanded to form ca .",
"40 , 000 projections of light-sensitive plasma membrane ( microvilli ) that form the rhabdomere .",
"Photons that are absorbed trigger G-protein-coupled phospholipase C ( PLC ) activity that culminates in the activation of the plasma membrane channels TRP and TRPL; the resulting Ca2+ influx triggers an electrical response to light ( Hardie and Raghu , 2001 ) .",
"Additionally , photon absorption by rhodopsin1 ( Rh1 ) also triggers the rhodopsin cycle [reviewed in ( Raghu et al . , 2012 ) ] .",
"Following photon absorption , Rh1 undergoes photoisomerization to meta-rhodopsin ( M ) .",
"M is phosphorylated at its C-terminus , binds β-arrestin and this complex is removed from the microvillar membrane via clathrin-dependent endocytosis to be either recycled back to the microvillar plasma membrane ( Wang et al . , 2014 ) or trafficked to the lysosomes for degradation ( Chinchore et al . , 2009 ) [reviewed in ( Xiong and Bellen , 2013 ) ] .",
"Tight regulation of this process is critical for rhabdomere integrity during illumination as mutants defective in any of the several steps of the rhodopsin cycle undergo light-dependent collapse of the rhabdomere [reviewed in ( Raghu et al . , 2012 ) ] .",
"However , the process that couples endocytosis of rhabdomere membrane to plasma membrane recycling remains poorly understood .",
"Phospholipase D ( PLD ) is an enzyme that hydrolyzes phosphatidylcholine ( PC ) to generate phosphatidic acid ( PA ) .",
"In yeast , loss of PLD ( spo14 ) results in a sporulation defect , failure to synthesize PA ( Rudge et al . , 2001 ) and accumulation of undocked membrane vesicles on the spindle pole body ( Nakanishi et al . , 2006 ) .",
"The v-SNARE Spo20p binds PA in vitro ( Nakanishi et al . , 2004 ) and is required to dock Spo20p to target membranes; in this setting , PA generated by PLD appears to regulate a vesicular transport process .",
"The potential role of PA in controlling vesicular transport also arose from observations in vitro that Arf proteins , key regulators of vesicular transport , stimulate mammalian PLD activity ( Brown et al . , 1993; Cockcroft et al . , 1994 ) .",
"Overexpression of PLD1 in a range of neuronal ( Cai et al . , 2006; Vitale et al . , 2001 ) and non-neuronal cells ( Choi et al . , 2002; Cockcroft et al . , 2002; Huang et al . , 2005 ) suggests that PLD can regulate vesicular transport .",
"A previous study showed that elevated PA levels during development of Drosophila photoreceptors disrupts rhabdomere biogenesis with associated endomembrane defects ( Raghu et al . , 2009 ) that were Arf1-dependent .",
"However , the mechanism underlying the role of PLD in regulating membrane transport has remained unclear , and to date , no study in metazoans has demonstrated a role , if any , for endogenous PLD in regulating vesicular transport in vivo .",
"In this study , we show that during illumination in Drosophila photoreceptors , rhabdomere size is regulated through the turnover of apical plasma membrane via RLVs .",
"We find that photoreceptors have a light-regulated PLD activity that is required to maintain PA levels during illumination and support apical membrane size .",
"PLD works in coordination with retromer function and Arf1 activity to regulate apical membrane size during illumination .",
"Thus , PLD is a key regulator of plasma membrane turnover during receptor activation and signaling in photoreceptors ."
],
[
"We quantified rhabdomere size of Drosophila photoreceptors during illumination by transmission electron microscopy ( TEM ) followed by volume fraction analysis .",
"When wild-type flies are grown in white light for 48 hr ( hrs ) post-eclosion , the volume fraction ( Vf ) of the cell occupied by the rhabdomere in peripheral photoreceptors R1-R6 was reduced ( Figure 1A , B ) .",
"This reduction in Vf occurred prior to the onset of any obvious vesiculation or rhabdomere degeneration; the Vf of rhabdomere R7 that expresses UV-sensitive rhodopsin ( that does not absorb white light ) did not change ( Figure 1A , B ) .",
"This reduction in rhabdomere size was accompanied by changes in the localization of Rh1 , the rhodopsin isoform expressed in R1-R6 .",
"With just 12 hr of illumination , there was an increase in the number of RLVs in the cell body ( Figure 1C , D ) .",
"A subset of these RLVs co-localize with the early and late endocytic compartment markers Rab5 and Rab7 , respectively ( Figure 1E , F ) .",
"Over a period of 4 days , illumination results in a reduction in total Rh1 protein levels ( Figure 1G ) and manifests functionally as a reduction in sensitivity to light ( Figure 1H ) . 10 . 7554/eLife . 18515 . 003Figure 1 . Rhabdomere size regulation during illumination in Drosophila photoreceptors .",
"( A ) TEM images of single rhabdomere from wild-type photoreceptors ( PRs ) of 2-day-old flies post-eclosion reared in constant dark ( CD ) , 12 hr light , 12 hr dark ( 12 h L/D ) and constant light ( CL ) .",
"Scale bar: 1 µm .",
"( B ) Quantification of rhabdomere volume in PRs reared in various conditions .",
"The peripheral PRs represent R1 to R6 rhabdomeres .",
"The X-axis represents the rearing condition and the Y-axis represents the volume fraction ( Vf ) of rhabdomere expressed as a % with respect to total cell volume .",
"n = 90 rhabdomeres taken from three separate flies .",
"( C ) Longitudinal section ( LS ) of retinae from control stained with rhodopsin 1 ( Rh1 ) antibody .",
"Flies were dissected after 0–6 hr ( day 0 ) and 12 hr of bright light illumination ( 12 h CL ) post-eclosion .",
"Scale bar: 5 µm .",
"( D ) Quantification of RLVs from LS of retinae from control .",
"The X-axis represents the time point and rearing condition .",
"Y-axis shows the number of RLV’s per ommatidium .",
"n = 10 ommatidia taken from three separate preps .",
"( E ) LS of retinae from control stained with Rh1 and Rab5; Rh1 and GFP ( for Rh1>GFP::Rab7 ) .",
"Rearing condition is same as mentioned in ( panel C ) .",
"Scale bar: 5 µm .",
"( F ) Quantification of RLVs from LS of retinae from control .",
"The X-axis represents the population of vesicles positive for mentioned protein .",
"Y-axis shows the number of RLVs per ommatidium .",
"n = 10 ommatidia taken from three separate preps .",
"( G ) Western blot from head extracts of control flies reared in various conditions as indicated on the top of the blot .",
"The blot was probed with antibody to rhodopsin .",
"Tubulin levels were used as a loading control .",
"( H ) Intensity response function of the light response from 4-day-constant-light ( DAY 4 CL ) and 4-day-constant-dark ( DAY 4 CD ) old control flies .",
"The X-axis represents increasing light intensity in log units and Y-axis the peak response amplitude at each intensity normalized to the response at the maximum intensity .",
"n=separate flies .",
"Data are presented as mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 18515 . 003 We generated loss-of-function mutants in dPLD using homologous recombination ( Gong and Golic , 2003 ) ( Figure 2—figure supplement 1A ) .",
"Multiple alleles were isolated of which dPLD3 . 1 is described in detail .",
"To test if dPLD3 . 1 represents an animal with no residual PLD activity , we used the transphosphatidylation assay that exploits the ability of PLD to use primary alcohols as nucleophilic acceptor .",
"Flies were starved for 12 hr , allowed to feed for 6 hr on 10% ethanol/sucrose and the formation of phosphatidylethanol ( PEth ) was monitored using LC-MS ( Wakelam et al . , 2007 ) .",
"Under these conditions , multiple species of PEth were detected in wild-type flies , no PEth could be detected in dPLD3 . 1 extracts under the equivalent conditions ( Figure 2—figure supplement 1C , D ) .",
"Thus , dPLD3 . 1 mutants have no residual PLD activity .",
"dPLD3 . 1 flies are homozygous viable as adults .",
"At eclosion , photoreceptor ultrastructure in dPLD3 . 1 was indistinguishable from controls ( Figure 2A ) .",
"Following exposure to 2000 lux white light for 48 hr , as expected , Vf occupied by peripheral rhabdomeres was reduced in wild-type flies ( Figure 2B ) , whereas Vf of R7 was unaffected .",
"In dPLD3 . 1 , rhabdomere Vf reduced following illumination ( Figure 2B ) ; however , the reduction was substantially greater than in wild type ( Figure 2C ) . 10 . 7554/eLife . 18515 . 004Figure 2 . dPLD is required to support rhabdomere volume during illumination .",
"( A ) TEM images showing single ommatidium from control and dPLD3 . 1 .",
"PRs of 0- to 12-hr-old flies post eclosion .",
"Scale bar: 1 µm ( B ) Quantification of the rhabdomere volume of control and dPLD3 . 1 .",
"PRs reared in constant dark and constant light for 2 days post-eclosion .",
"n = 90 rhabdomeres taken from three separate flies .",
"( C ) Quantification of fold reduction in rhabdomere volume of control and dPLD3 . 1 in light compared to dark .",
"Genotypes are indicated on the X-axis and the Y-axis represents the percentage volume fraction ( Vf ) of the rhabdomere with respect to cell .",
"( D ) LS of retinae stained with rhodopsin one from dPLD3 . 1 .",
"Rearing conditions are indicated .",
"Scale bar: 5 µm .",
"( E ) Quantification of RLVs from LS of retinae from control and dPLD3 . 1 .",
"n = 10 ommatidia taken from three separate preps .",
"( F ) Quantification of RLVs from LS of retinae from control and dPLD3 . 1 reared in 12 hr CL .",
"The X-axis represents the population of vesicles positive for mentioned protein .",
"Y-axis shows the number of RLVs per ommatidium .",
"n = 10 ommatidia taken from three separate preps .",
"( G ) Western blot from head extracts of control ( C ) and dPLD3 . 1 ( P ) of matched eye color .",
"Rearing conditions as indicated on the top of the blot .",
"The blot was probed with antibody to rhodopsin .",
"Tubulin levels were used as a loading control .",
"( H ) Quantification of fold reduction of rhodopsin seen in dPLD3 . 1 normalized to controls .",
"The X-axis shows the genotype .",
"Y-axis represents the fold reduction in rhodopsin .",
"n = 3 .",
"( I ) Representative ERG responses of 0- to 12-hr-old flies to a single 2 s flash of green light .",
"Genotypes are indicated .",
"X-axis represents the time in seconds ( s ) and the Y-axis represents the amplitude of response in mV .",
"The duration of light pulse is indicated .",
"( J ) Intensity response function of the light response of 0- to 12-hr-old flies .",
"Responses from control and dPLD3 . 1 flies with matched eye color are shown .",
"The X-axis represents increasing light intensity in log units and Y-axis the peak response amplitude at each intensity normalized to the response at the maximum intensity .",
"n= five separate flies .",
"Data are presented as mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 18515 . 00410 . 7554/eLife . 18515 . 005Figure 2—figure supplement 1 . Characterization of dPLD3 . 1 loss-of-function allele .",
"( A ) Schematic diagram representing the method used to generate the knockout of Drosophila phospholipase D ( dPLD3 . 1 ) using homologous recombination .",
"The mutant allele generated with respect to the wild-type locus is shown .",
"The domains of dPLD ( PX , PH , catalytic HKD1 and HKD2 and PIP2-binding domains ) are shown .",
"The C-terminal domain is marked in red .",
"A Pw+ insertion ( red box ) that disrupts the HKD1 domain with stop codons in all three frames on both strands ( white boxes ) is indicated as P[w+] .",
"The last three amino acids at the C-terminus that have been mutated are shown as a black box .",
"T5 , T6 and T7 marks the primers designed in the HKD1 motif of dPLD .",
"( B ) PCR analysis for presence of HKD1 motif from crude genomic DNA extracts of WT ( 1 ) dPLD3 . 1 ( 2 ) dPLD3 . 1/Df ( 2R ) ED1612 ( 3 ) and water control ( 4 ) .",
"One the left side of the gel picture , primer pairs used are mentioned and on the right side , the product lengths are indicated .",
"( C ) Total amounts of various phosphatidylcholine ( PC ) species extracted and measured from flies used for the transphosphatidylation assay experiment .",
"The X-axis shows acyl chains species that were detected .",
"Y-axis represents the mole percent of phosphatidylethanol species .",
"Species measured from wild type and dPLD3 . 1 with ( 10% ) and without ( 0% ) ethanol are shown .",
"( D ) The generation of phosphatidylethanol ( P-EtOH ) by dPLD ( via the enzyme’s transphosphatidylation activity ) was measured .",
"The X-axis shows acyl chains species that were detected .",
"Y-axis represents the mole percent of phosphatidylethanol species .",
"Species measured from wild type and dPLD3 . 1 with ( 10% ) and without ( 0% ) ethanol are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 18515 . 00510 . 7554/eLife . 18515 . 006Figure 2—figure supplement 2 . Normal levels and localization of other apical domain proteins in dPLD3 . 1 . ( A ) Western blot from head extracts of control ( C ) and dPLD3 . 1 ( P ) reared in various conditions as indicated on the top of the blot .",
"The blot was probed with antibody to NORPA and TRP .",
"Tubulin was used as loading control .",
"( B ) Confocal images of transverse section of retinae stained with an antibody to TRP in control and dPLD3 . 1 .",
"Cross-sections of the rhabdomere stained in red are shown .",
"Scale bar 5 µmDOI: http://dx . doi . org/10 . 7554/eLife . 18515 . 00610 . 7554/eLife . 18515 . 007Figure 2—figure supplement 3 . Electrophysiological characterization of dPLD3 . 1 . ( A ) Representative ERG responses of 0- to 12-hr-old flies to a single 10 s flash of green light .",
"Genotypes are indicated .",
"X-axis represents the time in seconds ( s ) and the Y-axis represents the amplitude of response in mV .",
"The duration of light pulse is indicated .",
"( B ) Quantification of the light response .",
"Y-axis represents the ratio of final ( Af ) and initial ( Ai ) amplitude of single trace during the stimulus in percentage .",
"X-axis represents the genotypes .",
"n = 3 separate flies ( C ) Representative ERG responses of 0- to 12-hr-old flies to a 1 s flash of green light train - five pulses .",
"Genotypes are indicated .",
"X-axis represents the time in seconds ( s ) and the Y-axis represents the amplitude of response in mV .",
"The duration of light pulse is indicated .",
"( D ) Quantification of the light response .",
"Y-axis represents the ratio of final ( Af # 5 pulse ) and initial ( Ai # 1 pulse ) amplitude during the stimulus in percentage .",
"X-axis represents the genotypes .",
"n = 3 separate flies Data are presented as mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 18515 . 007 We visualized RLVs in photoreceptors by Rh1 immunolabeling and counted them .",
"These analyses were done at 0 days post-eclosion , prior to the onset of any obvious ultrastructural change in dPLD3 . 1 .",
"In dark-reared flies , the number of RLVs in dPLD3 . 1 was greater than in wild-type photoreceptors ( Figure 2D , E ) .",
"Following illumination for 12 hr , the number of RLVs increases in both controls and dPLD3 . 1; however , the increase was greater in dPLD3 . 1 ( Figure 2E ) .",
"Further , while the number of RLVs that were Rab5-positive was not significantly different between controls and dPLD3 . 1 , the number of Rab7-positive RLVs were significantly greater in dPLD3 . 1 compared to controls ( Figure 2F ) .",
"Thus , during illumination there is enhanced accumulation of RLVs in a Rab7 positive compartment in dPLD3 . 1 .",
"We measured Rh1 protein levels using Western blotting in flies exposed to bright illumination for four days post-eclosion .",
"As expected , levels of Rh1 decreased when wild-type flies were reared in bright light compared to dark-reared controls ( Figure 2G ) .",
"In dark reared flies , Rh1 levels are equivalent in controls and dPLD3 . 1 ( Figure 2G ) ; following illumination Rh1 levels decrease in both genotypes but the reduction seen in dPLD3 . 1 is much greater than in wild-type flies of matched eye color ( Figure 2G , H ) .",
"Consistent with this , we found that dPLD3 . 1 photoreceptors were less sensitive to light compared to controls of matched eye color on eclosion ( Figure 2J ) .",
"These findings demonstrate that during illumination , the turnover of Rh1 , an apical membrane protein of photoreceptors is altered in dPLD3 . 1 .",
"However , such changes were not seen in the levels or localization of TRP and NORPA , two other apical membrane proteins , during illumination ( Figure 2—figure supplement 2A , B ) .",
"dPLD3 . 1 photoreceptors did not exhibit a primary defect in the electrical response to light in electroretinograms ( Figure 2I , Figure 2—figure supplement 3 A-D ) .",
"We grew flies in constant illumination following eclosion .",
"Under these conditions , control photoreceptors maintain normal structure; however , dPLD3 . 1 undergoes light-dependent retinal degeneration .",
"The degeneration starts by day 5 post-eclosion and all six peripheral photoreceptors degenerate by day 14 ( Figure 3A , B ) .",
"This degeneration is strictly dependent on illumination as dPLD3 . 1 not exposed to light retains normal ultrastructure up to day 14 ( Figure 3A , B ) .",
"Photoreceptor degeneration is underpinned by a collapse of the apical microvillar membrane as well as the accumulation of membranous whorls within the cell body ( Figure 3C ) .",
"Retinal degeneration was also seen in a trans-heterozygote combination of two independently isolated alleles dPLD3 . 1 and dPLD3 . 3 ( Figure 3—figure supplement 1A , B ) .",
"No degeneration was seen in either dPLD3 . 1/+ or dPLD3 . 3/+ ( Figure 3—figure supplement 1A ) excluding a dominant negative or neomorphic effect of these alleles .",
"The light-dependent degeneration was also seen when the dPLD3 . 1 allele was placed over a deficiency chromosome for the dPLD gene region; in dPLD3 . 1/Df ( 2R ) ED1612 , retinal degeneration was comparable and was no worse than in dPLD3 . 1 homozygotes ( Figure 3—figure supplement 1C ) , suggesting that dPLD3 . 1 is a null allele .",
"Light-dependent degeneration in dPLD3 . 1could be rescued by a wild-type transgene [dPLD3 . 1;Hs>dPLD] but not by a lipase dead transgene [dPLD3 . 1;Hs>dPLDK/R] ( Figure 3D , E , F ) .",
"These results demonstrate that dPLD enzyme activity is required to support normal photoreceptor ultrastructure during illumination . 10 . 7554/eLife . 18515 . 008Figure 3 . dPLD is essential to support rhabdomere structure during illumination .",
"( A ) Representative optical neutralization ( ON ) images showing rhabdomere structure from control and dPLD3 . 1 .",
"The age and rearing conditions are mentioned on the top of the panels .",
"( B ) Quantification of rate of PR degeneration of control and dPLD3 . 1 reared in bright light .",
"The X-axis represents age of the flies and the Y-axis represents the number of intact rhabdomeres visualized in each ommatidium .",
"n = 50 ommatidia taken from at least five separate flies .",
"( C ) TEM images showing a single ommatidium from control and dPLD3 . 1 PRs reared in bright illumination for 6 days post eclosion .",
"* indicates the collapsed rhabdomere and the arrow head indicate whorl like membranes accumulated in the cell body .",
"Scale bar 1 µm .",
"( D ) Representative ON images showing ommatidia from dPLD3 . 1;Hs>dPLD and dPLD3 . 1;Hs>dPLDK/R .",
"The age and rearing conditions are indicated on the top of the image .",
"( E ) Quantification of rate of PR degeneration of control , dPLD3 . 1 , dPLD3 . 1;Hs>dPLD and dPLD3 . 1;Hs>dPLDK/R reared in bright light .",
"n = 50 ommatidia taken from at least five separate flies .",
"( F ) TEM images showing a single ommatidium from control , dPLD3 . 1 , dPLD3 . 1;Hs>dPLD and dPLD3 . 1;Hs>dPLDK/R PRs reared in light for 10 days post-eclosion .",
"Scale bar 1 µm .",
"Data are presented as mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 18515 . 00810 . 7554/eLife . 18515 . 009Figure 3—figure supplement 1 . Genetic validation of the retinal degeneration phenotype of dPLD3 . 1 . ( A ) Quantification of retinal degeneration in dPLD3 . 1/+ , dPLD1 . 1/+ , dPLD3 . 1/ dPLD1 . 1 .",
"The X-axis represents age of the flies and the Y-axis represents the number of rhabdomere visualized in each ommatidium .",
"Error bars represents mean ± SEM from 50 ommatidia taken from at least five separate flies .",
"( B ) Quantification of retinal degeneration in control , dPLD3 . 1 , dPLD3 . 1/ dPLD1 . 1 .",
"The X-axis represents age of the flies and the Y-axis represents the number of rhabdomere visualized in each ommatidium .",
"Error bars represents mean ± SEM from 50 ommatidia taken from at least five separate flies .",
"( C ) Quantification of retinal degeneration in Df ( 2R ) ED1612/+ , dPLD3 . 1 , dPLD3 . 1/Df ( 2R ) ED1612 .",
"The X-axis represents age of the flies and the Y-axis represents the number of rhabdomere visualized in each ommatidium .",
"Error bars represents mean ± SEM from 50 ommatidia taken from at least five separate flies . DOI: http://dx . doi . org/10 . 7554/eLife . 18515 . 009 In order to understand the biochemical basis of retinal degeneration of dPLD3 . 1 , we measured levels of PC and PA from retinal extracts using direct infusion mass spectrometry ( Schwudke et al . , 2011 ) .",
"We found that levels of PC were not significantly different between controls and dPLD3 . 1 ( Figure 4A ) .",
"By contrast , there was a significant decrease in total PA levels in dPLD3 . 1 ( Figure 4B ) .",
"At the level of molecular species , this reduction was associated with changes in the levels of PA species with longer acyl chain lengths ( Figure 4C ) .",
"Rescue of retinal degeneration in dPLD3 . 1 by reconstitution with Hs>dPLD was associated with restoration in PA levels back to that of controls ( Figure 4D ) .",
"Reconstitution with Hs>dPLDK/R that failed to rescue degeneration also did not restore PA levels in dPLD3 . 1 ( Figure 4D ) .",
"These results show that retinal degeneration in dPLD3 . 1 is correlated with reduced PA levels . 10 . 7554/eLife . 18515 . 010Figure 4 . Phosphatidic acid levels and retinal degeneration in dPLD3 . 1 . ( A ) Total PC level in retinae of control and dPLD3 . 1 .",
"The X-axis represents the genotypes and the Y-axis shows the level of PC as pmole/µmole of total lipid phosphate present in the sample .",
"n = 3 .",
"( B ) Total PA level in retinae of control and dPLD3 . 1 .",
"The X-axis represents the genotypes and the Y-axis shows the level of PA as pmole/µmole of total lipid phosphate present in the sample .",
"n = 3 .",
"( C ) Molecular species of PA in retinae of control and dPLD3 . 1 .",
"X-axis shows the acyl chain composition of each species predicted from its monoisotopic peaks and corresponding elemental composition constraints .",
"Y-axis shows the abundance of each species as pmole/µmole of total lipid phosphate present in the sample .",
"n = 3 .",
"( D ) PA levels in heads extracts of control , dPLD3 . 1 , dPLD3 . 1;Hs>dPLD and dPLD3 . 1;Hs>dPLDK/R .",
"n = 3 .",
"( E ) Quantification of retinal degeneration seen in control , laza22 , dPLD3 . 1 and dPLD3 . 1;laza22 .",
"n= 50 ommatidia taken from at least five separate flies .",
"( F ) PA levels in heads extracts of control , laza22 , dPLD3 . 1 and dPLD3 . 1;laza22 n = 3 .",
"( G ) PA levels from retinal extracts of Gq1 and Gq1 , dPLD3 . 1 .",
"Flies were reared in complete darkness and post ecclosion one set of flies were shifted to bright illumination for 12 hr while the others kept in darkness for 12 hr . n = 3 .",
"( H ) LS of retinae stained with Rh1 from Rh1>dPLD and Rh1>dPLDK/R .",
"Rearing conditions are indicated at the top of panels .",
"Scale bar:5 µm .",
"( I ) Quantification of RLVs from LS of retinae from control , Rh1>dPLD and Rh1>dPLDK/R .",
"n = 10 ommatidia taken from three separate preps .",
"Data are presented as mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 18515 . 01010 . 7554/eLife . 18515 . 011Figure 4—figure supplement 1 . Rescue of dPLD3 . 1 phenotypes by overexpression of DGK .",
"( A ) Quantification of retinal degeneration in control , Rh1>rdgA , dPLD3 . 1 , dPLD3 . 1;Rh1>rdgA .",
"The X-axis represents age of the flies and the Y-axis represents the number of rhabdomere visualized in each ommatidium .",
"Error bars represents mean ± SEM from 50 ommatidia taken from at least five separate flies .",
"( B ) PA levels in heads extracts .",
"Genotypes indicated on X-axis .",
"Y-axis shows the total PA as pmole/µmole of total lipid phosphate present in the sample .",
"Error bars indicate the mean ± SEM from three separate analyses . DOI: http://dx . doi . org/10 . 7554/eLife . 18515 . 011 We hypothesized that if the retinal degeneration in dPLD3 . 1 is due to reduced PA levels , elevating PA levels in dPLD3 . 1 retinae by methods independent of dPLD activity should rescue this phenotype .",
"It is reported that in laza22 photoreceptors , lacking Type II PA phosphatase activity , PA levels rise during exposure to light ( Garcia-Murillas et al . , 2006 ) .",
"We generated double mutants dPLD3 . 1;laza22 and studied retinal degeneration in these flies .",
"We found that dPLD3 . 1;laza22 photoreceptors did not undergo light dependent-degeneration ( Figure 4E ) .",
"To test if this was due to restoration of PA levels , we measured PA levels in all these genotypes .",
"As previously reported , we found that PA levels were elevated in laza22; importantly , the reduced levels of PA seen in dPLD3 . 1 was restored in dPLD3 . 1;laza22 ( Figure 4F ) .",
"We also overexpressed rdgA , encoding the major diacylglycerol kinase ( DGK ) activity in photoreceptors .",
"Overexpression of rdgA has previously been shown to elevate PA levels without affecting retinal ultrastructure ( Raghu et al . , 2009 ) .",
"When rdgA is overexpressed in dPLD3 . 1 ( dPLD3 . 1;Rh1>rdgA ) , retinal degeneration was completely rescued and the reduced PA levels seen in dPLD3 . 1 were reverted back to wild type levels ( Figure 4—figure supplement 1A , B ) .",
"Collectively , these observations suggest that reduced PA levels underlie the retinal degeneration phenotype of dPLD3 . 1 .",
"The finding that dPLD3 . 1undergoes light-dependent retinal degeneration suggests that dPLD might be activated during illumination .",
"When Drosophila photoreceptors are illuminated , a key source of PA is the sequential activity of PLCβ and DGK ( Inoue et al . , 1989; Yoshioka et al . , 1983 ) and PA is also metabolized by the PA phosphatase laza ( Garcia-Murillas et al . , 2006 ) .",
"In order to uncover a potential dPLD generated pool of PA , we exploited dGq1 mutants in which the failure to activate PLCβ results in a suppression of PA production via DGK ( Garcia-Murillas et al . , 2006 ) .",
"We compared PA levels in retinal extracts from dGq1with dGq1 , dPLD3 . 1 both in the dark and following illumination with 12 hr of light .",
"PA levels from both genotypes were comparable in dark reared flies; however , PA levels rise in dGq1mutants following illumination presumably reflecting production from a non-PLCβ-DGK source ( Figure 4G ) .",
"This rise in PA levels was suppressed in dGq1 , dPLD3 . 1 flies ( Figure 4G ) .",
"Thus , illumination induces dPLD dependent PA production in Drosophila photoreceptors .",
"dPLD was overexpressed in adult photoreceptors ( Rh1>dPLD ) .",
"Following 12 hr of white light illumination , the number of RLVs increases in the cell body of wild type ( Figure 4H , I ) .",
"However , in Rh1>dPLD the number of RLVs did not increase ( Figure 4H , I ) ; this effect was not seen on overexpression of Rh1>dPLDK/R ( Figure 4H , I ) suggesting that the ability of dPLD to regulate RLV turnover is dependent on its catalytic activity .",
"Together , these observations suggest that during illumination dPLD activity can support RLVs turnover .",
"RLV numbers in the cell body are an outcome of the balance between ongoing clathrin- dependent endocytosis of Rh1 containing rhabdomere membrane as well as mechanisms that remove these from the cell body .",
"To understand the mechanism underlying the increased RLV number in dPLD3 . 1 , we exploited the temperature-sensitive allele of dynamin , shits1 .",
"At the permissive temperature of 18°C , where dynamin function is normal , we exposed flies to a 5-min pulse of bright white light to trigger Rh1 isomerization to M and trigger its endocytosis .",
"Under these conditions , the number of RLVs generated in cells with and without PLD function was indistinguishable ( Figure 5A , B ) .",
"Following this , animals were rapidly shifted to 25°C , incubated for various time periods , retinae were fixed , processed and RLVs counted .",
"Under these conditions , in shits1 , where there is no further ongoing endocytosis , RLV numbers fall rapidly , presumably reflecting the removal of previously endocytosed vesicles ( Figure 5A ) .",
"By contrast , in shits1;dPLD3 . 1 , following the shift to 25°C post-illumination , there was no drop in RLV number with time implying a defect in mechanisms that remove RLVs from the cell body ( Figure 5B ) . 10 . 7554/eLife . 18515 . 012Figure 5 . dPLD activity supports the removal of RLVs from the cell body during illumination .",
"( A ) Quantification of RLVs from LS of retinae from control and shits1 .",
"n = 10 ommatidia taken from three separate preps .",
"( B ) Quantification of RLVs from LS of retinae from shits1 and shits1;dPLD3 . 1 .",
"n = 10 ommatidia taken from three separate preps .",
"( C ) LS of retinae stained with Rh1 from norpAP24 and norpAP24;Rh1>dPLD .",
"Rearing condition is indicated at the top of each panel .",
"Scale bar: 5 µm .",
"( D ) Quantification of RLVs from LS of retinae from control , norpAP24 and norpAP24;Rh1>dPLD .",
"n = 10 ommatidia taken from three separate preps .",
"( E ) TEM images showing single ommatidium from control , norpAP24 , Rh1>dPLD and norpAP24;Rh1>dPLD PRs of flies .",
"* indicates the degenerated rhabdomere .",
"Rearing condition is indicated on the top of the image .",
"Scale bar: 1 µm .",
"( F ) Quantification of retinal degeneration in control , norpAP24 and norpAP24;Rh1>dPLD done using TEM images .",
"The Y-axis represents the number of rhabdomeres visualized in each ommatidium .",
"n = 50 ommatidia taken from at least two separate flies .",
"( G ) Quantification of RLVs from LS of retinae from Rh1>dPLD in dark vs light ( 12 h CL ) .",
"The X-axis represents the population of vesicles positive for mentioned protein .",
"Y-axis shows the number of RLV’s per ommatidium .",
"n = 10 ommatidia taken from three separate preps .",
"Data are presented as mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 18515 . 012 We counted RLVs in norpAP24 subjected to bright illumination; as previously reported , we found RLV numbers were elevated ( Chinchore et al . , 2009 ) .",
"This elevation in RLV number could be suppressed by the overexpression of dPLD ( Figure 5C , D ) .",
"We also found that the light-dependent retinal degeneration in norpAP24 that is reported to depend on RLV accumulation in a Rab7 compartment ( Chinchore et al . , 2009 ) ( Wang et al . , 2014 ) could be partially suppressed by overexpressing dPLD ( Figure 5E , F ) .",
"Interestingly , we found that during illumination , in Rh1>dPLD , there was a significant reduction in the number of Rab7-positive RLVs but not in the number of Rab5-positive RLVs ( Figure 5G ) .",
"Collectively , these findings show that dPLD supports a process that can clear RLVs from the cell body of photoreceptors during illumination .",
"The retromer complex plays a central role in removing endocytosed transmembrane proteins from the lysosomal pathway and targets them to other cellular compartments ( Gallon and Cullen , 2015 ) .",
"We tested the effect of manipulating core members of the retromer complex in photoreceptors .",
"RNAi downregulation of vps35 results in an increase in RLV numbers both in the dark and following 12 hr illumination ( Figure 6A , B ) .",
"We tested the effect of overexpressing vps35 in photoreceptors during illumination; in an otherwise wild-type fly , this did not result in changes in RLV number ( Figure 6D ) or caused retinal degeneration ( Figure 6C ) .",
"However , in dPLD3 . 1 photoreceptors , overexpression of vps35 results in two key outcomes:",
"( i ) the increased numbers of RLVs seen in dPLD3 . 1 are reduced back to wild type levels ( Figure 6D ) and",
"( ii ) the retinal degeneration of dPLD3 . 1 is suppressed ( Figure 6C ) . 10 . 7554/eLife . 18515 . 013Figure 6 . dPLD regulates clearance of RLVs via retromer function .",
"( A ) LS of retinae stained with Rh1 from Rh1>Dicer , vps35RNAi .",
"Rearing conditions are indicated at the top of panels .",
"Scale bar:5 µm .",
"( B ) Quantification of RLVs from LS of retinae control and Rh1>Dicer , vps35RNAi .",
"n = 10 ommatidia taken from three separate preps .",
"( C ) Quantification of retinal degeneration in control , dPLD3 . 1 , Rh1>vps35 and dPLD3 . 1;Rh1>vps35 .",
"n = 50 ommatidia taken from at least five separate flies .",
"( D ) Quantification of RLVs from LS of retinae from control , dPLD3 . 1 , Rh1>vps35 and dPLD3 . 1;Rh1>vps35 .",
"n = 10 ommatidia taken from three separate preps .",
"( E ) Longitudinal section of retinae stained with Rh1 Rh1>dPLD; Dicer , vps35RNAi .",
"Rearing condition is indicated at the top of each panel .",
"Scale bar: 5 µm .",
"( F ) Quantification of RLVs from longitudinal section of retinae from control , Rh1>Dicer , vps35RNAi , Rh1>dPLD and Rh1>dPLD; Dicer , vps35RNAi .",
"n = 10 ommatidia taken from three separate preps .",
"Data are presented as mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 18515 . 013 During illumination , overexpression of dPLD results in a reduction of RLV number in a lipase-dependent manner ( Figure 4H , I ) .",
"We tested the requirement of intact retromer function for the ability of dPLD to clear RLVs .",
"We found that in cells where vps35 was downregulated , overexpression of dPLD could not reduce RLV numbers ( Figure 6E , F ) .",
"These findings suggest that intact retromer function is required for dPLD to support the clearance of RLVs during illumination .",
"We overexpressed garz , the Drosophila ortholog of the guanine nucleotide exchange factor ( GBF1 ) of Arf1 ( Cox et al . , 2004 ) .",
"In adult photoreceptors , garz overexpression does not impact rhabdomere structure during illumination ( Figure 7B , C ) , although RLV numbers were reduced ( Figure 7A ) .",
"When garz is overexpressed in dPLD3 . 1 , it completely rescues retinal degeneration ( Figure 7B , C ) .",
"These findings strongly suggest that retinal degeneration in dPLD3 . 1 may be due to reduced ARF1 activity .",
"If this model is true then reducing garz activity in wild-type flies should phenocopy dPLD3 . 1 .",
"To test this , we down-regulated garz in photoreceptors; this resulted in light-dependent retinal degeneration , the kinetics of which were comparable to that of dPLD3 . 1 ( Figure 7D , E ) .",
"Finally , we found that overexpression of garz reduced RLV number in dPLD3 . 1 back toward wild-type controls ( Figure 7F ) . 10 . 7554/eLife . 18515 . 014Figure 7 . Arf1 activity and retinal degeneration in dPLD3 .",
".",
"( A ) Quantification of RLVs from longitudinal section of retinae from control and Rh1>garz .",
"n = 10 ommatidia taken from three separate preps .",
"( B ) TEM images showing single ommatidium from Rh1>garz and dPLD3 . 1;Rh1>garz PRs of flies .",
"Rearing condition is indicated on the top of the image .",
"Scale bar: 1 µm .",
"( C ) Quantification of retinal degeneration in control , dPLD3 . 1 , Rh1>garz and dPLD3 . 1;Rh1>garz .",
"n = 50 ommatidia taken from at least five separate flies .",
"( D ) TEM images showing single ommatidium from Rh1>garzRNAi PRs of flies .",
"Rearing condition is indicated on the image .",
"Scale bar:1 µm .",
"( E ) Quantification showing the retinal degeneration in control , dPLD3 . 1 and Rh1>garzRNAi .",
"n = 50 ommatidia taken from at least five separate flies .",
"G ) Quantification of RLVs from longitudinal section of retinae from control , dPLD3 . 1 , dPLD3 . 1;Rh1>garz .",
"n = 10 ommatidia taken from three separate preps .",
"Data are presented as mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 18515 . 014 Since both garz and dPLD play a role in RLV clearance during illumination ( Figure 7 and Figure 4 ) , we tested the requirement of each molecule on the other for this function .",
"We found that the ability of Rh1>dPLD to clear RLVs required intact garz function .",
"When garz is also depleted ( Rh1>garzRNAi ) in Rh1>dPLD cells , the reduction in RLV number seen in Rh1>dPLD alone was attenuated ( Figure 8A ) .",
"This finding suggests that a garz-dependent step is required to support RLV clearance by dPLD during illumination . 10 . 7554/eLife . 18515 . 015Figure 8 . dPLD and garz are required for RLV clearance during illumination .",
"( A ) Quantification of RLVs from longitudinal section of retinae from control , Rh1>dPLD , Rh1>dPLD; Dicer , Rh1>garzRNAi .",
"n = 10 ommatidia taken from three separate preps .",
"( B ) Quantification of RLVs from longitudinal section of retinae from control , Rh1>garz , Rh1>Dicer , vps35RNAi and Rh1>garz;Dicer , vps35RNAi .",
"n = 10 ommatidia taken from three separate preps .",
"( C ) TEM images showing single ommatidium from control and dPLD3 . 1 , Rh1>Arf1CA and dPLD3 . 1;Rh1>Arf1CA PRs of day 0-old flies post-eclosion .",
"Scale bar: 1 µm ( D ) A model of the light activated turnover of rhabdomere membranes in Drosophila photoreceptors .",
"The cross-section of a PR is shown .",
"The area indicated by the red box is enlarged to the left .",
"PC-phosphatidylcholine , PA-phosphatidic acid , dARF1-GTP- GTP bound active ARF1 , dARF1-GDP-GDP bound inactive Arf1 , brown star indicates retromer , blue RLVs indicate endocytic compartment while orange RLVs indicate recycling compartment . DOI: http://dx . doi . org/10 . 7554/eLife . 18515 . 015 We also explored the route by which garz activity clears RLVs .",
"When Rh1>garz is performed in photoreceptors where retromer function is depleted ( Rh1>vps35RNAi ) , the reduction in RLV number seen in Rh1>garz alone is substantially blocked ( Figure 8B ) .",
"Thus , the ability of garz to support RLV clearance from the cell body requires intact retromer function .",
"Since our observations indicate a role of dPLD and its product PA in the context of Arf1-GTP activity , we tested the requirement for dPLD in regulating the biological activity of Arf1 .",
"In photoreceptors , overexpression of constitutively active Arf1 , Arf1CA ( Rh1>Arf1CA ) , results in ultrastructure defects in the rhabdomere ( Figure 8C ) .",
"We expressed Rh1>Arf1CA in dPLD3 . 1 and studied its effect on ultrastructure .",
"In the absence of dPLD function , the effect of Rh1>Arf1CA on ultrastructure was substantially reduced ( Figure 8C iii versus iv ) .",
"This finding suggests that PA produced by dPLD is required to mediate the effects of Arf1 in vivo ."
],
[
"Although the importance of plasma membrane turnover in determining cellular responses to external stimuli is well appreciated , the mechanisms that regulate this process remain unclear .",
"In photoreceptors , change in size of photosensitive membranes during illumination represents a special example of the broad principle of plasma membrane turnover following receptor-ligand interaction .",
"In dPLD3 . 1 photoreceptors , the process of light-induced membrane turnover is exaggerated; these photoreceptors show larger reductions in rhabdomere volume and greater reductions in Rh1 levels than is seen in wild-type flies .",
"The physiological consequence of this is that dPLD3 . 1 photoreceptors are less sensitive to light than controls when reared in light ( Figure 2J ) .",
"During illumination , RLVs are generated and mature through Rab5 and Rab7 endocytic compartments .",
"We found that",
"( i ) photoreceptors contain a light-stimulated PLD activity ,",
"( ii ) Loss of dPLD activity results in enhanced numbers of Rab7-positive RLVs in the cell body during illumination ,",
"( iii ) overexpression of catalytically active dPLD was able to clear light-induced Rab7-positive RLVs in wild-type cells and",
"( iv ) dPLD overexpression was able to reduce the enhanced RLV number and partially suppress retinal degeneration in norpAP24 , a mutant that shows enhanced Rab7 positive RLVs during illumination .",
"Thus , dPLD represents an enzyme activity that couples the generation of RLVs by light-induced endocytosis to their removal from the cell body .",
"It has previously been reported that the Rh1 that accumulates in Rab7 compartment is targeted for degradation , thus leading to retinal degeneration ( Chinchore et al . , 2009 ) .",
"Accumulation of Rh1 in Rab7-positive endosomes may explain the progressive microvillar collapse and reduced Rh1 protein levels in the cell body of dPLD3 . 1 .",
"Both , the microvillar degeneration and reduced PA levels of dPLD3 . 1 retinae were rescued by a dPLD transgene with intact lipase activity and elevation of PA levels was sufficient to rescue this phenotype .",
"Collectively , our observations strongly suggest that photoreceptors depend on a light-activated dPLD to generate PA to maintain apical membrane turnover during illumination .",
"They also suggest that protein-protein interactions of PLD , independent of its catalytic activity , may not be a primary mechanism underlying the function of this enzyme in cells .",
"In principle , the number of RLVs seen in a photoreceptor following illumination is a balance between ongoing endocytosis and processes that remove endocytosed RLVs either by recycling to the microvillar membrane or targeting to the late endosome-lysosome system for degradation .",
"Using the temperature-sensitive allele of dynamin shits1 , we were able to uncouple RLV endocytosis from their removal from the cell body and found that the generation of RLVs during illumination was not dependent on dPLD activity ( Figure 5I ) and the number of Rab5-positive RLVs was not increased in dPLD3 . 1 photoreceptors ( Figure 2F ) .",
"Collectively , these observations suggest no primary defect in clathrin dependent endocytosis in dPLD3 . 1 .",
"However , we found that in dPLD3 . 1 , the clearance of endocytosed RLVs was dramatically slower than in controls implying that dPLD supports a process that clears RLVs from the cell body .",
"These RLVs were Rab7-positive suggesting that they accumulate in late endosomes .",
"Conversely , in Rh1>dPLD , the number of Rab7-positive RLVs was fewer than in wild-type cells .",
"Together , these observations strongly suggest that dPLD activity supports a process that clears RLVs post endocytosis .",
"Following endocytosis , endosomes containing trans-membrane proteins ( such as Rh1 ) can be targeted for lysosomal degradation or be retrieved for recycling to other membranes through retromer-dependent processes .",
"The enhanced RLV numbers as well as retinal degeneration in dPLD3 . 1 could be rescued by enhancing retromer activity and the ability of dPLD overexpression to reduce RLV number during illumination required intact retromer activity .",
"Together , these observations suggest that during illumination dPLD stimulates RLV clearance through a retromer-dependent mechanism .",
"We found enhanced number of Rab7-positive RLVs in dPLD3 . 1 and Rh1>dPLD had reduced number of Rab7-positive RLVs .",
"A previous study has reported that retromer activity can clear RLVs from a Rab7-positive compartment ( Wang et al . , 2014 ) .",
"Together , our findings suggest that in the absence of dPLD the sorting of RLVs away from Rab7 endosomes into retromer-dependent recycling is inefficient .",
"Why might cargo sorting in dPLD3 . 1 be abnormal ?",
"Sorting reactions in vesicular transport often involve a small GTPase working in conjunction with a lipid-metabolizing enzyme .",
"Altering garz function , presumably altering Arf1-GTP levels , has three consequences:",
"( i ) in wild-type cells , enhancing garz levels results in fewer RLVs and blocks the rise in RLVs seen during light exposure .",
"( ii ) enhancing garz levels reduces RLV accumulation in dPLD3 . 1",
"( iii ) enhancing garz levels suppresses degeneration in dPLD3 . 1 while depleting garz in wild-type flies results in light-dependent retinal degeneration with a time course similar to that seen in dPLD3 . 1 .",
"Thus , an Arf1-GTP dependent step is required for both RLV turnover and maintaining apical domain size in photoreceptors .",
"Our finding that the ability of Rh1>dPLD to modulate RLV number requires intact garz function ( Figure 8A ) is consistent with this model .",
"These findings imply that Arf1-GTP levels positively regulate a step that enhances RLV recycling to the microvillar plasma membrane in the face of ongoing light-induced endocytosis , presumably through retromer complex activity .",
"In support of this idea , we found that the ability of Rh1>garz to reduce RLV numbers during illumination depends on intact retromer function ( Figure 8B ) .",
"We propose , that during illumination , rhabdomere size is maintained by the balance between clathrin-dependent endocytosis generating RLVs and an Arf1-GTP dependent sorting event that recycles RLVs to the plasma membrane via retromer activity ( Figure 8D ) .",
"dPLD , specifically its product PA is likely able to balance these two reactions by coupling light-induced endocytosis to Arf1-dependent sorting of RLVs into the recycling pathway .",
"Previous studies have identified proteins from brain cytosol that bind PA in vitro and are known to regulate membrane transport events; prominent among these was Arf1 ( Manifava et al . , 2001 ) although the in vivo significance of this binding is unknown .",
"Our findings that the biological activity of Arf1CA in photoreceptors requires intact dPLD activity and that the ability of increased garz ( Arf1-GEF ) levels to clear RLVs requires intact dPLD function suggests that Arf1 is a key target of PA generated by dPLD in mediating sorting and recycling of RLVs in photoreceptors .",
"It has been reported that EHD1 an ATPase required to generate tubular recycling endosomes is recruited by MICAL-L1 and the BAR domain protein syndapin2 both of which bind PA ( Giridharan et al . , 2013 ) .",
"It is possible that these proteins are also targets of PA generated by dPLD .",
"In the absence of PA , RLV sorting into the recycling pathway is impaired in dPLD3 . 1 , some fraction of the endocytosed RLVs accumulates in Rab7 endosomes and is targeted for degradation leading to the reduction in Rh1 levels .",
"These reduced Rh1 levels likely account for the reduced light sensitivity of dPLD mutants reported both in this study as well as in a previous analysis ( LaLonde et al . , 2005 ) .",
"What is the transduction pathway between photon absorption and dPLD activation ?",
"dPLD3 . 1 photoreceptors show normal electrical responses to light and the microvillar degeneration of dPLD3 . 1 could not be suppressed by a strong hypmorph of dGq ( Scott et al . , 1995 ) that is required for PLCβ dependent phototransduction .",
"We also found that light-activated elevation of PA levels was dependent on dPLD activity but did not require Gq-PLCβ signalling .",
"Collectively , these findings imply that dPLD activity is dispensable for Gq-PLCβ mediated activation of TRP channels and that the light-dependent degeneration of dPLD3 . 1 is not a consequence of abnormal TRP channel activation .",
"Our findings suggest that M activates dPLD without the requirement of Gq function , although the molecular mechanism remains to be determined .",
"dPLD has been reported to be localized in the submicrovillar cisternae ( LaLonde et al . , 2005; Raghu et al . , 2009 ) , a specialization of the smooth endoplasmic reticulum that is positioned ca .",
"10 nm from the plasma membrane at the base of the microvilli ( Yadav et al . , 2016 ) .",
"At this location , dPLD might bind to the C-terminal tail of M either before or after it is endocytosed into RLV; binding of mammalian PLD1 has been reported to the C-terminal tail of several rhodopsin superfamily GPCRs including the 5-HT2a , muscarinic and opioid receptors [ ( Barclay et al . , 2011 ) and references therein] .",
"It is possible that PA produced by dPLD bound to the C-terminus of Rh1 may then stimulate recycling to the apical membrane .",
"Thus , the control of apical membrane turn over by dPLD during illumination may represent an example by which ligand bound GPCRs signal without a direct involvement for heterotrimeric G-protein activity .",
"More generally in the brain , neurons and glial cells express GPCRs ( 5-HT2a , mGluR and opioid receptors ) of key functional importance .",
"Controlling these GPCR numbers on the plasma membrane during receptor stimulation ( of which rhodopsin turnover during illumination is a prototypical example ) is of critical importance to brain function and mechanisms that regulate this process will likely be crucial for the understanding and treatment of neuropsychiatric syndromes ."
],
[
"Flies were reared on medium containing corn flour , sugar , yeast powder and agar along with antibacterial and antifungal agents .",
"Flies were maintained at 25°C and 50% relative humidity .",
"There was no internal illumination within the incubator , and the flies were subjected to light pulses of short duration only when the incubator door was opened .",
"When required , flies were grown in an incubator with constant illumination from a white light source ( intensity ~2000 lux ) .",
"The wild type used for all experiments was Red Oregon-R .",
"GAL4-UAS system was used to drive expression of transgenic constructs .",
"The following transgenic lines were obtained from the Bloomington Stock Center: UAS-GFP::Rab5 ( B#43336 ) , UAS-YFP::Rab7 ( B#23270 ) .",
"UAS-garzRNAi ( V# 42140 ) was obtained from the Vienna Drosophila RNAi Center .",
"Dicer;UAS-vps35RNAi was obtained from Miklós Sass ( Eötvös Loránd University , Budapest , Hungary ) and UAS-vps35::HA was obtained from Prof . Hugo Bellen ( Baylor College of Medicine , Howard Hughes Medical Institute , Houston ) .",
"Flies were cooled on ice , decapitated using a sharp blade , and fixed on a glass slide using a drop of colorless nail varnish .",
"Imaging was done using 40X oil objective of Olympus BX43 microscope .",
"In order to obtain a quantitative index of degeneration , atleast five flies were scored for each time point .",
"A total of 50 ommatidia were assessed to generate degeneration index .",
"To quantify degeneration , a score of one was assigned to each rhabdomere that appeared to be wild type .",
"Thus , wild-type ommatidia will have a score of 7 .",
"Mutants undergoing degeneration will have a score between 1 and 7 .",
"Score were expressed as mean ± SEM .",
"Flies were anesthetized on ice and immobilized at the end of a disposable pipette tip using a drop of nail varnish .",
"The recording electrode ( GC 100 F-10 borosilicate glass capillaries , 1 mm O . D and 0 . 58 mm I . D from Harvard apparatus filled with 0 . 8% w/v NaCl solution ) was placed on the surface of eye and the reference electrode was placed on the neck region/thorax .",
"Flies were dark adapted for 5 min followed by ten repeated green light flashes of 2 s duration , each after an interval of 10 s .",
"Stimulating light was delivered from a LED light source placed within a distance of 5 mm of the fly's eye through a fiber optic guide .",
"Calibrated neutral density filters were used to vary the intensity of the light over five log units .",
"Voltage changes were amplified using a DAM50 amplifier ( WPI ) and recorded using pCLAMP 10 . 2 .",
"Analysis of traces was performed using Clampfit ( Axon Laboratories ) .",
"Heads from 1-day-old flies ( unless otherwise specified ) were decapitated in 2X SDS-PAGE sample buffer followed by boiling at 95°C for 5 min .",
"For detection of rhodopsin , samples were incubated at 37°C for 30 min and then subjected to SDS-PAGE and western blotting .",
"The following antibodies were used: anti-rhodopsin ( 1:250-4C5 ) , anti-α-tubulin ( 1:4000 , E7c ) , anti-TRP ( 1:4000 ) and anti-NORPA ( 1:1000 ) .",
"All secondary antibodies ( Jackson Immunochemicals ) were used at 1:10000 dilution .",
"Quantification of the blot was done using Image J software from NIH ( Bethesda , MD , USA ) .",
"For immunofluorescence studies retinae from flies were dissected under low red light in phosphate buffer saline ( PBS ) .",
"Retinae were fixed in 4% paraformaldehyde in PBS with 1 mg/ml saponin at room temperature for 30 min .",
"Fixed eyes were washed three times in PBST ( 1X PBS + 0 . 3% TritonX-100 ) for 10 min .",
"The sample was then blocked in a blocking solution ( 5% Fetal Bovine Serum in PBST ) for 2 hr at room temperature , after which the sample was incubated with primary antibody in blocking solution overnight at 4°C on a shaker .",
"The following antibodies were used: anti-Rh1 ( 1:50 ) , anti-TRP ( 1:250 ) and anti-GFP ( 1:5000 , abcam [ab13970] ) .",
"Appropriate secondary antibodies conjugated with a fluorophore were used at 1:300 dilutions [Alexa Fluor 488/568/633 IgG , ( Molecular Probes ) ] and incubated for 4 hr at room temperature .",
"Wherever required , during the incubation with secondary antibody , Alexa Fluor 568-phalloidin ( Invitrogen ) was also added to the tissues to stain the F-actin .",
"After three washes in PBST , sample was mounted in 70% glycerol in 1X PBS .",
"Whole mounted preps were imaged on Olympus FV1000 confocal microscope using Plan-Apochromat 60x , NA 1 . 4 objective ( Olympus ) .",
"Whole mount preparations of photoreceptors stained with anti-Rh1 were imaged on Olympus FV1000 confocal microscope using Plan-Apochromat 60X , NA 1 . 4 objective ( Olympus ) .",
"The RLV’s per ommatidium were counted manually across the Z-stacks using Image J software from NIH ( Bethesda , MD , USA ) .",
"Samples for TEM were prepared as mentioned in previous publication ( Garcia-Murillas et al . , 2006 ) .",
"Briefly samples were bisected in ice cold fixative solution ( For 1 ml: 0 . 5 ml of 0 . 2 M PIPES ( pH:7 . 4 ) , 80 µl of 25% EM grade glutaraldehyde , 10 µl of 30% H2O2 and 0 . 41 ml water ) .",
"After over-night fixation at 4°C , samples were washed in 0 . 1M PIPES ( thrice 10 min . each ) and then fixed in 1% osmium tetroxide ( 15 mg Potassium Ferrocyanide , 500 µl 0 . 2M PIPES , 250 µl 4% Osmium tetroxide and 250 µl of distilled water ) for 30 min .",
"The eyes were then washed with 0 . 1M PIPES ( thrice 10 min . each ) and then stained in en-block ( 2% Uranyl acetate ) for 1 hr .",
"Eyes were dehydrated in ethanol series and embedded in epoxy .",
"Ultrathin sections ( 60 nm ) were cut and imaged on a Tecnai G2 Spirit Bio-TWIN ( FEI ) electron microscope .",
"For volume fraction analysis , TEM images of Drosophila retinae were acquired and analyzed using the ADCIS Stereology toolkit 4 . 2 . 0 from the Aperio Imagescope suite .",
"A grid probe was used whose probe intersections were accurate to about 200–300 points .",
"The volume fraction ( Vf ) of the rhabdomere with respect to its corresponding photoreceptor cell was calculated as:Vf=NumberofpointsfallingontherhabdomereTotalnumberofpointsonthecell Volume fractions were calculated separately for R1–R6 and R7 .",
"TEM images were acquired using Tecnai G2 Spirit Bio-TWIN ( FEI ) electron microscope .",
"To quantify degeneration , a score of one was assigned to each rhabdomere that appeared to be wild type and a score of 0 . 5 was assigned to each rhabdomere that appeared to be partially degenerated .",
"Pure preparations of retinal tissue were collected using previously described methods ( Fujita et al . , 1987 ) .",
"Briefly , 0- to 12-hr-old flies ( unless otherwise specified ) were snap frozen in liquid nitrogen and dehydrated in acetone at −80°C for 48 hr .",
"The acetone was then drained off and the retinae dried at room temperature .",
"They were cleanly separated from the head at the level of the basement membrane using a scalpel blade .",
"Ten heads or 100 retinae per sample ( dissected from 1-day-old flies ) were homogenized in 0 . 1 ml methanol containing internal standards ) using an automated homogenizer .",
"The methanolic homogenate was transferred into a screw-capped tube .",
"Further methanol ( 0 . 3 ml ) was used to wash the homogenizer and was combined in the special tube .",
"0 . 8 ml chloroform was added and left to stand for 15 min . 0 . 88% KCl ( 0 . 4 ml ) was added to split the phases .",
"The lower organic phase containing the lipids were dried , re-suspended in 400 µl of chloroform:methanol 1:2 and was ready for analysis .",
"Total lipid phosphate was quantified from each extract prior to infusion into the mass spectrometer .",
"Mass spectrometer analyses were performed on a LTQ Orbitrap XL instrument ( Thermo Fisher Scientific ) using direct infusion method .",
"Stable ESI-based ionization of glycerophospholipids was achieved using a robotic nanoflow ion source TriVersa NanoMate ( Advion BioSciences ) using chips with the diameter of spraying nozzles of 4 . 1 μm .",
"The ion source was controlled by Chipsoft 8 . 3 . 1 software .",
"Ionization voltages were +1 . 2 kV and −1 . 2 kV in positive and negative modes , respectively; back pressure was set at 0 . 95 psi in both modes .",
"The temperature of ion transfer capillary was 180°C .",
"Acquisitions were performed at the mass resolution Rm/z400 = 100 , 000 .",
"Dried total lipid extracts were re‐dissolved in 400 μl of chloroform:methanol 1:2 .",
"For the analysis , 60 μl of samples were loaded onto 96‐well plate ( Eppendorf ) of the TriVersa NanoMate ion source and sealed with aluminum foil .",
"Each sample was analyzed for 20 min in positive ion mode where PC was detected and quantified .",
"This was followed by an independent acquisition in negative ion mode for 20 min where PA was detected and quantified .",
"Lipids were identified by LipidXplorer software by matching m/z of their monoisotopic peaks to the corresponding elemental composition constraints .",
"Molecular Fragmentation Query Language ( MFQL ) queries compiled for all the aforementioned lipid classes .",
"Mass tolerance was 5 p . p . m . and intensity threshold was set according to the noise level reported by Xcalibur software ( Thermo Scientific ) .",
"One-day-old flies were starved for 12 hr and then fed on 10% ethanol in sucrose for 6 hr .",
"Following this lipids were extracted ( with appropriate internal standards ) and phosphatidylethanols detected and quantified by HPLC/MS method ( Wakelam et al . , 2007 ) .",
"Data were tested for statistics using unpaired t-test .",
"*** denotes p<0 . 001; ** denotes p<0 . 01; * denotes p<0 . 05 and ns denotes not significant ."
]
] | [
"During illumination , the light-sensitive plasma membrane ( rhabdomere ) of Drosophila photoreceptors undergoes turnover with consequent changes in size and composition .",
"However , the mechanism by which illumination is coupled to rhabdomere turnover remains unclear .",
"We find that photoreceptors contain a light-dependent phospholipase D ( PLD ) activity .",
"During illumination , loss of PLD resulted in an enhanced reduction in rhabdomere size , accumulation of Rab7 positive , rhodopsin1-containing vesicles ( RLVs ) in the cell body and reduced rhodopsin protein .",
"These phenotypes were associated with reduced levels of phosphatidic acid , the product of PLD activity and were rescued by reconstitution with catalytically active PLD .",
"In wild-type photoreceptors , during illumination , enhanced PLD activity was sufficient to clear RLVs from the cell body by a process dependent on Arf1-GTP levels and retromer complex function .",
"Thus , during illumination , PLD activity couples endocytosis of RLVs with their recycling to the plasma membrane thus maintaining plasma membrane size and composition ."
] | [
"Certain cells in the eye contain a receptor protein known as rhodopsin that enables them to detect light .",
"Rhodopsin is found in distinct patches on the membrane surrounding each of these “photoreceptor” cells and the number of rhodopsin molecules present controls how sensitive the cell is to light .",
"In humans , vitamin A deficiency or genetic defects can decrease the number of rhodopsin molecules on the membrane , leading to difficulty in seeing in dim light .",
"Fruit fly eyes also contain rhodopsin .",
"Exposure to normal levels of light triggers parts of the membranes of fly photoreceptor cells to detach and move into the interior of the cell .",
"These internalized pieces of membrane have two possible fates: they can either be destroyed or recycled back to the cell surface .",
"This membrane turnover adjusts the size of the membrane surrounding the cell and the number of rhodopsin molecules in it to regulate the cell’s sensitivity to light .",
"It is crucial that turnover is tightly regulated in order to maintain the integrity of the cell membrane .",
"However , it is not clear how the process is regulated during light exposure .",
"Thakur et al . set out to address this question in fruit flies .",
"The experiments show that an enzyme called phospholipase D is activated when photoreceptors are exposed to light .",
"Active phospholipase D – which generates a molecule called phosphatidic acid – coordinates the internalization of pieces of membrane with the recycling of rhodopsin back to the cell surface .",
"Thakur et al . generated fly mutants that lacked phospholipase D and in these animals the internalized rhodopsin was not transported back to the cell membrane .",
"This caused the membrane to shrink in size and decreased the number of rhodopsin molecules in it .",
"As a result , the photoreceptor cells became less sensitive to light .",
"The findings of Thakur et al . show that in response to normal levels of light , phospholipase D balances membrane internalization and recycling to maintain the size and rhodopsin composition of the membrane .",
"Future challenges will be to work out exactly how phospholipase D is activated and how phosphatidic acid tunes membrane internalization and recycling ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"genetics and genomics"
] | A Myt1 family transcription factor defines neuronal fate by repressing non-neuronal genes | elife-46703-v1 | [
[
"Transcriptional repressors such as RE1-silencing transcription factor ( REST ) and Hairy/Enhancer of Split ( Hes ) repress neuronal genes in non-neuronal cells ( Ballas et al . , 2005; Chen et al . , 1998; Chong et al . , 1995; Grill et al . , 2012; Ishibashi et al . , 1995; Ohsako et al . , 1994; Schoenherr and Anderson , 1995 ) .",
"However , it is unknown whether transcriptional repressors of non-neuronal genes are required in neuronal precursors to specify neuronal fate during development .",
"The Myt1 family of C2HC-type zinc finger transcription factors contributes to fibroblast to neuron reprogramming in vitro by repressing Notch signaling ( Bellefroid et al . , 1996; Mall et al . , 2017; Vasconcelos et al . , 2016; Vierbuchen et al . , 2010 ) .",
"The Myt1 family factors were first shown to regulate neurogenesis in Xenopus gastrula embryos , where X-MyT1 is expressed in neuronal precursors along with classical proneural genes ( Bellefroid et al . , 1996 ) .",
"Mammalian Myt1 family proteins , Myt1 , Myt1l , and St18 , are also highly expressed in developing nervous systems and are required for proper migration of neuronal precursors into the subventricular zone and cortical plate ( Mall et al . , 2017; Vasconcelos et al . , 2016 ) .",
"Myt1 transcriptionally represses Notch signaling , primarily by repressing the transcription factor Hes1 , which inhibits neuronal cell fate ( Mall et al . , 2017; Vasconcelos et al . , 2016 ) .",
"The ability of Notch intracellular domain to repress neurogenesis is neutralized by overexpression of Myt1 family proteins ( Bellefroid et al . , 1996; Mall et al . , 2017 ) .",
"Based on these results , it has been proposed that Myt1 family proteins counteract lateral inhibition and subsequently commit neuronal progenitors to terminal differentiation .",
"Recent in vitro studies showed that Myt1l , together with the proneural gene Ascl1 and the neuronal transcription factor Brn2 , are sufficient to induce transdifferentiation ( TD ) into neurons from various cell types ( Masserdotti et al . , 2016; Vierbuchen et al . , 2010; Wapinski et al . , 2013 ) .",
"Interestingly , a number of non-neuronal mouse embryonic fibroblast ( MEF ) signature genes were also found to be repressed by Myt1l during neuronal transdifferentiation .",
"Furthermore , co-expression of Myt1l reduced efficiency of MyoD-induced myocyte differentiation in vitro ( Mall et al . , 2017 ) .",
"Consistent with a role for Mytl1 in transcriptional repression during neuronal transdifferentiation , Myt1l was found to be associated with transcriptional corepressor complexes , including the Sin3 histone deacetylase complex ( Sin3-HDAC ) , to mediate repression of non-neuronal genes ( Romm et al . , 2005 ) .",
"Redundancy between Myt1 family proteins has prevented mouse models from providing insight into the developmental functions of Myt1 ( Wang et al . , 2007 ) .",
"As a result , the in vivo functions of Myt1 family proteins during development remain poorly understood .",
"In C . elegans , ZTF-11 is the sole Myt1 family homolog containing the characteristic C2HC zinc finger domains .",
"The DNA-binding zinc finger domains of ZTF-11 exhibit a high degree of conservation in amino acid sequence compared to other Myt1 family members ( Figure 1—figure supplement 1 ) .",
"Both ZTF-11 and vertebrate Myt1 family proteins recognize the same consensus sequence ( AAGTT ) in vitro ( Mall et al . , 2017; Narasimhan et al . , 2015; Vasconcelos et al . , 2016 ) .",
"Apart from the zinc finger domains , ZTF-11 and other Myt1 family proteins are poorly conserved in sequence , including regions that interact with the Sin-3-HDAC complex ( Mall et al . , 2017; Romm et al . , 2005 ) ( Figure 1—figure supplement 1 ) .",
"Here , we demonstrate that a Myt1 family protein is required for in vivo developmental neurogenesis of specific lineages as well as transdifferentiation by characterizing the in vivo functions of ztf-11 .",
"We found that ZTF-11 is expressed exclusively in neuronal precursors at single-cell resolution during embryonic and postembryonic neurogenesis .",
"Remarkably , ZTF-11 is required for epithelial-to-neuronal transdifferentiation during C . elegans development , suggesting that in vivo transdifferentiation utilizes genetic programs similar to those required for neuronal reprogramming in vitro .",
"We also found that ZTF-11 is necessary and sufficient for postembryonic neurogenesis from a non-neuronal precursor .",
"In these lineages , we show that ztf-11 represses expression of non-neuronal genes to allow establishment of neuronal identity .",
"Unexpectedly , ztf-11 does not function as a repressor of the Hes1 ortholog lin-22 in this context .",
"Instead , our genetic data support the model that ZTF-11 acts downstream of Hes1 to promote neuronal differentiation .",
"We further show that ZTF-11 mediates transcriptional repression through directly binding to MuvB co-repressor complex , but not the Sin3-HDAC complex .",
"Taken together , our results indicate that neurogenesis requires repression of non-neuronal programs by Myt1 family proteins in addition to activation of neuronal programs ."
],
[
"To investigate the role of ztf-11 in development , we first examined the expression pattern of ZTF-11 by endogenously tagging ztf-11 with a C-terminal GFP via CRISPR/Cas9 genome editing .",
"To facilitate Cre recombinase-mediated conditional knock-outs , we used the same approach to insert two loxP sites in the first intron and 3’UTR of ztf-11::gfp ( Figure 1A ) .",
"Insertion of GFP and loxP sites in the ztf-11 locus did not yield any overt phenotypes .",
"In comparison , a deletion spanning one of two zinc-finger domains ( tm2315 ) , which likely abolished DNA binding , was homozygous lethal and produced severely paralyzed , developmentally arrested L1 larvae when maintained with the hT2 balancer chromosome ( data not shown ) .",
"As expected , we found that ZTF-11::GFP fluorescence was predominantly localized to nuclei ( Figure 1B ) .",
"Myt1 family transcription factors have been shown to be expressed early in neural precursors in the neural plate of Xenopus gastrula embryos and the developing CNS of rat embryos ( Bellefroid et al . , 1996; Kim et al . , 1997 ) .",
"As with vertebrate orthologs , we found that ZTF-11::GFP is expressed in neural precursors starting in the mid-gastrula embryo ( ~100-cell stage ) ( Figure 1B and Video 1 ) .",
"ZTF-11::GFP expression was strongest during the late gastrula to lima-bean embryonic stages , coinciding with the birth of most embryonically generated neurons .",
"ZTF-11::GFP expression became weaker in subsequent stages of embryogenesis .",
"Around the time of hatching , ZTF-11::GFP could only be detected in a small number of neuronal nuclei .",
"Post-embryonic ZTF-11::GFP expression showed a similar pattern; ZTF-11::GFP expression was transiently observed in postembryonic neuroectoblasts , such as Pn , Q , and V5 cells , but was quickly extinguished in postmitotic neurons ( Figure 1—figure supplement 2 ) .",
"Together , these data suggest that ZTF-11 is transiently expressed in neuronal precursors and postmitotic neurons .",
"We next performed embryonic lineage tracing with 4-D microscopy to further characterize ztf-11 expression with single-cell resolution .",
"The invariant cell division patterns during embryogenesis made it possible to reliably track cell lineage with a nuclear marker ( Bao et al . , 2006; Sulston et al . , 1983 ) .",
"Examining ZTF-11::GFP fluorescence throughout embryonic development , we found that ztf-11 is expressed in the vast majority of lineages that generate neurons but is rarely expressed in lineages that do not produce neurons ( Figure 1C , also see Supplementary file 1 for full lineage diagram ) .",
"At the 350-cell stage , 126 of 145 ( 87% ) neuronal precursor cells expressed ZTF-11::GFP .",
"All six neuroectoblasts ( P7/8 , P5/6 , and P3/4 ) that did not express ZTF-11 at the 350-cell stage showed postembryonic expression of ZTF-11 .",
"In contrast , only 15 of 195 ( 7% ) non-neuronal precursor cells expressed ZTF-11::GFP .",
"In C . elegans , the majority of neurons are generated from the neuroectodermal AB lineage , while a small number of neurons are produced by other lineages ( Sulston et al . , 1983 ) .",
"Strong correlations between ztf-11 expression and neuronal cell fate were evident in all branches of the lineages at single-cell resolution ( Figure 1D ) .",
"These observations suggest that ZTF-11 plays a broad role in neurogenesis .",
"The expression pattern of the Myt1 family proteins closely follows proneural bHLH ( basic helix-loop-helix ) genes in vertebrate systems ( Bellefroid et al . , 1996; Kim et al . , 1997 ) .",
"The vertebrate Myt1 family proteins are direct transcriptional targets of proneural genes , including Ascl1 ( Mall et al . , 2017; Vasconcelos et al . , 2016; Wapinski et al . , 2013 ) .",
"C . elegans proneural genes are conserved through evolution and act as master regulators of neurogenesis .",
"These proneural genes are expressed in neuronal precursors and differentiating neurons ( Frank et al . , 2003; Hallam et al . , 2000; Murray et al . , 2012; Zhao and Emmons , 1995 ) .",
"To investigate whether neural precursor-specific expression of ZTF-11 was directly controlled by proneural genes in C . elegans , we first asked whether proneural genes are required for ZTF-11 expression .",
"All C . elegans proneural genes , including HLH-3/Achaete-Scute , LIN-32/Atonal , NGN-1/neurogenin , and CND-1/NeuroD , form heterodimers with HLH-2/Daughterless and bind to canonical E-boxes ( CANNTG ) to regulate transcription of target genes ( Grove et al . , 2009 ) .",
"We tested whether hlh-2 functions with proneural genes to regulate ztf-11 expression .",
"Since hlh-2 is linked to ztf-11 , we constructed a ztf-11 transcriptional reporter fusion by placing the ztf-11 promoter upstream of Histone::GFP ( HIS::GFP ) and confirmed that the transcriptional reporter reproduces the endogenous expression pattern of ZTF-11 ( Figure 2—figure supplement 1 ) .",
"We found that hypomorphic hlh-2 ( tm1768 ) mutant showed a strong reduction of ztf-11 transcriptional reporter signal ( 78% ) in comparison to wild type ( Figure 2A ) , suggesting that hlh-2 is required for proper expression of ztf-11 .",
"In contrast to the essential hlh-2 , the proneural dimer partners of HLH-2 are redundantly expressed at early stages of neuronal development ( Grove et al . , 2009; Murray et al . , 2012 ) .",
"Consistent with this redundancy , endogenous ZTF-11::GFP expression was largely unperturbed in single mutants of hlh-3 ( ot354 ) , cnd-1 ( ju29 ) or ngn-1 ( ok2200 ) ( data not shown ) .",
"We found that lin-32 ( n372 ) mutant showed loss of ZTF-11 expression in the postembryonic postdeirid lineage , where LIN-32 functions to generate sensory neurons ( Figure 8A ) .",
"Furthermore , we identified multiple canonical E-box sequences upstream of the ztf-11 coding region ( Figures 1A and 2B ) .",
"Mutating these E-box sequences ( CANNTG to ACNNAG ) ( ∆E-box ) caused a severe reduction ( 79% ) of ∆E-box reporter signal compared to wild-type reporter ( Figure 2B ) .",
"These results are consistent with findings in vertebrates and suggest that proneural genes and HLH-2 together activate the expression of ztf-11 in neuronal precursors through the E-boxes in the ztf-11 promoter .",
"We next investigated the requirement of ztf-11 for neuronal fate determination in three contexts: in vivo transdifferentiation , postembryonic neurogenesis , and embryonic neurogenesis .",
"An epithelial-to-neuronal transdifferentiation event occurs invariantly during normal C . elegans development ( Jarriault et al . , 2008 ) , providing a model for investigating genetic pathways involved in neuronal transdifferentiation in vivo .",
"The rectal epithelial Y cell undergoes an epithelial-to-neuronal transdifferentiation event to form the motor neuron PDA in a stepwise process; epithelial identity is first lost and neuronal identity is subsequently acquired ( Jarriault et al . , 2008; White et al . , 1986 ) ( Figure 3A ) .",
"At the L1 stage , the Y cell is part of the rectal epithelium .",
"During the L2 stage , the Y cell gradually loses its epithelial fate ( Y . 0 ) while migrating anteriorly and gaining neuronal markers ( Y . 1 ) .",
"In the L3 stage , the Y cell becomes the PDA neuron and extends an axon while the P12 . pa cell replaces the Y cell in the anal epithelium ( Jarriault et al . , 2008; Sulston and Horvitz , 1977; White et al . , 1986 ) ( Figure 3A–B ) .",
"We first asked whether ZTF-11 functions during the Y-PDA transdifferentiation event .",
"Using the endogenously-tagged ZTF-11::GFP , we found that ztf-11 was expressed in the Y cell in early L2 animals at the start of the transdifferentiation process ( Figure 3C ) .",
"ZTF-11 expression coincided with the initial withdrawal of the Y cell from the rectum , suggesting that ZTF-11 may mediate the early dedifferentiation step of transdifferentiation ( Y . 0 ) .",
"We next asked if ztf-11 is required for transdifferentiation by generating a conditional deletion strain , in which we used egl-26::Cre to delete ztf-11 from the Y cell in the postembryonic lineage .",
"Conditional deletion of ztf-11 in the Y cell led to the loss of PDA neuronal markers , including EXP-1 and COG-1 , suggesting that PDA was not generated ( Figure 3D , F , and Figure 3—figure supplement 1 ) .",
"We next asked whether the transdifferentiation defect is due to a failure to eliminate epithelial identity or to acquire neuronal identity .",
"We found that epithelial markers in the Y cell ( EGL-26 and COL-34 ) persisted throughout development in ztf-11 cKO animals ( Figure 3E and Figure 3—figure supplement 1 ) .",
"Moreover , the persistent Y cell in ztf-11 cKO animals retained its original rectal niche location and morphology during development ( Figure 3E–F ) .",
"These results argue that ztf-11 functions to eliminate epithelial identity in the Y cell and allows for subsequent acquisition of neuronal PDA identity .",
"This results are in accordance with Myt1l’s function in in vitro neuronal transdifferentiation ( Mall et al . , 2017; Vierbuchen et al . , 2010 ) .",
"While mammalian models of in vivo neuronal transdifferentiation have not yet been described , Myt1 family factors may function as key evolutionarily conserved repressive factors in transdifferentiation events .",
"Neurogenesis via developmental transdifferentiation is rare in the animal kingdom .",
"Most neurons are generated through asymmetric cell divisions and quickly adopt a neuronal cell fate after mitosis .",
"We next assessed ztf-11’s function in this more common cellular pathway of neurogenesis by studying a postembryonic neuronal lineage .",
"The C . elegans postdeirid is a simple sensory organ comprised of two morphologically and functionally distinct sensory neurons , PVD and PDE , and a pair of sheath ( PDEsh ) and socket ( PDEso ) glia that support the PDE sensory dendrite ( White et al . , 1986 ) .",
"These four cells are born postembryonically from the V5 seam cell , which forms part of the lateral epidermis ( Figure 4A–B and Figure 4—figure supplement 2A–B ) .",
"Unlike V5 or tail T lineages , parallel lateral epidermal seam cells of V lineages ( V1-4 and V6 ) do not give rise to any neural progeny ( Sulston and Horvitz , 1977 ) .",
"Previous genetic studies have identified mutations in lin-32 , the homolog of Drosophila atonal and mammalian Atoh1 , which cause the V5 lineage to lose postdeirid neuroblast cell fate and instead to adopt a V1-4-like cell fate , establishing LIN-32 as the master regulator of postdeirid development ( Kenyon , 1986; Zhao and Emmons , 1995 ) .",
"It is interesting to note that homologs of the atonal family of proneural bHLH factors function in the development of various mechanosensory modalities , including mammalian inner ear hair cells ( Bermingham et al . , 1999 ) , Drosophila chordotonal organs ( Jarman et al . , 1993 ) , and the C . elegans postdeirid and male sensory rays ( Zhao and Emmons , 1995 ) , suggesting that atonal family bHLH genes drive a conserved genetic program .",
"To investigate the expression pattern of ZTF-11 in V5 postdeirid lineage in detail , we followed the V5 lineage using two fluorescent markers , endogenously labeled ZTF-11::GFP and HIS:mCherry driven by the lin-32 promoter .",
"Starting from mid L1 , ZTF-11::GFP was observed in the posterior daughter cell of V5 , which gives rise to the neurons , but not in V1-4 nor in the anterior daughter of V5 , which generate epidermal cells ( hyp7 ) and seam cells .",
"Within the V5 lineage , ZTF-11::GFP was maintained in neurons and glia but turned off in the non-neuronal precursors ( Figure 4A–B and Figure 4—figure supplement 1C ) .",
"Unexpectedly , the lin-32 transcriptional reporter showed dynamic expression in the V5 lineage .",
"lin-32 expression was detected prior to expression of ZTF-11::GFP .",
"However , lin-32 could not be detected in late L1 , while ZTF-11 expression was maintained throughout .",
"lin-32 reappeared again in the postdeirid neuroblast V5 . pa , but not in epithelial sister V5 . pp ( Figure 4B and Figure 4—figure supplement 1C ) .",
"While the significance of the lin-32 expression dynamics remains unclear , both LIN-32 and subsequent ZTF-11 expression were correlated with neuronal and glial cell fate .",
"To investigate the role of ZTF-11 in postdeirid neurogenesis , we generated a seam cell-specific ztf-11 conditional knock-out ( cKO ) by expressing Cre recombinase under the seam-cell-specific nhr-81 promoter to excise the ZTF-11::GFP locus .",
"To determine efficiency of the ztf-11 cKO , we measured ZTF-11::GFP intensity in the postdeirid lineage .",
"We found near complete loss of ZTF-11::GFP expression in 70% of cKO animals .",
"However , 30% of cKO animals showed only partial knock-down that fell within the wild type range of ZTF-11 expression , likely due to perdurance of ztf-11 mRNA or protein ( Figure 4—figure supplement 1A–B ) .",
"Partial penetrance observed in subsequent phenotypic analyses of ztf-11 cKO was most likely attributable to these limitations of the cKO approach .",
"We first scored neuronal reporters to examine whether the postdeirid neurons , PVD and PDE , could adopt a neuronal fate in the absence of ZTF-11 .",
"We found that approximately 50% of PVD and PDE neurons had lost their neuronal fate , as reflected by loss of the respective cell-type-specific markers , ser-2 and dat-1 , as well as loss of the pan-neuronal rab-3 marker ( Figure 4C and E , and Figure 4—figure supplement 1C–E ) .",
"Additionally , we found a similar loss of glial markers from the socket and sheath glia that function with the PDE neuron ( Figure 4—figure supplement 1F ) , suggesting that ZTF-11 is also required for glial fate in the postdeirid lineage .",
"The number of LIN-32-expressing V5 lineage cells was unchanged in ztf-11 cKO animals ( Figure 4C–D ) , indicating that V5 lineage cells still undergo the stereotyped cell divisions that would generate neurons and glia in wild-type animals .",
"This is in contrast to lin-32 mutants , which do not go through postdeirid cell divisions , and instead exclusively adopt epithelial V1-4-like lineages ( Kenyon , 1986; Zhao and Emmons , 1995 ) .",
"Since the neurons and glia of the postdeirid originate from an epithelial precursor , proper differentiation into their terminal fate likely requires both the loss of epithelial identity and the acquisition of neuronal/glial identities , similar to transdifferentiation .",
"In wild-type animals , the expression of the seam cell fate marker SCM::GFP is invariably lost in lin-32-positive postdeirid cells as they acquire neuronal/glial fate .",
"Strikingly , in ztf-11 cKO animals , we observed that some lin-32-positive postdeirid cells retained seam cell fate marker expression ( Figure 4D and F ) , suggesting that ztf-11 was required for the removal of epithelial identity preceding the acquisition of neuronal identity .",
"These data are consistent with the notion that ZTF-11 plays a role in eliminating epithelial fate in differentiating V5 lineage cells .",
"We next asked whether ztf-11 was sufficient to produce neurons .",
"We ectopically expressed ZTF-11 in non-neurogenic V1-4 seam cells where ztf-11 is not normally expressed .",
"Remarkably , we found that ectopic expression of ZTF-11 led to transformation into a neuronal lineage .",
"In 45% of transgenic animals expressing ZTF-11 in seam cells , we found additional cells expressing the PVD cell marker anterior to the wild-type PVD ( Figure 5A ) .",
"In addition , ectopic PVD-like cells showed the characteristic ‘dendritic menorah’ morphology of PVD neurons ( Albeg et al . , 2011 ) .",
"The positions of the ectopic PVDs were consistent with positions of V1-4 seam cell precursors ( Figure 5A–B ) .",
"Similarly , additional PDE-like cells were identified based on the presence of the PDE cell marker and PDE morphology ( Figure 5C–D ) .",
"In contrast to the proneural activity of ZTF-11 , additional glia-like cells could not be identified ( Figure 5D ) , suggesting additional requirements for glia development .",
"Our genetic data suggested that ZTF-11 is required to eliminate epithelial identity in developing neurons and glial cells .",
"We next asked whether ztf-11 is sufficient to eliminate epithelial identity by ectopically expressing ZTF-11 in seam cell lineages .",
"In animals ectopically expressing ZTF-11 in seam cells , we indeed found that some seam cells lost their identity marker ( Figure 5E–F ) .",
"Seam cells fuse in adult C . elegans to form a continuous syncytium ( Sulston and Horvitz , 1977 ) .",
"Using the apical junction marker AJM-1::GFP , we found that the loss of seam cell identity resulted in ‘gaps’ in the seam cell syncytium ( Figure 5E–F ) , suggesting that ztf-11 is capable of eliminating epithelial identity and function .",
"To investigate the proneural mechanism of ZTF-11 further , we tested whether ZTF-11 requires LIN-32 for its proneural activity in V1-4 lineages .",
"Ectopic PVD-like cells could not be generated by ZTF-11 overexpression in the lin-32 ( u282 ) loss of function background ( Figure 5B ) , suggesting that the proneural activity of ZTF-11 depends on proneural bHLH function .",
"Consistent with the requirement for LIN-32 , we found that the ectopic neurons induced by misexpression of ZTF-11 turned on lin-32 transcriptional reporter , whereas ZTF-11::GFP-positive non-neuronal cells did not ( Figure 5—figure supplement 1 ) , suggesting that ZTF-11 overexpression can induce expression of LIN-32 to drive neuronal fate .",
"In contrast , we found that ZTF-11 continued to eliminate epithelial identity in the lin-32 ( u282 ) mutant ( Figure 5F ) .",
"These results indicate that ZTF-11 can induce LIN-32 to specify neuronal and glial cell fate in certain circumstances .",
"While LIN-32 promotes the ‘neuronal’ features , ZTF-11 helps to erase epithelial identity from prospective neuronal/glial daughters of the V5 lineage ( Figure 5G ) .",
"Our genetic analysis revealed that ZTF-11 was important for eliminating epithelial identity during transdifferentiation of PDA neuron and neurogenesis from a neuroectoblast V5 lineage .",
"We set out to assess whether ZTF-11 is required for neurogenesis from different postembryonic neuroectoblast lineages .",
"We first asked whether postembryonic neurons generated during L1 larval development are present in ztf-11 ( tm2315 ) null mutant animals .",
"QR/L neuroectoblast lineages contribute six postembryonic neurons ( SDQR/L , AVM , PVM , AQR , and PQR ) during the mid L1 stage ( Sulston and Horvitz , 1977 ) .",
"Among them , AVM and PVM could be unambiguously identified as UNC-86 expressing nuclei based on their solitary positions ( Finney and Ruvkun , 1990; Serrano-Saiz et al . , 2018 ) .",
"We found that UNC-86 expression in the respective positions of AVM and PVM nuclei was invariantly lost in ztf-11 ( tm2315 ) late L1 animals ( Figure 6B–D ) , suggesting that ZTF-11 is required for both AVM and PVM postembryonic neuronal fates .",
"In contrast , UNC-86 expression was not lost in embryonic neurons such as ALM ( Figure 6B ) .",
"Additionally , G1 and K neuroectoblast lineages give rise to RMH and DVB neurons respectively during late L1 stage ( Sulston and Horvitz , 1977 ) .",
"We again found loss of the respective cell fate markers for RMH ( Figure 6B , E ) and DVB ( Figure 6B , F ) , SEM-2 ( Vidal et al . , 2015 ) and LIM-6 ( Hobert et al . , 1999 ) , in ztf-11 ( tm2315 ) late L1 animals .",
"We next examined the postembryonic ventral cord motor neurons ( VMNs ) .",
"P1-12 ( Pn ) cells form the ventral epidermis of the newly hatched animal .",
"During the late L1 stage , Pn cells give rise to postembryonic VMNs of the VA , VB , AS , VD , and VC classes ( Sulston and Horvitz , 1977 ) .",
"To ask whether ZTF-11 is required for generating postembryonic VMNs , we examined condition knockouts of ZTF-11 in Pn lineages by expressing Cre in the epidermis ( Kage-Nakadai et al . , 2014 ) .",
"The VMNs can be further classified based on their respective neurotransmitters , acetylcholine , GABA , or monoamine ( serotonin ) .",
"We counted the total number of VMNs expressing each neurotransmitter marker .",
"Aminergic VMNs ( two serotonergic VC4-5 neurons ) are exclusively postembryonically born ( Duerr et al . , 1999; Sulston and Horvitz , 1977 ) .",
"We found that the aminergic neuron marker CAT-1 was largely lost ( 89% of animals ) in VC4-5 , suggesting that ZTF-11 is required for VC4-5 fates .",
"In contrast to amingergic VMNs , cholinergic or GABAergic VMNs are comprised of both embryonic and postembryonic neurons ( McIntire et al . , 1993; Pereira et al . , 2015; Sulston and Horvitz , 1977; Sulston et al . , 1983 ) .",
"However , any loss of neuronal markers in this experiment was likely exclusively due to postembryonic neuron defects , as ZTF-11 was conditionally knocked out in only Pn lineages .",
"With cholinergic ( CHO-1 ) and GABAergic ( UNC-47 ) neuronal markers , we found more subtle decreases in total cholinergic ( 19% ) or GABAergic ( 5% ) VMNs in ztf-11 cKO animals .",
"Unlike cholinergic or GABAergic postembryonic VMNs , VC4-5 neurons do not mature until the late L4 stage and maintain expression of ZTF-11 into adulthood ( data not shown ) , which may account for their stronger requirement for ZTF-11 .",
"Taken together , our loss-of-function analysis suggest that ZTF-11 functions in multiple neuroectoblast lineages to specify postembryonic neuronal identities .",
"In many postembryonic lineages , neurons are generated from precursor cells , which are differentiated cells such as the rectal epithelial Y cell or the V5 precursor cell ( seam cell ) .",
"In contrast , the majority of embryonic neurons are generated from short-lived precursor cells through rapid cell divisions ( Sulston et al . , 1983 ) .",
"We next investigated the role of ztf-11 in embryonic neurogenesis .",
"Using a pan-neuronal RAB-3 marker , we found that most embryonic neurons are born and obtain neuronal fate in ztf-11 mutants ( Figure 7A ) .",
"The small size of the L1 animals made it difficult to determine the exact number of RAB-3 expressing nuclei , especially amongst densely packed neurons in cephalic ganglia .",
"We instead counted the number of embryonic motor neurons in the ventral cord , which could be unambiguously identified from rab-3 expressing nuclei along the length of the animal ( White et al . , 1976 ) .",
"We found that there was a small ( 2% ) loss of rab-3 expressing nuclei in the ventral cord , suggesting that ZTF-11 is mostly dispensable for neuronal fate acquisition during embryogenesis ( Figure 7B ) .",
"To account for potential maternal contribution of ZTF-11 , we additionally knocked down ZTF-11 in ztf-11 ( tm2315 ) /hT2 heterozygote mothers by feeding RNAi and found that their ztf-11 ( tm2315 ) homozygote progeny still generated a normal number of embryonic VMNs ( Figure 7B ) .",
"To further examine the requirement of ZTF-11 for embryonic neurogenesis , we counted the number of respective head neurons of four major neurotransmitter types ( acetylcholine , glutamate , GABA , and monoamines ) in wild type and ztf-11 ( tm2315 ) early L1 animals .",
"Unfortunately , many cholinergic or glutamatergic head neurons were tightly clustered in early L1 animals , which could introduce systematic errors in counting .",
"With this caveat , we did not find significant changes in cholinergic or glutamatergic head neurons in ztf-11 ( tm2315 ) mutant animals , suggesting that ZTF-11 might indeed be dispensable for neuronal fates of major neurotransmitter types .",
"Taken together , these results indicate that ztf-11 is particularly important for neurons that are generated from epidermal lineages that have fully differentiated in both morphology and function .",
"Despite the near normal cell number , the ztf-11 deletion mutants showed near complete loss of movement .",
"When maintained with the hT2 balancer chromosome , ztf-11 ( tm2315 ) heterozygous mothers produced homozygous mutant individuals that were completely immobile in bacterial lawns after hatching .",
"ztf-11 ( tm2315 ) mutant individuals also invariantly did not develop any further after hatching , potentially due to feeding deficits .",
"To measure defects in motility , we performed a thrashing assay in M9 buffer .",
"We found that homozygous ztf-11 ( tm2315 ) mutant individuals showed near complete loss of thrashing motion and severely uncoordinated swimming motion ( Figure 7—figure supplement 1 ) .",
"In comparison , heterozygous ztf-11 ( tm2315 ) /hT2 individuals did not show a significant change in the number of thrashes compared to wild type ( N2 ) ( Figure 7—figure supplement 1 ) .",
"These results raise the possibility that ztf-11 may be required for proper function of embryonic neurons .",
"Next we investigated how ztf-11 specifies neuronal fate .",
"Previous studies suggested that proneural genes induce neuronal fate while Notch signaling inhibits neurogenesis by inhibiting proneural genes ( Bertrand et al . , 2002; Lewis , 1998; Heitzler et al . , 1996; Takebayashi et al . , 1997 ) .",
"Myt1 family factors are induced by proneural genes and act as transcriptional repressors of Notch signaling , including the Notch effector gene Hes1 ( Dhanesh et al . , 2016; Mall et al . , 2017; Vasconcelos et al . , 2016 ) .",
"Repressing Hes1 transcription is in turn thought to de-repress proneural bHLHs such as Ascl1 , mediating exit from a proliferative neural stem cell fate and subsequent neuronal differentiation ( Mall et al . , 2017; Vasconcelos et al . , 2016 ) .",
"The C . elegans orthologs of Hes1 , lin-22 , and its target proneural gene lin-32/Atoh1 , function in postdeirid development ( Kenyon , 1986; Portman and Emmons , 2000; Wrischnik and Kenyon , 1997 ) .",
"lin-22/Hes1 is expressed in seam cells , including V1-4 , but not in V5 ( Katsanos et al . , 2017 ) .",
"In lin-32/Atoh1 mutants , no PVD or PDE cells were generated , while in lin-22 mutants , additional PVD and PDE neurons were generated in each of the V1-4 lineages , suggesting that lin-22 represses proneural gene lin-32 in V1-4 , but not in the V5 lineage ( Portman and Emmons , 2000; Wrischnik and Kenyon , 1997 ) .",
"We set out to use this evolutionarily conserved genetic circuit to investigate whether ZTF-11 also acts through repressing lin-22 .",
"We first investigated the effect of ztf-11 cKO on lin-22 expression by examining a transcriptional reporter for lin-22 .",
"Consistent with previous studies , we found that the LIN-22 transcriptional reporter was invariantly excluded from V5 lineage cells during postdeirid development ( Figure 8B , top panel; Figure 8C ) .",
"However , in the ztf-11 cKO , the LIN-22 reporter remained undetectable in V5 lineages .",
"In addition , we detected no change in the expression pattern or level of the LIN-22 reporter in V1-4 ( Figure 8B , bottom panel; Figure 8C ) .",
"This result suggests that lin-22 is unlikely to be a transcriptional target of ZTF-11 .",
"To additionally test whether ZTF-11 functions through repression of LIN-22 activity , we performed a genetic analysis of ztf-11 cKO and lin-22 ( n372 ) .",
"The lin-22 ( n372 ) single mutant generates ectopic neurons in the V1-4 lineages .",
"If ztf-11 acts upstream to repress lin-22 , we predicted that ztf-11 cKO; lin-22 double mutant would show a lin-22 single mutant phenotype , with ectopic neurons generated from the V1-4 lineages .",
"Contrary to this prediction , we observed that ztf-11 cKO; lin-22 double mutant resulted in a loss of the PVD cell marker in V1-4 as well as V5 lineages ( Figure 8D–E ) .",
"Both the genetic and expression data argue against the proposed model in which ZTF-11 acts via repression of LIN-22 .",
"Next , we investigated whether ZTF-11 de-represses lin-32 transcription .",
"To understand ztf-11’s relationship with lin-32 , we examined lin-32 expression in ztf-11 cKO mutant and found that lin-32 is still expressed in the V5 lineage at the level of wild-type controls ( Figure 8F–G ) .",
"However , ZTF-11 expression in the V5 lineage is completely eliminated in the lin-32 mutant ( Figure 8A ) , suggesting that ztf-11 is turned on by LIN-32 .",
"This is consistent with the fact that the E-box sequences in the promoter region of ztf-11 are required for its expression ( Figure 2 ) .",
"Additionally , the loss of lin-32 did not alter the ability of ectopically expressed ZTF-11 to reprogram epithelial identity ( Figure 5B ) , suggesting that ztf-11 acts downstream of both lin-22 and lin-32 to repress epithelial fate .",
"We next measured the fluorescence intensity of lin-32 transcriptional reporter during postdeirid development .",
"If ZTF-11 acts as a de-repressor of lin-32 , we would expect a loss or reduction of lin-32 transcriptional activity .",
"However , we did not observe a significant change in fluorescence intensity between wild type and ztf-11 cKO .",
"Together , these results argue against the existing model in which ztf-11 acts through repressing lin-22 and instead support a linear genetic model in which lin-22/Hes1 represses lin-32/Atoh1 , which in turn activates ztf-11 ( Figure 8A ) .",
"To understand how ztf-11 promotes neuronal fate , we performed transcriptome analysis in ztf-11 knockdown animals during development .",
"RNAi hypersensitive eri-1 ( mg366 ) ; ztf-11::gfp worms were fed with bacteria expressing dsRNA against ZTF-11 ( Ahringer RNAi collection ) or empty feeding RNAi vector as a control .",
"ztf-11::gfp fluorescence was strongly reduced in embryos fed with ztf-11 RNAi , confirming the knockdown efficiency ( data not shown ) .",
"Consistent with this observed reduction of ZTF-11::GFP , we found that ztf-11 transcript levels were reduced by 72% in ztf-11 knockdown embryos ( Supplementary file 2 ) .",
"Differential expression analysis revealed that 419 genes were significantly dysregulated in ztf-11 KD embryos ( FDR < 0 . 1 ) ( Figure 9A; Supplementary file 2 ) .",
"The majority ( 88% ) of the differentially expressed genes were upregulated in ztf-11 KD , consistent with the hypothesis that ztf-11 acts a transcriptional repressor ( Figure 6A ) .",
"Notably , among the upregulated genes , the vast majority were non-neuronal genes , including genes specific to epidermis ( collagens ) or muscle ( sarcomere components ) ( Figure 9B ) .",
"GO-term enrichment analysis revealed that epidermal and muscular genes were significantly enriched among upregulated genes .",
"In contrast , we did not find significant changes in expression of most neuronal genes , including those involved in neurodevelopment , synaptic transmission , axon guidance , and neurotransmitter synthesis ( Figure 9C ) , likely reflecting our findings that embryonic neurons are still generated in ztf-11 null mutant .",
"These findings are consistent with our genetic analysis , which demonstrated that ZTF-11 acts as a repressor of epithelial identity rather than a direct driver of neuronal fate .",
"Our transcriptomic analysis suggested that ZTF-11 mostly represses gene expression .",
"To further test if ZTF-11 indeed functions as a transcriptional repressor , we fused its DNA binding Zinc-finger ( ZF ) domains with either a transcriptional activator ( VP64 ) or repressor ( EnR ) domain .",
"We found that the expression of the transcriptional repressor fusion protein ( EnR::ZF ) in seam cells resulted in proneural activity similar to overexpression of the native ZTF-11 protein .",
"In contrast , expression of the transcriptional activator fusion protein ( VP64::ZF ) showed a dominant negative effect and blocked postdeirid neurogenesis ( Figure 10A ) .",
"Based on these results , we conclude that ZTF-11 , like vertebrate Myt1 family proteins , indeed functions as a transcriptional repressor to promote neuronal fate ( Mall et al . , 2017; Vasconcelos et al . , 2016 ) .",
"Transcription factors repress gene expression by recruiting corepressor complexes .",
"Corepressor complexes modify chromatin into a more repressed state by catalyzing posttranslational modification of histone tails .",
"Histone chaperones RbAp46/48 mediate interaction with the histone and thus form the core histone-binding subunits of several histone post-translational modifying complexes ( Huang et al . , 1991; Loyola and Almouzni , 2004; Qian et al . , 1993 ) .",
"To investigate the mechanism of transcriptional repression by ZTF-11 , we first examined the role of histone chaperone RbAp46/48 homologs , RBA-1 and LIN-53 , in postdeirid neurogenesis where ZTF-11 is required .",
"In rba-1 or lin-53 single mutants , approximately 20% of the PVD neurons are missing while another 40% of PVDs showed severe morphological defects ( Figure 10C–D ) .",
"This result suggests that the histone chaperone RbAp46/68 homologs , rba-1 and lin-53 , were required for proper postdeirid neurogenesis and that ZTF-11 likely functions through a corepressor complex containing histone chaperones RbAp46/48 .",
"Vertebrate Myt1 interacts with the Sin3 histone deacetylase corepressor complex ( Sin3-HDAC ) , which contains RbAp46/48 , to repress target genes during transdifferentiation in vitro ( Mall et al . , 2017; Romm et al . , 2005 ) .",
"It is unclear whether Myt1 family factors also function with the Sin3-HDAC complex in developmental contexts .",
"We examined the role of Sin3-HDAC components in postdeirid neurogenesis and observed no defects resulting from the loss of sin-3 , the sole Sin3 homolog in worms ( Choy et al . , 2007 ) ( Figure 10C–D ) .",
"We next tested whether other corepressor complexes that contain RbAp46/48 are involved in postdeirid neurogenesis .",
"We found components of the MuvB core of the DRM ( DP/Rb/MuvB ) corepressor complex , lin-9 , lin-52 , and lin-54 ( Harrison et al . , 2006 ) , to be required for robust postdeirid neurogenesis ( Figure 10C–E and Figure 10—figure supplement 1 ) .",
"These genetic results suggest that the MuvB repressor complex , rather than the Sin3-HDAC complex , is required for V5 neurogenesis in vivo .",
"Consistent with this result , the proneural activity of ectopically expressed ZTF-11 is strongly ( 80% ) reduced by the loss of lin-52 , suggesting that ZTF-11 functions through the MuvB complex ( Figure 10F ) .",
"We next tested whether ZTF-11 directly binds to MuvB complex components using a single molecule pull-down ( SiMPull ) analysis , a quantitative , imaging-based co-immunoprecipitation assay ( Jain et al . , 2011 ) .",
"We used CRISPR/Cas9 to endogenously tag candidate co-repressor subunits with mCherry , and ztf-11 was tagged with GFP .",
"ZTF-11::GFP was pulled down by anti-GFP antibody and displayed on a microscopy slide .",
"Co-precipitation was quantified by counting the number of mCherry-tagged co-repressor molecules on the slide .",
"We found that LIN-9 , LIN-52 , and the RbAp46/48 homolog RBA-1 could be pulled down by ZTF-11 , suggesting that ZTF-11 binds to DRM co-repressor complex in vivo ( Figure 10G ) .",
"Interestingly , lin-37 mutant did not show obvious neurogenesis defects ( Figure 10C–D ) , suggesting that LIN-37 may be dispensable for association of MuvB complex with ZTF-11 .",
"Unexpectedly , we found weak co-precipitation of SIN-3 with ZTF-11::GFP , suggesting that ZTF-11 may still function with SIN-3 in other in vivo contexts .",
"Taken together , these results also provide the first evidence that the evolutionarily conserved MuvB co-repressor complex ( LINC in vertebrates and dREAM/MuvB complex in flies , see Figure 10—figure supplement 1 ) ( Lewis et al . , 2004; Schmit et al . , 2007 ) functions in neurogenesis .",
"It has previously been shown that MuvB genes are required to repress germline genes in somatic cells ( Petrella et al . , 2011; Wang et al . , 2005 ) .",
"We propose that ZTF-11 binds to MuvB complex to similarly repress many non-neuronal genes during neuronal development ."
],
[
"Although Myt1 family factors were discovered more than two decades ago , the neurogenic role of Myt1 family factors is only starting to be unraveled on a molecular level .",
"In particular , advances in in vitro transdifferentiation have provided crucial insights in recognizing Myt1 family factors as key drivers of neurogenesis .",
"Here we studied the physiological and developmental functions of ZTF-11 in neurogenesis in vivo .",
"Comparing our results to the in vitro transdifferentiation literature , there are interesting similarities and differences .",
"First , in both systems , ZTF-11 and Myt1l are critical drivers of neural cell fate .",
"Examining neurogenesis in different classes of neurons in C . elegans , we found that ZTF-11 is particularly important for the generation of postembryonic neurons , which are derived from epidermal cells .",
"Most interestingly , a developmentally occurring epithelial-to-neuronal transdifferentiation event requires ZTF-11 to reprogram epithelial identity , further bridging the in vivo and in vitro neurogenic functions of Myt1 factors .",
"In contrast , embryonic neurons generated by rapid cell division from precursor cells are less dependent on ZTF-11 .",
"It is conceivable that ZTF-11 is required to ‘turn off’ the established epidermal/epithelial cell fate before neurons can be generated .",
"This could explain the particular requirement of Myt1l in transdifferentiation .",
"Second , both ZTF-11 and Myt1l function as transcriptional repressors .",
"This was evident from RNA-seq experiments from both C . elegans and transdifferentiating mammalian neurons ( Mall et al . , 2017 ) , where non-neuronal genes are suppressed by this family of proteins .",
"Third , the neuronal expression of ZTF-11 in developing neurons is activated by proneural bHLH genes , which is likely dependent on the conserved E-boxes in the promoter elements of ztf-11 and Myt1l .",
"Together , these similarities build a strong case that the Myt1 family transcription factors play conserved functions in neuronal specification by repressing the expression of non-neuronal genes .",
"These results also suggest that repression of non-neuronal genes is an important aspect of neurogenesis across species .",
"We also identified two key differences between ZTF-11 and Myt1 .",
"First , our genetic analysis shows that ZTF-11 does not repress the Hes1 homolog lin-22 .",
"How can this result be reconciled with previous results that Myt1 family factors repress lateral inhibition ?",
"One possible explanation for this discrepancy is that ZTF-11 may have lost its ability to repress Notch signaling .",
"However , it is also possible that vertebrate Myt1 family factors gained the ability to repress Hes1 .",
"Second , our data also suggest that the MuvB complex , but not the Sin3-HDAC complex , plays an important role in neurogenesis as a co-repressor complex that functions with ZTF-11 .",
"This result is interesting but unsurprising considering the sequence divergence of Myt1 family proteins outside the conserved DNA-binding zinc-fingers ( Figure 1—figure supplement 1 ) .",
"Myt1 family proteins may have diverged through evolution to function with different co-repressor complexes .",
"It is also noteworthy that ZTF-11 retained weak binding with SIN-3 ( Figure 10G ) , suggesting this interaction is conserved through evolution in other developmental contexts .",
"Examination of Myt1 family factors in other invertebrate model systems is likely to shed light on these intriguing questions .",
"During evolution , ancestral neurons likely arose from non-neuronal cells .",
"Consistent with this hypothesis , cnidarian neurons are generated from endodermal interstitial stem cells or epithelial precursors , rather than dedicated neural precursors ( Rentzsch et al . , 2017 ) .",
"As with proneural genes , Myt1 family factors are conserved throughout metazoan evolution with the exception of porifera ( sponges ) and ctenophora ( comb jellies ) , which either lack a nervous system or are thought to have independently evolved a nervous system ( Moroz et al . , 2014 ) .",
"MuvB complex genes are conserved in all animals regardless of presence of the nervous system , suggesting that Myt1 family proteins evolved later and recruited MuvB as their co-repressor .",
"It is now tempting to speculate that Myt1 family factors , alongside MuvB co-repressor complex , may comprise an ancestral core module for generating neurons from non-neuronal cells ."
],
[
"Wild-type strains were C . elegans variety Bristol , strain N2 .",
"Worms were maintained by standard methods as previously described ( Brenner , 1974 ) .",
"Worms were grown at 20°C on nematode growth media ( NGM ) plates seeded with bacteria ( Escherichia coli OP50 ) as a food source .",
"Transgenic strains were generated as previously described by gonadal injection ( Mello and Fire , 1995 ) .",
"The epidermal Cre strain ( FX15987 ) was kindly provided by Dr . Shohei Mitani .",
"The list of all mutant and transgenic strains used in this study is available in Supplementary file",
"3 . DNA plasmid constructs were generated by PCR amplification using Pfusion DNA polymerase followed by isothermal assembly or restriction digest and subsequent ligation using T4 DNA ligase ( NEB ) .",
"ztf-11 cDNA was amplified using C . elegans ORFeome library ( Lamesch et al . , 2004 ) .",
"ztf-11 promoter ( pZTF-11 ) was cloned via PCR amplification of 2740 bp fragment upstream of ztf-11 tss .",
"pZTF-11 ∆E-box mutations were generated with gBlock synthesis ( IDT ) followed by isothermal assembly into wild type pZTF-11 vector .",
"EnR and VP64 DNA were kindly provided by Dr . Mauritz Mall .",
"Unless otherwise indicated , worm lysate genomic DNA was used as the template for PCR amplification .",
"A complete list of DNA constructs and oligos is available in Supplementary file",
"4 . Two CRISPR/Cas9 genome editing protocols were used for this study .",
"In brief , for insertion of GFP or mCherry into endogenous genetic loci , GFP or mCherry was PCR amplified with primers containing homology arms for insertion sites as donor DNA for homologous recombination .",
"Donor DNA was co-injected with gRNA ( IDT ) , crRNA ( IDT ) , and Cas9 enzyme ( IDT ) as previously described ( Paix et al . , 2017 ) .",
"F1 generation animals were visually screened for presence of GFP or mCherry signals using Axioplan2 Fluorescence microscope ( Carl Zeiss ) .",
"GFP or mCherry-positive animals were then homozygosed in F2 generation and verified by Sanger sequencing .",
"For insertion of loxP sites into ztf-11 locus , a co-conversion strategy was used as previously described ( Arribere et al . , 2014 ) .",
"Synthesized loxP sequence ssDNA with 60 bp homology arms flanking insertion sites ( IDT ) was used as donor DNA for homologous recombination .",
"Donor DNA was co-injected with Cas9 expressing plasmid ( pJW1259 , kindly provided by Dr . Jordan Ward ) , sgRNA expressing vectors , dpy-10 targeting-gRNA , and dpy-10 donor DNA .",
"F1 animals were screened by PCR amplification of the loxP inserts .",
"Embryos were collected from gravid hermaphrodites and mounted with polystyrene beads ( Polysciences Inc ) as described ( Du et al . , 2015 ) .",
"Embryos were imaged on a Zeiss AxioObserver Z1 inverted microscope frame with Yokogawa CSU-X1 spinning disk and an Olympus UPLSAPO 60xs silicone oil immersion objective .",
"GFP and mCherry channels were acquired simultaneously on a pair of aligned EMCCD cameras ( C9100-13 ) .",
"Image acquisition was performed using MetaMorph software ( Molecular Devices ) .",
"Embryos were imaged every 75 s , with 30 z slices at 1 μm apart .",
"Lineage tracing and quantification of marker expression were done with the StarryNite and AceTree software as described ( Du et al . , 2015 ) .",
"Hermaphrodite animals were anesthetized using 2 . 5 mM levamisole , mounted on 3% agar pads , and imaged using a Zeiss LSM710 confocal microscope ( Carl Zeiss ) with a Plan-Apochromat 40x/1 . 3 NA objective or 63x/1 . 4NA objective .",
"Z stacks and maximum-intensity projections were generated using ImageJ ( NIH ) .",
"The imaging was not done by an experimenter blind to the experimental condition .",
"Fluorescence intensity measurements ( Figure 2 , Figure 8 , and Figure 4—figure supplement",
"2 ) were quantified using ImageJ ( NIH ) .",
"Quantification for cell identity markers were performed using Axioplan2 fluorescence microscope ( Carl Zeiss ) with a Plan-Apochromat 40x/1 . 3 NA objective .",
"When quantifying any cKO animals , presence of Cre-expressing transgene was checked after each animal’s phenotype was determined to prevent potential bias .",
"Worms grown on NGM plates were transferred to room temperature at least 1 hr prior to the assay .",
"Individual L1 animals were carefully transferred to a small drop of M9 media on a glass slide .",
"Following a minute of incubation in M9 , the number of thrashing events ( defined by one cycle of alternating ‘C’ bends ) were then counted for a minute .",
"The genotype of each assayed animals were determined after each counting to circumvent potential bias .",
"Samples for RNA-seq was prepared by feeding RNAi ( Timmons et al . , 2001 ) .",
"In brief , animals were harvested from NGM plates and eggs were collected by bleaching .",
"Eggs were hatched overnight in M9 media to get synchronized L1 larval culture ( Porta-de-la-Riva et al . , 2012 ) .",
"Synchronized L1 cultures were inoculated on plates expressing feeding RNAi clones for ztf-11 ( Kamath and Ahringer , 2003 ) ( Dr . Julie Ahringer ) or a control vector ( L4440 ) .",
"After 64 hr , adult worms bearing eggs were harvested and eggs were collected by careful bleaching .",
"Eggs were incubated in M9 for 3 hr to allow them to develop into gastrula stages .",
"Egg shell was disturbed with chymotrypsin ( Sigma-Aldrich ) and chitinase ( Sigma-Aldrich ) as previously described ( Edgar and Goldstein , 2012 ) and lysed by centrifuging through QiaShredder columns ( Qiagen ) following the manufacturer’s instructions .",
"RNA was isolated from the eggs using RNeasy Plus Micro Kit ( Qiagen ) following the manufacturer’s instructions .",
"Resulting RNA samples were quality controlled by Agilent Bioanalyzer 2100 and only the RNA samples with RIN of 9 or higher were submitted for library preparation .",
"mRNA libraries were prepared by Stanford Genome Sequencing Center using TruSeq Stranded mRNA Library Preparation kit ( Illumina ) .",
"Four biological replicates representing independent cultures of C . elegans on independently prepared feeding RNAi were performed for each sample in this study .",
"RNA-seq and computational analysis mRNA libraries were pooled and paired-end sequenced for 100 bp , resulting in 40 million raw reads per sample .",
"Raw reads were trimmed of adaptor sequences using fastx ( http://hannonlab . cshl . edu/fastx_toolkit/ ) and mapped to C . elegans reference genome ( ce10 ) using Tophat2 ( Kim et al . , 2013 ) and featureCounts ( Liao et al . , 2014 ) .",
"Uniquely mapped reads were used to calculate expression level of genes .",
"Differential expression analysis was performed using DeSeq2 ( Love et al . , 2014 ) .",
"GO-term enrichment analysis of significantly upregulated or downregulated genes ( FDR < 0 . 1 ) were performed through PANTHER gene ontology tool ( Thomas et al . , 2003 ) .",
"Raw sequencing data is accessible through NCBI GEO ( Accession code: GSE125694 ) .",
"Full DESeq2 output and GO-term enrichment analysis results can be found on Supplementary file 2 .",
"SiMPull assays were performed as previously described ( Zou et al . , 2016 ) .",
"In brief , C . elegans grown on twenty 15 cm dishes were collected and washed , then dropped in liquid nitrogen to form ‘worm pearls . ’ Worm pearls ( 50 mg wet weight ) were thawed in 250 ul lysis buffer ( 50 mM HEPES pH 7 . 7 , 50 mM KCl , 2 mM MgCl2 , 250 mM Sucrose , 1 mM EDTA pH 8 . 0 , with protease inhibitors ) .",
"After brief sonication on ice ( 3’ pulse with 30’ pause , six cycles ) to break the cuticle , 100 mM NaCl and 1% Triton X-100 were added into solution and samples were rotated at 4°C for 1 hr .",
"After centrifugation at 16 , 000 g for 15 min , supernatants were transferred to new tubes and measured by BCA assay ( Thermo Fisher Scientific ) for total protein concentration .",
"Worm lysates from different samples were adjusted to 7 mg/ml concentration by lysis buffer and used for SiMPull .",
"Briefly , normalized lysates were incubated on quartz slides pre-coated with or without biotinylated anti-GFP antibodies ( Rockland immunochemicals ) to pull down ZTF-11::GFP , after washing away unbound sample , mCherry signals were recorded to visualize captured ZTF-11 binding partners .",
"mCherry tagged proteins immobilized on the slides were visualized by a TIRF microscope equipped with excitation laser 561 nm , and DV2 dichroic 565dcxr dual-view emission filters ( 520/30 nm and 630/50 nm ) .",
"Mean spot counts per image and standard deviation were calculated from images taken from 5 to 17 different regions ."
]
] | [
"Cellular differentiation requires both activation of target cell transcriptional programs and repression of non-target cell programs .",
"The Myt1 family of zinc finger transcription factors contributes to fibroblast to neuron reprogramming in vitro .",
"Here , we show that ztf-11 ( Zinc-finger Transcription Factor-11 ) , the sole Caenorhabditis elegans Myt1 homolog , is required for neurogenesis in multiple neuronal lineages from previously differentiated epithelial cells , including a neuron generated by a developmental epithelial-to-neuronal transdifferentiation event .",
"ztf-11 is exclusively expressed in all neuronal precursors with remarkable specificity at single-cell resolution .",
"Loss of ztf-11 leads to upregulation of non-neuronal genes and reduced neurogenesis .",
"Ectopic expression of ztf-11 in epidermal lineages is sufficient to produce additional neurons .",
"ZTF-11 functions together with the MuvB corepressor complex to suppress the activation of non-neuronal genes in neurons .",
"These results dovetail with the ability of Myt1l ( Myt1-like ) to drive neuronal transdifferentiation in vitro in vertebrate systems .",
"Together , we identified an evolutionarily conserved mechanism to specify neuronal cell fate by repressing non-neuronal genes ."
] | [
"The human body contains many cell types that each have different job and can look very different from each other .",
"However , each of the cells in an individual’s body contains almost exactly the same genes , because all of them share the same DNA inherited from the individual’s parents .",
"Cells therefore become different from one another by controlling the activity of sets of genes .",
"They do this by using proteins called transcription factors , which find specific genes and turn them either on or off .",
"Nerve cells or neurons form and develop in a process called neurogenesis .",
"During neurogenesis , some genes including those specific to neurons need to be switched on while other non-neuronal genes need to be switched off .",
"The “off-switch” is particularly important when neurons are generated by conversion from skin cells , which sometimes happens in animals .",
"Before these cells can become mature nerve cells , they require transcription factors to ensure that skin-specific genes are off .",
"The transcription factors turning on nerve cell-specific genes are well-understood , but far less is known about those that turn off other genes .",
"Lee et al . therefore set out to search for transcription factors that might switch off non-neuronal genes during neurogenesis and focused on one transcription factor that is known to be important for the development of nerve cells in a variety of animal species .",
"Experiments using the worm C . elegans revealed that this transcription factor – called ZTF-11 in worms – was present in all cells destined to be nerve cells , but not in cells that would assume other roles .",
"These experiments are possible with C . elegans because the final role , or ‘fate’ , of each cell in the body are already known , all the way from the fertilized egg to the adult .",
"Further work , using genetically engineered worms revealed that ZTF-11 worked by turning off genes that are related to the development of non-nerve cells .",
"Deleting the gene for ZTF-11 in immature nerve cells allowed these cells to turn on different sets of genes and resulted in adult worms with fewer mature nerve cells than normal worms .",
"On the other hand , forcing other cell types ( which would not normally become part of the nervous system ) to produce ZTF-11 was sufficient to convert them into nerve cells .",
"These results are an important step forward in understanding how nerve cells are built in the developing body , especially how nerve cells can be made from other cell types .",
"In the future , this knowledge could be used to help people with diseases of the nervous system , such as Parkinson’s disease ."
] | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"neuroscience"
] | Chronology-based architecture of descending circuits that underlie the development of locomotor repertoire after birth | elife-42135-v2 | [
[
"At the time of birth , most animals are only capable of a limited set of reflexive behaviors that ensure their immediate survival .",
"However , as development progresses , their behavioral repertoire rapidly diversifies and they become capable of producing increasingly refined and cognitive behaviors ( Kagan and Herschkowitz , 2006; Harlow and Harlow , 1965; Fox , 1965; Drapeau et al . , 2002 ) .",
"Concurrent with such changes , many new connections are rapidly being formed in the nervous system ( Gilmore et al . , 2018 ) ; Semple et al . ( 2013 ) ; Levitt , 2003 ) , suggesting that the formation of new connections is linked to the development of the behavioral repertoire .",
"However , despite many studies that carried out circuit-level examination of the postnatal formation of new connections ( Morrie and Feller , 2016; Polley et al . , 2013; Stein and Stanford , 2013; Kano and Watanabe , 2013 ) very little is known still of how nascent connections are organized to support a behavioral repertoire that grows by including increasingly more sophisticated behaviors while retaining vital reflexive behaviors .",
"Recent studies in developing zebrafish have shed some light on the neural underpinnings of behavior development .",
"As in most animals , the behavioral repertoire of zebrafish quickly diversifies after birth ( hatching ) to include more sophisticated behaviors ( Drapeau et al . , 2002; McLean and Fetcho , 2009 ) .",
"At the time of birth , a zebrafish is mostly quiescent but in response to a tactile stimulus exhibits crude locomotor behaviors that entail powerful and large bends of the body such as seen during escape and struggle ( Figure 1A , Escape , Struggle ) ( McLean and Fetcho , 2009; Liao and Fetcho , 2008 ) .",
"This indicates that both of these early-born behaviors require strong activation of axial muscles across a large portion of the fish’s body despite their differences with respect to the the direction of motion and tail beat frequency ( escape is forward locomotion with fast tail beat frequency while struggle is backward locomotion with slow tail beat frequency ) .",
"However , by 2 days after birth , the fish also spontaneously exhibit a more refined locomotor behavior wherein only the caudal portion of the tail moves ( Figure 1A , Spontaneous swim ) with relatively weaker and slower wave-like undulating movements ( McLean and Fetcho , 2009 , Figure 1A; Mirat et al . , 2013 ) .",
"Such movements are indicative of slow propagation of moderate axial muscle activity from the rostral to the caudal end of the moving part of the tail .",
"With this type of tail-restricted locomotion , the fish is able to move forward by keeping its head , and therefore gaze , stable .",
"This ability may be critical for the subsequent development of visually guided behaviors such as prey capture .",
"Escape , one of the crude locomotor patterns that emerge early after the birth of a zebrafish , is controlled by spinal interneurons that are born well before the animal’s birth ( McLean and Fetcho , 2009; Kimura et al . , 2006; Eklöf-Ljunggren et al . , 2012 ) , whereas the more refined and weaker locomotion pattern that emerges after an animal’s birth is controlled by spinal interneurons that are born around the time of an animal’s birth ( McLean et al . , 2007; Satou et al . , 2012; McLean and Fetcho , 2009 ) .",
"This indicates that the sequential emergence of new and more sophisticated locomotor patterns during development is mediated at the level of the spinal cord by the addition of distinct functional groups of neurons rather than by fine control of a single functional group ( Fetcho and McLean , 2010 ) .",
"Similarly in the mammalian spinal cord , it has been shown more recently that distinct functional groups emerge in sequence during development ( Tripodi et al . , 2011 ) .",
"This finding has led to the idea that even in mammals , the development of the locomotor repertoire is supported by the addition of new functional groups ( Tripodi and Arber , 2012 ) .",
"However , it has yet to be revealed how the connections from the brain to emerging spinal functional groups are established so that new and existing spinal groups can be recruited appropriately by the brain to support the diversification of the locomotor repertoire .",
"Locomotion-related signals from the brain reach the spinal cord by way of excitatory reticulospinal neurons of the hindbrain ( Dubuc et al . , 2008; Grillner and Georgopoulos , 1996; Jordan et al . , 2008; Roberts et al . , 2008 ) .",
"V2a neurons in the hindbrain , similar to the ones in the spinal cord , are glutamatergic and project their axons ipsilaterally ( Cepeda-Nieto et al . , 2005; Kinkhabwala et al . , 2011; Kimura et al . , 2013; Bouvier et al . , 2015 ) .",
"They include excitatory reticulospinal neurons ( Cepeda-Nieto et al . , 2005; Kimura et al . , 2013; Bouvier et al . , 2015 ) and are capable of initiating and terminating locomotion ( Kimura et al . , 2013; Bouvier et al . , 2015 ) .",
"Interestingly , the hindbrain V2a neurons in larval zebrafish show a topographical organization that is related to differentiation time such that newly-born neurons stack dorsally to pre-existing ones ( Kinkhabwala et al . , 2011 ) .",
"Furthermore , in a subset of the hindbrain V2a neurons , it has been shown that the ventral , and therefore early-born , population gets recruited during escape-like locomotion with fast tail beat frequency while the dorsal , and therefore late-born , population gets recruited during spontaneous swim-like locomotion with slow tail beat frequency ( Kinkhabwala et al . , 2011 ) .",
"Even though it is unknown whether this finding holds true for the entire hindbrain V2a population , it suggests that , even in the hindbrain V2a neurons , distinct functional groups emerge in sequence and contribute to the development of locomotor behaviors .",
"Such hindbrain organization raises the possibility that a new hindbrain functional group connects selectively to an age-matched spinal functional group to produce a novel locomotion pattern .",
"However , it is also possible that hindbrain functional groups emerging in succession provide increasingly finer excitatory drive to all spinal groups , but each hindbrain group recruits only the age-matched spinal group because of the higher excitability of the latter compared to its predecessors ( McLean et al . , 2007 ) .",
"Indeed , in the case of midbrain descending pathways , the recruitment of spinal neurons is determined not by selectivity of the descending inputs these neurons receive but by the biophysical properties they express ( Wang and McLean , 2014 ) .",
"Thus , it remains to be resolved how the the hindbrain descending neurons establish connections to the spinal functional groups during development to support the postnatal diversification and increased sophistication of locomotor patterns .",
"Here , we examined the development of spinal pathways from the hindbrain V2a neurons and the role of these pathways in the development of locomotion .",
"We show that early-born V2a descending neurons that project to the spinal cord early in development are only recruited during the stimulus-elicited crude and powerful locomotor patterns that appear early in development whereas their late-born counterparts that project to the spinal cord late in development are recruited during the more refined and weaker spontaneous locomotion that appears later in development .",
"Moreover , the spinal projections of these two populations form spatially-distinct neuropil layers and give rise to parallel pathways instead of forming a series of non-selective pathways that differ in the strengths of their connections .",
"Furthermore , these parallel pathways differ in their connectivity patterns and biophysical properties in a manner suitable for the locomotor behavior they participate in .",
"The early-born group makes direct connections to a class of motoneurons capable of producing strong axial muscle activity across a large extent of the spinal cord .",
"The early-born group also expresses biophysical properties with fast time constants .",
"Both these features are well-suited for producing the crude types of locomotion that appear early in development in that they support the near simultaneous activation of axial muscles across a large extent of the fish’s body .",
"On the other hand , the late-born group connects to the late-born spinal interneurons that are active during refined locomotion and express biophysical properties with slow kinetics .",
"These interneurons innervate to a class of motoneurons that produce weaker axial muscle activity in the caudal spinal cord .",
"These features make the late-born group more suitable for producing the refined locomotion that appears later in development in that they support the moderate axial muscle activity in the caudal portion of the tail .",
"Indeed , ablation of each group produced deficits in distinct motor patterns: ablation of the early-born group weakened the sensory-elicited crude and fast locomotion whereas ablation of the late-born group affected the more refined and slower spontaneous locomotion .",
"Altogether , we reveal in the descending circuits a chronologically-layered parallel architecture underlying the diversification and increased sophistication of motor patterns in larval zebrafish .",
"Even though chronotopic neuropil organization similar to what we have described here has been observed in many neural systems ( Espinosa and Luo , 2008;Tripodi et al . , 2011; Voigt et al . , 1993; Walsh and Guillery , 1985; Kulkarni et al . , 2016; Brierley et al . , 2009; Brierley et al . , 2012 ) , it has not been demonstrated in these systems how such organization relates to the postnatal development of behaviors .",
"The findings we report here suggest that the chronological layering of parallel circuits and the systematic variation in the associated connectivity patterns and biophysical properties are fundamental attributes of the nervous system that form the basis for an animal’s ability to exhibit new and increasingly sophisticated behaviors after birth while maintaining vital reflexive behaviors ."
],
[
"To link the development of the locomotor repertoire to the development of hindbrain V2a neurons and their spinal pathways , we systematically examined the ontogeny of these neurons , their topography and the onset of their projections to the spinal cord until 120 hr post-fertilization ( hpf ) when larvae are capable of exhibiting both stimulus-evoked crude locomotion that appears by 48 to 72 hpf and more refined spontaneous locomotion that appears by 96 to 120 hpf ( Figure 1A ) ( Drapeau et al . , 2002; McLean and Fetcho , 2009 ) .",
"We first examined the ontogeny of hindbrain V2a neurons from 24 hr post fertilization ( hpf ) to 120 hpf with time lapse imaging ( Figure 1B , n = 8 for each timepoint ) of a transgenic line expressing EGFP under the control of the promoter of vsx2 , a transcription factor specific to V2a neurons .",
"This transcription factor has been shown to become active after the final cell division ( Kimura et al . , 2008 ) .",
"Therefore , the onset of EGFP expression in a V2a neuron in our transgenic line indicates the time of differentiation - which we use synonymously with the time of birth - of this neuron .",
"At 24 hpf , one or two pairs of V2a neurons appeared in a segmental fashion ( Figure 1B iii , iii’ , 24 hpf ) , and then the number of V2a neurons increased dramatically until 60 hpf ( Figure 1B iii , iii’ , 60 hpf ) .",
"After 72 hpf , the V2a cluster showed no drastic visible changes in the overall shape ( Figure 1B iii , iii’ , 72–120 hpf ) .",
"As a fish is still only capable of generating crude forms of locomotion at 72 hpf , this suggests that the connectivity of its neurons and their electrophysiological properties need to mature to support refined locomotion .",
"We then examined where V2a neurons born at different time points resided in the hindbrains of 120 hpf fish that are capable of generating both the crude and refined forms of locomotion .",
"We did this by photoconverting Kaede , a fluorescent photoconvertible protein , which was expressed under the control of the vsx2 promoter .",
"We converted Kaede throughout the fish at different time points ( 24–96 hpf ) in different groups of fish and imaged each of these groups at 120 hpf ( Figure 2—figure supplement 1A i , n = 8 for each timepoint ) .",
"Based on the presence of converted Kaede ( shown in magenta ) , we distinguished neurons that already had Kaede at the time of photoconversion from those that started to express Kaede after the time of photoconversion ( Kimura et al . , 2006; Caron et al . , 2008 ) .",
"In most rhombomeres , V2a neurons born by 24 hpf were located in the most lateral portions of the brain region occupied by this group of neurons ( Figure 2—figure supplements 1C and 24 hpf conversion , magenta cells ) , whereas neurons born afterwards gradually filled up the space medial to the pre-existing ones ( Figure 2—figure supplement 1C and 36–72 hpf conversion ) .",
"Compared to photoconversion at earlier time points , photoconversion at 72 hpf revealed relatively few , if any , unconverted green cells , except in the rostral hindbrain ( Figure 2—figure supplements 1C and 72 hpf conversion ) .",
"This indicated that the neurogenesis of hindbrain V2a neurons was mostly complete by 72 hpf when fish are still only capable of producing the crude locomotion , suggesting that further developments of these neurons are required if they are to contribute to the development of locomotor behaviors .",
"Hindbrain V2a neurons with spinal projections ( Video 1 ) are more likely to play roles in the development of the locomotor repertoire , therefore we examined the times of birth of these neurons in further detail .",
"First , we focused on large spinal projecting neurons ( reticulospinal neurons ) that were labeled by dye injection in the spinal cord ( Figure 2C–E , n = 4 for each timepoint ) .",
"These neurons are identifiable across animals and are named based on their rostrocaudal ( Figure 2C , left ) and dorsoventral positions ( Figure 2C , right ) ( Kimmel et al . , 1982; Mendelson , 1986 ) .",
"The dorsal subpopulation of reticulospinal neurons ( Figure 2C , MiD2i , MiD3i , RoM2 , RoM3 and dorsal MiV1 ) corresponded to V2a neurons born by 24 hpf ( Figure 2D ) , while the majority of ventral reticulospinal neurons ( RoV3 , ventral MiV1 and MiV2 ) corresponded to V2a neurons born by 36 hpf ( Figure 2E; Video 2 ) .",
"This birth order is consistent with previous birthdating analysis of reticulospinal neurons based on the incorporation of a marker into replicating DNA ( Mendelson , 1986 ) .",
"Other than the large spinal projection neurons that are labeled consistently by dye injection , a majority of the V2a neurons in the caudal hindbrain have also been shown to project to the spinal cord ( Kimura et al . , 2013 ) ( Video 1 ) .",
"In this region , the cell bodies of the earliest-born V2a neurons were located lateral to the populations born afterwards ( Figure 2F ) .",
"This clear topographical organization allows us to readily identify the earliest-born population and the later-born population in the caudal hindbrain .",
"Notably , this region contained the youngest group of neurons among the hindbrain V2a descending neurons we examined , suggesting their roles in the refined locomotion that develops later ( Figures 2F and 48 hpf conversion ) .",
"For the hindbrain V2a neurons to contribute to the development of the locomotor repertoire , they first need to establish connections to spinal neurons .",
"To examine the development of spinal projections we optically back labeled V2a descending neurons from the spinal cord ( ‘optical backfill’ ) using photoconversion restricted to the rostral spinal cord ( Kimura et al . , 2013 ) .",
"This procedure was repeated at multiple developmental time points to examine the developmental sequence of spinal projections ( Figure 3A , n = 6 for each timepoint ) .",
"The optical backfill at 36 hpf labeled the dorsal V2a but not the ventral V2a reticulospinal neurons ( Figure 3C ) while the backfill at 60 hpf labeled both the dorsal and ventral reticulospinal neurons ( Figure 3D ) , indicating that these neurons had projected to the rostral spinal cord in sequence based on their respective differentiation times .",
"The V2a subpopulation in the caudal hindbrain showed a pattern similar to that indicated by the birthdating analysis ( Figure 3E ) .",
"This indicates that the sequential development of spinal projections based on differentiation time holds true for the caudal hindbrain V2a subpopulation as well .",
"Among these descending neurons , the caudal medial V2a neurons were the last to project to the spinal cord .",
"As these neurons had descending processes in the rostral spinal cord by 84 hpf , it is plausible that they contribute to the refined locomotion that appears as early as 96 hpf .",
"Collectively , the results of our developmental analysis showed the timeline of neurogenesis and spinal projections of hindbrain V2a descending neurons relative to the development of the locomotor repertoire .",
"Furthermore , we found that the birth order of these neurons dictated the positions of their cell bodies and the order of their spinal projections .",
"Most importantly , this analysis established a way to identify the birthdates of these neurons based on the positions of their cell bodies .",
"This set the stage for functional analysis of these neurons , wherein we characterized how neurons of different age were recruited during the distinct locomotor patterns that fish develop in sequence so as to examine the functional links between the development of V2a neurons and the development of locomotor behaviors .",
"To identify functional cell groups related to the crude and refined locomotor behaviors that appear in sequence during development , we examined the recruitment of hindbrain V2a neurons expressing the calcium indicator GCaMP6s ( Chen et al . , 2013 ) using whole-hindbrain two-photon volumetric imaging at 2 Hz in 120–132 hpf fish , which are capable of generating both forms of locomotion .",
"Fish were paralyzed and axial motor activity was monitored using glass pipettes attached to motor nerves innervating axial muscles on both sides ( Figure 4; Video 3 ) .",
"In this condition , fish spontaneously exhibited episodes of rhythmic axial motor activity ( Figure 4A i; Figure 4B",
"i ) .",
"The maximum beat frequencies of these episodes matched those of slow spontaneous swims generated through tail-restricted movements in unparalyzed fish ( 12 fish , Figure 4C ) ( Mirat et al . , 2013 ) ; Marques et al . , 2018 ) , suggesting that these episodes are fictive analogs of refined locomotor behavior .",
"To elicit the crude locomotor behaviors , we used two stimulus paradigms ( six fish for each paradigm ) .",
"The first paradigm made use of a transient electrical pulse delivered to one side of the head to evoke locomotor episodes similar to escapes in unparalyzed condition ( Figure 4A",
"i ) .",
"These episodes started with strong axial motor activity on the side contralateral to stimulation , which is consistent with the powerful whole-body bending away from the stimulus observed at the start of escape ( Liu and Fetcho , 1999; Koyama et al . , 2016 ) .",
"Maximum beat frequencies during these episodes was also significantly higher than those during spontaneous rhythmic activity ( Figure 4C , p<0 . 001 ) .",
"This is consistent with fast rhythmic bending observed during escape ( Mirat et al . , 2013 ) ; Marques et al . , 2018 ) , further suggesting that these episodes correspond to escapes .",
"The second paradigm employed a gradual mechanical stimulus applied to the front of the head to evoke locomotor episodes similar to struggles ( Liao and Fetcho , 2008 ) .",
"These episodes exhibited powerful bursting activity ( Figure 4B",
"i ) , consistent with the powerful whole-body bending observed during struggles ( Liao and Fetcho , 2008 ) .",
"The maximum beat frequencies of these powerful bouts of bursting activity were clearly lower than those of putative escapes ( Figure 4C , p<0 . 001 ) and also statistically lower than those of spontaneous slow swims ( Figure 4C , p<0 . 001 ) .",
"They also matched the frequencies previously reported for struggles ( Liao and Fetcho , 2008 ) .",
"Peripheral motor nerve recordings from a single site on each side did not allow us to determine if the rostrocaudal patterns of axial motor activity were organized similarly to those observed in unparalyzed fish , but beat frequency and relative strength of axial motor activity were similar to those of axial movements observed in unparalyzed fish .",
"With these three distinct locomotor patterns , we examined not only how hindbrain V2a descending neurons are recruited during crude forms of locomotion that develop early and more refined form of locomotion that develop later but also how the recruitment of these neurons changes in relationship to beat frequency and strength of axial motor activity .",
"To examine neuronal recruitment , we first analyzed imaging data , focusing on the V2a descending neurons we had identified based on development .",
"In the electrical shock paradigm ( Figure 4A ) , almost all the identifiable dorsal neurons were recruited during putative escapes but primarily on the side ipsilateral to the leading axial motor activity ( Figure 4A iii , blue traces ) .",
"This is consistent with the primarily ipsilateral projections of these neurons ( Kinkhabwala et al . , 2011; Kimmel et al . , 1982 ) .",
"On the other hand , the ventral MiV1 and caudal medial neurons showed stronger activity during weak spontaneous swimming .",
"In the gradual mechanical stimulus paradigm , a subset of the neurons recruited during putative escapes were recruited when there was strong ‘struggle’ like motor activity on the ipsilateral side ( Figure 4B iii-iv , blue traces close to the end ) .",
"The ventral MiV1 and caudal medial cells were again recruited during weak spontaneous swimming .",
"When we sorted these cell types based on their birthdate and examined their responses during these motor patterns , it became apparent that there was a systematic relationship between recruitment and birthdate ( Figures 4C and 6 fish for each paradigm ) .",
"The early-born group ( Figure 4C , <24 hpf ) did not show any clear response during spontaneous weak swimming , but many neurons in this group showed significant responses during putative struggles that exhibited strong locomotor activity but with low beat frequency .",
"The responses in a few of these neurons were even stronger during putative escapes that exhibited strong locomotor activity with high beat frequency ( Figure 4C , MiD2i , RoM2 and RoM3 ) .",
"The recruitment of the early-born group during both putative struggles and escapes indicates that this group of neurons underlies powerful whole-body movements observed in both struggles and escapes in the unparalyzed condition .",
"The further recruitment of this group during putative escapes also indicates that such enhanced activity may lead to fast cyclic movements of the whole-body as observed in escapes .",
"On the other hand , the intermediate-aged group ( Figure 4C 24-36 hpf ) showed mixed recruitment patterns; the subgroup in the rostral hindbrain ( Figure 4C , RoV3 ) showed comparably strong activity during putative escapes and putative struggles but not during weak spontaneous swims whereas the subgroup in the middle hindbrain ( Figure 4C , ventral MiV1 ) showed comparably strong activity in all three conditions .",
"In contrast , the late-born group in the caudal hindbrain ( Figure 4C 36-60 hpf ) showed more activity only during weak spontaneous swimming .",
"Interestingly , during stimulus-evoked strong swims , these neurons showed decreased activity instead , raising the possibility of inhibition during strong swims .",
"Taken together , our findings indicate that a recruitment pattern that is based on the time of differentiation holds true for all the hindbrain V2a descending neurons that we identified based on development; the early-born group is recruited during the crude forms of locomotion ( escapes and struggles ) that appear early in development whereas the late-born group is recruited during the refined locomotion that appears later in development .",
"This finding , along with the finding that spinal projections develop sequentially , suggests that distinct functional groups emerge in developmental sequence and contribute to the development of locomotor behaviors .",
"Furthermore , the active set of hindbrain V2a neurons switched based on the type of ongoing motor activity , which is reminiscent of the recruitment of spinal interneurons ( McLean et al . , 2008 ) .",
"This raises the possibility that distinct functional groups of hindbrain V2a neurons may provide excitatory drive to corresponding spinal functional groups .",
"To see if our observations about neuronal recruitment extend to the complete set of hindbrain V2a neurons , we performed regression analysis ( Miri et al . , 2011b ) ( Figure 5 ) .",
"In the electrical pulse paradigm , we used two regressors to distinguish the activity related to shock-induced putative escapes from activity related to weak spontaneous swimming ( Figure 5A",
"ii ) .",
"The activity map for the shock-evoked putative escapes ( Figure 5A iii , left; Figure 5A iv , magenta; Video 4 , magenta ) revealed a large population of recruited neurons on the side contralateral to the stimulus ( ipsilateral to the leading side of motor activity ) , consistent with their primarily ipsilateral projections ( Kinkhabwala et al . , 2011 ) .",
"This includes the dorsal early-born group and the rostral intermediate group ( RoV3 ) in the rostral hindbrain ( Figure 5A v , magenta ) and a large number of the lateral early-born neurons in the caudal hindbrain ( Figure 5A vi , magenta ) .",
"The activity map for spontaneous weak swimming revealed the middle intermediate group ( ventral MiV1 ) in the rostral hindbrain ( Figure 5A iii , right; Video 4 , green ) and a large number of the medial late-born neurons in the caudal hindbrain ( Figure 5A iii , right; Figure 5A vi , cyan; Video 4 , green ) .",
"For the gradual mechanical stimulus paradigm that produced putative struggles that varied in latency , we constructed three regressors based on the strength of the axial motor activity: two regressors for the strong motor activity for each side to capture the struggle related activity and one regressor for the weak motor activity to capture the activity related to spontaneous weak swims ( Figure 5B",
"ii ) .",
"The activity maps for the strong motor activity ( Figure 5B iii , left and right; Figure 5B iv , yellow and magenta; Video 5 , yellow and magenta ) revealed a large population of neurons ipsilateral to the side of motor activity ( Figure 5B iii , left and right ) consisting of a subpopulation of the dorsal early-born group , the rostral intermediate group ( RoV3 ) in the rostral hindbrain ( Figure 5B",
"v ) , and a large number of the early-born lateral neurons in the caudal hindbrain ( Figure 5B vi , magenta ) .",
"The map for spontaneous swimming revealed the same set of the neurons shown for spontaneous swimming occurring in the electrical pulse paradigm ( Figure 5B iii , middle; Figure 5B iv , cyan; Video 5 , cyan ) .",
"These regression maps were reproducible across fish ( Figure 5—figure supplement 1 ) and further support the differentiation time dependent recruitment pattern revealed by the ROI analysis .",
"The activity maps for putative escapes and struggles were surprisingly similar , especially in the caudal hindbrain ( Figure 5—figure supplement 1 ) .",
"This supports the idea that the early-born population contributes to the crude whole-body movements observed in both struggles and escapes .",
"However , this analysis also made it clear that the activity of the early-born group is further enhanced during escapes compared to struggles , especially in the rostral hindbrain ( Figure 5—figure supplement 1 ) , suggesting again that further activation of this population leads to fast cyclic movements of the whole-body , as observed in escapes .",
"Interestingly , these maps also revealed a striking functional separation of neuropil in the caudal hindbrain ( Figure 5A vi; Figure 5B vi; Figure 5—figure supplement 1C ) .",
"The neuropil active during weak spontaneous swimming was located lateral to the neuropil active during putative struggles ( Figure 5B vi , filled arrowheads; Figure 5—figure supplement 1C , Struggle , arrowheads ) .",
"This functional segregation is the opposite of the functional segregation of cell bodies in the caudal hindbrain ( Figure 5B vi , open arrowheads ) .",
"This neuropil separation was maintained when additional cells were recruited for shock-induced escapes ( Figure 5A vi , filled arrowheads; Figure 5—figure supplement 1C , Escape , arrowheads ) , suggesting that these additional cells also followed this functional separation .",
"Over all , this raises the possibility that these functional neuronal groups project their descending axons through distinct regions in the neuropil .",
"To this point , we showed a correspondence between the birth order of hindbrain V2a neurons and their involvement in distinct forms of locomotion that appear in sequence .",
"This suggests that distinct functional cell groups emerge in the hindbrain and project to the spinal cord in sequence and contribute to the diversification of locomotor repertoire .",
"Furthermore , the similarity in the recruitment of hindbrain and spinal functional groups as well as the spatial segregation of the neuropil of the hindbrain functional groups both raise the possibility that parallel pathways connect matching sets of hindbrain and spinal functional groups .",
"Having established a link between the birth order and function of hindbrain V2a neurons , we next examined the spinal organization of descending processes from hindbrain functional groups .",
"To understand how the spinal projections from hindbrain V2a neurons are organized to support the development of the locomotor repertoire , we examined if the aforementioned sequentially generated functional groups of hindbrain V2a neurons exhibit distinct innervation patterns to the spinal cord .",
"First , we examined the overall neuropil organization of hindbrain and spinal V2a neurons based on the time of differentiation by photoconverting early-born V2a neurons at 24 and 36 hpf ( Figure 6A , n = 6 fish for each timepoint ) .",
"In the hindbrain ( Figure 6A",
"iii ) , and throughout the spinal cord ( Figure 6A iv-vi ) , the neuropil from V2a neurons born before 24 hpf ( Figure 6A iii-vi , magenta arrowheads ) was located medial to the neuropil from neurons born after 24 hpf ( Figure 6A iii-vi , green arrowheads ) , consistent with the functional segregation of neuropil we observed in the caudal hindbrain .",
"The neuropil from V2a neurons born before 36 hpf was still located medial to the neuropil from neurons born after 36 hpf ( Figure 6A viii-xi , arrowheads ) .",
"However , it extended more toward the lateral surface than the neuropil from V2a neurons born before 24 hpf ( Figure 6A xii ) .",
"This suggested that axonal processes from new neurons were continuously being added laterally to preexisting processes , forming layers based on the time of differentiation .",
"To see if this neuropil organization was maintained in the descending processes of hindbrain V2a neurons , we photoconverted V2a neurons only in the hindbrain ( Figure 6B ) .",
"The Gal4-UAS system was used to gain enough expression of Kaede to be able to visualize the spinal processes of hindbrain V2a neurons .",
"Putative presynaptic terminals were also labeled with synaptophysin-GFP driven by the Gal4-UAS system .",
"To visualize the spinal projections from only the early-born and intermediate-aged hindbrain V2a groups , UV light was shone on the hindbrain at 42 hpf ( Figure 6B ii-v , n = 8 fish ) .",
"This conversion time was selected to account for the delay in the expression of Kaede driven by the Gal4-UAS system and to match the age groups labeled by the photoconversion of Tg ( vsx2:Kaede ) at 36 hpf .",
"The descending processes from the early-born and intermediate-aged groups reached the caudal part of the spinal cord ( Figure 6B",
"v ) and were in the medial part of the neuropil in the rostral and middle spinal cord ( Figure 6B iii-iv , magenta arrowheads ) .",
"To visualize the spinal projections from almost all the hindbrain V2a neurons , we photoconverted Kaede in the hindbrain at 96 hpf and imaged them at 120 hpf ( Figure 6Bvi-ix , n = 8 fish ) .",
"The descending processes from hindbrain V2a neurons covered almost all the neuropil region formed by V2a neurons ( Figure 6B vii-ix , magenta arrowheads ) .",
"Comparison of the lateral extents of the descending processes of the early-born and intermediate-aged groups ( Figure 6B x , blue lines ) and all groups ( Figure 6B x , orange lines ) indicated that the descending processes from the late-born group were most prominent in the lateral neuropil in the rostral and middle spinal cord .",
"Taken together , these results indicated that the spinal projections from hindbrain V2a neurons were also layered based on the time of differentiation and that the late-born group had shorter descending processes than the early-born and intermediate-aged groups .",
"The difference in the axon length across different age groups is consistent with the pattern previously revealed by single-cell labeling ( Kinkhabwala et al . , 2011 ) .",
"These observations support the notion that the sequentially generated hindbrain functional groups provide parallel spinal pathways .",
"To examine the pattern of connections made by each pathway onto possible postsynaptic spinal neurons , we highlighted the descending processes of early-born and intermediate-aged hindbrain V2a neurons by photoconversion in the hindbrain , and additionally backfilled the following two spinal cell types with a far-red dye .",
"The first cell type is primary motoneurons ( PMN ) which are among the earliest-born neurons in the spinal cord ( Myers et al . , 1986 ) ( Figure 6B xi , n = 8 fish ) .",
"They are only recruited during the strongest of movements in contrast to secondary motoneurons ( SMN ) which are also recruited during weaker movements ( McLean et al . , 2007; Menelaou and McLean ( 2012 ) ; Wang and Brehm , 2017 ) .",
"The second cell type is multipolar commissural neurons ( MCoD ) , which are later-born excitatory interneurons in the rostral spinal cord ( Figure 6B xii , n = 8 fish ) .",
"They are only active during slow swimming and have monosynaptic connections to SMNs in the caudal spinal cord ( McLean et al . , 2008; Fetcho and McLean , 2010 ) .",
"We found that PMNs were located close to the medial neuropil region occupied by the spinal projections from the early-born hindbrain V2a population , whereas MCoDs were located close to the lateral neuropil region occupied by the spinal projections from the late-born population .",
"Thus , these results support the notion that there are parallel pathways between the hindbrain and the spinal cord that are separable by differentiation time .",
"The innervation patterns exhibited by these hindbrain V2a age groups are consistent with the type of axial muscle activity observed during the locomotor patterns that these age groups participate in: direct activation of PMNs throughout the spinal cord by the early-born group is expected to lead to the powerful whole-body bends observed during the crude and strong forms of locomotion ( escape and struggle ) , whereas indirect activation of SMNs in the caudal spinal cord through MCoDs should lead to slow bending of the caudal tail observed during the more refined and weaker locomotion .",
"Thus , these observations raise the possibility that each age group contributes to kinematics specific to the locomotor pattern in which this group participates .",
"So far , we experimentally investigated the overall organization of the hindbrain V2a population and the results suggest that birthdate-dependent parallel circuit organization underlies the development of locomotor behaviors .",
"To examine this more rigorously and to gain deeper insights about the biophysical properties of V2a neurons and their descending pathways , we examined various subsets of V2a reticulospinal neurons in depth .",
"Systematic characterization of the development , recruitment , and connectivity of V2a descending neurons led us to hypothesize that early-born hindbrain V2a descending neurons contribute to the powerful whole-body movements observed during the stimulus-evoked locomotion that develops early in development while their late-born counterparts contribute to the weaker tail-restricted movements observed during the spontaneous locomotion that develops later .",
"To test this hypothesis , we separately ablated either the early-born or the late-born hindbrain V2a descending neurons and at 5 days post-fertilization ( dpf ) examined the effects of each of these ablations on a few distinct patterns of locomotion ( Figure 10 ) .",
"To examine a wide range of swimming patterns in the freely swimming condition , we employed two experimental conditions: auditory-evoked escape responses ( Figure 10A",
"ii ) and spontaneous swimming ( Figure 10A",
"iii ) .",
"In each condition , we monitored movements of the fish using a high-speed camera ( Figure 10A",
"i ) .",
"Auditory stimulus-elicited escape episodes consisted of large and fast changes in head orientation as well as body bends ( Figure 10A",
"ii ) , these being characteristic of the crude locomotion that develops early .",
"On the other hand , spontaneous swim episodes mostly consisted of small changes in head orientation , and slow and small bends restricted to the caudal portion of the tail ( Figure 10A",
"iii ) , and these are typical of the more refined locomotion that develops later .",
"The distinctiveness of these two swim types became quite apparent when for each swim episode the maximum angular velocity ( see Materials and methods ) was plotted against the maximum body bend amplitude ( Figure 10A",
"iv ) .",
"In this plot , the points corresponding to escapes and slow swims clearly segregated into distinct clusters .",
"Consistent with the findings of a previous study ( Burgess and Granato , 2007 ) , when we looked at the distribution of the amplitude of the first total body bend in each spontaneous swim episode we found it to be multimodal , suggesting that spontaneous swim episodes consisted of distinct subtypes ( Figure 10A",
"v ) .",
"A Gaussian mixture model fit indicated that the best fit to the distribution was using a linear combination of three distinct gaussian distributions , suggesting that there were potentially three subcategories of spontaneous swim episodes ( see Materials and methods ) .",
"The timeseries of body bends and head orientation in each category showed consistent motor patterns ( Figure 10A",
"vi ) , further supporting the idea that fish at 5 dpf exhibit at least three distinct motor patterns spontaneously .",
"We referred to these patterns as ‘scoot’ , ‘routine turn’ and ‘high-angle turn’ based on the nomenclature adopted in previous studies ( Burgess and Granato , 2007; Marques et al . , 2018 ) .",
"Combined with auditory-evoked escape responses , we examined four distinct motor patterns in total ( Figure 10A",
"vi ) .",
"We focused on two age groups of hindbrain V2a descending neurons based on their times of differentiation and their recruitment patterns .",
"The first group consisted of neurons born before 24 hpf ( 10 fish for the ablated group; 10 fish for the control group ) .",
"We targeted all these neurons in the hindbrain as the optical backfill and Ca2+ imaging indicated that all of them were descending neurons involved in strong locomotion .",
"We visualized these neurons by photoconverting Tg ( vsx2:Kaede ) at 24 hpf , and at 60 hpf we ablated these neurons by femtosecond laser pulses ( Figure 10B i; 131 ± 17 . 3 cells/fish ) .",
"We delivered multiple pulses to each neuron ( typically 5–10 pulses/neuron ) until the fluorescence signal suddenly decreased .",
"Neurons labeled with photoconverted Kaede disappeared with no clear unintended damage to the nearby cells and processes ( Figure 10B",
"ii ) .",
"To confirm that these neurons did not recover before the behavior experiments , we imaged them again at four dpf ( Figure 10—figure supplement 1A ) .",
"Comparison with the control group at 4 dpf clearly showed that these early-born neurons did not recover from ablation .",
"The second group of neurons targeted for ablation consisted of neurons born from 42 hpf to 60 hpf ( Figure 10B iii; 10 fish for the ablated group; 10 fish for the control group ) .",
"We focused on the neurons in the dorsocaudal hindbrain because the optical backfill and Ca2+ imaging indicated these neurons are descending neurons involved in spontaneous slow locomotion .",
"We identified these V2a neurons based on the lack of photoconverted Kaede at 60 hpf after the photoconversion at 42 hpf ( Figure 10B",
"iii ) and their relative position to the nearby early-born neurons ( 508 ± 15 cells/fish; see Materials and methods ) .",
"Femtosecond laser pulses ablated these neurons with no clear unintended damage to the nearby early-born neurons ( Figure 10B",
"iv ) .",
"Comparison with the control group at 4 dpf clearly showed the reduction of the late-born neurons in the dorsocaudal hindbrain ( Figure 10—figure supplement 1B ) .",
"To assess if ablations had resulted in global changes in swim episodes , we examined total swim distance per episode , maximum swim velocity per episode , mean swim velocity per episode , episode duration , and total number of bends per episode ( see Materials and methods ) for each of the four distinct swim types ( Figure 10—figure supplement 2 ) .",
"We found that for scoots all of the foregoing global parameters were significantly reduced in the late-born V2a neuron ablation group ( Figure 10—figure supplement 2B , p<0 . 01 , corrected for multiple comparisons , Holm test ) .",
"For routine turns , these ablations produced a slight reduction in swim distance and mean swim velocity , but at a lower significance level ( Figure 10—figure supplement 2B , p<0 . 05 , corrected for multiple comparisons , Holm test ) .",
"This is consistent with our prediction that the late-born V2a neuron contributes to the weaker tail-restricted movements observed during spontaneous swims .",
"On the other hand , ablation of early-born V2a neurons did not result in changes in any of these global parameters .",
"Neither ablation of the early-born or the late-born neurons resulted in a change in the onset latency of escape ( Figure 10—figure supplement 2A ) .",
"The lack of phenotype for the early-born group ablation is perhaps not surprising as the recruitment and connectivity of the early-born V2a neurons suggest that the this group is likely to contribute most to the powerful body bends observed during the initial phase of escapes .",
"To examine the effect of ablations on a bend-by-bend basis , for each swim type , we compared the amplitudes and durations of body bends between ablated and matched control groups ( Figure 10C ) .",
"This analysis is based on the assumption that each swim type is so stereotypical that one could compare identically-numbered bends across episodes .",
"We assess this assumption based on the timing of each bend with respect to the onset of the episode ( Figure 10—figure supplement 3 ) : we examined how discriminable the time of maximum amplitude of each bend is from that of the preceding bend ( see Materilas and methods ) .",
"We found that the discriminability during escapes dropped suddenly after the sixth bend but for other swim types discriminability decreased more gradually until the tenth bend which marked the end to most of these episodes ( Figure 10—figure supplement 3B",
"ii ) .",
"Based on this observation , we decided to perform bend-by-bend analysis up to the 10th bend across all swim types for consistency .",
"Relative to the control group , the early-born V2a ablation group exhibited significant decreases in the amplitudes of the first , second and fourth bends , as well as a decrease in the duration of the first bend of escape ( Figure 10C i , Escape; p<0 . 01 , corrected for multiple comparisons , Holm test ) .",
"However , we observed no statistically significant changes in the three other slower locomotor patterns ( Figure 10C i , Scoot , R-turn and High-angle turn ) .",
"On the other hand , in the late-born V2a ablation group we saw no clear changes in the escape response ( Figure 10C ii , Escape ) .",
"Instead , this group showed a decrease in the amplitudes of bends in the later phase of scoot ( Figure 10C ii , Scoot; p<0 . 01 , corrected for multiple comparisons ) but not the other two spontaneous swim patterns involving turning ( Figure 10C ii , Routine turn and High-angle turn ) .",
"These observations comport well with the recruitment pattern and the spinal projections we observed earlier for the neurons we ablated here .",
"Furthermore , the fact that we observed significant changes only in the late phase of scoots suggests that late-born V2a neurons may play a role in maintaining the excitatory drive in the spinal cord during slow forward locomotion .",
"To gain assurance that we compared the same set of spontaneous locomotor behaviors between the control and ablated groups , we compared the distributions of the first body bend we used to subcategorize spontaneous locomotor behavior ( Figure 10—figure supplement 4 ) .",
"Although there were slight differences in the peak amplitudes of the probability distributions across the groups , the overall shapes of the distributions were consistent , indicating that we compared the same sets of locomotor behaviors across the control and ablated groups .",
"To examine this more closely for scoot behavior , which showed significant deficits upon ablation of late-born caudal hindbrain neurons , we examined the time course of the amplitude of body bending during this behavior ( Figure 10—figure supplement 5 ) .",
"Traces from all the episodes of scoot from the ablated fish ( Figure 10—figure supplement 5 , orange ) and the control fish ( Figure 10—figure supplement 3 , blue ) are overlaid for each ablation group .",
"In both ablation groups , the ablated and control groups both exhibited the small amplitude body bends with slow left-right alternation ( Figure 10—figure supplement 5 ) that is characteristic of scoot behavior ( Figure 10A",
"vi ) .",
"Furthermore , the overlaid traces of the late-born V2a ablation and control groups clearly revealed that the bend amplitudes of scoots decreased in the later phase of scoot in the ablated group ( Figure 10—figure supplement 5A ) .",
"This further strengthened the idea that late-born V2a neurons of the caudal hindbrain play a role in the maintenance of excitatory drive during slow forward locomotion .",
"To summarize , our ablations did not change the set of distinct locomotor behaviors fish exhibit spontaneously .",
"While examining the results of ablations , we noticed that the early-born and the late-born control groups exhibited significantly different bend amplitudes during scoot ( Figure 10C i , Scoot; Figure 10C ii , Scoot , p<0 . 01 ) .",
"Comparison to an independent control group that was not subjected to photoconversion suggested that photoconversion at 42 hpf impacted the kinematics of scoot at five dpf ( data not shown ) .",
"Although this does not undermine the observed significant decrease in bend amplitudes of scoot in the late-born ablation group , it does raise some concerns about the lack of significant effects in the early-born ablation group .",
"For instance , it is conceivable that ablation of early-born neurons does not reveal a decrease in scoot bend amplitudes because of a floor effect , wherein the observed bend amplitudes somehow represent the lowest values that the fish can inherently display in order to produce a scoot ( Figure 10C i , Scoot ) .",
"However , we think this is unlikely because we noticed a slight increase rather than decrease in bend amplitudes after ablation of the early-born neurons ( Figure 10C i , Scoot ) .",
"In any case , the effects of photoconversion itself further justifies our use of matched control for each ablation group .",
"In conclusion , with ablation experiments we confirmed our hypothesis about the functional roles of early-born and late-born V2a neurons in freely swimming fish .",
"Moreover , the double dissociation of the two locomotor patterns indicates that the underlying V2a descending pathways are largely independent , in accordance with their parallel circuit arrangement resulting from the layered growth of their spinal projections .",
"This suggests that the layered development of new connections in the nervous system underlies its ability to integrate new circuits into existing circuitry in such a way as to allow the different circuits to operate autonomously .",
"Interestingly , previous studies showed that the ablation of ventral reticulospinal neurons , the hindbrain V2a neurons we classified as the intermediate-aged group , affects slow but large turns with no clear kinematic effects on forward locomotion and suggested that the descending pathway for turning behaviors is independent of the one for forward locomotion ( Huang et al . , 2013; Orger et al . , 2008 ) .",
"Collectively , this suggests that the array of locomotor behaviors fish display after birth are supported by sequentially developing parallel hindbrain-spinal cord circuits , with each circuit accounting for a specific range of kinematics and dynamics through its unique connectivity pattern and biophysical properties ."
],
[
"We examined hindbrain V2a neurons and their descending pathways and uncovered a chronology-based architecture that explains the postnatal development of locomotor behaviors ( Figure 11 ) .",
"Hindbrain V2a descending neurons born early are also the first to project to the spinal cord where they innervate similarly early-born motoneurons throughout the spinal cord with fast conducting axons and synapses that exhibit fast dynamics .",
"Consistent with their connectivity pattern and biophysical properties , they contribute to the crude forms of locomotion that consist of powerful bends of the whole-body .",
"Hindbrain V2a descending neurons born later give rise to a more laterally located layer of descending pathways that function in parallel to existing pathways: through similarly late-born premotor neurons in the rostral spinal cord , these V2a neurons polysynaptically connect to caudal motoneurons via slow conducting axons and synapses that exhibit slow dynamics .",
"Consistent with the connectivity pattern and biophysical properties , they contribute to the more refined and weaker locomotion that appears later and that consists of weaker bends of the caudal tail .",
"In sum , our results reveal that the chronological layering of parallel circuits and the systematic variation in the connectivity patterns and biophysical properties of these circuits underlie the diversification and increase in sophistication of the locomotor repertoire .",
"Such chronology-based incorporation of new motor circuits in parallel to existing circuits may provide distinct advantages over a non-parallel organization such as exists within the midbrain descending pathways ( Wang and McLean , 2014 ) .",
"First , it allows the motor patterns produced by these circuits to be controlled independently of one another .",
"Such independent control may be critical for some behaviors such as prey capture , for instance , that require flexible coordination of multiple motor patterns .",
"Indeed , during the final approach phase of prey capture , zebrafish can increase the speed of forward swimming to values approaching those seen during fast behaviors established earlier in development , but without displaying the large lateral head displacements seen in these earlier-established behaviors ( Patterson et al . , 2013 ) .",
"This suggest that fish can generate fast forward swimming by strongly driving only the late-born pathway that predominantly drives tail movements .",
"Second , this parallel organization allows incorporation of new circuits controlling increasingly sophisticated motor patterns in an open-ended way .",
"Interestingly , the development of the corticospinal pathway in mammals appears to fit into this view; in this pathway , spinal tracts are established postnatally in the lateral part of the spinal cord after the prenatal development of other descending pathways ( Lakke , 1997; KUYPERS , 1962 ) , and these tracts contribute to skilled forelimb movements that animals gradually develop postnatally ( Martin , 2005; Alstermark and Isa , 2012 ) .",
"Further analysis will be necessary , however , to assess the degree to which the corticospinal pathway can function without contributions from other descending pathways ( see Esposito et al . , 2014 ) .",
"In sum , chronology-based parallel incorporation of new motor circuits provides certain advantages over non-parallel organization .",
"There is some evidence that suggests that such chronology-based parallel incorporation of new circuits extends beyond motor systems .",
"In the lateral line system of zebrafish , sensory afferents in the hindbrain are topologically organized based on their birthdate ( Pujol-Martí et al . , 2012 ) .",
"In the cerebellar molecular layer in mice , parallel fiber axons from granule cells line up in order of age ( Espinosa and Luo , 2008 ) .",
"In the fly visual system , the growth cones of photoreceptor afferents are segregated based on their birth order ( Kulkarni et al . , 2016 ) .",
"Also , in the mouse hippocampus , age-matched subpopulations of principal neurons are interconnected ( Deguchi et al . , 2011 ) .",
"Although the link between age-dependent layered circuit organization and the development of behaviors remains to be demonstrated in these systems , given how widely this pattern is observed , we suspect that it is a fundamental organizing principle in the brain that allows for implementation of new circuits in parallel to existing ones to meet the demands of an increasingly diverse behavioral repertoire following an animal’s birth .",
"The systematic changes in connectivity patterns and biophysical properties we observed in the chronologically layered parallel circuits fit well with the general development pattern of movements observed in many vertebrates ( Kagan and Herschkowitz , 2006; Harlow and Harlow , 1965; Fox , 1965; Drapeau et al . , 2002 ) .",
"For example , in humans , global and fast body movements such as startle develop first followed by isolated limb movements and fine motor skills ( de Vries et al . , 1982; Gallahue et al . , 2012 ) .",
"Broad and direct connectivity patterns equipped with fast biophysical properties would be suitable for the global and fast body movements that develop early .",
"Localized and indirect connectivity patterns equipped with slow biophysical properties would be suitable for the finer movements that develop later .",
"It would be interesting to see if the neural pathways underlying such behaviors indeed exhibit systematic changes in connectivity patterns and biophysical properties .",
"The aforementioned systematic changes in biophysical properties and connectivity patterns may not be restricted to motor systems .",
"Indeed , correlations between morphological and physiological properties and the time of differentiation have also been reported in the mammalian cortex ( Butt et al . , 2005; Miyoshi et al . , 2007 ) and hippocampus ( Picardo et al . , 2011; Marissal et al . , 2012 ) .",
"Although the computational and behavioral roles of these systematic changes in morphological and physiological properties remains to be answered , it seems likely that they represent a general strategy utilized by nervous systems to support the wide range of neural processes required for the complete behavioral repertoire of animals: from the fast and global sensorimotor processing observed in innate reflexive behaviors to the more sustained and finer processing presumed to happen in deliberate and sophisticated cognitive behaviors that animals acquire later .",
"The hindbrain contains a series of cell types that are arranged in columns that run rostrocaudally throughout the hindbrain ( Kinkhabwala et al . , 2011; Koyama et al . , 2011; Gray , 2013 ) .",
"Each cell type exhibits a specific combination of transcription factor expression , neurotransmitter identity and axonal projection pattern .",
"Hindbrain V2a neurons comprise one such cell type .",
"As these cell types span across various sensorimotor circuits in the hindbrain , it has been proposed that each cell type may provide a fundamental neural operation that is broadly useful to many of these circuits ( Kinkhabwala et al . , 2011; Koyama and Pujala , 2018 ) .",
"Indeed , there is some evidence suggestive of the involvement of hindbrain V2a neurons in other hindbrain motor circuits besides locomotor circuits .",
"In mice , genetic ablation of all V2a neurons in the nervous system produces deficits in respiration ( Crone et al . , 2012 ) .",
"A class of premotor neurons in the horizontal eye movement circuit partly corresponds to putative V2a neurons in larval zebrafish ( Lee et al . , 2015 ) .",
"These neurons show persistent activity correlated to eye position but the time constant of persistent activity varies across cells ( Miri et al . , 2011a ) .",
"This raises the possibility that , as in locomotor movements , eye movements are controlled by a series of V2a neurons that show systematic changes in biophysical properties based on the time of differentiation .",
"Perhaps one fundamental role of hindbrain V2a neurons is to provide to hindbrain motor circuits a series of excitatory drives that vary in duration so that these circuits can result in a behavioral repertoire containing movements with a wide range of temporal dynamics .",
"Precise functional mappings and detailed circuit analyses will be necessary to examine if this is indeed the case .",
"Previous studies of spinal V2a neurons in larval zebrafish employed tail beat frequency as a readout of locomotor speed and revealed that distinct age groups are recruited as a function of locomotor speed: the early-born group is active during fast locomotion while the late-born group is active during slow locomotion ( Kimura et al . , 2006; McLean et al . , 2008 ) .",
"This seems at odds with the recruitment of early-born hindbrain V2a descending neurons during struggles which generate powerful whole-body bends but at low tail beat frequencies .",
"However , in the context of forward locomotion , the strength of body bends also increases as locomotor speed increases ( McLean et al . , 2008 ) .",
"Thus , it is possible that spinal V2a neurons are recruited based on the strength of body bends similar to hindbrain V2a descending neurons .",
"Consistent with this idea , the earlier-born spinal V2a neurons have longer axons than the later-born ones ( Menelaou et al . , 2014 ) , suggesting that the early-born population contributes to large body bends through its innervation to motoneurons across many segments .",
"Indeed , the activity of CiD interneurons , putative early-born V2a population , is enhanced when fish exhibit a larger initial bend during escape ( Bhatt et al . , 2007 ) .",
"Previous studies in tadpole also identified putative spinal V2a neurons recruited during struggles ( Li et al . , 2007 ) .",
"These neurons can also fire during fast beat frequencies ( Li et al . , 2007 , Figure 4E; Li , 2015 , Figure 7A ) , suggesting that they correspond to the early-born population active during fast swimming in zebrafish .",
"Taken together , this suggests that like the hindbrain V2a population we examined , spinal V2a neurons may also exhibit recruitment pattern based on the strength of movements .",
"However , direct examination of the early-born spinal V2a neurons during struggles will be necessary to fully resolve this issue .",
"A shared set of early-born hindbrain V2a descending neurons is recruited during two crude forms of locomotion , struggles and escapes .",
"This suggests that the early-born V2a descending pathway is in use during both behaviors and generates strong activation of axial muscles along the whole body as observed in both behaviors .",
"However , these behaviors are distinct behaviors: escape is forward locomotion with fast tail beat frequency while struggle is backward locomotion with slow tail beat frequency .",
"How do these differences emerge ?",
"Within the early-born V2a descending neurons , majority of the cells show stronger activity during escapes .",
"This suggests that the further recruitment of the early-born pathway underlies the transition from struggles to escapes .",
"However , it is also possible that non-V2a descending neurons that were not examined in this study show activity only during struggles and recruit spinal neurons active only during struggles such as CoLA ( Liao and Fetcho , 2008 ) .",
"Comprehensive functional and anatomical examination of all descending neurons will be necessary to understand how these two distinct behaviors are generated .",
"In principle , it is possible for nervous systems to acquire new patterns of activity corresponding to new behaviors through the development of neuromodulatory systems because neuromodulation permits a fixed complement of neurons to give rise to many different patterns of activity ( Bargmann , 2012; Marder , 2012 ) .",
"Indeed , in larval zebrafish blocking dopamine signaling through D4 receptors leads to less frequent but longer swim episodes ( Lambert et al . , 2012 ) .",
"It remains to be seen if these episodes correspond to the crude form of swims observed in embryos ( McLean and Fetcho , 2009 ) or to the refined form of swims but with a larger number of tail beats .",
"Nevertheless , this indicates that D4 receptor signaling can either suppress or modify the late-born pathway we described , suggesting that the development of both new circuits and neuromodulatory systems is required for proper development of locomotor behaviors .",
"Although the parallel arrangement of differentially aged circuits that control distinct behaviors can explain how these behaviors can be controlled independently of one another , it does not explain how animals can coherently string together these behaviors .",
"The coordination between early-born and late-born neurons has been described in a few neural systems .",
"For example , through their widespread axonal arborizations , early-born ‘pioneer’ GABAergic neurons in the hippocampus exert a huge impact on the activity of neurons born afterwards , and in this way , these neurons play a crucial role in the synchronization of activity observed in the developing hippocampus ( Picardo et al . , 2011 ) .",
"Although these early-born neurons have been shown to exist in the adult hippocampus and to project heavily to the septum ( Villette et al . , 2016 ) , it has yet to be revealed how their control over late-born neurons influences behavior in adulthood .",
"The reticulospinal system in the zebrafish could serve as a good avenue for understanding the interactions that occur among age groups during behavior because it is composed of a relatively small number of identifiable neurons , many of which are known to be recruited during escape behavior ( Kimmel et al . , 1982; Gahtan et al . , 2002 ) .",
"During escapes , fish transition from fast and crude whole-body movements to slower and refined tail-restricted movements ( Mirat et al . , 2013; Marques et al . , 2018 ) .",
"The age-related recruitment pattern we observed in V2a descending neurons suggests that neural activity shifts from the early-born neurons contributing to the crude whole-body movements to the late-born reticulospinal neurons contributing to the refined tail-restricted movements .",
"This raises the possibility that the recruitment of the early and late-born reticulospinal neurons are coordinated to ensure the smooth transition from the crude to refined locomotor patterns .",
"Indeed , in adult goldfish and zebrafish , it’s been shown that the activation of Mauthner cell , the earliest born reticulospinal neuron , influences ongoing slow locomotor activity in the spinal cord ( Svoboda and Fetcho , 1996; Song et al . , 2015 ) , further supporting the presence of such coordination .",
"Thus , the reticulospinal system may permit detailed analyses of the interactions among age groups and their behavioral roles .",
"In any case , detailed circuit analyses of early-born and late-born neurons in the context of behavioral development will be essential to understand how nervous systems organize the interactions between early-born and late-born circuits , and thus allow animals to produce coherent behaviors ."
],
[
"Zebrafish larvae were obtained from an in-house breeding colony of wild-type adults maintained at 28 . 5°C on a 14–10 hr light-dark cycle .",
"Embryos were raised in a separate incubator but at the same temperature and on the same light-dark cycle .",
"Embryos were staged ( Kimmel et al . , 1995 ) and only the ones with normal development were used in the study .",
"All experiments presented in this study were conducted in accordance with the animal research guidelines from the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee and Institutional Biosafety Committee of Janelia Research Campus ( 16-145 ) .",
"The following previously published transgenic lines were propagated to Casper background and used in this study ( see Supplementary file 1 ) : TgBAC ( vsx2:GFP ) ( Kimura et al . , 2006 ) ; TgBAC ( vsx2:Kaede ) ( Kimura et al . , 2006 ) ; TgBAC ( vsx2:Gal4 ) ( Kimura et al . , 2013 ) ; Tg ( UAS:Kaede ) ( Davison et al . , 2007 ) ; Tg ( UAS:synaptophysin-EGFP ) ( Heap et al . , 2013 ) ; Tg ( UAS:GCaMP6s ) ( Muto et al . , 2017 ) ; Tg ( mnx1:TagRFPT ) ( Jao et al . , 2012 ) ; TgBAC ( islet1:GFP ) ( Higashijima et al . , 2000 ) ; Tg ( Dbx1b:Cre , vglut2a:lRl-GFP ) ( Koyama et al . , 2011 ) .",
"For examining cholinergic transmission in the Mauthner cell , we used the paralytic mutant , relaxed , to avoid the use of the cholinergic blocker α-bungarotoxin for the induction of paralysis ( Koyama et al . , 2011 ) .",
"TgBAC ( vsx2:EGFP ) was anesthetized in tricaine methanesulfonate ( MilliporeSigma , E10521 , St Louis , MO ) dissolved in system water at 160 mg/L ( hereafter referred as MS-222 ) and then embedded in 1 . 6% low melting point agar ( MilliporeSigma , 2070-OP , MO ) .",
"Volumetric images of hindbrain V2a neurons were acquired with a custom two-photon microscope .",
"25 × 1 . 1 NA objective lens ( Nikon Instruments , CFI Apo LWD 25XW 1300 nm , NY ) was used and EGFP was excited at 940 nm ( MKS Instruments , Mai Tai HP , Deep See , MA ) .",
"For each developmental time point , a separate group of fish was used to avoid the potential developmental effects of the imaging procedure ( >6 fish per time point ) .",
"For time lapse imaging , fish were kept in 0 . 5% agar and temperature was maintained at 28 . 5°C with a temperature controller ( Luigs and Neumann , TC07 , Germany ) .",
"Fish expressing the photoconvertible fluorescent protein , Kaede , in V2a neurons ( TgBAC ( vsx2:Kaede ) ) were photoconverted at 24 , 36 , 48 , 60 , 72 and 96 hr post fertilization ( hpf ) to identify V2a neurons that existed at a given developmental time point using a procedure similar to the one described previously ( Kimura et al . , 2006; Caron et al . , 2008 ) .",
"Briefly , UV light was shone on around 10 fish within a drop of system water for 30–60 s using a stereo dissection microscope ( Olympus , MVX10 , PA ) equipped with a DAPI filter ( Chroma Technology , 49901 , VT ) and a halogen bulb ( Excelitas Technologies , X-Cite 120PC Q , MA ) .",
"Then , the fish were transferred back into the incubator and housed in a box that filtered out UV wavelengths from the light within the incubator .",
"This prevented photoconversion of additional developing neurons by the ambient light in the incubator .",
"The fish remained in the incubator until 120 hpf , at which point they were imaged with a confocal microscope ( Zeiss , LSM710 , Germany ) with 20 × 1 . 0 NA objective lens ( Zeiss , W Plan-Apochromat 20x/1 . 0 , Germany ) ( >8 fish per time point ) .",
"A group of fish converted at 24 and 36 hpf ( six fish per time point ) were injected with a dye in the spinal cord ( Liu and Fetcho , 1999 ) at 4 days post-fertilization ( dpf ) to label reticulospinal neurons .",
"A far red dye was used to avoid contaminating the red channel used for imaging photoconverted Kaede ( Alexa Fluor 680 dextran , MW: 10 , 000 , Thermo Fisher Scientific , MA ) .",
"These neurons are identifiable across animals and are named based on their rostrocaudal positions ( Figure 2C , left ) and dorsoventral positions ( Figure 2C , right ) ( Kimmel et al . , 1982; Mendelson , 1986 ) .",
"We used the same naming convention used previously except for a V2a reticulospinal neuron that was born by 24 hpf in rhombomere 4 .",
"This cell was the most dorsal cell within V2a reticulospinal neurons in rhombomere four but was slightly ventral to MiM1 .",
"Just as MiM1 refers to a single identifiable cell that is just ventromedial to the Mauthner cell ( Mendelson , 1986 ) , we decided to refer to this neuron as dorsal MiV1 .",
"Fish expressing Kaede in V2a neurons through the Gal4-UAS system were photoconverted in the rostral spinal cord ( muscle segment 5 to 7 ) at 36 , 60 , 84 and 108 hpf to identify hindbrain V2a neurons that had spinal projections at a given developmental time point as described previously ( Kimura et al . , 2013 ) .",
"Briefly , fish were anesthetized in MS-222 and embedded ventral-side up in 0 . 5% low-melting point agar and then the rostral spinal cord ( muscle segment 5 to 7 ) was illuminated for 2 min with 405 nm LED ( Lumencor , Spectra X , OR ) in an inverted microscope ( Nikon Instruments , Eclipse Ti-E , NY ) using a 60x objective lens ( Nikon Instruments , CFI Plan Apo VC 60XWI , NY ) .",
"This procedure was repeated three to five times with an interval of 15–20 min .",
"The fish were then kept in the dark until 120 hpf and imaged with a confocal microscope ( Zeiss , LSM 710 , Germany ) ( >6 fish per time point ) .",
"The expression of Kaede in the earliest born hindbrain V2a neurons , driven by the Gal4/UAS system was delayed roughly 6 hr relative to the one driven directly by the V2a promoter ( data not shown ) .",
"Theoretically , it is possible that some descending neurons are not labeled with this procedure due to 1 ) inefficient transport on small axons , or 2 ) fast turnover back to unconverted Kaede .",
"However , it is unlikely that we are missing cells because of insufficient diffusion time ( 36 hr to 84 hr ) because the converted Kaede diffused to the cell bodies of the caudal V2a neurons that are previously speculated to have thin axons in less than 12 hr ( Video 1 ) .",
"Turnover time of converted Kaede was more than 96 hr in the early-born population ( see Figure 2—figure supplement 1 for example ) .",
"This is longer than the diffusion time we used for our optical backfill experiments ( 36 hr to 84 hr ) .",
"Thus , the second possibility is also unlikely .",
"The genetically encoded calcium indicator GCaMP6s ( Chen et al . , 2013 ) was expressed in hindbrain V2a neurons using the Gal4-UAS system ( Tg ( vsx2:Gal4; UAS:GCaMP6s ) ) .",
"Fish at 120–132 hpf were paralyzed with α-bungarotoxin ( MilliporeSigma , 203980 , MO ) and then embedded in 1 . 6% low-melting point agar .",
"The agar around the tail and the nostrils was released using a fine tungsten pin .",
"Axial motor nerve activity was recorded through fire-polished glass pipettes placed on the dorsal intermyotomal cleft on both sides .",
"A gentle suction ( ~15 mm Hg ) was applied through a pneumatic device to gently break the skin .",
"The signal was amplified ( 1000x ) and bandpass filtered ( 100–1000 Hz ) through an extracellular amplifier ( NPI , EXT-02B , Germany ) and digitized at 6 kHz with PCIe-6363 ( National Instruments , TX ) using a custom C# program ( Source Code File 1 ) .",
"Clear motor activity was typically observed within 20 min .",
"The whole hindbrain was imaged with a custom two-photon microscope equipped with a resonant scanner ( Thorlabs , MPM-2PKIT ) and a piezo objective scanner ( PI , P-725K129 , Germany ) at a volume rate of 2 Hz ( 512 × 256 , 30 slices , 7 μm z step ) using ScanImage ( Vidrio Technologies , VA ) .",
"940 nm , 80 MHz femtosecond laser pulses were used to excite GCaMP6s ( MKS Instruments , Mai Tai HP , Deep See , MA ) .",
"A gradual mechanical stimulus was delivered by gradually contacting the rostral part of the head with a fire polished glass pipette controlled by a motorized manipulator ( Luigs and Neumann , Junior RE , Germany ) .",
"A brief electrical stimulus ( 0 . 5–1 ms in duration , 2–10 V ) was delivered through a glass electrode placed on the side of the head using a stimulus isolator ( Digitimer Ltd . , DS-2 , England ) .",
"The inter-stimulus interval was from 50 s to 2 min .",
"The stimulus amplitude was adjusted to evoke strong motor activity consistently .",
"Each experiment lasted between 20 and 40 min and thus contained 2400–4800 stacks .",
"Two-photon data was first corrected for mismatch between the odd and even scan lines by cross correlation if necessary and then the shift of the sample was corrected by cross correlation to a reference volume .",
"Then GCaMP6s signal from identifiable V2a neurons were examined in relationship to fictive swim bouts .",
"The regions of interest ( ROI ) for V2a-positive reticulospinal ( RS ) neurons ( Mid2i , Mid3i , RoM2 , RoM3 , RoV3 , dorsal MiV1 , and ventral MiV1/2 ) were drawn based on their distinct segmental distribution , dorsoventral position and soma morphology .",
"First , dorsal early-born V2a RS neurons were identified based on their large and laterally displaced cell bodies and used as a landmark for each rhombomere ( RoM2 , RoM3 , dorsal MiV1 and Mid2i , Mid3i ) ( Video 1 ) .",
"Then , the younger ventral V2a RS neurons in each rhombomere were identified based on their smaller cell bodies ventral to the dorsal early-born neuron .",
"This was confirmed further with spinal backfill ( Kimmel et al . , 1982 ) .",
"The ROIs for caudal hindbrain V2a neurons were drawn based on their stereotypical positions as examined in the birthdating analysis: the early-born caudal neurons were displaced laterally from the rest of caudal V2a neurons while the late-born caudal neurons were located dorsally , close to the midline .",
"Then the fluorescence time course ( F",
"( t ) ) for each ROI was extracted and the baseline fluorescence ( F0 ) was estimated as the bottom 20th percentile of the whole timeseries .",
"Then ΔF",
"( t ) /F0 was calculated as follows .",
"ΔF",
"( t ) F0 = F",
"( t ) -F0F0 To detect weak spontaneous swimming activity reliably , the axial motor nerve activity was processed by taking a windowed standard deviation of the original signal with a 10 ms moving window as described previously ( Ahrens et al . , 2012 ) .",
"Then , a threshold for swimming activity was selected to detect the weak spontaneous swimming activity reliably from the transformed signal .",
"In the gradual mechanical stimulus experiment , the latency of strong swimming activity from the onset of the stimulus was not consistent across trials .",
"Thus , we used the following procedure to detect the strong bursting activity .",
"For each swim bout , the maximum burst amplitude of the transformed signal was extracted independently for left and right channels ( Figure 3B",
"i ) .",
"Then , the distribution of the maximum burst amplitude was log-transformed to reduce skew in the distribution and fitted as a Gaussian mixture distribution with two components to estimate the distribution of the maximum burst amplitude that corresponds to weak spontaneous swims .",
"This estimated probability distribution corresponding to weak spontaneous swims was used to compute a value whose probability of falling in this distribution is less than 0 . 1% ( Figure 4B",
"i ) .",
"The swim episodes with the maximum burst amplitude above this value were categorized as strong swims induced by the stimulus ( Figure 4B i; Figure 3C , ‘Push’ ) .",
"The detected episodes matched with the slow and strong bursting activity ( as assessed by eye ) .",
"In the electrical pulse stimulus experiment , fast swim episodes were elicited reliably by the stimulus and were categorized as shock-induced fast swims ( Figure 3C , ‘Shock’ ) .",
"The remaining spontaneous swim episodes were categorized as spontaneous swims ( Figure 3C , ‘Sponta swim’ ) .",
"To derive the ΔF/F0 response related to these swim events ( Figure 3C ) , ΔF/F0 signal was aligned at the onsets of a given swim type and averaged over trials for each cell .",
"Only the fictive motor signal ipsilateral to the cell being examined was considered for the following reasons: 1 ) hindbrain V2a neurons are primarily ipsilaterally projecting neurons ( Kinkhabwala et al . , 2011; Cepeda-Nieto et al . , 2005 ) .",
"2 ) most of the neurons related to the strong swims responded only when there is a strong axial motor activity on the ipsilateral side ( Figure 3A and B ) .",
"The peak of the mean ΔF/F0 response was detected for each neuron and pooled for a given cell type to test if they showed activity consistently during a given swim type ( Figure 3C , top row ) .",
"To test if swim types had effects on the activity of a given cell type , an one-way ANOVA with a factor for swim types was used .",
"All possible comparisons were tested with Tukey’s multiple comparison test ( Figure 3C , bottom row ) .",
"To map the hindbrain V2a neurons recruited during these swim bouts in an unbiased way , voxel-level regression analysis was done with regressors representing the distinct types of swim bouts .",
"Each regressor was constructed by convolving the corresponding fictive motor signal ( as defined above ) with a GCaMP6s impulse response function modeled as the rise and decay exponentials ( 0 . 5 s rise and 2 s decay ) and then standardized for a given experimental session by subtracting its mean from its values and then dividing these new values by its standard deviation ( Figure 4A ii , 4B",
"ii ) .",
"Voxel time course ( Y ) was fitted with these standardized regressors ( X ) using the following linear model . Y=Xβ+ε The standardized coefficient ( β ) was estimated by ordinary least square and then T value for each voxel was calculated based on standardized coefficient ( β ) and residual ( ε ) .",
"Correction for multiple comparisons was done with the false discovery rate ( FDR ) ( Miri et al . , 2011b ) .",
"The threshold for T maps was set at PFDR <0 . 05 .",
"Maps of birthdate , descending projection and activity of hindbrain V2a neurons were registered to Zebrafish Brain Browser ( ZBB ) atlas ( Marquart et al . , 2015; Marquart et al . , 2017 ) using the procedure described in Marquart et al . ( 2017 ) with minor modifications .",
"Briefly , the volume of vsx2:Gal4 provided by ZBB was used as a reference for all the registration of V2a population .",
"Then the reference images for each map was created using either the summed volume of unconverted and converted Kaede for the birthdate and descending projection maps , or the time-averaged volume for activity maps .",
"Affine and symmetric diffeomorphic registrations were performed using Advanced Normalization Tools ( Avants et al . , 2008; Avants et al . , 2011 ) using the parameters described for live samples ( Marquart et al . , 2017 ) .",
"The photoconvertible protein , Kaede , and GFP fused to synaptophysin were expressed in V2a neurons using the Gal4/UAS system ( Tg ( vsx2:Gal4;UAS:Kaede;UAS:synaptophysin-EGFP ) ) .",
"Hindbrain V2a neurons were photoconverted with the same set up used for optical backfill but with a shorter total exposure time ( 1 round of 1 min exposure ) .",
"The photoconversions were done at 42 and 96 hpf to visualize the spinal projections of hindbrain V2a neurons that existed at a given developmental time point .",
"The fish were then kept in the box that filtered out UV until the imaging at 120 hpf .",
"A series of confocal images were taken in the hindbrain and the spinal cord from the dorsal side ( Zeiss , LSM710 , Germany ) .",
"In a separate set of experiments , hindbrain V2a neurons were photoconverted at 42 hpf and spinal motoneurons in the rostral spinal cord were labeled by the injection of a far-red dye ( Thermo Fisher Scientific , Alexa Fluor 680 dextran , MW: 10 , 000 , MA ) in the axial muscles at 108 hpf .",
"In yet another set of experiments , spinal interneurons in the rostral spinal cord were labeled instead by the injection of the far red dye in the caudal spinal cord ( 28th to 30th muscle segment ) in a manner described previously ( Hale et al . , 2001 ) .",
"Volumetric images of V2a neurons labeled with converted and unconverted Kaede in the hindbrain and the spinal cord were 3D-rendered in Imaris ( Bitplane , Switzerland ) .",
"Then maximum intensity projection views from the dorsal side were created in the area of interest .",
"The views were carefully oriented based on anatomical landmarks such as axons from V2a neurons to minimize the error from tilt of the 3D volume .",
"Then the intensity distribution in the neuropil along the mediolateral axis in the projection images was calculated in Fiji ( Fiji is just Image J ) from a line profile extending from the lateral surface of the cell bodies of the most laterally located V2a neurons to the lateral surface of the neuropil from V2a neurons .",
"The line thickness was set to 20 μm in Fiji to average the intensity profiles over 20 μm in the rostrocaudal direction .",
"The intensity of converted Kaede and the mediolateral position were normalized in each fish and the average intensity profiles and their standard errors were plotted as a function of the normalized mediolateral position in the neuropil .",
"Patch-clamp recordings of hindbrain neurons were performed with a procedure similar to the one described previously ( Kimura et al . , 2013 ) , but with some modifications .",
"Five-day-old fish were paralyzed with α-bungarotoxin ( MilliporeSigma , 203980 , MO ) that was dissolved in system water ( 1 mg/ml ) .",
"After successful paralysis , larvae were anesthetized with MS-222 and then secured with etched tungsten wires ( pins ) through the notochord to a Sylgard-coated glass-bottom dish containing extracellular solution ( 134 mM NaCl , 2 . 9 mM KCl , 1 . 2 mM MgCl2 , 2 . 1 mM CaCl2 , 10 mM HEPES , and 10 mM glucose , adjusted to pH 7 . 8 with NaOH ) .",
"Then , the head was rotated and secured ventral side up with tungsten pins placed through the ears and the rostral part of the jaw ( Figure 7A",
"ii ) .",
"The ventral surface of the hindbrain was carefully exposed by removing the notochord using an tungsten pin and fine forceps .",
"The skin of the middle region of the body was removed with a pair of forceps to gain access to the peripheral nerves from spinal motoneurons .",
"Locations of motor nerve recordings were from the 10th to 15th muscle segment .",
"Neurons were targeted based on fluorescence and scanned Dodt gradient contrast images acquired with a custom two-photon microscope .",
"40x ( Nikon Instruments , CFI Apo 40XW NIR , NY ) or 25x ( Leica , HCX IRAPO L 25x/0 . 95 W , Germany ) objective lens was used .",
"Descending hindbrain neurons were identified by the fluorescence signal from Tg ( vsx2:EGFP ) , Tg ( vsx2:Kaede ) or spinal backfill ( see above ) .",
"MiV1 neurons were located in rhomobomere 4 , medial and ventral to Mauthner cells which can be easily identified by Dodt gradient contrast .",
"The subclass of MiV1 neurons was targeted based on photoconversion of Kaede ( see above ) and its stereotypical position .",
"We also quantified dorso-ventral position based on volumetric images acquired after the recording as a proxy of birthdate , taking advantage of the fine-scale birthdate-related topographical organization .",
"First , we made a coronal optical section including all the neurons in this cluster .",
"The plane of the section was carefully adjusted so that it was parallel to the V2a processes running lateral to MiV1 neurons .",
"Then , we identified dorsal MiV1 based on its close proximity to M-cell , its soma larger than the nearby younger cells , and the presence of photoconverted Kaede .",
"We then defined normalized dorso-ventral position with ‘0’ being the dorsal edge of dorsal MiV1 and ‘1’ being the ventral edge of the most ventral MiV1 .",
"The cells other than dorsal MiV1 with the position smaller than 0 . 5 were categorized as middle MiV1 , while those with the position larger than 0 . 5 were categorized as ventral MiV1 .",
"Whole-cell recordings were established using the standard procedure and the signals were recorded using EPC 10 Quadro amplifier ( HEKA Electronik , Germany ) and PatchMaster ( HEKA Electronik ) .",
"The resistance of the electrode was 8 to 12 MOhm .",
"The intracellular solution contained ( in mM ) 125 mM K-gluconate , 2 . 5 mM MgCl2 , 10 mM EGTA , 10 mM HEPES and 4 mM Na2ATP adjusted to pH 7 . 3 with KOH .",
"A junction potential using this intracellular and extracellular solution ( see above ) has been calculated at 16 mV , which would result in a shift of measured potentials 16 mV in the negative direction .",
"Because this would not affect our conclusions , we did not correct for it .",
"The solution also contained 0 . 01% of Alexa Fluor 568 hydrazide or Alexa Fluor 647 hydrazide ( Thermo Fisher Scientific ) .",
"Z stacks were acquired with a custom two-photon microscope to confirm morphology and locations of the recorded neurons .",
"Input resistances were calculated from an average of 5 hyperpolarizing square current pulses between 20 and 80 pA .",
"This range of current injection produces a linear current-voltage response in all the MiV1 neurons examined .",
"Electrophysiological analysis of recruitment of MiV1 neurons was performed in a manner similar to the previous studies ( McLean et al . , 2007; McLean et al . , 2008 ) .",
"Fast fictive swimming was induced by a brief electrical stimulus ( <1 ms in duration at 1–10 V ) delivered via a pair of tungsten electrodes ( A-M systems , WA ) placed near the tail .",
"Slow swimming often occurs spontaneously but is also induced by flashes of light .",
"Cycle frequencies of axial motor activity were computed by taking the reciprocals of the time interval between each burst and the next one .",
"The spiking and subthreshold activity that precede each cycle were extracted and their relationship to cycle frequency was examined as follows .",
"Spikes were detected based on the threshold determined for each cell .",
"Subthreshold activity was examined based on the lowpass-filtered ( 60 Hz ) voltage trace , which effectively isolated rhythmic subthreshold activity during swimming .",
"The maximum depolarization for each cycle was calculated by subtracting its peak from the baseline which was defined as the minimum voltage in the 200 ms time window before the swim onset .",
"In order to statistically test if each age group changes spiking and subthreshold activity as a function of cycle frequency , each cycle was classified as fast ( >35 Hz ) or slow ( <35 Hz ) cycle and fitted with the following linear mixed models with a random effect for cell using the nlme package in R ( https://cran . r-project . org/web/packages/nlme/index . html ) .",
"Number of spikes was log-transformed to satisfy the assumptions of the linear model .",
"The effect of cycle speed was tested by comparing the activity for each age group with correction for multiple comparisons using the multcomp package in R ( https://cran . r-project . org/web/packages/multcomp/index . html ) .",
"log ( numberofspikes ) ∼cyclespeed∗agemembranedepolarization∼cyclespeed∗age Electroporation of Alexa Fluor 647 dextran dye ( MW 10 , 000; Thermo Fisher Scientific ) into single neurons was performed as described previously ( Koyama et al . , 2011 ) .",
"MiV1 neurons were targeted based on GFP expression in Tg ( vsx2:GFP ) and/or spinal backfill with Texas Red dextran dye ( MW 10 , 000; Thermo Fisher Scientific ) along with scanned Dodt gradient contrast under a custom two-photon microscope at 5 days old .",
"One day after electroporation , the hindbrain was imaged with a confocal microscope ( Zeiss , LSM 710 ) from the dorsal side to confirm the dorsoventral position of the electroporated MiV1 neurons .",
"Multiple z stacks from the side were also imaged from hindbrain to the caudal end of spinal cord to cover the entire spinal projection .",
"Stacks were then stitched together with stitching plugin available through Fiji ( Preibisch et al . , 2009 ) .",
"When spinal backfill was used to visualize descending neurons , the spinal segments caudal to the injection site ( 21st to 23rd muscle segment ) were excluded from imaging .",
"Stitched stacks were 3D rendered in Imaris ( Bitplane , Switzerland ) .",
"To visualize the spinal projection in coronal view , axonal arborization was reconstructed with neuTube ( http://www . neutracing . com/ ) and then visualized in Imaris .",
"Three to four muscle segments were used to render coronal views in the rostral spinal cord ( 6th to 9th muscle segment ) and in the middle spinal cord ( 14th to 17th muscle segment ) .",
"All the cells examined in the study had their main axon in the ventral spinal cord and had collaterals innervating the dorsal spinal cord .",
"The dorsal extent of the axonal arborization was quantified relative to the total dorsoventral extent of spinal cord as described previously ( McLean et al . , 2007 ) .",
"To statistically examine if the extent of spinal arborization depends on hindbrain cell type and the position in the spinal cord , the dorsal extent of arborization was fitted using the following linear mixed effects model with a random effect for cell using nlme package in R . Dorsalextentofaxoncollaterals∼hindbrainneurontypes∗positioninthespinalcord+ ( 1|CellID ) Putative presynaptic terminals were identified based on their characteristic puncta-like structure .",
"Then their apposition to fluorescently labeled spinal neurons was examined .",
"Primary motoneurons were identified using Tg ( mnx1:TagRFPT ) .",
"Secondary motoneurons were identified using TgBAC ( islet1:EGFP ) .",
"Spinal V2a neurons ( also called circumferential ipsilateral descending neuron ( CiD ) ) were identified using TgBAC ( vsx2:EGFP ) .",
"Multipolar commissural neurons ( MCoD ) were identified by spinal backfill from the caudal spinal cord ( see above ) .",
"To statistically examine if hindbrain cell type explained the variance in their spinal innervation patterns , the presence of innervation to the spinal neurons was fitted with the following generalized linear mixed effects model with a random effect for cell and a logit link function for a binomial distribution using lme4 package in R ( https://cran . r-project . org/web/packages/lme4/index . html ) .",
"Presenceofinnervation∼hindbrainneurontypes∗spinalneurontypes+ ( 1|CellID ) Whole-cell recordings from hindbrain neurons were done as described above .",
"The spinal cord was also exposed by removing the muscles overlying the cell of interest using the standard procedure described previously ( Drapeau et al . , 1999 ) .",
"Dorsal and ventral MiV1 neurons were targeted as described above .",
"Mid3i was identified based on the GFP signal from Tg ( vsx2:EGFP ) , its stereotypical segmental location ( rhombomere 6 ) and morphology ( laterally elongated soma and its position relative to nearby reticulospinal neurons in the same segment ) .",
"Mauthner cell was identified based on its segmental position ( rhombomere 4 ) and its large soma which is clearly visible under Dodt gradient contrast .",
"Spinal motoneurons and interneurons ( MCoDs ) were targeted with Dodt gradient contrast and/or fluorescence either from backfill ( see above ) or from transgenic lines ( Tg ( mnx1:TagRPFT ) for motoneurons; Tg ( Dbx1b:Cre , vglut2a:lRl-GFP ) for MCoDs ) .",
"Primary motoneurons are dorsally located large cells close to the lateral surface of the spinal cord .",
"MCoDs are ventrally located smaller cells but also close to the lateral surface of the spinal cord .",
"PMNs were sampled at muscle segment 17 to 19 .",
"MCoDs were sampled at muscle segments 11 to 13 .",
"To examine if a given connection is monosynaptic , extracellular solution with a high concentration of divalent cations ( 4x Mg2+ and 4x Ca2+ ) was perfused to increase the firing threshold of potential relay neurons .",
"Only the connection between Mauthner cell and MCoD was almost completely abolished , which is also consistent with its long latency .",
"Thus , this connection was excluded from subsequent analysis .",
"To examine the composition of electrical and chemical synapses for a given connection , chemical synapses were blocked by a cocktail of D-AP5 ( Tocris , 0106 , MN ) and NBQX ( Tocris , 0373 , MN ) for glutamatergic synapses and by mecamylamine ( Tocris , 2843 , MN ) for cholinergic synapses .",
"To statistically examine if hindbrain and spinal cell types had significant effects on the presence of monosynaptic connections , the presence of monosynaptic connection was fitted with the following generalized linear model with a logit link function for a binomial distribution using lme4 package in R . Presenceofmonosynapticconnection∼hindbrainneurontypes∗spinalneurontypes The differences in conduction velocity , postsynaptic potential ( PSP ) amplitude and PSP half decay time across connected pairs of the monosynaptic responses were statistically examined with Kruskal-Wallis test .",
"Post-hoc comparisons were done with Dunn’s multiple comparison test .",
"We ablated two groups of V2a neurons based on the developmental and recruitment analyses we performed ( Figures 2–4 ) .",
"The first group consisted of the early-born V2a neurons that were recruited during strong swims .",
"Procedurally , they were defined as hindbrain neurons that contained photoconverted Kaede at 60 hpf after photoconversion at 24 hpf in Tg[vsx2:Kaede] ( Figure 10B",
"i ) .",
"The second group consisted of the late-born V2a neurons in the caudal hindbrain that were recruited during spontaneous weak swims .",
"Procedurally , they were defined as neurons that did not contain photoconverted Kaede at 60 hpf after photoconversion at 42 hpf and met the following spatial criteria ( Figure 10B iv ) .",
"First , their somata were caudal to rhombomere six where the early-born neurons were located in dorsal hindbrain ( Kinkhabwala et al . , 2011 ) Second , they were dorsal to the early-born neurons in the caudal hindbrain .",
"These neurons born after 42 hpf probably do not include all the caudal neurons active during weak swims ( see Figure 5A vi and Figure 2F ) .",
"Thus , we may underestimate the contribution of the caudal V2a neurons to spontaneous weak swims .",
"However , we chose this to make sure there is no unintended damage to the most lateral early-born neurons so that we can see distinct locomotion phenotypes for each ablation group .",
"The time of ablation was matched in the two groups to minimize the difference in the procedure .",
"For every fish that was randomly selected for ablation from the pool of fish that had been photoconverted , one or two more fish were chosen in the same way to serve as a negative control",
"( s ) .",
"The fish to be ablated was anesthetized in MS-222 for 2 min before being mounted dorsal-side up in 1 . 6% low melting point agarose and placed under our custom two-photon microscope where the ablations took place .",
"Both the fish in which the ablations were carried out and the fish chosen as control were kept under anesthesia for the entire duration of the ablations .",
"Before proceeding to ablations , we first imaged hindbrain V2a neurons labeled with unconverted and converted Kaede using 1020 nm , 80 MHz femtosecond laser pulses ( MKS Instruments , Insight , Deep See , MA ) with a 25x objective lens ( Leica , HCX IRAPO L 25x/0 . 95 W , Germany ) .",
"Then , we proceeded to ablations starting from the most ventral target cells to increasingly more dorsal cells to minimize light scattering that could result from preceding ablations .",
"For a given depth , each target cell was exposed to femtosecond laser pulses for 5 ms with spiral scans centered on its soma ( outer diameter , 3 um ) before targeting the next target after a minimum 5 s interval using ScanImage ( Vidrio Technologies , VA ) .",
"We used 920 nm or 1020 nm laser ( 196 mW or 142 mW after objective lens , respectively ) .",
"This procedure was repeated up to ten times until the cells at a given depth were deemed ablated based on visual inspection .",
"After ablating all the cells of interest , we imaged hindbrain V2a neurons again to assess the quality of ablation .",
"Then , the fish was gently removed from the embedding agarose and both this fish and the control fish were housed singly in separate dishes until later use on the day of behavior imaging , which was at 5 dpf .",
"To examine the potential recovery of target cells after the ablation , a subset of the ablated fish and the control fish was selected for imaging at four dpf .",
"From the time of photoconversion till the day of behavior , the dishes containing the fish were enclosed in our UV-opaque box to prevent further conversion of Kaede .",
"At 5 dpf , ablated and control fish were transferred singly and in random sequence to a 50 mm diameter circular arena where they were allowed to swim freely while being imaged from above with a high-speed camera ( Miktrotron , MC-1362 , Germany ) .",
"To confine the movements of the fish to the yaw ( x-y ) plane , the depth of the water in the arena was limited to 2 mm .",
"Before the start of imaging , each fish was allowed to acclimate to the arena for a minimum of 20 min before their behavior was imaged .",
"If even after 20 min the fish spent a majority of the time being stationary or swimming very close to the edge of the arena ( as they tend to do soon after being transferred into the arena from their home dish ) , then the wait period was increased until the fish started showing frequent swims in trajectories that took them far from the arena edge .",
"Following this acclimation period , fish were first imaged at 300 frames per second ( fps ) for a total 5 min while they swam spontaneously .",
"After this 5-min period , fish were imaged continuously while they were presented with a series of alternating vibration and dark flash stimuli - although in this paper we did not use data from dark flash responses - at 1 min intervals .",
"A time period of 100 ms before and 1400 ms after each stimulus were imaged at 500 fps , whereas the intervening 1 min periods between stimuli were imaged at 30 fps .",
"The vibration stimulus was delivered via a sound speaker in contact with the acrylic base plate upon which the fish arena was placed .",
"The sound stimulus consisted of two cycles of 500 Hz sine wave at ( peak-to-peak acceleration≈100 ms-2 ) .",
"For the dark flash stimulus , the ambient light was turned off for 300 ms . Images collected during the behavior sessions were first sorted by frame rate .",
"For images collected at 30 fps we only tracked the fish’s head position , whereas for images collected at 300- and 500 fps , we estimated the fish’s head orientation and body curvature as well .",
"To extract the fish’s head position , we first computed a background image and subtracted this from all images to remove background from them .",
"The background image was computed by averaging a set of 1000 temporally uniformly spaced images from the set of all images .",
"After background subtraction , for convenience , the pixel values were multiplied by -1 so that the fish’s eyes would go from being the darkest to the brightest spots in the image .",
"The images were then smoothed by convolving each image with a 1 mm wide 2D Gaussian kernel .",
"The convolved images were then segmented using an automatically estimated threshold ( multithresh function in MATLAB ) to isolate within each image a blob ( contiguous set of pixels ) of the brightest pixels that included the fish’s eyes as well as the swim bladder .",
"The centroid of these isolated pixels was used as the fish’s head position .",
"The centroid obtained in this manner was reliably located between the eyes and the swim bladder in all image frames and for frames in which the fish did not move , the centroid typically shifted by less than 0 . 17 mm ( ≈3 pixels ) between frames ( confidence interval of 95% ) .",
"For quantifying the fish’s tail curvature , we needed to reliably isolate as many pixels on the fish’s body as possible so that the movements of the tail could be accurately tracked during swimming .",
"For this , we used a custom MATLAB script ( available upon request ) that used an iterative procedure to obtain a threshold for image binarization and isolation of fish pixels .",
"Following the detection of fish pixels , the images were binarized by setting fish pixels to 1 and the rest of the pixels to 0 .",
"The MATLAB function bwmorph was then used on the binary images to 'thin' the fish pixels down to a set of connected pixels that spanned the length of the fish and coarsely bisected it into two lateral halves .",
"To transform these 'midline' pixels into a smoother curve that better tracked the spine of the fish , we followed the procedure described in ( Huang et al . , 2013 ) , wherein the integer-valued image coordinates of the midline pixels were weighted by the intensities of the surrounding pixels to obtain a new decimal-valued set of pixels .",
"The curve obtained by this procedure was then interpolated using cubic splines to obtain a final smooth curve ( C ) of 50 points .",
"To characterize swimming , we looked at changes in the body posture and head orientation over time .",
"We used the midline curve ( C ) obtained using the procedure described above to characterize body posture .",
"First , we computed tangent vectors i^ along the points on C and used the angle differences between adjacent tangent vectors to compute local curvatures .",
"We then cumulatively summed these curvatures along the tail , starting from the point closest to the head centroid .",
"This way we obtained a set of angles along the midline that represented the angular difference between the tangent at a given point along the midline and the tangent at head centroid .",
"The total curvature ( K ) was computed as the sum of all the local curvature along the tail .",
"To obtain the head orientation , the first 10 points ( the 10 points closest to the head centroid ) of the midline curve Cwere sampled and a straight line ( L ) was fit to those points .",
"The head orientation ( ϕH ) was then estimated as the absolute angle of the vector extending from the point on L farthest from the head centroid to the point on L closest to the head centroid .",
"To identify distinct swim episodes , we used the total curvature time series , Κ",
"( t ) , after low pass filtering it at 70 Hz to remove high-frequency noise .",
"We then used a custom MATLAB script to identify the times corresponding to the onset and offset of each swim episode .",
"Briefly , the absolute function of the filtered curvature timeseries ( |Κ",
"( t ) | ) was convolved with a 200 ms gaussian kernel and in the first step the onsets of swim episodes were identified as the points where the transformed timeseries ( S",
"( t ) ) crossed and stayed above a threshold of 4o for at least 50 ms . Then , for each onset a corresponding offset was identified as the first point in S",
"( t ) after this onset where the signal returns and stays below the threshold for at least 50 ms . Then , in a second step to improve on the loss of temporal resolution resulting from the convolution of K",
"( t ) to get S",
"( t ) , we updated each onset by replacing it with the time of the peak in the second derivative of S",
"( t ) that occurred immediately before this onset .",
"To sort spontaneous swim episodes into distinct subtypes we used the amplitudes of the first bend within each swim episode because it was shown in a previous study ( Burgess and Granato , 2007 ) that this single metric could be used to effectively categorize spontaneous swims into at least two distinct groups .",
"To obtain the decision boundaries for sorting spontaneous swimming episodes from our dataset into distinct swim categories we extracted the first bend amplitude from each episode and fit a Gaussian Mixture ( GM ) model to the data using the scripts available in the scikit-learn library ( http://scikit-learn . org/stable ) in Python .",
"For training the GM model , we used data from 15 fish ( 6056 swim episodes ) in which V2a neurons had not been photoconverted ( Figure 10A , iv-v ) because photoconversion could have potentially altered swimming characteristics .",
"Our model incorporated three Gaussian components because this resulted in the lowest value for the Akaike Information Criterion ( AIC ) ( Figure 10A , iv bottom ) , indicating that our dataset consisted of three distinct spontaneous swim types .",
"Finally , we used the trained GM model on the entire spontaneous swimming dataset to assign each episode to one of the three categories .",
"To visually inspect the swim episodes from each category we used a custom script to temporally align all the episodes ( curvature time series ) within a category in such a way as to produce the highest correlations among them , and then plotted the traces atop of each other ( Figure 10A , vi ) .",
"For aligning escape episodes which have highly nonstationary beat frequencies , correlations were computed using a restricted time window of 80 ms after the episode onset .",
"As with the training of the GM model , we only used data from non-photoconverted fish to make these plots .",
"The plots showed that the swim episodes within each category were quite similar not just with respect to the amplitudes of their first bends , which we had used to predict swim categories , but with respect to subsequent bends as well .",
"Furthermore , the overlaid traces revealed that swim episodes within a category were very similar to each other , but swim episodes across categories were quite distinct .",
"This confirmed that as in the study by Burgess and Granato ( 2007 ) , we could use the amplitude of just the first bend of each swim episode to effectively categorize spontaneous swims into distinct subtypes .",
"In the case of escape swims , we treated them as belonging to a single category because they were all produced in response to the same vibration stimulus and because they are very rarely , if ever , observed in the absence of a stimulus .",
"Thus , for comparing the individual bend amplitudes and periods between control and ablated fish , we did not sort escapes in the same way as spontaneous swims .",
"To evaluate the effects of ablations on swimming , we computed two types of swim parameters from each swim episode and compared these across ablated and control groups of fish .",
"The first type of parameters were global in that each of these parameters described some aspect of a swim episode using a single number .",
"In this study , the global swim parameters we examined were total swim distance per episode , episode duration , mean swim velocity per episode , maximum swim velocity per episode , total number of body bends per episode , and onset latency of the episode ( only for escapes because these were triggered using a stimulus of very short duration ) .",
"The other type of swim parameters we computed were local in that they were a set of numbers that described a swim episode at different stages .",
"Here , the local parameters we examined are the amplitudes and durations ( or periods ) of the different body bends that comprise a swim episode .",
"To estimate the traveling distance per episode , we tracked the spatial position of a point on the fish’s body just caudal to the swim bladder .",
"We chose to track this point instead of the head centroid because it captured the fish’s translation in space without being susceptible to oscillations of the head , such as those conspicuously observed during escapes .",
"The total distance per episode was then computed as the sum of the distances traveled by the tracked point from one frame to another over all the image frames containing a single swim episode .",
"The duration of an episode was simply the time elapsed from the detected onset to the offset of the episode ( see above ) , and the mean swim velocity was obtained by dividing the total swim distance per episode by the duration of the episode .",
"To compute the maximum swim velocity , the temporal derivative of the fish’s spatial position was first computed to obtain the instantaneous swim velocity .",
"The maximum of this timeseries during the course of an episode was the maximum swim velocity per episode .",
"The number of bends per episode was computed by detecting and counting the total number of peaks in the curvature timeseries corresponding to a single swim episode .",
"Bend amplitudes and bend periods within a swim episode were computed after detecting peaks within the curvature timeseries as follows:An=Κtn-Κton when n=1A ( n ) =|K ( tn+1 ) −K ( tn ) | whenn>1Pn= tn-ton when n=1P ( n ) =tn+1−tn whenn>1where n corresponds to bend number , An and Pn are the nth bend amplitude and period respectively , tn is the time at the nth peak , and ton is the time of swim onset .",
"Peak angular velocities within a swim episode were computed in a similar manner from the angular velocity time series ω",
"( t ) , which is the temporal derivative of the timeseries K",
"( t ) .",
"To quantitatively examine how the stereotypy of swim episodes within each swim category changes over the course of an episode , which was one of the criteria used to limit the analysis of bend amplitude and periods first 10 bends of an episode ( see Results ) , we utilized ROC ( Receiver Operating Characteristic ) analysis .",
"We first computed the probability distributions of the peak times of each of the bends of a swim episode ( Figure 10—figure supplement 3A ) for all swim categories .",
"Here , the peak time of a bend refers to the time elapsed from the onset of the episode to the time when that bend reaches peak amplitude .",
"Then , from these probability distributions , we estimated the discriminability of pairs of adjacent bends using ROC analysis .",
"For each pair of adjacent bends up to the 10th bend , we used the probability distributions of their peak times to compute ROC curves ( Figure 10—figure supplement 3B",
"i ) .",
"The area under the curve ( AUC ) for an ROC curve generated for a given bend pair ( say , the second and third bend ) indicated how discriminable that pair of bends was with respect to their peak times .",
"To examine how discriminability of pairs of bends changes for each pair of successive bends , we plotted the AUC values computed for all the bend pairs against bend pairs ordered in succession ( Figure 10—figure supplement 3B",
"ii ) .",
"To determine if the global and local swim parameters we computed were significantly different in control and ablated fish , we carried out statistical tests using software packages implemented in R . The data was first fit using a mixed effects model ( nlme package ) in which the swim parameter of interest ( swim distance , bend amplitude , bend period , etc ) was treated as the response variable while the categorical variables treatment , ablation group , and swim type were treated as the explanatory variables .",
"The variable treatment took two values that indicated if the source of a given data sample was an ablated or a control fish .",
"The variable ablation group also assumed two values that indicated if a data point had been collected from a fish in which old ( birth-time < 24 hpf ) or young ( 42 hpf <birth time<60 hpf ) V2a neurons had been ablated .",
"Finally , the variable swim type indicated which of the aforementioned swim categories the data point belonged to .",
"In addition to the above explanatory variables , the identity of the fish was modeled as a random variable; the values within this last variable uniquely identified each fish that contributed to our dataset .",
"The model also took into account interactions between the independent variables .",
"We used this model-based approach to separately assess the effects of ablation on any of the variables of interest to us .",
"This approach requires that the variables of interest be normally distributed , so to check for normality , we generated quantile-quantile ( Q-Q ) plots of the data , fit a straight line to the points and computed the R2 value to assess how good the fit was .",
"R2 values greater than 0 . 8 were deemed good .",
"Based on this criteria , all the swim parameters we looked at except escape onset latencies followed a normal distribution .",
"However , when latencies were log-transformed and normality assessed on the transformed data , the R2 value exceeded our cutoff value of 0 . 8 .",
"So , the statistical test was carried out on log-transformed escape latencies .",
"Significance was assessed at p<0 . 01 , and the Holm correction for multiple comparisons was made . swimparameter∼treatment∗ablationgroup+swimtype+ ( 1/fishID ) Here , swim parameter can be any of the following: total swim distance per episode , episode duration , mean swim velocity per episode , maximum swim velocity per episode , total number of bends per episode , onset latency of escape , bend amplitude , bend period ."
]
] | [
"The emergence of new and increasingly sophisticated behaviors after birth is accompanied by dramatic increase of newly established synaptic connections in the nervous system .",
"Little is known , however , of how nascent connections are organized to support such new behaviors alongside existing ones .",
"To understand this , in the larval zebrafish we examined the development of spinal pathways from hindbrain V2a neurons and the role of these pathways in the development of locomotion .",
"We found that new projections are continually layered laterally to existing neuropil , and give rise to distinct pathways that function in parallel to existing pathways .",
"Across these chronologically layered pathways , the connectivity patterns and biophysical properties vary systematically to support a behavioral repertoire with a wide range of kinematics and dynamics .",
"Such layering of new parallel circuits equipped with systematically changing properties may be central to the postnatal diversification and increasing sophistication of an animal’s behavioral repertoire ."
] | [
"Newborn babies have limited abilities .",
"Indeed , most of our actions shortly after birth are the result of reflexes that serve our most basic need: to stay alive .",
"As we get older , however , our behaviour gradually becomes more sophisticated .",
"During this time , the billions of cells in our brain form new connections to build intricate ‘circuits’ of neurons that allow for more complicated thoughts and actions .",
"It is clear that the brain circuits that support new behaviours must develop in a way that does not interfere with the existing circuits that are vital for survival .",
"However , the challenge has been to find a way to peer into a brain as it develops to see how these new circuits form .",
"In recent years , zebrafish have revolutionised research into neuronal circuits in animals .",
"Developing over the course of a few days , these small transparent fish provide a window into the brain during the earliest stages of development .",
"Indeed , the circuits of neurons that descend from the brain and connect to the spinal cord have already been mapped in these animals .",
"Now , Pujala and Koyama have begun to follow the careful development of these ‘descending’ neurons , and relate it to the appearance of new behaviours in young zebrafish .",
"Time-lapse imaging with a fluorescent protein that is active only in specific descending neurons revealed that new circuits are laid down over existing ones , like the growth rings in a tree .",
"Next , at different timepoints in zebrafish development , Pujala and Koyama traced these neurons backwards from the spine to the brain to identify which connections formed first .",
"This showed that the spinal connections develop one after the other , in the same order that the neurons mature .",
"Next , Pujala and Koyama asked how the activity of neurons that mature early or late in development relates to specific behaviours in young zebrafish .",
"Early-born circuits connect to neurons that produce powerful , reflex-driven , whole-body movements such as an escape response .",
"The later circuits connect to different neurons through slower , less direct pathways; the late-born neurons also generate the refined movements that are acquired later in a zebrafish’s development and help the fish to explore its environment .",
"These findings show that descending circuits in zebrafish run parallel to each other , but with distinct connections and properties that allow them to control different kinds of movements .",
"While this study was conducted using an animal model , a better understanding of how such circuits develop and the movements they control may one day aid the treatment of patients with neurodegenerative diseases or injuries where connections have been lost ."
] | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"tools and resources",
"neuroscience"
] | Chemoptogenetic ablation of neuronal mitochondria in vivo with spatiotemporal precision and controllable severity | elife-51845-v1 | [
[
"High-resolution intravital imaging , coupled with transgenic expression of fluorescent reporters , provides opportunities to analyze the biology of the intact nervous system .",
"This approach has been particularly successful in larval zebrafish , which combine optical transparency ( White et al . , 2008 ) with vertebrate CNS structure and genetics ( Burton , 2014 ) , allowing advances in understanding whole-brain activity patterns at cellular resolution ( Portugues et al . , 2014; Ahrens et al . , 2013 ) , the formation of neural circuits ( Tay et al . , 2011 ) , neuronal mitochondrial trafficking ( Dukes et al . , 2016 ) and neuronal autophagy ( Khuansuwan et al . , 2019 ) .",
"Recent developments in light-sensitive channel proteins ( Cosentino et al . , 2015 ) and genetically-encoded photosensitizers ( Buckley et al . , 2017; He et al . , 2016 ) provide new opportunities to extend these studies from observations to experimental manipulation of the CNS in vivo , by light-induced activation ( Ljunggren et al . , 2014 ) , inhibition ( Bergeron et al . , 2015 ) or ablation ( Del Bene et al . , 2010 ) of genetically-defined neuronal populations .",
"In particular , genetically-encoded photosensitizers should offer the means to analyze neuronal biology with stringent spatial resolution , by damaging or ablating defined neuronal populations , cellular subcompartments , organelles or even specific molecules precisely , to determine their roles in nervous system function and pathophysiology .",
"Genetically-encoded photosensitizers often show limited photostability , decreasing the amount of oxidative damage that can be induced by light exposure ( He et al . , 2016 ) .",
"In addition , they are typically excited by blue or green light , which shows limited penetration through living tissues and can cause direct , untargeted , phototoxicity ( He et al . , 2016 ) .",
"Furthermore , constitutively-active photosensitizers allow chronic phototoxicity from passive absorption of ambient light .",
"Chemogenetic systems enable selective targeting of genetically-defined cells by expression of a designer receptor that is only activated after administration of a small molecule ligand that binds the receptor specifically ( Roth , 2016 ) .",
"Far-red light-sensitive channel proteins enable activation in deep tissues – even through the intact skull ( Chuong et al . , 2014 ) .",
"For optogenetic applications in vivo , it would be highly advantageous to combine on-demand chemical activation of genetically-targeted cells with photoactivation using far-red light .",
"dL5** is a modified single-chain antibody that can function as a fluorogen-activating protein ( FAP ) by binding malachite green ( MG ) fluorogens with picomolar affinity and activating their fluorescence thousands-fold ( Szent-Gyorgyi et al . , 2008; Szent-Gyorgyi et al . , 2013 ) .",
"It was recently reported that a di-iodine substituted MG derivative , MG2I , becomes a potent photosensitizer on binding to dL5** ( He et al . , 2016 ) .",
"The dL5**-MG2I complex showed maximal excitation in the highly tissue-penetrant far-red light range , and produced singlet oxygen ( 1O2 , molecular dioxygen in its first electronically excited state ) through inter-system crossing , with high quantum yield and with little photobleaching ( He et al . , 2016 ) .",
"Importantly , neither dL5** nor MG2I alone produced detectable 1O2 under far-red illumination , and the dL5**-MG2I complex did not produce 1O2 in the absence of light .",
"This system offers several key advantages as a photosensitizer for use in neuroscience applications in vivo .",
"1O2 is highly reactive with organic molecules and thus reacts within a short radius ( <20 nm ) of its site of formation in living systems ( its lifetime is approximately 4μs in water [Wilkinson et al . , 1995] , but may be as low as 100ns in cells [Moan and Berg , 1991] ) .",
"Consequently , oxidative damage can be provoked with a high degree of spatial precision by fusing dL5** to an appropriate protein or targeting sequence , to direct its expression to a specific sub-cellular region , organelle or protein complex ( He et al . , 2016 ) .",
"For example , dL5** fused to TRF1 , a component of the shelterin complex , was employed recently to induce 8-oxoguanine formation specifically in the telomeric DNA of cultured cells ( Fouquerel et al . , 2019 ) .",
"Furthermore , as 1O2 is generated only during far-red illumination , the onset of oxidative damage from dL5**-MG2I is temporally well-defined , and its severity and rate of induction can be regulated by adjusting light exposure time and power .",
"Finally , dependence of chemoptogenetic production of 1O2 on the presence of MG2I means that dL5**-expressing transgenic animals and cell lines can be generated , bred , handled and shipped in the absence of MG2I , without having to house them in darkness; this is a significant advantage over constitutively active genetic photosensitizers .",
"Alterations in neuronal mitochondrial function have been strongly linked to several neurodegenerative diseases ( Nguyen et al . , 2019; Swerdlow , 2018; Carmo et al . , 2018 ) and there is significant interest in understanding how mitochondrial homeostasis and bioenergetics are maintained in neurons under physiological conditions and following mitochondrial damage .",
"Prior work showed that dL5** could be expressed within the mitochondria of cultured HEK293 cells in vitro when fused to an appropriate targeting signal ( Qian et al . , 2019 ) .",
"Treatment of these cells with MG2I and exposure to far-red light resulted in decreased oxygen consumption , loss of respiratory chain activity , mitochondrial depolarization , compensatory glycolysis , and secondary ROS generation that was sufficient to cause oxidative telomere damage and cell cycle arrest ( Qian et al . , 2019 ) .",
"Unlike transformed cells in culture , however , neurons are generally dependent on mitochondrial function for ATP generation ( reviewed in Van Laar and Berman , 2013 ) and this is known to influence the responses of neuronal mitochondria to perturbing stimuli ( Van Laar et al . , 2011 ) .",
"To investigate the consequences of mitochondrial damage in neurons in vivo , we expressed dL5** in the neuronal mitochondria of transgenic zebrafish .",
"In the presence of MG2I , far red light provoked specific disruption of mitochondrial structure , respiration and ATP synthesis , resulting in dramatic neurophysiological and neurobehavioral abnormalities .",
"This highly innovative approach has numerous applications as a tool for understanding neuronal bioenergetics and mitochondrial homeostasis , investigating mitochondrial mechanisms in disease pathogenesis , and manipulating neural circuitry to understand the biological basis of behavior ."
],
[
"We generated transgenic zebrafish expressing the fluorogen-activating protein ( FAP ) dL5** ( Figure 1A; Szent-Gyorgyi et al . , 2008; Szent-Gyorgyi et al . , 2013 ) fused to mitochondrial targeting sequences from human COX4 and COX8 ( Telmer et al . , 2015 ) , and a fluorescent reporter , mCerulean3 , to allow visualization of transgene expression in vivo ( Figure 1B; dL5** and mCerulean3 are separated by a flexible GGGGSGGGGS linker to allow correct folding of each domain of the fusion protein ) .",
"Transgenic lines were made using the bipartite Gal4/UAS system ( Asakawa et al . , 2008 ) , so that mitochondrially-targeted dL5**-mCerulean3 could be expressed in any tissue or cell population of interest by crossing Tg ( UAS:COX4-COX8-dL5**-mCer3 ) zebrafish with an appropriate tissue-specific Gal4 driver line , thereby enhancing the utility of this line for multiple future applications .",
"For the present study , we generated a new Tg ( eno2:gal4FF ) line that expresses Gal4FF ( an engineered Gal4-VP16 fusion protein with attenuated toxicity; Asakawa et al . , 2008 ) widely in neurons , using a 12 kb eno2 regulatory element that we reported previously ( Bai et al . , 2007 ) .",
"Double transgenic Tg ( eno2:gal4FF ) pt425; Tg ( UAS:COX4-COX8-dL5**-mCer3 ) pt427 zebrafish ( referred to as ‘NeuMitoFAP’ zebrafish for brevity ) showed strong mCerulean3 expression throughout the nervous system ( Figure 1C ) .",
"At high magnification , punctate mCerulean3-labeled structures corresponding to individual mitochondria were visible within axons of live NeuMitoFAP larval neurons ( Figure 1D ) .",
"Tissue sections revealed extensive co-localization of mCerulean3 with TOM20 ( a mitochondrial marker ) in CNS neurons of NeuMitoFAP zebrafish ( Figure 1E ) .",
"Pixel-by-pixel analysis of single confocal planes showed that the mCerulean3 signal correlated strongly with the mitochondrial TOM20 signal , but not with the nuclear DAPI signal ( Figure 1F , G ) .",
"These data show that dL5**-mCerulean3 is expressed in the neuronal mitochondria of NeuMitoFAP zebrafish .",
"The excitation spectrum of the FAP-MG2I complex ( He et al . , 2016 ) is shown in Figure 1H , and summarized in Table 1 , in comparison with emission spectra of the light sources employed in this study .",
"A light stand was constructed ( Figure 1—figure supplement",
"1 ) to expose zebrafish larvae to far-red light ( λ = 661 ± 9 nm , peak ±half width at half height; Table",
"1 ) near the major FAP-MG2I excitation peak ( λ = 666 ± 30 nm; Figure 1—figure supplement 2; Table 2 ) , with adjustable power up to ≈160 mW/cm2 , and without transferring heat to the water bath ( Figure 1—figure supplement 3 ) .",
"Green LED safe lights ( λ = 516 ± 18 nm ) allowed MG2I-exposed NeuMitoFAP zebrafish to be handled , and behavioral responses provoked ( Burton et al . , 2017 ) , without activating 1O2 production from the FAP-MG2I complex ( Figure 1H; Figure 1—figure supplement 2; Tables 1 and 2 ) .",
"Infrared light sources that did not activate FAP-MG2I provided illumination for videography , while quantifying zebrafish motor function ( Zhou et al . , 2014 ) ( λ = 877 ± 25 nm ) and during electrophysiological recordings ( λ = 775 ± 32 nm ) .",
"By 5 days post-fertilization ( dpf ) , zebrafish larvae show spontaneous locomotor activity and evoked behavioral responses that are easily quantified by video tracking in 96-well plates ( Figure 2; Zhou et al . , 2014 ) .",
"We previously demonstrated that the visual motor response ( VMR ) , a series of stereotyped changes in motor activity provoked by abrupt alterations in ambient illumination ( Burgess and Granato , 2007 ) , can be elicited by green light at a wavelength that does not excite FAP-MG2I ( Burton et al . , 2017 ) .",
"Four experimental groups ( WT , WT-MG2I , NeuMitoFAP , NeuMitoFAP-MG2I ) were generated by growing non-transgenic and NeuMitoFAP zebrafish in embryo water containing MG2I or no additive under green light illumination from 3 dpf ( Figure 2 ) .",
"Zebrafish from all four experimental groups showed normal motor activity ( Figure 2B ) and morphology ( Figure 2—figure supplement",
"1 ) at 5dpf .",
"Robust responses to abrupt green light – dark transitions were apparent in all four experimental groups ( Figure 2B , left panel; Figure 2C , upper graphs ) prior to far-red light exposure .",
"However , motor responses were eliminated acutely in NeuMitoFAP-MG2I zebrafish , but not controls , following exposure to 60 J/cm2 far-red light ( mean ± SE swimming speed in dark phase of VMR , pre- versus post-exposure: WT , 1 . 80 ± 0 . 10 vs . 1 . 70 ± 0 . 08 mm/s; WT-MG2I , 1 . 69 ± 0 . 09 vs . 1 . 60 ± 0 . 10 mm/s; NeuMitoFAP , 2 . 02 ± 0 . 10 vs . 2 . 13 ± 0 . 08 mm/s; NeuMitoFAP-MG2I 2 . 36 ± 0 . 09 vs . 0 . 08 ± 0 . 02 mm/s , p<10−15 , 2-way ANOVA with Šidák multiple comparisons test; Figure 2B-D; Video 1 ) .",
"Loss of motor function in NeuMitoFAP-MG2I zebrafish was dependent on the amount of light energy delivered ( Figure 2—figure supplement 1 ) , but independent of the rate of delivery between 16–160 mW/cm2 at a fixed total exposure of 60 J/cm2 ( Figure 2—figure supplement 2 ) .",
"Motor function during FAP-MG2I activation was examined by using far-red light to both elicit the VMR and excite FAP-MG2I simultaneously ( Figure 2—figure supplement 3 ) .",
"In NeuMitoFAP-MG2I zebrafish , far-red light initially caused transient hyperkinesia , which was followed by progressive loss of motor function with cumulative light exposure; these abnormalities were not observed in controls .",
"Although NeuMitoFAP-MG2I zebrafish exposed to far-red light lost spontaneous and evoked motor function , their heart rate and circulation were preserved ( Figure 2E; Video",
"2 ) and there were no gross morphological changes ( Figure 2—figure supplement 4 ) , showing that the larvae remained alive and that abnormalities were restricted to the nervous system .",
"Together , these data show a dramatic neurological phenotype that was dependent on all three components of the chemoptogenetic system ( far-red light exposure in the presence of both dL5** and MG2I ) , and therefore attributable to 1O2 formation at the site of dL5** expression in neuronal mitochondria .",
"Furthermore , the severity of the abnormalities was determined by the amount of far-red light energy delivered .",
"Electrophysiological recordings were carried out to elucidate the basis for the abrupt loss of neurological function in NeuMitoFAP-MG2I zebrafish exposed to far-red light ( Figure 3; Figure 3—figure supplement 1 ) .",
"Large sensory neurons of the posterior lateral line ganglion express the eno2 driver strongly and are located superficially , allowing continuous whole-cell patch clamp recordings in intact zebrafish larvae during light exposure .",
"In control zebrafish , lateral line sensory neurons showed similar stable baseline membrane potentials that changed minimally during 20–30 min of far-red light exposure ( baseline versus final membrane potential: WT-MG2I , −66 . 9 ± 1 . 4 vs . −63 . 2 ± 1 . 2 mV; NeuMitoFAP , −64 . 4 ± 1 . 2 vs . −60 . 8 ± 2 . 5 mV; mean ± SE; Figure 3A-C ) .",
"Sensory neurons in NeuMitoFAP-MG2I larvae showed similar baseline membrane potential to controls but depolarized progressively during exposure to far-red light , initially reaching threshold potential and firing high-frequency trains of action potentials , then depolarizing further to become refractory ( baseline versus final membrane potential −60 . 8 ± 2 . 4 mV vs . −29 . 4 ± 4 . 5 mV , p<0 . 0001 , 2-way ANOVA with Tukey multiple comparison test; Figure 3A-C ) .",
"To determine whether bioenergetic depletion could account for these findings , identical recordings were obtained from NeuMitoFAP-MG2I neurons , but with the addition of phosphocreatine to the patch pipette solution .",
"Phosphocreatine is a substrate for cytoplasmic creatine kinase , allowing the regeneration of ATP from ADP and thereby providing a non-mitochondrial source of ATP .",
"The baseline resting membrane potential was unaltered by phosphocreatine , but its presence prevented depolarization of NeuMitoFAP-MG2I neurons during far-red light exposure ( baseline versus final membrane potential: −62 . 7 ± 1 . 4 vs . −59 . 6 ± 2 . 8 mV; Figure 3A-C ) .",
"Overall , 10/10 NeuMitoFAP-MG2I neurons depolarized by ≥20% of their baseline membrane potential following a mean exposure of 44 . 3 ± 6 . 3 J/cm2 far-red light , whereas in the presence of phosphocreatine , only 1/6 neurons depolarized after exposure to >80 J/cm2 ( p=0 . 0014 , Fisher’s exact test ) .",
"Transient exposures to smaller amounts of far-red light energy caused slower and more modest levels of depolarization ( Figure 3—figure supplement 2 ) .",
"Together , these data show that the acute neurological deficits provoked in NeuMitoFAP-MG2I zebrafish by exposure to far-red light were caused by neuronal depolarization , resulting from depletion of ATP that is necessary to drive the active ionic transporters that maintain transmembrane ionic gradients underlying the resting membrane potential .",
"We next evaluated mitochondrial function in NeuMitoFAP-MG2I zebrafish .",
"A bioluminescence assay was used to measure ATP concentration in lysates from NeuMitoFAP-MG2I larvae .",
"Far-red light exposure caused a ≈10% decrease in whole animal ATP content ( pre-light 4 . 12 ± 0 . 10 vs . post-light 3 . 69 ± 0 . 11 μMol ATP/g protein; mean ± SE; p=0 . 0060 , 2-tailed t-test; Figure 4A ) .",
"Mitochondrial respiration was analyzed using a flux analyzer to measure whole larval O2 consumption rate ( OCR ) .",
"In order to allow stable recordings , and to prevent changes in muscle O2 consumption secondary to altered motor activity from obscuring differences between experimental groups , larvae were anesthetized with tricaine and paralyzed with d-tubocurarine prior to OCR quantification ( Figure 4B; Figure 4—figure supplement 1 ) .",
"NeuMitoFAP zebrafish exposed to far-red light in the presence of MG2I showed a ≈24% decrease in whole larval OCR compared with controls that were not treated with MG2I ( NeuMitoFAP 170 . 7 ± 8 . 9 vs . NeuMitoFAP-MG2I 129 . 2 ± 7 . 0 pMol/min; mean ±SE; p=0 . 00053 , unpaired t-test; Figure 4C , D ) .",
"In contrast , WT controls exposed to far-red light showed similar stable OCRs regardless of MG2I treatment ( WT , 194 . 8 ± 11 . 4; WT-MG2I , 189 . 1 ± 14 . 1 pMol/min; mean ± SE; Figure 4E ) .",
"Bath acidification rate was unaltered in NeuMitoFAP-MG2I zebrafish exposed to far-red light ( Figure 4—figure supplement 2 ) , providing no evidence of compensatory glycolysis following abrogation of neuronal mitochondrial function .",
"Cultured cells , unlike whole zebrafish , can be treated with chemical inhibitors to evaluate specific aspects of mitochondrial function .",
"Consequently , we next evaluated dissociated brain cells derived from NeuMitoFAP zebrafish .",
"Cells were treated with MG2I and exposed to far-red light after dissociation ( Figure 4F ) .",
"Under these conditions , NeuMitoFAP-MG2I cells , but not controls , showed a dramatic , far-red light dose-dependent , decrease in maximal respiration ( measured by comparing OCR following exposure to the uncoupling agent FCCP with OCR following exposure to the complex I and III inhibitors rotenone and antimycin-A; Figure 4F , G ) .",
"Together , these data show that the combination of MG2I and far-red light caused significant disruption of mitochondrial bioenergetic and respiratory functions in NeuMitoFAP zebrafish .",
"We next investigated mitochondrial morphology by intravital microscopy .",
"The lateral line nerve runs superficially along the larval body axis in a rostro-caudal direction , allowing confocal imaging at sufficiently high magnification to visualize the morphology of mCerulean3-labeled mitochondria within the axons of live , intact NeuMitoFAP zebrafish ( Figure 5A ) .",
"Image stacks were acquired through the entire medio-lateral extent of the nerve and mitochondrial features analyzed quantitatively .",
"At baseline , mitochondria were elongated in shape and distributed regularly along axons ( length 4 . 89 ± 0 . 34 μm; circularity 0 . 29 ± 0 . 017; number of mitochondria per field of view 75 . 50 ± 6 . 84; mean ± SE; Figure 5A-D ) .",
"Length , shape and distribution were unaffected by either far-red light or MG2I alone ( Figure 5A–D ) .",
"However , in the presence of MG2I , an increased number of small , rounded mitochondria were seen in NeuMitoFAP axons immediately following far-red light exposure ( length 2 . 00 ± 0 . 12 μm , p<0 . 0001; circularity 0 . 60 ± 0 . 014 , p<0 . 0001; number 131 . 70 ± 10 . 57 , p<0 . 001; NeuMitoFAP-MG2I post-light compared indivdually with each control group , 1-way ANOVA with Tukey multiple comparisons test; Figure 5A-D ) .",
"These data suggest that the combination of NeuMitoFAP , MG2I and far-red light caused fragmentation of axonal mitochondria through mitochondrial 1O2 production .",
"Transmission electron microscopy was employed to investigate mitochondrial ultrastructure in the brains of NeuMitoFAP zebrafish , immediately following far-red light exposure .",
"In the absence of MG2I , neurons showed normal morphology; their mitochondria were elongated in shape and filled with densely-stacked tubular and lamellar cristae ( Figure 5E ) .",
"In the presence of MG2I , far-red light exposure caused the widespread appearance of lucent areas in neuronal cytoplasm at low magnification ( Figure 5F , left panel , arrows ) .",
"These areas corresponded to swollen , rounded mitochondria with nearly complete elimination of cristae ( Figure 5F; Figure 5—figure supplement 1 ) .",
"The proportion of abnormal mitochondria showing rounded shape and absent or reduced cristae was determined in 12 fields of view per experimental group by a blinded observer ( 6 . 14 ± 2 . 84 mitochondria per field , mean ± SD ) .",
"No abnormal mitochondria were observed in NeuMitoFAP zebrafish prior to far-red light exposure .",
"In the absence of MG2I , a single abnormal mitochondrion was seen immediately following far-red light exposure .",
"In contrast , in the presence of MG2I , far-red light caused extensive disruption to neuronal mitochondrial ultrastructure .",
"NeuMitoFAP-MG2I zebrafish showed 93 . 3 ± 3 . 5% abnormal mitochondria immediately post-exposure , and 97 . 0 ± 3 . 0% abnormal mitochondria at 24 hr post-exposure ( p<10−15 compared with no chemical control group at same time point; 2-way ANOVA with Šidák multiple comparisons test; Figure 5G ) .",
"Together , these data show that far-red light exposure in the presence of MG2I caused severe structural deficits in the neuronal mitochondria of NeuMitoFAP zebrafish that were attributable to damage caused by mitochondrial chemoptogenetic 1O2 production .",
"Finally , we determined the downstream consequences of neuronal mitochondrial damage .",
"The immediate loss of motor function observed in NeuMitoFAP-MG2I larvae following far-red light exposure did not recover over the subsequent 4 days , even though the zebrafish appeared morphologically unremarkable ( Figure 2—figure supplement 4 ) , underwent normal somatic , cardiac and vascular development and showed similar heartbeat and circulation to controls ( Figure 6A , B; Videos 3 and 4 ) .",
"In order to clarify the basis for this persistent neurological deficit , we employed acridine orange ( AO , a DNA intercalating agent that labels degenerating cells in which the plasma membrane has become permeable ) , in combination with intravital microscopy , to quantify cell death in live , intact larvae ( Figure 6C , D; Figure 6—figure supplement 1 ) .",
"Baseline developmental cell death was observed in the spinal cords of all zebrafish ( WT-MG2I , 9 . 5 ± 0 . 7; NeuMitoFAP , 10 . 1 ± 0 . 6; NeuMitoFAP-MG2I , 18 . 5 ± 2 . 1 AO-labeled cells/spinal cord; mean ± SE ) .",
"Immediately following far-red light exposure , cell death started to increase steadily in NeuMitoFAP-MG2I larvae but not controls , peaking at 24 hr post-exposure .",
"At this time point , over 30-fold more AO-labeled cells were found in NeuMitoFAP-MG2I larvae than controls ( WT-MG2I , 9 . 7 ± 0 . 8; NeuMitoFAP , 9 . 7 ± 1 . 1; NeuMitoFAP-MG2I , 311 . 7 ± 14 . 8 AO cells/spinal cord; p<10−15 NeuMitoFAP-MG2I vs . NeuMitoFAP , p<10−15 NeuMitoFAP-MG2I vs . MG2I , p<10−15 NeuMitoFAP-MG2I at 24 hr vs . baseline , 2-way ANOVA with Tukey multiple comparisons test; Figure 6C , D ) .",
"Transmission electron microscopy was employed to examine the underlying ultrastructural changes ( Figure 6E ) .",
"Prior to far-red light exposure , NeuMitoFAP-MG2I neurons showed normal morphology with homogenous nuclei , prominent nucleoli and elongated mitochondria with densely packed cristae .",
"Immediately after far-red light exposure , severe and widepread mitochondrial abnormalities were observed in neurons , as described above .",
"Remarkably , however , despite these dramatic mitochondrial changes , the adjacent nuclear membrane , Golgi apparatus and endoplasmic reticulum appeared normal at this time point .",
"By 2 hr post-exposure , scattered neurons started to show nuclear chromatin condensation and other morphological changes , including nuclear membrane separation .",
"At 24 hr post-exposure , numerous apoptotic bodies were visible , along with neurons showing morphological features suggesting ongoing apoptosis , and other neurons showing signs suggestive of necrosis ."
],
[
"We have demonstrated dramatic changes in the neurological function of an intact living vertebrate organism following chemoptogenetic targeting of mitochondrial function with organelle-level spatial precision .",
"Our data show a direct link between neuronal respiration , bioenergetics and physiology; quantify neuronal respiration and its bioenergetic contributions in vivo; and demonstrate that neuronal death following mitochondrial damage is delayed and presumably dependent on secondary mechanisms .",
"The new transgenic lines and methods we report will provide powerful tools for investigating mitochondrial homeostasis and pathophysiology , and for understanding the neural basis of behavior .",
"The requirement of all three components of the chemoptogenetic system ( dL5** , MG2I and far-red light ) shows that the observed phenotypes were caused by singlet oxygen .",
"The subcellular localization of the dL5**-mCerulean3 fusion protein in NeuMitoFAP zebrafish resulted in spatially-restricted 1O2 production within neuronal mitochondria .",
"Correspondingly , initial damage was confined to mitochondria , as evidenced by preservation of cellular ultrastructure immediately adjacent to severely damaged mitochondria directly after light exposure .",
"Potentially , 1O2 can react with all mitochondrial macromolecules and the biochemical targets of this reactive oxygen species in the mitochondrion are not yet fully resolved .",
"In cultured HEK293 cells expressing mitochondrially-targeted dL5**-mCerulean3 , exposure to MG2I and far-red light decreased the activity of mitochondrial respiratory chain complexes I , III and IV ( Qian et al . , 2019 ) .",
"COX4 and COX8 encode subunits of complex IV ( CIV , cytochrome C oxidase ) , and it is possible that the fusion protein is directed to CIV by the COX8 sequence remaining after cleavage of the signal peptide .",
"In this case , localized 1O2 damage to the CI-CIII2-CIV super-complex ( ‘respirasome’ ) ( Letts and Sazanov , 2017 ) is predicted to decrease electron transport and thereby dissipate the inner membrane proton gradient , resulting in loss of ATP production and mitochondrial swelling .",
"Although this would account for the loss of respiration and bioenergetic collapse we observed , it is unclear whether respiratory chain function is disrupted by 1O2 directly .",
"In HEK293 cells expressing mitochondrially-targeted dL5**-mCerulean3 , electron transport and respiration following light exposure were more severely disrupted by a secondary wave of ROS produced by damaged mitochondria ( Qian et al . , 2019 ) .",
"Consequently , the presence of oxidative damage does not necessarily imply that a molecule is a primary target of 1O2 .",
"Furthermore , the severe ultrastructural changes we observed immediately after light exposure raise the possibility that changes in mitochondrial function could occur as a consequence of disrupted mitochondrial architecture .",
"Mitochondrial cristae are dynamic structures whose organization strongly influences assembly of respiratory chain super-complexes and electron transport function ( Cogliati et al . , 2016 ) .",
"Primary 1O2-induced damage to proteins involved in forming or regulating cristae , for example OPA1 ( Frezza et al . , 2006; Patten et al . , 2014 ) or components of the MICOS complex ( Kozjak-Pavlovic , 2017 ) , or direct damage to inner membrane lipids , could cause indirect changes in respiratory function by disrupting the topological organization of electron transport chain complexes in the inner mitochondrial membrane ( Cogliati et al . , 2013 ) .",
"Additional studies will be necessary to determine the initial , direct targets of 1O2 in this model .",
"FAP-MG2I zebrafish showed a large decrease in whole-animal respiration following exposure to far-red light .",
"The 12 kb eno2 regulatory element is expressed in the majority of CNS and PNS neurons , most strongly in large projection neurons such as retinal ganglion cells , reticulospinal neurons and motor neurons , that are predicted to be most metabolically active ( Bai et al . , 2007; Bai et al . , 2009 ) .",
"If it is presumed that light exposure fully disrupted cellular respiration in NeuMitoFAP-MG2I cells , it can be inferred that eno2-expressing neurons account for nearly 25% of the baseline O2 consumption in an immobilized zebrafish larva .",
"The dramatic loss of respiratory activity following light exposure resulted in profound bioenergetic consequences .",
"The overall 10% decrease in whole-animal ATP levels suggests a much larger ATP deficit in eno2-expressing neurons that comprise a relatively small fraction of the total cells in a larval zebrafish .",
"It has been estimated that up to 50% energy expenditure in neurons is devoted to maintaining the transmembrane ionic gradients underlying the resting membrane potential ( Howarth et al . , 2012 ) .",
"Progressive depolarization of NeuMitoFAP-MG2I neurons during light exposure was rescued by phosphocreatine , a substrate for creatine kinase ( CK ) that allows ATP generation from ADP , independent of oxidative phosphorylation .",
"This observation formally establishes that loss of neural function in this model was attributable to bioenergetic crisis and highlights the importance of mitochondrial respiration for providing the ATP necessary for physiological functions such as active ion transport in neurons .",
"In addition , these findings provide further support to the specificity of the initial oxidative insult .",
"A cysteine residue within the active site of CK is necessary for enzymatic activity , which has been shown in other experimental systems to be readily inactivated by oxidants including dopamine ( Van Laar et al . , 2008 ) , superoxide ( Yuan et al . , 1992 ) and peroxynitrite ( Konorev et al . , 1998 ) .",
"The preservation of CK function in NeuMitoFAP zebrafish neurons sufficient to allow rescue of cellular bioenergetics by phosphocreatine suggests that 1O2-mediated damage did not inactivate cytoplasmic CK isoforms , even in the presence of severe mitochondrial disruption .",
"The persistence of acute neurobehavioral deficits in the days following light exposure was attributable to cell death .",
"This was maximal 24 hr after light exposure , well after acute neurobehavioral changes were first observed .",
"We predict that the initial mitochondrial damage provoked delayed loss of cellular viability through secondary mechanisms , several of which may be involved .",
"First , ATP depletion is a well-recognized cause of cellular necrosis .",
"In this regard , the reliance of neuronal ATP synthesis on oxidative phosphorylation may be a critical factor distinguishing neurons from cultured cells , which showed compensatory glycolysis and cell cycle arrest , but not loss of viability , following dL5**-MG2I-induced mitochondrial damage ( Qian et al . , 2019 ) .",
"Second , it is likely that cellular Ca2+ homeostasis was disrupted in NeuMitoFAP-MG2I neurons following far-red light exposure .",
"Cytosolic Ca2+ levels are maintained by ATP-dependent transport of Ca2+ into the endoplasmic reticulum and extracellular space , and by mitochondrial Ca2+ buffering that is dependent on the inner mitochondrial membrane electrochemical potential and its structural integrity .",
"Each of these processes is likely to have been impaired in NeuMitoFAP-MG2I neurons following light exposure .",
"Depending on severity and subcellular distribution , elevated cytosolic Ca2+ can trigger either necrotic or apoptotic cell death ( Pinton et al . , 2008 ) .",
"Finally , compromised mitochondrial structure may allow release of pro-apoptotic mediators .",
"For example , cytochrome c is retained physiologically within cristae folds; consequently , the obliteration of mitochondrial cristae observed in this model is predicted to allow cytochrome c efflux into the cytoplasm , where it can initiate Apaf-1-dependent apoptosome assembly and activation of the intrinsic apoptotic pathway ( Kroemer et al . , 2007 ) .",
"Since morphological markers of both apoptosis and necrosis were visible , it seems unlikely that a single downstream mechanism was responsible for the observed cell death .",
"The bioenergetic requirements of individual neurons vary according to their morphology and activity .",
"Further , dL5** expression varies between different neuronal populations in NeuMitoFAP zebrafish , because the eno2 regulatory element is expressed differentially in discrete cell types ( Bai et al . , 2007; Bai et al . , 2009 ) .",
"We predict that the mechanisms by which neurons die in this model depend on the relationship between bioenergetic demand and loss of ATP , similar to other cell types ( Lieberthal et al . , 1998 ) .",
"For example , a cell with high ATP requirements that sustains a severe mitochondrial injury might undergo necrotic cell death rapidly , whereas less prominent damage in a cell with more modest ATP demands might trigger signaling events culminating in delayed programmed cell death .",
"Targeting neuronal mitochondrial function with dL5**-MG2I provides new experimental opportunities in vivo , and the tools reported here will be useful for multiple downstream applications .",
"Use of Gal4-UAS genetics will greatly expand the utility of the approach , because the dL5**-mCerulean3 fusion protein can be expressed in the mitochondria of any cell type in vivo , by crossing Tg ( UAS:COX4-COX8-dL5**-mCer3 ) pt427 zebrafish to a tissue-specific Gal4 driver line .",
"Many relevant transgenic driver lines are available that express Gal4 in dopaminergic neurons ( Fujimoto et al . , 2011 ) or glial cells ( Frøyset et al . , 2018 ) of interest to disease pathogenesis , in addition to larger libraries of enhancer trap Gal4 insertions useful for functional neuroanatomy studies ( Bergeron et al . , 2015; Kawakami et al . , 2010; Marquart et al . , 2015 ) .",
"Furthermore , by exploiting the dependence of neurons on oxidative phosphorylation , our approach provides several improvements on current technology for targeted cell ablation to analyze neural circuits underlying behavior ( Bergeron et al . , 2015; Godoy et al . , 2015 ) The most widely applied method for chemogenetic ablation in zebrafish models relies on metronidazole-induced DNA crosslinking in transgenic animals expressing the bacterial enzyme nitroreductase ( Curado et al . , 2007 ) .",
"DNA damage in this model takes many hours , and sometimes days , to accumulate to a level sufficient to ablate the targeted cell groups .",
"In contrast , the rapid light-induced neuronal depolarization caused by mitochondrially-targeted dL5**-MG2I provides temporal certainty regarding lesion onset , avoids pharmacokinetic uncertainties inherent in chemical approaches , and occurs with sufficient rapidity to prevent compensatory changes in circuity that may obscure the resulting neurobehavioral consequences .",
"Furthermore , spatial specificity can be enhanced by directing the activating light to particular neurons or subcellular regions of interest , for example by using a far-red laser beam rather than a diffused light source to excite dL5**-MG2I .",
"This will be of value for future investigations into the compartmentalization of bioenergetics and maintenance of functional mitochondrial biomass in neurons in vivo .",
"Illuminating individual topological domains of NeuMitoFAP neurons may allow direct exploration of how mitochondrial spatial distribution contributes to maintaining bioenergetic requirements and ionic gradients throughout the dendritic arborization , cell body and axonal projection .",
"In addition , mitochondrial quality control has been implicated in the pathophysiology of neurological diseases including Parkinson’s disease , but the use of chemicals that non-selectively depolarize all mitochondria simultaneously precludes investigation of the underlying mechanisms in specific neurons .",
"dL5**-MG2I provides several advantages over other genetically-encoded photosensitizers such as KillerRed ( Bulina et al . , 2006 ) for these applications , including minimal photobleaching , high quantal yield of 1O2 , and excitation by tissue-penetrant far-red light ( He et al . , 2016 ) .",
"In addition , the necessity for addition of a chemical fluorogen to photosensitize FAP transgenic animals circumvents the practical challenges inherent in generating and propagating transgenic animals expressing constitutively active photosensitizers , which must be raised and handled in the dark .",
"Precision subcellular ablation is likely to have broad applications in neuroscience beyond investigation of mitochondrial function and zebrafish models .",
"Future development of this approach will include generation of constructs that direct dL5** to other cellular components , such as nuclear subdomains , lysosomal proteins or key components of pre- or post-synaptic terminals , allowing resolution of their specific contributions to cellular physiology and pathophysiology .",
"In addition , the system is fully portable to other experimental systems , including mammalian models ."
],
[
"pT2KSAGFF ( Asakawa et al . , 2008 ) and pT2-5UASMCS ( Asakawa et al . , 2008 ) were gifts from Dr . Koichi Kawakami , National Institute of Genetics , Tokyo , Japan .",
"To generate the driver construct , a 325 bp EcoRI/BglII restriction fragment encoding Gal4FF was released from pT2KSAGFF and , after Klenow blunting the BglII overhang , ligated into the EcoRI and Klenow-blunted PacI sites of pBS-I-Sce1-GFP-eno2-5’−3’-arm ( Bai et al . , 2007 ) .",
"The resulting plasmid was linearized with HindIII , and the 12 kb eno2 regulatory sequence , encompassing exons 1 and 2 and genomic flanking region , was captured from BAC zC51M24 by gap repair recombination , as described in our prior work ( Bai et al . , 2007 ) to yield pBS-I-Sce1-eno2:Gal4FF .",
"To generate the responder construct , a 1 . 74 kb PmeI/XhoI restriction fragment was released from pcDNA3 . 1-cox4-cox8-dL5-2xG4S-mCerulean3 ( Qian et al . , 2019; Telmer et al . , 2015 ) ( Addgene 73208 ) and ligated into the XhoI and Klenow-blunted EcoR1 sites of pT2-5UASMCS to yield pTol2-5UAS:cox4-cox8-dL5-2xG4S-mCerulean3 ( 2xG4S encodes a flexible GGGGSGGGGS linker between dL5** and mCerulean3 to allow correct folding of both protein domains ) .",
"Both plasmids were verified by DNA sequencing and restriction digest .",
"Zebrafish embryos were raised in E3 buffer ( 5 mM NaCl , 0 . 17 mM KCl , 0 . 33 mM CaCl2 , 0 . 33 mM MgSO4; unless otherwise stated , all chemicals were supplied by Sigma , St . Louis , MO ) at 28 . 5°C , under cyclic illumination comprising 14 hr green light:10 hr dark .",
"Transgenic zebrafish were generated as described in our previous work , using the I-Sce1 meganuclease method ( Bai et al . , 2007 ) for Tg ( eno2:gal4ff ) and the Tol2 transposon method ( Dukes et al . , 2016 ) for Tg ( UAS:COX4-COX8-dL5**-mCer3 ) .",
"Multiple transgenic F1 founders were identified by PCR genotyping ( primer sequences: eno2-GFF-F , 5’-GTCTTCTATCGAACAAGCATGC-3’; eno2-GFF-R , 5’-CATGTCAAGGTCTTCTCGAGG-3’; mitoFAP-F , 5’-CCGTCGTTACCCAAGAACC-3’; mitoFAP-R , 5’-TCCTGAGTCACCACAGCC-3’ ) and lines were selected for analysis based on robust transgene expression and minimal variegation .",
"The lines reported here show Mendelian inheritance of single transgene insertions and have been assigned allele designations Tg ( eno2:gal4ff ) pt425 and Tg ( UAS:COX4-COX8-dL5**-mCer3 ) pt427 .",
"Analyses were carried out using F3 and later generations after backcrosses to WT zebrafish .",
"A minimum of three independent biological replicates were completed for all experiments .",
"Double transgenic NeuMitoFAP animals were identified by epifluorescence microscopy for the mCerulean3 reporter .",
"Tricaine-anesthetized larvae were positioned in 3% methylcellulose for acquisition of live epifluorescence images using an Olympus MVX-10 stereo zoom microscope and SPOT camera ( Olympus , Center Valley , PA ) .",
"Confocal images of tricaine-anesthetized larvae , mounted in low melting point agarose in contact with the coverslip glass of a MatTek dish ( MatTek Corporation , Ashland , MA ) , were acquired using an Olympus IX-81 inverted microscope and Fluoview confocal system ( Olympus ) .",
"Acridine Orange-labeled neurons were visualized using an inverted epifluorescence microscope ( Olympus IX-71 ) and counted manually or imaged by confocal microscopy as above .",
"For intravital imaging of mitochondria , zebrafish larvae were anesthetized in tricaine , exposed to far-red light if appropriate and then mounted on their sides in low melting point agarose at the bottom of a Mattek dish , so that the skin was in contact with the glass coverslip allowing visualization of the lateral line nerve .",
"Images were acquired using a Leica SP8 confocal microscope with HC PL APO 1 . 30 NA 93x glycerol-immersion objective ( Leica Microsystems , Buffalo Grove , IL ) .",
"mCer3 was excited at 405 nm and emitted light collected from 447 to 699 nm with temporal gating between 0 . 6–6 ns using a Leica Acousto-Optical Beam Splitter .",
"Images were taken with a 0 . 600 ms dwell time and 4x line averaging .",
"Images were analyzed using NIS-Elements ( Nikon Instruments , Melville , NY ) , by reducing Z-stacks to 2D images using extended depth focus , then binarizing the resulting images to show regions of CFP fluorescence , allowing automated measurements of circularity , size and number .",
"Larvae were fixed in 4% paraformaldehyde in PBS at 4°C for 4 hr . 14μm-thick cryosections were incubated with primary antibody ( chicken anti-GFP #ab13970 , 1:5000 , Abcam , Cambridge , MA; rabbit anti-TOM20 sc-11415 , 1:1000 , Santa Cruz , Dallas , TX ) at 4°C for 16 hr , washed three times in PBS and incubated in secondary antibody ( Alexa 488 goat anti-chicken A11039 , 1:10000 , and Alexa 555 goat anti-rabbit A11034 , 1:10000 , Thermo Fisher , Waltham , MA ) .",
"After three further washes , sections were incubated in DAPI ( 200 ng/mL in PBS ) and mounted in Gelmount ( Electron Microscopy Sciences , Hatfield , PA ) for confocal imaging as detailed above .",
"Colocalization analysis was carried out using Olympus FluoView software .",
"500 nM MG2I ( synthesized as described previously He et al . , 2016 ) was added to larval E3 buffer at 72hpf ( hours post fertilization ) .",
"After treatment with MG2I , NeuMitoFAP zebrafish were housed and handled under green LED safelight illumination ( LSM-G3 × 3 , SuperBrightLEDs , St . Louis , MO ) .",
"Tricaine-anesthetized zebrafish were exposed to far-red light in 35 mm Mat-Tek dishes at 5dpf .",
"A Chanzon 100W Deep Red LED array ( Amazon , Seattle , WA ) was mounted on a heatsink and fan and suspended from a custom-built light stand ( Figure 1—figure supplement 1 ) , allowing the 35 mm dish to be illuminated from below without heat transfer to the water ( Figure 3—figure supplement 1 ) .",
"The LED power circuit was controlled using a microcontroller board ( Arduino Uno , Amazon , Seattle , WA ) for precise timing of exposure and adjustable power up to 160 mW/cm2 ( Figure 1—figure supplement 1 ) .",
"The emission spectra of all light sources were verified using a spectrometer ( BLK-CXR , Stellarnet , Tampa , FL ) .",
"Locomotor function was analyzed as reported in our prior work ( Zhou et al . , 2014 ) .",
"Larvae were transferred to 96 well plates at 5dpf under green LED illumination using a large-bore Pasteur pipette with a flame-polished aperture , then acclimatized to the recording chamber for 30 min at 28 . 5°C .",
"The visual motor response was elicited using green light ( Burton et al . , 2017 ) and recorded with a USB 3 . 0 camera ( #FL3-U3-13Y3M-C , Point Gray Research , Richmond , BC , Canada ) under infrared illumination ( #BL812-880 , Spectrum Illumination , Montague , MI ) .",
"Video recordings were analyzed offline using our published open-source MATLAB applications LSRtrack and LSRanalyze ( Cario et al . , 2011 ) .",
"All data were derived from recordings with <5% total tracking errors .",
"Heart rates of tricaine-anesthetized zebrafish were counted manually by direct visualization of cardiac contractions through a dissecting microscope .",
"Larvae were anesthetized in 0 . 02% tricaine , paralyzed by exposure to 1 mg/mL α-bungarotoxin for 10 min , then secured through the notochord to a layer of silicone ( Sylguard , Corning ) in the bottom of a 35 mm culture dish , using 0 . 025 mm tungsten pins .",
"The preparation was continuously superfused with extracellular solution ( NaCl 131 mM , KCl 2 mM , KH2PO4 1 . 25 mM , NaHCO3 20 mM , MgCl2 2 mM , CaCl2 2 . 5 mM , Glucose 10 mM , bubbled with 95% oxygen/5% CO2 , pH 7 . 4 ) at rate of 2 mL/min throughout recording .",
"A flap of skin was reflected using a fine tungsten probe to expose the posterior lateral line ganglion .",
"The region was imaged during recording using an Olympus BX51WI microscope with 40x water immersion objective and infrared DIC optics , illuminated with an Olympus U-LH100 IR halogen light source with 32BP775 IR bandpass filter ( Olympus , Center Valley , PA ) , and visualized using a USB 3 . 0 video camera ( #FL3-U3-20E4M-C , Point Gray Research , Richmond , BC , Canada ) .",
"Glass microelectrodes with resistance of 6–10 MΩ were filled with intracellular solution ( KGlu 126 mM , KCl 15 mM , NaCl 10 mM , HEPES 10 mM , MgCl2 2 mM , pH 7 . 2; phosphocreatine 10 mM was added for one experimental group ) and connected to a headstage ( HS-9A × 0 . 1 U , Molecular Devices , San Jose , CA ) mounted on a motorized micromanipulator ( MP-225 , Sutter Instrument , Novato , CA ) .",
"Patch clamp recordings were made from posterior lateral line ganglion neurons in a whole-cell configuration , using a microelectrode amplifier ( Axoclamp 900A , Molecular Devices , San Jose , CA ) in current clamp mode .",
"Signals were digitized at 4 kHz sampling rate ( Digidata 1440A , Molecular Devices ) .",
"A far-red LED light source ( Chanzon 100W Deep Red LED array , Amazon , Seattle , WA ) was mounted on a heatsink under the recording chamber .",
"Baseline membrane potential was recorded for 10 min , following which the LED was activated and recording continued for a further 20–30 min .",
"Data were analyzed offline ( Clampfit 10 . 7 module of pCLAMP , Molecular Devices ) .",
"Oxygen consumption rate ( OCR ) and bath acidification rate ( BAR ) were measured using a Seahorse XF24 Extracellular Flux Analyzer ( Agilent , Santa Clara , CA ) .",
"Zebrafish larvae were anesthetized in 0 . 015% tricaine and then a small cut made in the distal caudal fin to facilitate absorption of drugs from the bath .",
"After incubation in 40 μM d-tubocurarine for 5–10 min until spontaneous and touch-evoked muscle contractions were lost , larvae were transferred into the wells of a Seahorse XF24 Islet microplate ( Agilent , Santa Clara , CA ) containing 800 μL of E3 buffer , positioned in the center of each well using an eyelash , and then secured by islet plate capture screens .",
"Four buffer-only wells on each plate served as negative controls .",
"After sensor calibration , basal OCR and BAR were quantified over 24 cycles , each consisting of 1 . 5 min mixing and 4 min measurement .",
"For measurements on dissociated cells , freshly-dissected adult NeuMitoFAP zebrafish brains were incubated with 0 . 25% trypsin/EDTA ( Thermo Fisher , Waltham , MA ) at 37°C for 20 min , which was inactivated by adding 5 volumes of 10% fetal bovine serum ( FBS ) in PBS .",
"Samples were pipetted to dissociate cells , centrifuged at 2000 rpm , washed with 2% FBS in PBS and passed through a 40 μm cell strainer ( BD Falcon , Corning , NY ) , following which cells were incubated with 100 nm MG2I in the dark for 20 min at 20°C then washed and exposed to far-red light using the same apparatus as used for zebrafish .",
"3 × 105 cells in DMEM ( # D7777 , Sigma , St , Louis , MO ) were plated into each well of a 96-well Seahorse Xfe96 FluxPak plate ( Agilent , Santa Clara , CA ) and equilibrated at 28°C for one hour prior to running the Seahorse assay .",
"Inhibitors and final concentrations were: Oligomycin ( 1 μg/mL; Sigma , #O4876 ) , FCCP ( 300 nM; Sigma , #C2920 ) , rotenone ( 1 μM; Sigma , R8875 ) , antimycin-A ( 1μM; Sigma , A8674 ) .",
"2 hr after exposure to far-red light , groups of 5 larvae were collected and homogenized in 50 µL lysis buffer ( tris 30 mM , urea 9M , CHAPS 2% w/v , pH 7 . 4 ) on ice , then centrifuged at 10 , 000 g for 15 min .",
"ATP concentration was measured in the supernatant using a bioluminescence assay ( ATP Determination Kit , Invitrogen , Carlsbad , CA ) and a luminometer ( LMax II , Molecular Devices , San Jose , CA ) , in comparison with a reference curve of known ATP concentrations .",
"Values for each sample were then normalized to protein concentration , measured using a Bradford assay ( BioRad , Hercules , CA ) and microplate reader ( SPECTRAmax PLUS384 , Molecular Devices , San Jose , CA ) in comparison with a reference curve of known bovine albumin concentrations .",
"A 100 mg/mL stock solution of AO in ethanol was stored at 4°C and diluted in E3 buffer immediately before use to a final concentration of 5 μg/mL .",
"Zebrafish larvae were incubated in the resulting solution at 28°C for 30 min in the dark , washed twice for 10 min each in E3 buffer , anesthetized in 0 . 015% tricaine , and mounted in low melting point agarose against the coverslip glass of a MatTek dish for imaging .",
"Zebrafish larvae were fixed in Karnovsky fixative at 4°C for 16 hr , the skull and surrounding tissues dissected to expose the brain , then fixed for a further 24 hr .",
"Samples were then rinsed in PBS , post-fixed in 1% osmium tetroxide with 1% potassium ferricyanide , rinsed in PBS , dehydrated through a graded series of ethanol and propylene oxide and embedded in Poly/Bed 812 ( Polysciences , Warrington , PA ) .",
"300nm-thick sections were stained with 0 . 5% Toluidine Blue in 1% sodium borate and examined under a light microscope to identify regions of interest .",
"65 nm-thick sections were stained with uranyl acetate and Reynold’s lead citrate , then imaged using a JEOL 1011 transmission electron microscope with a side mount AMT 2K digital camera ( Advanced Microscopy Techniques , Danvers , MA ) .",
"All experiments were repeated in at least three independent biological replicates ( different cohorts of larval zebrafish bred on different days ) .",
"Data from replicate experiments whose results did not differ significantly were combined for clarity of presentation , as indicted in individual figure legends .",
"Experimental groups were defined firstly by genotype , then larvae allocated randomly to receive MG2I or no chemical ."
]
] | [
"Mitochondrial dysfunction is implicated in the pathogenesis of multiple neurological diseases , but elucidation of underlying mechanisms is limited experimentally by the inability to damage specific mitochondria in defined neuronal groups .",
"We developed a precision chemoptogenetic approach to target neuronal mitochondria in the intact nervous system in vivo .",
"MG2I , a chemical fluorogen , produces singlet oxygen when bound to the fluorogen-activating protein dL5** and exposed to far-red light .",
"Transgenic zebrafish expressing dL5** within neuronal mitochondria showed dramatic MG2I- and light-dependent neurobehavioral deficits , caused by neuronal bioenergetic crisis and acute neuronal depolarization .",
"These abnormalities resulted from loss of neuronal respiration , associated with mitochondrial fragmentation , swelling and elimination of cristae .",
"Remaining cellular ultrastructure was preserved initially , but cellular pathology downstream of mitochondrial damage eventually culminated in neuronal death .",
"Our work provides powerful new chemoptogenetic tools for investigating mitochondrial homeostasis and pathophysiology and shows a direct relationship between mitochondrial function , neuronal biogenetics and whole-animal behavior ."
] | [
"Most life processes require the energy produced by small cellular compartments called mitochondria .",
"Many internal and external factors can harm these miniature powerhouses , potentially leading to cell death .",
"For instance , in patients with Parkinson’s or Alzheimer’s disease , dying neurons often show mitochondrial damage .",
"However , it is unclear exactly how injured mitochondria trigger the demise of these cells .",
"Gaining a better understanding of this process requires studying the impact of mitochondrial damage in live neurons , something that is still difficult to do .",
"As a response to this challenge , Xie , Jiao , Bai , Ilin et al . designed a new tool that can specifically injure mitochondria in the neurons of live zebrafish larvae at will , and fine-tune the amount of damage inflicted .",
"The zebrafish are genetically engineered so that the mitochondria in their neurons carry a protein which can bind to a chemical compound called MG2I .",
"When attached to each other , MG2I and the protein respond to far-red light by locally creating highly damaging chemicals .",
"This means that whenever far-red light is shone onto the larvae , mitochondria in their neurons are harmed – the brighter the light , the stronger the damage .",
"Zebrafish larvae exposed to these conditions immediately stopped swimming: mitochondria in their neurons could not produce enough energy and these cells could therefore no longer communicate properly .",
"The neurons then started to die about 24 hours after exposure to the light , suggesting that the mitochondrial damage triggered other downstream processes that culminated in cell death .",
"This new light-controlled tool could help to understand the consequences of mitochondrial damage , potentially revealing new ways to rescue impaired neurons in patients with Parkinson’s or Alzheimer’s disease .",
"In the future , the method could be adapted to work in any type of cell and deactivate other cell compartments , so that it can be used to study many types of diseases ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"physics of living systems",
"computational and systems biology"
] | Optogenetics enables real-time spatiotemporal control over spiral wave dynamics in an excitable cardiac system | elife-41076-v2 | [
[
"Self-organization of macroscopic structures through atomic , molecular or cellular interactions is characteristic of many non-equilibrium systems .",
"Such emergent dynamic ordering often reveals fundamental universalities ( Cross and Hohenberg , 1993 ) .",
"One example is the occurrence of rotating spiral waves .",
"Spiral waves are found in diverse natural systems: from active galaxies ( Schulman and Seiden , 1986 ) , to simple oscillatory chemical reactions ( Belousov , 1985; Zhabotinsky , 1991 ) , to social waves in colonies of giant honey bees ( Kastberger et al . , 2008 ) , to Min protein gradients in bacterial cell division ( Caspi and Dekker , 2016 ) , and to the formation of vortices in fluids flowing past obstacles ( Karman , 1937 ) .",
"While being beneficial to some systems , for example slime molds , where they guide morphogenesis , such activity has detrimental consequences for other systems including the heart , where they underlie lethal cardiac arrhythmias ( Davidenko et al . , 1990 ) .",
"Understanding the dynamics of spiral waves in order to establish functional control over a system , has intrigued researchers for many decades .",
"It has been reported that irrespective of the nature of the excitable medium , spiral wave activity organizes around an unexcitable center ( core ) , whose properties determine its overall dynamics ( Krinsky , 1978; Beaumont et al . , 1998 ) .",
"Theorists attribute such particle-like behavior of a spiral wave to an underlying topological charge , which controls its short-range interaction , annihilation , and the ability to form intricate bound states with other spirals ( Ermakova et al . , 1989; Schebesch and Engel , 1999; Steinbock et al . , 1992 ) .",
"Rotational activity similar to spiral waves can also occur around small structural or functional heterogeneities ( e . g . areas of conduction block ) .",
"In this case , the dynamics of the rotating wave and its spatial position are determined by the location and properties of the heterogeneity .",
"Thus , in theory , by controlling the position and size of spiral wave cores , one can precisely and directly control the dynamics of spiral waves in general .",
"In order to achieve such control , it is therefore logical , to consider as a first step , possible core-targeting via the conversion of a free spiral wave to an anchored rotational activity .",
"To this end , a detailed mechanistic study was performed by Steinbock et al . ( 1993 ) , who demonstrated the possibility to forcibly anchor meandering spiral waves in an excitable light-sensitive Belousov-Zhabotinsky ( BZ ) reaction system .",
"Furthermore , Ke et al . ( 2015 ) demonstrated in a three-dimensional BZ reaction setting , that forced anchoring of scroll waves to thin glass rods , followed by subsequent movement of the rods themselves , could enable scroll wave relocation .",
"On a broader perspective , this could have significant meaning for the heart , where controlling the dynamics of scroll waves could add to the treatment of cardiac arrhythmias sustained by such waves .",
"In cardiac tissue , the analogs of a classical spiral wave and a wave rotating around a heterogeneity , are , respectively , functional and anatomical reentry , both of which are recognized as drivers of arrhythmias .",
"Interestingly , functional and anatomical reentrant waves are closely related to each other .",
"Seminal findings by Davidenko et al . ( 1991 ) demonstrated that a drifting spiral wave could anchor to an obstacle and thereby make a transition from functional to anatomical reentry .",
"Conversely , Ripplinger et al . ( 2006 ) showed that small electric shocks could unpin a reentrant wave rotating around an obstacle , bringing about the reverse transition from anatomical to functional reentry .",
"Nakouzi et al . ( 2016 ) and Zykov et al . ( 2010 ) demonstrated that the transitions between anchored and free spiral states may be accompanied by hysteresis near the heterogeneities .",
"Furthermore , Defauw et al . ( 2014 ) showed that small-sized anatomical heterogeneities could attract spiral waves from a close distance , and even lead to their termination if located near an unexcitable boundary .",
"However , to date , all studies dedicated to spiral wave attraction and anchoring involved the presence of anatomically predefined , permanent heterogeneities , or continuous-in-time processes , thereby making it impossible to manipulate spiral wave cores in a flexible , systematic and dynamical manner .",
"In the present study , we propose a new phenomenological concept to demonstrate real-time spatiotemporal control over spiral wave dynamics through discrete , systematic , manipulation of spiral wave cores in a spatially extended biological medium , that is cardiac tissue .",
"We establish such control through optogenetics ( Boyden et al . , 2005; Bi et al . , 2006; Deisseroth , 2015; McNamara et al . , 2016 ) , which allows the creation of spatially and temporally predefined heterogeneities at superb resolution at any location within an excitable medium .",
"Previous studies for example by Arrenberg et al . ( 2010 ) ; Bruegmann et al . ( 2010 ) ; Jia et al . ( 2011 ) ; Bingen et al . ( 2014 ) ; Entcheva and Bub ( 2016 ) and Burton et al . ( 2015 ) , demonstrate the power of optogenetics in cardiac systems .",
"Thus , the same technology was chosen to strategically exploit fundamental dynamical properties of spiral waves , like attraction , anchoring and unpinning , to discretely and effectively steer spiral wave cores along any desired path within an excitable monolayer of cardiac cells .",
"These findings are highly relevant for understanding non-linear wave dynamics and pattern formation in excitable biological media , as they enable , for the first time , real-time discrete dynamic control over processes that are associated with self-sustained spiraling phenomena , for example reentrant electrical activity , cAMP cycles and movement of cytosolic free Ca2+ , to name a few .",
"In particular , in the heart , tight control of spiral waves may allow restoration of normal wave propagation ."
],
[
"Self-sustained spiral waves can be actively generated in most natural excitable media .",
"In this study , we induced spiral waves of period 60±5 ms in silico and of period 63±11 ms in vitro in confluent monolayers of optogenetically modified neonatal rat atrial cardiomyocytes ( see Materials and methods for details ) .",
"Targeted application of light to the monolayers led to the sequential occurrence of two events:",
"( i ) creation of a spatially predefined temporal heterogeneity ( i . e . a reversible conduction block ) near the core of a spiral wave , and",
"( ii ) emergence of a wave from the spot of illumination .",
"In a previous study ( Feola et al . , 2017 ) , we demonstrated the possibility to create such a temporal heterogeneity with optical control over the size , location and duration of the block .",
"Here , we show how creation of a light-induced block close to the core of a spiral wave , can attract the spiral wave tip , causing it to eventually anchor to the block .",
"Subsequent movement of the light spot to different locations within the monolayer , results in dragging of the spiral wave along a predefined pathway of illumination , thereby giving rise to , what we call Attract-Anchor-Drag-based ( AAD ) control .",
"Figure 1A illustrates a schematic diagram of the experimental setup that we used for our in vitro studies , whereas the sequence of light patterns used for real-time AAD control is described in Figure 1B .",
"Figure 1 ( C1-4 ) and 1 ( D1-4 ) show representative examples of AAD control of spiral wave dynamics in silico and in vitro , respectively , where the spiral wave core is dragged along a predefined 9-step triangular path ( see also Video 1 ) .",
"Our results demonstrate the possibility to capture a spiral wave , to manipulate its trajectory in a spatiotemporally precise manner ( Figures 1 C2–3 and D2 , and , finally , to release it in order to rotate freely again ( Figure 1 C4 , D4 ) .",
"Thus , we prove that it is indeed possible to directly and precisely manipulate the spatial location of a stable spiral wave core , and thereby overcome the constraints imposed by internal and external factors that shape the natural meander and drift trajectories .",
"Such control is very important because it provides a direct and effective handle over the spatiotemporal patterning of the system , which would affect its general functionality .",
"Natural systems currently suffer from the lack of such a handle ( Mikhailov and Showalter , 2006; Mikhailov and Loskutov , 1996 ) .",
"Spiral wave dragging seeks to fill this lacuna at the simplest level .",
"An immediate application of such a process , that follows dynamic manipulation of spiral waves , involves using this technique to terminate complex spiral wave activity .",
"We first focus on removing a single spiral wave in silico ( Figures 2 A1–5; see also Video 2 ) .",
"With a five-step drag sequence of circular light spots of 0 . 275 cm diameter and 250 ms illumination time per spot , we demonstrate the possibility to remove a spiral wave from a monolayer , by capturing its core somewhere near the middle of the simulation domain and subsequently dragging it all the way to the unexcitable boundary on the left , causing the phase singularity to collide with the border and annihilate .",
"The representative trajectory of the spiral wave , as it is dragged to termination , is shown in Figure 2 ( A2-4 ) with dashed red lines .",
"The direction of movement of the spiral is indicated in each frame with red arrows .",
"Inspired by the outcome of our in silico experiments , we tested the same principle in vitro , with a five-step drag sequence of circular light spots with similar characteristics as those applied in silico .",
"Our in vitro findings corroborate the in silico results ( Figures 2 B1–5; see also Video 2 ) .",
"The representative trajectory of this spiral wave , as it is dragged to termination , is shown in Figure 2 ( B2-4 ) with dashed white lines .",
"In each frame of Figure 2 , the location of the light spot is marked with a transparent blue circle .",
"Having proven the possibility to drag single spiral waves to unexcitable tissue borders in favor of termination , we attempt to develop an in-depth in silico qualitative understanding of the parameters that play a crucial role in the dragging process .",
"We focus on the previously described case: linear five-step dragging from the center of the simulation domain to a point on the periphery , with continuous illumination , and start by investigating the dependence of the probability of successful dragging ( Pdrag ) , on the diameter ( d ) of a continuously illuminated moving spot ( i . e . without a finite time gap between successive applications of light ) .",
"Our study reveals a positive correlation between Pdrag and d .",
"At d≲3 mm , the minimum time ( τmin ) required for relocation of a spiral wave to the next position decreases abruptly with increasing d .",
"However , at d>3 mm , τmin decreases slowly , trending towards a possible saturation value .",
"These results are illustrated in Figure 3 .",
"Traces of the spiral tip trajectory ( indicated by means of red lines on top of a representative frame depicted in Figure 3 A1–A3 ) demonstrate that the ease with which a spiral core can be dragged , from the center of the domain to a border , increases with increasing d .",
"At small d , the spiral tip is dragged along a cycloidal path , in which , the diameter of the loop is O ( d ) .",
"As d increases , the spiral tends to translate linearly , without executing rotational movement about the core .",
"To investigate the conditions that allow spiral wave dragging with discrete light pulses , we apply a series of circular light spots of duration τlight .",
"These spots appear sequentially in time , and are physically separated in space , with gradually decreasing distance from the boundary .",
"For simplicity , we apply all spots along the same line , drawn from the center of the simulation domain to the boundary .",
"We define the time gap between successive light pulses ( when the light is off everywhere ) as the dark interval τdark .",
"Thus , effectively , our optical stimulation protocol is periodic in time ( with period τlight+τdark ) , but not in space .",
"We perform two sets of studies for the case of linear dragging .",
"In the first set , we fix τlight+τdark at 200 ms and tune τdark .",
"In the second set , we fix τlight ( three different values ) and tune τdark .",
"Our results indeed demonstrate the occurrence of AAD control with discrete illumination ( τdark≠0 ms ) .",
"Figure 3D shows , for example , that at τdark=0 , 80 , 120 , and 180 ms , respectively , Pdrag decreases from 80% to 20% to 10% .",
"Interestingly , τdark=80ms appears to be as effective as continuous illumination ( τdark=0 ms , Figure 3D ) .",
"Thus , our findings confirm that , for a fixed cycle length of optical stimulation by a spot of light of diameter d=0 . 275 cm , Pdrag increases with decreasing τdark .",
"If , however , we tune to explore the dependence of Pdrag on τlight at flexible cycle lengths , there is no clear relationship between Pdrag and τlight ( Figure 3E ) , despite an increase in the ease of attraction of the spiral tip towards the illuminated spot at short τdark .",
"In general , τlight≃O ( 0 . 1τdark ) does not lead to effective AAD control , whereas , τlight≃𝒪 ( 10τdark , τdark≠0 ) does .",
"However , exceptional cases also exist .",
"For example ,",
"( i ) Pdrag can be large ( i . e . 70% ) when a short light pulse ( τlight=20 ms ) is followed by a long dark interval ( τdark=100 ms ) ; and ,",
"( ii ) combinations of abbreviated τlight and τdark ( i . e . τlight=20 ms , τdark=20 ms ) can lead to lower Pdrag than pulse combinations with shorter or longer τdark .",
"We find that probability of attraction also depends on the phase of the spiral wave rotation at the moment of light exposure .",
"This dependence , in simple terms , can be quantified as dependence on the angle between the vector along the instantaneous direction of motion of the spiral tip a→ and the vector r→ of the displacement of the center of the circular light spot , in polar coordinate system .",
"This is illustrated in Figure 3F .",
"Our results demonstrate that spiral wave dragging occurs most efficiently if the spot of light is applied sufficiently close to the location of the spiral tip and within an angular spread of ΔθX in the direction of drift of the spiral core ( Figure 3G ) .",
"Here , X denotes the cutoff probability for the occurrence of dragging , that is Δθ50% refers to an angular spread of Δθ for which Pdrag≥50% .",
"A spiral wave cannot be dragged when r > 5 . 63 mm .",
"When 4 . 7 mm < r < 5 . 63 mm , Δθ50% = 150∘ .",
"Pdrag increases to 240∘ when 3 . 44 mm < r < 4 . 7 mm , and to 360∘ when r < 3 . 44 mm .",
"Furthermore , to develop a detailed understanding of the physical mechanisms involved in spiral wave dragging , the nature of the process itself is analyzed .",
"Our in silico findings indicate that successful control necessitates the spiral wave to make alternate transitions between functional ( free ) and anatomical ( anchored ) types of reentry .",
"When a spot of light is applied reasonably close to the core of a spiral wave , within the allowed Δθ , it creates a region of light-induced depolarization that attracts and anchors the core of the free spiral , thereby effectuating a transition from functional to anatomical reentry .",
"When the light spot is now moved to a different location , still within the basin of attraction of the first light spot , the previously depolarized region recovers , forcing the anchored spiral to unpin and make a reverse transition to functional reentry .",
"The larger the value of τdark , the more visible the transition .",
"Finally , at the new location of the light spot , a zone of depolarization is created , which either attracts and anchors the spiral tip , or produces a new wave of excitation that replaces the existing spiral wave with a new one , still followed by attraction and anchoring .",
"A sequence of in silico voltage maps in support of the proposed mechanism , is shown in Figure 3H .",
"With a detailed understanding of the parameters involved in the process of spiral wave dragging , we explore the possibility to apply this phenomenon to tackle more challenging problems , such as , termination of complex patterns of reentry .",
"We specifically target two patterns:",
"( i ) figure-of-eight type reentry , and",
"( ii ) reentry characterized by multiple spiral waves , that is multiple phase singularities .",
"In silico , we find that figure-of-eight type reentry can be removed efficiently by dragging the cores of both spiral waves towards each other , till their phase singularities collide , leading to self-annihilation .",
"Figure 4 ( A1-6 ) ( see also Video 3 ) show subsequent steps in the process of removal of a figure-of-eight type reentry via AAD control , when the reentry pattern comprises two spirals of opposite chirality , rotating in phase with each other .",
"Figure 4 ( B1-6 ) ( see also Video 3 ) show successful experimental ( in vitro ) validation of our in silico findings , following the same termination protocol .",
"When the figure-of-eight type reentry comprises two spirals of opposite chirality that do not rotate exactly in phase , the strategy should be to capture the cores of the pair of spirals with light spots of unequal sizes , so as to compensate for the difference in phases , at the very first step of the control method .",
"A representative example of such a scenario is presented in the Figure 4—figure supplement",
"1 . In order to terminate reentry characterized by multiple phase singularities , we try different approaches .",
"Figure 5 ( A1-7 ) and ( B1-7 ) ( see also Video 4 ) illustrate two examples of such attempts in silico with reentry characterized by three and seven phase singularities , respectively .",
"With three spiral cores , we follow a strategy which is a combination of the cases presented in Figures 4 and 2 that is we capture all three cores ( Figure 5A2 ) , drag them toward each other ( Figure 5A3 ) , reduce complexity via annihilation of two colliding phase singularities ( Figure 5 A4–5 ) , and drag the remaining spiral core to termination via collision of its phase singularity with the unexcitable boundary ( Figures 5 A6–7 ) .",
"However , with higher complexity , there is no unique optimal approach that leads to the termination of reentry .",
"In the example presented in Figure 5 ( B1-7 ) , we rely on reducing the complexity of the reentrant pattern itself , via strategic application of two light spots , prior to the actual dragging process .",
"New waves emerging from the locations of the applied light spots interact with the existing electrical activity in the monolayer to produce new phase singularities , which collide with some of the preexisting ones to lower the complexity of the reentrant pattern .",
"With reduced complexity , we drag the spiral cores toward each other , till they merge to form a single two-armed spiral , anchored to the applied light spot .",
"We then drag this spiral to the boundary of the monolayer causing its termination .",
"A similar strategy applied in vitro enabled successful termination of a complex reentry pattern characterized by four phase singularities .",
"This is illustrated in Figure 5 ( C1-7 ) .",
"At the instant when the first set of light spots is applied , reentry is characterized by four phase singularities ( Figure 5C1 ) .",
"Note that , of the four phase singularities , two coincide with the locations of two of the applied light spots and two are located away from the third light spot ( Figure 5C2 ) .",
"These first two phase singularities anchor immediately to the applied light spots .",
"However , the other two interact with the new wave generated by the third applied light spot and reduce to one phase singularity , which remains free ( unanchored ) ( Figure 5C3 ) .",
"When the next set of light spots are applied ( in a complex pattern of three overlapping circles ) , this phase singularity displays attraction to the new light spot , thus effectively producing a three-armed spiral ( Figure 5C4 ) .",
"Finally , when the complex light pattern is replaced by a smaller circular spot of light , the three-armed spiral loses two of its arms through interaction with the emergent new wave from the location of the applied light spot ( Figure 5C5 ) .",
"The single spiral can then be dragged to termination ( Figures 5 C6–7 ) as in Figure",
"2 . Taken together , our results show that core positions of spiral waves can be strategically manipulated with the help of optogenetics to establish direct spatiotemporal control over a highly nonlinear system .",
"We demonstrate that application of well-timed and spaced light pulses can result in successful dragging of a spiral wave from one location to another within a monolayer , along any desired path and even to cause its termination .",
"Spiral wave dragging can also be employed to terminate complex reentrant patterns characterized by multiple phase singularities , through appropriate AAD control .",
"However , the probability of its occurrence is subject to two key parameters:",
"( i ) size d and",
"( ii ) drag angle ( Δθ ) ."
],
[
"Since we demonstrate AAD control method in a cardiac tissue system , a logical question would be , how to envision the application of this principle to the real heart in order to treat arrhythmias ?",
"Currently this topic faces major challenges .",
"The practical application of optogenetics in cardiology is , in itself , a debatable issue .",
"However , with recent advances in cardiac optogenetics ( Nussinovitch and Gepstein , 2015; Crocini et al . , 2016; Nyns et al . , 2017; Bruegmann et al . , 2018; Boyle et al . , 2018 ) , the future holds much promise .",
"Firstly , we envision the usage of AAD control method in treating arrhythmias that are associated with scroll waves .",
"Since the penetration depth of light in cardiac tissue is relatively short ( Bruegmann et al . , 2016 ) , full transmural illumination might be challenging , particularly in ventricles of large mammals like pig , monkey or human .",
"There , AAD control may provide a powerful tool to regulate scroll wave dynamics by epicardial or endocardial illumination .",
"Furthermore , we expect the method to prove most useful when dealing with ‘hidden’ spiral waves , that is spiral waves in remote locations of the heart that are unaccessible by ablation catheters .",
"Ideally , one should build upon the concept introduced by Entcheva and Bub ( 2016 ) .",
"With live spacetime optogenetic actuation of the electrical activity in different parts of the heart , the first step is to detect the location of the instability .",
"Next , one can use a catheter with an in-built LED to attract the scroll wave filament and steer it towards the nearest tissue border for termination .",
"The advantage of this method lies in that one does not require to ablate , and thereby destroy , excitable cardiac tissue , thus avoiding the possibility to create permanent damage to the heart .",
"In addition , as our study demonstrates , anchoring of the spiral core ( scroll filament ) can occur even if the ‘precise’ location of the core is not identified .",
"Lastly , the discrete nature of our method allows temporal flexibility in steering the spiral core ( scroll filament ) , in that , temporary loss of communication between the catheter and the spiral core will not lead to failure of the technique in general .",
"The reversible nature of the AAD control technique makes it unsuitable for terminating arrhythmias that rely on the establishment of a permanent conduction block as can be produced via conventional catheter ablation .",
"However , this special feature of AAD may come with certain unique advantages that should be explored in more detail in future studies .",
"For example , in younger patients that are expected to undergo periodic repetitive ablation for termination of reoccurring arrhythmias of unknown origin , the non-destructive nature of AAD may prove to be more desirable than the cumulative widespread destruction of cardiac tissue by radiofrequency or cryoballoon ablation .",
"Alternatively , other methods could be developed and explored for AAD control without the need of optogenetic modification , while still relying on the creation of spatiotemporally controlled heterogeneities for attraction , anchoring and dragging of spiral waves .",
"In this study , we focus on spiral waves in cardiac excitable media , as these abnormal waves have been associated with lethal heart rhythm disturbances , while their management and termination remain a serious challenge .",
"The insights gained from our results , as well as the AAD control method itself , may not only improve our understanding of spiral wave’s dynamics in favor of restoring normal cardiac rhythm , but also create incentive to explore these principles in other excitable media prone to spiral wave development ."
],
[
"All animal experiments were reviewed and approved by the Animal Experiments Committee of the Leiden University Medical Center and conformed to the Guide for the Care and Use of Laboratory Animals as stated by the US National Institutes of Health .",
"Monolayers of neonatal rat atrial cardiomyocytes expressing Ca2+-translocating channelrhodopsin ( CatCh ) were established as previously described ( Feola et al . , 2017 ) .",
"Briefly , the hearts were excised from anesthetized 2-day-old Wistar rats .",
"The atria were cut into small pieces and dissociated in a solution containing collagenase type I ( Worthington , Lakewood , NJ ) and 18 , 75 Kunitz/ml DNase I ( Sigma-Aldrich , St . Louis , MO ) .",
"The resulting cell suspension was enriched for cardiomyocytes by preplating for 120 min in a humidified incubator at 37∘C and 5% CO2 using Primaria culture dishes ( Becton Dickinson , Breda , the Netherlands ) .",
"Finally , the cells were seeded on round glass coverslips ( d=15 mm; Gerhard Menzel , Braunschweig , Germany ) coated with fibronectin ( 100 μg/ml; Sigma-Aldrich ) to establish confluent monolayers .",
"After incubation overnight in an atmosphere of humidified 95% air- 5% CO2 at 37∘C , these monolayers were treated with Mitomycin-C ( 10 μg/ml; Sigma-Aldrich ) for 2 hr to minimize proliferation of the non-cardiomyocytes .",
"At day 4 of culture , the neonatal rat atrial cardiomyocyte monolayers were incubated for 20 – 24 hr with CatCh-encoding lentiviral particles at a dose resulting in homogeneous transduction of nearly 100% of the cells .",
"Next , the cultures were washed once with phosphate-buffer saline , given fresh culture medium and kept under culture conditions for 3 – 4 additional days .",
"Optical voltage mapping was used to investigate optogenetic manipulation of spiral wave dynamics in the CatCh-expressing monolayers on day 7 of culture by using the voltage-sensitive dye di-4-ANBDQBS ( 52 . 5 μM final concentration; ITK diagnostics , Uithoorn , the Netherlands ) , as described previously ( Feola et al . , 2017 ) .",
"In summary , optical data were acquired using a MiCAM ULTIMA-L imaging system ( SciMedia , Costa Mesa , CA ) and analyzed with BrainVision Analyzer 1101 software ( Brain vision , Tokyo , Japan ) .",
"Only monolayers characterized by uniform AP propagation at 1 Hz pacing and homogeneous transgene expression were included for the following optogenetic investigation .",
"CatCh was locally activated by using a patterned illumination device ( Polygon400; Mightex Systems , Toronto , ON ) connected to a 470-nm , high-power collimator light-emitting diode ( LED ) source ( type-H , also from Mightex Systems ) .",
"PolyLite software ( Mightex Systems ) was used to control the location and movement of the areas of illumination .",
"Before local optogenetic manipulation , reentry was induced by light-based stimulations ( n = 18 ) .",
"Reentrant waves that were stable for >1 s were exposed to a circular light spot ( d=3 mm ) at different targeted locations within the monolayer for 200−250 ms at 0 . 3 mW/ mm2 .",
"Optical voltage signal was processed with spatial and temporal derivative filters to allow visualization of electrical wave propagation during the application of the light spots .",
"The data were sampled according to the location of the applied light spot .",
"Next , for each sample set , we obtained the lowest background intensity recorded by the individual pixels over time and constructed a two-dimensional array with these values as elements .",
"Next , we subtracted this array from each frame in the sample set and reassembled the processed datasets to make a complete video ."
]
] | [
"Propagation of non-linear waves is key to the functioning of diverse biological systems .",
"Such waves can organize into spirals , rotating around a core , whose properties determine the overall wave dynamics .",
"Theoretically , manipulation of a spiral wave core should lead to full spatiotemporal control over its dynamics .",
"However , this theory lacks supportive evidence ( even at a conceptual level ) , making it thus a long-standing hypothesis .",
"Here , we propose a new phenomenological concept that involves artificially dragging spiral waves by their cores , to prove the aforementioned hypothesis in silico , with subsequent in vitro validation in optogenetically modified monolayers of rat atrial cardiomyocytes .",
"We thereby connect previously established , but unrelated concepts of spiral wave attraction , anchoring and unpinning to demonstrate that core manipulation , through controlled displacement of heterogeneities in excitable media , allows forced movement of spiral waves along pre-defined trajectories .",
"Consequently , we impose real-time spatiotemporal control over spiral wave dynamics in a biological system ."
] | [
"From a spinning galaxy to a swarm of honeybees , rotating spirals are widespread in nature .",
"Even within the muscles of the heart , waves of electrical activity sometimes rotate spirally , leading to irregular heart rhythms or arrhythmia – a condition that can be fatal .",
"Irrespective of where they occur , spiral waves organize around a center or core with different biophysical properties compared to the rest of the medium .",
"The properties of the core determine the overall dynamics of the spiral .",
"This means that , theoretically , it should be possibly to completely control a spiral wave just by manipulating its core .",
"Now , Majumder , Feola et al . have tested this long-standing hypothesis using a combination of computer modeling and experiments with single layers of rat heart cells grown in a laboratory .",
"First , the heart cells were genetically modified so that their electrical properties could be altered with light; in other words , the cells were put under optical control .",
"Next , by using of a narrow beam of light , Majumder , Feola et al . precisely controlled the electrical properties of a small number of cells , which then attracted and supported a rotating spiral wave by acting as its new core .",
"Moving the light beam allowed the core of the spiral wave to be shifted too , meaning the spiral wave could now be steered along any desired path in the cell layer .",
"Majumder , Feola et al . hope that these underlying principles may one day provide the basis of new treatments for irregular heartbeats that are more effective and less damaging to the heart than existing options .",
"Yet first , more work is needed to translate these findings from single layers of cells to actual hearts ."
] | 2018 |
[
"Introduction",
"Results and discussion",
"Materials and methods"
] | [
"medicine"
] | Metabolic response of blood vessels to TNFα | elife-54754-v2 | [
[
"Tumor necrosis factor-α ( TNFα ) is a central mediator of the inflammatory response ( Sedger and McDermott , 2014 ) .",
"TNFα can be generated by monocytes or macrophages and activates endothelial cells at sites of tissue injury or infection through TNF receptor-1 ( TNFR1 ) ( Chimen et al . , 2017; Torres-Castro et al . , 2016; Green et al . , 2016 ) .",
"Following activation , the endothelium elicits a multitude of local responses such as vascular leakage , leukocyte adhesion and coagulation that together are essential to the physiological homeostatic responses to anti-microbial immunity .",
"However , chronic exposure to adverse metabolic and hemostatic risk factors ( Masi et al . , 2018 ) , obesity ( Engin , 2017 ) , or disease states such as kidney disease ( Rabelink et al . , 2010 ) or rheumatoid arthritis ( van Zonneveld et al . , 2010 ) are all associated with a systemic inflammatory condition and elevated circulating levels of TNFα .",
"As a consequence , TNFα signaling induces the generation of high levels of free radicals in the vascular endothelium that , when excessive , can deplete the cellular anti-oxidant defense systems and lead to a state of oxidative stress and vascular dysfunction ( Pisoschi and Pop , 2015 ) .",
"Mechanistically , TNFα signaling in endothelial cells involves the activation of NFκB and results in the increased synthesis of reactive oxygen species ( ROS ) from a number of sources such as mitochondria , NADPH oxidase , uncoupled eNOS , xanthine oxidase , and peroxidases ( Cai and Harrison , 2000; Blaser et al . , 2016 ) .",
"On its turn , elevated ROS can lead to the generation of bioactive lipids directly or indirectly , such as prostaglandins , isoprostanes , lysophosphatidic acid classes , sphingolipids and platelet activating factor ( PAF ) .",
"Under physiologic conditions , in concert with the transcriptional regulation of a plethora of inflammatory genes ( Poussin et al . , 2020 ) , these bioactive lipids are critically involved in the first response of endothelial cells to environmental changes , controlling vascular permeability and platelet- and leukocyte adhesion .",
"Also , in fore mentioned patients , isoprostanoids such as 8-epiPGF2α are generated by peroxidation during conditions of oxidative stress ( Morrow et al . , 1990 ) and serve as gold standard oxidative stress plasma markers ( Ridker , 2004 ) associating with an increased risk for cardiovascular disease and its underlying causes ( Moutzouri et al . , 2013; Vassalle et al . , 2004 ) .",
"The central role of TNFα in many disease states has identified this cytokine as an important therapeutic target to counteract vascular inflammation ( Sedger and McDermott , 2014; Esposito and Cuzzocrea , 2009 ) and several TNFα blockers have been approved by the FDA and have been effective for the suppression of immune-system diseases , such as Crohn’s disease , ulcerative colitis , rheumatoid arthritis , ankylosing spondylitis , psoriatic arthritis and plaque psoriasis ( Ackerman et al . , 2016 ) .",
"Following the success of the TNFα antagonists , a deeper understanding of the impact of oxidative stress on the homeostasis of the human vascular endothelium may yield more specific targets for inflammatory diseases affecting the vasculature .",
"To that end , many in vitro mechanistic studies have relied on static , two-dimensional ( 2D ) cultures of primary endothelial cells such as those derived from human umbilical veins ( HUVECs ) ( Jaffe et al . , 1973 ) .",
"However , in recent years it has become increasingly apparent that these cultures reflect a ‘stressed’ endothelial phenotype due to the lack of their native environmental cues .",
"In vitro , endothelial cells are usually cultured on surfaces such as plastics and glass that are much stiffer than natural substrates such as the extracellular matrix .",
"Recent studies demonstrated that primary endothelial cells on a hard substrate adopt a pro-inflammatory phenotype ( Stroka and Aranda-Espinoza , 2011; Huveneers et al . , 2015 ) .",
"Vascular stiffness is strongly associated with vascular disorders such as arterial hypertension , kidney disease and atherosclerosis ( Huveneers et al . , 2015 ) .",
"Likewise , native microvessels need laminar shear to maintain a quiescent phenotype and the lack of laminar shear stress in static cultures converts endothelial cells to a pro-inflammatory ‘diseased’ phenotype ( Baeyens et al . , 2016 ) .",
"Novel microfluidics-based perfused three-dimensional ( 3D ) microvessels-on-a-chip models provide a unique opportunity to generate endothelial microvessels in a more physiological environment .",
"We employed a gelatin coated 3D microvessels-on-a-chip model in which endothelial cells are organized in a tube-like architecture , with densities of cells and area to volume ratios that are closer to a physiological condition than those in typical 2D culture .",
"To assess whether these 3D microvessels display a more anti-inflammatory phenotype , we used an optimized targeted liquid chromatography–tandem mass spectrometry measurements of a panel of pro- and anti-inflammatory bioactive lipids and generated expression profiles both in TNFα treated microvessels under flow as well as in 2D endothelial cell cultures under static condition .",
"We demonstrate bioactive lipid profiles can be readily detected from single microvessels and display a more dynamic , less inflammatory response to TNFα , that resembles more the human situation , compared to classical 2D endothelial cell cultures ."
],
[
"In this paper , we present a novel set-up to measure the metabolic response of 3D endothelial microvessels to TNFα , including pro- and anti-inflammatory markers .",
"We cultured 96 perfused microvessels against extracellular matrix ( ECM ) using the microvessels-on-a-chip platform technology recently developed by using the OrganoPlate platform of MIMETAS ( Wevers et al . , 2018; van Duinen et al . , 2017 ) .",
"The microchannels in the OrganoPlate were coated with gelatin , preventing endothelial cells from growing on glass and enabling them to form stable microvesseels .",
"The shear stress in the microvessels , calculated based on a previous work , ranges from 1 to 5 dyne/cm2 ( van Duinen et al . , 2017 ) .",
"In vivo , the shear stress ranges from 95 . 5 dyne/cm2 at the smallest capillaries to 2 . 8 dyne/cm2 at the postcapillary venules ( Koutsiaris et al . , 2007 ) .",
"Conditioned medium perfused through TNFα treated and control ( untreated ) microvessels was sampled , pooled and measured with a UPLC-MS/MS metabolomics method developed recently by us to study inflammation and oxidative stress ( Figure 1; Schoeman et al . , 2018 ) .",
"As a metabolic read-out for TNFα signaling , we measured prostaglandins , isoprostanes , LPAs , lysosphingolipids and PAF and first assessed the concentrations of these metabolites in the basic EGM2 medium and compared their concentrations after 18 hr incubation to condition the medium in the microvessels-on-a-chip cultures .",
"As shown in Table 1 , the peaks detected demonstrated that , while members of the prostaglandins and isoprostanes clearly increased after conditioning of the medium , most of the LPA metabolites were readily detectable in the medium and actually displayed a significant decreased concentration .",
"When the EGM2 medium was incubated for 18 hr without exposure to the microvessels no significant changes in the concentrations of these metabolites was observed .",
"Therefore , we conclude that during the conditioning of the medium , prostaglandins and isoprostanoids are excreted from the endothelial cells and that the LPA metabolites are consumed or actively degraded by the cells .",
"When we assessed the biolipid composition of the conditioned medium of TNFα stimulated microvessels , 33 measured metabolites passed the quality control ( QC ) thresholds .",
"Figure 2 shows examples of the chromatograms of prostaglandin E2 and different isoprostanes isomers that were secreted by the microvessels after exposure to TNFα for 18 hr compared to the control samples showing a marked increase in abundance of a number of these metabolites .",
"The control samples are microvessels without exposure to TNFα .",
"Bar plots of the absolute concentrations of prostaglandins are presented in Figure 3a , showing a significant difference between untreated and TNFα treated microvessels .",
"While at physiological concentration of TNFα ( 0 . 4 ng/ml ) there was an increase in the excretion of PGF1α , PGF2α , PGF3α , PGE2 , PGD2 and 13 , 14-dihydro-PGF2α , however , no significant difference between the untreated and TNFα treated microvessels was evident , except for PGF1α .",
"At 15 ng/ml a stronger differential response was observed for selected isoprostanes , several LPAs , sphingolipids and PAF ( Figure 3b–e ) .",
"The relative concentrations of the bioactive lipids found in the microvessels-on-a-chip are strikingly similar with those found in normal human blood vessels ( Table 2 ) .",
"Phorbol 12-myristate 13-acetate ( PMA ) is an activator of protein kinase C ( PKC ) , hence of NFκB , that relates to TNFα signaling and causes a wide range of effects in cells .",
"As it is a known and potent up-regulator of cyclooxygenase-2 ( COX-2 ) ( Chang et al . , 2005 ) , we next measured the effect of PMA on the secretion of the biolipids to compare the TNFα response to a condition of maximal stimulation .",
"When our combined results were plotted in a heatmap , marked differences are observed between control- and TNFα or PMA-treated microvessels ( Table 3 ) .",
"As shown in Table 3 , overnight exposure to TNFα and PMA shows increase in the release of the prostaglandins PGF1α , PGF2α , PGF3α , PGE2 , PGD2 and 13 , 14-dihydro-PGF2α from the TNFα treated microvessels ( Figure 3a and Table 3 ) .",
"During inflammation , ROS contributes to the increased PGE2 , PGF2α , PGD2 and 13 , 14-dihydro-PGF2α production through the release of arachidonic acid and COX-2 activation , having a pro-inflammatory effect in the endothelium ( Wong and Vanhoutte , 2010; Dworski et al . , 2001 ) .",
"At the same time , anti-inflammatory prostaglandins PGF1α , PGF3α , PGE1 , and PGA2 are also secreted by the endothelium ( Trebatická et al . , 2017; Gezginci-Oktayoglu et al . , 2016 ) .",
"PGE1 and PGA2 are known to suppress TNFα induced NFκB activation and production of ROS ( Ohmura et al . , 2017 ) .",
"Relating to these two prostaglandins , no differences were detected in our system between untreated and TNFα treated microvessels for 18 hr .",
"When we focus on the compounds produced by the reaction of free radicals with arachidonic acid , the isoprostanes , high levels of 8-iso-13 , 14-dihydro-PGF2α and 8-iso-PGF2α were detected in the supernatant of TNFα treated microvessels ( Figure 3b and Table 3 ) .",
"These metabolites inhibit platelet aggregation and induce monocyte adhesion to endothelial cells ( Rokach et al . , 1997; Duracková , 2010 ) .",
"We also detected 8-iso-PGE2 , 5-iPF2α , 8 , 12-iPF2α IV and 8-iso-PGE1 in the control sample and TNFα induced microvessels .",
"8-iso-PGE1 is recognized as vasoconstrictor with a similar effect as PGF2α ( Nakano and Kessinger , 1970 ) .",
"However , no significant difference between the two groups was evident after incubating the microvessels for 18 hr with TNFα .",
"Looking at lipids that mediate diverse biological actions , the LPA classes , sphingosine and PAF are appropriate markers to take along in our metabolic read-out , because of their diverse biological actions .",
"The LPA classes consist of LPAs and cyclic-lysophosphatidic acids ( cLPAs ) .",
"They are formed by activated platelets and oxidation of low-density lipoproteins ( LDLs ) ( Karshovska et al . , 2018 ) .",
"Once an inflammatory response is triggered , LPAs can activate platelets ( Khandoga et al . , 2008 ) and lead to endothelial dysfunction by activating NFκB ( Biermann et al . , 2012; Yang et al . , 2016; Ninou et al . , 2018; Palmetshofer et al . , 1999 ) .",
"On the other hand , cLPAs inhibit pro-inflammatory cytokine expression in the endothelium ( Tsukahara et al . , 2014 ) .",
"In our data , we saw high concentrations of several LPAs in the control sample compared to TNFα treated microvessels .",
"Similar results were seen in the levels of sphingosine-1-phosphate ( S-1-P ) and PAF ( Table 3 and Figure 3c–e ) .",
"In TNFα signaling , S-1-P binds to TNF receptor-associated factor 2 ( TRAF2 ) to activate NFκB , while PAF induces vascular permeability ( Alvarez et al . , 2010; Palur Ramakrishnan et al . , 2017 ) .",
"To assess whether these three-dimensional microvessels display a more anti-inflammatory phenotype , we compared the bioactive lipid response of the 3D microvessels-on-a-chip to TNFα to that of 2D endothelial cell monolayers .",
"In addition , we made a detailed inventory of the reported action of the individual lipids on inflammation , platelet activation , vascular tone and angiogenesis ( for references see Supplementary file 1 ) .",
"When the TNFα-induced biolipids profiles are listed in relation to their biological activities ( Table 4 ) , we conclude that the 3D microvessels-on-a-chip display a more dynamic , less inflammatory response to TNFα , that resembles more the human situation , compared to classical 2D endothelial cell cultures .",
"In particular , the anti-inflammatory prostaglandins PGF1α , PGF3α , and PGD2 are increased to a larger extent and the anti-inflammatory lysophosphatidic acids are maintained or decreased to a lesser extent .",
"In concert , the pro-inflammatory lipids PGE2 , 8-iso-PGE2 and 8-iso-PGF2α are present at higher levels in the medium of the TNFα exposed 2D endothelial monolayer culture .",
"The elevated levels of the oxidative stress markers PGE2 , 8-iso-PGF2α ( Ridker , 2004 ) , LPA C22:5 and LPA C22:6 ( Ackerman et al . , 2016 ) confirm the increased inflammatory status of the 2D cultures making it tempting to speculate that an increased production of ROS in these cells may underlie these responses .",
"The response of the microvasculature to inflammatory cytokines such as TNFα is often directly associated with inhibition of platelet activation ( to maintain patency of the microvessel ) , a vasoconstrictive response and a pro-angiogenic response characterized by the loss of endothelial cell-cell contacts and microvascular leakage .",
"Many of these activities are also driven by the bioactive lipids in our panel ( Supplementary file 1 ) , and it is interesting to note that concomitantly to the less inflammatory nature of the profiles of the conditioned media derived from the 3D microvessel the profile also suggests to be more restrictive of platelet activation , less vasoconstrictive and less angiogenic .",
"It should be noted that we did not compare the excretion of bioactive of unstimulated 2D cell cultures and 3D vessels as the normalization on the number of endothelial cells would not have been straightforward .",
"The observed differences between the 2D and the 3D chip-based platforms may be attributed to the mechanical properties of the two systems ( Lee et al . , 2009 ) .",
"The microvessels-on-a-chip are surrounded by an ECM layer and the 3D configuration allows intensified cell-cell interactions , resembling the in vivo situation .",
"Moreover , vascular endothelial cells in vivo are influenced by distinct hemodynamic forces and this applies also to the endothelial cells in our microvessels-on-a-chip .",
"Evidence suggest that shear stress activates phospholipids turnover that is involved in the production of free arachidonic acid ( Bhagyalakshmi et al . , 1992 ) .",
"This might also explain the differences we see between the increase/decrease of fatty acids in the microvessels-on-a-chip and the 2D cell culture .",
"As shear stress influences RhoA activity and stress fiber formation , the regulation of fatty acids , RhoA might be important in this process ( Liu et al . , 2014 ) .",
"In addition , the environmental changes in the 3D configuration could impact on the expression of the TNF receptors .",
"Several reports showed that oxidative stress induces endothelial dysfunction , which plays a central role in vascular diseases .",
"It can promote the expression of pro-inflammatory and pro-coagulant factors , apoptosis and impair the release of nitric oxide ( Schulz et al . , 2011; Shiraki et al . , 2012 ) .",
"This study set out with the aim of using metabolomics as a readout of endothelial function in microvessels-on-a-chip exposed to TNFα , to trigger inflammatory responses seen in vasculopathy .",
"For the first time we show that the regulation of prostaglandins , isoprostanes , LPAs , sphingolipids and PAF can be measured in our microfluidic system , even though they cause profound physiological effects at very dilute concentrations that serve as early-stage markers of oxidative stress and inflammation ( Ryu et al . , 2015 ) .",
"The findings support the model that TNFα signaling induces ROS production that causes changes in signal transduction and gene expression , which leads to release of oxidative stress and inflammatory markers ( Figure 4 ) .",
"Further research should be undertaken to confirm the results in gene and protein levels .",
"We demonstrate bioactive lipid profiles can be readily detected from minor volumes of <1 µl of conditioned medium from microvessels-on-a-chip and display a more dynamic , less inflammatory response to TNFα compared to classical two-dimensional endothelial cell cultures .",
"We can conclude that the response to TNFα resembles for the microvessels-on-a-chip more the human situation as described in the literature than the 2D endothelial cell culture .",
"As the physiological readout of endothelial function is a critical aspect in using microvessels-on-a-chip for disease and drug research , the results suggest that the metabolic readout using metabolomics is more informative compared to morphological changes studied with imaging analyses of phenotypic changes .",
"But it is the combination of both techniques , metabolic readout using metabolomics and imaging analysis that may facilitate mechanistic studies and the detection and validation of biomarkers for microvascular disease at the systemic level .",
"Furthermore , it will provide the information needed to understand microvascular destabilization and will generate a knowledge base for developing and testing personalized therapeutic interventions ."
],
[
"Human umbilical vein endothelial cells ( HUVECs ) were isolated from umbilical cord of newborns , collected with informed consent , by an adaption of the method developed by Jaffe et al . , 1973 .",
"Although denoted as veins , umbilical veins carry oxygenated blood and thus the phenotype of their endothelium is similar to arterial endothelial cells .",
"The umbilical cord was severed from the placenta soon after birth and placed in a sterile container filled with phosphate-buffered saline ( PBS; Fresenius Kabi , The Netherlands ) and held at 4°C until processing .",
"The cord was inspected and at both ends a piece of 1 cm was cut off to remove damaged tissue from clamping .",
"Subsequently , the umbilical vein was cannulated and perfused with PBS to wash out the blood and allowed to drain .",
"When clear fluid flow was observed , the vein was filled with trypsin/EDTA solution ( CC-5012 , Lonza , USA ) , placed in the container filled with PBS and incubated at 37°C for 20 min .",
"After incubation , the trypsin-EDTA solution containing the endothelial cells was flushed from the cord with air and afterwards PBS .",
"The effluent was collected in a sterile 50 ml tube containing 20 ml Endothelial Cell Growth Medium 2 ( EGM2; C-39216 , PromoCell , Germany ) supplemented with antibiotics and the cell suspension was centrifuged at 1200 rpm for 7 min .",
"The cell pellet was resuspended in 10 ml EGM2 and cultured on 1% gelatin-coated T75 flasks .",
"Cells were maintained in a 37°C incubator with 5% CO2 and the medium was refreshed every other day .",
"After 80% confluency , cells were spilt at 1:3 ratio and cultured in new 1% gelatin-coated T75 flasks .",
"The isolated cells were positive for the endothelial cell markers , including platelet endothelial cell adhesion molecule ( PECAM-1 ) and von Willebrand factor ( vWF ) ( Figure 5 ) .",
"All experiments using HUVECs were repeated six times using cells from three different male donors at passage 3 .",
"For 2D experiments , we cultured 50 ·103 cells/ml in 24-well plates overnight at 37°C in humidified air containing 5% CO2 .",
"The following day , the cells were incubated with 15 and 50 ng/ml TNFα ( H8916 , Sigma-Aldrich , The Netherlands ) and 20 ng/ml phorbol 12-myristate 13-acetate ( PMA; P8139 , Sigma-Aldrich , The Netherlands ) for 18 hr .",
"The medium was collected and stored in −80°C .",
"We used the OrganoPlate ( 9603-400-B , MIMETAS , The Netherlands ) for all microfluidic cell culture experiments .",
"The microvascular and extracellular matrix ( ECM ) channels were separated by phaseguides ( Vulto et al . , 2011 ) .",
"Before seeding the cells , 4 mg/ml rat tail collagen type 1 ( 3440-005-01 , Trevigen , USA ) neutralized with 10% 37 g/L Na2CO3 ( S5761 , Sigma-Aldrich , The Netherlands ) and 10% 1 M HEPES buffer ( 15630-056 , Gibco , The Netherlands ) was added in the ECM channels .",
"Subsequently , the collagen was let to polymerize by incubating the device for 10 min in the incubator at 37°C and 5% CO2 .",
"The observation windows were filled with 50 µl Hank’s Balanced Salt Solution with calcium and magnesium buffers ( HBSS+; 24020117 , Life Technologies , The Netherlands ) for optical clarity and to prevent gel dehydration .",
"Using a repeater pipette , 2 µl of 1% gelatin was added into the inlet of each microvascular channel and the device was put in the incubator at 37°C for 30 min .",
"We trypsinized cells at 80-90% confluency and seeded 15·106 cells/ml in the outlet of the microvascular channels of the OrganoPlate .",
"Afterwards , the cells were incubated at 37°C and 5% CO2 for one hour to allow microvascular formation .",
"After incubation , 50 μl of culture medium was added to the inlets and outlets of the microvascular channels .",
"The device was placed on a rocker platform with a 7° angle of motion and an eight-minute timed operation to allow continuous flow of minor volumes of medium in the microvessels .",
"The microvascular channels typically contain volumes of <1 µl .",
"After 24 hours , the medium was refreshed , and the HUVECs were cultured for an additional 3-4 days .",
"The microvessels were treated with TNFα ( 0 . 4 , 15 and 50 ng/ml ) and PMA ( 20 ng/ml ) for 18 hours .",
"Subsequently , medium of four microvessels were pooled to form one sample to allow analyses of metabolites at low concentrations due to low cell numbers .",
"This still allowed us to create three biological replicates with 4 – 6 technical replicates data per experimental condition for metabolomics analyses .",
"The samples were stored in -80°C .",
"For immunofluorescence staining , HUVECs were fixed using 4% paraformaldehyde ( PFA ) in HBSS+ for 10 min at room temperature .",
"The fixative was aspirated , and the cells were rinsed once with HBSS+ .",
"Next , the cells were permeabilized for 2 min with 0 . 2% Triton X-100 in HBSS+ and washed once with HBSS+ .",
"The cells were blocked in 5% BSA in HBSS+ for 30 min and incubated with the primary antibody solution overnight at 4°C .",
"Mouse anti-human CD144 ( 1:150; 555661 , BD Biosciences , USA ) , sheep anti-human CD31 ( 1:150; AF806 , R and D Systems , The Netherlands ) and rabbit anti-human vWF ( 1:1000; A0082 , Agilent Dako , USA ) were used as the primary antibodies .",
"The cells were washed with HBSS+ , followed by an one-hour incubation with Hoechst ( 1:2000; H3569 , Invitrogen , USA ) , rhodamine phalloidin ( 1:200; P1951 , Sigma-Aldrich , The Netherlands ) and the secondary antibody solution , containing Alexa Fluor 488-conjugated goat anti-mouse ( 1:250; R37120 , ThermoFisher , USA ) , Alexa Fluor 488-conjugated donkey anti-sheep ( 1:250; A11015 , ThermoFisher , USA ) and Alexa Fluor 647-conjugated goat anti-rabbit ( 1:250; A27040 , ThermoFisher , USA ) antibodies .",
"The cells were washed three times with HBSS+ .",
"High-quality Z-stack images of the stained cells were acquired using a high-content confocal microscope ( Molecular Devices , ImageXpress Micro Confocal ) .",
"All samples were measured using an oxidative and nitrosative stress profiling platform which has been developed and validated in our lab ( Schoeman et al . , 2018 ) .",
"This platform covers various isoprostane classes , signaling lipids from the sphingosine and sphinganine classes and their phosphorylated forms , as well as three classes of lysophosphatidic acids: lysophosphatidic acids , alkyl-lysophosphatidic acids and cyclic-lysophosphatidic acids ( all ranging from C14 to C22 chain length species ) .",
"For metabolite extraction , sample preparation procedure was according to the in-house experimental protocol which has been standardized and published; extra samples were pooled for internal quality control ( QC ) ( Schoeman et al . , 2018 ) .",
"Briefly , cell media ( 150 μl ) were thawed on ice and added with 5 μl antioxidant solution and 10 μl internal standards ( ISTDs ) .",
"Acidified with citric acid/phosphate buffer ( pH 4 . 5 ) , all samples were then dealt with liquid-liquid extraction ( LLE ) with 1 mL of butanol and ethyl acetate ( 1:1 v/v ) .",
"Samples were vortexed and centrifuged and then the organic phase was collected and dried .",
"After reconstitution with ice-cold 70% MeOH injection solution , each sample was again vortexed and centrifuged and the supernatant was transferred to the insert in a glass vial .",
"Ultra-performance liquid chromatography tandem mass spectrometry ( UPLC-MS/MS ) based analysis was then applied for low-pH measurement ( Shimadzu LCMS-8060 , Japan ) and high-pH measurement ( Shimadzu LCMS-8050 , Japan ) respectively .",
"Standard stock solutions were prepared in MeOH containing butylated hydroxytoluene ( 0 . 4 mg/ml ) .",
"A calibration stock was made with concentrations found in Supplementary file 2 for the prostaglandins , isopostranes , LPAs , sphingolipids and PAF available base standards and was labeled ‘C9’ .",
"This solution was diluted to levels C8 to C1 and from these mixes , 20 µl was added to 150 ul sample to construct the calibration curves .",
"LabSolutions ( Shimadzu , Version 5 . 91 ) was applied to accomplish all the peak determination and integration .",
"For each metabolite , the response ratio was obtained by calculating the ratio of peak area of the target compound to the peak area of the assigned internal standard .",
"After QC evaluation , metabolites of which QC samples had an RSD less than 30% were used for further statistical analysis .",
"Finally , the absolute concentration of the targets was determined using the calibration curves .",
"Heatmaps and bar plots were created with GraphPad Prism 7 ( GraphPad Software ) .",
"The fold change was calculated by normalizing the conditions to the control group .",
"Subsequently , the data were log2 transformed and used for the heatmaps .",
"The absolute concentrations of those compounds were visualized in the bar plots .",
"We used IBM SPSS Statistics 23 ( IBM ) for statistical analyses .",
"Bar plots were plotted as mean ± s . e . m . of three biological replicates per condition; n = 4–6 technical replicates .",
"Significance levels were set at *p<0 . 1 , **p<0 . 05 , ***p<0 . 01 , ****p<0 . 001 using the unpaired Student’s t-test ."
]
] | [
"TNFα signaling in the vascular endothelium elicits multiple inflammatory responses that drive vascular destabilization and leakage .",
"Bioactive lipids are main drivers of these processes .",
"In vitro mechanistic studies of bioactive lipids have been largely based on two-dimensional endothelial cell cultures that , due to lack of laminar flow and the growth of the cells on non-compliant stiff substrates , often display a pro-inflammatory phenotype .",
"This complicates the assessment of inflammatory processes .",
"Three-dimensional microvessels-on-a-chip models provide a unique opportunity to generate endothelial microvessels in a more physiological environment .",
"Using an optimized targeted liquid chromatography–tandem mass spectrometry measurements of a panel of pro- and anti-inflammatory bioactive lipids , we measure the profile changes upon administration of TNFα .",
"We demonstrate that bioactive lipid profiles can be readily detected from three-dimensional microvessels-on-a-chip and display a more dynamic , less inflammatory response to TNFα , that resembles more the human situation , compared to classical two-dimensional endothelial cell cultures ."
] | [
"In a range of conditions called autoimmune diseases , the immune system attacks the body rather than foreign elements .",
"This can cause inflammation that is harmful for many organs .",
"In particular , immune cells can produce excessive amounts of a chemical messenger called tumor necrosis factor alpha ( TNFα for short ) , which can lead to the release of fatty molecules that damage blood vessels .",
"This process is normally studied in blood vessels cells that are grown on a dish , without any blood movement .",
"However , in this rigid 2D environment , the cells become ‘stressed’ and show higher levels of inflammation than in the body .",
"This makes it difficult to assess the exact role that TNFα plays in disease .",
"A new technology is addressing this issue by enabling scientist to culture blood vessels cells in dishes coated with gelatin .",
"This allows the cells to organize themselves in 3D , creating tiny blood vessels in which fluids can flow .",
"However , it was unclear whether these ‘microvessels-on-a-chip’ were better models to study the role of TNFα compared to cells grown on a plate .",
"Here , Junaid et al . compared the levels of inflammation in blood vessels cells grown in the two environments , showing that cells are less inflamed when they are cultured in 3D .",
"In addition , when the artificial 3D-blood vessels were exposed to TNFα , they responded more like real blood vessels than the 2D models .",
"Finally , experiments showed that it was possible to monitor the release of fatty molecules in this environment .",
"Together , this work suggests that microvessels-on-a-chip are better models to study how TNFα harms blood vessels .",
"Next , systems and protocols could be develop to allow automated mass drug testing in microvessels-on-a-chip .",
"This would help scientists to quickly screen thousands of drugs and find candidates that can protect blood vessels from TNFα ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression"
] | Therapeutic effects of telomerase in mice with pulmonary fibrosis induced by damage to the lungs and short telomeres | elife-31299-v2 | [
[
"Mammalian telomeres are protective structures at ends of chromosomes that consist of TTAGGG repeats bound by a six-protein complex known as shelterin ( Blackburn , 2001; de Lange , 2005 ) .",
"A minimum length of telomeric repeats is necessary for shelterin binding and telomere protection ( Blackburn , 2001; de Lange , 2005 ) .",
"Telomerase is an enzyme composed of two subunits , the telomerase reverse transcriptase ( TERT ) and the RNA component ( Terc ) , which is used as template for the de novo addition of telomeric repeats to chromosome ends ( Greider and Blackburn , 1985 ) .",
"Adult tissues , including the stem cell compartments , do not have sufficient telomerase activity to compensate for the progressive telomere shortening associated with cell division throughout lifespan ( Canela et al . , 2007; Flores et al . , 2008; Harley et al . , 1990; Vera et al . , 2012 ) .",
"When telomeres reach a critically short length , this triggers activation of a persistent DNA damage response at telomeres and the subsequent induction of cellular senescence or apoptosis .",
"Indeed , this progressive shortening of telomeres with increasing age is considered one of the hallmarks of aging both in mice and humans ( López-Otín et al . , 2013 ) .",
"In particular , critical telomere shortening at the stem cell compartments results in the loss of the regenerative capacity of these compartments eventually compromising tissue renewal and homeostasis ( Blasco , 2007; Flores et al . , 2005; Povedano et al . , 2015 ) .",
"Interestingly , the rate of telomere shortening throughout lifespan has been shown to be influenced both by genetic factors ( ie . , mutations in genes necessary for telomere maintenance ) and environmental factors ( ie . , cigarette smoke has a negative effect ) ( Armanios , 2013; King et al . , 2011 ) .",
"In support of critical telomere shortening being a determinant of aging and longevity , increased TERT expression in the context of cancer resistant transgenic mice was sufficient to delay aging and extend mouse longevity by 40% ( Tomás-Loba et al . , 2008 ) .",
"More recently , these findings have been translated into a potential therapeutic strategy by using adeno-associated vectors ( AAV ) to transiently activate telomerase in adult tissues ( Bär et al . , 2014; Bernardes de Jesus et al . , 2012 ) .",
"In particular , treatment with Tert gene therapy using non-integrative AAV9 vectors of adult mice was able to delay aging and increase longevity by decreasing age-related pathologies such as osteoporosis , glucose intolerance , as well as neuromuscular and cognitive decline .",
"Furthermore , the onset of cancer was also delayed in the Tert treated mice ( Bernardes de Jesus et al . , 2012 ) .",
"More recently , AAV9-Tert delivery specifically to the heart was sufficient to significantly increase mouse survival and heart function upon myocardial infarction , which was concomitant with decreased fibrosis and increased cardiac myocyte proliferation ( Bär et al . , 2014 ) .",
"These findings support the notion that telomere shortening is at the origin of age-related diseases and that , by delaying or reverting this process with telomerase , it is possible to delay and treat more effectively age-associated diseases , such as heart infarct .",
"Extreme telomere shortening can occur prematurely in individuals with mutations in telomerase and other telomere maintenance genes causing the so-called telomere syndromes , which include dyskeratosis congenita , aplastic anemia and pulmonary fibrosis , among others ( for a review see [Armanios and Blackburn , 2012] ) .",
"These syndromes are characterized by premature loss of the regenerative capacity of tissues , affecting both high and low proliferation tissues ( Armanios and Blackburn , 2012; Holohan et al . , 2014 ) .",
"Among the telomere syndromes , idiopathic pulmonary fibrosis ( IPF ) is the most common condition associated with telomere dysfunction in humans ( Armanios , 2013; Armanios and Blackburn , 2012 ) .",
"Both familial and sporadic cases have been linked to telomerase mutations , either in TERT or TERC ( Alder et al . , 2008; Armanios et al . , 2007 ) .",
"In particular , mutations in TERT and TERC account for 8–15% of familial and 1–3% of sporadic cases ( Alder et al . , 2008; Armanios , 2013; Armanios et al . , 2007 ) .",
"Interestingly , sporadic cases of IPF , not associated with telomerase mutations , also show shorter telomeres compared to age-matched controls , with 10% of the patients showing telomeres as short as the telomerase mutation carriers ( Alder et al . , 2008 ) .",
"Telomerase mutations have also been found in up to 1% of smokers showing chronic obstructive pulmonary disease ( COPD ) , also leading to abnormally short telomeres ( Stanley et al . , 2015 ) .",
"Unfortunately , in spite of its prevalence , idiopathic pulmonary fibrosis is still a life-threatening lung degenerative disease , with few available therapeutic options ( King et al . , 2011 ) .",
"As an example , the recently FDA-approved drugs , nintedanib and pirfenidone , show anti-inflammatory and anti-fibrotic activity ( Ahluwalia et al . , 2014; Karimi-Shah and Chowdhury , 2015; King et al . , 2014 ) , and slow IPF progression but are not curative ( Hunninghake , 2014; Karimi-Shah and Chowdhury , 2015; King et al . , 2014 ) .",
"Indeed , to date , lung transplantation is the only curative therapeutic option in less than 5% of IPF patients with severe disease ( Lama , 2009 ) .",
"Thus , development of new , more effective , therapeutic strategies aimed against treating the origin of the disease is urgently needed .",
"An important limitation to the development of new therapeutic strategies has been the lack of appropriate pre-clinical mouse models .",
"Induction of acute pulmonary fibrosis with high doses of bleomycin in mice has been the most widely used preclinical model , although the disease spontaneously reverses in this model after 2–3 weeks ( Mouratis and Aidinis , 2011 ) .",
"Furthermore , telomerase-deficient mice with short telomeres do not spontaneously develop pulmonary fibrosis ( Alder et al . , 2011 ) , suggesting that additional insults contribute to the disease in addition to the genetic defects .",
"In support of this notion , we recently demonstrated that treatment with low doses of bleomycin ( 0 . 5 mg/kg BW ) , which normally do not lead to pulmonary fibrosis in wild-type mice , however , results in full-blown progressive pulmonary fibrosis in telomerase deficient mice ( Povedano et al . , 2015 ) .",
"Thus , this model shows that short telomeres are at the molecular origin of pulmonary fibrosis and could represent a useful pre-clinical tool to test the challenging hypothesis of whether therapeutic strategies based on telomerase activation maybe effective in the treatment of the disease .",
"Here , we tested this hypothesis by using a Tert based gene therapy in mice diagnosed with pulmonary fibrosis owing to treatment with low doses of the lung-damaging agent bleomycin in the context of short telomeres , a scenario that resembles pulmonary fibrosis in humans associated with short telomeres .",
"Our findings demonstrate that Tert treatment significantly improves pulmonary function , decreases inflammation , and accelerates fiber disappearance in fibrotic lungs as early as 3 weeks after viral treatment , resulting in a more rapid improvement or disappearance of the fibrosis .",
"At the molecular level , AAV9-treatment results in telomere elongation and increased proliferation of ATII cells , also significantly decreasing DNA damage , apoptosis , and senescence in these cells .",
"Further supporting these findings , telomerase treatment induces gene expression changes indicative of increased proliferation , lower inflammation and decreased fibrosis in isolated ATII cells ."
],
[
"Here , we set to address whether telomerase treatment of adult mouse lungs by using AAV9-Tert vectors could effectively prevent the progression of pulmonary fibrosis provoked by damage to the lungs ( ie . , low-dose bleomycin ) and the presence of short telomeres ( Povedano et al . , 2015 ) , a scenario that resembles both familiar and sporadic cases of the human disease ( Alder et al . , 2008; Armanios , 2013; Armanios et al . , 2007 ) .",
"To this end , we used our previously described mouse model of progressive pulmonary fibrosis induced by a low bleomycin dose in the context of short telomeres ( Povedano et al . , 2015 ) .",
"In particular , owing to the fact that short telomeres per se in the context of the telomerase-deficient mouse model are not sufficient to induce pulmonary fibrosis in mice ( Alder et al . , 2011 ) , we previously generated a mouse model for pulmonary fibrosis associated with short telomeres by treating telomerase-deficient mice from the second ( G2 ) and fourth ( G4 ) generation , G2-G4 Tert-/- with a low dose of bleomycin ( 0 . 5 mg/kg body weight ) .",
"This low dose of bleomycin is not sufficient to induce pulmonary fibrosis in wild-type mice , but leads to progressive pulmonary fibrosis in the G2-G4 Tert-/- ( Povedano et al . , 2015 ) .",
"It is relevant to note that this is in contrast to the widely used mouse model of pulmonary fibrosis using a much higher dose of bleomycin ( 2 mg/kg body weight ) , which leads to pulmonary fibrosis in wild-type mice but does not recapitulate the short telomere phenotype present in human patients ( see Figure 1—figure supplement 1A ) .",
"In particular , we show here that male wild-type mice inoculated either with vehicle or with the standard high-dose bleomycin protocol did not show any significant telomere length changes 4 weeks after bleomycin challenge compared to vehicle inoculated mice ( Figure 1—figure supplement 1A ) , suggesting that this mouse model of pulmonary fibrosis does not recapitulate one of the molecular features of the human disease ( ie , the presence of short telomeres ) .",
"Further supporting this notion , treatment of these mice with AAV9-Tert did not show any significant decrease in the amount of fibronectin compared to the empty vector-treated lungs ( Figure 1—figure supplement 1B ) .",
"To test the efficacy of telomerase gene therapy in our mouse model of pulmonary fibrosis induced by DNA damage to the lungs ( ie . , low-dose bleomycin ) and the presence of short telomeres ( Povedano et al . , 2015 ) , we selected AAV9 serotype owing to its high viral transduction of the lungs , and its low immunogenicity ( Bell et al . , 2011; Zincarelli et al . , 2008 ) .",
"In particular , we previously showed that AAV9-Tert transduced lungs cells showed Tert mRNA over-expression for at least 8 month post-infection of the vector , as well as resulted in re-activation of telomerase as determined by Telomerase Repeated Amplification Protocol ( TRAP ) in adult lungs ( Bernardes de Jesus et al . , 2012 ) .",
"To determine the transduction efficiency of the lungs in our current study , we intravenously ( IV ) injected wild-type adult mice with AAV9-eGFP and determined eGFP expression in the lungs 2 weeks later .",
"We found transduction of 3% ( GFP positive cells ) of total lung cells ( Figure 1A ) .",
"Next , we addressed which adult lung cell types were being transduced with the AAV9 vector .",
"We previously described that alveolar type II cells ( ATII ) cells are a key cell type in the origin of pulmonary fibrosis owing to dysfunctional telomeres ( Povedano et al . , 2015 ) .",
"Thus , we performed double immunofluorescence against eGFP and the surfactant protein C ( Sftpc ) , a specific marker of ATII cells .",
"We first observed that 13 . 4% of total lung cells were ATII cells ( Sftpc-positive cells ) ( Figure 1A ) , which is in line with the 12–15% reported abundance of ATII cells in whole lung cell population ( Dobbs , 1990; Van der Velden et al . , 2013 ) .",
"Importantly , we observed that 17% of total ATII cells were transduced by AAV9-eGFP ( GFP-positive ) ( Figure 1A ) .",
"Indeed , more than 80% of all the GFP-positive lung cells were ATII cells ( Figure 1A ) , indicating that AAV9 has a specific tropism for these cells .",
"Next , we treated telomerase-deficient male mice from the second generation , G2 Tert-/- mice , with the low bleomycin dose ( 0 . 5 mg/kg BW ) ( Povedano et al . , 2015 ) .",
"Two weeks after bleomycin treatment , we performed computed tomography ( CT ) to identify those mice with abnormal radiological images of the lungs , indicative of inflammation and pulmonary fibrosis ( Figure 1B ) .",
"Approximately 50% of the mice showed an abnormal radiographic pattern presenting reticular opacities suggestive of pulmonary fibrosis ( Povedano et al . , 2015 ) .",
"These mice with an abnormal CT pattern were divided in two random groups , one group was intravenously ( IV ) injected with AAV9-Tert and the other group was injected with the empty vector , as control placebo group .",
"Disease progression was followed longitudinally in both cohorts both by performing weekly spirometry during the first 3 weeks after viral treatment , to measure lung function , as well as by CT imaging at 1 , 2 , 4 and 7 weeks post viral treatment to follow progression of the abnormal radiographic patterns ( Figure 1B ) .",
"Interestingly , only one week after viral treatment , CT imaging showed that all abnormal radiological images in AAV9-Tert treated mice regressed in size , while they further increased in size in mice treated with the empty vector ( Figure 1C , D ) .",
"After the second week of treatment , we observed a regression of the affected CT lung volume in both groups , although at all time points analyzed the AAV9-Tert treated mice showed significantly smaller volume of the CT lesions as compared to mice treated with the empty vector ( Figure 1C , D ) .",
"Importantly , at week seven after treatment with the viral vectors ( week nine after the induction of fibrosis with bleomycin ) , the affected CT lung volume in the AAV9-Tert treated mice corresponds to only 5% of total lung volume , while at this point mice treated with the empty vector still exhibited 40% of total lung volume affected as indicated by CT ( Figure 1C , D ) .",
"Given the small size of mouse lungs , CT imaging as PF diagnose is not fully accurate since inflammation can also give rise to abnormal CT pattern .",
"As an independent longitudinal non-invasive indicator of improvement of pulmonary lesions in the treated mice , pulmonary function was determined by using plethysmography ( spirometry ) that measures the amount of air left in the lung after deep inhalation and forced exhalation , both previous to viral treatment and during the first 3 weeks after treatment .",
"We observed that lung function measured as the ratio between lung resistance and dynamic compliance worsen in the AAV9-Empty treated mice compared to the AAV9-Tert controls already the first week after viral treatment .",
"Importantly , pulmonary function in AAV9-Tert treated mice became similar to that of healthy mice non-treated with bleomycin at two weeks post-viral treatment and was maintained thereafter , illustrating the efficacy of the treatment at restoring lung health .",
"In contrast , mice treated with AAV9-Empty show significant higher LR/Cdyn values as compared to healthy and to AAV9-Tert , indicating a worsened pulmonary function ( Figure 1E ) .",
"Finally , in order to confirm the areas of the lung affected with fibrosis , at week eight after viral treatment with the vectors , all mice were sacrificed for histopathological , biochemical , and molecular analysis of the lungs .",
"First , we confirmed increased expression of Tert mRNA by qPCR in the lungs of AAV9-Tert treated mice compared to mice treated with the empty vector , which lacked detectable Tert mRNA expression ( Figure 1F ) ( Bär et al . , 2014; Bernardes de Jesus et al . , 2012 ) .",
"We next used Masson´s trichrome staining to quantify the lung fibrotic areas by histochemistry .",
"We considered ‘severe fibrosis’ when more than 30% of the lung parenchyma was affected by fibrosis; ‘mild fibrosis’ when less than 10% of the lung parenchyma was affected; and ‘non-fibrotic lungs’ when no signs of fibrosis were found .",
"We found that at week 8 after treatment with the viral vectors ( week 10 after the induction of fibrosis ) , all mice treated with the empty vector showed severe fibrosis as indicated by more than 30% of the lung parenchyma affected by fibrosis ( Figure 1G , H ) .",
"In contrast , none of the AAV9-Tert treated mice showed severe fibrosis at this point .",
"Instead , 50% of Tert-treated mice presented mild fibrosis lesions and 50% were completely free of fibrotic lesions ( Figure 1G–H ) .",
"Thus , 50% AAV9-Tert treated mice showed undetectable fibrosis as determined by Masson´s trichrome staining at 8 weeks post-viral treatment , while all empty vector treated mice still showed severe fibrotic lesions .",
"Picrosirius red staining of lungs to determine collagen deposition , confirmed that AAV9-Tert treated mice presented one-third less collagen deposition compared to mice treated with the empty vector ( Figure 2A , B ) .",
"As an independent biochemical method , we determined collagen peptides containing hydroxyproline on whole lung tissue at 8 weeks after treatment with the viral vectors using liquid chromatography-tandem mass spectrometry ( LC-MS/MS ) which has been previously validated to quantify collagen ( Chaerkady et al . , 2013; Montgomery et al . , 2012; Ono et al . , 2009; Qiu et al . , 2014; Taga et al . , 2014 ) .",
"As validation of the technique in our experimental setting , we determined collagen peptides containing hydroxyproline in non-fibrotic lungs from Tert-deficient mice that had not been treated with bleomycin and in fibrotic lungs from Tert-deficient mice treated with bleomycin at 5 weeks post-bleomycin treatment ( 0 . 5 mg/kg BW ) ( Figure 2C , left panel ) .",
"The results show that bleomycin treated lungs present a 2-fold increase in the amount of collagen peptides containing hydroxyproline compared to control lungs not treated with bleomycin ( w/o bleomycin ) in agreement with induction of fibrosis by bleomycin in Tert-deficient mice ( Figure 2C , left panel ) , thus validating this method for quantification of fibrosis .",
"At 8 weeks after viral treatment ( 10 weeks post bleomycin treatment ) , AAV9-Tert treated lungs showed 2-fold lower content in collagen peptides containing hydroxyproline compared to AAV9-Empty treated lungs , indicating that telomerase treatment improves collagen removal ( Figure 2C , right panel ) .",
"Analysis of total procollagen levels in the lung using western blot analysis also showed approximately 50% and 30% lower levels of procollagen in AAV9-Tert treated mice compared to the controls at 3 and 8 weeks post-viral treatment , respectively , suggesting that Tert gene therapy leads to a more rapid removal of fiber deposition ( Figure 2D , E ) .",
"In line with lower collagen , AAV9-Tert treated lungs also showed significantly less αSMA-positive myofibroblasts compared to empty vector-treated mice , in agreement with the fact that these cells are associated with collagen deposition in human IPF patients ( Figure 2F , G ) , thus suggesting an inactivation of fibrotic foci upon Tert treatment .",
"Finally , also in agreement with fibrosis regression and tissue healing in mice treated with telomerase , AAV9-Tert treated mice showed significantly less macrophage infiltrates as detected by F4/80 staining in their remaining fibrotic areas compared with AAV9-Empty treated mice ( Figure 2H , I ) , suggestive of decreased inflammation .",
"We confirmed lower inflammation in the AAV9-Tert treated mice compared to the empty vector treated group by quantification of a large panel of cytokines including BLC , C5/C5A , G-CSF , I-309 , IL-1a , IL-1b , IL-1ra , IL-2 , IL-3 , IL-4 , IL-5 , IL-6 , IL-7 , IL-16 , IL-17 , IL-23 , IL-27 , IP-10 , I-TAC , KC , M-CSF , JE , MCP-5 , MIG , MIP-1a , MIP-1b , MIP-2 , RANTES , SDF-1 , TIMP-1 , TNF-a and TREM-1 by Elisa .",
"In particular , already at 3 weeks after viral treatment , AAV9-Tert treated mice showed significantly lower levels of these cytokines compared to the cohort treated with the empty vector and this was maintained at 8 weeks after viral treatment , indicating the efficacy of the therapy in dampening the inflammatory response ( Figure 2J , K ) .",
"Thus , these results demonstrate lower inflammation in the lungs of Tert treated mice compared to the controls .",
"Short dysfunctional telomeres have been previously shown to trigger a persistent DNA damage response ( DDR ) characterized by increased γH2AX foci , increased expression of p21 and p53 cell arrest and senescence markers , as well as induction of apoptosis ( Hemann et al . , 2000; Meier et al . , 2007 ) .",
"Indeed , we previously described that mice with pulmonary fibrosis owing to short telomeres also show increased γH2AX foci , increased expression of p21 and p53 senescence markers , as well as induction of apoptosis in the lungs ( Povedano et al . , 2015 ) .",
"Interestingly , analysis of these molecular markers in the lungs of treated mice at two time points after viral treatment shows that the lungs of AAV9-Tert treated mice have a significant reduction of DNA damage already at 3 weeks post-viral treatment which is also maintained at 8 weeks post-viral treatment ( endpoint of the experiment ) , as indicated by lower percentage of cells positive for γH2AX compared to mice treated with empty vector ( Figure 3A , B ) .",
"Consistently , we also found a significant reduction in the abundance of p21 and p53-positive cells in AAV9-Tert treated mice compared to those treated with the empty vector as early as 3 weeks after viral treatment and again these lower levels were maintained at 8 weeks post-viral treatment ( Figure 3A , B ) .",
"Moreover , we also observed a significant decrease in caspase 3-positive cells at both time points ( 3 and 8 weeks post-viral treatment ) in the lungs of Tert-treated mice compared to the empty-treated cohort ( Figure 3A , B ) , indicative of decreased apoptosis .",
"Together , these results indicate that Tert expression in the lungs of adult mice with pulmonary fibrosis is sufficient to decrease DNA damage and apoptosis , as well as to decrease the levels of p21 and p53 , as early as 3 weeks after viral treatment and this is maintained until the end-point of the experiment at 8 weeks post-viral treatment when the fibrosis was reverted or cured in a significant proportion of mice treated with telomerase .",
"In order to specifically address presence of senescent cells in the lungs , we performed whole mount staining for SA-β-galactosidase assay in mice diagnosed with pulmonary fibrosis and treated with either AAV9-Tert or -Empty vectors .",
"While senescence epithelial cells were readily detected in mice diagnosed with pulmonary fibrosis and treated with the empty vector , they were undetectable in the residual fibrotic areas present in few AAV9-Tert treated lungs at the end of the experiment ( Figure 3A , B ) .",
"Of note , macrophages and fibroblasts were discarded from the analysis based on cell morphology .",
"These findings indicate that Tert gene therapy rescues DNA damage , apoptosis and cellular senescence in mice diagnosed with pulmonary fibrosis owing to critically short telomeres .",
"However , as these histological analyses do not permit distinguishing among different cell types , to specifically address the DNA damage burden in ATII cells we performed double immunohistochemistry staining with anti-SFTPC to mark ATII cells and anti-γH2AX to mark cells with DNA damage ( Figure 3C , D ) .",
"The results clearly show that the amount of damaged ATII cells in AAV9-Tert treated lungs is reduced by 3-fold compared to control mice treated with the AAV9-Empty vector at 3 weeks post-viral treatment ( Figure 3C , D ) .",
"To further understand the molecular mechanisms by which Tert gene therapy results in significant remission and healing of pulmonary fibrosis owing to short telomeres , we next studied telomere length specifically in the ATII cells of mice treated with either AAV9-Tert or the empty vector .",
"To this end , we performed an Immuno-FISH using a telomeric PNA probe and a Sftpc antibody to specifically mark ATII cells in lung samples at 8 weeks post-viral treatment .",
"We analyzed telomere intensity in both Sftpc positive ( ATII ) and negative ( non-ATII ) cells .",
"We found that ATII cells have shorter telomeres than non-ATII cells in telomerase-deficient mice in agreement with our previous findings indicating that these cells are important for the regeneration of lung damage induced by dysfunctional telomeres , as they have undergone more cell divisions ( Povedano et al . , 2015 ) .",
"Interestingly , ATII cells from mice treated with AAV9-Tert showed the same telomere length than the surrounding non-ATII cells , suggesting that telomerase treatment is preserving telomeres in these cells in the context of lung fibrosis ( Figure 3E–G ) .",
"Indeed , the percentage of short telomeres of ATII cells compared to non-ATII in empty vector-treated mice ( ATII/non-ATII ratio = 1 . 4 ) is significantly higher than in AAV9-Tert treated mice ( ATII/non-ATII ratio = 0 . 98 ) .",
"We considered short telomeres those spots with an intensity ≤30 a . u . corresponding to 20th percentile .",
"The percentage of short telomeres from ATII cells was normalized to non-ATII cells to avoid inter-individual variability ( Figure 3E , G ) .",
"In summary , these results indicate that specific telomerase targeting to ATII cells results in improved telomere length maintenance and a consequent reduction in DNA damage burden of these cells compared to ATII cells from mice treated with the empty vector .",
"Next , we addressed the effects of Tert treatment on the ability of ATII cells to proliferate and regenerate the damaged lung tissue upon diagnosis of fibrosis .",
"To this end , double immunofluorescence against the ATII cells-specific marker Sftpc and the proliferation marker Ki67 was performed in lung samples at 8 weeks post-viral treatment .",
"We found that Tert treated mice showed a 2-fold increase in Ki67 positive cells in whole lung tissue compared to controls ( Figure 3H ) .",
"When specifically looking at ATII cells , we observed a 2-fold increase in the total number of Sftpc positive cells and 2 . 5-fold increase in Ki67 positive ATII cells in AAV9-Tert treated lungs compared to the empty vector controls ( Figure 3I–K ) .",
"Although we cannot distinguish between the AAV9-Tert infected and non-infected ATII cells , the fact that 80% of the total AAV9-infected lung cells are ATII cells ( Figure 1A ) , suggests that AAV9-Tert treatment is resulting in increased proliferation of these cells leading to a higher potential for lung regeneration and the remission of lung fibrosis .",
"Thus , the higher number of proliferating ATII cells are in agreement with the significant lower percentage of short telomeres in ATII cells in AAV9-Tert treated lungs as well as with the significant decrease in the number of p21 and p53 positive cells in AAV9-Tert treated lungs ( Figure 3B–J ) .",
"Next , we studied gene expression changes induced by Tert expression in the context of lung fibrosis .",
"To this aim , we first performed DNA microarray analysis from the post-caval lung lobe from mice diagnosed with pulmonary fibrosis which were treated either with AAV9-Tert or with the empty vector at 8 weeks post viral treatment with the vectors ( 5 mice were included per group ) .",
"We found that only 53 genes were significantly upregulated ( False Discovery Rate , FDR < 0 . 05 ) in AAV9-Tert treated mice compared to mice treated with the empty vector ( Supplementary file 1 ) .",
"This FDR cutoff highlights the significance of the gene expression changes observed , as only 5% of the hits are expected to be false positive .",
"Gene set enrichment analysis ( GSEA ) showed significantly deregulated pathways between both groups ( Figure 4A–B ) .",
"Those pathways found upregulated in AAV9-Tert treated lungs presented a signature related with DNA replication and mitosis , apoptosis , DNA repair , the leukocyte transendothelial migration pathway , and extension of telomeres ( Figure 4A , Figure 4—figure supplement 1A ) .",
"Upregulation of the ‘extension of telomeres pathway’ is in line with improved telomere maintenance in ATII cells treated with Tert ( Figure 4A ) .",
"Similarly , upregulation of DNA replication and mitosis pathways is in line with increased proliferation in ATII cells ( Figure 4A , Figure 4—figure supplement 1A ) .",
"In contrast , pathways downregulated in AAV9-Tert compared to AAV9-Empty treated lungs were related to fibroblast growth factor receptors , Wnt and TGF-β pathways ( Figure 4B , Figure 4—figure supplement 1A ) .",
"Of interest , four of the pathways downregulated by Tert are related with fibroblast growth factor receptors; that is , the FGFR2C , FGFR4 , FGFR and the FGFR1 ligand binding and activation cascades ( Figure 4B ) .",
"As FGF1-FGFRc over-expression has been shown to contribute to pathogenesis in IPF patients ( MacKenzie et al . , 2015 ) , these findings suggest that Tert impairs fibroblast activation , thus facilitating fibrosis regression .",
"In line with this , we also found a downregulation of the TGF-β pathway in the AAV9-Tert treated lungs .",
"This is in agreement with our previous findings that Tert overexpression in mouse embryonic fibroblasts ( MEFs ) induces downregulation of TGF-β ( Geserick et al . , 2006 ) .",
"TGF-β pathway has been linked to fibroblast activation and differentiation to myofibroblast ( Pedroza et al . , 2016 ) .",
"Indeed , pirfenidone , a drug that blocks TGF-β can significantly slow pulmonary fibrosis progression ( Hunninghake , 2014; Karimi-Shah and Chowdhury , 2015; King et al . , 2014 ) .",
"By using qRT-PCR , we validated a random selection of the more differentially expressed genes within these pathways: Apc , Ctnnb1 , Fzd5 , Lrp6 , Lrp5 , Bim , Flir , Bid , Mcl1 , Mmp-9 and Cenpq ( Figure 4C ) .",
"Of note , downregulation of the Wnt pathway in AAV9-Tert treated adult lungs is in contrast with the notion that TERT can activate Wnt/β-catenin pathway during development ( Park et al . , 2009 ) .",
"Instead , our results go in line with recent findings showing that high levels of the Wnt pathway genes Lrp5 and Lrp6 are linked to bad prognosis for IPF patients ( Lam et al . , 2014 ) .",
"Interestingly , we found Mmp9 upregulation in Tert treated lungs , in line with the fact that Mmp9 overexpression attenuates fibrosis in bleomycin-induced IPF ( Cabrera et al . , 2007 ) .",
"Next , we set to analyze whether the observed gene expression changes corresponded to lung epithelial cells .",
"To this end , we compared the AAV9-Tert lung signature with the genes normally expressed in different lung populations ( The Gene Expression Barcode 3 . 0 ) .",
"Most upregulated genes ( 0 < Fc < 1 ) corresponded to genes specifically expressed by ATII cells ( Figure 4—figure supplement 1B ) , with a minority of the genes being normally expressed in leukocytes or embryonic fibroblasts .",
"Similar findings were found for the downregulated genes ( −1 < Fc < 0 ) ( Figure 4—figure supplement 1C ) .",
"Finally , we find of interest the fact that similar findings were found by us on the amelioration of heart function after infarct in mice treated with AAV9-Tert ( Bär et al . , 2014 ) .",
"In particular , Tert treatment lead to lower fibrotic scarring of the heart and increased cardiac myocyte proliferation concomitant with transcriptional changes suggestive of a regenerative signature ( Bär et al . , 2014 ) .",
"Interestingly , the gene expression changes in AAV9-Tert treated lungs correlate with the regenerative heart signature described in neonatal mice ( Haubner et al . , 2012 ) as well as those reported by us in the context of improved cardiac regeneration upon infarct by AAV9-Tert treatment ( Bär et al . , 2014 ) ( Figure 4—figure supplement 2 ) .",
"To specifically address the gene expression changes stemmed from Tert upregulation in ATII cells , we isolated ATII cells at one week after treatment of fibrotic lungs with AAV9-Tert and performed transcriptional profiling .",
"ATII cells were identified as EpCAM+ LysoTracker+ cells and non-ATII cells as EpCAM+ LysoTracker- ( Figure 5A ) , and expression of the ATII-specific marker Sftpc by RT-PCR was used to validate the FACS sorting ( Figure 5B ) .",
"FACS-sorted ATII cells from AAV9-Tert treated mice showed Tert mRNA expression while it was undetectable in FACS-sorted ATII cells from empty vector-treated controls ( Figure 5C ) .",
"We also validated decreased p53 and p21 mRNA expression by RT-PCR in ATII cells from Tert treated mice compared with empty vector treated mice ( Figure 5D , E ) , in agreement with lower senescence and DNA damage in Tert treated mice ( see Figure 3A , B ) .",
"Importantly , upon gene expression analysis of isolated ATII cells from Tert-treated mice , GSEA analysis showed downregulation of p53 signaling and apoptotic pathways ( Figure 5F , G ) , consistent with lower DNA damage in lungs from Tert-treated mice compared to those from empty vector-treated mice ( see Figure 3A , B ) .",
"Also in line with lower fibrosis in the lungs from Tert- treated mice , ATII cells showed downregulation of several inflammation related pathways including the TGF-β , NF-KappaB , IL2 and TNF signaling pathways ( Figure 5H–K ) .",
"Together , these results further demonstrate that ATII cells are transduced by AAV9-Tert , leading to increased telomerase expression , as well as to downregulation of DNA damage and fibrotic pathways ."
],
[
"In spite of recent therapeutic advances for the treatment of pulmonary fibrosis , most patients still face a fatal outcome , where the only curative treatment is lung transplantation .",
"As an example , the recently FDA approved drugs nintedanib and pirfenidone can significantly reduce the progression of pulmonary fibrosis in patients although no full-remissions have been observed ( Hunninghake , 2014; Karimi-Shah and Chowdhury , 2015; King et al . , 2014 ) .",
"Thus , new therapeutic strategies aimed to cure the disease are still needed .",
"As short telomeres have been shown to be at the origin of both sporadic and familial cases of pulmonary fibrosis ( Alder et al . , 2008; Armanios et al . , 2007; Povedano et al . , 2015 ) , here , we set out to address the potential of telomerase gene therapy in the treatment of these cases of idiopathic pulmonary fibrosis ( IPF ) .",
"To this end , we used a pre-clinical mouse model of pulmonary fibrosis induced by damage to the lungs ( ie . , treatment with a low bleomycin dose ) and the presence of short telomeres ( Povedano et al . , 2015 ) , a scenario that resembles both familiar and sporadic cases of the human disease which are associated with the presence of short telomeres ( Alder et al . , 2008; Armanios et al . , 2007 ) .",
"It is important to note that only the presence of short telomeres per se in mice deficient for telomerase does not lead to pulmonary fibrosis ( PF ) ( Alder et al . , 2011 ) .",
"We generated a new , more ‘humanized’ mouse model for pulmonary fibrosis by subjecting Tert-/- mice with short telomeres to small doses of bleomycin ( 0 . 5 mg/kg body weight ) .",
"This low dose of bleomycin is not sufficient to induce pulmonary fibrosis in wild-type mice , but synergizes with short telomeres in the context of Tert-/- mice leading to full-blown , progressive pulmonary fibrosis , recapitulating many of the features of the human disease , including the presence of short telomeres ( Blasco et al . , 1997 ) .",
"We further show here that the most widely used mouse model of pulmonary fibrosis , which is based on treating wild-type mice with a high dose of bleomycin ( Adamson and Bowden , 1974 ) , do not present short telomeres in the lung .",
"Thus , to our knowledge the mouse model used here is the only available mouse model to date that develops pulmonary fibrosis as a consequence of telomere length defects as it is also the case of human patients with pulmonary fibrosis associated to short telomeres ( Alder et al . , 2008; Armanios et al . , 2007 ) .",
"Here we extensively demonstrate by using biochemical , functional , and histochemistry analysis , that Tert gene therapy ( AAV9-Tert ) in mice diagnosed with pulmonary fibrosis leads to a more rapid regression of pulmonary fibrosis and improves pulmonary function as early as weeks 1 and 3 after treatment and this is maintained at the end-point of the experiment at week 8 , when a significant percentage of mice show curation of the fibrosis .",
"Interestingly , we found that the major lung cell type transduced by AAV9 are ATII cells , previously shown by us to be at the origin of pulmonary fibrosis owing to dysfunctional telomeres ( Povedano et al . , 2015 ) .",
"ATII cells have been also proposed to be involved in lung regeneration upon injuries ( Serrano-Mollar et al . , 2007 ) .",
"Tert increased expression in ATII cells results in improved telomere maintenance and proliferation of these cells , which is concomitant with lower DNA damage as well as decreased presence of apoptotic and senescence cells already at week 3 after AAV9-Tert treatment .",
"As a consequence , Tert-treated mice show a better pulmonary function as well as decreased inflammation and decreased fibrosis ( lower collagen depots ) .",
"These results are in line with the notion that short telomeres can impair the ability of stem cells to regenerate tissues ( Blasco , 2007; Flores et al . , 2008 ) , and with recent findings suggesting that IPF is the result of defective regeneration upon repetitive epithelial cell injury ( Hinz et al . , 2007; Ryu et al . , 2014 ) .",
"Of clinical relevance is the observation that these beneficial effects of Tert gene therapy are achieved with a transduction efficiency of 3% of total lung cells and 17% of ATII cells .",
"Of relevance , we show here that Tert expression in fibrotic lungs leads to downregulation of pathways involved in fibroblast activation and , in particular , of the TGF-β pathway .",
"Indeed , gene expression analysis of isolated ATII cells from Tert-treated mice showed downregulation of p53 signaling , apoptotic pathways and of several inflammation-related pathways including the TGF-β , NF-KappaB , IL2 and TNF signaling pathways as early as 1 week after Tert treatment .",
"Dampening of inflammation upon Tert treatment was further demonstrated by decreased levels of a large number of cytokines already at 3 weeks post-viral treatment that were maintained all throughout the experiment .",
"These pathways are known to be important players in IPF , and are targeted by the currently approved treatments for this disease , such as pirfenidone ( Inomata et al . , 2014; Oku et al . , 2008 ) .",
"Importantly , in contrast to the available IPF treatments , pirfenidone and nintedanib , which are not able to induce disease remission neither in patients nor in preclinical mouse models ( Inomata et al . , 2014; Oku et al . , 2008; Tanaka et al . , 2012 ) , we show here that AAV9-Tert therapy effectively accelerates the regression of pulmonary fibrosis in mice .",
"We would like to propose that this might be due to the fact that AAV9-Tert therapy targets one of the molecular causes of the disease , namely short telomeres ( Alder et al . , 2008; Armanios et al . , 2007; Povedano et al . , 2015 ) , which in turn we show here that results in decreased DNA damage and improved proliferative potential of the ATII cells , and subsequently in decreased fibrosis and inflammation ( Figure 6 ) .",
"In agreement with this , it was shown that treatment with GRN510 , a small molecule activator of telomerase , suppresses the development of fibrosis and accumulation of senescent cells in the lung in a model of bleomycin-induced fibrosis ( Le Saux et al . , 2013 ) .",
"In contrast , pirfenidone and nintedanib might be acting on downstream events , particularly on reducing fibrosis , while molecular damage at the origin of the disease ( ie . damaged telomeres ) , as well as the subsequent impairment of the regenerative potential of epithelial cells persists ( Alder et al . , 2008; Armanios et al . , 2007; Povedano et al . , 2015 ) .",
"Future ATII lineage tracing experiments would be of interest to ultimately demonstrate that defective regenerative potential of ATII associated to short telomeres is a key molecular event in PF development .",
"As a note of caution regarding the use of AAV vectors , it has been shown that transcriptional active host loci and DNA repair factors impact on rAAV vector integration in the host genome as well as on vector maintenance as linear or circular episomes , affecting thereby the duration of expression and mutagenic potential of the vector ( Inagaki et al . , 2007; Nakai et al . , 2003; Song et al . , 2001; Song et al . , 2004 ) .",
"Thus , further work is needed to address the potential effects of the PF disease on vector genome processing .",
"In addition , although we have not observed increased cancer incidence by systemic administration of the AAV9-Tert vector in different mouse models previously studied in the lab , such as AAV9-Tert treatment to delay organismal aging and increase longevity ( Bernardes de Jesus et al . , 2012 ) , AAV9-Tert treatment in mouse models of heart infarct ( Bär et al . , 2014 ) , and AAV9-Tert treatment in mouse models of aplastic anemia ( Bar et al . , 2016 ) , further work is needed to address its potential tumorigenic effects in cancer prone scenarios , or in the context of severely damaged tissues , where senescence and apoptosis may be acting as tumor suppressive mechanisms .",
"In summary , the findings described here demonstrate the therapeutic clinical potential of Tert gene therapy to efficiently improve pulmonary fibrosis associated with short telomeres ."
],
[
"Tert heterozygous mice generated as previously described ( Liu et al . , 2000 ) were backcrossed to >98% C57/BL6 background .",
"Tert+/- mice were intercrossed to generate first generation ( G1 ) homozygous Tert-/- knock-out mice .",
"G2 Tert-/- mice were generated by successive breeding of G1Tert-/- .",
"8 to 10 weeks old male G2 Tert-/- mice were intratracheally inoculated with 0 . 5 mg/kg body weight bleomycin as previously described ( Povedano et al . , 2015 ) .",
"Mice within experimental groups were allocated randomly .",
"Blind analysis of the samples was performed throughout this work .",
"All mice were produced and housed at the specific pathogen-free barrier area of the CNIO , Madrid .",
"All animal procedures were approved by the CNIO-ISCIII Ethics Committee for Research and Animal Welfare ( CEIyBA ) ( PROEX 42/13 ) and conducted in accordance to the recommendations of the Federation of European Laboratory Animal Science Associations ( FELASA ) .",
"Viral vectors were generated as described ( Matsushita et al . , 1998 ) and purified as previously described ( Ayuso et al . , 2014 ) .",
"Vectors were produced through triple transfection of HEK293T .",
"Cells were grown in roller bottles ( Corning , NY , USA ) in DMEM medium supplemented with fetal bovine serum ( 10% v/v ) to 80% confluence and then co-transfected with the following plasmids: plasmid_1 carrying the expression cassette for gene of interest flanked by the AAV2 viral ITRs; plasmid_2 carrying the AAV rep2 and cap9 genes; plasmid_3 carrying the adenovirus helper functions ( plasmids were kindly provided by K . A . High , Children’s Hospital of Philadelphia ) .",
"The expression cassettes were under the control of the cytomegalovirus ( CMV ) promoter and contained a SV40 polyA signal for EGFP and the CMV promoter and the 3’-untranslated region of the Tert gene as polyA signal for Tert .",
"AAV9 particles were purified following an optimized method using two caesium chloride gradients , dialysed against PBS , filtered and stored at 80°C until use .",
"Mice were injected via tail vein IV with 100 μL of rAVV9 viral genome particle ( 2 . 5*1013 vg/mL ) .",
"Histopathological analysis of paraffin-embedded lungs was performed in lung sections stained with nuclear fast red and Masson´s trichrome using standard procedures .",
"To quantify collagen deposition picosirius red staining was performed on deparaffinised slides for 1 hr ( Broytman et al . , 2015 ) .",
"Immunohistochemistry staining were performed with the following primary antibodies: rat monoclonal to p53 ( POE316A/E9; CNIO histopathology core unit ) , rat monoclonal to p21 ( HUGO-291H/B5; CNIO histopathology core unit ) , mouse monoclonal to phospho–Histone H2AX ( Ser139 ) ( Merck Millipore , Germany ) , rat monoclonal to F4/80 ( ABD serotec , UK ) , p19ARF ( 5-C3-1 Santa Cruz Biotecchnology , Dallas , Texas ) , rabbit polyclonal to Sftpc ( AB3786 , Merck Millipore ) and activated-caspase-3 ( R&D systems , Minneapolis , Minesota ) .",
"For immunofluorescence , the antibodies used were goat polyclonal anti Sftpc ( C-19; Santa Cruz Biotechnology ) , αSMA ( CME 305; Biocare Medical , Concord , California ) , anti GFP ( Roche , Switzerland ) and rabbit monoclonal anti Ki67 ( 0003110QD; Master Diagnostica , Spain ) .",
"Images were obtained using a confocal ultraspectral microscope ( TCS-SP5 , Leica , Germany ) .",
"Fluorescence intensities were analyzed with Definiens software .",
"For each analysis , 3–5 lung sections and 10 visual fields/section were scored .",
"The acquisition was made on a high-resolution CT system ( CT Locus , GE Healthcare ) specially designed for small laboratory animals .",
"Mice were anesthetized with a 4% rate of isoflurane ( IsoVet Braun ) during the induction and 2% during the maintenance period ( scanning time ) .",
"Micro-CT image acquisition consisted of 400 projections collected in one full rotation of the gantry in approximately 14 min in a single bed focused on the legs , with a 450 μA/80kV X-ray tube .",
"2-D and 3-D images were obtained and analysed using the software program MicroView ( GE Healthcare ) .",
"Pulmonary function was determined by plethymosgraphy using a pulmonary plethysmograph for sedated animals ( Emka Technologies ) .",
"The ratio between lung resistance and dynamic compliance ( LR/Cdyn ) was used as a measurement of pulmonary fitness .",
"All procedures were carried out according to the European Normative of Welfare and Good Practice ( 2010/63/UE ) .",
"Proteins were extracted from lung samples in 8M urea/2M thiourea inTris pH 8 . 2 buffer using a Precelys disruptor and digested subsequently on a 30 KDa MWCO filter with LysC and Trypsin .",
"Resulting peptides were resuspended in 1% TFA and desalted and concentrated using a homemade SCX Stage TIP ( 3M Empore ) .",
"The samples were vacuum dried and dissolved in 100 µL of loading buffer ( 0 . 5% formic acid ) and were analysed by LC-MS/MS in a Q-q-TOF Impact ( Bruker Daltonics ) .",
"The Impact was coupled online to a nanoLC Ultra system ( Eksigent ) , equipped with a CaptiveSpray nanoelectrospray ion source supplemented with a CaptiveSpray nanoBooster operated at 0 . 2 bar/minute with isopropanol as dopant .",
"Samples ( 5 µL ) were loaded onto a reversed-phase C18 , 5 µm , 0 . 1 × 20 mm trapping column ( NanoSeparations ) and washed for 10 min at 2 . 5 µl/min with 0 . 1% FA .",
"The peptides were eluted at a flow rate of 250 nl/min onto an analytical column packed with ReproSil-Pur C18-AQ beads , 2 . 4 μm , 75 μm x 50 cm ( Dr . Maisch ) , heated to 45°C .",
"Solvent A was 4% ACN in 0 . 1% FA and Solvent B acetonitrile in 0 . 1% FA .",
"The gradient used was a 150 min curved gradient from 2% B to 33 . 2% B in 130 min .",
"The MS acquisition time used for each sample was 150 min .",
"The Q-q-TOF Impact was operated in a data dependent mode .",
"The spray voltage was set to 1 . 35 kV ( 1868 nA ) and the temperature of the source was set to 160°C .",
"The MS survey scan was performed at a spectra rate of 2 . 5 Hz in the TOF analyzer scanning a window between 150 and 2000 m/z .",
"The minimum MS signal for triggering MS/MS was set to a normalized threshold of 500 counts .",
"The 20 most abundant isotope patterns with charge ≥2 and m/z > 350 from the survey scan were sequentially isolated and fragmented in the collision cell by collision induced dissociation ( CID ) using a collision energy of 23–56 eV as function of the m/z value .",
"The m/z values triggering MS/MS with a repeat count of 1 were put on an exclusion list for 60 s using the rethinking option .",
"For protein identification and quantification raw data were analyzed by MaxQuant interrogating a database containing mouse Uniprot Canonnical/TrEmbl sequences plus the most common contaminats ( 43936 entries ) , with Metionine and Proline Oxidation ( HydroxyProline ) allowed as variable modifications .",
"A normalization factor was calculated to correct for variations in total protein content in each sample .",
"Hydroxyproline-containing collagen peptides were quantified and their intensity values were normalized to the total peptide intensity for each sample .",
"Cytokine levels in lungs were analyzed by Mouse Cytokine Array ( ProteomeProfiler mouse Cytokine Array Panel A from R&D Systems ) following the manufacturer’s instructions .",
"The pixel density was determined by Image J Software .",
"Q-FISH determination on paraffin-embedded tissue sections was performed as described previously ( González-Suárez et al . , 2000 ) .",
"After deparaffinization , tissues were post-fixed in 4% Formaldehyde 5 min , washed 3 × 5 min in PBS and incubated at 37°C 15 min in pepsin solution ( 0 . 1% Porcine Pepsin , Sigma; 0 . 01M HCl , Merck ) .",
"After another round of washes and fixation as above-mentioned , slides were dehydrated in a 70%–90–100% ethanol series ( 5 min each ) .",
"After 10 min of air-drying , 30 μl of telomere probe mix ( 10 mM TrisCl pH7 , 25 mM MgCl2 , 9 mM Citric Acid , 82 mM Na2HPO4 , 70% Deionised Formamide –Sigma- , 0 . 25% Blocking Reagent –Roche- and 0 . 5 μg/ml Telomeric PNA probe -Panagene ) were added to each slide .",
"A cover slip was added and slides incubated for 3 min at 85°C , and for further 2 hr at RT in a wet chamber in the dark .",
"Slides were washed 2 × 15 min in 10 mM TrisCl pH7 , 0 . 1% BSA in 70% formamide under vigorous shaking , then 3 × 5 min in TBS 0 . 08% Tween20 and then incubated in a 4` , 6-diamidino-2-phenylindole ( DAPI ) bath ( 4 μg/ml DAPI ( Sigma ) in PBS ) before mounting samples in Vectashield ( VectorTM ) .",
"Confocal image were acquired as stacks every 1 μm for a total of 3 μm using a Leica SP5-MP confocal microscope and maximum projections were done with the LAS-AF software .",
"Telomere signal intensity was quantified using Definiens software .",
"RNA was extracted from post-caval lobe frozen lungs with RNeasy kit following manufacturer instruction ( Qiagen , cat . N° 73504 ) and RNA integrity analyzed in an Agilent Bioanalyzer .",
"cDNA was synthesised and analyzed on Agilent´s Mouse Genome DNA microarray , following the manufacturer´s instructions .",
"Microarray background subtraction was carried out using normexp method .",
"To normalize the dataset , we performed loess within arrays normalization and quantiles between arrays normalization .",
"Differentially expressed genes were obtained by applying linear models with R limma package ( Smyth GK ) ( Bioconductor project , http://www . bioconductor . org ) .",
"To account for multiple hypotheses testing , the estimated significance level ( p value ) was adjusted using Benjamini and Hochberg False Discovery Rate ( FDR ) correction .",
"Those genes with FDR < 0 . 05 were selected as differentially expressed between the AAV9-treated and non-treated groups .",
"This standard FDR threshold assumes a 5% of false positives in most impactful genes obtained in the differential expression analysis .",
"The raw data have been deposited in GEO database ( accession number GSE93869 ) .",
"Gene set enrichment analysis ( GSEA ) was applied using annotations from Biocarta , KEGG , NCI pathways and Reactome .",
"Genes were ranked based on limma moderated t statistic .",
"After Kolmogorov-Smirnoff testing , those gene sets showing FDR < 0 . 05 , a well-established cut-off for the identification of biologically relevant gene sets ( Subramanian et al . , 2005 ) , were considered enriched between classes under comparison .",
"Cells were isolated from mouse lungs of both groups AAV9-Tert and AAV9-empty vector .",
"Lungs were extracted and introduced in HBBS buffer with antibiotic and 1% BSA .",
"Separate the lobules of the lung on a dish mince them with a scalpel .",
"Transfer them to a GentleMacs tube with HBBS , antibiotics , , 1% BSA , DNAse I ( 60 units/mL ) ( Sigma , DN25 ) and collagenase type I ( 70 units/mL ) ( GIBCO , Cat . Number 17100 ) .",
"Then we run the GentleMac program ‘lung 1’ , after the program incubate the sample at 37°C for 30 min and at the end we run the GentleMac program ‘lung 2’ .",
"Cell suspension was filtered through a 40 μm stainer and then centrifuge 1200 rpm 5 min .",
"Cells were resuspended in 2 mL ACK Lysis Buffer to lyse red blood cells .",
"Incubate 4 min . at room temperature .",
"We added DMEM without serum to wash , centrifugate and discard supernatant .",
"At the end , resuspend cells in PBS with EDTA ( 1 mM ) , Hepes ( 25 mM ) and 3% FBS to start staining with LysoTracker as described in commercial protocol ( Molecular Probes , LysoTracker Green DND-26 , Cat . Num . L7526 ) and the following antibodies from Pharmingen ( BD Biosciences , San Jose CA ) : PE antimouse CD45 , PE antimouse CD31 , APC antimouse EpCAM .",
"DAPI ( Sigma , St Louis MO ) was used to identify dead cells .",
"Data was collected and the defined populations ( CD45-CD31-EpCAM + LysoTracker + and LysoTracker- ) were sorted using an InFlux cell sorted ( BD , San Jose CA ) , we excluded cell aggregates by using pulse processing in the scatter signals and dead cells in the basis of DAPI staining .",
"All data was analyzed using FlowJo software v9 . 8 . 5 ( Treestar , Ahsland OR ) ."
]
] | [
"Pulmonary fibrosis is a fatal lung disease characterized by fibrotic foci and inflammatory infiltrates .",
"Short telomeres can impair tissue regeneration and are found both in hereditary and sporadic cases .",
"We show here that telomerase expression using AAV9 vectors shows therapeutic effects in a mouse model of pulmonary fibrosis owing to a low-dose bleomycin insult and short telomeres .",
"AAV9 preferentially targets regenerative alveolar type II cells ( ATII ) .",
"AAV9-Tert-treated mice show improved lung function and lower inflammation and fibrosis at 1–3 weeks after viral treatment , and improvement or disappearance of the fibrosis at 8 weeks after treatment .",
"AAV9-Tert treatment leads to longer telomeres and increased proliferation of ATII cells , as well as lower DNA damage , apoptosis , and senescence .",
"Transcriptome analysis of ATII cells confirms downregulation of fibrosis and inflammation pathways .",
"We provide a proof-of-principle that telomerase activation may represent an effective treatment for pulmonary fibrosis provoked or associated with short telomeres ."
] | [
"Idiopathic pulmonary fibrosis ( or IPF for short ) is a rare disease that scars the lungs .",
"The condition gets worse over time , making it harder and harder to breathe , and eventually leading to death .",
"Patients typically only survive for a few years after being diagnosed with IPF .",
"This is because , as yet , there is no cure; the available treatments only act to lessen the symptoms .",
"Several risk factors have linked to the development of IPF , among them , the presence of short telomeres .",
"Like the plastic tips on shoelaces , telomeres are protective structures at the ends of chromosomes .",
"Telomeres shorten with age , and when they become too short the cell stops dividing and often dies in a process known as apoptosis .",
"IPF can develop when the telomeres in the cells that repair everyday wear and tear in the lungs ( known as ATII cells ) become too short .",
"This means that the damage goes unrepaired , triggering an immune reaction and uncontrolled scarring .",
"Telomerase is an enzyme that can lengthen short telomeres , and Povedano , Martínez et al . set out to develop a new treatment approach that would use this enzyme to correct the short telomeres , and cure the scarring seen in IPF .",
"Gene therapy was used to introduce the gene for telomerase into mice that had scarring in their lungs due to short telomeres .",
"Povedano , Martínez et al . found that , when injected into the mice , the telomerase gene therapy was able to reach ATII cells and could help to heal the lungs .",
"At the level of individual cells , mice treated with telomerase had longer telomeres , meaning that more of their ATII cells stayed alive and kept dividing to regenerate the lung tissue .",
"Consistent with previous studies , the telomerase gene therapy caused no negative side effects in the mice; for example , there was no increased risk of cancer .",
"These findings may possibly lead to new treatments for those patients suffering from IPF associated with short telomeres .",
"Developing this approach into a clinical trial could in the future benefit many IPF patients who currently have very limited treatment options ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology",
"immunology and inflammation"
] | Induction of homologous recombination between sequence repeats by the activation induced cytidine deaminase (AID) protein | elife-03110-v1 | [
[
"The activation induced cytidine deaminase ( AID ) is essential for all types of B cell-specific immunoglobulin ( Ig ) gene diversification—somatic hypermutation ( SH ) , gene conversion ( GC ) , and class switch recombination ( CSR ) ( Muramatsu et al . , 2000; Revy et al . , 2000; Arakawa et al . , 2002; Harris et al . , 2002 ) .",
"AID likely initiates these processes by the deamination of deoxycytidines to uracils , as inactivation of the DNA Uracil Glycosylase ( UNG ) gene changed the SH spectrum at C/G bases toward transitions and impaired GC and CSR ( Di Noia and Neuberger , 2002; Rada et al . , 2004; Saribasak et al . , 2006 ) .",
"Abasic sites resulting from the excision of AID-induced uracils are most likely removed by the apurinic/apyrimidinic endonuclease 1 ( Masani et al . , 2013 ) , but how the nicked DNA strands are further processed to initiate alternatively CSR , GC , or SH is poorly understood .",
"AID-dependent double-strand breaks—believed to be generated by deamination of nearby cytidines on both strands of sequence repeats within switch regions—have been detected during CSR ( Wuerffel et al . , 1997; Petersen et al . , 2001; Rush et al . , 2004; Schrader et al . , 2005 ) .",
"These breaks are normally joined by non-homologous end joining leading to the deletion of the intervening DNA sequence , but erroneous repair may lead to chromosomal translocations ( reviewed by Boboila et al . , 2012 ) .",
"It is uncertain , however , whether double-strand breaks routinely accompany SH , since breaks within hypermutating V segments were subsequently found to be AID independent ( Papavasiliou and Schatz , 2002; Bross and Jacobs , 2003 ) .",
"Similarly , it remains unclear whether GC is initiated by a double strand-break or by a single-strand nick ( Yabuki et al . , 2005; Nakahara et al . , 2009 ) .",
"A number of studies have suggested that AID-mediated DNA lesions could be repaired by homologous recombination .",
"Phosphorylated AID was reported to interact with Replication Protein A ( RPA ) , a protein also required for recombination , which then recruited AID to single-stranded DNA and facilitated the deamination of cytidines ( Basu et al . , 2005 ) .",
"The RPA that accumulated at sites of AID-mediated DNA damage was subsequently shown to be associated with the RAD51 recombination protein ( Yamane et al . , 2013 ) , suggesting the formation of homologous recombination intermediates .",
"Notably , inhibition of homologous recombination decreased the viability of B cells expressing AID and induced widespread double-strand breaks and genomic instability ( Hasham et al . , 2010 ) .",
"The chicken B-cell line DT40 , easily modified by targeted gene integration ( Buerstedde and Takeda , 1991 ) , is a useful model to study AID-mediated GC and SH ( Di Noia and Neuberger , 2002; Arakawa and Buerstedde , 2009 ) .",
"DT40 modifies its rearranged Ig light chain gene primarily by uni-directional gene conversion using nearby pseudo-V genes as conversion donor sequences ( Buerstedde et al . , 1990 ) .",
"However , GC decreases and SH increases if homologous recombination is impaired by inactivation of RAD51 paralogues ( Sale et al . , 2001 ) , upon deletion of the upstream pseudo V genes that act as GC donor sequences ( Arakawa et al . , 2004 ) or following inactivation of the UNG gene ( Saribasak et al . , 2006 ) .",
"SH of the Ig light chain gene , as well as a green fluorescence protein ( GFP ) transgene were found to be strongly increased in the presence of a nearby Diversification Activator ( DIVAC ) sequence ( Blagodatski et al . , 2009 ) .",
"It was recently shown that this DIVAC consists of the chicken enhancer and enhancer-like elements and that it can be replaced by the human Ig lambda ( hIgλE ) and Ig heavy intron ( IgHiE ) enhancers ( Buerstedde et al . , 2014 ) .",
"CSR has been extensively studied using the murine B cell line CH12 ( Whitmore et al . , 1991; Kinoshita et al . , 1998 ) .",
"This model system responds to the appropriate stimulation by increasing both Ig switch region transcription and AID expression ( Muramatsu et al . , 2000 ) and by recombining its endogenous Ig heavy chain locus or transfected switch region constructs by CSR .",
"While AID has been known to induce homologous recombination in the form of unidirectional gene conversion ( Arakawa et al . , 2002; Harris et al . , 2002 ) , there was previously no direct evidence that AID could mediate more complex rearrangements of sequence repeats .",
"Using both DT40 and CH12 cells , we demonstrate here that AID can induce homologous recombination of repeats ( RR ) leading to frequent deletions between direct repeats ."
],
[
"Transfection of GFP-based hypermutation reporter constructs into DT40 suggested that in rare instances in which transgenes had undergone multi-copy integration there was frequent copy number contraction during cell clone expansion ( data not shown ) .",
"To investigate this unexpected form of genomic instability in AID expressing cells , we decided to develop dual color reporters to reveal transgene recombination by changes in cellular fluorescence .",
"As initial controls , single red fluorescence protein ( RFP ) reporters similar to the previously described GFP2 ( Blagodatski et al . , 2009 ) were made encoding either the tdTomato ( tdT ) or DS-Red Express ( DsR ) gene ( Figure 1A ) .",
"The RFP genes were efficiently translated due to the presence of two in-frame ATG start codons , one at the beginning of the 5′ untranslated exon and one at the beginning of the RFP open reading frame ( marked by arrows in Figure 1A ) .",
"These constructs as well as all others used in our study were integrated in a targeted manner into the IgL ( − ) DT40 cell line at the position of the deleted IgL locus ( Blagodatski et al . , 2009 ) .",
"FACS analysis of subclones of the transfectants revealed a trailing cloud of cells with decreased red fluorescence ( Figure 1B , C ) .",
"Red fluorescence loss was stimulated 10- to 30-fold in the presence of the human Ig lambda enhancer ( hIgλE ) DIVAC ( Buerstedde et al . , 2014 ) ( compare Figure 1B2 to Figure 1B3 , Figure 1B4 to Figure 1B5; and Figure 1C ) .",
"These results , combined with our previous extensive analyses of GFP-based reporter constructs ( Blagodatski et al . , 2009; Buerstedde et al . , 2014 ) , suggested that the loss of red fluorescence was due to inactivation of the RFP transgenes by AID dependent , DIVAC-stimulated hypermutation events .",
"Interestingly , a discrete cell population of intermediate red fluorescence was seen in transfectants of the tdT but not the DsR gene constructs ( Figure 1B2 and Figure 1B3 , circled ) .",
"Since tdT is encoded by a direct tandem repeat of a RFP gene sequence ( Shaner et al . , 2004 ) , the intermediate red fluorescence possibly reflected the loss of one of the tdT repeats by intragenic recombination . 10 . 7554/eLife . 03110 . 003Figure 1 . FACS analysis of DT40 transfectants expressing red fluorescence reporter constructs .",
"( A ) Diagrams of the constructs .",
"Sequences important for the behavior of the construct are labeled and color coded: hIgλE—human Igλ enhancer; RSV—rous sarcoma virus promoter; u-exon-intron-exon—upstream splice cassette; tdT and DsR–Tomato and DsRed open reading frames , respectively; IRES-bsr–internal ribosome entry site followed by the blasticidin resistance open reading frame; SV40 polyA—SV40 polyadenylation signal .",
"The names of the constructs indicate the presence of DIVACs , the promoter , the fluorescence genes and sequence repeats .",
"The tandem repeats of tdT are marked by lines with arrows .",
"( B ) Two color FACS dot plots of the non-transfected IgL ( − ) cell line and representative subclones derived from primary transfectants .",
"The levels of green and red fluorescence are plotted according to the x-axis ( FL1 ) and y-axis ( FL2 ) , respectively .",
"The number of the plot and the name of the transfected construct are indicated above .",
"Gates and the percentage of gated cells are indicated within the plots .",
"Populations of intermediate red fluorescence are circled .",
"( C ) Graphs showing the percentages of gated cells for all subclones of each independent transfectant .",
"The median percentage of gated cells is indicated by the bar and numerically displayed above the graph for each transfectant . DOI: http://dx . doi . org/10 . 7554/eLife . 03110 . 003 We then proceeded to reporters containing two fluorescent reporter genes: an upstream tdT which was efficiently translated due to the presence of the same in-frame ATG start codons as in the single RFP control constructs ( marked by arrows in Figure 2A ) , and a downstream GFP which lacked an ATG start codon .",
"For simplicity and consistency , the names of the dual color reporter constructs reflected only the RFP gene ( either tdT or DsR ) , they contained and the presence of DIVACs and sequence repeats .",
"In the first of these constructs , DIVAC_tdT , the upstream tdT and the downstream GFP genes shared no sequence homology and were driven by different promoters , RSV and Ubiquitin C , respectively ( Figure 2A ) .",
"Transfectants of the DIVAC_tdT construct gave rise to cell populations of decreased red fluorescence in the R3 gate and of intermediate fluorescence ( Figure 2B1 , Figure 2C ) identical to the ones seen for single tdT gene transfectants ( Figure 1B3 , Figure 1C ) . 10 . 7554/eLife . 03110 . 004Figure 2 . FACS analysis of DT40 transfectants expressing dual tdT/GFP fluorescence reporter constructs .",
"( A ) Diagrams of the constructs .",
"Sequences important for the behavior of the construct are labeled and color coded as in Figure 1 and explained in the following: hIgHiE—human Ig heavy chain intron enhancer; UB promoter—human Ubiquitin C promoter; d-exon-intron-exon—downstream splice cassette; GFP—GFP open reading frame; BGH polyA—Bovine Growth Hormone polyadenylation signal .",
"The names of the dual fluorescence constructs indicate only the presence of DIVACs , the RFP gene and sequence repeats .",
"( B ) FACS dot plots of representative subclones derived from primary transfectants .",
"The number of the plot and the name of the transfected construct are indicated above .",
"Plots of subclones in which the AID expression cassette have been deleted are labeled AID− .",
"Gates and the percentage of gated cells are indicated within the plots .",
"( C ) Graphs for each gate showing the percentages of gated cells for all subclones of each independent transfectant .",
"The median percentage of gated cells is indicated by the bar and numerically displayed above the graph for each transfectant . DOI: http://dx . doi . org/10 . 7554/eLife . 03110 . 004 To test whether the presence of sequence repeats induced instability , a 344 bp direct repeat sequence ( iHS ) was inserted into the introns of both the tdT and the GFP genes yielding the DIVAC_iHS_tdT_iHS construct ( Figure 2A ) .",
"Subclones of transfectants showed , in addition to cells in the R3 gate ( hereafter , R3 cells ) , green fluorescence positive , red fluorescence negative cells in the R1 gate at median frequencies of about 0 . 8% ( Figure 2B2 , Figure 2C ) .",
"Cells of this phenotype were expected to arise if homologous recombination between the iHS sequences deleted the tdT gene and activated GFP translation due to the gain of the first ATG start codon previously located upstream of tdT .",
"In other constructs , both the tdT and GFP genes were driven by RSV promoters and they either lacked ( RSV_tdT_RSV ) or contained ( DIVAC_RSV_tdT_RSV and DIVAC2_RSV_tdT_RSV ) a DIVAC element ( Figure 2A ) .",
"Surprisingly , subclones gave rise to red fluorescence negative cells of intermediate green fluorescence in the R2 gate ( note that homologous recombination between the RSV promoters does not provide GFP with a start codon and hence would not be expected to yield cells with strong GFP fluorescence in the R1 gate ) .",
"The appearance of these cells was strongly enhanced in the presence of either the hIgλE or hIgHiE DIVAC ( compare Figure 2B4 to Figure 2B5 , B7; and Figure 2C ) .",
"Removal of the AID expression cassette from the DIVAC_iHS_tdT_iHS and DIVAC_RSV_tdT_RSV transfectants resulted in stable red fluorescence only expression ( Figure 2B3 , 2B6 , Figure 2C ) indicating that all fluorescence variation depended on AID .",
"Similar results were obtained after transfection of constructs containing the DsR gene instead of the tdT gene ( Figure 3A–C ) . 10 . 7554/eLife . 03110 . 005Figure 3 . FACS analysis of DT40 transfectants expressing dual DsR/GFP fluorescence reporter constructs .",
"( A ) Maps of the constructs .",
"( B ) FACS dot plots of representative subclones derived from primary transfectants .",
"( C ) Graphs for each gate showing the percentages of gated cells for all subclones of each independent transfectant .",
"The median percentage of gated cells is indicated by the bar and numerically displayed above the graph for each transfectant . DOI: http://dx . doi . org/10 . 7554/eLife . 03110 . 005 The blasticidin resistance gene ( bsr ) , which is positioned between the RFP and GFP genes ( Figure 2A ) , would be lost if the iHS or the RSV promoter repeats of the dual fluorescence constructs underwent homologous recombination of repeats ( RR ) .",
"Addition of blasticidin to the culture indeed eliminated R1 cells from the DIVAC_iHS_tdT_iHS transfectant and strongly reduced R2 cells of RSV repeat transfectants ( Figure 4A , compare upper to lower panel ) consistent with the deletion of bsr in R1 and R2 cells .",
"In contrast , R3 cells and cells of intermediate red fluorescence remained blasticidin resistant ( Figure 4A , lower panel ) , as expected if these events reflected inactivation of the RFP gene by hypermutation and intragenic recombination of the tdT repeats , respectively . 10 . 7554/eLife . 03110 . 006Figure 4 . Homologous recombination of sequence repeats in gated cells .",
"( A ) FACS profiles of primary transfectants 20 days after transfection .",
"Cells analyzed in the upper row were cultured in the absence of blasticidin , those in the lower row in the presence of blasticidin .",
"( B ) Representative FACS profiles of subclones derived from a cell of intermediate red fluorescence or from R2- and R3-gated cells .",
"The transfected constructs are indicated above the profiles by name , with the origin of the precursor cell indicated by the suffix .",
"( C ) Top: agarose gel electrophoresis of PCR products amplified from DNA of the DIVAC_RSV_tdT_RSV transfectant and two of its subclones .",
"The first subclone is derived from a cell of intermediate red fluorescence , the second from a R2 cell .",
"The primers used are indicated above the numbered lanes .",
"Bands representing amplifications of the un-rearranged and rearranged tdT genes are marked by asterisks in lane 2 .",
"Below: the diagram shows the positions of the primers and the expected sizes of the PCR products for the transfected construct and its recombinants .",
"The increased GFP expression of recombinants is indicated by color changes of the rectangle representing the GFP open reading frame . DOI: http://dx . doi . org/10 . 7554/eLife . 03110 . 006 Subclones derived from cells showing altered fluorescence continued to generate variant cell populations during expansion ( Figure 4B ) .",
"A subclone derived from a cell of intermediate red fluorescence still generated R2 and R3 cells presumably due to ongoing RSV repeat recombination and hypermutation of the recombined tdT gene respectively ( Figure 4B1 ) .",
"R2 subclones still generated R3 cells presumably due to deleterious hypermutation of the rearranged GFP gene ( Figure 4B2 and 2B3 ) , and a R3 subclone still generated R2 cells as expected for ongoing recombination of the RSV repeat ( Figure 4B4 ) .",
"To detect rearrangements of the constructs , genomic DNA of a DIVAC_RSV_tdT_RSV primary transfectant as well as subclones derived thereof from an intermediate red fluorescence cell or an R2 cell was amplified by PCR using various combinations of primers ( Figure 4C ) .",
"Whereas the 1/3 primer pair amplified two fragments of about 1 . 7 kb and 2 . 5 kb from the primary transfectant ( Figure 4C , lane 2 marked by asterisks ) , amplification from the intermediate red fluorescence subclone yielded only the lower fragment consistent with the deletion of one of the tdT repeats in cells of intermediate fluorescence ( Figure 4C , lane 6 ) .",
"The 1/2 and 1/3 primer pairs did not amplify the DNA of the R2 subclone ( Figure 4C , lane 9 and 10 ) and the 1/4 and 1/5 primer pairs amplified only fragments of about 1 . 1 kb and 1 . 9 kb ( Figure 4C , lane 11 and 12 ) , but not the larger fragments of 4–5 kb size seen in addition to the lower size fragments in amplifications from the primary transfectant ( Figure 4C , lane 3 and",
"4 ) and the intermediate red fluorescence subclone ( Figure 4C , lanes 7 and 8 ) .",
"This was consistent with a large deletion that included one RSV repeat and the intervening sequences in R2 cells .",
"The 1 . 9-kb fragment amplified by primer pair 1/5 from sorted DIVAC_RSV_tdT_RSV R2 cells was subcloned and sequenced .",
"12 sequences showed deletions of one RSV repeat and the intervening sequence with no additional nucleotide changes when compared to the sequence of the transfected construct ( data not shown ) .",
"These analyses demonstrate that AID induces intragenic homologous recombination between the tdT repeats and intergenic recombination between the RSV promoter sequences at high frequencies .",
"However , as the deletion in R2 cells included the ATG codons upstream of the tdT gene , the reason for the increased green fluorescence of R2 cells remains unclear .",
"It might be related to enhanced GFP transcription or the gain of an alternative GFP translation start codon in the recombinants .",
"To better understand AID-mediated deletions , we made the DIVAC_uHS_tdT_dHS construct ( Figure 5A ) in which an upstream ( uHS ) and downstream ( dHS ) homeologous sequence differed about every 50 base pairs by single nucleotide substitutions , deletions , or insertions .",
"The duplicated sequence consisted of the RSV promoter and a 560-bp sequence encompassing the first exon , the intron , and the beginning of the second exon .",
"As only uHS provided in-frame ATG start codons , the unmutated form of the construct supported tdT but not GFP expression .",
"However , recombination events accompanied by a crossover downstream of the first ATG start codon would place this codon upstream of the GFP gene , causing loss of red fluorescence and gain of green fluorescence . 10 . 7554/eLife . 03110 . 007Figure 5 . FACS profiles of transfectants of the DIVAC_uHS_tdT_dHS construct .",
"( A ) Diagram of the construct .",
"The upstream and downstream homeologous sequence repeats ( uHS and dHS , respectively ) are highlighted .",
"( B ) FACS profile of a representative subclone .",
"( C ) Graphs showing the percentages of gated cells for subclones of three independent transfectants .",
"The median percentage of gated cells of all subclones is indicated by the bar and numerically displayed above the graph for each transfectant . DOI: http://dx . doi . org/10 . 7554/eLife . 03110 . 007 DIVAC_uHS_tdT_dHS transfectants generated cells in the R1 , R2 , and R3 gates , as well as cells of intermediate red fluorescence in the R4 gate ( Figure 5B , C ) as expected from the behavior of previous constructs .",
"The median percentage of R1 and R2 events was lower , however ( Figure 5B , C ) , most likely due to inhibition of homologous recombination by the heterologies of the uHS/dHS sequences .",
"At least two other minor populations of increased green or red fluorescence were seen in the R5 and R6 gates , respectively ( Figure 5B , C ) .",
"A 6-week culture of a primary DIVAC_uHS_tdT_dHS transfectant was sorted for cells in the R1–R6 gates ( Figure 6A ) , and the sorted populations were subcloned .",
"Only subclones derived from R1- and R2-gated cells were killed upon addition of blasticidin , indicating that these cells but not the cells in other gates had deleted the bsr gene ( data not shown ) .",
"Subclones derived from gated cells in turn generated subpopulations with characteristic patterns of fluorescence .",
"Subclones derived from R1 ( Figure 6B1 ) and R2 ( Figure 6B2 ) cells produced double negative R3 cells presumably due to the inactivation of the rearranged GFP gene by hypermutation .",
"Subclones derived from double negative R3 cells ( Figure 6B3 ) gave rise to green fluorescence positive cells , presumably due to uHS/dHS repeat recombination , but also to cells having regained red fluorescence presumably due to repair of their mutated tdT gene .",
"R5 subclones generated R2 and R3 cells , as well as cells of intermediate red fluorescence relative to R5 cells , but no events in the R1 gate ( Figure 6B5 ) .",
"Finally , R6 subclones produced a FACS profile similar to that of the DIVAC_uHS_tdT_dHS primary transfectant with the difference that all cell populations displayed increased green fluorescence ( Figure 6B6 and data not shown ) . 10 . 7554/eLife . 03110 . 008Figure 6 . Recombination of the uHS/dHS repeat in R1 and R2 cells of the DIVAC_uHS_tdT_dHS transfectant .",
"( A ) FACS profile of a DIVAC_uHS_tdT_dHS transfectant showing the gates used for preparative sorts .",
"( B ) FACS plots of representative subclones .",
"The gate from which the precursor cell of the subclone is derived is shown above the plot .",
"( C ) Agarose gel electrophoresis of PCR products amplified from DNA of subclones which were derived for gated cells as indicated on top of the gel image .",
"The primer pairs used for the amplifications are shown above the lanes .",
"The lower scheme shows the positions of the primers and the expected sizes of the PCR products for the transfected construct and its recombinants . DOI: http://dx . doi . org/10 . 7554/eLife . 03110 . 008 Genomic DNA of two subclones of each gated population was analyzed by PCR ( Figure 6C ) .",
"While the 1/3 primer pair did not amplify DNA from R1 ( Figure 6C , lane 1 and",
"3 ) and R2 ( Figure 6C , lane 5 and 7 ) subclones , the 1/5 primer pair produced only a single fragment of about 2 . 3 kb as expected for uDS/dHS recombination in the precursor cell of the R1 and R2 subclones ( Figure 6C , lane 2 , 4 , 6 and 8 ) .",
"Amplification from R4 subclones by the 1/3 primer pair produced a single fragment of about 2 . 2 kb ( marked by an asterisk in Figure 6C , lane 13 and 15 ) consistent with the deletion of one tdT repeat in R4 cells .",
"PCR amplifications from the R3 , R5 , and R6 subclones produced band patterns similar to one another , showing fragments of the size expected for cells carrying the non-rearranged construct as well as smaller fragments expected from cells having undergone either tdT ( detected by the 1/3 primer pair ) or uHS/dHS repeat ( detected by 1/5 primer pair ) recombination during subclone expansion ( Figure 6C , lanes 9–12 and 17–24 ) .",
"This indicated that R1 and R2 cells had recombined uHS/dHS , and R4 cells had recombined the tdT repeat , whereas R3 , R5 , R6 cells did not carry detectable rearrangements .",
"If the deletions in R1 and R2 cells occurred by homologous recombination , the nucleotide differences distributed along the entire length of uHS and dHS ( Figure 7A ) would allow the mapping of crossover sites into an interval between two polymorphic positions .",
"Analysis of recombined sequences amplified by the 1/5 primer pair from gated R1 and R2 cells indeed provided evidence for variable crossover positions ( Figure 7B ) .",
"All R1 sequences had maintained uHS-specific sequence at least until the single nucleotide insertion polymorphism ( marked by two vertical arrows in Figure 7A ) that puts the upstream ATG codon in frame with tdT prior to recombination and in frame with GFP after recombination .",
"This explains the strong increase in green fluorescence seen in R1 cells .",
"In contrast , all R2 sequences maintained dHS-specific sequence at this position ( Figure 7B ) suggesting again that modest increase of green fluorescence in R2 cells is either due to enhanced GFP transcription or the gain of an alternative GFP translation start codon . 10 . 7554/eLife . 03110 . 009Figure 7 . Sequences derived from gated cells of the DIVAC_uHS_tdT_dHS transfectant .",
"( A ) Map of the construct showing the polymorphisms of the aligned uHS and dHS sequences .",
"The types of polymorphism are coded above the uHS sequence .",
"Asterisks indicate nucleotide substitutions while triangles pointing up and down indicate single nucleotide insertions and deletions , respectively , within uHS .",
"The positions of the first and second ATG start codon of uHS are highlighted by single arrows , and the position of the single nucleotide deletion that puts the first ATG in frame with tdT is indicated by stacked arrows .",
"In all parts of the figure vertical lines within the sequence bars indicate uHS specific sequence .",
"( B ) Schematic representation of the sequences of uHS/dHS recombinants amplified from sorted R1 and R2 cells .",
"uHS specific nucleotides downstream of dHS nucleotides are marked by red circles .",
"( C ) uHS sequences amplified from sorted R5 cells .",
"( D ) A dHS sequence amplified from a R6 subclone . DOI: http://dx . doi . org/10 . 7554/eLife . 03110 . 009 Five R1 sequences ( S1 , S2 , S3 , S4 , and S7 ) maintained uHS sequence down to the last polymorphic nucleotide in front of the GFP reading frame and five R2 sequences ( S14 , S20 , S23 , S26 , S30 ) maintained dHS sequence up to the last polymorphic nucleotide downstream of the hIgλE sequence , suggesting preferential crossover events near the boundaries of sequence homology .",
"Interestingly , six sequences from R2 cells ( S9 , S10 , S12 , S13 , S18 , S21 ) contained either single or multiple dHS-specific nucleotides upstream of uHS-specific nucleotides ( marked by red circles in these sequence in Figure 7B ) , most easily explained by mismatch repair of recombination associated heteroduplexes .",
"Four sequences amplified from gated R4 cells by the 1/3 primer pair showed deletion of one tdT repeat and the 69-bp intervening sequence between tdT repeats with no other sequence changes confirming that cells of intermediate red fluorescence were due to intragenic tdT repeat recombination ( data not shown ) .",
"Two uHS sequences amplified by the 1/3 primer pair from R5 subclones showed that the middle part had converted to dHS-specific sequence thereby erasing the first ATG start codon ( Figure 7C ) .",
"If the canonical tdT protein translated from the second ATG codon were more active than the artificial tdT fusion protein translated from the first ATG codon , this would explain the increased red fluorescence of R5 cells .",
"The loss of the first ATG start codon is also consistent with the observation that R5 subclones were unable to generate R1 cells .",
"The observed sequence changes likely reflected unidirectional dHS-templated gene conversions , as an increase of green fluorescence was not observed in R5 cells , but would have been expected if a reciprocal exchange had introduced the first ATG codon into dHS .",
"Surprisingly , dHS sequences amplified from an R6 subclone ( with very high green fluorescence ) by the 1/5 primer pair showed the insertion of seven uHS specific nucleotides including the downstream ATG start codon upstream of the GFP coding sequence ( Figure 7D ) .",
"This change—explaining the strongly increased green fluorescence of R6 cells—was most likely enabled by a stretch of 12 identical nucleotides at the beginning of the tdT and GFP coding sequences .",
"These results show that the appearance of R5 cells can be explained by dHS-templated gene conversion leading to the loss of the first ATG codon in uHS whereas the appearance of R6 cells reflect uHS-templated gene conversion leading to the gain of the second ATG codon at the very end of dHS .",
"Gene conversions within the uHS/dHS repeat occurred about 3–10 times less frequently than deletions by RR , based on the analysis of subclones of three independent DIVAC_uHS_tdT_dHS transfectants ( compare the medians of R1 and R2 vs R5 and R6 events in Figure 5C ) .",
"To determine whether RR can accompany CSR in chicken DT40 and murine CH12 cells , we designed the DIVAC_RSV_Sμ_tdT_RSV_Sα construct in which both the tdT and the GFP genes were driven by RSV promoters and switch region sequences were placed within intron sequences ( Figure 8A ) .",
"In this construct , CSR between the two S regions should generate R1 cells ( high GFP due to the start codon upstream of the recombined S regions ) , whereas RR of the RSV promoter regions should generate R2 cells ( low GFP ) , as described above . 10 . 7554/eLife . 03110 . 010Figure 8 . RSV repeat and class switch recombination after transfection of CH12 cells .",
"( A ) Diagram of the CH12 DIVAC_RSV_Sμ_tdT_RSV_Sα construct .",
"Sμ and Sα—portions of the murine Sμ and Sα switch regions .",
"( B ) FACS profile of a representative DT40 transfectant as well as an uninduced and induced CH12 transfectant .",
"( C ) FACS profile of the induced CH12 transfectant showing the gates of the preparative sorts .",
"( D ) Top: agarose gel electrophoresis of PCR products amplified by the 1/5 primer pair from DNA of sorted DT40 R1 cells , the induced CH12 transfectant and sorted CH12 R1 and CH12 R2 cells .",
"Bottom: the diagrams show the positions of the primers and the expected sizes of the PCR products for the transfected construct and its recombinants . DOI: http://dx . doi . org/10 . 7554/eLife . 03110 . 010 When DIVAC_RSV_Sμ_tdT_RSV_Sα was introduced into DT40 by targeted integration , transfectants accumulated cells in the R1 and R2 gates at median frequencies of about 0 . 15% and 5% respectively after 12 days culture ( Figure 8B1 and data not shown ) .",
"Transfectants of CH12 varied in their FACS profiles most likely due to integration of the transfected construct at variable chromosomal positions .",
"However , about one in three transfectants generated a low number of cells similar to R1 and R2 cells of the DT40 transfectants .",
"The frequency at which R1 and R2 cells were generated was strongly increased when factors known to increase both AID expression and Ig CSR ( Kinoshita et al . , 1998 ) were added to the culture of CH12 transfectants ( compare Figure 8B2 to Figure 8B3 and data not shown ) .",
"Whereas the DT40 transfectant showed about 50 times fewer R1 than R2 cells , the frequency of cells in the two gates was roughly equal in CH12 transfectants indicating increased CSR in CH12 as compared to DT40 cells .",
"CH12 transfectants also generated strong subpopulations of R3 cells ( Figure 8B3 ) , but almost all of these cells disappeared when blasticidin was added to the cultures ( data not shown ) suggesting that these cells arose through transcriptional silencing of the tdT gene and not inactivation by hypermutation .",
"R1 cells of a DT40 transfectant as well as R1 and R2 cells of a stimulated CH12 transfectant ( Figure 8C ) were sorted yielding the cell populations DT40 R1 , CH12 R1 , and CH12 R2 , respectively .",
"DNA from the sorted populations as well as from the unsorted CH12 transfectant were amplified by the 1/5 primer pair .",
"Amplification from DT40 R1 ( Figure 8D , lane",
"1 ) and CH12 R1 ( Figure 8D , lane",
"3 ) cells produced a smear of fragments in the range of about 2 . 5–3 . 2 kb as expected for diverse switch region recombination events .",
"In contrast , a discrete fragment of about 3 . 1 kb was amplified from CH12 R2 cells ( Figure 8D , lane",
"4 ) and to a lesser degree from CH12 unsorted cells ( Figure 8D , lane",
"2 ) as expected for RSV repeat recombination .",
"Fragments amplified from DT40 R1 and CH12 R1 cells were subcloned and sequenced .",
"Sequences from both DT40 and CH12 cells had joined the upstream Sμ to downstream Sα switch regions at variable positions displaying unusually long junctional microhomologies ( Figure 9A , B ) similar to recombination events previously reported using similar constructs ( Kinoshita et al . , 1998; Okazaki et al . , 2002 ) .",
"To confirm recombination of RSV repeats in CH12 cells , we subcloned and sequenced the 3 . 1 kb band amplified from sorted CH12 R2 DNA .",
"Ten sequences showed uniform deletions of one RSV repeat and the intervening sequence with no other sequence changes as expected for faithful RR events .",
"These results demonstrate that DT40 and CH12 cells carried out both RSV and switch region recombination , but the absolute and relative frequencies of switch recombination were about 5 and 30-fold higher , respectively , in induced CH12 cells . 10 . 7554/eLife . 03110 . 011Figure 9 . Sequences of switch region recombinants recovered from sorted DT40 R1 and CH12 R1 cells .",
"( A ) Deletions induced by the joining of Sμ and Sα switch regions are indicated below in the map of the DIVAC_RSV_Sμ_tdT_RSV_Sα construct by horizontal arrows .",
"( B ) Switch region junctions are aligned to the sequence of Sμ and Sα .",
"Aligned Sμ and Sα sequences of the construct are shown above and below , respectively , the sequence of each switch recombinant .",
"Junctional microhomologies are marked by a line above the sequence . DOI: http://dx . doi . org/10 . 7554/eLife . 03110 . 011"
],
[
"The finding that AID can induce chromosomal deletions by homologous recombination of repeats ( RR ) adds another activity to the remarkable array of AID-induced mutation and recombination events .",
"RR mediated deletions could be visualized after chromosomal integration of fluorescent reporter constructs by virtue of changes in the fluorescence profiles of individual cells within expanding cultures .",
"Deletions occurred at high frequencies in different sequence contexts and in both the chicken DT40 and the murine CH12 cell lines .",
"Interestingly , transfectants of DT40 recombined the duplicated RSV promoters and tdT repeats as frequently by RR as they inactivated the RFP genes by somatic hypermutation ( SH ) .",
"Although DT40 diversifies its Ig genes by gene conversion ( GC ) , an artificial homeologous sequence upstream of the tdT and GFP genes was recombined more often by RR than it was modified by GC .",
"Similarly , class switch recombination ( CSR ) active CH12 cells rearranged a construct containing duplicated RSV promoters and intronic switch regions as often by RR as by CSR .",
"AID-induced RR does not seem to involve error prone DNA synthesis because precise deletions , but no non-templated nucleotide changes , were encountered in a large number of recombinant sequences .",
"Our data indicate that AID-mediated RR can be a remarkably efficient process .",
"To explain RR , we propose that the single strand nick—believed to occur after the excision of an AID-induced uracil ( Figure 10B ) —is further processed into a homologous recombination intermediate .",
"This could either be a single strand gap generated by nuclease mediated resection of the nicked strand ( Model 1 , Figure 10C–F ) or a double strand break believed to occur when the replication fork collapses at the site of the unrepaired single strand nick within one of the template strands ( Kowalczykowski , 2000; Petermann and Helleday , 2010 ) ( Model 2 , Figure 10G–K ) .",
"Since recombination occurred at high frequency between the RSV repeats , which are upstream of the transcription start site and hence likely to be hundreds of bases away from the peak of AID-induced cytidine deamination ( Saribasak et al . , 2006 ) , both models invoke substantial DNA resection to expose single stranded DNA distant from the nicks ( Figure 10C , I ) .",
"The fact that RPA ( Hakim et al . , 2012 ) as well as RPA together with RAD51 ( Yamane et al . , 2013 ) accumulated at multiple chromosomal positions within activated murine B cells in an AID-dependent fashion is consistent with the formation of recombination competent nucleoprotein filaments .",
"Later stages of the models postulate heteroduxplex DNA and Holliday junctions ( Figure 10E , I ) , which are consistent with the sequence analysis of homeologous repeat recombinants ( Figure 7 ) .",
"Nevertheless , the choice , validity , and details of the two alternative models remain speculative .",
"Further insight into the RR reaction will likely be provided by the analysis of constructs containing inverted repeats and the behavior of constructs in cells with specific recombination and repair factor deficiencies ( Hu et al . , 2013; Willis et al . , 2014 ) . 10 . 7554/eLife . 03110 . 012Figure 10 . Models for AID induced Recombination of Repeats ( RR ) assuming initiation of homologous recombination by a single strand gap or a double strand break .",
"( A ) The diagrams show two neighboring genes containing direct sequence repeats marked in red and brown .",
"The transcription start sites and the direction of transcription are indicated by horizontal arrows .",
"Deamination of a cytidine by AID within the transcribed sequence of the first gene , marked in blue , leads to an uracil/guanidine base pair ( ‘U’ opposite to ‘G’ ) .",
"( B ) Removal of the uracil and cleavage of the abasic site results in a single-strand nick that is postulated to be a common intermediate for the two models .",
"( C ) The first model ( Model 1 , C–F ) assumes that 5′ to 3′ resection of the nick produces a gapped DNA duplex and that the unpaired , continuous strand initiates the search for homology .",
"( D ) D-loop formation at the downstream repeat sequence .",
"( E ) Following strand exchange DNA synthesis shown by the dashed lines fills in the gap in the DNA duplex .",
"( F ) Cleavage of the Holliday junctions in one plane creates a chromosome containing a deletion and a circular DNA molecule of the deleted sequence .",
"Cleavage of the Holliday junctions in the other plane would result in a chromosome with no deletion ( not shown ) .",
"( G ) The second model ( Model 2 , G–K ) assumes replication arrest at the AID-induced nick .",
"( H ) The nick is converted into a single ended DSB due to replication fork collapse .",
"( I ) Upon resection the DSB erroneously re-initiates the replication fork at the position of the downstream repeat sequence .",
"( J ) The Holliday junction is cleaved and replication continues .",
"( K ) Different sister chromatids , one carrying a deletion and one without a deletion , are produced by completion of replication . DOI: http://dx . doi . org/10 . 7554/eLife . 03110 . 012 RR needs to be thought about within the context of Ig locus diversification and the repair of AID-induced DNA damage .",
"For example , it could be responsible for V gene replacements that are not associated with cryptic V ( D ) J signal recognition sequences , as these were observed in AID expressing germinal center B cells ( Darlow and Stott , 2005 ) .",
"Similar to RR , GC , which diversifies the rearranged Ig genes of B cells in chickens and many mammalian species ( Reynaud et al . , 1987; Butler 1998 ) , requires the interaction of nearby homeologous sequences on the same chromosome ( Arakawa et al . , 2004; Sale , 2004 ) .",
"Intriguingly , the two processes might involve the same intermediates leading to sequence alignment and strand invasion ( Figure 10C , D ) and later diverge when the Holliday junctions ( Figure 10E ) are resolved by cleavage during RR , but by convergent branch migration during GC .",
"How GC and RR are controlled during Ig repertoire development remains unresolved .",
"Both of the fluorescent reporter genes in our constructs were transcribed and expected to accumulate AID induced nicks which might facilitate RR .",
"On the contrary , the pseudo V genes within the chicken Ig loci are unlikely to be transcribed , and the absence of transcription or their less accessible chromatin configuration may favor GC .",
"While possibly involved in the repair of AID-induced DNA damage , RR is also likely to add to the mutation signature of AID by introducing deletions and inversions of repetitive sequences .",
"AID expressing B cells are prone to transformation ( Robbiani and Nussenzweig , 2013 ) and mutation signatures of APOBEC homologues are found in the genomes of many cancer cells ( Alexandrov et al . , 2013; Burns et al . , 2013; Taylor et al . , 2013 ) .",
"Cis-acting sequences that activate SH in nearby transcribed sequences and which have been termed DIVersification ACtivators ( DIVACs ) ( Buerstedde et al . , 2014 ) , strongly stimulated the frequency of RR , revealing an additional risk to genome integrity posed by DIVAC-like sequences outside of the Ig loci .",
"However , RR-mediated deletions were also detectable after transfection of ‘no DIVAC’ containing control constructs ( e . g . , R2 events arising from the RSV_tdT_RSV substrate; Figure 2B , C ) .",
"Thus , AID and perhaps other cytidine deaminases represent a general threat to transcribed regions of the genome via multiple mechanisms including RR , as reported here .",
"Given the highly repetitive nature of mammalian genomes , even a low level of RR could significantly contribute to genome instability and B cell transformation ."
],
[
"The RSV promoter , GFP , IRES-Bsr , and SV40 polyA sequences used for the RFP gene expression cassettes and the hIgλE and hIgHiE DIVACs had been previously described ( Buerstedde et al . , 2014 ) .",
"The u-exon-intron-exon sequence was derived from the leader-intron-V gene sequence of the chicken IgL gene .",
"The tdT , DsR , Ubiquitin C promoter , d-exon-intron-exon sequence , and the BGH polyA signal were amplified from Addgene plasmids pcDNA3 . 1 ( + ) /Luc2 = tdT , pDsRed-Sensor , pUB-GFP , pCIFlagPCAF , and pcDNA3 . 1 ( + ) /Luc2 = tdT , respectively .",
"The iHS and the uHS sequence was custom synthesized .",
"The dHS sequence was identical to the RSV promoter and the d-exon-intron-exon sequence .",
"The partial Sμ and Sα switch regions of about 1 . 3 kb and 1 . 2 kb size were amplified from the plasmid SCI ( μ , α ) ( Okazaki et al . , 2002 ) .",
"All cloning steps , sometimes including additions or deletions of restrictions sites , were done by the Infusion Cloning Kit ( Clontech , Mountain View , CA ) after PCR amplifications of fragments using Q5 High-Fidelity DNA Polymerase ( NEB , Ipswich , MA ) .",
"DT40 transfectants having integrated the fluorescence reporter constructs targeted at the position of the deleted IgL locus of the IgL ( − ) variant cell line were identified as previously described ( Blagodatski et al . , 2009 ) .",
"All transfectants were initially selected by blasticidin , but cultured subsequently in the absence of blasticidin .",
"Subcloning was also performed in the absence of blasticidin .",
"To test for blasticidin sensitivity , cultures were split and one half was cultured for 3 days in the presence of blasticidin , the other half in the absence of blasticidin .",
"Transfection of CH12 cells was done using a Gene Pulser Xcell ( BioRad , Hercules , CA ) electroporator and a square wave protocol of 230 V and 20 ms . Transfectants were initially selected in medium containing blasticidin at a concentration of 15 μg/ml .",
"Stimulation of CH12 transfectants was performed in medium containing 5 ng/ml IL4 , 0 . 2 μg/ml anti-CD40 antibody , and 0 . 1 ng/ml TGFβ for 5 days .",
"FACS analysis of transfectants carrying the red and dual fluorescence constructs was similar to the previously described analysis of transfectants carrying green fluorescence reporters ( Blagodatski et al . , 2009; Buerstedde et al . , 2014 ) .",
"Green ( FL1 ) and red fluorescence ( FL2 ) were plotted on the y-axis and x-axis respectively using a FACSCalibur ( BD Biosciences , San Jose , CA ) .",
"Excitation was by the 488 nm laser and appropriate FL1/FL2 and FL2/FL1 settings were used for compensation .",
"Despite likely suboptimal excitation of tdT by the 488 nm laser , the red fluorescence of tdT expressing cells was very bright , causing difficulty in displaying positive and negative cells on the same screen .",
"FACS settings were optimized for the analysis of either tdT or DsR expressing cells and then consistently used for the analysis of respective primary transfectants and subclones .",
"Primary transfectants were analyzed 20 days after transfection ( about 12 days in blasticidin free culture medium ) , subclones were analyzed 12 days after subcloning .",
"Over 20 subclones of each transfectant were usually analyzed .",
"Preparative FACS sorts were performed on Beckman Coulter MoFlo using the following parameters: 488 nm excitation , 100 mW power laser for GFP; 532 nm , 200 mW laser for RFP expression .",
"PCR amplifications were performed using Q5 High-Fidelity DNA Polymerase ( NEB , Ipswich , MA ) and genomic DNA isolated either from primary transfectants , from sorted cell populations or from subclones .",
"PCR reactions were analyzed by electrophoresis on 0 . 8% agarose gels .",
"For sequence analysis , PCR fragments were excised from the agarose gels and cloned into the linearized pUC19 provided with In-Fusion Cloning Kit ( Clontech , Mountain View , CA ) .",
"Sequences of the subcloned PCR fragments were compared to the sequence of the transfected construct using ‘Align two sequences’ blastn searches ( http://blast . ncbi . nlm . nih . gov/ ) ."
]
] | [
"The activation induced cytidine deaminase ( AID ) protein is known to initiate somatic hypermutation , gene conversion or switch recombination by cytidine deamination within the immunoglobulin loci .",
"Using chromosomally integrated fluorescence reporter transgenes , we demonstrate a new recombinogenic activity of AID leading to intra- and intergenic deletions via homologous recombination of sequence repeats .",
"Repeat recombination occurs at high frequencies even when the homologous sequences are hundreds of bases away from the positions of AID-mediated cytidine deamination , suggesting DNA end resection before strand invasion .",
"Analysis of recombinants between homeologous repeats yielded evidence for heteroduplex formation and preferential migration of the Holliday junctions to the boundaries of sequence homology .",
"These findings broaden the target and off-target mutagenic potential of AID and establish a novel system to study induced homologous recombination in vertebrate cells ."
] | [
"Mutation can be harmful because changes to genes can disrupt vital processes or even cause diseases such as cancer .",
"However , some genetic mutations can also be beneficial .",
"Cells of the immune system , for example , need to create antibodies that attack a huge diversity of invading microbes .",
"To do this , immune cells introduce changes into their genes to increase the diversity of the proteins that make up the antibodies .",
"An enzyme called AID is thought to play a crucial role in increasing the diversity of our antibodies by changing specific letters of the genetic code .",
"Now , Buerstedde et al . have shown that the AID enzyme can also cause sections of DNA to be deleted from the genome .",
"Buerstedde et al . constructed pieces of DNA that include , in order , a gene that makes cells glow red , a gene that makes cells resistant to an antibiotic , and a gene that could make cells glow green .",
"However , the very start of the ‘green’ gene was missing , which meant that it was switched off .",
"Stretches of DNA were repeated in front of the ‘red’ and ‘green’ genes in some of the ‘constructs’ .",
"After inserting this DNA into cells from chickens or mice , most cells glowed red , but some started to glow green instead .",
"Green cells were killed by the antibiotic; and were only seen when cells carried the constructs with the repeating DNA .",
"Cells that lacked the AID enzyme only glowed red , regardless of which DNA construct they carried .",
"Buerstedde et al . showed that AID causes the DNA constructs to align and re-arrange at the repeated sequences .",
"As such , when the cells divide and their DNA is separated and packaged into newly formed cells , the DNA between the repeating sequences can be deleted .",
"Thus , cells started to glow green because the ‘on’ switch at the start of the red gene ended up at the start of the green gene when the region in between was deleted .",
"This also explains why green cells always died when exposed to the antibiotic , because this deletion removed the resistance gene too .",
"Buerstedde et al . suggest that when a cell attempts to correct the errors caused by the AID enzyme changing the letters in the DNA , it actually can trigger the exchange and deletion of repeated sequences .",
"Future work is now needed to understand how this new role for the AID enzyme is regulated , and whether this role beneficial or harmful to the immune cells ."
] | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | Regulator of G protein signaling 12 enhances osteoclastogenesis by suppressing Nrf2-dependent antioxidant proteins to promote the generation of reactive oxygen species | elife-42951-v1 | [
[
"Osteoporosis is a pervasive disorder characterized by skeletal fragility and microarchitectural deterioration that predisposes individuals to bone fractures .",
"The disease has a significant global impact , affecting an estimated 200 million people worldwide and exerts a heavy economic burden .",
"Moreover , disease prevalence is projected to rise by approximately 50% within the next ten years ( Amin et al . , 2014 ) .",
"Therefore , understanding the pathogenesis of osteoporosis is an urgent matter to develop better treatments for this debilitating disease .",
"Bone remodeling is carried out by the coordinated actions of the bone-forming osteoblasts ( OBs ) and the bone-resorbing osteoclasts ( OCs ) .",
"Disorders of skeletal deficiency such as osteoporosis are typically characterized by enhanced osteoclastic bone resorption relative to bone formation , thereby resulting in net bone loss .",
"Although significant progress has been made in understanding the pathological role of OCs , the molecular pathways that drive OC differentiation remains an area needing further investigation .",
"Regulators of G-protein Signaling ( RGS ) are a family comprised of more than thirty proteins that share the eponymous and functionally conserved RGS domain; these proteins play a classical role in attenuating G protein-coupled receptor signaling through its GTPase-accelerating protein activity to inactivate the Gα subunit ( Neubig and Siderovski , 2002; Keinan , 2014 ) .",
"RGS proteins are multifunctional proteins that can contribute to various cellular processes including cell differentiation .",
"Rgs12 is unique in that it is the largest protein in its family .",
"In addition to the RGS domain , Rgs12 contains a PSD-95/Dlg/ZO1 ( PDZ ) domain , a phosphotyrosine-binding ( PTB ) domain , a tandem Ras-binding domain ( RBD1/2 ) , and a GoLoco interaction motif .",
"The multi-domain architecture of Rgs12 is thought to facilitate its role as a scaffolding protein in complexes in which multiple signaling pathways might converge ( Snow et al . , 2002; Sambi et al . , 2006; Snow et al . , 1998; Willard et al . , 2007; Schiff et al . , 2000 ) .",
"Reactive oxygen species ( ROS ) are produced as a normal byproduct of cellular metabolism ( Callaway and Jiang , 2015 ) and forms the basis Denham Harman’s free radical theory of aging , which perhaps is the most enduring model of aging .",
"The theory of aging postulates that the gradual accumulation of damage inflicted by ROS eventually manifests as degenerative diseases associated with aging ( Harman , 1956; Krause , 2007 ) .",
"In addition to longevity , ROS have been implicated in the management and prevention of cancers , cardiovascular diseases , macular degeneration , Alzheimer’s disease , arthritis , and many other tissues—to which the bone is no exception ( Naka et al . , 2008; Domazetovic et al . , 2017 ) .",
"More recent studies have shown that RANKL-induced ROS are indispensable for OC differentiation ( Callaway and Jiang , 2015; Lee et al . , 2005; Kim et al . , 2010; Bartell et al . , 2014 ) .",
"ROS at high levels induce oxidative stress , which if left unchecked becomes deleterious to cell .",
"At low concentrations , however , ROS have been shown to participate in signaling events in OCs , including the RANKL-dependent activation of mitogen-activated protein kinases ( MAPKs ) , phospholipase C gamma ( PLCγ ) , nuclear factor kappa B ( NFκB ) , and [Ca2+] oscillations; all of which contribute to the activation of nuclear factor of T-cells ( NFAT ) , the master regulator of OC differentiation .",
"Multiple lines of evidence have consistently shown that suppression of ROS by various means could inhibit OC differentiation ( Lee et al . , 2005; Kim et al . , 2010; Bartell et al . , 2014 ) .",
"In particular , RANKL-dependent activation of PLCγ , [Ca2+] oscillations , and NFAT were abrogated when OC precursors were treated with the antioxidant N-acetylcysteine ( NAC ) ( Kim et al . , 2010 ) .",
"Furthermore , our previous studies demonstrated that Rgs12 silencing could inhibit PLCγ activation , [Ca2+] oscillations , and the expression of NFATc1 and its downstream factors ( Yang and Li , 2007 ) .",
"Hence , these findings led us to hypothesize that Rgs12 may play a role in regulating the cellular redox state , thereby controlling OC differentiation ."
],
[
"To assess the role of Rgs12 in OC differentiation and bone remodeling in vivo , we generated a conditional gene knockout mouse model by crossing Rgs12flox/flox mice with Lysozyme M-cre ( LyzMCre ) transgenic mice ( Rgs12 cKO ) .",
"The LyzM promoter-driven Cre expression targets Rgs12 gene deletion to cells of the myeloid lineage , including monocytes/macrophages ( Abram et al . , 2014; Clausen et al . , 1999 ) .",
"Micro-CT analysis of the distal femurs obtained from Rgs12flox/flox and Rgs12+/+;LyzMCre showed no statistically difference in bone histomorphometry ( Figure 1—figure supplement 1 ) .",
"Rgs12flox/flox mice were used as controls .",
"The Cre-lox-mediated deletion of the Rgs12 gene was confirmed by PCR amplification of spleen genomic DNA ( Figure 1A ) and qPCR to measure Rgs12 transcripts in isolated bone marrow macrophages ( BMMs ) ( Figure 1B ) , thereby confirming our mouse Rgs12 cKO model .",
"Rgs12 cKO mice exhibited increased trabecular bone mass , evident in the hematoxylin and eosin ( H and E ) -stained sections of the proximal tibia ( Figure 1C ) and 3D micro-computed tomography ( micro-CT ) visualization of the femoral trabecular bone morphology and microarchitecture ( Figure 1D ) .",
"Quantitative micro-CT measurements further demonstrated statistically significant increases in bone volume ( VOX-BV/TV ) , accompanied by increases in both trabecular number ( Tb . N ) and thickness ( Tb . Th ) , and reduced trabecular separation ( Tb . Sp ) ( Figure 1E–I ) .",
"Therefore , the targeted deletion of Rgs12 in mice resulted in increased bone mass .",
"To determine whether the high bone mass was a consequence of decreased osteoclast numbers or increased osteoblast numbers in vivo , we quantified the cell numbers from distal femurs stained for tartrate-resistant acid phosphatase ( TRAP ) ( Figure 1J ) .",
"The histological assessment clearly demonstrates no difference in osteoblast numbers between Rgs12 cKO and control bone tissues ( Figure 1K ) whereas the number of TRAP+ multinucleated cells were markedly reduced in Rgs12-deficient samples ( Figure 1L and M ) .",
"Furthermore , a dynamic histomorphometric analysis by double calcein labeling to measure bone growth over time shows that the rate of bone formation was comparable between Rgs12 cKO and control mice ( Figure 1N–P ) .",
"Consequently , the results collectively support the specific role of Rgs12 in OCs and emphasizes the gene’s importance in bone remodeling .",
"To further evaluate the role of Rgs12 in osteoclastogenesis , OC precursors isolated from wild-type mice ( Rgs12+/+ ) were differentiated using macrophage colony-stimulating factor ( M-CSF ) and receptor activator of nuclear factor κB ligand ( RANKL ) , the two cytokines necessary and sufficient to induce osteoclast formation .",
"Rgs12 protein and transcript levels were dramatically upregulated upon stimulation by the differentiation factors , and seem to consistently increase into OC maturity at day 5 ( Figure 2A–B ) .",
"A comparison of osteoclastogenic potential between precursors derived from Rgs12 cKO and control mice show that while control BMMs differentiated into large , TRAP+ multinucleated OCs , Rgs12-deficient precursor cells showed a reduction in the number of OCs containing 6–9 nuclei and 10+ nuclei , which were also visibly smaller ( Figure 2C–D ) .",
"We also probed into the overall bone resorptive activity and found that Rgs12-deficient OCs have significantly reduced ability to resorb calcium phosphate surfaces ( Figure 2E–F ) .",
"Complementing our Rgs12 knockout model , we generated an Rgs12 overexpression OC model in which the transformed murine macrophage-like RAW264 . 7 cells were stably-transfected with a vector carrying a recombinant N-terminus FLAG-tagged Rgs12 gene ( Flag-Rgs12 ) .",
"Rgs12 overexpression in RAW264 . 7 cells was confirmed by western blotting ( Figure 2G ) .",
"Using this cell model , we next determined whether Rgs12 overexpression could promote OC formation .",
"Contrasting our findings in Rgs12 cKO primary cells , we found that the overexpression of Rgs12 in RAW264 . 7 cells led to an increased number of OCs with 10+ nuclei ( Figure 2H–I ) .",
"We also observed significantly decreased numbers of smaller OCs containing 3–5 and 6–9 nuclei in Rgs12 overexpressing cells , presumably because most of the smaller OCs have fused to form large OCs containing 10+ nuclei .",
"A previous study investigating the relationship between OC size and state of resorptive activity found that a greater proportion of large OCs were active whereas non-resorbing OCs were on average smaller ( Lees et al . , 2001 ) .",
"In our study , the quantification of the mean areas of OCs with 10+ nuclei revealed that Rgs12-overexpressing OCs were significantly larger as compared to empty vector-transfected controls ( Figure 2J ) .",
"To determine whether increased OC size translated to increased bone resorptive activity in our study , RAW264 . 7 cells were similarly cultured on calcium phosphate surfaces ( Figure 2K–L ) .",
"Consistent with our overall findings , ectopic overexpression of Rgs12 in OCs potently increased bone resorption activity .",
"Our findings therefore demonstrate the importance of Rgs12 in promoting OC formation and activity , which is consistent with the osteopetrotic phenotype observed in the Rgs12-deficient mouse model .",
"To uncover the role of Rgs12 in OC differentiation , we employed the IonStar liquid chromatography tandem mass spectrometry ( LC-MS/MS ) -based quantitative proteomics strategy ( Shen et al . , 2018 ) to profile the temporal dynamics in the global protein levels in Rgs12 cKO and control BMMs at 0 , 1 , 3 , and 5 days of OC differentiation ( Figure 3 ) .",
"Proteomics analysis identified 3714 quantifiable proteins that are present in all samples ( no missing data ) , using a highly stringent identification criteria of ≥2 peptides per protein and 1% false discovery rate ( Figure 3A ) .",
"Within this dataset , we identified 83 and 61 unique proteins that were significantly up- and downregulated , respectively , in Rgs12 cKO OCs relative to control .",
"Proteins were considered significantly altered if they exceeded the empirically-determined thresholds set at p<0 . 05 and>0 . 3 log2-transformed ratio ( Figure 3B ) .",
"Most of the protein expression changes in Rgs12-deficient cells were captured at 3 and 5 days of OC differentiation ( Figure 3A ) .",
"Interestingly , the proteomic disturbances as a result of Rgs12 deletion closely coincided with the pattern of endogenous Rgs12 protein expression during OC differentiation ( Figure 2A ) .",
"To determine the biological significance of these altered proteins , we performed gene ontology analysis to identify the canonical pathways involved ( Figure 3C ) .",
"Classically processes related to OC differentiation ( e . g . ‘NFAT Signaling’ , ‘RANK Signaling in OCs’ , and ‘Role of OCs in Rheumatoid Arthritis’ ) were enriched at 3 and 5 days of OC differentiation .",
"Closer inspection showed that OC marker proteins including metalloproteinase-9 ( Mmp9 ) , TRAP , ATPase H+ transporting V0 subunit D2 ( Atp6v0d2 ) , and integrin β3 ( Itgb3 ) were significantly downregulated in Rgs12 cKO OCs ( Figure 3D ) .",
"Additionally , the analysis revealed several biological functions related to ROS homeostasis that were impacted by Rgs12 deletion ( e . g . ‘Production of ROS’ , ‘Superoxide Radical Degradation’ , and NRF2-mediated Stress Response’ ) ( Figure 3C ) .",
"Inspection of the proteins involved in these pathways showed a significant upregulation of numerous Nrf2-dependent antioxidant enzymes responsible attenuating oxidative stress , including: peroxiredoxin 1/4 ( Prdx1/4 ) , thioredoxin 1/2 ( Trxr1 ) , glutathione reductase ( Gshr ) , and NAD ( P ) H dehydrogenase quinone 1 ( Nqo1 ) ( Figure 3E ) .",
"Upstream regulator ( transcription factor ) analysis by the Ingenuity Pathway Analysis software function identified that the antioxidant enzymes upregulated in Rgs12 cKO OCs share the common upstream regulator Nrf2 , a key transcription factor that regulates cellular redox balance through the expression of protective antioxidant and phase II detoxification proteins .",
"( Venugopal and Jaiswal , 1996; Itoh et al . , 1997 ) .",
"Although the upstream regulator analysis predicted an upregulation of Nrf2 activity , the transcription factor itself was not detected by our proteomics analysis .",
"Proteins of typically low abundance such as cytokines , signal regulatory molecules , and transcription factors tend to be ‘crowded out’ during MS analysis by more highly abundant proteins such those proteins involved in glycolysis and purine metabolism , protein translation , and cytoskeletal components ( Beck et al . , 2011 ) .",
"Nonetheless , our proteomics-based discovery tool allowed us to generate the hypothesis that Nrf2 is aberrantly activated by Rgs12 deletion , causing excessive clearance of ROS by antioxidant enzymes and in turn disrupting OC differentiation .",
"Based on our proteomics analysis , we hypothesized that Rgs12 is needed to suppress Nrf2 activity and facilitate the formation of ROS , which has been previously shown to play a critical role in OC differentiation ( Lee et al . , 2005; Kanzaki et al . , 2013 ) .",
"To test this hypothesis , we assessed Nrf2 activity and the expression of Nrf2 and Keap1 in Rgs12 cKO and control precursor cells ( Figure 4 ) .",
"Western blotting of Nrf2 in day 3 OCs also showed increased levels of Nrf2 in Rgs12 cKO cells ( Figure 4A–B ) .",
"Keap1 , however , which is known to suppresses Nrf2 activity by facilitating its degradation via the proteasome pathway , was unexpectedly elevated in Rgs12-deficient cells .",
"Furthermore , immunofluorescence staining of Nrf2 demonstrated increased nuclear translocation of the transcription factor in Rgs12 cKO cells ( Figure 4C , upper panel ) .",
"The elimination of ROS with N-acetylcysteine ( NAC ) , a precursor to the antioxidant glutathione , was able to completely suppress Nrf2 nuclear translocation in both Rgs12 cKO and control BMMs ( Figure 4C , middle panel ) .",
"Conversely , induction of oxidative stress using the peroxide tert-buthylhydroxyperoxide ( tBHP ) potently induced Nrf2 nuclear translocation ( Figure 4C , bottom panel ) .",
"To further test whether elevated Nrf2 activity in Rgs12-deficient OCs could result in reduced intracellular ROS levels , we detected intracellular ROS levels .",
"As expected , the RANKL-dependent ROS induction observed in control cells was suppressed in Rgs12 cKO OCs ( Figure 4D ) .",
"These findings demonstrate an abnormal upregulation of Nrf2 activity and expression in Rgs12-deficient cells , indicating that Rgs12 may be required to suppress Nrf2 to facilitate osteoclastogenesis .",
"Under basal conditions ( i . e . absence of cellular stress ) , Nrf2 remains inactive through its interaction with Keap1 , which causes its continual ubiquitination and degradation via the proteasome pathway ( Stewart et al . , 2003; Zhang and Hannink , 2003 ) .",
"A variety of stress conditions can induce conformational changes in Keap1 , thereby releasing Nrf2 from the ubiquitin-proteasome pathway , allowing it to accumulate and translocate into the nucleus ( Itoh et al . , 2003; Kensler et al . , 2007 ) .",
"To better understand the mechanism by which Rgs12 suppresses Nrf2 activity , we therefore first determined whether the ability of Rgs12 to suppress Nrf2 activity relies on this canonical mechanism ( Figure 5A ) .",
"Given that Rgs12 deletion resulted in elevated Nrf2 expression and nuclear translocation , we first determined whether Rgs12 overexpression could exert an opposite effect ( Figure 5A–B ) .",
"We measured Nrf2 protein levels in RAW264 . 7 cells stably transfected with the Rgs12-His or empty vector and found no difference when cells are at their un-induced , basal state .",
"Stimulation of RAW264 . 7 cells with tert-buthylhydroquinone ( tBHQ ) , which is known to directly bind Keap1 and attenuate its inhibitory effect on Nrf2 ( Abiko et al . , 2011 ) , caused a robust induction of Nrf2 protein levels in a dose-dependent manner ( Figure 5A–B ) .",
"More importantly , RAW264 . 7 cells overexpressing Rgs12 showed a significant reduction of Nrf2 protein levels that resulted from Keap1 inhibition compared to those in the control cells .",
"Moreover , the ability of Rgs12 to facilitate Nrf2 degradation despite the inhibition of Keap1 suggests that Rgs12 functions downstream of Keap1 , either by controlling the ubiquitination or proteasomal degradation of Nrf2 .",
"Given the possibility that the reduction of Nrf2 levels in Rgs12 overexpression cells may be a result of increased Nrf2 degradation , we further tested whether inhibiting the proteasome , a step downstream of Keap1 , could attenuate the ability of Rgs12 to facilitate Nrf2 degradation ( Figure 5D and E ) .",
"Similar to tBHQ , preventing Nrf2 degradation using the proteasome inhibitor MG-132 caused Nrf2 protein to substantially accumulate ( Figure 5D , left panel ) .",
"Interestingly , when Nrf2 protein levels were artificially induced , we observed the presence of a lower molecular weight band , which could correspond to a different post-translational modification state ( e . g . unphosphorylated or non-ubiquitinated ) .",
"Furthermore , we did not observe any changes in Keap1 protein levels .",
"In the previous scenario wherein Rgs12 overexpression could still promote Nrf2 degradation in spite of tBHQ treatment , this was not the case when using MG-132 .",
"In fact , inhibiting the proteasome was able to reverse the ability of Rgs12 to promote Nrf2 degradation , indicating the requirement for Rgs12 in the proteasome’s function .",
"We subsequently repeated this experiment in RAW264 . 7 cells differentiated for 3 days with RANKL ( Figure 5D ) .",
"Interestingly , RANKL treatment correlated with reduced Nrf2 levels in OCs ( Figure 5D , right panel ) .",
"Our observation corroborates with previous findings documenting the suppressive effect of RANKL on Nrf2 expression , which was attributed to reduced transcriptional activity ( Kanzaki et al . , 2013; Hyeon et al . , 2013 ) .",
"We confirmed this effect by measuring the Nrf2 transcript levels by qPCR ( Figure F ) .",
"More importantly , we demonstrate that Rgs12 overexpression could suppress Nrf2 protein levels , but inhibition of the proteasome using MG-132 reversed this effect ( Figure 5D , right panel ) .",
"To confirm that Rgs12 inhibits Nrf2 through a post-translational mechanism , we measured Nrf2 transcript levels by qPCR and found no difference between vector control and Rgs12-overexpressing cells ( Figure 5F ) .",
"Overall , our data collectively indicate that Rgs12 suppresses Nrf2 activity by facilitating its degradation through the proteasome-dependent pathway .",
"It was previously demonstrated that ROS could act as an intracellular signal mediator OC differentiation , and is required for the RANKL-dependent activation of p38 mitogen-activated protein kinase ( MAPK ) , extracellular signal-regulated kinase ( ERK ) , and NFκB ( Lee et al . , 2005; Ha et al . , 2004 ) .",
"Given our findings that Rgs12 could suppress the activity of Nrf2 and thereby promoting intracellular ROS , we hypothesized that Rgs12 could promote RANKL-dependent signaling , and that this effect would be abrogated by the addition of an antioxidant ( Figure 6A–B ) .",
"As expected , RAW264 . 7 cells overexpressing Rgs12 demonstrated a more robust activation of ERK1/2 and NFκB phosphorylation but not p38 MAPK .",
"Pretreating Rgs12 overexpressing cells with the antioxidant NAC diminished ERK1/2 activation , and almost completely abrogated NFκB activation .",
"These results support the role of Rgs12 in promoting ROS that is important OC signaling , likely through the suppression of Nrf2 activity ."
],
[
"The importance of ROS in osteoclasts has been underlined by the growing corpus of evidence demonstrating that ROS increased with aging or during inflammation can stimulate bone resorption and exacerbate bone loss ( Callaway and Jiang , 2015 ) .",
"Targeting ROS in diseases of excess bone resorption such as osteoporosis could therefore represent a novel therapeutic strategy .",
"An important mechanism of cellular ROS clearance relies on the Keap1-Nrf2 pathway , which has been well characterized especially in the context of cancer biology ( Kansanen et al . , 2013 ) ; however , the upstream signaling molecules that could regulate the Keap1-Nrf2 axis in OCs remains unknown .",
"Targeting this gap in knowledge , our study uncovered a novel role of the signaling protein Rgs12 in regulating Nrf2 , thereby controlling cellular redox state and OC differentiation .",
"Within this study , we first demonstrated the essential role of Rgs12 in OC differentiation such that myeloid cell-targeted Rgs12 knockout mice exhibited an osteopetrotic phenotype , evidenced by reduced OC numbers but no changes to OB numbers nor bone formation ( Figure 1 ) .",
"Furthermore , OC precursors isolated from these mice showed reduced in vitro OC differentiation and bone resorptive activity ( Figure 2 ) .",
"On the contrary , forced overexpression of Rgs12 in RAW264 . 7 cells significantly promoted OC formation and increased the size of the resultant OCs , which was also associated with increased bone resorptive activity .",
"However , the mechanism by which Rgs12 regulates OC differentiation remains unclear , to which we investigated more deeply using a leading-edge and high-throughput proteomics technique .",
"Proteomics is a powerful tool that has led to numerous discoveries of proteins and biological processes that drive OC differentiation ( Segeletz and Hoflack , 2016 ) .",
"Notably , this technique was recently used to map the podosome proteome which helped to advance our understanding of determinants in the macrophage multinucleation process ( Rotival et al . , 2015; Cervero et al . , 2012 ) , and how metabolism and energy is redirected towards bone resorption in OCs ( An et al . , 2014 ) .",
"Proteomics can therefore provide a broad yet informative overview of the systemic changes in the differentiating OC .",
"To discover the cellular function of Rgs12 in OCs , we employed a robust and high-throughput quantitative proteomics approach to characterize the global protein changes of OCs derived from Rgs12 cKO and cKO BMMs ( Figure 3 ) .",
"The analysis revealed that most perturbations to the proteome was related to day 3 and 5 of OC differentiation , which coincides with the timing of Rgs12 upregulation in wild-type OCs ( Figure 2A–B ) .",
"This overlap suggests that Rgs12 may be more important to later phases of OC differentiation , including cell-cell fusion and bone resorption .",
"The analysis also identified the upregulation of a collection of antioxidant enzymes that are all transcriptionally regulated by the antioxidant response element ( ARE ) within the promoter region , which is activated by the transcription factor Nrf2 ( Nguyen et al . , 2009 ) .",
"While the upregulation of these genes would lead one to surmise that Nrf2 should be similarly increased , the transcription factor was not detected in the proteomics analysis , which is likely result of technical limitations inherent to current LC-MS capabilities .",
"It was previously reported that transcription factor protein levels are notoriously difficult to obtain due to many having relatively low expression levels that are generally below current mass spectrometer detection limits ( Simicevic and Deplancke , 2017 ) .",
"The quantitation of transcription factors typically entails an upstream enrichment/separation step before LC-MS analysis .",
"Therefore , the absence of Nrf2 in our proteomics analysis is not an unusual happenstance from a technical standpoint .",
"Alternatively , we confirmed our proteomics findings by showing increased Nrf2 protein levels and nuclear translocation activity in Rgs12-deficient osteoclasts ( Figure 4A–C ) .",
"Based on our preliminary evidence , we further investigated the role of Rgs12 in Nrf2 signaling and found that Nrf2 protein levels and nuclear translocation were increased in OC precursors in which Rgs12 was depleted ( Figure 4 ) .",
"Because our data showed that Rgs12 deficiency upregulated Nrf2 , we expected that Keap1 levels should be reduced in order to facilitate the increased Nrf2 activity .",
"On the contrary , Keap1 levels were upregulated in Rgs12-deficient cells , to which we speculate that Rgs12-deficient cells may be overcompensating Keap1 expression in order to rein back the increased Nrf2 activity .",
"Nevertheless , consistent with the upregulation of Nrf2 and its corresponding antioxidant enzymes , the RANKL-dependent induction of ROS was attenuated in Rgs12-deficient cells .",
"Through an orthogonal approach , we demonstrated that Rgs12 overexpression in OC precursors could also enhance RANKL-mediated activation of ERK1/2 and NFκB ( Figure 6 ) , which are established to be dependent on ROS ( Hyeon et al . , 2013 ) .",
"Furthermore , inhibition of intracellular ROS blocked the effect of Rgs12 overexpression , indicating that Rgs12 promotes RANKL-dependent signaling by facilitating ROS production .",
"Overall , the data collectively demonstrate that Rgs12 promotes osteoclastogenesis by facilitating ROS generation through the suppression of Nrf2 and its target antioxidant genes .",
"The transcription factor Nrf2 is constitutively expressed but its activity is inhibited through its interaction with Keap1 .",
"Under basal conditions , Nrf2 is restricted to the cytoplasm where it is continually depleted through the proteosomal degradation pathway .",
"When bound to Nrf2 , Keap1 recruits the cullin 3 ( Cul3 ) -dependent E3 ubiquitin ligase complex , which ubiquitinates and targets Nrf2 for degradation by the 26S proteasome ( Zhang and Hannink , 2003; Itoh et al . , 2003; Nguyen et al . , 2005; McMahon et al . , 2003 ) .",
"Keap1 protein contains multiple reactive cysteine residues that serve as redox sensors ( Abiko et al . , 2011 ) that are sensitive to stressor conditions including oxidative stress causes the electrophilic modification of Keap1 , inducing conformational changes , causing the protein to dissociate from Nrf2 and thereby allowing the transcription factor to enter the nucleus ( Stewart et al . , 2003 ) .",
"We therefore determined how Rgs12 regulates this well-defined mechanism ( Figure 5 ) .",
"tBHQ is a selective inhibitor of Keap1 activity by covalently binding the protein’s reactive thiols and as a result activate Nrf2 and its downstream proteins in RAW264 . 7 cells ( Abiko et al . , 2011 ) .",
"tBHQ was also previously shown to inhibit OC differentiation via the upregulation of heme oxygenase-1 , a Nrf2-dependent antioxidant enzyme ( Yamaguchi et al . , 2014 ) .",
"In this study , we determined whether Rgs12 could suppress the tBHQ-dependent upregulation of Nrf2 ( Figure 5B ) .",
"We reasoned that if Rgs12 relies on a Keap1-dependent mechanism , then the inhibition of Keap1 by tBHQ should prevent the ability of Rgs12 to suppress Nrf2 .",
"However , we observed that Rgs12 was still able to suppress Nrf2 despite the pharmacological blockade of Keap1 activity , thereby indicating that Rgs12 functions downstream of Keap1 .",
"Following Keap1-mediated ubiquitination of Nrf2 , the targeted protein should be degraded by the proteasome .",
"Again , we reasoned that if Rgs12 is dependent on the proteasomal degradation pathway , then inhibition of this pathway using the proteasome inhibitor MG-132 should prevent Rgs12-mediated suppression of Nrf2 .",
"Indeed , we found that Inhibiting the proteasome was able to reverse the Rgs12-mediated degradation of Nrf2 , which places Rgs12 in between Keap1 and the proteasome in the Nrf2 degradation pathway ( Figure 5D–E ) .",
"Thus , Rgs12 could either regulate the Cul3-dependent E3 ubiquitin ligase complex to facilitate the ubiquitination of Nrf2 , or directly control proteasome activity .",
"While Keap1 certainly plays a canonical role in Nrf2 suppression , the evidence herein suggest that Keap1 is not involved in the Rgs12 function .",
"It is also interesting to note that NFκB activation is also dependent on the proteasomal degradation of inhibitor of κB ( IκB ) , which otherwise sequesters NFκB to the cytoplasm ( Boyce et al . , 2015 ) .",
"If Rgs12 could modulate proteasome activity , it is possible that Rgs12 could directly promote NFκB by facilitating the degradation of IκB .",
"However , the fact that antioxidant treatment to suppress ROS could almost completely block the phosphorylation of NFκB points to an important involvement of ROS , and not just simply the proteasomal degradation of IκB in NFκB activation ( Figure 6 ) .",
"Nonetheless , this potential crosstalk between the NFκB and Nrf2 pathways will need to be evaluated in future studies .",
"ROS is an inevitable byproduct of the mitochondrial electron transport chain ( oxidative phosphorylation , OXPHOS ) during ATP synthesis .",
"Microscopy analyses have noted the high abundance of mitochondria in osteoclasts as early as 1961 ( Gonzales , 1961 ) , but interest in the metabolic profile that supports OC differentiation has made a resurgence in recent years .",
"As examples , we and others through the proteomic study of OCs have reported that proteins involved in ATP synthesis were elevated in mature osteoclasts as compared to precursors ( An et al . , 2014; Ng et al . , 2018 ) .",
"Additionally , it was demonstrated that mature OCs exhibit larger mitochondria , higher levels of enzymes of the electron transport chain , and higher oxygen consumption rates ( Lemma et al . , 2016 ) .",
"The evidence collectively point towards OXPHOS as a main bioenergetic source for osteoclast differentiation , and can therefore be considered an integral aspect of the process .",
"As a consequence of increased mitochondrial biogenesis , ROS would similarly be elevated due to the ‘leakiness’ of the electron transport chain , which in turn contributes to the ROS that promotes OC differentiation .",
"As such , an increase in ROS has been associated with mitochondria biogenesis in osteoclasts ( Ishii et al . , 2009 ) , and mitochondrial ROS is essential for osteoclast formation ( Bartell et al . , 2014 ) .",
"Therefore , the analysis of OXPHOS and glycolytic patterns should not be discounted in a study that focuses on redox biology .",
"To address this concern , we evaluated the molecular subsets in the proteome involved in the energy metabolism pathways including glycolysis , tricarboxylic acid ( TCA ) cycle , and OXPHOS pathways ( Figure 3—figure supplement 1 and Figure 3—source data 2 ) .",
"The loss of Rgs12 in BMMs led to the inhibition of RANKL-dependent ROS , which we demonstrated was a function of increased Nrf2 activity and elevated levels of its target antioxidant enzymes .",
"At the same time , the proteomics analysis identified a reduction of proteins involved in the TCA cycle and OXPHOS ( Figure 3—source data 2 ) .",
"Interestingly , cytochrome b5 type A ( CYB5A ) was against the general trend and was significantly increased by ~30% in Rgs12-deficient BMMs .",
"The TCA cycle is responsible for the consumption of acetyl-CoA to generate NADH which is fed into the OXPHOS pathway to power ATP synthase and catalyze the formation of ATP .",
"The impediment of OXPHOS , which requires molecular oxygen as co-factor , can shift ATP production towards anaerobic glycolysis under hypoxic and other conditions .",
"However , the proteomic analysis revealed that protein levels of phosphofructokinase ( PFK ) , the rate-limiting enzyme of glycolysis , were unaffected by Rgs12 deletion ( Figure 3—figure supplement 1 and Figure 3—source data 2 ) .",
"In fact , all enzymes we detected that are involved in the glycolytic pathway with the exception of enolase 3 ( a . k . a . phosphopyruvate hydratase ) were unaffected by the loss of Rgs12 .",
"On one hand , the loss of Rgs12 was associated with reduced OXPHOS , which would theoretically contribute to lower ROS levels .",
"Conversely , the inhibition of OC differentiation could lead to reduced mitochondrial biogenesis , which could also explain lowered ROS levels in Rgs12-deficient BMMs .",
"Therefore , mitochondrial ROS could be a contributing factor in Rgs12 function—albeit likely as a secondary effect .",
"Although OXPHOS enzymes were reduced in Rgs12-deficient osteoclasts , only 19/83 proteins identified ( 22 . 9% ) were altered , as compared to 26/26 proteins ( 100% ) identified as Nrf2 targets ( Figure 3—figure supplement 1 and Figure 3—source data 2 ) .",
"Additionally , the magnitude of protein expression fold-changes was markedly higher in Nrf2 response genes as compared to OXPHOS genes .",
"Therefore , the higher degree of intra-group concordance and magnitude of expression of Nrf2 genes both indicate a more targeted response , whereas the weak pattern of expression of OXPHOS genes suggests an equivocal effect .",
"Secondly , Nrf2 antioxidant proteins act downstream of mitochondrial ROS formation by acting directly on ROS .",
"This effect is best exemplified by the fact that the ectopic expression of mitochondria-targeted catalase was able to suppress ROS and inhibit OC differentiation ( Bartell et al . , 2014 ) .",
"In the case of Rgs12 overexpression , it could be expected that increased mitochondrial ROS production would also need the concomitant suppression of Nrf2 to raise the overall intracellular ROS level .",
"In the current work , we demonstrate that Rgs12 could promote Nrf2 degradation .",
"Therefore , the inhibition of RANKL-dependent ROS associated with loss of Rgs12 was unlikely to be the direct result of glycolytic shift or reduced OXPHOS .",
"While changes in mitochondrial ROS could be an minor aspect of Rgs12 function , but Nrf2 remains an essential component in the overall scheme .",
"In conclusion , we identified a new gene that could modulate the Nrf2-proteasome axis , which forms the crux of redox homeostasis in many biological contexts .",
"Our study also points to a novel role of Rgs12 in OC redox biology , thus forming the molecular basis for developing therapies to modulate ROS for osteoporosis and other diseases of bone loss .",
"Outside of its role in bone homeostasis , Rgs12 has also been shown to hold diverse and significant roles in other clinical contexts including pathological cardiac hypertrophy ( Huang et al . , 2016 ) , tumor suppression in African American prostate cancer ( Wang et al . , 2017 ) , and psychostimulant-induced increases in dopamine levels in the brain ( Gross et al . , 2018 ) .",
"We speculate that the ability of Rgs12 to regulate Nrf2 could be an important clue to better understand the role of Rgs12 in the context of cardiac disease and tumorigenesis , in which ROS is known to have significant implications .",
"Therefore , our research would have widespread appeal to audiences of different research backgrounds ."
],
[
"Rgs12flox/flox mice were crossed with LyzMCre transgenic mice to generate Rgs12 cKO mice specific to the myeloid lineage in a C57BL/6J background .",
"The methodology for generating Rgs12flox/flox and LyzMCre mice and genotyping are previously described ( Clausen et al . , 1999; Yang et al . , 2013; Yuan et al . , 2015 ) .",
"Mice used for experiments were 10–12 weeks old , as indicated .",
"All animal studies were approved by the University at Buffalo and University of Pennsylvania Institutional Animal Care and Use Committees ( IACUC ) .",
"Quantitative analysis of bone morphology and microarchitecture was performed using a micro-CT system ( USDA Grand Forks Human Nutrition Research Center , Grand Forks , ND , USA ) .",
"Fixed femur from 10-week-old Rgs12 cKO and control mice were analyzed and 3D reconstruction was used to determine bone volume to tissue volume ( BV/TV ) , structure model index ( SMI ) , trabecular thickness ( Tb . Th , μm ) , trabecular number ( Tb . N , /mm ) , and trabecular separation ( Tb . Sp , μm ) .",
"Mouse tibiae and femurs from 10-week-old mice were excised and fixed in 4% PFA for 24 hr and decalcified in a 10% EDTA for 3–4 weeks at 4°C .",
"The samples were embedded in paraffin , sectioned at 8 μm , stained with hematoxylin and eosin ( H and E ) or tartrate-resistant acid phosphatase ( TRAP ) using the Acid Phosphatase , Leukocyte ( TRAP ) Kit ( Sigma-Aldrich , St . Louis , MO , USA ) , and imaged using a Leica inverted microscope ( DMI6000B , Leica , Germany ) .",
"To assess the rate of bone formation , mice were intraperitoneally injected with calcein ( 25 mg/kg ) twice at postnatal day 90 and day 96 .",
"Mice were euthanized and harvested 2 days after last injection .",
"Femurs were dissected and fixed in 4% PFA for 24 hr , dehydrated in ethanol , and embedded in optimal cutting temperature ( OCT ) compound for cryosection without decalcification .",
"By applying Cryofilm tape ( Section Lab , Hiroshima , Japan ) , 8 μm longitudinal sections were cut from distal femurs using a microtome ( CM1950 , Leica , Germany ) .",
"Measurements of surface-based histomorphometric indices were performed using the OsteoMeasure analysis system ( OsteoMetrics , Decatur , GA ) .",
"These indices were used to calculate bone formation rate per bone surface ( BFR/BS , μm3/um−2 per day ) , mineral apposition rate ( MAR , μm per day ) , OB number per bone perimeter ( N . Ob/B . Pm , mm−1 ) and OC number per bone perimeter ( N . Oc/B . Pm , mm−1 ) as previously described ( Yuan et al . , 2016; Li et al . , 2019 ) .",
"Full length Rgs12 ( Accession: NM_173402 . 2 ) cDNA was cloned into the p3XFLAG-myc-CMV-26 expression vector ( Sigma-Aldrich , St . Louis , MO , USA ) .",
"Briefly , HindIII sites were incorporated into both termini of the Rgs12 cDNA using restriction-site-generating PCR , and the restriction sites were used to insert the Rgs12 sequence into the expression vector containing an N-terminus FLAG tag sequence ( Flag-Rgs12 ) .",
"The primer walking method was used to validate the correct directionality of the insert .",
"Additionally , a vector expressing C-terminus His-tagged Rgs12 ( Rgs12-His ) was generated by subcloning the Rgs12 cDNA into the pcDNA3 . 1 ( + ) -c-His vector ( Genscript , Piscataway , NJ , USA ) .",
"HindIII and EcoRV sites were introduced by PCR and the restriction sites were used to insert Rgs12 into the pcDNA3 . 1 ( + ) -c-His vector .",
"All vector constructs were confirmed by DNA sequencing ( Eurofins Genomics , Louisville , KY , USA ) .",
"RAW264 . 7 cells were purchased from ATCC which was confirmed to be free of mycoplasma contamination .",
"RAW264 . 7 cells were seeded at 2 × 106 cells per 6-well and transfected using FuGENE HD reagent ( Promega , Madison , WI , USA ) according to manufacturer’s instructions at a 1:3 DNA to transfection reagent ratio .",
"After 48 hr post-transfection , cells were treated with 0 . 4 mg/mL geneticin ( G418 , Thermo Fisher Scientific , Waltham , MA , USA ) for 2 weeks until antibiotic-resistant colonies are formed .",
"Stably transfected cells were thereafter maintained in media containing 0 . 4 mg/mL G418 .",
"The vector encoding the recombinant mRANKL-His ( K158-D316 ) construct and a modified E . coli strain Origami B ( DE3 ) cells ( EMD Millipore , Billercica , MA , USA ) co-expressing chaperone proteins that was used to express the recombinant RANKL were generous gifts from Dr . Ding Xu at the University at Buffalo .",
"The protocol for expressing and purifying mRANKL-His was described previously ( Li et al . , 2016a ) .",
"Endotoxins were removed using the Pierce High Capacity Endotoxin Removal Resin ( Thermo Fisher Scientific , Waltham , MA , USA ) .",
"BMMs were obtained from the tibiae and femurs of 10-week-old mice as described previously ( Yang and Li , 2007 ) .",
"For in vitro osteoclastogenesis experiments , BMMs were seeded at 2 × 106 cells per 24-well plates and stimulated with 100 ng/mL RANKL and 20 ng/mL M-CSF ( R and D Systems , Minneapolis , MN , USA ) for 5 days to generate mature OCs .",
"RAW264 . 7 cells were seeded at 1 . 35 × 104 cells per 24-well and stimulated with M-CSF and RANKL for 5 days .",
"Prior to fixing and staining , RAW264 . 7-derived OCs were gently but thoroughly rinsed with PBS to remove mononuclear cells that tend to obscure OCs during imaging .",
"TRAP staining was performed using the Acid Phosphatase , Leukocyte ( TRAP ) kit ( Sigma-Aldrich , St . Louis , MO , USA ) .",
"Cells were imaged using the Cytation 5 Cell Imaging Multi-Mode Reader ( BioTek , Winooski , VT , USA ) using the montage function .",
"Osteoclasts were quantified by counting the number of TRAP+ , multinucleated cells ( MNCs , ≥3 nuclei/cell ) per well .",
"Average osteoclast area was determined by measuring total TRAP+ area using the ImageJ software ( US National Institute of Health , Bethesda , MA , USA ) and dividing the value by total osteoclast number .",
"For bone resorptive activity experiments , the method above was applied except that cells were seeded on Osteo Assay Surface plates ( Corning , Corning , NY ) and allowed to differentiate and resorb the calcium phosphate surface for 5–6 days .",
"A 10% bleach solution was added to each well for 5 min to remove cells , followed by rinsing with deionized water , and air-dried .",
"Resorption pits were visualized and captured under a light microscope ( DMI6000B , Leica , Germany ) and analyzed using ImageJ software as previously described ( Li et al . , 2016b ) .",
"Total RNA was isolated from cultured BMMs and OCs using Trizol reagent ( Invitrogen , Carlsbad , CA , USA ) following manufacturer’s instructions .",
"cDNA was reverse transcribed from 2 μg total RNA using the RNA to cDNA EcoDry Premix kit ( Clontech , Palo Alto , CA , USA ) .",
"Primers were designed using Primer-BLAST ( Ye et al . , 2012 ) and obtained from IDT ( Integrated DNA Technologies , San Diego , CA , USA ) .",
"Rgs12 ( F: 5’-AAGATCCATTCCCTAGTGACC-3’ , R: 5’-ACCTCCACTTTCCCACCCTG-3’ , 587 bp ) , Nrf2 ( F: 5’-GCCCACATTCCCAAACAAGAT-3’ , R: 5’-CCAGAGAGCTATTGAGGGACTG-3’ , 172 bp ) , Keap1 ( F: 5’-TGCCCCTGTGGTCAAAGTG-3’ , R: 5’-GGTTCGGTTACCGTCCTGC-3’ , 104 bp ) , β-actin ( F: 5’-CTAGGCACCAGGGTGTGAT-3’ , R: 5’-TGCCAGATCTTCTCCATG TC-3’ , 148 bp ) , GAPDH ( F: 5’-AGGTCGGTGTGAACGGATTTG-3’ , R: 5’-TGTAGACCATGTAGTTGAGGTCA-3’ , 123 bp ) .",
"qPCR was performed using the 2x SYBR Green qPCR Master Mix following manufacturer’s instructions ( Bimake , Houston , TX , USA ) .",
"All reactions were performed in triplicate and normalized to the housekeeping gene β-actin or GAPDH as indicated .",
"Data analysis was performed using the CFX Maestro software ( Bio-Rad , Hercules , CA , USA ) .",
"The Rac1-GTP pulldown assay was performed following manufacturer instructions in the Rac1 activation assay kit ( Cytoskeleton , Denver , CO , USA ) .",
"To measure ROS production , BMMs were seeded into black , glass-bottom 96-well plates and cultured with M-CSF for 48 hr until confluence .",
"Cells were loaded with 20 μM 2’7’-dichlorofluorescein diacetate ( DCFDA , Sigma ) at 37°C for 30 min and washed using PBS .",
"The cells were swapped into complete phenol red-free MEM ( Gibco ) containing RANKL/M-CSF .",
"Fluorescence intensity was measured using the Cytation five plate reader ( BioTek ) with excitation wavelength at 488 nm and emission wavelength at 535 nm .",
"Background signals ( cells not loaded with DCF-DA ) were subtracted .",
"Experiments were carried out in quintuplicate wells .",
"Cells were harvested using ice-cold lysis buffer ( 50 mM Tris-formic acid , 150 mM NaCl , 0 . 5% sodium deoxycholate , 1% SDS , 2% NP-40 , pH 8 . 0 ) with protease inhibitor ( cOmplete , Mini , EDTA-free; Roche , Mannheim , Germany ) .",
"Samples were prepared for MS analysis using an established method ( Shen et al . , 2018; Shen et al . , 2017 ) .",
"The ‘IonStar’ LC-MS experimental pipeline was developed and optimized in a previous study ( Shen et al . , 2018; Shen et al . , 2017 ) .",
"A stringent set of criteria including a low peptide and protein false discovery rate ( FDR ) of <1% and≥2 peptides per protein was used for protein identification .",
"An ion current-based quantification method ( IonStar processing pipeline ) was described previously ( Shen et al . , 2018; Shen et al . , 2017 ) .",
"Ingenuity Pathway Analysis ( Qiagen , Redwood City , CA , USA ) was used to perform gene ontology enrichment analysis .",
"Hierarchical clustering analysis and heat map visualizations were performed using the agnes function in R Package cluster and ggplot2 with the viridis color palette , respectively .",
"For the Nrf2 nuclear translocation experiment , BMMs were cultured on coverslips and treated with RANKL and M-CSF for 72 hr , 5 mM NAC for 16 hr , or 50 μM tBHP for 16 hr .",
"Coverslips were fixed with 4% paraformaldehyde solution in PBS for 10 min at room temperature and permeabilized using 0 . 1% Triton X-100 for 5 min at room temperature .",
"Coverslips were blocked using Image-iT FX signal enhancer ( Thermo Fisher Scientific ) for 1 hr at room temperature , stained with the primary antibody in 1% BSA/TBST overnight at 4°C , and stained with the secondary antibody for 1 hr at room temperature .",
"4 , 6-diamidino-2-phenylindole ( DAPI ) ( Sigma ) was used as a counterstain for nuclei .",
"The coverslips were mounted using ProLong Gold antifade mountant ( Thermo ) and images were obtained using a fluorescence microscope ( Leica , Wetzlar , Germany ) .",
"For experiments studying the Keap1-Nrf2 pathway , cells were cultured in 6-well plates and pre-treated with the indicated concentrations of tBHQ or 25 µM MG-132 for 4 hr .",
"For MAPK and NFκB activation experiments , stable-transfected RAW264 . 7 cells were cultured in 6-well plates and starved in serum-free medium containing 5 mM NAC for 16 hr .",
"Cells were subsequently induced with RANKL ( 200 ng/mL ) and M-CSF ( 100 ng/mL ) for the indicated times .",
"Western blotting was performed as described previously ( Yuan et al . , 2016 ) .",
"The primary antibodies used in this study were as follows: Nrf2 ( H-300 ) and Nrf2 ( C-20 ) ( 1:100 , Santa Cruz Biotechnology , Dallas , TX , USA ) , Keap1 ( E-20 ) ( 1:100 , SCBT ) , phospho-p38 ( Thr180/Tyr182 ) ( 1:1000 , Cell Signaling Technology ) , p38 ( 1:1000 , CST ) , phospho-p44/42 MAPK ( Erk1/2 ) ( Thr202/Tyr204 ) ( 1:1000 , CST ) , ERK1/2 ( 1:1000 , CST ) , phospho-NFκB p65 ( Ser536 ) ( 1:1000 , CST ) , NFκB p65 ( 1:1000 , CST ) , and β-actin ( 1:4000 , SCBT ) .",
"Densitomety analysis was performed using ImageJ ( Schindelin et al . , 2012 ) and normalized to the β-actin signal .",
"Relative phosphorylation of was presented as the ratio between the phosphorylated normalized to the non-phosphorylated/total protein .",
"NAC , tBHQ , and tBHP were obtained from Sigma-Aldrich ( St . Louis , MO , USA ) , and MG-132 was obtained from Selleck Chemicals ( Houston , TX , USA ) ."
]
] | [
"Regulators of G-protein Signaling are a conserved family of proteins required in various biological processes including cell differentiation .",
"We previously demonstrated that Rgs12 is essential for osteoclast differentiation and its deletion in vivo protected mice against pathological bone loss .",
"To characterize its mechanism in osteoclastogenesis , we selectively deleted Rgs12 in C57BL/6J mice targeting osteoclast precursors using LyzM-driven Cre mice or overexpressed Rgs12 in RAW264 . 7 cells .",
"Rgs12 deletion in vivo led to an osteopetrotic phenotype evidenced by increased trabecular bone , decreased osteoclast number and activity but no change in osteoblast number and bone formation .",
"Rgs12 overexpression increased osteoclast number and size , and bone resorption activity .",
"Proteomics analysis of Rgs12-depleted osteoclasts identified an upregulation of antioxidant enzymes under the transcriptional regulation of Nrf2 , the master regulator of oxidative stress .",
"We confirmed an increase of Nrf2 activity and impaired reactive oxygen species production in Rgs12-deficient cells .",
"Conversely , Rgs12 overexpression suppressed Nrf2 through a mechanism dependent on the 26S proteasome , and promoted RANKL-induced phosphorylation of ERK1/2 and NFκB , which was abrogated by antioxidant treatment .",
"Our study therefore identified a novel role of Rgs12 in regulating Nrf2 , thereby controlling cellular redox state and osteoclast differentiation ."
] | [
"Human bodies change with age , and the skeleton is among the parts of the body most visibly affected .",
"This is because bone tissue tends to decrease as the skeleton gets older .",
"For example , people often get shorter as they get older , mostly because they lose bone mass in areas of the skeleton that support posture .",
"Severe bone loss can also lead to osteoporosis , a debilitating condition where bones become brittle and fracture easily .",
"Human skeletons contain cells , called osteoclasts , which break down bone tissue .",
"Osteoclasts normally cooperate with other cells that add new bone , which helps maintain the balance between ‘bone-eating’ and ‘bone-building’ responsible for sculpting a healthy skeleton .",
"This balance is disrupted during old age when the body starts producing too many ‘hyperactive’ osteoclasts , and bone formation cannot keep up with bone loss .",
"Reactive oxygen species ( ROS ) are unstable , potentially toxic molecules that have been linked with diseases of aging .",
"Recent research has shown that low amounts of ROS can also drive the formation of new osteoclasts .",
"Ng , Li et al . therefore wanted to determine how exactly ROS did this – specifically , whether ROS works together with the cell signaling mechanisms involved in bone loss controlled by a gene called Rgs12 .",
"Initial experiments , using genetically altered mice , showed that removing Rgs12 from immature osteoclasts was enough to stop them from maturing .",
"The bones of these mice were also stronger and thicker than usual .",
"In contrast , forcing osteoclasts to produce large amounts of the protein encoded by Rgs12 heightened their bone-eating ability .",
"Analysis of the proteins made by cells without Rgs12 revealed that the cells had turned on the Nrf2 gene , a molecular ‘master switch’ that helps produce the enzymes capable of counteracting ROS ( termed antioxidants ) .",
"These cells therefore contained abnormally high amounts of antioxidants and low levels of ROS .",
"However , osteoclasts where the Rgs12 gene was present were able to generate ROS by switching off the Nrf2 gene , and were thus able to reach maturity .",
"These results shed new light on the molecular signals that direct the development and activity of osteoclasts .",
"In the future , a better understanding of these mechanisms could help us prevent them going wrong during aging , or even lead to better therapies for osteoporosis and other skeletal disorders ."
] | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | IK1 channels do not contribute to the slow afterhyperpolarization in pyramidal neurons | elife-11206-v1 | [
[
"In 1980 , Hotson and Prince described a long lasting hyperpolarization ( AHP ) that followed current-induced repetitive firing of action potentials in CA1 pyramidal neurons ( Hotson and Prince , 1980 ) .",
"Since this initial finding the role of the slow afterhyperpolarization ( sAHP ) has been elucidated particularly in the hippocampus where its activity profoundly impacts learning .",
"Thus , for example , the sAHP increases with aging and ovarian hormone deficiency ( Wu et al . , 2011 ) and this may underlie cognitive deficits in older or hormone deficient individuals .",
"In 1982 , Madison and Nicoll demonstrated that this sAHP was blocked by application of noradrenaline , via protein kinase A ( PKA ) , since the effect is prevented by PKA antagonists and mimicked by application of cyclic AMP or the PKA catalytic subunit ( Madison and Nicoll , 1982; Pedarzani and Storm , 1993 ) .",
"Blocking the sAHP eliminated spike-frequency adaptation and resulted in dramatically enhanced repetitive firing ( Madison and Nicoll , 1982 ) .",
"This same effect can be observed following application of other neurotransmitter receptors , such as histamine ( Haas and Konnerth , 1983 ) , dopamine ( Benardo and Prince , 1982a; Pedarzani and Storm , 1995 ) , cholinergic agonists ( Benardo and Prince , 1982b; Benardo and Prince , 1982c; Cole and Nicoll , 1984 ) , and various peptides ( Haug and Storm , 2000 ) .",
"In 1984 , these same authors reported that loading cells with the Ca2+ chelator , EGTA , or extracellular application of the voltage-gated Ca2+ channel blocker , Cd2+ , blocked the sAHP ( Madison and Nicoll , 1984 ) .",
"In 1986 , Lancaster and Adams used a hybrid clamp technique to record the sAHP in current clamp and the underlying current in voltage clamp .",
"This showed a characteristic slowly activating current , the IsAHP , with a reversal potential that shifted with the concentration of extracellular K+ in a manner consistent with a K+ current ( Lancaster and Adams , 1986 ) .",
"In 2002 , Power et al . showed that activation of the IsAHP was voltage independent ( Power et al . , 2002 ) .",
"Taken together , these and other reports strongly support the proposal that the sAHP was due to the activity of a Ca2+-dependent , voltage-independent K+ channel .",
"Given the importance of the sAHP the identity of the underlying channel ( s ) and associated molecular components are of great interest .",
"In 1996 , Kohler et al . reported the cloning and expression of three Ca2+-activated , voltage independent K+ channels , the SK ( small conductance ) channels ( SK1-3; KCNN1-3 ) ( Kohler et al . , 1996 ) and in 1997 the fourth member of this family , SK4 ( IK1; KCNN4; intermediate conductance ) was reported ( Ishii et al . , 1997; Joiner et al . , 1997 ) .",
"The pharmacological fingerprints of these channels are distinct .",
"Apamin , an 18 amino acid peptide isolated from honeybee venom selectively blocks SK2 and SK3 channels , while SK1 is less apamin sensitive and IK1 is not apamin sensitive ( Adelman et al . , 2012 ) .",
"Importantly , the sAHP is not blocked by apamin .",
"IK1 ( SK4 ) is blocked by charybdotoxin ( ChTX ) a 37 amino acid peptide isolated from scorpion venom that also blocks BK channels ( Ishii et al . , 1997 ) .",
"The antimycotic agent , clotrimazole also blocks IK1 ( Ishii et al . , 1997 ) , but this is also a P450 inhibitor .",
"However , the related triarlymethane , TRAM-34 , potently and specifically blocks IK1 channels ( Wulff et al . , 2000 ) .",
"Sensitivity to TRAM-34 has therefore been taken as the signature of IK1 channels .",
"The biophysical and pharmacological properties of the SK/IK channels fulfill many of the expected characteristics of the channels underlying the sAHP in CA1 and basolateral amygdala ( BLA ) pyramidal neurons: Ca2+-activated , voltage-independent , K+ selective .",
"In situ hybridization and immunohistochemistry suggest that SK1-3 are widely expressed in overlapping yet distinct patterns in the brain , including hippocampus and amygdala ( Stocker and Pedarzani , 2000; Sailer et al . , 2002 ) .",
"IK1 mRNA expression was detected in some limited brain areas but hippocampus and amygdala were ambiguous ( http://mouse . brain-map . org/experiment/show/130911; http://mouse . brain-map . org/experiment/show/119686 ) .",
"Knock out mice for each of the three SK channels revealed that the apamin-insensitive IsAHP was not affected in any of the SK null mice , eliminating them as candidates for the sAHP channel ( Bond , 2004 ) .",
"However , a recent report used an IK1 reporter mouse to show that the IK1 promoter was active in several brain regions including hippocampus ( Turner et al . , 2015 ) .",
"Turner and colleagues further reported that the sAHP and the IsAHP in CA1 pyramidal neurons were greatly reduced by application of TRAM-34 , and the sAHP was absent in CA1 pyramidal neurons from IK1 null mice , strongly suggesting that IK1 channels underlie the sAHP ( King et al . , 2015 ) .",
"In line with this , a recent paper reported that IK1 channels were suppressed by direct phosphorylation by PKA ( Wong and Schlichter , 2014 ) .",
"Given the disparity on the role of IK1 channels , we have examined the sensitivity of the sAHP in pyramidal neurons from area CA1 of the hippocampus and the BLA to TRAM-34 and found that this compound did not significantly affect the current underlying the sAHP measured in voltage clamp .",
"TRAM-34 also had no effect on the sAHP amplitude or intrinsic excitability measured in current clamp .",
"Moreover , IK1 null mice express a characteristic IsAHP .",
"Together our results indicate that IK1 channels do not mediate the sAHP in pyramidal neurons ."
],
[
"As previously described ( Pedarzani and Storm , 1993; Madison et al . , 1987; Gerlach et al . , 2004 ) , a robust IsAHP was recorded in whole-cell voltage clamp configuration ( 22–24˚C ) from CA1 pyramidal neurons in freshly prepared hippocampal slices from 6–8 week old rats .",
"From a holding potential of -63 mV , a 200 ms voltage command to +7 mV was delivered to promote Ca2+ influx through voltage-gated Ca2+ channels .",
"Repolarization to -63 mV elicited a characteristic slowly decaying outward tail current , the IsAHP that decayed over several seconds with a time constant of 2 . 9 ± 0 . 2 s ( n = 20 ) .",
"Apamin was included in the bath solution to eliminate the SK channel contribution that overlaps with the initial decay phase of the IsAHP ( Bond , 2004 ) .",
"The IsAHP was measured as the current at 1 sec after the voltage step .",
"The tail current protocol was repeated every 30 sec for 25 min , and showed modest rundown of the IsAHP in control cells , being reduced to 0 . 74 ± 0 . 17 of the initial current amplitude ( n = 12 , P < 0 . 001 ) .",
"In some experiments carbachol ( CCh; 1 μM ) , a muscarinic agonist that potently blocks the IsAHP , was applied after 25 min ( Figure 1B-D ) .",
"To test the effects of TRAM-34 , control tail currents were first obtained for 5 min in the absence of drug .",
"The average amplitude of the IsAHP in this baseline control period was 209 . 3 ± 27 . 9 pA ( n = 11 ) , not different for control cells ( 195 . 0 ± 28 . 3 pA , n = 12 ) ( Figure 1C , D ) .",
"TRAM-34 ( 1 μM ) was added to the bath solution and the tail current protocol was continued .",
"After 25 min in TRAM-34 , the IsAHP relative to control baseline was 0 . 79 ± 0 . 18 ( n = 11 ) , not different than rundown in control cells ( Figure 1E ) .",
"As in control , subsequent addition of CCh abolished the IsAHP ( Figure 1C ) .",
"While TRAM-34 rapidly blocks native and cloned IK1 channels when applied in the bath solution , the binding site for TRAM-34 is internal ( Wulff , 2001 ) .",
"Therefore , TRAM-34 was also applied through the patch pipette ( Figure 1A , D-F ) .",
"With intracellular dialysis of TRAM-34 , the relative amplitude of the IsAHP measured 25 min after dialysis was not different from external TRAM-34 application ( 0 . 92 ± 0 . 13 of the initial current , n = 4 ) ; CCh treatment eliminated the IsAHP .",
"The sensitivity of the IsAHP to 5 μM TRAM-34 was also tested and this increased concentration of TRAM-34 was without effect ( 0 . 83 ± 0 . 07 , n = 9 ) .",
"ChTX ( 100 nM ) was also tested and as for TRAM-34 , ChTX did not affect the IsAHP ( relative IsAHP 0 . 83 ± 0 . 11 , n = 10 ) .",
"To be certain that the drugs , TRAM-34 and ChTX , were active each was bath-applied to HEK293 cells transiently expressing cloned IK1 channels .",
"TRAM-34 ( 1 μM ) produced a rapid block within 30 sec of the IK1 current ( relative current after TRAM-34 compared to baseline = 0 . 07 ± 0 . 03 , n = 3 ) ( Figure 2 ) .",
"Similarly , ChTX ( 100 nM ) blocked cloned IK1 currents ( relative current after ChTX = 0 . 05 ± 0 . 01 , n = 8; not shown ) .",
"These data show that ChTX or TRAM-34 does not block the IsAHP but they do block IK1 channels . 10 . 7554/eLife . 11206 . 003Figure 1 . TRAM-34 ( 1 µM; 22-24˚C ) does not affect the IsAHP .",
"( A ) Time course of the normalized amplitude of the IsAHP from control rundown ( ctrl , closed black symbols ) , or TRAM-34 treated cells either bath applied ( closed red symbols ) or internally delivered ( open red symbols ) in CA1 pyramidal neurons .",
"( B , C , D )",
"Representative CA1 pyramidal neuron tail currents elicited by the voltage protocol shown above the traces for control rundown ( B ) , bath applied TRAM-34 ( C ) and internally delivered TRAM-34 ( D ) at −5 to 0 min ( black ) , 25-30 min ( red ) and 10– 15 min after CCh application ( blue ) .",
"Vertical dash line at 1 sec after the pulse indicates time point for IsAHP measurement in time course plot of ( A ) .",
"( E ) Bar plot of the amplitudes of the IsAHP during a 5 min baseline period for control rundown ( Ctrl ) , bath applied TRAM-34 and internal TRAM-34 .",
"( F ) Bar plot of the IsAHP measured at 25–30’ ( red shaded area panel A ) relative to 5 min baseline ( black shaded area panel A ) for control rundown ( Ctrl; n = 10 ) and bath applied TRAM-34 ( n = 11 ) and internal TRAM-34 ( n = 4 ) .",
"Error bars are ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 11206 . 00310 . 7554/eLife . 11206 . 004Figure 2 . TRAM-34 blocks cloned IK1 channels .",
"( A ) Time course of TRAM-34 block of IK1 channels expressed in HEK293 cells ( n = 3 ) .",
"( B ) Representative whole-cell recordings with 10 µM Ca2+ in the patch pipette .",
"Currents were evoked from HEK293 cells expressing IK1 by voltage ramp commands ( 0 . 16 mV/ms ) in control bath solution ( black ) and after TRAM-34 application ( red ) ( 1 µM; 22– 24 ˚C ) .",
"( C ) Scatter plot of TRAM-34 block of IK1 current ( n = 3 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11206 . 004 Somatic whole-cell current clamp recordings ( 33˚C ) were obtained from CA1 pyramidal neurons in rat hippocampal slices .",
"A brief spike train was evoked by depolarizing current injection ( 200 pA for 100 ms ) , and was followed by characteristic medium ( m ) and slow ( s ) AHPs ( Figure 3A ) .",
"The peak AHP amplitudes recorded in normal aCSF were 4 . 36 ± 0 . 39 mV for the mAHP , and 2 . 79 ± 0 . 41 mV for the sAHP ( n = 6 ) .",
"The cells treated with TRAM-34 for 30 min ( n = 7 ) showed similar AHP amplitudes: 4 . 46 ± 0 . 33 mV for the mAHP and 3 . 17 ± 0 . 27 mV for the sAHP ( Figure 3B ) .",
"In a different set of neurons ( n = 5 ) TRAM-34 application for at least 25 min did not affect the sAHP ( Figure 3D ) , but the sAHP was rapidly blocked by subsequent bath application of noradrenaline ( 10 μM ) ( Figure 3C , D , E ) .",
"Combined bath application of XE991 to block Kv7/KCNQ/M channels and apamin to block SK channels , a combination also used by King et al . ( 2015 ) , for at least 30 min effectively eliminated the mAHP , but did not significantly affect the sAHP ( Figure 4A , B ) .",
"The same combination including TRAM-34 also did not affect the sAHP ( Figure 4A , B ) .",
"Similar results were obtained either using acute hippocampal slices or hippocampal slice cultures , so the data were pooled together ( Figure 5 ) .",
"Finally , intrinsic excitability was examined in CA1 pyramidal cells in acute hippocampal slices .",
"Spike trains were evoked by a series of depolarizing current pulses ( 0-400 pA; 1s ) in control or TRAM-34 containing bath solution ( Figure 5A ) .",
"There was no difference in spike rates between the two groups of cells ( Figure 5B ) .",
"These data indicate that TRAM-34 does not affect the sAHP or intrinsic excitability . 10 . 7554/eLife . 11206 . 005Figure 3 . TRAM-34 has no significant effects on the medium afterhyperpolarization ( mAHP ) or slow afterhyperpolarization ( sAHP ) in CA1 pyramidal cells .",
"( A ) Brief spike trains ( 7 spikes/100 ms ) were evoked by depolarizing current pulses from a holding potential of -70 mV .",
"Black and red traces represent recordings from two pyramidal cells , in control medium and after incubation with TRAM-34 ( 1µM ) for 30 minutes .",
"Inset , showing mAHP ( ⚫ ) and sAHP ( ▲ ) at an enlarged scale .",
"( B ) mAHP and sAHP amplitudes were not significantly different between control and TRAM-34 treated groups .",
"Data are given as , mean ± SEM .",
"( C ) Representative traces of the effect of TRAM-34 ( 1 µM , red ) and noradrenaline ( NA , 10 µM , blue ) on the mAHP and sAHP in CA1 pyramidal neurons .",
"( D ) Time course of the sAHP amplitude measured in panel A . Bath application of TRAM-34 ( 1 µM ) for 25 min did not significantly reduce the sAHP ( n = 5 ) .",
"However , subsequent application of noradrenaline ( NA , blue line ) rapidly eliminated the sAHP .",
"( E ) Summary bar plots showing the sAHP amplitude averaged over a period of five minutes during control ( black bar ) , TRAM-34 ( red bar ) and NA ( blue bar ) application .",
"The averaged periods are represented by color bottom lines in panel D . DOI: http://dx . doi . org/10 . 7554/eLife . 11206 . 00510 . 7554/eLife . 11206 . 006Figure 4 . Incubation of acute hippocampal slices and organotypic slice cultures with TRAM-34 ( 1 μM ) did not reduce the sAHP in CA1 pyramidal neurons .",
"( A ) Example traces showing the mAHP and sAHP in three different conditions tested .",
"Cells in the control group ( black trace ) were recorded in normal ACSF .",
"Cells in the second group ( XE991+Apamin , green trace ) were recorded after incubation for at least 30 minutes in ACSF with XE991 ( 10 μM ) and apamin ( 100 nM ) .",
"Cells in the third group ( XE991+Apamin+TRAM-34 , red trace ) were recorded after incubation for at least 30 minutes in ACSF with XE991 ( 10 μM ) , apamin ( 100 nM ) and TRAM-34 ( 1 μM ) .",
"( B ) Summary of the results from slices ( open circles ) and organotypic cultures ( open squares ) .",
"The sAHP amplitude did not differ between the three groups of cells: ( 1 ) control ( n = 10 ) , ( 2 ) XE991+Apamin ( n = 11 ) , and ( 3 ) XE991+Apamin+TRAM-34 ( n = 12 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11206 . 00610 . 7554/eLife . 11206 . 007Figure 5 . TRAM-34 ( 1 µM ) had no significant effect on the excitability of CA1 pyramidal cells .",
"( A ) Representative spike trains evoked by 1s long depolarizing current ( 200 pA ) injections from -76 mV , recorded from pyramidal cells in control medium ( left , black trace ) and after incubation of TRAM-34 ( right , red trace ) .",
"( B ) Comparison of spike rates ( spikes/s ) between control ( black ) and TRAM-34 ( red ) treated groups evoked by depolarizing , 1 s long current pulses ( 0–400 pA ) .",
"Mono-exponential fits were used to compare spike rates in control medium and TRAM-34 treated groups , by using the function: f ( X ) = A[exp ( -x/τ ) ]+ y0 .",
"No significant differences were found between control and TRAM-34 treated groups [control , τ: 263 ( 58 ) pA; TRAM-34 , τ: 371 ( 65 ) pA , N . S , p = 0 . 161 , t-test after Box-Cox transformation ( Minitab 17 ) ] .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 11206 . 007 Pyramidal neurons of the basolateral amygdala ( BLA ) have been shown to express an IsAHP and sAHP that are indistinguishable from those observed in hippocampal CA1 pyramidal neurons ( Power et al . , 2011 ) , suggesting that the same molecular components underlie the sAHP .",
"BLA pyramidal neurons were first recorded in whole-cell voltage clamp ( Figure 6A , B ) .",
"From a holding potential of −50 mV a depolarizing command to 10 mV was given for 100 ms . Upon return to −50 mV a characteristic outward tail current , IsAHP , was observed ( Figure 6A ) .",
"Neurons were recorded in the absence ( n = 8 ) or presence ( n = 10 ) of TRAM-34 ( 1 μM ) in the internal pipette solution .",
"The IsAHP showed modest rundown when examined at 2 , 9 and 17 min after whole-cell formation but TRAM-34 was without effect ( Figure 6B ) .",
"In either condition , subsequent addition of noradrenaline ( 10 μM ) abolished the IsAHP ( n = 1 in the absence and n = 2 in the presence of TRAM-34 ) .",
"BLA pyramidal neurons were also recorded in current clamp mode ( Figure 6C , D ) .",
"A train of action potentials was evoked every 10 sec by an 800 ms depolarizing current injection .",
"Whether recorded in the absence ( n = 6 ) or presence ( n = 7 ) of TRAM-34 in the internal solution the numbers of action potentials at 1 min or 18 min were not significantly different ( Figure 6D , top ) .",
"In addition , TRAM-34 did not affect action potential half width ( Figure 6D , bottom ) or resting membrane potential ( not shown ) .",
"These results show that TRAM-34 does not affect the IsAHP or excitability in BLA pyramidal neurons . 10 . 7554/eLife . 11206 . 008Figure 6 . TRAM-34 did not block the IsAHP in BLA pyramidal neurons . Whole-cell voltage clamp recording from BLA pyramidal neurons .",
"( A ) Representative current traces for IsAHP current evoked from a holding potential of −50 mV under control conditions ( upper traces ) and with 1 µM TRAM-34 added to the pipette solution .",
"Insets show the effects of 10 µM noradrenaline .",
"( B ) The peak IsAHP current , measured 500 ms after the voltage step is plotted under the two conditions in control and TRAM-34 loaded neurons at the indicated times after onset of the whole-cell recording configuration ( break-in ) .",
"The lower panel shows the same data normalized to the amplitude 2 minutes after break-in .",
"( C ) Current clamp recordings from the neurons shown in ( A ) , discharge evoked by a 600 ms current injection .",
"( D ) Plotted are the action potential half width and number of evoked action potentials evoked by the current injection at the indicated times in control and TRAM-34 . DOI: http://dx . doi . org/10 . 7554/eLife . 11206 . 008 Whole-cell voltage clamp recordings were made from CA1 pyramidal neurons in freshly prepared hippocampal slices from IK1 null mice ( Si , 2006 ) or strain-matched wild type mice , as for those in rat ( above ) .",
"In wild type mice , the amplitude of the slow component of the outward tail current measured at 1 s following repolarization to − 63 mV was 78 . 2 ± 16 . 5 pA ( n = 9 ) , and was blocked by subsequent application of CCh ( Figure 7A , C ) .",
"The IsAHP elicited from CA1 pyramidal neurons of IK1 null mice was 74 . 9 ± 13 . 8 pA ( n = 12 ) and was potently blocked by CCh .",
"Current subtraction yielded the CCh-sensitive IsAHP current with the characteristic slow rising onset and slow decay ( Figure 7B , C ) .",
"Thus , CA1 pyramidal neurons from IK1 null mice express an IsAHP that seems indistinguishable from that of wild type mice . 10 . 7554/eLife . 11206 . 009Figure 7 . The IsAHP in IK1 null mice is not different from wild type .",
"( A , B )",
"Tail currents elicited by the voltage protocol from −63 to 7 mV shown above the traces for wild type ( A ) and IK1 null ( B ) .",
"Black traces are control , red traces are after CCh application , and blue traces are the subtracted CCh sensitive currents .",
"( C ) Scatter plot of the IsAHP amplitude for wild type and IK1 null mice .",
"Mean indicated by horizontal line . DOI: http://dx . doi . org/10 . 7554/eLife . 11206 . 009"
],
[
"The sAHP conductance in CA1 pyramidal neurons is a powerful modulator of intrinsic excitability and many neurotransmitters activate second messenger pathways that converge on the sAHP , suppressing the sAHP and increasing intrinsic excitability ( Haug and Storm , 2000 ) .",
"Modulation of the sAHP has been implicated in behavioral learning: animals with a smaller sAHP in CA1 pyramidal neurons learn hippocampus-dependent tasks better than those with a larger sAHP ( Moyer et al . , 2000; Tombaugh , 2005 ) , and a reduction of the sAHP is observed after successful learning ( Moyer et al . , 1996; Oh , 2003 ) .",
"The sAHP increases with age ( Landfield and Pitler , 1984 ) , and this may underlie cognitive deficits in older animals ( Deyo et al . , 1989; Knuttinen et al . , 2001 ) .",
"Thus , understanding the molecular basis of the sAHP is important and may lead to novel therapeutic approaches to manage cognitive decline .",
"Compelling evidence suggests that the sAHP reflects the activity of Ca2+-dependent , voltage-independent K+-selective channels .",
"Members of the SK ( KCNN ) channel family share many features that are similar to those that mediate the sAHP .",
"Thus , all four members ( KCNN1-4; SK1-3 , IK1 ) are voltage independent and are Ca2+-gated via constitutively bound calmodulin ( Adelman et al . , 2012 ) .",
"The fourth member of the family , IK1 , has a larger unitary conductance than the other family members ( ~40 pS vs ~10 pS in symmetrical K+ ) ( Kohler et al . , 1996; Ishii et al . , 1997; Joiner et al . , 1997 ) and , importantly , the SK and IK channels are pharmacologically distinct .",
"The peptide toxin , apamin potently and selectively blocks SK2 and SK3 channels ( Adelman et al . , 2012 ) , while IK1 channels are apamin-insensitive but are selectively blocked by TRAM-34 ( Wulff et al . , 2000 ) .",
"IK1 is additionally blocked by the scorpion peptide charybdotoxin ( ChTX ) ( Ishii et al . , 1997 ) that also blocks BK channels .",
"In situ hybridization and immunohistochemistry data indicate that SK1-3 are expressed in overlapping but distinct patterns in the CNS .",
"In hippocampal CA1 pyramidal neurons and BLA pyramidal neurons , SK2 is heavily expressed while SK1 and SK3 are expressed at lower levels ( Stocker and Pedarzani , 2000; Sailer et al . , 2002 ) .",
"IK1 mRNA is expressed in peripheral tissues such as smooth muscle endothelium , gastrointestinal tract , lung , and salivary glands , but brain expression is limited ( Begenisich et al . , 2004 ) .",
"Indeed , recent transcriptome profiling using single CA1 hippocampal pyramidal neurons did not detect significant IK1 expression ( Zeisel et al . , 2015 ) .",
"In contrast , a recent study using both immunohistochemistry and a GFP reporter mouse suggested that IK1 expression was significant in both cortex and hippocampus , including CA1 pyramidal neurons ( Turner et al . , 2015 ) .",
"Using a monoclonal antibody to IK1 , immunohistochemistry detected IK1 throughout the hippocampal formation ( Turner et al . , 2015 ) .",
"However this antibody also recognized a band on Western blots using tissue derived from either of two independently generated IK1 knockout mice ( Turner et al . , 2015 ) .",
"Therefore , the specificity of the monoclonal antibody is questionable .",
"For both of these transgenic IK1 gene disruption mouse lines previous work reported the loss IK1 mRNA expression ( Si , 2006; Begenisich et al . , 2004 ) , and for one line , the line employed here , the loss of IK1 protein expression as well ( Si , 2006 ) .",
"Indeed , Si et al . ( 2006 ) used a different IK1 antibody that detected robust expression in Western blots with red blood cells prepared from wild type but did not detect a band in protein samples from the null mice , engendering confidence in this IK1 null mouse .",
"We used these IK1 null mice to record the characteristic IsAHP in CA1 pyramidal neurons .",
"Additionally , Turner et al . ( 2015 ) generated an IK1 promoter GFP-reporter mouse .",
"In this mouse GFP was detected throughout the hippocampus .",
"Assuming that the BAC employed contained all of the requisite regulatory sequences to direct normal IK1 expression , these results suggest that the IK1 promoter is active in hippocampus , but stands in contrast to transcriptome results ( Zeisel et al . , 2015 ) .",
"In red blood cells , IK1 has been shown to be the ‘Gardos' channel , responsible for Ca2+-dependent K+ efflux . The channel protein is presumably very stable as erythrocytes live for ~120 days after enucleation . Thus it is possible that a very brief ‘pulse’ of IK1 mRNA could give rise to channels that are stable long after the mRNA has been degraded .",
"CA1 pyramidal neurons from SK1 , SK2 , or SK3 knockout mice do not show alterations in the IsAHP , suggesting that none of these channels individually contribute to the sAHP , particularly SK1 that is less apamin sensitive ( Bond , 2004 ) .",
"In primary afferent neurons of the gastrointestinal tract there is a late AHP that follows the action potential that bears many similarities to the sAHP in CA1 pyramidal neurons .",
"IK1 is prominently expressed in these myenteric neurons and the late AHP is blocked by TRAM-34 ( Nguyen et al . , 2007 ) .",
"Thus , it is likely that IK1 channel activity is responsible for this late AHP .",
"A recent report showed that the sAHP in CA1 pyramidal neurons was at least partially blocked by the signature agent , TRAM-34 , and that the sAHP was absent in IK1 null mice .",
"The authors concluded that IK1 channel activity is also responsible for the sAHP in CA1 pyramidal neurons ( King et al . , 2015 ) .",
"Consistent with this , PKA has been shown to inhibit IK1 channel activity via direct phosphorylation of the channel ( Wong and Schlichter , 2014 ) .",
"We re-examined the sAHP in hippocampal CA1 pyramidal neurons and found that TRAM-34 or ChTX did not block it , and TRAM-34 had no effect on intrinsic excitability .",
"Similar results were obtained from pyramidal neurons of the BLA .",
"We noted that the average amplitude of the IsAHP in Figure 1 is higher than previously recorded in CA1 pyramidal cells ( Gu et al . , 2005 ) .",
"There are two aspects to this .",
"First , the IsAHP increases with age , and for the IsAHP in CA1 pyramidal neurons we used older ( 8 weeks ) rats .",
"Second , the amplitude of the IsAHP is sensitive to changes in temperature ( Lancaster and Adams , 1986; Sah and Isaacson , 1995 ) that may explain the larger IsAHP amplitude in our present study ( recorded at 22–23° ) .",
"However , this is unlikely to affect our overall interpretation , since IsAHP may be increased at lower temperatures and therefore any IK1 component might be even bigger .",
"Importantly , IK1 null mice showed a prominent IsAHP .",
"Moreover , IK1 channels have been reported to have a single channel conductance of ~12 pS ( Joiner et al . , 1997 ) , however , while not directly recorded , channels underlying the IsAHP have been proposed to have a much lower single channel conductance of 2– 5 pS ( Sah and Isaacson , 1995 ) .",
"Our data show no effect of TRAM-34 in contrast to King et al . ( 2015 ) This discrepancy may be related to a combination of several factors:",
"1 ) run-down of IsAHP during whole-cell recordings has been observed in several previous studies and cannot be excluded in absence of time course plots .",
"2 ) The Ca2+ influx and IsAHP may differ during synaptic stimulation ( King et al . , 2015 ) or somatic current pulses by recruitment of different Ca2+ sources .",
"This may affect activation of IK1 depending on its specific subcellular localization .",
"Regardless , data from three different laboratories using different conditions failed to find an effect of TRAM-34 .",
"Therefore , we conclude that the channel ( s ) underlying the sAHP is not IK1 and has not yet been identified .",
"Several possibilities remain .",
"For example , the sAHP might not be due to the activity of one particular type of K channel , but rather an ensemble of channels ( Andrade et al . , 2012 ) .",
"It has also been proposed that the sAHP is mediated at least in part by Kv7/KCNQ ( M ) channels with Ca2+ sensitivity endowed by the calcium binding protein , hippocalcin ( Tzingounis et al . , 2010; Tzingounis et al . , 2007 ) , although the Kv7/KCNQ ( M ) channel blocker XE991 fails to block the sAHP of CA1 pyramidal cells ( Gu et al . , 2005 ) ( see Figure 4 ) .",
"Alternatively , the K+ channel superfamily contains several subunits that do not express functional channels on their own .",
"In either case , the pore-forming subunits may be co-assembled into larger signaling complexes that endow Ca2+ sensitivity and characteristic slow activation kinetics .",
"Recapitulating the sAHP using cloned components , together with gene targeting and pharmacology will eventually reveal the molecular details of the sAHP ."
],
[
"All procedures were done in accordance with the guidelines of the Institutional Animal Care and Use Committee ( IACUC ) of the Oregon Health & Science University ( IACUC: IS00002421 ) , the Animal Care and Use Committee of Institute of Basic Medical Sciences of the University of Oslo ( FOTS ID 5676 ) , and the Animal Ethics Committee ( AEC ) of the University of Queensland ( QBI/551/12/NHMRC/ARC ) .",
"Acute hippocampal slices were prepared from 3–8 week-old Wistar rats , IK1 null mice ( Si , 2006 ) or C57BL/6J mice .",
"Acute amygdala slices were prepared from 3–4 week-old C57BL/6J mice .",
"Slices from rats and mice were prepared as previously described ( Gu et al . , 2005; Lin et al . , 2008; Faber et al . , 2005 ) .",
"Organotypic hippocampal slice cultures were prepared from postnatal day 5–6 Wistar rats and used for recordings 14–21 d after preparation .",
"Pups were decapitated and the brains placed in a solution containing ( in mM ) : NaCl 137 , KCl 5 , NaH2PO4 0 . 85 , CaCl2 1 . 5 , KH2PO4 0 . 22 , MgSO4 0 . 28 , MgCl2 1 , NaHCO3 2 . 74 and glucose 45 , dissolved in tissue grade water .",
"Each hippocampus was individually dissected out and cut into 400 μm thick transverse slices using a McIlwain tissue chopper .",
"Slices were placed on filter membranes ( 0 . 4 um Hydrophilic PTFE filters , Millipore , Billerica , MA ) , and cultivated in 6-wells plates at 36 °C for 21 d .",
"Each of the wells contained 1 ml culture medium replaced first after 24 h , and then every 3–4 days .",
"The culture medium consisted of Basal Medium Eagle with HBSS ( 50% ) and Hanks balanced salt solution , HBSS ( 25% ) ( both from AMIMED , UK ) , heat inactivated horse serum ( 25% ) , penicillin/streptomycin ( 100 U/ml ) and ( in mM ) : L-glutamine 1 , glucose 20 and NaHCO3 6 .",
"For voltage clamp recordings , CA1 and BLA pyramidal neurons from acutely prepared brain slices were visualized with infrared–differential interference contrast optics ( Zeiss Axioskop 2FS or Olympus BX50-WI ) and a CCD camera ( Sony , Tokyo , Japan or Dage-MTI , Michigan City , IN ) .",
"Whole-cell patch-clamp recordings were obtained from CA1 pyramidal cells using an Axopatch 1D amplifier ( Molecular Devices , Sunnyvale , CA ) , digitized with an ITC-16 analog-to-digital converter interface ( Heka Instruments , Bellmore , NY ) and transferred to computer using Patchmaster ( Heka Instruments , Bellmore , NY ) or a Multiclamp 700B amplifier ( Molecular Devices , Sunnyvale , CA ) , digitized using a Digidata 1440A interface ( Molecular Devices , Sunnyvale , CA ) , and transferred to a computer using pClamp10 software ( Molecular Devices , Sunnyvale , CA ) .",
"Recordings were performed at 22– 23˚C .",
"For CA1 pyramidal neurons , apamin ( 100 nM ) was added to minimize contribution of SK currents , and SR95531 ( 2 µM ) and CGP55845 ( 1 µM ) were present to block GABAA and GABAB receptors , respectively .",
"For BLA pyramidal neurons whole-cell patch-clamp recordings were obtained using a Multiclamp 700A amplifier ( Molecular Devices , Sunnyvale , CA , USA ) , digitized with an ITC-16 analog-to-digital converter interface ( Heka Instruments , Bellmore , NY ) and transferred to computer using Axograph-X ( Axograph Scientific , New South Wales , Australia ) .",
"Recordings were performed at 32˚C .",
"Patch pipettes ( 2 . 5–3 . 5 MΩ ) for IsAHP recordings in CA1 pyramidal neurons were filled with a KMeSO4 internal solution containing ( in mM ) KMeSO4 140 , NaCl 8 , MgCl2 1 , HEPES 10 , MgATP 5 , Na3GTP 0 . 4 , EGTA 0 . 05 , ( pH 7 . 3 ) .",
"For IsAHP recordings in BLA neurons , patch pipettes ( 4–6 MΩ ) were filled with a KMeSO4 internal solution containing ( in mM ) KMeSO4 135 , NaCl 8 , HEPES 10 , Mg2ATP 2 , Na3GTP 0 . 4 , Spermine 0 . 1 , Phosphocreatine 7 , EGTA 0 . 2 , ( pH 7 . 3 with KOH; osmolarity ~ 290 ) .",
"Currents were recorded in whole-cell voltage clamp mode .",
"For CA1 and BLA pyramidal neurons the membrane potential was held at −63 mV ( CA1 ) and −50 mV ( BLA ) , and IAHP currents were evoked in CA1 pyramidal neurons by depolarizing voltage commands to +7 mV for CA1 neurons for 200 ms and 10 mV for BLA neurons for 100 ms followed by a return to baseline potential for 10 sec where the current underlying the sAHP was measured .",
"All cells had a resting membrane potential more hyperpolarized than −60 mV and input resistances of 150–350 MΩ for CA1 pyramidal neurons and 75–325 MΩ for BLA pyramidal neurons .",
"Input resistance was determined from a −5 mV ( 100 ms ) hyperpolarizing pulse applied at the beginning of each sweep .",
"Access resistance was 80% electronically compensated and stable at <20 MΩ .",
"IAHP recordings were filtered at 3 kHz and digitized at a sampling frequency of 10 kHz .",
"Voltages in CA1 and BLA pyramidal neurons were corrected for the liquid junction potential .",
"Data were analyzed using Igor Pro ( WaveMetrics , Lake Oswego , OR ) or Excel ( Microsoft , Seattle , WA ) .",
"Data are expressed as mean ± SEM .",
"Paired t-tests or Wilcoxon-Mann-Whitney 2-sample rank test was used to determine significance; P < 0 . 05 was considered significant .",
"For current clamp recordings , organotypic hippocampal slice cultures or acutely cut hippocampal slices were transferred to a recording chamber perfused with aCSF ( 34 °C ) of the following composition ( in mM ) : NaCl 125 , KCl 3 . 5 , MgCl2 1 , NaH2PO4 1 . 25 , NaHCO3 25 , CaCl2 1 . 6 , glucose 25 .",
"Acutely cut BLA brain slices were transferred to a recording chamber perfused with aCSF ( 34°C ) of the following composition ( in mM ) : NaCl 118 , KCl 2 . 5 , MgCl2 1 . 3 , NaH2PO4 1 . 2 , NaHCO3 25 , CaCl2 2 . 5 , glucose 10 .",
"CA1 and BLA pyramidal neurons were visually identified using infrared-differential interference contrast ( IR-DIC ) optics on an Olympus BX-51WI or BX-50WI microscope .",
"For hippocampal neurons the intracellular recording solution contained ( in mM ) : KGluconate 120 , KCl 20 , Na2 . phosphocreatine 5 , HEPES 10 , MgATP 4 , Na2GTP 0 . 4 , EGTA 0 . 1 ( pH 7 . 2 adjusted with KOH ) .",
"For BLA neurons the intracellular recording solution contained in ( in mM ) KMeSO4 135 , NaCl 8 , HEPES 10 , Mg2ATP 2 , Na3GTP 0 . 4 , Spermine 0 . 1 , Phosphocreatine 7 , EGTA 0 . 2 , ( pH 7 . 3 with KOH ) .",
"Whole-cell current clamp recordings were obtained using a Multiclamp 700A or B amplifier , signals were low-pass filtered at 10 KHz and digitized at 20 KHz .",
"Access resistances , typically 15–35 MΩ , were monitored and compensated throughout the experiments .",
"Potentials were corrected for liquid junction potential ( −14 mV ) .",
"Origin 9 . 1 ( Hearne Scientific Software , Victoria , AU ) was used for statistical analysis and graphical representations .",
"In all hippocampal experiments , 6 , 7-dinitroquinoxaline-2 , 3-dione ( DNQX , 10 μM ) , DL-2-amino-5-phosphono-pentanoic acid ( DL-AP5 , 50 μM ) and gabazine ( SR-95531 , 5 μM ) were added to the aCSF to block spontaneous synaptic transmission .",
"In Figure 4 , XE991 ( 10 μM ) , apamin ( 100 nM ) and TRAM-34 ( 1 μM ) were added to the ACSF and slices were perfused for 25 minutes before recordings started .",
"The mAHP and sAHP values were measured by averaging a time window ( 20 and 100 ms , respectively ) around the peak of each one ( 20–50 ms and 0 . 17–0 . 4 s after a train of 7–8 spikes , respectively ) and subtracted from the baseline voltage .",
"Statistical analysis was done in Minitab 17 ( Minitab UK ) .",
"Group data are expressed as mean and standard error ( SEM , in parentheses ) , and the sample size of cells ( n ) , and number of rats ( N ) used in the individual experiments is given .",
"Data were checked for normal distribution according to a normal probability plot of their residuals and 2-tailed t tests were performed for independent samples ( control group vs . drug group ) using a critical level of significance α=0 . 05 .",
"HEK293 cells ( ATCC CRL-1573 ) were transfected using lipofectamine with a plasmid directing expression of IK1 cDNA ( Ishii et al . , 1997 ) ."
]
] | [
"In pyramidal neurons such as hippocampal area CA1 and basolateral amygdala , a slow afterhyperpolarization ( sAHP ) follows a burst of action potentials , which is a powerful regulator of neuronal excitability .",
"The sAHP amplitude increases with aging and may underlie age related memory decline .",
"The sAHP is due to a Ca2+-dependent , voltage-independent K+ conductance , the molecular identity of which has remained elusive until a recent report suggested the Ca2+-activated K+ channel , IK1 ( KCNN4 ) as the sAHP channel in CA1 pyramidal neurons .",
"The signature pharmacology of IK1 , blockade by TRAM-34 , was reported for the sAHP and underlying current .",
"We have examined the sAHP and find no evidence that TRAM-34 affects either the current underling the sAHP or excitability of CA1 or basolateral amygdala pyramidal neurons .",
"In addition , CA1 pyramidal neurons from IK1 null mice exhibit a characteristic sAHP current .",
"Our results indicate that IK1 channels do not mediate the sAHP in pyramidal neurons ."
] | [
"Neurons carry signals in the form of electrical impulses called action potentials .",
"These nerve impulses result from ions flowing through proteins called ion channels in the neuron’s membrane , and they determine how the neuron communicates with neighboring neurons .",
"The number of action potentials a neuron can produce can vary over a wide range .",
"In the brain , a particular kind of ion channel limits the number of action potentials that many neurons produce via a negative feedback mechanism .",
"That is to say , nerve impulses activate this ion channel and the activated channel then makes the neuron less able to send further nerve impulses for a while . The activity of this ion channel increases with age and it may be responsible for some forms of age-related decline in cognitive abilities .",
"However , the exact identity of the ion channel responsible was unclear .",
"Recent research has suggested the ion channel in question was a protein called IK1 .",
"This conclusion was largely based on how this ion channel responded to drugs in the laboratory .",
"Wang , Materos-Aparico et al . sought to verify this conclusion and , in contrast with the previous reports , found that the IK1 ion channel did not respond to these drugs in the same way when it was in neurons in the brains of mice .",
"In further experiments , mice that had been engineered to lack the IK1 ion channel still showed the characteristic negative feedback that regulates the firing of action potentials .",
"Thus , Wang , Materos-Aparico et al . found no evidence to support the previous conclusion , and instead conclude that the exact identity of this important ion channel in the brain has yet to be defined ."
] | 2016 |
[
"Introduction",
"Results and discussion",
"Materials and methods"
] | [
"structural biology and molecular biophysics"
] | Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5′ splice site recognition | elife-04986-v1 | [
[
"Removal of introns from pre-messenger RNA ( pre-mRNA ) is an essential step in eukaryotic gene expression .",
"This process is catalysed by a large and dynamic RNA-protein assembly called the spliceosome , which consists of five small nuclear ribonucleoprotein particles ( U1 , U2 , U4 , U5 and U6 snRNPs ) and numerous non-snRNP proteins ( Will and Lührmann , 2011 ) .",
"U1 snRNP recognizes a short sequence at the 5′-splice site ( 5′SS ) of pre-mRNA through basepairing between the 5′-end of U1 snRNA and the 5′SS sequence ( Lerner et al . , 1980; Zhuang and Weiner , 1986; Siliciano and Guthrie , 1988; Séraphin et al . , 1988 ) and promotes an ordered assembly of the four remaining snRNPs to form the spliceosome , which then undergoes extensive conformational and compositional remodelings to become catalytically active ( Will and Lührmann , 2011 ) .",
"During activation , the interaction between U1 snRNP and the 5′SS is disrupted by RNA helicase Prp28 ( Staley and Guthrie , 1999 ) and then the 5′SS intron sequence base-pairs with part of the ACAGAGA box in U6 snRNA ( Sawa and Abelson , 1992; Sawa and Shimura , 1992; Wassarman and Steitz , 1992; Kandels-Lewis and Séraphin , 1993; Lesser and Guthrie , 1993; Sontheimer and Steitz , 1993 ) ; whilst the 5′ exon interacts with U5 snRNA loop I for the first trans-esterification reaction ( Newman and Norman , 1992 ) .",
"U1 snRNP is also an important regulator of mRNA 3′ end cleavage and polyadenylation ( Almada et al . , 2013; reviewed in Spraggon and Cartegni ( 2013 ) ) .",
"Human U1 snRNP comprises U1 snRNA , seven Sm proteins ( SmB/SmB′ , SmD1 , SmD2 , SmD3 , SmE , SmF and SmG ) and three U1-specific proteins ( U1-70K , U1-A and U1-C ) ( Hinterberger et al . , 1983; Bringmann and Lührmann , 1986 ) .",
"We reported the structure of the functional core of U1 snRNP ( Pomeranz Krummel et al . , 2009 ) based on an experimental electron density map at 5 . 5 Å resolution to which we fitted previously-determined structures of protein components ( Kambach et al . , 1999; Muto et al . , 2004; Leung , 2005 ) .",
"The most striking feature of the structure is the N-terminal region of U1-70k , which extends from its RRM through a long α-helix and wraps around the Sm protein assembly so that its N-terminus makes contact with U1-C protein , thus accounting for the requirement of U1-70k for U1-C binding ( Nelissen et al . , 1994; Hilleren et al . , 1995 ) .",
"In this crystal the 5′-end of U1 snRNA pairs with its symmetry-related counterpart , mimicking the binding of the 5′SS of pre-mRNA to U1 snRNP .",
"The Zn-finger domain of U1-C is located adjacent to this RNA duplex but the low-resolution map was insufficient for analysis of the RNA-protein contacts in atomic detail and hence it was not clear how U1-C contributes to the recognition of the 5′SS .",
"The structure of U1 snRNP from HeLa cells , treated with chymotrypsin , was subsequently reported ( Weber et al . , 2010 ) .",
"Although this crystal had a DNA oligonucleotide with the 5′SS consensus sequence bound to the 5′-end of U1 snRNA , the N-terminal end of U1-70k together with U1-C protein were lost by protease treatment .",
"Hence neither of these structures revealed molecular details of 5′SS recognition by U1 snRNP .",
"In order to gain crucial insight into the mechanism of 5′SS recognition we continued our attempts to grow crystals of U1 snRNP diffracting to high resolution but this proved unsuccessful because the inherent mobility of long RNA helices arranged as a 4-way junction prevented the formation of well-diffracting crystals ( Oubridge et al . , 2009; Weber et al . , 2010 ) .",
"Hence we designed two sub-structures of U1 snRNP , with exclusively human sequences , based on our 5 . 5 Å resolution structure and determined their crystal structures at high resolution .",
"Yeast U1 snRNP , when compared to the human particle , contains a larger and more complex snRNA , which is associated with many protein factors ( Prp39 , Snu71 , Prp40 , Prp42 , Nam8 , Snu56 , Urn1 and Prp5 ) , which have no counterparts in human U1 snRNP ( Neubauer et al . , 1997 ) .",
"However , despite these differences , the sequence of the 5′-single stranded region of U1 snRNA ( nts 1–10 ) is invariant from yeast to human ( http://rfam . sanger . ac . uk/ ) and the amino acid sequence of the Zn-finger of U1-C ( yeast Yhc1 ) is also highly conserved ( Muto et al . , 2004 ) .",
"Hence the 5′SS of pre-mRNA is expected to make exactly the same contacts with the 5′ end of U1 snRNA and U1-C in human and yeast U1 snRNPs .",
"However , some positions of the 5′SS have quite different nucleotide bias in yeast and human genes ( Burge et al . , 1999 ) .",
"In human the 5′SS sequences processed by major spliceosomes are degenerate but show significant overall complementarity to the sequence of the 5′ end of U1 snRNA ( Lerner et al . , 1980 ) .",
"In contrast the 5′SS intron sequence of yeast pre-mRNA is stringently conserved to be GUAUGU ( Burge et al . , 1999 ) .",
"Upon activation of the yeast spliceosome , the intron sequence , UGU ( +4 , +5 and +6 ) , pairs with ACA within the ACAGAGA sequence in U6 snRNA and hence these nucleotides are selected to be nearly invariant ( Sawa and Abelson , 1992; Kandels-Lewis and Séraphin , 1993 ) .",
"Cross-linking studies revealed interaction of the same regions of U6 snRNA and the 5′SS sequence in human but the sequence requirement is less obvious ( Sawa and Shimura , 1992; Wassarman and Steitz , 1992 ) .",
"Prp8 is also known to influence the selection of the 5′SS nucleotide ( reviewed in Grainger and Beggs ( 2005 ) ; Galej et al . ( 2013 ) ) .",
"Furthermore in humans constitutive or alternative splicing factors facilitate the binding of U1 snRNP to weak splice sites .",
"As discussed above , the 5′SS is subjected to multiple selections which differ in yeast and human and give rise to different nucleotide biases at 5′SS .",
"The strength of variant 5′SS sequences is assessed by relative usage of competing 5′SS ( Roca et al . , 2005 , 2012 ) which is determined not only by the affinity of 5′SS to U1 snRNP but also by multiple factors ( Roca et al . , 2013 ) .",
"Our two new crystals together reveal the structures of the substantial parts of U1 snRNP at high resolution and provide crucial insights into the mechanism of pre-mRNA recognition by U1 snRNP .",
"In particular , we find that U1-C makes no base-specific contacts with the 5′SS sequence .",
"Also , by measuring the intrinsic affinity of recombinant U1 snRNP for various 5′SS sequences we disentangle the role played by the U1 snRNP from the other complexities of 5′SS recognition , and assess the relative contributions of U1 snRNA and U1-C protein in light of our crystal structure ."
],
[
"The first 215 residues of U1-70k are conserved well from yeast to human ( 48% sequence similarity ) , suggesting that this region has an evolutionarily conserved essential function , whereas the C-terminal region , predicted to be poorly structured , has diverged considerably .",
"Some alternative splicing factors are known to bind to this region ( Labourier et al . , 2001; Ignjatovic et al . , 2005; Cho et al . , 2011 ) .",
"The U1A70kF-RNA crystal structure ( Figure 1B ) reveals the interaction between stem-loop I and U1-70k in detail , illustrating a new mode of RRM-RNA interaction .",
"The canonical RRM domain is known to bind with RNA through RNP1 and RNP2 motifs as first observed in the U1A- stem loop II complex structure ( Oubridge et al . , 1994 ) .",
"The RRMs of U1-A and U1-70k have very similar structures ( the β-strands and α-helices of the two RRMs superimpose with an rmsd of 0 . 70 Å ) ( Figure 5A–B ) whereas the RNA loops bound to these RRMs have strikingly different structures ( Figure 5C–D ) .",
"The U1-A bound RNA loop has an open structure with ten nucleotide bases splayed out ( Figure 5B , D ) ( Oubridge et al . , 1994 ) .",
"Bases of the first seven loop nucleotides show stacking interactions , either with adjacent bases or with protein side chains , while the last three nucleotides are poorly ordered .",
"In contrast , the U1-70k bound RNA loop 1 with 11 loop nucleotides ( Figure 1—figure supplement 1C ) is stabilized by base stacking interactions and basepairing of nucleotides within the loop and hence it is effectively a five-nucleotide loop ( Figure 5A–C ) .",
"In U1-A the polypeptide loop between β2 and β3 ( loop 3 ) protrudes through the RNA loop , stabilising it in an open conformation ( Figure 5B ) whereas loop 3 of U1-70k forms a β-turn and embraces C33 and G34 ( Figure 5A ) .",
"The bases of C33 and G34 stack with each other and are sandwiched between the side chains of Arg191 and Lys138 , which form salt-bridges with the phosphate groups of C33 and G34 ( Figure 6A ) .",
"The second residue of RNP2 motif ( Phe106 ) and the fifth residue of RNP1 motif ( Phe148 ) show stacking interactions with the bases of C31 and A32 ( Figure 6B ) as commonly observed in the RRM-RNA complexes ( Oubridge et al . , 1994 ) .",
"The RNA loop is closed by a trans WC/Hoogsteen base pair formed between A29 and A36 ( Figure 6C ) .",
"The base of G28 , instead of forming a base pair with G37 , flips out from the RNA helix and is sandwiched between the side chains of Arg172 and Tyr112 , while the guanidinium group of Arg200 fills the gap ( Figure 6D ) .",
"The stacking interaction between G28 and Tyr112 accounts for the UV-crosslinking of these residues in U1 snRNP ( Urlaub et al . , 2000 ) .",
"On the opposite strand the bases A35 , A36 , G37 and G38 continuously stack ( Figure 6C–D ) .",
"U30 is packed against the side chain of Leu175 and forms a hydrogen bond with the side chain of Asp177 and the exocyclic amino group of the adjacent C31 ( Figure 6E ) .",
"The stacking interaction between U30 and Leu175 is consistent with the UV-crosslinking of these residues ( Urlaub et al . , 2000 ) . 10 . 7554/eLife . 04986 . 012Figure 5 . RRMs of U1-A and U1-70k show distinct recognition modes of stem-loop I and II .",
"( A ) Interaction of stem-loop I with U1-70k RRM .",
"( B ) Interaction of stem-loop II with U1-A RRM .",
"( C ) Schematic representation of RNA-protein contacts between U1-70k RRM and stem-loop I of U1 snRNA .",
"( D ) Schematic representation of detailed RNA-protein contacts between U1-A RRM and stem-loop II .",
"( E ) Regions of U1-70k flanking the RRM folds onto RNA loop and make extensive contacts with RNA .",
"( F ) Apical loop I is completely covered by U1-70k . DOI: http://dx . doi . org/10 . 7554/eLife . 04986 . 01210 . 7554/eLife . 04986 . 013Figure 6 . Detailed RNA-protein contacts between U1-70k RRM and stem-loop I of U1 snRNA .",
"( A ) C33 and G34 embraced by U1-70k loop 3 .",
"( B ) C31 and A32 stack onto Phe106 and Phe148 residues of the beta sheet .",
"( C ) The last three loop nucleotides stack continuously on G38 of the loop-closing base pair .",
"( D ) The base of G28 is flipped out from the RNA helix and its place is taken by Arg200 , which , along with A29 and Arg190 , continues the helical stacking of the stem .",
"( E ) U30 is packed against the hydrophobic side chains of Leu175 and Leu196 .",
"In all cases nitrogen atoms are shown in blue , oxygen in red and phosphorus in magenta .",
"Hydrogen bonds are represented as dashed lines .",
"Carbon atoms are coloured grey in RNA , orange in U1-70k . DOI: http://dx . doi . org/10 . 7554/eLife . 04986 . 013 The most unusual feature of the U1-70k complex is that the regions flanking the RRM fold , which have no apparent secondary structural elements , make extensive interactions with the RNA loop bound on the surface of the β-sheet of the RRM , almost completely burying the RNA loop ( Figure 5E–F ) .",
"The C-terminal region following the RRM folds onto the RNA and runs along the shallow minor groove by forming an extensive network of hydrogen bonds .",
"Similarly , the region between helix 0 and the RRM folds onto the RNA .",
"The crystal structure of the minimal U1 snRNP has revealed in detail molecular contacts between U1 snRNP and a 5′SS RNA with the consensus sequence .",
"The role of U1-C in stabilising the 5′SS binding was first shown by Heinrichs et al . ( 1990 ) using 172 nucleotide pre-mRNA .",
"We measured binding of U1 snRNP to a [32P]-labelled 5′SS oligonucleotide by filter-binding assay ( Figure 7A ) .",
"The affinity of U1 snRNP without U1-C ( U1 snRNP[ΔU1-C] ) for the wild type 5′SS oligonucleotide increases by between three and fourfold on addition of U1-C ( Figure 7A ) .",
"In order to assess the contribution of the molecular contacts revealed by the crystal structure in 5′SS sequence selection we next assayed binding of variant 5′SS oligonucleotides to U1 snRNP containing uncapped , but otherwise fully authentic , U1 snRNA . 10 . 7554/eLife . 04986 . 014Figure 7 . Influence of nucleotide substitutions at the 5′-splice site on U1 snRNP binding .",
"( A ) Filter-binding results for U1 snRNP reconstituted with and without U1-C to [32P]-labelled 5′ splice site oligonucleotide .",
"By curve fitting , the Kd with U1-C is 4 . 7 ± 0 . 8 nM and without U1-C is 15 . 8 ± 2 . 5 nM .",
"CPM , counts per minute .",
"( B ) Nucleotides found at each position of the 5′-splice site of the U2-type introns .",
"Adapted from Roca et al . ( 2008 ) .",
"A , green; C , blue; G , black; U , red .",
"Numbers for highly conserved positions are highlighted in red .",
"( C ) Competition assays of mutant 5′SS RNA binding to U1 snRNP containing U1C and uncapped but fully modified U1 snRNA .",
"The 5′SS oligonucleotide with +1C , +5C , −1C and +2C substitutions compete weakly with the wild type oligonucleotide .",
"In panels C–F , mP is an arbitrary unit of fluorescence polarization and error bars indicate standard error .",
"( D ) Competition assay with 5′SS oligonucleotides with +3G , +4G , +3U , +4U substitution and the wild type .",
"5′SS oligonucleotide with +2C substitution is included for comparison .",
"( E ) Same as in B except that U1 snRNP lacks U1-C .",
"( F ) Same as in C except that U1 snRNP lacks U1-C . DOI: http://dx . doi . org/10 . 7554/eLife . 04986 . 014 Within the 5′SS of human genes processed by the major spliceosome , the most frequently observed nucleotides at each position of pre-mRNA from −3 to +6 form a Watson-Crick basepair with a nucleotide of the 5′-end of U1 snRNA ( Figure 4; Figure 7B ) .",
"The observed frequency tends to be higher in the middle and taper off towards both ends because mismatches affect the stability of the duplex less when they are further away from the middle .",
"The first two intron nucleotides , which pair with C8 and A7 of U1 snRNA , are nearly invariantly GU ( Burge et al . , 1999; Sheth et al . , 2006 ) ( Figure 4C; Figure 7C ) .",
"The nucleotides at +3 and +4 pair with Ψ6 and Ψ5 of U1 snRNA , respectively , and A/G are found at +3 but A is found predominantly at +4 .",
"G is preferred at positions −1 and +5 ( Figure 7B ) , which pair with C9 and C4 of U1 snRNA , whilst U is preferred at +6 .",
"As the filter-binding assay described above requires large amounts of U1 snRNP , we studied binding of different 5′SS sequences to U1 snRNP by competition assay using a short , fluorescently-labelled oligonucleotide as a reference ( Table 2 ) .",
"We found that a labelled consensus sequence oligonucleotide bound too tightly to be competed off by the weaker competitor oligonucleotides at concentrations that could be feasibly achieved in the experiment .",
"Hence we used a mismatched labelled oligonucleotide ( 5SS-F ) .",
"This does not influence the experiment's capacity to show whether one oligonucleotide competes better or worse than another .",
"Single nucleotide substitutions at highly conserved positions substantially reduce the affinity of the 5′SS oligonucleotide for U1 snRNP ( Figure 7C ) .",
"For example , substitution of G at −1 , +1 and +5 with C drastically reduces the binding of the competitor oligonucleotide ( Figure 7C ) as C does not form a stable basepair with C ( Leontis et al . , 2002 ) .",
"Furthermore , substitution of +2U with an A severely reduces the binding affinity , presumably because an A–A mismatch distorts and destabilizes the duplex itself ( Leontis et al . , 2002 ) ( Figure 7C ) .",
"The affinity of +2C 5′SS RNA is higher than that of +2A ( Figure 7C ) presumably because cytosine could form a wobble basepair with a protonated A ( Gao and Patel , 1987; Jang et al . , 1998; Wild et al . , 2001 ) .",
"This is consistent with the fact that C is found at +2 position , although very infrequently .",
"G at +3 and +4 could form a wobble basepair with Ψ6 and Ψ5 , respectively , and substitution of A with G at +3 or +4 position has only a moderate effect on the affinity ( Figure 7D ) .",
"The 5′SS with a G − Ψ wobble basepair at +4 position ( +4G ) has higher affinity than the one at +3 position ( +3G ) ( Figure 7C ) whilst +3G occurs more frequently in human genes ( Figure 7B ) than +4G .",
"Roca et al . ( 2012 ) reported the effect of three single base substitutions in the 5′SS on the melting temperature of the duplex between 5′SS oligonucleotides and an oligonucleotide representing the 5′-end of U1 snRNA .",
"+1A and +2C both had severe effects on melting temperature , in agreement with our results .",
"Most other mutations they studied introduced a bulged nucleotide .",
"Here we did not include such 5′SS oligonucleotides in our analysis , as it is hard to predict the effect of the bulged nucleotide on the molecular contacts observed in our crystal structure .",
"We next studied binding of the same set of the 5′SS oligonucleotides to U1 snRNP in the absence of U1-C ( U1 snRNP[ΔU1-C] ) to see how U1-C contributes to the selection of 5′SS oligonucleotides ( Figure 7E , F ) .",
"To a first approximation these 5′SS mutants show very similar affinities relative to the wild type both in the absence and presence of U1-C , enforcing the notion that 5′SS are selected primarily by U1 snRNA .",
"In the absence of U1-C , the mutant 5′SS oligonucleotides with +3G or +4G compete well with the wild type as in the presence of U1-C but other oligonucleotides with +3U , +4U , +1C , −1C or +5C mutations compete better in the presence of U1-C than in its absence .",
"This shows that U1-C fine-tunes the affinity of these mismatched oligonucleotides , in most sequences studied here , to provide extra stabilisation relative to the wild type .",
"For example , in the presence of U1-C the oligonucleotides with +3U or +4U , which introduce U–U mismatches , compete better with the wild type than they do in the absence of U1-C .",
"Different types of U–U basepair have been reported ( Lietzke et al . , 1996; Leontis et al . , 2002; Kiliszek et al . , 2009; Sheng et al . , 2013 ) .",
"Our crystal structure showed that U1-C forms hydrogen bonds exclusively with sugar-phosphate backbone between −2 to +3 positions of pre-mRNA and at C9 and G11 of U1 snRNA ( Figure 4B–C ) .",
"When extra hydrogen bonding groups are provided by U1-C , the U–U pair could easily switch from one type to another .",
"It is conceivable that these amino acid residues of U1-C could provide extra stabilization for these non-canonical basepairs , possibly by altering helical geometry and/or minor groove width . 10 . 7554/eLife . 04986 . 015Table 2 . 5′splice site bindingDOI: http://dx . doi . org/10 . 7554/eLife . 04986 . 015Oligo nameSequence*5ss-FAGGAAAGUAU-F†WTCAAAGGUAAGUUGGA−1CCAAACGUAAGUUGGA+1CCAAAGCUAAGUUGGA+2ACAAAGGAAAGUUGGA+2CCAAAGGCAAGUUGGA+3UCAAAGGUUAGUUGGA+3GCAAAGGUGAGUUGGA+4UCAAAGGUAUGUUGGA+4GCAAAGGUAGGUUGGA+5CCAAAGGUAACUUGGA*Bold nucleotides highlight the position of mismatch .",
"†F denotes 3′-fluorescein label .",
"It has been proposed that U1 snRNP selects the same 5′SS sequence even in the absence of the 5′-end of U1 snRNA ( Du and Rosbash , 2002; Lund and Kjems , 2002 ) , and that U1C in isolation can recognize the 5′SS sequence .",
"This conclusion is mainly based on a SELEX experiment , which may have been prone to an artefact caused by incomplete removal of the 5′-end of U1 snRNA .",
"We observed no binding of 5′SS oligonucleotide by human U1-C protein , which was shown to be properly folded by NMR ( Muto et al . , 2004 ) .",
"Schwer and Shuman ( 2014 ) investigated the role of conserved basic and hydrophilic residues in yeast U1-C: they mutated residues that form hydrogen bonds with the backbone atoms of either 5′SS ( Thr11 , Thr14 , Tyr12 and His24 ) or U1 snRNA ( His15 and Ser19 ) , form a salt-bridge with SmD3 ( Arg21 ) or lie near the phosphate backbone ( Lys22 and Arg28 [Lys28 in yeast] ) in our crystal structure ( Figure 4 ) .",
"Substitution of any one of these residues with Ala had no effect on yeast growth at any temperature indicating that these mutations do not have a major influence on 5′SS selectivity , in agreement with our conclusion .",
"The structures of two sub-domains of U1 snRNP reported here have revealed an intricate network of interactions between the components of U1 snRNP .",
"U1-70k N-terminal peptide binds to the subunit interfaces between SmD2 and SmF , and between SmD3 and SmB , and hence only the fully-formed core domain can induce its binding , which in turn enables U1-C to bind the core domain .",
"Our structure also revealed the molecular contacts between U1 snRNP and the 5′SS of pre-mRNA and provided new insights into the molecular mechanism of 5′SS selection .",
"U1-C makes no contacts with nucleotide bases and U1 snRNP selects 5′SS sequences primarily by thermodynamic stability of the RNA duplex between the 5′-end of U1 snRNA and the 5′SS .",
"However U1-C fine-tunes the affinity to stabilize the binding of some mismatched 5′SS oligonucleotides relative to the canonical 5′SS ."
],
[
"The SmE/SmF/SmG trimer , the SmD1/SmD2 dimer , the SmB/SmD3 dimer , U1-A , U1-70k and U1-C were prepared as described previously ( Oubridge et al . , 1994; Kambach et al . , 1999; Muto et al . , 2004; Pomeranz Krummel et al . , 2009; Leung et al . , 2011 ) .",
"All proteins are based on human sequences , unless otherwise indicated .",
"The coding sequence of thioredoxin , ( His ) 6-tag , tobacco etch virus ( TEV ) protease cleavage site , and a U1-70K fragment ( residues Thr 2–Arg 59 ) was PCR-amplified from the U1-70K expression vector and inserted at the initiation codon of SmD1 in the SmD1/SmD2 coexpression vector ( Kambach et al . , 1999 ) to create the U1-70kSmD1/SmD2 expression vector ( Figure 1—figure supplement 1 ) .",
"A ( Gly–Ser ) 3 sequence was included in the PCR primer to link the U1-70k fragment to SmD1 .",
"Escherichia coli BL21 ( DE3 ) pLysS cells were transformed with the expression vector ( Studier et al . , 1990 ) .",
"The cells were grown at 37°C in 2xTY medium with ampicillin ( 100 μg/ml ) and , when A600 nm = 0 . 4 − 0 . 8 , protein expression was induced by addition of 0 . 5 mM IPTG and the culture was continued at 15°C overnight .",
"Cell pellets were resuspended in Ni-A buffer ( 20 mM Tris-Cl pH7 . 4 , 1 M NaCl , 1 M urea , 10 mM 2-mercaptoethanol ) supplemented with EDTA-free protease inhibitor cocktail ( Roche , Basel , Switzerland ) .",
"The cells were lysed by sonication and clarified lysate was loaded onto a Ni-NTA column .",
"The protein was eluted with a gradient of imidazole to 300 mM .",
"The thioredoxin and ( His ) 6-tags of the protein were cleaved by His-tagged TEV protease during dialysis against Ni-A buffer before passing through the Ni-NTA column again .",
"The flowthrough fractions were diluted fivefold with Na-0 buffer ( 20 mM Tris-Cl pH7 . 4 , 1 M urea , 10 mM 2-mercaptoethanol ) and loaded onto a HiTrap heparin column ( GE Healthcare , Little Chalfont , UK ) equilibrated with heparin-A buffer ( 20 mM Tris pH7 . 4 , 200 mM NaCl , 1 M urea , 10 mM 2-mercaptoethanol ) .",
"The U1-70kSmD1/SmD2 heterodimer was eluted by a NaCl gradient and peak fractions were pooled , concentrated to ∼300 µM , rapidly frozen in liquid nitrogen and stored at −80°C .",
"A coding sequence for U1A70kF , ( Figure 1—figure supplement 1B ) consisting of a ( His ) 6-tag , TEV-protease cleavage site , residues 2–111 of U1-A protein , a linker of six Gly–Ser repeats and residues 60–216 of U1-70k protein , was constructed from PCR fragments and synthetic oligonucleotides , and ligated into the pET13 vector .",
"E . coli BL21 ( DE3 ) pLysS cells were transformed with the expression vector ( Studier et al . , 1990 ) and cells were grown in 2xTY medium with ampicillin ( 50 μg/ml ) and chloramphenicol ( 34 μg/ml ) at 37°C until A600 nm was approximately 0 . 7 .",
"Then , the temperature was lowered to 20°C and protein expression induced by addition of 0 . 5 mM IPTG .",
"After 8–10 hr cells were harvested by centrifugation and resuspended in lysis buffer ( 20 mM Na+-Hepes pH 7 . 5 , 25 mM imidazole , 0 . 5 M NaCl , 0 . 5 M urea ) .",
"The cells were lysed by sonication , clarified and loaded onto a Ni-NTA column .",
"The U1A70kF protein was eluted by a linear gradient of 25–500 mM imidazole in the same buffer .",
"The His-tag was cleaved off with His-tagged TEV protease and the uncleaved protein was removed by passing the protein through a second Ni-NTA column .",
"The protein was loaded onto a heparin-Sepharose column and eluted with a linear NaCl gradient ( 120–1000 mM ) in 20 mM Na+-Hepes pH 7 . 5 , 25 mM Imidazole , 60 mM Na Phosphate pH 7 . 4 , 1 M urea .",
"Peak fractions were concentrated by ultrafiltration , buffer exchanged into 20 mM Na . Hepes , 25 mM imidazole , 0 . 3 M NaCl , pH 7 . 5 , rapidly frozen in liquid nitrogen and stored at −80°C .",
"DNA templates for in vitro transcription ( Figure 1—figure supplement 2 ) were assembled by ligating overlapping oligonucleotides , which included the T7 promoter , into pUC18 vector .",
"All RNAs are based on human sequences , unless otherwise indicated .",
"Genes for full-length and minimal U1 snRNAs lacking the first 10 nucleotides were also cloned into pUC18 together with T7 promoter .",
"These truncated RNAs were transcribed in the presence of 2 mM GMP to facilitate ligation of modified 5′ end oligonucleotides .",
"The 5′ fragment of U1 snRNA with post-transcriptional modifications ( 5′-AmUmACΨΨACCU-3′ or 5′-AmUmACUUACCU-3′ where Ψ = pseudo-uridine , Am , Um = 2′-O-methyl nucleotides ) were purchased from Dharmacon ( GE Healthcare , Little Chalfont , UK ) and ligated to the truncated U1 snRNAs by splint-assisted ligation with T4 DNA ligase ( Ohkubo et al . , 2013 ) .",
"The plasmid template preparation , in vitro transcription and purification of RNA were carried out as described ( Price et al . , 1995 ) .",
"The full-length U1 snRNA at 8 µM was incubated in 50 mM KCl at 80°C for 2 min and annealed by snap cooling on ice .",
"Three Sm protein sub-complexes were diluted to 100 μM each in reconstitution buffer ( RB: 250 mM KCl , 20 mM K+-HEPES pH 7 . 5 , 5 mM DTT ) .",
"RNA was mixed with Sm proteins so that the solution contained 4 μM of RNA , 6 μM of each of the Sm proteins , 10 mM of DTT , and 40 units/ml of RNasin ( Promega , Fitchburg , Wisconsin , USA ) in RB .",
"The mixture was incubated at 30°C for 30 min , and then at 37°C for 15 min .",
"U1-70k was diluted to 12 μM with RB .",
"The Sm protein-RNA complex was mixed with an equal volume of the U1-70k solution and incubated on ice for 15 min .",
"U1-A protein was added to 4 μM final concentration .",
"The final reconstitution mix contained 2 μM of RNA , 3 μM each of the Sm proteins , 4 μM of U1-A , and 6 μM of U1-70k protein in RB .",
"The solution was incubated on ice overnight and applied to a monoQ column ( GE Healthcare , Little Chalfont , UK ) .",
"The reconstituted complex was eluted with a gradient of KCl from 250 mM to 1 M in RB buffer .",
"U1-C protein was added to U1 snRNP by buffers supplemented with 1 . 5 μM of U1-C protein to ensure an excess of U1-C in binding assays .",
"2 µM of SmKCm RNA ( Figure 1—figure supplement 2B ) was mixed with 1 . 5-fold molar excess of U1-70kSmD1/SmD2 , SmD3/SmB and SmE/SmF/SmG sub-complexes in RB supplemented with 2 M urea .",
"After incubation at 37°C for 1 hr , the sample was dialyzed against RB buffer at 4°C overnight .",
"U1-C protein was added to a final concentration of 6 µM and incubated at 30°C for 15 min and at 4°C for at least 1 hr .",
"The complex was purified on a monoQ column as described for full-length U1 snRNP .",
"U1A70kF protein was added slowly to 10 μM SL1·SL2 RNA in 0 . 4 M NaCl , 40 mM Na+-Hepes pH 7 . 5 , 50 mM imidazole at room temperature for a final protein concentration of 15 μM .",
"After 20 min the mixture was diluted with an equal volume of water and incubated for a further 10 min .",
"The complex was purified on a monoQ column equilibrated with 20 mM Na+-Hepes pH 7 . 5 , 150 mM NaCl and eluted with a NaCl gradient ( 150 mM–1 M ) .",
"Peak fractions were pooled , concentrated to 6 mg/ml with a centrifugal concentrator , and buffer exchanged into 0 . 2 M NaCl , 20 mM Na+-Hepes pH 7 . 5 , 25 mM imidazole .",
"Crystals of the SmKCm complex were obtained by sitting-drop vapour diffusion at 277K .",
"The purified U1 snRNP complex was mixed with 1 . 2-fold molar excess of 5′SS RNA oligo ( 5′-AGGUAAGUCC-3′ ) purchased from Dharmacon ( GE Healthcare , Little Chalfont , UK ) and 0 . 15 mM of polyamine-9 ( Sauter et al . , 1999 ) .",
"The complex solution ( 4 mg/ml in 300 mM KCl , 20 mM K+-Hepes , pH 7 . 5 , 5 mM MgCl2 , 1 mM DTT ) was mixed with an equal volume of reservoir solution ( 7% MPD , 180 mM KCl , 5 mM MgSO4 , 50 mM Na+-Hepes pH 6 . 4 ) .",
"The crystals were improved by streak-seeding from another crystallization drop using a feline whisker 3 min after mixing the drops .",
"Crystals suitable for data collection appeared after 1 month , reaching maximum dimensions of 0 . 35 × 0 . 05 × 0 . 05 mm3 .",
"Crystals were transferred to cryo-protection buffer ( 20% MPD , 200 mM KCl , 5 mM MgSO4 , 50 mM Na+-Hepes pH 6 . 4 , 25% PEG4000 ) in three steps with 30 min equilibration after each transfer and flash frozen in liquid nitrogen .",
"The U1A70kF RNA complex was mixed with an equal volume of 40% MPD , 0 . 15 M NaCl , 0 . 1 M sodium acetate pH 4 . 2 and equilibrated with the same solution by sitting drop vapour diffusion at 20°C .",
"Crystals of type I ( wedge-shaped ) grew within 16–48 hr whereas crystals of type II ( plate-like ) grew in the same drops after several weeks .",
"Only the latter type diffracted to sufficient resolution for a structure determination .",
"Diffraction data were collected at Diamond Light Source beamlines I02 , I03 , I04 and I04-1 .",
"Data were integrated in iMosflm ( Leslie and Powell , 2007 ) and scaled with Scala ( Collaborative Computational Project Number 4 , 1994 ) .",
"For the minimal U1 snRNP ( SmKCm complex ) an initial molecular replacement solution was found in Phaser ( McCoy et al . , 2007 ) using the U4 snRNP core domain proteins ( Leung et al . , 2011 ) .",
"These form similar ring structures in both U1 and U4 snRNP , and have a total mass of ∼70 kDa , which is more than half the mass of the minimal U1 snRNP ( ∼120 kDa ) .",
"Phases were improved by density modification using Parrot ( Cowtan , 2010 ) .",
"This allowed us to build the remainder of the model using Coot ( Emsley et al . , 2010 ) .",
"The N-terminal region of U1-70k was built de novo and we manually placed the zinc finger domain of U1-C protein ( Muto et al . , 2004 ) and HIV1 kissing loop structure ( Ennifar et al . , 2001 ) into electron density .",
"The structure was refined by Refmac ( Murshudov et al . , 1997 ) ( Table 1 ) .",
"For the U1A70kF-RNA complex , an initial molecular replacement solution was found in Phaser ( McCoy et al . , 2007 ) using part of the structure of U1-A complex with SL2 RNA ( Oubridge et al . , 1994 ) and a homology model of the U1-70k RRM , which was built with Modeller ( Mart-Renom et al . , 2000 ) using multiple templates and manually refined alignment as an input .",
"The remainder of the model was built into the MR map using Coot ( Emsley et al . , 2010 ) , and the structure was refined by Refmac ( Murshudov et al . , 1997 ) ( Table 1 ) .",
"Figures of molecular structures were drawn using Pymol ( www . pymol . org ) .",
"Pre-mRNA oligonucleotide substrates containing 5′ splice-site sequences were purchased from Dharmacon ( GE Healthcare , Little Chalfont , UK ) ( Table 2 ) .",
"All the components were diluted in binding assay buffer ( BAB: 10 mM K+-Hepes pH7 . 5 , 200 mM KCl , 2 mM MgCl2 , 0 . 5 mM DTT , 100 µg/ml tRNA , 50 µg/ml BSA ) , and 1 . 5 µM U1-C protein was added to BAB to ensure saturation of U1 snRNP with U1-C protein under assay conditions .",
"In competition assays , each reaction ( 16 μl ) containing 1 nM fluorescein-labeled reference RNA oligo ( 5ss-F ) and 35 nM U1 snRNP reconstituted with U1 + Ψ RNA ( Figure 1—figure supplement 2 ) , was titrated with non-labelled competitor 5′SS RNA oligos .",
"Binding curves were measured by fluorescence polarization using Pherastar ( BMG Labtech , Ortenberg , Germany ) .",
"The assays were carried out in triplicate .",
"The values of fluorescence polarization in the absence of competitor RNA oligo were plotted at 1 pM for reference .",
"In the filter-binding assay , a 5′SS RNA oligonucleotide ( 5′-32P- CAAAGGUAAGAUGGA-3′ , 10 fmol ) , was mixed with various concentrations of U1 snRNP , with or without U1-C protein , in Filter-Binding Buffer ( FBB: 200 mM KCl , 2 mM MgCl2 , 0 . 5 mM DTT , 10 mM Hepes , pH 7 . 5 , 100 µg/ml tRNA , 50 µg/ml BSA ) in a final volume of 40 µl and incubated at 22°C for 2 hr .",
"The binding reaction was passed , under vacuum , through a Schleicher & Schuell ( Dassel , Germany ) NC45 , 25 mm diameter filter that had been pre-wetted with FBB .",
"The filter was washed with 0 . 5 ml FBB and then dried .",
"The proportion of radiolabeled oligonucleotide retained on the filter was determined by scintillation counting .",
"The experiment was performed in triplicate ."
]
] | [
"U1 snRNP binds to the 5′ exon-intron junction of pre-mRNA and thus plays a crucial role at an early stage of pre-mRNA splicing .",
"We present two crystal structures of engineered U1 sub-structures , which together reveal at atomic resolution an almost complete network of protein–protein and RNA-protein interactions within U1 snRNP , and show how the 5′ splice site of pre-mRNA is recognised by U1 snRNP .",
"The zinc-finger of U1-C interacts with the duplex between pre-mRNA and the 5′-end of U1 snRNA .",
"The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone around the splice junction but U1-C makes no base-specific contacts with pre-mRNA .",
"The structure , together with RNA binding assays , shows that the selection of 5′-splice site nucleotides by U1 snRNP is achieved predominantly through basepairing with U1 snRNA whilst U1-C fine-tunes relative affinities of mismatched 5′-splice sites ."
] | [
"Genes are made up of long stretches of DNA .",
"The regions of a gene that code for proteins ( known as exons ) are interrupted by stretches of non-coding DNA called introns .",
"To produce proteins from a gene , the DNA is ‘transcribed’ to form pre-mRNA molecules , from which the introns must be removed in a process called splicing .",
"The remaining exons are then joined together to form a mature mRNA molecule that contains the instructions to build a protein .",
"Errors in the splicing process can lead to numerous diseases , such as cancer .",
"A molecular machine known as a spliceosome is responsible for splicing the pre-mRNA molecules .",
"This consists of five different complexes called small nuclear ribonucleoprotein particles ( snRNPs ) , which are in turn made up from numerous proteins and RNA molecules .",
"The spliceosome assembles anew every time it splices , and an early step in this assembly process involves the interaction of an snRNP called U1 with the start of an intron in the pre-mRNA .",
"This interaction then stimulates the assembly of the rest of the spliceosome .",
"In 2009 , researchers reported the structure of the U1 snRNP , but the structure did not contain enough detail to reveal how the snRNP recognizes the start of an intron .",
"Kondo , Oubridge et al . , including some of the researchers involved in the 2009 work , now present the crystal structure of the human version of the U1 snRNP in more detail .",
"High-quality crystal structures of the complete U1 snRNP molecule could not be obtained because the arrangement of the RNA molecules in the snRNP prevented a regular crystal from forming .",
"Kondo , Oubridge et al . instead engineered two subcomponents of U1 snRNP that each crystallized well , and determined their structures .",
"This revealed that the interactions between the various parts of the U1 snRNP form a complex network .",
"A protein present in the U1 snRNP , known as U1-C , had previously been reported to be able to recognize introns on its own—without requiring the complete U1 snRNP .",
"Kondo , Oubridge et al . reveal that this is not the case and that U1-C does not read the intron RNA sequence directly .",
"Instead , U1 snRNP is able to find the start of the intron because the U1 RNA can stably bind to this site .",
"The U1-C protein can however adjust the strength of this binding to ensure that the spliceosome can operate with a variety of intron start sequences ( or signals ) ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"structural biology and molecular biophysics"
] | 3.3-Å resolution cryo-EM structure of human ribonucleotide reductase with substrate and allosteric regulators bound | elife-31502-v1 | [
[
"Ribonucleotide reductase ( RNR ) , an essential enzyme in all organisms , catalyzes the reduction of ribonucleotides into deoxyribonucleotide precursors for replication and repair of DNA .",
"Because RNR is vital for cell proliferation and genome maintenance , drugs that target human RNR are used against some of the most aggressive and challenging to treat cancers , including refractory lymphoblastic leukemia , metastatic ovarian and pancreatic cancers , and melanoma ( Aye et al . , 2015 ) .",
"Human RNR is a class Ia RNR , represented by eukaryotes and some prokaryotes that includes the well-studied homologs from Escherichia coli and Saccharomyces cervisiae ( Cotruvo and Stubbe , 2011; Brignole et al . , 2012; Hofer et al . , 2012; Minnihan et al . , 2013b ) .",
"In the class Ia RNRs two homodimeric subunits work together to reduce diphosphate forms of all four canonical ribonucleotides ( NDPs ) to their deoxyribonucleotide counterparts ( Figure 1A–D ) ( von Döbeln and Reichard , 1976; Brignole et al . , 2012; Hofer et al . , 2012 ) .",
"The smaller β subunit houses a stable diferric-tyrosyl radical cofactor generated by oxidation of a di-iron cofactor ( Atkin et al . , 1973; Sjöberg et al . , 1978; Larsson and Sjöberg , 1986; Bollinger et al . , 1991; Cotruvo and Stubbe , 2011 ) ( Figure 1C , D ) .",
"The larger α subunit contains the active site and two allosteric regulatory sites ( Brown and Reichard , 1969b; von Döbeln and Reichard , 1976; Eriksson et al . , 1997 ) ( Figure 1C , D ) .",
"Long-distance radical transfer ( RT ) from β2 to α2 generates a transient active site thiyl radical in α2 that catalyzes NDP reduction and reverse radical transfer from α2 to β2 regenerates the tyrosyl-radical in β2 on every turnover ( Licht et al . , 1996; Minnihan et al . , 2013b ) ( Figure 1A ) .",
"This remarkable inter-subunit RT minimally requires formation of an α2β2 subunit complex ( Brown and Reichard , 1969a; Rofougaran et al . , 2006; Rofougaran et al . , 2008 ) .",
"Crystal structures of individual dimeric subunits from E . coli , yeast , mouse , and human RNRs have revealed general structural similarity ( Nordlund et al . , 1990; Uhlin and Eklund , 1994; Voegtli et al . , 2001; Strand et al . , 2004; Xu et al . , 2006; Smith et al . , 2009; Fairman et al . , 2011 ) .",
"Differences include a conspicuous three-helix insert in eukaroytic α ( Xu et al . , 2006 ) that is absent in E . coli ( Figure 1C , D ) .",
"Additionally , the C-termini of α2 and β2 have not been detected in the available crystal structures , due to flexibility essential to their function .",
"The unstructured C-terminal β2 tail interacts with a specific binding site on α2 and participates in inter-subunit RT .",
"The α2 C-terminus emanates from a strand in the active site bearing two redox active residues on the RT pathway ( Tyr737 and Tyr738 in human α , equivalent to Tyr730 and Tyr731 in E . coli α ) , which becomes disordered shortly following these Tyr residues , preventing direct observation of a pair of Cys residues that are required for shuttling reducing equivalents to the active site ( Figure 1C , D ) .",
"A species-specific combination of allosteric , transcriptional , post-translational , and subcellular locational controls regulates RNR activity to maintain appropriate concentrations and proportions of deoxyribonucleotides and ensures fidelity of DNA synthesis and repair ( Hofer et al . , 2012; Guarino et al . , 2014 ) .",
"Allosteric regulation depends on two nucleotide-binding sites in α2 that , through modulation of α2 conformation and subunit oligomerization , tune activity and substrate specificity , as described below .",
"The allosteric binding site that regulates substrate preference is located at the α-dimerization interface on one face of a conserved structural element , called loop 2 , whose opposite face makes contacts with the substrate base ( Figure 1C , D ) .",
"dATP binding to this effector site results in a preference for CDP and UDP reduction; TTP for GDP reduction; and dGTP for ADP reduction ( Brown and Reichard , 1969b; von Döbeln and Reichard , 1976 ) ( Figure 1B ) .",
"Recent crystal structures of E . coli RNR with all four specificity effector-substrate pairs ( dATP-CDP , dATP-UDP , TTP-GDP , and dGTP-ADP ) reveal the molecular basis of specificity regulation in this archetypal class Ia RNR ( Zimanyi et al . , 2016 ) .",
"Although the rules of allosteric regulation of specificity ( Figure 1B ) appear to be conserved , whether the molecular features of specificity regulation are the same in eukaryotic RNRs remains an open question ( Xu et al . , 2006; Ahmad et al . , 2012; Zimanyi et al . , 2016 ) .",
"Overall RNR activity can also be allosterically regulated .",
"For class Ia RNRs , dATP binding to the cone domain at the N-terminus of α2 ( Figure 1C , D ) has an inhibitory effect , whereas ATP binding reverses this inhibition ( Brown and Reichard , 1969b; Rofougaran et al . , 2006; Rofougaran et al . , 2008 ) .",
"In the prototypical system from E . coli , binding of the allosteric inhibitor dATP within the cone domain results in an α4β4 ring-shaped structure that holds the α2 and β2 subunits in a configuration incompatible with RT ( Ando et al . , 2011; Zimanyi et al . , 2012 ) ( Figure 2A ) .",
"ATP binding shifts the equilibrium from the inactive α4β4 state to the active α2β2 state ( Rofougaran et al . , 2008; Ando et al . , 2011 ) .",
"Thus , activity regulation for E . coli RNR involves oligomeric state changes that are modulated by the ratio of dATP to ATP in the cell .",
"Eukaryotic RNRs do not appear to form α4β4-ring structures ( Fairman et al . , 2011; Aye et al . , 2012; Ando et al . , 2016 ) .",
"Instead human α2 has the propensity to form α6 rings in the presence of both ATP and dATP ( Rofougaran et al . , 2006; Fairman et al . , 2011; Ando et al . , 2016 ) and the stability of the α6 ring appears to regulate activity ( Ando et al . , 2016 ) ( Figure 2B ) .",
"Rings formed with dATP are stable , showing no oligomeric state change upon addition of β2 , whereas α6 rings formed in the presence of ATP are unstable and disassemble into active state ( s ) upon addition of β2 ( Ando et al . , 2016 ) .",
"Support for the idea that ‘stable’ α6 rings correspond to an inhibited form of human RNR comes from studies with clofarabine ( ClF ) , an adenosine analog used for cancer treatment , and related analogs cladrabine and fludarabine ( Aye and Stubbe , 2011; Aye et al . , 2012; Wisitpitthaya et al . , 2016 ) .",
"In those studies , human RNR treated with CIF diphosphate ( ClFDP ) or triphosphate ( ClFTP ) lead to inactive enzyme and the formation of extremely stable ‘persistent’ α6 rings ( Aye and Stubbe , 2011; Aye et al . , 2012 ) .",
"Structural information about α6 rings has been limited to a 9-Å resolution crystal structure of human α obtained in the presence of dATP ( Ando et al . , 2016 ) , a 6 . 6-Å resolution crystal structure of yeast α with dATP ( Fairman et al . , 2011 ) , and a 28-Å resolution EM structure dATP-induced α6 from yeast in the presence of β2 ( Fairman et al . , 2011 ) .",
"These low-resolution structures were sufficient to establish the overall α6 subunit arrangement , but failed to reveal the molecular basis for inactivity of the α6 ring .",
"Here , we use state-of-the-art cryo-EM to determine the structure of human RNR α6 at the near-atomic resolution of 3 . 3 Å and probe the mechanisms of allosteric regulation of activity and specificity for the human class Ia RNR enzyme ."
],
[
"Human α forms ring-like particles composed of three α-dimers ( Figure 3A ) upon addition of 0 . 05 mM dATP ( Figure 3—figure supplement 1C , D ) as seen previously in the low-resolution crystal structures of human and yeast α ( Fairman et al . , 2011; Ando et al . , 2016 ) and in human RNR in the presence of ClFDP ( Aye et al . , 2012 ) ( Figure 3—figure supplement 1F ) .",
"These α6 rings formed with dATP alone showed some structural flexibility .",
"α6 rings were also the predominant form observed following incubation of α with 1 mM ATP , but some particles were incomplete or partially open rings , indicating that the subunit contacts are tenuous , consistent with the proposition that the rings formed in the presence of ATP are not stable ( Figure 3—figure supplement 1B ) .",
"Increasing ATP concentration to 3 mM resulted in an increase in the proportion of dissociated rings and free α-dimers .",
"At 10 mM ATP we observed fewer α6 rings and a filamentous form of α is now apparent .",
"Analysis of the filamentous oligomers showed that they are composed of α2 units chained together end-to-end , often only three units in length .",
"With TTP , the dimeric α2 form is present exclusively ( Figure 3—figure supplement 1A ) as seen in prior studies ( Thelander et al . , 1980; Reichard et al . , 2000; Rofougaran et al . , 2006; Aye and Stubbe , 2011; Fairman et al . , 2011; Scott et al . , 2001 ) .",
"Finally , we examined α in the presence of 0 . 05 mM dATP and 3 mM ATP , a combination expected to be physiological relevant .",
"In dividing cells where RNR is actively producing deoxynucleotides , the dATP concentration is approximately 0 . 024 mM and the ATP concentration is approximately 3 mM ( Traut , 1994 ) , thus the combination of 0 . 05 mM dATP and 3 mM ATP is expected to mimic cellular effector concentrations under which this higher dATP concentration inhibits RNR .",
"To verify that 0 . 05 mM dATP inhibits human RNR even in the presence of 3 mM ATP , enzyme assays were performed , which showed inactivation ( Figure 3—figure supplement 2 ) .",
"Under negative stain EM , this combination of effectors ( 0 . 05 mM dATP and 3 mM ATP ) yielded data sets in which α6 rings are preponderant and could be combined into a well-defined 3D structure with D3 point group symmetry ( Figure 3—figure supplement 1E ) .",
"Given that this combination of effectors provided the most structurally homogeneous preparation of α6 rings , we used it for the high-resolution cryo-EM analysis .",
"It is unfortunate that structural stability is so sensitive to nucleotide identity and concentration , since these constraints limit the ability to determine high-resolution structures of the human RNR with all combinations of substrate and effectors .",
"The near-atomic resolution structure described below was determined in the presence of substrate CDP and effectors dATP and ATP .",
"We obtained a near-atomic resolution structure of the human α subunit of RNR in the presence of 0 . 05 mM dATP , 3 mM ATP and substrate CDP .",
"Based on the lack of enzymatic activity under these conditions , this first near-atomic resolution structure of human α6 is in a dATP-inhibited state ( Figure 3A ) .",
"Imaging of the cryo-EM samples as dose-fractionated movie frames from a direct electron detector allowed for correction of specimen movement and compensation for radiation damage ( Table 1 ) .",
"To eliminate concerns about model bias , particles were selected automatically , clustered using Iterative Stable Alignment and Clustering ( ISAC ) ( Yang et al . , 2012 ) , and an initial 3D map was generated de novo using stochastic hill climbing refinement ( Elmlund et al . , 2013 ) implemented in SPARX ( Hohn et al . , 2007 ) .",
"The initial map was used as reference for refinement of image alignment parameters using SPARX , to obtain a final map that has an overall resolution of approximately 3 . 3 Å , and a maximum resolution of 3 . 15 Å around the α-α dimer interface ( Figure 3—figure supplements 3 and 4 ) .",
"At this resolution densities can be distinguished clearly for the spiraling backbone of α-helices , the strands of β-sheets , residue side chains , and bound substrate and effector nucleotides ( Figure 3A ) .",
"The final model contains six α subunits with residues 1 through 743 of the 792 amino acids .",
"Although both ATP and dATP were present , dATP was the nucleotide included in the final model , because it is a better fit to the density in both allosteric sites and is also expected to have higher affinity for both sites ( Brown and Reichard , 1969b; Reichard et al . , 2000; Kashlan and Cooperman , 2003 ) .",
"Thus , each α subunit contained one CDP in the active site , one dATP in the specificity site and one dATP in the activity site .",
"Standard metrics of model quality show excellent statistics and fit to the cryo-EM map ( Table 1 ) .",
"Six α subunits , organized as three dimeric units , make contacts through their cone domains to assemble an α6 ring that resembles the overall arrangement seen previously in lower resolution crystal structures of dATP-inhibited human ( Ando et al . , 2016 ) and yeast α ( Fairman et al . , 2011 ) ( Figure 3A ) .",
"The α6 ring is ~180 Å in diameter and ~80 Å thick with a central hole that is constricted to 60 Å near each opening but widens to ~80 Å midway through the ring’s interior .",
"Each α subunit from the α6 ring is similar to the 2 . 4-Å resolution crystal structure of human α2 ( Fairman et al . , 2011 ) with an RMSD over 738 Cα atoms of 2 . 25 Å .",
"Differences are localized to three regions ( Figure 3—figure supplement 5 ) , the cone domain , a β-hairpin loop ( residues Ile624–Val637 ) adjacent to the cone domain , and loop 2 .",
"The largest change is rotation of the cone domain by 20° , which is necessary for the three α dimers to interact with the requisite geometry to assemble a closed α6 ring ( Figure 3B , C ) .",
"Density consistent with Mg2+-dATP is observed within the cone domain close to the subunit interface ( Figure 3D ) , where the nucleotide effector makes similar contacts to those reported in crystal structures of human α2 and E . coli α4β4 ( Fairman et al . , 2011; Zimanyi et al . , 2012; Zimanyi et al . , 2016 ) .",
"In crystal structures of E . coli RNR ( Zimanyi et al . , 2012 , 2016 ) , His59 forms a hydrogen bond with the deoxyribose 3′-hydroxyl group of dATP .",
"The equivalent residue in human α , Asp57 , was not observed in the human α2 crystal structure with dATP bound ( Fairman et al . , 2011 ) , but density corresponding to it is apparent in the EM map and modeling of the Asp57 side chain shows that its position would allow it to hydrogen-bond with dATP .",
"The observation of an interaction between Asp57 and dATP in the human RNR EM structure is an important finding , as mutation of Asp57 to Asn results in loss of inhibition by dATP in eukaryotic RNRs ( Caras and Martin , 1988; Reichard et al . , 2000 ) .",
"The interface between α2 subunits in the α6 ring involves cone domain helices 1 and 2 ( Figure 3C ) .",
"Remarkably , the same two helices of the cone domain make α-β contacts in the E . coli α4β4 dATP-inhibited ring , although the residues involved are not conserved ( Figure 3E ) .",
"740 Å2 of solvent-accessible surface area is buried per subunit upon α6 ring formation , which is somewhat more extensive than the 575 Å2 per subunit buried surface in the E . coli α-β contact ( measured in 5CNS [Zimanyi et al . , 2016] ) .",
"In the human α6 ring structure , the α2-α2 inter-subunit contacts are primarily composed of a hydrophobic core with Phe15 and Ile44 positioned on the two-fold symmetry axes and the side chains of Met1 , Ala37 , Thr40 , Met41 , and Leu47 contributing additional contacts ( Figure 3C ) .",
"In addition to hydrophobic shape complementarity , Arg12 and Gln23 may contribute a hydrogen bond at the flanks of the interface .",
"Asp16 , a highly conserved residue that when mutated was found to disrupt α6 ring formation and reduce inhibition by dATP ( Fairman et al . , 2011 ) , also sits at the two-fold symmetry axis .",
"Unfortunately , the side chain of Asp16 has poorly defined density beyond Cβ of the side chain , making it difficult to fully explain the high degree of conservation .",
"Overall , however , the density quality is impressive ( Video 1 ) , providing the first near-atomic view of the molecular interactions responsible for human RNR inhibition by dATP .",
"One explanation for why a stable α6 ring is inactive is that β2 cannot access α2 for radical generation in this state .",
"To evaluate the interaction between human α and β , we prepared negative stained specimens of α and β with the same mixture of ATP , dATP and CDP used to determine our cryo-EM α6 structure .",
"Despite the fact that β was added in equimolar ratio with α , α6β6 complexes did not form .",
"Instead , we observed that only a fraction of α6 rings have a single , variably positioned additional density , which we attribute to a single β2 , consistent with earlier molecular mass observations of an α6β2 complex of mouse RNR ( Rofougaran et al . , 2006 ) .",
"The majority of rings showed no β2 density at all ( Figure 4A ) .",
"Similar results were obtained with CIFTP-inhibited RNR .",
"ISAC averages that result from cryo-EM specimens prepared with ClFTP exhibit a single additional β2 density protruding from the ring or no additional density ( Figure 4B ) .",
"Collectively , these results indicate that β2 subunits show limited interaction affinity for stable α6 rings .",
"A 3D reconstruction of an α6β2 complex calculated from images of CIFTP-inhibited RNR shows β2 density only above the ring plane , not within it ( Figure 4C ) .",
"Whether β2 density was above or within the ring was not clear from previous 2D images ( Fairman et al . , 2011 ) .",
"These new data allow us to establish that β2 density is above the ring and to consider why this is the case .",
"The dimensions of β2 and the α6 ring provide a simple explanation for exclusion of β2 from the inner ring cavity .",
"At ~80 Å wide on its longest edge , β2 would have difficulty navigating through the ~60 Å wide entrance to the cavity .",
"If β2 were able to access the interior of the ring , it would still need to be able to assume an RT-competent position with respect to α6 .",
"Docking ( based on a ~30-Å resolution EM structure of the α2β2 state of E . coli RNR [Brignole et al . , 2012; Uhlin and Eklund , 1994; Minnihan et al . , 2013a] ) suggests that the ability of β2 to assume a catalytically relevant position is constrained by cone domains and the three-helix insertion motif on α ( Figure 4D ) .",
"Additionally , the ‘cavity’ of the α6 ring is not empty .",
"As in all structures of RNRs , the 49-residue-long C-terminal tail of α is disordered and could not be directly detected in the 3 . 3-Å resolution cryo-EM structure .",
"However , the locations of the last visible residues suggest that the six disordered C-terminal tails of α6 are likely pointing into the central cavity .",
"Consistent with this location for the C-terminal tails , a 3D variance map shows partial densities protruding into the α6 ring ( Figure 4E ) .",
"Six 49-residue-long flexible tails would further impede ring access by β2 ( Figure 2B ) .",
"No high-resolution structures of RNRs in an active state , capable of inter-subunit RT to form the catalytic thiyl radical , have been reported .",
"However , it has not been necessary to crystallize an active state of RNR to visualize substrate and effector binding , given that substrates and effectors bind well to pre-catalytic states .",
"In fact , substrate/effector binding increases α2-β2 affinity five-fold ( Ingemarson and Thelander , 1996; Hassan et al . , 2008 ) , suggesting that substrate/effector binding precedes subunit association and RT .",
"That being said , crystal lattice contacts have historically complicated the analysis of substrate- and effector-bound structures of RNRs , resulting in poor quality density for either substrate , effector , or loop 2 , the loop that communicates between the substrate and effector-binding sites ( Zimanyi et al . , 2016 ) .",
"Here , using cryo-EM , we notably find clear density for both substrate and effector and are able to assess their binding interactions ( Figure 5A , Video 2 ) .",
"dATP can be modeled into the cryo-EM map at the specificity site located at the α2 dimer interface , where it makes contacts with loop 2 ( residues 286–295 ) and loop 1 ( residues 255–271 ) from the neighboring subunit ( Figure 5B ) .",
"Backbone amides of residues Ala263 and Gly264 in loop 1 establish contacts with the β- and γ-phosphates similar to those seen in crystal structures of yeast or human α with bound TTP or human α with dATP ( Xu et al . , 2006; Fairman et al . , 2011 ) .",
"As previously noted for those structures , the orientation of loop 1 in the eukaryotic α2 is different from that in E . coli α2 , but in both cases contacts are made by the loop to the effector phosphates .",
"As in other RNR structures ( Larsson et al . , 2004; Xu et al . , 2006; Fairman et al . , 2011 ) , Arg256 provides further stabilization of the γ-phosphate , Lys243 ( not on loop 1 or loop 2 ) interacts with the α- and β-phosphates , and the Asp226 side chain makes hydrogen bonds with the O3’ of the deoxyribose sugar .",
"As previously observed in E . coli , the backbone amide and carbonyl of loop 2 residue Asp287 ( human numbering; Ser293 in E . coli ) are involved in the specific recognition of the adenine base of effector dATP ( Zimanyi et al . , 2016 ) , positioning loop 2 such that the side chain of the adjacent Gln288 ( Gln294 in E . coli ) is directed into the active site where it can hydrogen bond to the base of substrate CDP ( Figure 5A ) The presence of an extra residue in loop 2 in human RNR ( compared to E . coli ) results in an additional contact to the dATP adenine base made by the Gly289 carbonyl , further stabilizing a ‘Gln-in’ conformation of loop 2 .",
"Since the presence of the Gln side chain in the active site would inhibit the binding of the larger purine bases , the “Gln-in “position confers preference for pyrimidines CDP and UDP over purines ADP and GDP .",
"This observation was first made for the E . coli RNR enzyme ( Zimanyi et al . , 2016 ) , and we now find the same molecular mechanism of substrate specificity in play with the human enzyme .",
"CDP density is clearly observed in the active site ( Figure 5A ) .",
"As with other RNRs , substrate binding does not require Mg2+ or other cations to counter balance the negative charge of the phosphates .",
"Instead , the phosphates of CDP are bound through contacts with the backbone and/or side chains of Ser202 , Thr604 , Ala605 , Ser606 , and Thr607 ( Figure 5C ) .",
"Additionally , Arg293 on loop 2 ( Arg298 in E . coli ) reaches over the cytosine base to hydrogen bond with the phosphates , providing both a stacking interaction with the base and a positive-counter charge to the negatively charged substrate phosphates .",
"The same interaction of Arg298 with the substrate phosphates is observed in E . coli for all four substrate-effector pairs ( Zimanyi et al . , 2016 ) , leading to the proposal that Arg298 is a molecular latch that seals the active site for radical chemistry when the cognate substrate-effector pairs are bound .",
"Consistent with this idea , mutation of Arg298 to Ala in E . coli abolishes activity for all four substrates ( Zimanyi et al . , 2016 ) .",
"The substrate ribose modeled in the 3′-endo state is positioned by contacts with Glu431 ( the catalytic acid/base ) ( Lawrence et al . , 1999; Licht and Stubbe , 1999 ) , Cys218 ( one of the cysteines that forms a disulfide during catalysis ) , the side chain of Asn427 , and the backbone carbonyl of Ser217 ( Figure 5C ) .",
"The Ser217 side chain also appears to contact the ribose O4′ , whereas a Ser rotamer faces away from the substrate in previously determined crystal structures of α from E . coli , yeast , and human ( Xu et al . , 2006; Fairman et al . , 2011; Zimanyi et al . , 2016 ) .",
"The Cys218-Cys444 disulfide appears to be reduced , and Cys429 ( the thiyl radical forming cysteine ) is ~4 . 3 Å from ribose C3′ , the site of hydrogen atom abstraction , which initiates catalysis ( Minnihan et al . , 2013b ) .",
"Cys429 is also within hydrogen bonding distance Tyr737 , which is adjacent to Tyr738 .",
"Both tyrosines are essential for RT ( Minnihan et al . , 2013b ) .",
"As mentioned above , recognition of the cytosine base is mediated through Gln288 ( Gln294 in E . coli ) ."
],
[
"The mechanisms of allosteric regulation of RNRs are both fascinating and complex .",
"Substrate specificity regulation is particularly intriguing -- how does the binding of dATP or ATP to the allosteric specificity site 15 Å away from the active site increase the preference of the active site for a pyrimidine substrate whereas TTP and dGTP binding promote purine substrates ?",
"Although the rules of regulation are conserved ( Figure 1B ) and the locations of the substrate and effector binding sites are conserved ( Figure 1C , D ) , it has not been clear , even for prokaryotic versus eukaryotic class Ia RNRs , if the molecular mechanisms that afford the ‘rules’ will be the same .",
"In part , the issue has been that obtaining structural data to visualize the molecular basis of allosteric specificity regulation has been nontrivial .",
"To date , there are only a handful of structures of RNRs that have clear density for the loop responsible for the allosteric communication ( loop 2 ) ( Xu et al . , 2006; Zimanyi et al . , 2016 ) , which has led to conflicting mechanistic proposals ( Xu et al . , 2006; Ahmad et al . , 2012; Zimanyi et al . , 2016 ) .",
"The near-atomic resolution structure presented here provides the first visualization of RNR structure from a eukaryotic RNR that has a well-ordered loop 2 .",
"Using our structure , which has CDP bound in the active site , we can investigate whether the molecular mechanism for pyrimidine substrate preference is conserved between human RNR and the well-studied E . coli enzyme , resolving a controversy as well as providing key insight into this captivating form of allosteric regulation .",
"The molecular basis for allosteric regulation of activity has been similarly enigmatic .",
"Prior to this study , we knew that human RNR forms an α6 state in the presence of allosteric activity effectors ATP and dATP , but it was not clear why the stable dATP-induced α6 state was inactive .",
"Here our 3 . 3-Å resolution cryo-EM structure of the human RNR enzyme in a dATP-inhibited α6 state has considerably surpassed the resolution of a previous 9-Å resolution crystal structure ( Ando et al . , 2016 ) and has allowed us to interrogate the molecular basis of dATP-associated inactivity for human RNR .",
"We also use these data , along with lower resolution EM structures of human RNR α6 rings in the presence of the anticancer drug CIFTP and the radical storage β2 subunit , to consider the implications of these findings for anticancer therapies .",
"Our near-atomic resolution structure of human α6 is the first structure of the human enzyme with the CDP/dATP substrate/effector pair bound .",
"As mentioned above , it also represents one of the few RNR structures with a well-ordered loop 2 , the region of the structure responsible for communication between the specificity effector and its cognate substrate .",
"The conformation of loop 2 observed between the base of the dATP specificity effector and the base of the CDP substrate indicates that communication of CDP binding preference from the specificity effector-binding site is conserved with E . coli α2 ( Figure 5D ) .",
"In both systems , backbone atoms of this loop ‘read out’ the adenine base and position Gln288 into the active site to recognize the cytosine of CDP .",
"With the CDP appropriately bound , Arg293 seals the active site for radical chemistry .",
"A previously solved structure of human α2 with dATP bound in the specificity site , but without substrate ( Fairman et al . , 2011 ) , shows Gln288 positioned for pyrimidine recognition ( CDP or UDP ) , but in the absence of substrate , Arg293 is disordered and the C-terminal portion of loop 2 has not yet adopted the conformation that would fully stabilize the substrate-effector pair ( Figure 5E ) .",
"Importantly , a crystal structure of yeast α2 with ATP analog AMPPNP and CDP bound exhibits a conformation unlike that seen in the human or E . coli structures ( Figure 5F ) .",
"Substantial differences between yeast and E . coli substrate-effector bound RNR structures have been previously reported ( Xu et al . , 2006; Ahmad et al . , 2012; Zimanyi et al . , 2016 ) and opposite roles for the conserved Gln of loop 2 have been proposed .",
"For E . coli , it is believed that the positioning of the Gln side chain into the active site promotes CDP/UDP binding through hydrogen bonding while hindering ADP/GDP binding due to steric bulk .",
"However , for yeast , it has been proposed the Gln side chain points into the active site to increase ( not decrease ) the affinity of ADP .",
"Thus , one goal of this work was to evaluate whether eukaryotic RNRs , such as human and yeast , use Gln and loop 2 differently from their prokaryotic counterparts .",
"In other words , we wished to establish the conformation of loop 2 and Gln for the CDP-dATP substrate-effector pair in the human enzyme and compare it to yeast and E . coli .",
"As described above , we find that the human RNR communicates CDP preference in an identical fashion as E . coli .",
"Therefore , whether or not the yeast enzyme does employ Gln differently , it is not the case that all eukaryotic RNRs will be different from prokaryotic RNRs .",
"Notably , with the addition of this CDP-dATP bound structure , there are now six RNR structures for which loop 2 is well ordered .",
"Four are from the E . coli class Ia RNR ( CDP-dATP , UDP-dATP , GDP-TTP , and ADP-dGTP , listed as substrate-specificity effector pair ) ( Zimanyi et al . , 2016 ) , one from Thermatoga maritima class II RNR ( GDP-TTP ) ( Larsson et al . , 2004 ) , and this one for human class Ia RNR ( CDP-dATP ) .",
"It was previously reported that the two GDP-TTP structures are highly similar despite being from different RNR species and classes .",
"Taken together with these results , we postulate that the molecular mechanism of allosteric specificity regulation will be conserved among class I and II RNRs; that structural differences observed to date are more likely due to crystal disorder/lattice contacts rather than real variations in mechanism .",
"Single particle cryo-EM , a technique for which lattice contacts are not an issue , thus provides valuable insight about how an unrestrained regulatory feature , such as loop 2 , responds to substrate/effector binding .",
"Moving forward , we suspect that cryo-EM will be increasingly used to investigate allosteric regulation .",
"Whereas our results support a conserved mechanism for allosteric regulation of specificity among class Ia RNRs from different species , they argue against conservation of the mechanism for allosteric activity regulation .",
"For the human enzyme in the presence of dATP , we see no evidence of E . coli-like α4β4 structures ( Figure 2A ) nor do we see anything that resembles the α4 oligomeric state that was recently reported for dATP-inhibited Pseudomonas aeruginosa RNR ( Johansson et al . , 2016 ) .",
"Instead , we observe α6 and some α6β2 .",
"In agreement with quantitative studies of human RNR by SAXS ( Ando et al . , 2016 ) , EM analysis shows that α6 rings form in the presence of both dATP and ATP , or ClFTP , and that addition of β2 does not disrupt these stable rings .",
"Additionally , our EM data show that β2 can sit in multiple positions around the outside of the α6 ring , but appears unable to penetrate into the inside of the ring structure where it could initiate chemistry on α2 .",
"Thus , instead of ‘holding β2 at arm’s length’ to prevent RT in the presence of dATP as in E . coli RNR , the human enzyme holds α2 subunits together in a circle in the presence of dATP to exclude β2 from accessing α2 ( Figure 2 ) .",
"The cryo-EM structure furthermore offers an explanation for β2 exclusion from the inside of the α6 ring .",
"We find that β2 access is impaired by the 60-Å constriction at the surface of the α6 ring and by the disordered C-terminal tails of α that extend into the ring .",
"Also , the ability of β2 to assume a catalytically relevant position with respect to α is restricted by the cone domains at the α2-α2 interface and the three-helix insertion motif of adjacent α2 subunits .",
"In short , when the ring is stable and cannot expand , β2 cannot fit and assume a catalytically relevant position .",
"Thus , the weak association between the cone domains in the presence of ATP should allow β2 to further destabilize the α6 ring by inserting itself into the central opening to form an active complex with α2 .",
"In contrast , tight association of the cone domains in the presence of dATP would preclude productive β2 interaction with α2 ( Figure 2B ) .",
"These structural results also offer insight into inhibition of RNR by the diphosphate and triphosphate forms of the therapeutics clofarabine ( ClFDP and ClFTP ) , cladribine ( ClADP and ClATP ) , and fludarabine ( FlUTP ) that are known to impart α hexamers with enhanced stability ( Aye and Stubbe , 2011; Aye et al . , 2012; Wisitpitthaya et al . , 2016 ) .",
"Given that the well-studied RNR inhibitors gemcitabine-diphosphate and hydroxyurea are known to target the active site on α and the tyrosyl radical on β2 , respectively ( van der Donk et al . , 1998; Offenbacher et al . , 2014 ) , the possibility that some RNR inhibitors might act by targeting the allosteric activity site on α was intriguing to investigate .",
"ClF and ClA are cytotoxic in a cell line expressing wild-type α , whereas a cell line expressing an α mutation in its allosteric activity site ( Asp57Asn ) is resistant to the drugs ( Wisitpitthaya et al . , 2016 ) , consistent with the allosteric activity site being targeted by these molecules .",
"From our data , we can now rationalize that molecules that stabilize the hexamer by binding to the cone domain will inhibit human RNR by impairing the ability of β2 to access α .",
"Thus , designing inhibitors to bind to the allosteric site in the cone domain appears to represent a new and attractive method for inhibiting human RNR in vivo .",
"In summary , advances of cryo-EM methodology have allowed us to obtain a near-atomic resolution structure that has provided remarkable insight into the allosteric regulation of the human RNR enzyme .",
"Although detailed structural information about the active α-β RNR complexes is still an important missing piece to the puzzle , our understanding of RNR inactive states has advanced substantially with views of beautiful yet distinct ring structures ."
],
[
"5-3H]-CDP was obtained from ViTrax .",
"hTrx1 and hTrxR1 were isolated as previously described ( Ando et al . , 2016 ) ."
]
] | [
"Ribonucleotide reductases ( RNRs ) convert ribonucleotides into deoxyribonucleotides , a reaction essential for DNA replication and repair .",
"Human RNR requires two subunits for activity , the α subunit contains the active site , and the β subunit houses the radical cofactor .",
"Here , we present a 3 . 3-Å resolution structure by cryo-electron microscopy ( EM ) of a dATP-inhibited state of human RNR .",
"This structure , which was determined in the presence of substrate CDP and allosteric regulators ATP and dATP , has three α2 units arranged in an α6 ring .",
"At near-atomic resolution , these data provide insight into the molecular basis for CDP recognition by allosteric specificity effectors dATP/ATP .",
"Additionally , we present lower-resolution EM structures of human α6 in the presence of both the anticancer drug clofarabine triphosphate and β2 .",
"Together , these structures support a model for RNR inhibition in which β2 is excluded from binding in a radical transfer competent position when α exists as a stable hexamer ."
] | [
"Cells often need to make more DNA , for example when they are about to divide or need to repair their genetic information .",
"The building blocks of DNA – also called deoxyribonucleotides – are created through a series of biochemical reactions .",
"Among the enzymes that accomplish these reactions , ribonucleotide reductases ( or RNRs , for short ) perform a key irreversible step .",
"One prominent class of RNR contains two basic units , named alpha and beta .",
"The active form of these RNRs is made up of a pair of alpha units ( α2 ) , which associates with a pair of beta units ( β2 ) to create an α2β2 structure .",
"α2 captures molecules called ribonucleotides and , with the help of β2 , converts them to deoxyribonucleotides that after futher processing will be used to create DNA .",
"As RNR produces deoxyribonucleotides , levels of DNA building blocks in the cell rise .",
"To avoid overstocking the cell , RNR contains an ‘off switch’ that is triggered when levels of one of the DNA building blocks , dATP , is high enough to occupy a particular site on the alpha unit .",
"Binding of dATP to this site results in three pairs of alpha units getting together to form a stable ring of six units ( called α6 ) .",
"How the formation of this stable α6 ring actually turns off RNR was an open question .",
"Here , Brignole , Tsai et al . use a microscopy method called cryo-EM to reveal the three-dimensional structure of the inactive human RNR almost down to the level of individual atoms .",
"When the alpha pairs form an α6 ring , the hole in the center of this circle is smaller than β2 , keeping β2 away from α2 .",
"This inaccessibility leads to RNR being switched off .",
"If RNR is inactive , DNA synthesis is impaired and cells cannot divide .",
"In turn , controlling whether or not cells proliferate is key to fighting diseases like cancer ( where ‘rogue’ cells keep replicating ) or bacterial infections .",
"Certain cancer treatments already target RNR , and create the inactive α6 ring structure .",
"In addition , in bacteria , the inactive form of RNR is different from the human one and forms an α4β4 ring , rather than an α6 ring .",
"Understanding the structure of the human inactive RNR could help scientists to find both new anticancer and antibacterial drugs ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"evolutionary biology",
"genetics and genomics"
] | Combinations of Spok genes create multiple meiotic drivers in Podospora | elife-46454-v1 | [
[
"The genomes of all Eukaryotes harbor selfish genetic elements that employ a variety of mechanisms to undermine the canonical modes of DNA replication and meiosis in order to bias their own transmission ( Werren et al . , 1988; Burt and Trivers , 2009 ) .",
"As the proliferation of these elements is independent of the regulated reproduction of the host organism , they can create conflict within the genome ( Rice and Holland , 1997 ) .",
"Such intragenomic conflict is predicted by theory to spur an arms race between the genome and the elements and , consequently , to act as a major driver of evolutionary change ( Werren , 2011 ) .",
"To understand the extent to which intragenomic conflict has shaped the evolution of genomes and populations , it is crucial to identify the selfish genetic elements that are able to impact the dynamics of natural populations .",
"One important class of selfish genetic elements are known as meiotic drivers .",
"These elements use a variety of mechanisms to hijack meiosis in order to bias their transmission to the gametes in proportions greater than 50% ( Sandler and Novitski , 1957 ) .",
"This segregation distortion of alleles can be difficult to observe unless it is linked to an obvious phenotype such as sex ( Sandler and Novitski , 1957; Helleu et al . , 2014 ) , thus the prevalence of meiotic drive in nature is probably underestimated .",
"Nevertheless , meiotic drive has been observed in many model systems , including Drosophila , Mus , Neurospora , and Zea mays , suggesting that it is widespread across all major Eukaryotic groups ( Lindholm et al . , 2016; Bravo Núñez et al . , 2018b ) .",
"In ascomycete fungi , meiotic drive occurs in the form of spore killing , which represents the most direct way to observe the presence of drive ( Turner and Perkins , 1991 ) .",
"When a strain possessing a driving allele mates with a compatible strain that does not carry the allele ( i . e . , a sensitive strain ) , the meiotic products ( ascospores ) that carry the driving allele will induce the abortion of their sibling spores that do not have the allele .",
"Spore killing is apparent in the sexual structures ( asci ) of the fungi because it results in half of the normal number of viable spores .",
"Owing to the haplontic life cycle of most fungi , spore killing is unusual among meiotic drivers as it is the only system in which the offspring of an organism are killed by the drive ( Lyttle , 1991 ) .",
"In addition , with few exceptions ( Hammond et al . , 2012; Svedberg et al . , 2018 ) , spore killer elements appear to be governed by single loci that confer both killing and resistance ( Grognet et al . , 2014; Nuckolls et al . , 2017; Hu et al . , 2017 ) , which contrasts with the other well-studied drive systems that comprise genomic regions as large as entire chromosomes ( Larracuente and Presgraves , 2012; Hammer et al . , 1989 ) .",
"Meiotic drivers are often expected to reach fixation or extinction in populations relatively rapidly ( Crow , 1991 ) , at which point the effects of the drivers will no longer be observable .",
"In agreement with this expectation , most drivers that have been described exhibit large shifts in frequencies in both time and space ( Lindholm et al . , 2016; Carvalho and Vaz , 1999 ) .",
"In the case of spore killers , multiple drivers have been found to coexist within a given species .",
"The evolutionary dynamics of multiple drivers within species has not been thoroughly explored , but two contrasting examples are known .",
"In the genomes of Schizosaccharomyces pombe , numerous copies of both functional and pseudogenized versions of the wtf driver genes are found ( Nuckolls et al . , 2017; Hu et al . , 2017; Eickbush et al . , 2019 ) .",
"By contrast , the two spore killers Sk-2 and Sk-3 of Neurospora intermedia have only been described in wild strains four times and once , respectively , whereas resistance to spore killing is widespread ( Turner , 2001 ) .",
"The impact of multiple drivers coexisting in a single population was not characterized in either of these cases .",
"Natural populations of the filamentous fungus Podospora anserina are known to host multiple spore killers ( Grognet et al . , 2014; van der Gaag et al . , 2000; Hamann and Osiewacz , 2004 ) , and hence provide an ideal system for the investigation of interactions among drivers at the population level .",
"The first spore killer gene to be described in P . anserina was het-s , a gene that is also involved in allorecognition ( Dalstra et al . , 2003 ) .",
"Another class of spore killer genes in Podospora are known as Spok genes .",
"Spok1 is only known from a single representative of P . comata , a species that is closely related to P . anserina , whereas Spok2 has been shown to exist in high frequency among strains of a French population of P . anserina ( Grognet et al . , 2014 ) .",
"Spok1 is capable of killing in the presence of Spok2 , but not vice versa , indicating a dominant epistatic relationship between the two genes .",
"In addition , seven spore killer genotypes have been identified through classical genetic analysis ( van der Gaag et al . , 2000 ) .",
"These are referred to as Psk-1 through Psk-7 and were defined by observing the presence , absence and frequency of killed spores in defined crosses among French and Dutch P . anserina strains ( Box 1—figure 1 ) .",
"At the onset of this study , it was not known whether the Psk elements represent independent meiotic drive genes , or whether they may be related to the Spoks and/or allorecognition loci .",
"The het-s gene itself is not associated with the Psks , but allorecognition is correlated with Psk spore killing ( van der Gaag et al . , 2003 ) .",
"On the other hand , the relationship between the Spoks is reminiscent of the hierarchy of killing among the Psks , suggesting a possible connection between the activity of Spok genes and Psks .",
"The primary goal of this study was to determine the identity of the genes that are responsible for the Psk spore-killer types found in P . anserina , and whether they relate to known meiotic drive genes .",
"We identified two novel Spok homologs ( Spok3 and Spok4 ) and showed that these two , together with the previously described Spok2 , represent the genetic basis of the Psk spore killers .",
"The new Spoks occur in large novel regions that can be found in different genomic locations in different strains .",
"Our results illuminate the underlying genetics of a polymorphic meiotic drive system and expand our knowledge regarding their mechanism of action ."
],
[
"To investigate the genetic basis of spore killing in P . anserina , we generated high-quality whole-genome assemblies using a combination of long-read ( PacBio and MinION Oxford Nanopore ) and short-read ( Illumina HiSeq ) technologies .",
"Table 1 lists the strains used for investigation .",
"In all cases , we sequenced single haploid monokaryons ( marked with + or - following the strain name , to designate their mating type; see 'Materials and methods' and Appendix 1 ) .",
"We selected strains from a natural population in Wageningen ( Wa ) , the Netherlands , and a few strains from France , representing six of the seven previously described Psk spore killer types ( Psk-1 , Psk-2 , Psk-3 , Psk-4 , Psk-5 and Psk-7; van der Gaag et al . , 2000 ) along with a strain of a novel killing type ( Wa100 ) , to which we assign the type Psk-8 , and strain Wa63 .",
"The reference strain of P . anserina , S , was not given a Psk designation previously , as it was not known to induce spore-killing .",
"However , Grognet et al . ( 2014 ) demonstrated that it can indeed induce spore-killing , so here we assign it to Psk-S along with Wa63 .",
"In addition , we acquired and sequenced strains from the closely related Podospora species P . pauciseta ( CBS237 . 71 ) and P . comata ( strain T ) .",
"A strain annotated as T was acquired from two different laboratories , one from the Wageningen collection ( referred hereafter as TD ) and one from Goethe University Frankfurt ( here as TG ) .",
"Our results revealed that these strains do not represent the same isolate , as previously thought ( Hamann and Osiewacz , 2004 ) , but are distinct .",
"TG is a Psk-5 strain of P . anserina and was sequenced with Nanopore and Illumina .",
"TD matches the P . comata epitype reported by Silar et al . ( 2019 ) and was sequenced with Illumina alone .",
"The final assemblies ( long-read technologies polished with Illumina HiSeq data ) recovered the expected seven chromosomes in their entirety for five strains , and in up to 13 scaffolds for the rest ( Supplementary file 1 ) .",
"BUSCO analyses of these assemblies reported 97–98% of 3725 Sordariomyceta-conserved genes ( Supplementary file 1 ) , which is concordant with the same analysis done in the reference assemblies of the P . anserina strain S+ ( hereafter referred to as Podan2; Espagne et al . , 2008 ) and of P . comata ( PODCO; Silar et al . , 2019 ) .",
"Notice that as the assemblies of each strain were produced from one haploid ( monokaryotic ) isolate , we will refer to specific genome assemblies with their strain name followed by their corresponding mating type; for example , the assembled genome of monokaryon Wa63+ ( derived from the strain Wa63 ) is called PaWa63p ( Supplementary file 1 ) .",
"In addition , all genomes sequenced with Illumina were assembled de novo using SPAdes .",
"The resulting assemblies consisted of between 222 and 418 scaffolds that were larger than 500 bp , with a mean N50 of 227 kbp ( Supplementary file 2 ) .",
"The alignment coverage of Podan2 ( Espagne et al . , 2008 ) was above 98% for all of the SPAdes assemblies of P . anserina .",
"When the filtered Illumina reads were mapped to Podan2 , all samples had a sequencing depth greater than 75x ( Supplementary file 2 ) .",
"Taken together , our genome assemblies , resulting from both long- and short-read data , are very comprehensive .",
"A NeighborNet split network of 1000 single-copy orthologs ( including introns ) showed that the P . anserina samples are remarkably similar to each other and distinct from those of both P . comata and P . pauciseta ( Figure 1A ) .",
"Nevertheless , the three taxa are very closely related: the average genic identity within P . anserina is 99 . 97% , whereas the genic distance between P . anserina and P . pauciseta is 99 . 10% , between P . anserina and P . comata is 98 . 87% , and between P . pauciseta and P . comata is 98 . 79% .",
"Accordingly , the whole-genome alignments recovered strongly conserved synteny at the chromosomal level when small ( <13 kb ) translocations ( presumably due to transposable elements ( TEs ) ) are excluded ( Figure 1B; Figure 1—figure supplement 1 ) .",
"Only three larger translocations were detected , of which two might be mis-assemblies in the reference genome of P . comata ( PODCO; see 'Materials and methods' ) .",
"By searching our assemblies for the Spok2 sequence ( presented by Grognet et al . , 2014 ) using BLAST , we confirm the presence of this Spok gene on the left arm of chromosome 5 in the majority of strains , in agreement with Grognet et al . ( 2014 ) .",
"Furthermore , on the basis of sequence similarity with Spok2 , we identified two novel homologs that we refer to as Spok3 and Spok4 .",
"These newly identified Spoks are found at different genomic locations depending on the strain .",
"Both Spok3 and Spok4 can be located on the left arm of chromosome 3 or on the left arm of chromosome 5 , and Spok3 can be found at an additional location on the right arm of chromosome 5 .",
"In addition , the BLAST searches recovered a pseudogenized Spok gene ( SpokΨ1 ) in the subtelomeric region of the right arm of chromosome 5 .",
"The Spok gene content of the strains investigated in this study is reported in Table 1 .",
"A schematic representation of the Spok homologs is shown in Figure 2A .",
"We considered the Spok2 sequence of S+ , and the Spok3 and Spok4 sequences of Wa87+ as reference alleles for each homolog .",
"Overall they show a high degree of conservation , including the 3' and 5' UTRs .",
"A nucleotide alignment of the Spok genes’ coding sequence revealed 130 variable sites out of 2334 total sites ( Figure 2—figure supplement 1 ) .",
"A relatively large proportion ( 67% , 87/130 ) of those variable sites result in amino acid changes and 74% are unique to one of the Spok homologs .",
"Table 2 displays pairwise comparisons of the amino-acid sequence of the SPOK proteins , revealing a high rate of non-synonymous substitutions , and a relatively high similarity between Spok1 and Spok4 .",
"There are six indels among all the Spok genes , including one at the 5' end of the ORF that represents a variable-length repeat region , and one at the 3' end of the ORF that is shared by Spok3 and Spok4 .",
"The 3' end indel induces a frameshift and changes the position of the stop codon ( Figure 2A ) .",
"SpokΨ1 has a missing 5' end , multiple stop codons , and a discoglosse ( Tc1/mariner-like ) DNA transposon ( Espagne et al . , 2008 ) inserted in the coding region .",
"Of particular interest , SpokΨ1 has no deletions relative to the other Spok homologs , suggesting that the indels in the functional Spok homologs represent derived deletions .",
"There is little allelic variation within the Spok homologs in the Wageningen population and the variants of the four homologs cluster phylogenetically ( Figure 2B and C ) .",
"The Spok2 gene in the Wageningen strains are identical to the two alleles described in Grognet et al . ( 2014 ) , with the exception of Spok2 from Wa58– which has a single SNP that results in a D358N substitution .",
"The Spok2 allele of the French strain A , which shows resistance without killing ( as reported by Grognet et al . , 2014 ) , was not found in any of the genomes investigated in this study .",
"Spok3 has five allelic variants , and the allelic variation of Spok4 is reminiscent of Spok2 , with only Wa100+ and Wa58– having a single synonymous SNP ( Figure 2C ) .",
"Lastly , the three copies of SpokΨ1 are all unique .",
"Notably , a number of the allelic variants of Spok3 show signatures of gene conversion events ( Lazzaro and Clark , 2001 ) .",
"Specifically , strain Y+ has three SNPs near the start of the gene that result in amino-acid changes and that match those in Spok2 exactly ( Figure 2—figure supplement 1 ) .",
"The Wa53+ allele of Spok3 has a series of SNPs ( a track of 205 bp ) that are identical to those in Spok4 but different from all other Spok3 sequences , and three additional SNPs near the 5' end that also match Spok4 ( Figure 2—figure supplement 1 ) .",
"The TG+ strain possesses two identical copies of Spok3 ( see 'Materials and methods' ) that share the aforementioned tract with Wa53+ , but which extends for an additional 217 bp ( Figure 2—figure supplement 1 ) .",
"These chimeric Spoks are recovered from the final assemblies ( pre- and post-Pilon polishing ) with high long-read coverage ( >30 x ) , suggesting that our finding is not a bioinformatic artifact .",
"The gene conversion events between Spok homologs are supported by the reticulation shown in a NeighborNet split network ( Figure 2B ) and by a significant recombination Phi test ( 199 informative sites , p=1 . 528e-12 ) .",
"A maximum likelihood phylogenetic analysis of the UTR sequences ( defined by conservation across homologs ) suggests that Spok3 and Spok4 are closely related ( Figure 2C ) , which is at odds with the high structural similarity of the coding sequences of Spok1 and Spok4 ( Figure 2A ) .",
"Therefore , we cannot make any strong inference about the relationships between the Spok homologs from the sequence data .",
"In the few strains with no copy of Spok2 , analysis of the region suggests that this is a result of a one-time deletion ( Figure 3 ) .",
"The annotation in the original reference genomes of TD and S is erroneous because of mis-assemblies and/or incomplete exon prediction , which were both corrected in our study using our own Illumina data and annotation pipeline , and then validated by the RNAseq expression data for TD .",
"First , the flanking gene P_5_20 ( marked as ( 1 ) in Figure 3 ) in P . pauciseta ( CBS237 . 71 ) and P . comata ( TD ) is considerably longer than the P . anserina ortholog , which is truncated by a discoglosse ( Tc1/mariner-like ) DNA transposon ( 2 ) .",
"In the strains without Spok2 ( Wa46 , Y , and TG ) , the discoglosse transposon itself is interrupted and the sequence continues on the 3' end of a fragmented crapaud ( gypsy/Ty3 ) long terminal repeat ( LTR ) element , which can be found in full length downstream of Spok2 in the other strains .",
"This configuration implies that the absence of Spok2 constitutes a deletion ( 3 ) , rather than the ancestral state within P . anserina .",
"An alternative scenario would require multiple additional insertions and deletions of TEs and Spok2 .",
"The Spok1 gene was previously identified from the P . comata strain TD ( Grognet et al . , 2014 ) .",
"No other strains investigated in this study were found to possess Spok1 , indicating that this gene is probably not present in P . anserina .",
"Remarkably , BLAST searches of the Spok2 gene ( including the UTR sequences ) revealed the presence of a small piece ( ~156-bp long ) of a presumably degraded Spok gene in the TD de novo assembly and on chromosome 4 of PODCO .",
"This piece overlaps with the last amino acids of the CDS 3' end and is flanked by an arthroleptis ( solo LTR ) retrotransposon on one side and by unknown sequence on the other .",
"Owing to the small size of this piece , it not clear whether it belongs to a novel Spok gene , but the location ( between genes PODCO_401390 and PODCO_401400 ) differs from those of the other known homologs .",
"The sequencing reveals that the genome of the P . pauciseta strain CBS237 . 71 contains both Spok3 and Spok4 ( Table 1; Figure 2B ) , but they are at a genomic location that differs from those in any of the P . anserina strains .",
"We constructed knock-in and knock-out strains to confirm that the newly discovered Spok homologs , Spok3 and Spok4 , can induce spore killing on their own ( Supplementary file 3 ) , as previously shown for Spok2 by Grognet et al . ( 2014 ) .",
"First , the Spok2 gene was deleted from the strain s to create a ΔSpok2 strain for use with the knock-ins .",
"A cross between s and the ΔSpok2 strain resulted in about 40% two-spored asci , as previously reported by Grognet et al . ( 2014 ) ( 80/197 , 40 . 6% ) ( Figure 4—figure supplement 2B ) .",
"The Spok3 and Spok4 genes were inserted separately at the centromere-linked PaPKS1 locus ( a gene controlling the pigmentation of spores [Coppin and Silar , 2007] ) .",
"As PaPKS1 is tightly linked to the centromere , we expected that if the genes are capable of meiotic drive , then crosses to the ΔSpok2 strain should yield nearly 100% two-spored asci with white ( unpigmented ) spores .",
"Accordingly , both Spok3::PaPKS1 ΔSpok2 x ΔSpok2 and Spok4::PaPKS1 ΔSpok2 x ΔSpok2 crosses yielded almost 100% two-spored asci with two white spores ( 118/119 , 99 . 1%; Figure 4—figure supplement 2C ) and ( 343/346 , 99 . 1%; Figure 4—figure supplement 2D ) , respectively .",
"These results show that Spok3 and Spok4 function as spore killers when introduced as a single copy at the PaPKS1 locus .",
"To determine whether there are any interactive effects between Spok2 , Spok3 , and Spok4 , we made use of the knock-in strains to assay pairwise interactions among them .",
"First , to determine the interaction between Spok3 and Spok4 , we crossed a strain bearing Spok4 at PaPKS1 with a strain bearing Spok3 .",
"Because crosses that are homoallelic for the PaPKS1 deletion have poor fertility , we constructed a strain in which Spok3 is inserted as a single copy at the PaPKS1 locus but just downstream of the coding region ( Spok3::PaPKS1d ) in order to yield strains with normal pigmentation and normal fertility in crosses to PaPKS1-deletion strains .",
"In control crosses , the Spok3::PaPKS1d strain showed killing when crossed with a strain lacking Spok3 but no killing when crossed with Spok3::PaPKS1 ( Figure 4—figure supplement 2E and F ) .",
"The cross between Spok3::PaPKS1d and Spok4::PaPKS1 yielded asci that had four aborted spores , indicating mutual killing of Spok3 and Spok4 ( Figure 4—figure supplement 2G ) .",
"To determine the killing relation between Spok2 and Spok3 , a cross was conducted between Spok3::PaPKS1 and strain s ( of the Psk-S type ) .",
"This cross yielded mostly two-spored asci with two unpigmented spores ( 163/165 asci: 98 . 8% ) ( Figure 4—figure supplement 2H ) , indicating that Spok3 kills in the presence of Spok2 .",
"Similarly , to determine the killing relation between Spok2 and Spok4 , a cross was conducted between Spok4::PaPKS1 and s , which resulted in 99:5% killing ( 216/217 asci ) ( Figure 4—figure supplement 2I ) .",
"Although these two crosses indicate that Spok2 does not confer resistance to Spok3 and Spok4 ( Spok3 and Spok4 both kill Spok2 ) , they do not allow us to determine whether Spok3 or Spok4 confer resistance to Spok2 .",
"To address this point , Spok2 killing was analyzed in a cross that was homoallelic for Spok3 ( Spok3::PaPKS1 x Spok3::PaPKS1d ΔSpok2 ) , which yielded 46% two-spored asci ( 143/310 ) , confirming that Spok2 killing occurs in the presence of Spok3 ( Figure 4—figure supplement 2J ) .",
"Finally , to determine whether Spok4 is resistant to Spok2 , we made a Spok4::PaPKS1 x Spok4::PaPKS1 ΔSpok2 cross , which resulted in 11/24 two-spored asci ( Figure 4—figure supplement 2K ) .",
"Although this genetic background is ill-suited for determining killing frequency ( because of the aforementioned effect of the homozygous PaPKS1 deletion on fertility ) , the presence of two-spore asci suggests that Spok4 does not confer resistance to Spok2 killing .",
"Overall , these results indicate that Spok2 , Spok3 , and Spok4 do not interact .",
"To evaluate whether the newly discovered Spok homologs represent the genes that underlie the Psk spore-killer types , we sequenced backcrossed laboratory strains using Illumina Hiseq technology .",
"A strain of each of Psk-1 , Psk-2 , Psk-5 and Psk-7 was previously backcrossed five times to the reference strain S ( van der Gaag et al . , 2000 ) .",
"The backcrossed strains are referred to here as Psk1xS5 , Psk2xS5 , Psk5xS5 , and Psk7xS5 ( Table 1 ) .",
"The backcrossed strains should maintain the killing percentage and mutual interactions of the dominant Psk parent .",
"Given previous studies , we do not expect S ( Psk-S ) to be dominant over the other Psks ( van der Gaag et al . , 2000 ) .",
"Notably , crossing results reveal that Psk5xS5 has neither a Psk-5 nor a Psk-S phenotype , but a Psk-1 phenotype ( Figure 4—source data 2 ) .",
"This is only possible if multiple killing loci are involved , which is concordant with the observation of multiple Spok genes in these strains .",
"Our Illumina data recovered a total of 41 , 482 filtered biallelic SNPs from the four S5 backcrosses and the parental strains .",
"All backcrossed strains show a few continuous tracts of SNPs from the dominant killer parent ( Figure 4—figure supplement 3 ) .",
"For example , Psk1xS5– has a long tract in chromosome 1 that represents the mat– mating type , which is to be expected because the published reference of S ( Podan2 ) , for which the SNPs are called , is of the opposite mating type ( mat+ ) .",
"Importantly , the location of Spok3 and/or Spok4 of each parental strain has a corresponding introgressed SNP tract in the corresponding S5 backcross , while all backcrossed strains possess the Spok2 gene from strain S ( Figure 4—figure supplement 3 ) .",
"The Psk-5 parental strain of Psk5xS5 ( strain Y ) does not possess Spok2 , whereas Psk5xS5 does .",
"Hence , the change in the killing phenotype of the backcrossed strain can be attributed to the presence of Spok2 ( see below ) .",
"Taken together , these data suggest that the total Spok gene content is responsible for the killer phenotype of Psk-1 , Psk-2 , Psk-5 , and Psk-7 ( Figure 4 ) .",
"In addition , we determined ( on the basis of experimental crosses ) that the newly described Psk-8 type can also be described by Spok gene content and position ( Figure 4—source data 2 and 3 ) .",
"Specifically , Psk-8 has the same Spok block position as Psk-7 , but does not possess Spok3 ( Figure 4 ) .",
"Our results from the crosses also identified inconsistencies with previous studies ( see also Appendix 2 ) .",
"Originally , Psk-4 was defined as a spore-killer ( van der Gaag et al . , 2000 .",
"However , the Psk-4 strain Wa46 has no intact Spok genes ( Table 1 ) .",
"The spore killing observed when this strain was crossed to S in previous publications ( or to Wa63 here ) is a result of Spok2-induced killing .",
"Hence , we recommend discontinuing the use of Psk-4 and that the term ‘naïve’ strain is used instead .",
"Moreover , our crossing data show that our representative strain of Psk-3 ( Wa21 ) ( van der Gaag et al . , 2000 ) is of Psk-2 killer type because it does not exhibit spore killing when crossed to Wa28 ( Psk-2 ) , because it has the expected spore-killing percentage when crossed to a Psk-S strain , and because its Spok content and location are representative of a Psk-2 strain .",
"Finally , our representative strain of Psk-6 ( Wa47 ) behaves as naïve ( Psk-4 ) , and does not exhibit the spore-killing reported by van der Gaag et al . ( 2000 ) in test crosses with Wa46 ( Figure 4—source data 2 ) .",
"As each isolate of the entire Wageningen collection was previously assessed to determine its Psk type ( van der Gaag et al . , 2000 ) , we can estimate the frequency of each Spok gene in the Wageningen population .",
"Isolates of Psk-1 , Psk-2 , Psk-4 , Psk-5 , and Psk-7 , as well as those previously considered as ‘sensitive’ ( now Psk-S ) , account for 92 of the 99 strains collected from Wageningen .",
"The seven remaining strains were identified as either Psk-3 or Psk-6 .",
"Following the rationale outlined in the previous paragraph , we assume that strains annotated as Psk-4 possess no functional Spok genes and omit all the Psk-3 strains ( except Wa21 ) and the Psk-6 strains ( except Wa47 ) from the analysis .",
"We estimate that Spok2 is in 98% , Spok3 in 17% , and Spok4 in 11% of the Wageningen strains .",
"A subsample of 11 strains from the 1937 French collection ( including strains Y , Z and TG ) have also been assessed for their Psk type , as have eight strains from a collection from Usingen ( Us ) , Germany ( Hamann and Osiewacz , 2004; van der Gaag et al . , 2000 ) .",
"Hence , we infer that Spok2 is present in all of the Us strains and in 73% of the analyzed French strains .",
"Spok3 and Spok4 is in 36% of the French strains , whereas Spok3 is in one Us strain and Spok4 is absent from the Us strains .",
"Although the Spok genes are often assembled into small fragmented contigs when obtained by using Illumina data alone , in the PacBio and MinION assemblies , Spok3 and Spok4 are fully recovered within an inserted block of novel sequence ( 74–167 kbp depending on the strain ) , hereafter referred to as the Spok block ( Figure 4 ) .",
"When present , the Spok block was found once per genome and always contained at least one Spok gene .",
"Whole-genome alignments revealed that the Spok block has clear boundaries , and is localized at different chromosomal positions on chromosome 3 or on either arm of chromosome 5 in different strains of P . anserina ( Table 1 ) .",
"Importantly , these positions correspond to a single SNP tract identified in the S5 backcrosses .",
"In P . pauciseta ( CBS237 . 71 ) , the Spok block is found on chromosome 4 .",
"This is evident in Figure 1B as the only large-scale translocation between P . anserina and P . pauciseta ( number 3 ) .",
"The Spok blocks of the different strains share segments and overall structure ( Figure 5 and Figure 5—figure supplement 2 ) , which suggests that they have a shared ancestry .",
"However , complex rearrangements are found when aligning the block between the genomes .",
"Within the Spok block , a given strain can harbor either or both of Spok3 and Spok4 , and the regions containing the Spok genes appear to represent a duplication event ( Figure 5 ) .",
"Strain TG+ shows an additional duplication that has resulted in a second copy of Spok3 ( Figure 5—figure supplement 2 ) .",
"When present , SpokΨ1 is surrounded by numerous TEs , and the region does not appear to be homologous to the Spok block ( Figure 5—figure supplements 1 and 3 ) .",
"To determine whether other components of the Spok block influence the interactions of the Spok genes and/or the Psks , we conducted crosses between selected strains and evaluated spore killing ( Figure 4—source data 1 , 2 and 3 ) .",
"Specifically , dikaryotic F1 progeny that are homoallelic for the killing locus were selected , backcrossed to both parental strains , and also allowed to self-fertilize ( Figure 4—figure supplement 4 ) .",
"On the basis of the results of these crosses , killing interactions were classified into one of the following categories .",
"As an example , in a cross between strains of Psk-1 and of Psk-7 killer types , there is spore-killing .",
"However the F1 progeny from this cross show no killing when crossed to either parent , nor when selfed , satisfying condition 2 .",
"Thus they are mutually resistant , which is consistent with the fact that they carry the same three Spok genes .",
"The reason spore killing is observed in the original cross is because the Spok block is located at different genomic positions .",
"As a result , the Spok block can co-segregate during meiosis , leaving two spores without any Spoks and making them vulnerable to killing ( see Appendix 1 for a detailed explanation ) .",
"An example in which the Psks show dominance is in the interaction between Psk-7 and Psk-8 , which is revealed by the absence of killing when an F1 progeny from a Psk-7 x Psk-8 cross is selfed or crossed to the Psk-7 parent , and the observation of killing when crossed to the Psk-8 parent .",
"This result indicates that the F1 progeny inherited its killing function from the Psk-7 parent , and is consistent with the idea that Spok gene interaction determine killing as , although Psk-8 and Psk-7 strains have a Spok block in the same location , the Psk-8 version of the block does not possess Spok3 ( Figure 4 ) .",
"The backcrossing method described above was not conducted for representative strains of all pairwise interactions among the Psks because of the unmanageable number of crosses that would be involved and the difficulty in mating some strains .",
"In cases where dominance was strongly suspected ( on the basis of Spok gene distribution , such as with crosses to Psk-S or naïve strains ) , the killing percentage was used as a proxy for dominance because in crosses involving dominance , the killing percentage should reflect that of the dominant Psk ( Figure 4—source data 2 and 3 ) .",
"Figure 4 displays these results as a killing hierarchy , in which strains of the Psk type at the top of the hierarchy are dominant to strains of the Psk type lower in the hierarchy and either mutually resistant to ( level 3 ) or mutual killers of ( level 2 ) strains on the same level .",
"The exception is Psk-5 , which exhibits mutual killing with Psk-S .",
"Our classifications , based on crosses , show that the killing hierarchy observed in the Wageningen population of P . anserina is an emergent property of the presence and absence of the various Spok homologs in the different genomes .",
"Hence , our data demonstrate that other components of the Spok block do not affect spore-killing .",
"Of note , crosses between Psk-1 and Psk-5 strains have a lower killing percentage than would be expected on the basis of the FDS of Spok2 ( ~25% instead of ~40% ) .",
"We confirmed that Spok2 is completely associated with two-spored asci in these crosses using a pooled sequencing approach ( Figure 4—figure supplement 5 ) , an observation that is in line with the backcrossing results .",
"However , we noted a high prevalence of three-spored asci ( which were excluded from the analyses ) .",
"We also observed three-spored asci in crosses between Psk-S and naïve strains .",
"Despite low germination rates , we have been able to isolate a spore from a three-spored ascus in a cross between Psk-S and a naïve strain that has no copy of Spok2 ( Appendix 2 ) .",
"Therefore , the three-spored asci are probably due to incomplete penetrance of the killing factor and support the conclusion that the spore killing observed in these crosses is caused by the same gene , Spok2 .",
"This result is consistent with findings presented in the study by van der Gaag ( 2005 ) that provided independent evidence for incomplete penetrance of spore-killing between S and Wa46 ( Psk-S and naïve ) .",
"The lower killing percentage is probably the result of asci with FDS of Spok2 , which contain three or four spores instead of two .",
"In contrast to the absence of epistatic interactions among the Spok genes of P . anserina , Spok1 of P . comata and Spok2 were shown previously to interact epistatically ( Grognet et al . , 2014 ) .",
"To determine whether Spok1 is also dominant to Spok3 and Spok4 , crosses were conducted between strain TD and strains of P . anserina .",
"Although TD shows low fertility with P . anserina ( Boucher et al . , 2017 ) , we were successful in mating TD to a number of P . anserina strains of different Psk spore-killer types ( Figure 4—source data 2 and 3 ) .",
"Often , only a few perithecia were produced with limited numbers of asci available to count , but despite this obstacle , the crosses clearly demonstrate that TD is dominant over Psk-S and Psk-2 strains , and is mutually resistant to a Psk-5 strain .",
"These results imply that Spok1 provides resistance to all of the Spok homologs in P . anserina and is capable of killing in the presence of Spok2 and Spok3 , but not Spok4 .",
"The mutual resistance with the Psk-5 strain also demonstrates that Spok4 provides resistance against Spok1 .",
"Additional crosses were also conducted with the P . pauciseta strain CBS237 . 71 , which were consistent for the Spok3 and Spok4 interactions ( Figure 4—source data 2 and 3 ) .",
"As both TD and CBS237 . 71 have unique spore-killing phenotypes , we assign them the types Psk-C1 and Psk-P1 , respectively .",
"To investigate whether the Spok genes are expressed during spore-killing , we conducted an additional nine backcrosses of the S5 strains to S , in order to generate S14 backcrossed strains ( see 'Materials and methods' ) .",
"We produced RNAseq data for self-killing S14 cultures and mapped the reads to the final assemblies of the dominant killer parental strains ( Figure 2—figure supplement 2A ) .",
"The expression of the Spok genes is evident in this data and supports the presence of an intron in the 5' UTR of the Spok homologs ( Figure 2 and Figure 2—figure supplement 2B–E ) .",
"Given its conservation across the Spok homologs and as the wtf spore-killer system in S . pombe was described as involving two alternate transcripts of the same gene ( Nuckolls et al . , 2017; Hu et al . , 2017 ) , the role of the intron in Spok3 spore-killing activity was investigated .",
"The intron was deleted in a plasmid bearing the Spok3::PaPKS1 deletion cassette by site-directed mutagenesis , and the modified plasmid was used to transform the ΔKu70 ΔSpok2 strain .",
"Three transformants bearing the Spok3 lacking the intron sequence ( Spok3 Δi ) were crossed to a ΔSpok2 strain .",
"As in the control cross with wildtype ( wt ) Spok3 , in which close to 100% killing was found , we observed that 109/109 of the asci contained two unpigmented spores ( Figure 4—figure supplement 2L ) .",
"Thus , Spok3 Δi displays wildtype killing activity .",
"We conclude from this experiment that the unspliced form of Spok3 is not required for normal killing activity , and neither does the killing and resistance function rely on an alternatively spliced form of this intron .",
"In order to gain insights into the molecular function of the SPOK proteins , domain identification was performed with HHPred and a HMM profile based on an alignment of 282 Spok3 homologs from various Ascomycota species .",
"The SPOK3 protein was predicted to be composed of three folded domains ( located at amino-acid positions ~ 40 to 170 , 210 to 400 , and 490 to 700 in the protein ) separated by two unstructured domains ( ~170 to 210 and 400 to 490 ) as shown in Figure 6 .",
"No functional identification was recovered for domain 1 , but a coiled-coil motif was found in the N-terminal 40 amino acids and predicted to form a parallel dimer , which corresponds to the variable length repeat of the nucleotide sequences ( Figure 2A ) .",
"Domain 2 showed homology to a class of phosphodiesterases of the PD- ( D/E ) XK superfamily ( ~214 to 325 ) with the catalytic residues forming the PD- ( D/E ) XK motif spanning positions 219 to 240 in the SPOK3 sequence ( Steczkiewicz et al . , 2012 ) .",
"The best hit in HHPred was to the HsdR subunit of a type-I restriction enzyme from Vibrio vulnificus ( Uyen et al . , 2009 ) .",
"The sequences align in the catalytic core region in the PD- ( D/E ) XK motif and also around a QxxxY motif ( 294 to 298 in SPOK3 ) that was found to be important for nucleic-acid binding and nuclease activity ( Sisáková et al . , 2008 ) ( Figure 6—figure supplement 2 ) .",
"Domain 3 was identified as a hypothetical kinase domain ( ~539 to 700 ) as predicted previously by Grognet et al . ( 2014 ) .",
"In addition , a motif with a cluster of three highly conserved cysteine residues together with histidine residues ( C-x3-C-x13-C-x5-H-x7-H ) that is reminiscent of the zinc-finger motifs was identified upstream of the kinase motif ( Figure 6 ) .",
"As previously reported for Spok2 , D667 was identified as the catalytic base residue in the catalytic loop ( subdomain VIb ) of the kinase domain .",
"Kinases often use other proteins as substrates , but they may also target small molecules ( Smith and King , 1995 ) .",
"Inspection of the VIb and VII functional regions , which are informative as regards to kinase substrate specificity , suggests that the Spok-kinase domain might be more closely related to eukaryotic-like kinases ( ELKs ) than to eukaryotic protein kinases ( ePKs ) , raising the possibility that this kinase domain is not necessarily a protein kinase domain and could phosphorylate other substrates ( Steczkiewicz et al . , 2012; Kannan et al . , 2007 ) .",
"The ability of the Spok genes to perform both killer and resistance functions with a single protein is unique among meiotic drive systems ( Bravo Núñez et al . , 2018b ) .",
"To investigate the role that the domains 1–3 may play in these two functions , we constructed a number of point mutations and truncation variants of Spok3 and assayed their ability to kill or provide resistance in vegetative cells .",
"We were able to determine that domain 2 is important for killing activity whereas domain 3 is important for resistance activity .",
"It had been shown previously that the predicted kinase domain of SPOK2 ( Figure 6 ) is involved in the resistance function ( Grognet et al . , 2014 ) .",
"We introduced a point mutation in a plasmid-cloned Spok3 gene that led to the replacement of the predicted catalytic aspartic acid residue of Spok3 by an alanine ( D667A ) .",
"The mutant allele was first used to transform a ΔSpok2 recipient strain .",
"This Spok3 D667A mutant allele leads to a drastic reduction in transformation efficiency ( Figure 6—source data 1 ) , whereas the Spok3 wildtypeallele only moderately affects the number of transformants .",
"As this approach results in random integration and potential multicopy insertion , we also attempted to introduce the mutant Spok3 D667A allele as a single copy at the PaPKS1 locus , as described above for wildtype Spok3 .",
"The initial transformants were heterokaryotic and displayed sectors of abnormal growth that corresponded to unpigmented mycelium , presumably containing nuclei with Spok3 D667A that inserted at PaPKS1 .",
"Monokaryotic transformants could be recovered and were tested for killing activity in a cross to a ΔSpok2 .",
"Four-spored asci with two white and two black spores were observed , suggesting that the D667A mutation abolishes spore killing .",
"However , when the integrated Spok3 allele was amplified by PCR and sequenced , it appeared that the allele presents a GAG to TAG mutation , leading to a premature stop codon in position 282 ( E282stop ) .",
"This result is consistent with the observation that Spok3 D667A affects transformation efficiency and is toxic .",
"Moreover , we detected expression of Spok2 and Spok1 in the vegetative cells of monokaryotic ( self-sterile ) cultures , suggesting that Spok activity is not restricted to the sexual cycle ( Figure 2—figure supplement 2C and D ) .",
"No further attempts to insert the mutant allele at PaPKS1 were made .",
"If the toxicity of the Spok3 D667A allele in vegetative cells is mechanistically related to spore-killing , it is expected that this toxicity should be suppressed by wildtype Spok3 .",
"Therefore , we assessed whether Spok3 D667A toxicity in vegetative cells is suppressed by co-expression with wildtype Spok3 .",
"Co-transformation experiments were set up with Spok3 D667A used as the transformation vector in the presence or absence of wt Spok3 .",
"As in the previous experiment , Spok3 D667A alone was found to affect transformation efficiency , but this effect was suppressed in co-transformations with Spok3 ( Figure 6—source data 1 ) .",
"This experiment confirms that Spok3 D667A is only toxic in the absence of Spok3 .",
"Therefore , the Spok-related killing and resistance activities can be recapitulated in vegetative cells .",
"We also analyzed the role of the conserved cysteine cluster just upstream of the predicted kinase domain .",
"Three plasmids with point mutations in that region were constructed ( a C493A C497A double mutant , and C511A and C511S point mutants ) , and the mutant alleles were used in transformation assays as previously described for Spok3 D667A .",
"All three mutants reduced transformation efficiencies as compared to the controls , and this effect was suppressed in co-transformations with wt Spok3 ( Figure 6—source data 1 ) .",
"These results suggest that the kinase domain and the cysteine-cluster region are both required for the Spok-related resistance function but not for the killing activity .",
"To test this , we constructed a truncated allele of Spok3 that lacks these two regions: Spok3 ( 1–490 ) ( see Figure 6—figure supplement 1 ) .",
"The Spok3 ( 1–490 ) allele drastically reduced transformation efficiencies and this effect was suppressed in co-transformations with wildtype Spok3 ( Figure 6—source data 1 ) .",
"If , as proposed here , the toxicity and suppression activities assayed in vegetative cells are mechanistically related to spore-killing , then domain 3 appears to be required for the resistance function but dispensable for the killing activity , which can be carried out by the N-terminal region of the SPOK3 protein ( domains 1 and 2 ) .",
"Next we analyzed the role of the predicted nuclease domain ( domain 2 ) in spore-killing activity .",
"We generated a plasmid with a point mutant that affects the predicted catalytic core lysine residue ( K240A ) .",
"Introduction of this point mutation in the Spok3 ( 1--490 ) allele abolished its killing activity in transformation assays ( Figure 6—source data 1 ) , suggesting that the predicted nuclease domain is required for killing activity .",
"The Spok3 K240A mutant was then inserted at the PaPKS1 locus and the resulting knock-in strain was crossed with a ΔSpok2 strain ( in order to assay killing ) and to a Spok3::PaPKS1d strain ( to assay resistance ) ( Figure 4—figure supplement 2M and N ) .",
"In the cross to ΔSpok2 , no killing was observed: the majority of the asci were four-spored with two white and two black spores ( 308/379 , 81 . 2% ) , indicating that the K240A mutation abolishes the spore-killing activity of Spok3 .",
"In the Spok3 K240D::PaPKS1 x Spok3::PaPKS1d cross , no killing was observed: the majority of the asci were four-spored with two white and two black spores ( 268/308 , 87% ) .",
"These crosses indicate that the Spok3 K240A allele has no killing ability but has retained resistance .",
"Grognet et al . ( 2014 ) reported that strain A bears a mutant allele of Spok2 that has affected killing ability but retains resistance .",
"The mutations in that allele fall within a conserved region of the predicted nuclease domain ( Figure 6 ) and map on predicted structural models in close vicinity to the catalytic lysine residue ( K240 in SPOK3 ) and the other catalytic residues ( Figure 6—figure supplement 2 ) .",
"Hence , the properties of the Spok2 allele of strain A provide independent evidence that the nuclease domain of SPOK proteins is involved in killing activity but dispensable for resistance .",
"A BLAST search for closely related homologs of the Spok genes across fungi revealed an uneven distribution of related proteins among taxa .",
"Among the available genomes from the Sordariales , we did not find any protein-coding sequences in addition to our newly described Spok genes of Podospora .",
"By contrast , we found protein-coding sequences with high similarity across other orders of the Sordariomycetes , namely the Xylariales , Glomerellales and Hypocreales , as well as in one species of the Eurotiomycetes , Polytolypa hystricis ( Poh; Figure 7 ) .",
"We used maximum likelihood analyses to construct phylogenies of the SPOK sequences and of an orthologous gene set of the strains for which we retrieved hits in the BLAST search ( Figure 7 ) .",
"These phylogenies reveal two notable patterns .",
"First , the SPOK phylogeny shows a high degree of incongruence with the species phylogeny .",
"Moreover , the SPOK phylogeny can be robustly divided into two clades: Clade I and II .",
"Clade I contains the Fs_82228 sequence from Fusarium solani ( old name Nectria haematococca ) .",
"This sequence was previously introduced into P . anserina , and the genetically modified strain produced empty asci when mated to a naïve strain , suggesting that it has a killing action ( Grognet et al . , 2014 ) .",
"Clade II contains the Podospora Spok homologs .",
"The sequences from the two clades are disparately distributed in the species phylogeny .",
"The second notable pattern is the distribution of the SPOK sequences within the genomes .",
"The sequences in Clade I are present in single copies in each strain , except for Fusarium oxysporum f .",
"sp .",
"pisi ( Fop ) , suggesting that they are all orthologs .",
"By contrast , many of the sequences in Clade II are present in multiple copies in each genome .",
"It is particularly interesting to note how many Spok homologs from Clade II are present across strains of F . oxysporum ( Fo ) and the number of copies that are found in each genome .",
"Several of the duplicate Spok homologs are present on the lineage-specific chromosomes of Fusarium that are often associated with pathogenicity ( Armitage et al . , 2018 ) .",
"Beauveria bassiana ( Bb ) also shows a high degree of variability in homolog content among the four strains that have homologs , indicating that the homologs are polymorphic in this species .",
"The insect pathogen Metarhizium rileyi ( Mr ) shows an interesting pattern in that it possesses four divergent homologs , which is in stark contrast to many of the other species ( including Podospora ) that have multiple , though nearly identical , copies .",
"The Clade II Spok homologs also appear to diversify within each strain or species in much the same way as the Spok genes do in Podospora , with variable lengths of the coil-coil repeat region and frameshift mutations near the 3′ end that relocate the stop codon .",
"A few of the sequences may also represent pseudogenes , as evidenced by premature stop codons and/or frameshifts , although these features might also be the result of unidentified introns ( Figure 7 ) ."
],
[
"The presence of the complex Spok block presents a unique feature among the known meiotic drive systems .",
"Often , meiotic drive elements occupy regions of suppressed recombination that span large tracts of chromosomes ( Turner and Perkins , 1979; Hammer et al . , 1989; Sandler et al . , 1959 ) and co-occur with complex rearrangements ( Harvey et al . , 2014; Silver , 1993; Dyer et al . , 2007; Svedberg et al . , 2018 ) .",
"In these well-studied cases , the elements of the drive mechanisms are encoded by separate genes within the region , and the rearrangements and suppression of recombination are expected to have evolved to ensure that the drive machinery ( e . g . , the toxin and antitoxin genes ) is inherited as one unit ( Lyttle , 1991; Bravo Núñez et al . , 2018b ) .",
"In Podospora , a single Spok gene is fully capable of driving , and thus no region of suppressed recombination is required .",
"Nevertheless , Spok3 and Spok4 are found in a large region that is not syntenic with the null allele .",
"Hence , had the Spok genes not been previously identified from more placid genomic regions , the entire Spok block may have been misidentified as a driving haplotype with multiple interacting components .",
"Considering that single-gene meiotic drivers might be more common than anticipated , it becomes necessary to question whether other drive systems that are located within complex regions , and for which the genetics are not well known , may also represent single gene drivers .",
"At this stage , our data strongly suggest that the Spok block is moving in the genomes as a unit , but nevertheless , the mechanism of movement remains unknown .",
"It may be hypothesized that movement of the block is achieved via an interaction with TEs at different genomic locations and non-allelic homologous recombination .",
"This hypothesis is supported by the observation that the Spok genes outside of the Spok block , including SpokΨ1 , are not located at the same position in different species , and that they are often surrounded by similar TEs .",
"Such movement may be under selection as matings between strains that have the same Spok genes but in different locations will result in spore-killing .",
"Furthermore , because of the idiosyncrasies of meiosis in Podospora , the position of the block may be under selection because the killing frequency is dependent on the frequency of crossing over with the centromere .",
"Alternatively , the TEs may simply accumulate around the Spok genes because of a reduced efficacy of purifying selection at regions linked to the driver genes; their presence per se might increase the chance of rearrangements .",
"As such , the role that TEs play in generating complex regions that are associated with meiotic drive should be investigated further in order to determine their importance in the evolution of drive .",
"Spore-killing systems display analogies to toxin-antitoxin ( TA ) systems in bacteria and it is interesting to note that many toxin families rely on nuclease activity ( Harms et al . , 2018 ) .",
"The contrast between the Spok system and TA systems , however , resides in the fact that Spok toxin and antitoxin activities appear to be supported by the same protein molecule .",
"The predicted kinase activity seems able to counter the toxic activity of the predicted nuclease domain both in cis and in trans .",
"Mechanistic spore-killing models have to explain:",
"( i ) how the Spok gene can affect spores that do not carry it , at a distance , with SPOK proteins being intracellular proteins ( Grognet et al . , 2014 ) ; and",
"( ii ) how asymmetry is brought about in this system if killing and resistance activities are carried by the same protein molecule .",
"It is premature to devise a mechanistic model for the molecular basis of Spok gene drive , yet it might be possible to conjecture about the substrates of the proposed kinase ( and nuclease ) activities of SPOK proteins .",
"A first way to explain how Spok genes might act at distance and affect spores not containing them is to hypothesize that toxicity relies on the production of a diffusible metabolite .",
"The predicted nuclease/phosphodiesterase activity would lead to the production of a diffusible toxic molecule that could be further detoxified by phosphorylation ( much in the way that bacterial phosphotransferases detoxify antibiotics ) ( Shi et al . , 2013 ) .",
"Isolation of the toxic intermediate from the detoxifying activity of the kinase would allow its accumulation specifically outside of the spores expressing SPOK proteins , and thus could bring about asymmetry in spore-killing .",
"Then , alternatively , spores lacking the Spok gene might be affected by SPOK proteins if some amount of SPOK protein expressed in the zygote or from sister nuclei is carried over at the time of spore delimitation .",
"The resistance function of the predicted kinase domain could be explained by hypothesizing that the nuclease activity can be inhibited by autophosphorylation of the SPOK proteins .",
"Alternatively , it could also be that it is the phosphorylation of a distinct macromolecule that nullifies toxicity .",
"In a simple model , the same molecule could be the target of both the kinase and nuclease activity , and the phosphorylation of the target would make it resistant to the toxic action of the predicted nuclease domain .",
"As stated above , models have to explain how killing could occur specifically in spores lacking the Spok gene .",
"This situation could occur if the proposed kinase activity is concentration-dependent and favored at higher SPOK protein concentrations ( for instance , the kinase activity might require a protein dimerization step that occurs specifically in spores expressing the Spok gene ) .",
"In addition to the yet unresolved mechanistic basis of killing and resistance , the characterization of Spok gene function described here poses another puzzle .",
"As all SPOK products have a predicted active kinase , it is not yet known what changes in sequence confer the hierarchical interactions among some Spok genes , or why not all SPOKs are able to provide resistance to one another .",
"One possibility is that the cellular targets for the proposed nuclease and kinase activities differ for the different SPOK proteins .",
"Studies of similar protein domains suggest that the coil-coiled domain is likely to be involved in protein–protein interactions ( van Maldegem et al . , 2015 ) .",
"The fact that Spok1 and Spok4 have the same length repeat in this domain could imply that the protein–protein interactions of this domain are important for resistance , as Spok1 and Spok4 are mutually resistant .",
"This model would agree somewhat with the results of reporter constructs from Grognet et al . ( 2014 ) , which showed an N-terminal mCherry tag on Spok2-produced empty asci .",
"It is possible that the functional divergence observed between the SPOK proteins is due to mutations in this portion of the protein .",
"In this model , domain 1 might be responsible for the target specificity of the nuclease ( and kinase ) activity .",
"The killing action itself is expected to be universal among the Spoks and is supported by the fact that this entire domain of Spok3 from TG is identical to Spok4 , yet appears to retain Spok3 functionality .",
"The identification of the role of the predicted nuclease domain in killing and of the predicted kinase domain in resistance provides a first mechanistic insight into the dual role of Spok genes .",
"However , further dissection of the molecular action of these proteins is required so that we can fully understand the molecular basis of Spok drive .",
"One of the main factors that stands out in the Podospora system , as compared to the other well-studied spore killers , is the lack of resistant strains .",
"Only one strain of P . anserina ( the French strain A ) has ever been described as resistant ( Grognet et al . , 2014 ) .",
"The point mutations of Spok3 that were induced in the laboratory imply that the creation of a resistant strain is a simple task , as only a single nucleotide change was required .",
"Likewise , the resistant strain A Spok2 is different from the reference allele by only two novel insertions .",
"Consequently , the lack of resistance does not appear to be the result of a mechanistic constraint .",
"Potentially , the current Spok gene distribution could be a relatively young phenomenon and resistance could evolve over time .",
"Another possibility is that resistance itself is somehow costly to the organism and selected against .",
"In addition , it is puzzling that none of the Spoks in P . anserina show cross resistance .",
"Intuitively , it would seem advantageous for novel Spok homologs to evolve new killing functions while maintaining resistance to the other Spok homologs .",
"Again , the lack of cross-resistance does not solely appear to be the result of functional constraints , as Spok1 , which is highly similar to Spok4 , is resistant to all other Spok homologs .",
"It is possible that it is more advantageous to combine multiple independent spore killers than to have a single broadly resistant gene .",
"This option is supported by two observations presented in this study: the occurrence of the killing hierarchy and the association of Spok3 and Spok4 .",
"The fact that Spok3 and Spok4 are present in the Spok block means that they are in tight linkage with each other .",
"It may be the case that the linkage was selected for because it provided strains with the ability to drive against strains with just Spok3 or just Spok4 .",
"However , this association could also be simply the result of a duplication without invoking selection .",
"Whether the killing hierarchy that we observe in P . anserina is due to a complex battle among the Spok homologs or a result of the existence of the Spok block will require further experimentation and mathematical modeling to resolve .",
"Some interesting aspects of meiotic drive in Podospora identified herein bear numerous features that parallel the wtf genes that are responsible for drive in Schizosaccharomyces pombe .",
"There is no sequence similarity or conserved domains between the Spok and wtf genes , and Podospora and Schizosaccharomyces are only distantly related ( ~500 million years diverged ) ( Wang et al . , 2009; Prieto and Wedin , 2013 ) .",
"Yet these systems display similar evolutionary dynamics within their respective species .",
"Both of these systems are built of multiple members of gene families , which appear to duplicate , rapidly diverge to the point where they no longer show cross reactions ( potentially with the aid of gene conversion ) , and then pseudogenize and become nonfunctional ( Nuckolls et al . , 2017; Bravo Núñez et al . , 2018a; Hu et al . , 2017 ) .",
"Both systems also have close associations with TEs ( Bowen et al . , 2003 ) .",
"Hu et al . ( 2017 ) invoke LTR-mediated non-allelic homologous recombination as a possible mechanism for wtf gene deletion in a lab strain of S . pombe .",
"We provide evidence for the deletion of Spok2 , but it does not fit with expectation that this deletion is LTR-mediated .",
"Nevertheless , as TEs are still accumulating in the region , other TE-related processes may have been involved in the deletion .",
"The factors that determine the abundance and diversity of multigene family meiotic drivers in a species are the rates of gene duplication and loss , and time since origin .",
"In the case of the Spok genes , we expect a low rate of deletion as they approach fixation because of the dikaryotic nature of Podospora .",
"Specifically , when first appearing , a deletion is only expected to be present in one of the two separate nuclear genomes maintained within a dikaryon .",
"Any selfing event should erase ( i . e . drive against ) the deletion , meaning that in order to become homoallelic for a deletion , the strain would have to outcross with another individual with no Spok genes or Spok genes that differ from its own .",
"Such outcrossing could allow deletions of Spok3 and Spok4 , but as Spok2 is nearly fixed in the population , any outcrossing event should also lead to the elimination of the deletion by the driving action of Spok2 .",
"A possible solution to the paradoxical finding that Spok2 appears to have been lost occasionally is that the incomplete penetrance of Spok2 may have allowed spores that were homoallelic for the deletion to survive and persist .",
"In this sense , Spok2 fits a model of driver turn over , wherein it is beginning to lose killing function after becoming fixed in the population .",
"SpokΨ1 is missing the portion of the gene that is responsible for killing and the small Spok fragment of P . comata also corresponds to the resistance part of the gene .",
"Both of these observations suggest that the killing domain may have been lost prior to these genes becoming fully pseudogenized and hints that they may have functioned as resistance genes .",
"It has been pointed out that spore-killing may be a weak form of meiotic drive , because the transmission advantage is relative to the number of spores produced in a given cross , but there is no absolute increase at the population level ( Lyttle , 1991 ) .",
"Hence , a spore killer was predicted by Nauta and Hoekstra ( 1993 ) to require an additional fitness advantage in order to reach fixation in a population .",
"It is thus striking that Spok2 is close to fixation in at least the European populations , bringing into question the direct fitness effects of the Spok genes .",
"On the other hand , the Spok block ( and hence Spok3 and Spok4 ) seems to be present at relatively low frequency .",
"It is possible that the rate at which the Spok block switches position is higher than the rate at which the Spoks can sweep to fixation .",
"Therefore , the dynamics of Spok genes within the Spok block might differ from the Spok2 life-cycle and might explain why spore-killing is observed to be polymorphic in P . anserina .",
"In addition , P . anserina is capable of selfing , which may slow down the rate of fixation of the genes .",
"Moreover , the vegetative and/or sexual expression of the Spok genes might be deleterious in itself , and hence natural selection might increase or maintain the frequency of strains without all Spok homologs .",
"Overall , this complex system requires population genetic modeling to resolve the factors affecting the frequency of the Spok genes in populations of this fungus .",
"The relationships among the Spok genes can provide insight as to the evolutionary history of the Spok block .",
"The observation that Spok3 and Spok4 are both present in the Spok block in a duplicate region suggests that these genes represent paralogs that formed via duplication .",
"Indeed , the phylogenetic analysis of the UTRs agrees with a duplication origin .",
"However , this scenario is contradicted by the finding that Spok4 shares many features with Spok1 of P . comata , but not Spok3 .",
"The four most likely evolutionary scenarios are outlined in Figure 8 .",
"If the relationship between Spok1 and Spok4 is a result of common descent ( orthology; Figure 8A ) , then after the duplication event that generated Spok3 and Spok4 , Spok3 would have to have had a much higher rate of change than both Spok1 and Spok4 in order to explain the observed divergence .",
"In addition , the inferred deletions in the Spok3 coding sequence would have to be reverted by gene conversion with an unknown Spok homolog , and new deletions would have to appear subsequently in Spok3 .",
"Alternatively , the diversification of the Spok genes may have been influenced by past hybridization , and we discuss a few possible scenarios here .",
"One possibility is that Spok4 was introduced into P . comata , which then diverged to become Spok1 after the duplication event ( Figure 8B ) .",
"This divergence would then have to be followed by gene conversion in order to account for the shared frameshift mutation in Spok3 and Spok4 .",
"On the other hand , Spok1 may have been transferred to P . anserina ( Figure 8C ) , and then duplicated to form Spok3 and Spok4 , but this scenario requires the same additional steps as the orthology scenario .",
"A final option is that Spok3 and Spok4 are not homologs formed via duplication but rather orthologs that evolved independently in separate populations ( Figure 8D ) .",
"Their current positions in the Spok block could be due to the fusion of ancestral blocks , followed by gene conversion .",
"In P . pauciseta ( CBS237 . 71 ) , Spok3 , Spok4 , and the Spok block are nearly identical to the same sequences in P . anserina , so in order to explain the current pattern of the Spok genes and the Spok block without interspecies transfer , the block would have had to remain virtually unchanged since the divergence of P . anserina and P . pauciseta .",
"This scenario seems highly unlikely given the relative divergence between the three species .",
"The broader Spok homolog phylogeny presented here is also not consistent with a simple vertical decent model , supporting the conclusion that the evolution of the Spok genes has involved inter-lineage transfers .",
"Such interspecies interactions that mediate the introgression of meiotic drive genes between species would not be a phenomenon that is unique to the Spok genes of Podospora , as meiotic drive genes in Drosophila have been observed to cross species boundaries and erode barriers of reproduction ( Meiklejohn et al . , 2018 ) .",
"Further analyses of the genomes of populations of multiple Podospora species is needed in order to resolve the history of the Spok genes and the block .",
"Grognet et al . ( 2014 ) demonstrated that proteins that are related to the SPOKs are distributed across a diverse group of Ascomycota , but the majority of them are very diverged .",
"Here , we have identified a group of more closely related homologs ( clade II ) from genome sequences that have been released since the Grognet et al . ( 2014 ) study , allowing us to analyze the evolutionary history at a finer resolution .",
"The phylogenetic distribution of the clade II Spok homologs supports the general hypothesis that the Spok genes are transferred horizontally among evolutionarily disparate groups , as suggested by Grognet et al . ( 2014 ) .",
"For example , the eurotiomycete Polytolypa hystricis possesses a homolog that is closely related to the Podospora Spok genes .",
"However , the phylogeny presented here shows that a subset of the clade II homologs agree with the relationships among closely related species ( Maharachchikumbura et al . , 2015 ) , suggesting an alternative hypothesis whereby the Spok genes are ancestral to the Sordariomycetes but lost frequently .",
"Such a scenario would imply that there are long-term consequences of possessing spore-killer genes , even if they are fixed in the population .",
"These two hypotheses are not mutually exclusive , and with our data , we are not able to disentangle their relative importance for the observed pattern .",
"The diversification pattern may also give insight into the possibility that SPOK homologs function as drivers in lineages other than Podospora .",
"The phylogeny presented here suggests that the clade I homologs do not represent meiotic drive genes because only one presumably orthologous copy is typically found .",
"By contrast , the numerous closely related Spok homologs of clade II may be driving .",
"For example , in F . oxysporum f .",
"sp .",
"cepae four nearly identical copies are found , resembling the distribution of the Spok genes in Podospora .",
"However , no sexual cycle has been observed in F . oxysporum .",
"Given that we demonstrate vegetative killing with Spok3 , it is possible that the Fusarium Spok genes operate in vegetative tissue to ensure the maintenance of the pathogenic-associated chromosomes .",
"Alternatively , as F . oxysporum strains have been found with both mating type alleles ( O'Donnell et al . , 2004 ) , there may be a cryptic sexual cycle in which the Spok homologs are active .",
"With this study , we have provided a robust connection between the phenotype and genotype of spore-killing in P . anserina .",
"We showed that meiotic drive in Podospora spp .",
"is governed by genes of the Spok family , a single locus drive system that confers both killing and resistance within a single protein , which synergize to create hierarchical dynamics by the combination of homologs at different genomic locations .",
"We define Psk-1 , Psk-2 , Psk-5 , Psk-7 , Psk-8 , and Psk-S in terms of Spok gene content and describe the interactions among them .",
"The Spok genes are prone to duplication , diversification and movement in the genome .",
"Furthermore , our results indicate that they probably evolved via cross-species transfer , highlighting the potential risks of the release of synthetic gene drivers for biological control invading non-target species .",
"Moreover , we present evidence that homologs of the Spok genes might have similar dynamics across other groups of fungi , including pathogenic strains of Fusarium .",
"Taken together , the Spok system provides insight into how the genome can harbor numerous independent elements that enact their own agendas and affect the evolution of multiple taxa ."
],
[
"The fungal strains used in this study are listed in Table 1 and were obtained from the collection maintained at the Laboratory of Genetics at Wageningen University ( van der Gaag et al . , 2000 ) and the University of Bordeaux .",
"Strains with the ‘Wa’ identifier were collected from the area around Wageningen between 1991 and 2000 ( van der Gaag et al . , 1998; van der Gaag et al . , 2000; Hermanns et al . , 1995 ) .",
"Strains S , Y , and Z were collected in France in 1937 ( Rizet , 1952; Belcour et al . , 1997 ) .",
"Strain S is commonly used as a wildtype reference , and an annotated genome ( Espagne et al . , 2008 ) is publicly available at the Joint Genome Institute MycoCosm website ( https://genome . jgi . doe . gov/programs/fungi/index . jsf ) as ‘Podan2’ .",
"A strain labeled T ( referred to herein as TG ) was kindly provided by Andrea Hamann and Heinz Osiewacz from the Goethe University Frankfurt and originates from the laboratory of Denise Marcou .",
"However , as the genome sequence of TG did not match that reported by Silar et al . ( 2019 ) , but instead is a strain of P . anserina , we included in our dataset another strain labeled T from the Wageningen Collection that was originally provided by the laboratory of Léon Belcour .",
"We referred to this strain as TD , and sequenced it using only Illumina HiSeq ( see Appendix 2 for further discussion ) .",
"It remains unclear where exactly TD and TG were collected , given the labeling confusion .",
"Representative strains of the Psk spore-killer types from the Wageningen collection were phenotyped to confirm the interactions described by van der Gaag et al . ( 2000 ) .",
"Strains Wa87 and Wa53 were selected as representative of the Psk-1 type , Wa28 for Psk-2 , Wa21 for Psk-3 , Wa46 for Psk-4 , Y for Psk-5 , Wa47 for Psk-6 , and Wa58 for Psk-7 .",
"Strains S and Wa63 were used as reference strains and are annotated as Psk-S .",
"Strain Wa58 mated poorly in general , so strain Z was also used as a mating tester for the Psk-7 spore-killer type .",
"For all crossing experiments and genome sequencing , we isolated self-sterile monokaryons ( i . e . , haploid strains containing only one nuclear type ) from spontaneously produced five-spored asci ( Rizet and Engelmann , 1949 ) , identified their mating type ( mat+ or mat– ) by crossing them to tester strains , and annotated them with + or – signs accordingly .",
"All crosses were performed on Petri dishes with Henks Perfect barrage medium ( HPM ) .",
"This media is a modified recipe of PASM2 agar ( van Diepeningen et al . , 2008 ) , to which 5 g/L of dried horse dung is added prior to autoclaving .",
"Strains were first grown on solid minimal medium , PASM0 . 2 .",
"For each cross , a small area of mycelium of each of two monokaryons was excised from the plates and transferred to HPM .",
"Perithecia ( fruiting bodies ) form at the interface between sexually compatible mat+ and mat– monokaryons .",
"Mature perithecia with fully developed ascospores were harvested after 8–11 days and the percentage of two-spored asci was evaluated to determine the killing percentage ( Box 1—figure 1 ) .",
"All cultures were incubated at 27°C under 70% humidity for a 12:12 light:dark cycle .",
"Barrage formation , whereby confrontations between mycelia of two different strains will produce a visible line of dead cells if they are vegetatively incompatible , was also evaluated on HPM .",
"For details , see van der Gaag et al . ( 2003 ) .",
"To determine the epistatic interactions between the different Psks , crosses were set up according to the following design ( Figure 4—figure supplement 4A ) .",
"Monokaryons of two parental strains ( P1 and P2 ) were confronted on Petri dishes with solid HPM media and perithecia were dissected upon maturation , which takes place after 9–12 days .",
"If only four-spored asci were observed , P1 and P2 are the same Psk , otherwise they represent different Psks .",
"A spore was selected from a two-spored ascus to generate an F1 strain for further crosses: selfing or backcrossing to the parental strains .",
"As most F1 strains from two-spored asci will be homokaryotic for a driver , they will result in four-spored asci when selfed , except in the case of mutual killing ( Figure 4—figure supplement 4B ) .",
"Mutual killing can also result in completely empty asci if the drivers are at the same locus .",
"By crossing the F1 strains to both + and – strains of P1 and P2 , we can distinguish between mutual killing , mutual resistance , and dominance .",
"If none of the crosses yield two-spored asci , there is mutual resistance ( Figure 4—figure supplement 4 ) .",
"In a dominance interaction , for example when P1 is dominant to P2 , the F1 strain will produce four-spored asci with both mating types ( +/– ) of P1 , but will have two-spored asci with both mating types of P2 .",
"If two-spored asci were observed in crosses to both P1 and P2 , or if there are two-spored asci when the F1 is selfed , then there is mutual killing .",
"For both DNA and RNA Illumina HiSeq reads , adapters were identified with cutadapt v . 1 . 13 ( Martin , 2011 ) and then trimmed using Trimmomatic 0 . 36 ( Bolger et al . , 2014 ) with the options ILLUMINACLIP:adapters . fasta:1:30:9 LEADING:20 TRAILING:20 SLIDINGWINDOW:4:20 MINLEN:30 .",
"Only filtered reads with both forward and reverse were kept for downstream analyses .",
"For short-read mapping , we used BWA v . 0 . 7 . 17 ( Li and Durbin , 2010 ) with PCR duplicate marking of Picard v . 2 . 18 . 11 ( http://broadinstitute . github . io/picard/ ) , followed by local indel re-aligning implemented in the Genome Analysis Toolkit ( GATK ) v . 3 . 7 ( Van der Auwera et al . , 2013 ) .",
"Mean depth of coverage was calculated with QualiMap v . 2 . 2 ( Okonechnikov et al . , 2016 ) .",
"The raw PacBio reads were filtered and assembled with the SMRT Analysis package and the HGAP 3 . 0 assembler ( Chin et al . , 2013 ) .",
"The resulting assembly was error-corrected ( polished ) with Pilon v . 1 . 17 ( Walker et al . , 2014 ) using the mapped filtered Illumina reads of the same monokaryotic strain .",
"The samples sequenced with MinION were assembled using Minimap2 v . 2 . 11 ( Li , 2018 ) and Miniasm v . 0 . 2 ( Li , 2018; Li , 2016 ) , polished twice with Racon v . 1 . 3 . 1 ( Vaser et al . , 2017 ) using the MinION reads , and further polished for five consecutive rounds of Pilon v . 1 . 22 using the Illumina reads as above .",
"Scaffolds were assigned to chromosome numbers on the basis of homology with Podan2 .",
"Small scaffolds ( <100 kb ) corresponding to rDNA and mitochondrial-derivatives were discarded .",
"Only the biggest mitochondrial scaffold was retained .",
"In addition , DNA Illumina reads were assembled de novo for each sample using SPAdes v . 3 . 12 . 0 ( Bankevich et al . , 2012; Antipov et al . , 2016 ) using the k-mers 21 , 33 , 55 , 77 and the –careful option .",
"BLAST searches of the scaffolds in the final assembly of the strain CBS237 . 71 revealed contamination by a Methylobacterium sp .",
"in the MinION data ( but not in the Illumina data set ) .",
"The scaffolds matching the bacterium were removed from the analysis .",
"Long-read assemblies were evaluated using BUSCO v . 3 . 0 . 2 ( Simão et al . , 2015; Waterhouse et al . , 2017 ) for the Sordariomyceta ortholog set with the following dependencies: BLAST suit 2 . 6 . 0+ ( Camacho et al . , 2009 ) , HMMER v , 3 . 1b2 ( Mistry et al . , 2013 ) , and AUGUSTUS v 3 . 2 . 3 ( Stanke and Waack , 2003 ) .",
"Short-read assemblies were evaluated using QUAST v . 4 . 6 . 3 ( Mikheenko et al . , 2016 ) .",
"The assembly of the Spok block was visually inspected by mapping the long reads ( using Minimap2 ) and the short reads ( BWA ) as above into the long-read polished assemblies .",
"As the MinION assemblies maintain some degree of sequencing error at repetitive regions that cannot be confidentially polished , we also assembled both types of reads into a hybrid assembly using SPAdes ( same options as above ) and , whenever different for short indels or SNPs but fully assembled , the sequence of the Spok genes was taken from the ( low-error ) hybrid assembly .",
"Assembly of the Spok block of the TG+ strain was particularly challenging because the recovered MinION reads were relatively short .",
"However , a few ( <10 ) reads were long enough to cover the tandem duplication that contains Spok3 ( albeit with high nucleotide error rate in the assembly ) .",
"The hybrid SPAdes assembly collapsed the duplication into a single copy .",
"We therefore mapped the short reads into the hybrid assembly , confirming that the Spok3 gene had doubled coverage and no SNPs , as expected for a perfect duplication .",
"Alignments of the assembled genomes were performed with the NUCmer program of the MUMmer package v . 4 . 0 . 0beta2 ( Kurtz et al . , 2004 ) using options –b 200 c 2000 –maxmatch , except when otherwise noted .",
"The figures showing alignments of chromosome 5 , the Spok block , and the Spok2 region ( Figure 3 , Figure 5 , and Figure 5—figure supplements 1 and 2 ) were generated by extracting the regions from each de novo assembly and aligning them in a pairwise fashion .",
"The NUCmer output was then visualized using a custom Python script .",
"The distribution of GC was plotted along the chromosomes with a custom Python script using 4-kb windows and steps of 2 kb .",
"In order to evaluate synteny across species , we aligned the chromosomal scaffolds of Wa58– ( P . anserina , Psk-7 ) , CBS237 . 71- ( P . pauciseta ) , and the reference genome of P . comata ( PODCO , Silar et al . , 2019 ) using NUCmer with –b 2000 in an all-vs-all fashion .",
"Given that the largest TE reported for P . anserina is around 12 kb ( Espagne et al . , 2008 ) , we filtered out alignments smaller than 13 kb and plotted the remaining alignments using Circos ( Krzywinski et al . , 2009 ) .",
"We further excluded alignments of missing data ( Ns ) tracks in chromosome 6 and 7 from PODCO .",
"See https://github . com/johannessonlab/SpokPaper ( Ament-Velásquez , 2019; copy archived at https://github . com/elifesciences-publications/SpokPaper ) for a Snakemake pipeline and Circos configuration files .",
"Notice that the Circos plot includes intrachromosomal alignments; for example , the rDNA operon in chromosome 3 is especially noticeable for Wa58– .",
"To evaluate the large-scale translocations in P . comata ( Figure 1B and Figure 1—figure supplement 1 ) , we mapped the short-reads of TD+ to PODCO and Podan2 , inferring mis-assemblies on the basis of the concordance of paired-end reads .",
"Translocation 1 is clearly a misassembly .",
"The translocation 2 in chromosome 4 however is complex because the corresponding boundaries in Podan2 start at a cluster of TEs at the 5′ end , and finish at the centromere on the 3′ end .",
"Indeed , Silar et al . ( 2019 ) could not verify this translocation using PCR .",
"Accordingly , the mapping of the paired-end reads does not support the translocation to the end of chromosome 4 in PODCO .",
"For annotation , we opted for gene prediction trained specifically on P . anserina genome features .",
"We used the ab initio gene prediction programs GeneMark-ES v . 4 . 32 ( Lomsadze et al . , 2005; Ter-Hovhannisyan et al . , 2008 ) and SNAP release 2013-06-16 ( Korf , 2004 ) .",
"All of the training process was performed on the sample Wa28– , for which all chromosomes were assembled ( see 'Results' ) .",
"The program GeneMark-ES was self-trained with the script gmes_petap . pl and the options –fungal –max_intron 3000 min_gene_prediction 120 .",
"SNAP was trained as instructed in the tutorial of the MAKER pipeline v . 2 . 31 . 8 ( Holt and Yandell , 2011; Campbell et al . , 2014 , and in the SNAP README file ) .",
"First , we use the Podan2 transcripts and protein models as sole evidence to infer genes with MAKER ( option est2genome = 1 ) and then we had a first round of SNAP training .",
"The resulting HMM file was used to re-run MAKER ( est2genome = 0 ) and to re-train SNAP , obtaining the final HMM training files .",
"A library of repetitive elements was constructed by collecting the reference P . anserina TEs described in Espagne et al . ( 2008 ) available in Genbank , and combining them with the fungal portion of Repbase version 20170127 ( Bao et al . , 2015 ) and the Neurospora library of Gioti et al . ( 2013 ) .",
"In order to produce transcript models , we used STAR v . 2 . 6 . 1b ( Dobin et al . , 2013 ) with maximum intron length of 1000 bp to map the RNAseq reads of all samples , followed by processing with Cufflinks v . 2 . 2 . 1 ( Trapnell et al . , 2010 ) .",
"For the final genome annotation , we used MAKER v . 3 . 01 . 02 along with GeneMark-ES v . 4 . 33 , SNAP release 2013-11-29 , RepeatMasker v . 4 . 0 . 7 ( http://www . repeatmasker . org/ ) , BLAST suit 2 . 6 . 0+ ( Camacho et al . , 2009 ) , Exonerate v . 2 . 2 . 0 ( Slater and Birney , 2005 ) , and tRNAscan-SE v . 1 . 3 . 1 ( Lowe and Eddy , 1997 ) .",
"After preliminary testing , we chose the transcripts of Psk7xS14 ( mapped to the PacBio assembly of Wa58– ) and Wa63– ( PacBio assembly of the same strain ) as expressed sequence tag ( EST ) evidence , and the Podan2 and TD ( Silar et al . , 2019 ) models as protein evidence .",
"The MAKER models of relevant regions were manually curated by comparing with RNAseq mapping and coding sequences ( CDS ) produced with TransDecoder v . 5 . 5 . 0 ( Haas et al . , 2013 ) on the Cufflinks models .",
"We used the blastn program to localize possible copies of Spok genes in all genome assemblies .",
"The Spok2 ( Pa_5_10 ) gene from Grognet et al . ( 2014 ) was selected as the query .",
"We named the new Spok genes ( Spok3 and Spok4 ) arbitrarily on the basis of sequence similarity , as reflected in the phylogenetic analyses ( see below ) .",
"Note that the existence of Spok3 had previously been hypothesized by Grognet et al . ( 2014 ) , but no DNA sequence was provided .",
"Moreover , the strain Y , in which they identified Spok3 , contains both Spok3 and Spok4 .",
"Backcrossed strains of the various spore-killer phenotypes were generated through five recurrent backcrosses to the reference strain S ( S5 ) by van der Gaag et al . ( 2000 ) .",
"In the original study , the strains selected as spore-killer parents were Wa53+ for Psk-1 , Wa28– for Psk-2 , Y+ for Psk-5 , and Wa58– for Psk-7 .",
"The S5 strains are annotated as Wa170 ( Psk-1 ) , Wa130 ( Psk-2 ) , Wa200 ( Psk-5 ) , and Wa180 ( Psk-7 ) in the Wageningen collection , but for the sake of clarity , we refer to them as Psk1xS5 , Psk2xS5 , Psk5xS5 , and Psk7xS5 .",
"We sequenced the S5 strains along with the reported parental strains using Illumina HiSeq 2500 .",
"We mapped the reads to Podan2 as described above , and then performed SNP calling using the HaplotypeCaller pipeline of GATK ( options: –ploidy 1 –newQual –stand_call_conf 20 . 0 ) .",
"We removed sites that had missing data , that overlapped with repeated elements as defined by RepeatMasker , or in which all samples were different from the reference genome , using VCFtools v . 0 . 1 . 16 ( Danecek et al . , 2011 ) , BEDtools v . 2 . 27 . 1 ( Quinlan and Hall , 2010 ) , and BCFtools v . 1 . 9 ( Danecek and McCarthy , 2017 ) , respectively .",
"We plotted the density of filtered SNPs across the genome with the R packages vcfR ( Knaus and Grünwald , 2017; Kamvar et al . , 2015 ) and poppr ( Knaus and Grünwald , 2017; Kamvar et al . , 2015 ) .",
"A full Snakemake ( Johannes and Rahmann , 2018 ) pipeline can be found at https://github . com/johannessonlab/SpokPaper .",
"Notice that we sequenced both monokaryons of our strain S to account for the mutations that might have had occurred since the separation of the reference S strain in the laboratory of Espagne et al . ( 2008 ) and our S strain from the Wageningen collection .",
"These mutations should be present in the backcrosses , but they are independent from the spore=killer elements .",
"Inspection of the introgressed tracks revealed that the variants of the backcross Psk1xS5 do not match Wa53+ ( the reported parent ) perfectly .",
"Given that the Spok content is the same as that in Wa53+ , the introgressed track co-occurs with the expected position of the Spok block on chromosome 3 , and the fact that the phenotype of this backcross matches a Psk-1 spore-killer type , we concluded that Wa170 ( Psk1xS5 ) in the collection actually belongs to another of the Psk-1 backcrosses described in the doctoral thesis of van der Gaag ( 2005 ) , probably backcrossed from Wa52 .",
"Puzzlingly , an introgressed track in the chromosome 3 of the Psk2xS5 strain does not match the expected parent ( Wa28 ) either , both in SNPs and het-gene alleles Figure 4—figure supplement 3 ) .",
"However , other tracks in different chromosomes , including that of chromosome 5 where the Psk-2 Spok block can be found , do match Wa28 .",
"Likewise , like Wa28 , Psk2xS5 only has Spok2 and Spok3 copies .",
"Hence , we concluded that our results are not affected by these inconsistencies .",
"As reported by van der Gaag et al . ( 2000 ) , the S5 strains were generated by selecting ascospores from two-spored asci of crosses between S and the spore killer parent .",
"This procedure ensures that the offspring will be homozygous for alleles of the spore-killer parent from the spore-killing locus to the centromere ( Box 1—figure 1 and Figure 4—figure supplement 3 ) .",
"To eliminate as much background as possible from the spore killer parents in the backcrossed strains , nine additional backcrosses were conducted where ascospores were selected from four-spored and two-spored asci in alternating generations .",
"Ascospores from the final generation were selected from two-spored asci to ensure that the strains would be homozygous at the spore-killing locus .",
"These strains are the result of 14 backcrosses to S ( S14 ) and are annotated as Psk1xS14 , Psk2xS14 , Psk5xS14 , and Psk7xS14 .",
"The S5 and S14 strains were phenotyped by crossing the strains to their parents as well as other reference spore-killer strains to confirm that the killing phenotypes remained unchanged after the backcrosses .",
"To knock-out Spok2 , a 459-bp and a 495-bp fragment flanking the Spok2 ORF downstream and upstream were obtained by PCR .",
"These fragments were then cloned flanking the hph gene in the SKhph plasmid as blunt end fragments in a EcoRV site and a SmaI site .",
"The deletion cassette was then amplified by PCR and used to transform a ΔKu70 strain ( El-Khoury et al . , 2008 ) .",
"Five transformants were screened for integration of the hph marker at Spok2 by PCR and crossed to s .",
"To purify the ΔSpok2 nuclei , a heterokaryotic binucleated ΔSpok2 spore was recovered from a two-spored ascus and used to fertilize the initial ΔSpok2 transformant ( which may or may not be heterokaryotic ) .",
"Uninucleated hygR-resistant spores were then recovered from this cross .",
"To replace the ORF of the centromere-linked Pa_2_510 ( PaPKS1 ) gene by one of the Spok3 or Spok4 genes ( see 'Results' ) , a disruption cassette was constructed as follows .",
"A DNA fragment corresponding to the 700-bp upstream region of the PaPKS1 ORF was amplified with oligonucleotides UpPks1_F and UpPks1_R .",
"This fragment was then cloned into a SKpBluescript vector ( Stratagene ) containing the nourseothricin-resistance gene Nat in the EcoRV site ( vector named P1 ) using the SacII/NotI restriction enzymes ( upstream from the Nat gene ) to produce the P1UpstreamPKS1 vector .",
"Then , the 770 bp downstream of the PaPKS1 ORF was amplified with oligonucleotides DownPks1_F and DownPks1_R .",
"This second fragment was cloned into the P1UpstreamPKS1 vector using the HindIII/SalI restriction enzymes ( downstream from the Nat gene ) to produce the P1UpstreamDownstreamPKS1 vector .",
"Finally , Spok3 was amplified from the Wa28 strain with oligonucleotides UpSpok3 and 4_F and DownSpok3 , and Spok4 was amplified from the Wa87 strain with oligonucleotides UpSpok3 and 4_F and DownSpok4 .",
"These genes were then cloned into the P1UpstreamDownstreamPKS1 vector using the NotI/XbaI restriction enzymes ( between PaPKS1 upstream region and Nat gene ) to produce the P1UpstreamDownstreamPKS1_Spok3 or the P1UpstreamDownstreamPKS1_Spok4 vector , so that the Spok3or Spok4 and Nat genes are flanked by the upstream and downstream regions of PaPKS1 ORF , allowing PaPKS1 ORF replacement by homologous recombination .",
"The Spok3 and Spok4 amplified Spok genes contain the ORFs flanked by the 983-bp upstream the start codon and the 460-bp downstream the stop codon for Spok3 , and by the 984-bp upstream the start codon and the 393-bp downstream the stop codon for Spok4 , allowing the expression of the Spok genes using their native promoter and terminator regions .",
"The disruption cassettes were then amplified from the final vectors using the most distal oligonucleotides 3′insidePsk1_F and 3′insidePsk1_R and named PKS1::Spok3_nat-1 and PKS1::Spok4_nat-1 .",
"See Supplementary file 4 for primer sequences .",
"Point mutations in the Spok3 gene were obtained by site-directed mutagenesis using the Q5 high-fidelity polymerase ( New England Biolabs ) and verified by Sanger sequencing .",
"The P . anserina ΔSpok2 ( ΔPa_5_10 ) strain was obtained after disruption of the gene Pa_5_10 and replacement of its ORF with the hygromycin-resistance gene hph in a ΔKu70 strain .",
"This strain was used as recipient strain for the disruption cassettes .",
"We used 5 µl of the cassettes for transfection and Nourseothricin-resistant transformants were selected .",
"As expected , most of the transformants were unpigmented and corresponded to the insertion of Spok3 or Spok4 by replacement of PaPKS1 .",
"Gene replacement was verified by PCR .",
"Prediction of unstructured regions was performed in SPOK3 with PrDOS with a 2% false-positive setting ( Ishida and Kinoshita , 2007 ) .",
"Coiled-coil prediction was performed with LOGICOIL ( Vincent et al . , 2013 ) , CCHMM_PROF ( Bartoli et al . , 2009 ) and Multicoil2 ( Wolf et al . , 1997 ) .",
"Domain prediction was performed using Gremlin ( Balakrishnan et al . , 2011 ) and RaptorX contact predict ( Ma et al . , 2015 ) .",
"Conserved residues were identified using Weblogo 3 ( Crooks et al . , 2004 ) with a Gremlin-generated alignment as input .",
"Domain identification was done with HHPred ( Zimmermann et al . , 2018 ) .",
"In order to compare the diversity at the nucleotide level with the protein models , we calculated the average pairwise nucleotide differences ( Nei and Li , 1979 ) for each bi-allelic site ( correcting by the number of sites ( n/ ( n –1 ) ) while ignoring sites with gaps ) on a Spok alignment ( see below ) , using overlapping windows of 100 bp and steps of 20 bp .",
"This procedure was performed on a selected representative of each Spok homolog ( Spok2 of S , Spok3 and Spok4 of Wa87 , and Spok1 from TD ) , or for all the alleles of each Spok within the P . anserina strains .",
"Values of dN/ds were calculated using the seqinr package in R ( Charif and Lobry , 2007 ) .",
"In order to infer the relationship between the three Podospora taxa investigated herein , we used OrthoFinder v . 2 . 2 . 6 ( Emms and Kelly , 2015; Emms and Kelly , 2019 ) to define orthogroups across all samples with long-read data and the reference genomes of P . anserina ( Espagne et al . , 2008 ) and P . comata ( Silar et al . , 2019 ) .",
"We randomly selected 1000 orthogroups of single-copy orthologs .",
"As OrthoFinder works with protein sequence , we used the Podan2 ortholog to do BLAST searches against each genome and extracted the corresponding best hit as nucleotide sequence ( including introns ) .",
"We then aligned each orthogroup using MAFFT v . 7 . 407 ( Katoh et al . , 2017 ) with options ––maxiterate 1000 –retree 1 –localpair .",
"We concatenated the 1000 alignments ( 1 , 652 , 987 sites , from which 27 , 000 were variable ) and used the resulting matrix as input for SplitsTree4 v . 4 . 14 . 16 , build 26 Sep 2017 ( Huson and Bryant , 2006 ) to construct an unrooted split network with a NeighborNet ( Bryant and Moulton , 2002 ) distance transformation ( uncorrected distances ) and an EqualAngle splits transformation .",
"To estimate the genetic ( gene ) distance between species , we averaged the identity across all P . anserina samples vs P . pauciseta ( CBS237 . 71 ) and vs . P . comata ( Silar et al . , 2019 ) , using the values produced in the distance matrix of SplitsTree .",
"The final gene models of all the Spok genes in Podospora spp .",
"were aligned along with the sequences of Spok2 and Spok1 from Grognet et al . ( 2014 ) using MAFFT online version 7 ( Katoh et al . , 2017 ) with default settings ( only one copy of Spok3 from TG was used ) .",
"The resulting alignment was manually corrected taking into account the reading frame of the protein .",
"As the UTRs seem to be conserved between paralogs , 654 ( 5' end ) and 250 ( 3' end ) bp of the flanking regions with respect to Spok2 were also included in the alignment .",
"An unrooted split network was constructed in SplitsTree4 as above .",
"SplitsTree4 was used likewise to perform a Phi test for recombination ( Bruen et al . , 2006 ) , using a windows size of 100 and k = 6 .",
"In addition , we used the BlackBox of RAxML-NG v . 0 . 6 . 0 ( Kozlov et al . , 2019 ) to infer maximum likelihood phylogenetic trees of the nucleotide alignment of the 5' UTR , the coding sequence ( CDS ) , and the 3' UTR of the Spok homologs .",
"We ran RAxML-NG with 10 parsimony and 10 random starting trees , a GTR +GAMMA ( four categories ) substitution model , and 100 bootstrap pseudo-replicates for each analysis .",
"In order to create a phylogeny of proteins that are closely related to the products of the Spok genes in Podospora ( and hence likely to be meiotic drivers ) , the protein sequence of Spok1 was used as a query against the NCBI genome database ( as of 14 November 2018 ) .",
"We collated all hits with e-values lower than Fs_82228 , which has been shown previously to have some spore-killing functionality in P . anserina ( Grognet et al . , 2014 ) , with hit coverage greater than 75% and no missing data ( Ns ) in the sequence .",
"The sequences were aligned using the codon-aware program MACSE v . 2 . 03 ( Ranwez et al . , 2018 ) , with the representative Podospora Spok genes set as ‘reliable’ sequences ( –seq ) , and the rest as ‘non reliable’ ( –seq_lr ) .",
"Many of the original gene models predict introns in the sequences , but no divergent regions were apparent in the alignment and , even if present , MACSE tends to introduce compensatory frame shifts .",
"The entire gene alignment was used for the analysis .",
"The resulting nucleotide alignment was corrected manually , translated into amino acids , and trimmed with TrimAl v . 1 . 4 . 1 ( Capella-Gutiérrez et al . , 2009 ) using the gappyout function .",
"A maximum likelihood tree was then produced using IQ-TREE v . 1 . 6 . 8 ( Kalyaanamoorthy et al . , 2017; Nguyen et al . , 2014 ) with extended model selection ( –m MFP ) and 1000 standard bootstrap pseudo-replicates .",
"The protein sequence Uv_5543 of Ustilaginoidea virens was selected as an outgroup on the basis of a BioNJ tree made with SeaView v . 4 . 5 . 4 ( Gouy et al . , 2010 ) of the Gremlin alignment described above .",
"For comparison , we performed a phylogenomic analysis of all of the strains that had at least one BLAST hit for the Podospora Spok homologs , as defined above .",
"Briefly , we recovered all the protein sequences for each genome from GenBank and ran OrthoFinder to recover orthogroups .",
"We obtained 288 single-copy orthogroups that were then processed with PREQUAL v . 1 . 02 ( Whelan et al . , 2018 ) and aligned with MAFFT .",
"Columns with more than 50% missing data were removed with TrimAl ( –gt 0 . 5 ) and all alignments were concatenated .",
"The supermatrix was analyzed with IQTree as above but with 100 standard bootstraps .",
"In order to confirm that Spok2 is responsible of the killing relationship between Psk-5 and Psk-1 , we conducted a cross between the strains Wa87 and Y . When perithecia started shooting spores , we replaced the lid of the cross plate with a water-agar plate upside-down , and let it sit for around an hour .",
"As the P . anserina spores from a single ascus typically land together , it is possible to distinguish spores that came from an ascus with no killing ( groups of four spores ) from those that survived killing ( groups of two spores ) .",
"To improve germination rates , we scooped spore groups of the same ascus type and deposited them together in a single plate of germination medium .",
"After colonies became visible , they were transferred into a PASM2 plate with a cellophane layer where they grew until DNA extraction , which was followed by pool-sequencing with Illumina HiSeq X . In total , 21 two-spore groups , and 63 four-spore groups were recovered .",
"The resulting short reads were quality controlled and mapped to Podan2 as above .",
"We used GATK to call variants from the parental strains ( treated as haploid ) and the two pool-sequencing databases ( as diploids ) .",
"We then extracted SNPs , removed sites with missing data , and attempted to quantify the coverage frequency of the parental genotypes for each variant .",
"The expectation was that spore killing ( two-spore asci ) would result in a long track of homozygosity ( only one parental genotype ) around Spok2 , as compared to the fully heterozygous four-spore asci .",
"A full Snakemake pipeline is available at https://github . com/johannessonlab/SpokPaper ."
]
] | [
"Meiotic drive is the preferential transmission of a particular allele during sexual reproduction .",
"The phenomenon is observed as spore killing in multiple fungi .",
"In natural populations of Podospora anserina , seven spore killer types ( Psks ) have been identified through classical genetic analyses .",
"Here we show that the Spok gene family underlies the Psks .",
"The combination of Spok genes at different chromosomal locations defines the spore killer types and creates a killing hierarchy within a population .",
"We identify two novel Spok homologs located within a large ( 74–167 kbp ) region ( the Spok block ) that resides in different chromosomal locations in different strains .",
"We confirm that the SPOK protein performs both killing and resistance functions and show that these activities are dependent on distinct domains , a predicted nuclease and kinase domain .",
"Genomic and phylogenetic analyses across ascomycetes suggest that the Spok genes disperse through cross-species transfer , and evolve by duplication and diversification within lineages ."
] | [
"In many organisms , most cells carry two versions of a given gene , one coming from the mother and the other from the father .",
"An exception is sexual cells such as eggs , sperm , pollen or spores , which should only contain one variant of a gene .",
"During their formation , these cells usually have an equal chance of inheriting one of the two gene versions .",
"However , a certain class of gene variants called meiotic drivers can cheat this process and end up in more than half of the sexual cells; often , the cells that contain the drivers can kill sibling cells that do not carry these variants .",
"This results in the selfish genetic elements spreading through populations at a higher rate , sometimes with severe consequences such as shifting the ratio of males to females .",
"Meiotic drivers have been discovered in a wide range of organisms , from corn to mice to fruit flies and bread mold .",
"They also exist in the fungus Podospora anserina , where they are called ‘spore killers’ .",
"Fungi are often used to study complex genetic processes , yet the identity and mode of action of spore killers in P . anserina were still unknown .",
"Vogan , Ament-Velásquez et al . used a combination of genetic methods to identify three genes from the Spok family which are responsible for certain spores being able to kill their siblings .",
"Two of these were previously unknown , and they could be found in different locations throughout the genome as part of a larger genetic region .",
"Depending on the combination of Spok genes it carries , a spore can kill or be protected against other spores that contain different permutations of the genes .",
"Copies of these genes were also shown to be present in other fungi , including species that are a threat to crops .",
"Scientists have already started to create synthetic meiotic drivers to manipulate how certain traits are inherited within a population .",
"This could be useful to control or eradicate pests and insects that transmit dangerous diseases .",
"The results by Vogan , Ament-Velásquez et al . shine a light on the complex ways that natural meiotic drivers work , including how they can be shared between species; this knowledge could inform how to safely deploy synthetic drivers in the wild ."
] | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"microbiology and infectious disease",
"immunology and inflammation"
] | High-resolution mapping of the neutralizing and binding specificities of polyclonal sera post-HIV Env trimer vaccination | elife-64281-v2 | [
[
"Mapping polyclonal antibody responses is central to understanding antigen-specific humoral immunity .",
"However , it is difficult to disentangle the multiple epitope specificities within polyclonal responses .",
"Often , serum neutralization assays or ELISAs with variant antigens are used to crudely map epitope specificities .",
"But these and other traditional serological mapping approaches do not provide high-resolution , residue-level information for the multiple components of polyclonal serum responses .",
"Cloning and characterizing many individual monoclonal antibodies ( Scheid et al . , 2009 ) have revolutionized our understanding of serum responses , but antibody cloning can be biased by the isolation strategy , is not proportional to antibody serum abundance or potency , and fails to characterize the entirety of the serum neutralization response .",
"Further advances in understanding polyclonal sera have been made through techniques that rely on high-throughput B-cell receptor sequencing ( Kreer et al . , 2020 ) , mass spectrometry-based approaches to directly sequence antibody proteins ( Lavinder et al . , 2014; Wine et al . , 2013 ) , or decomposing bulk serum-level measurements ( Ackerman et al . , 2017; Chung et al . , 2015; Georgiev et al . , 2013 ) .",
"Only recently have techniques been developed that directly measure the antibody specificity in polyclonal sera .",
"The first of these techniques , electron microscopy polyclonal epitope mapping ( EMPEM ) , directly images serum Fabs bound to an antigen of interest ( Barnes et al . , 2020; Bianchi et al . , 2018; Boyoglu-Barnum et al . , 2020 ) .",
"However , this approach characterizes the binding response , whereas it is the neutralizing antibody response that is most directly correlated with vaccine protection .",
"Here we combine EMPEM with a second complementary technique , mutational antigenic profiling ( Dingens et al . , 2017 ) , that quantifies the effect of all single amino-acid mutations to a viral entry protein on escape from serum neutralization .",
"For the purpose of this study , we mapped polyclonal anti-HIV antibody responses elicited with stabilized recombinant SOSIP Env trimers .",
"These trimers have been used extensively as immunogens because they recapitulate the native or near-native structure of Env on the virus surface ( Julien et al . , 2013; Lyumkis et al . , 2013; Pancera et al . , 2014; Sanders et al . , 2013; Sanders and Moore , 2017 ) .",
"In general , immunizing animals with prototypical SOSIP trimer variants based on the BG505 strain ( Wu et al . , 2006 ) induces autologous , tier-2 neutralizing antibody responses ( de Taeye et al . , 2015; Klasse et al . , 2016; Sanders et al . , 2015; Torrents de la Peña et al . , 2018 , Torrents de la Peña et al . , 2017 ) .",
"While such immunizations can protect against infection of simian–human immunodeficiency virus , bearing the matched BG505 Env in macaques ( Pauthner et al . , 2017; Pauthner et al . , 2019 ) , heterologous breadth has not been consistently achieved .",
"This lack of breadth highlights the need to understand the targets of vaccine-elicited neutralizing antibodies and re-focus responses to more broadly conserved epitopes .",
"Prior mapping of SOSIP trimer-induced antibody responses in animal models has revealed viral strain- and species-specific hierarchical responses , with BG505 trimer immunogenicity in rabbits serving as a well-characterized model system .",
"BG505-induced rabbit neutralizing antibodies predominantly target a BG505-specific glycan hole ( GH ) in Env’s glycan shield , centered on the broadly conserved glycosylation sites at residues 241 and 289 that are missing in BG505 .",
"Reintroducing glycans back in at these sites eliminates much of the neutralizing activity in many rabbit serum responses ( Klasse et al . , 2018; Klasse et al . , 2016 ) and mAbs isolated from immunized rabbits target this immunodominant GH ( McCoy et al . , 2016 ) .",
"Serum neutralization assays with large panels of pseudovirus point mutants identified a second frequently immunogenic site in rabbits as the C3/V5 epitope ( previously termed C3/465 ) , as well as a less immunodominant and less commonly targeted epitope in V1 ( Klasse et al . , 2018 ) .",
"These rabbit immunogenicity data were largely corroborated using EMPEM to directly visualize serum Fabs bound to BG505 trimer bait ( Bianchi et al . , 2018 ) .",
"In SOSIP trimer-vaccinated guinea pigs and non-human primates , antibody cloning or EMPEM has identified additional strain-specific responses to the C3/V4 , C3/V5 , V1 , and gp120/gp41 interface regions ( Cottrell et al . , 2020; Lei et al . , 2019; Nogal et al . , 2020; Nogal et al . , 2019 ) .",
"Additionally , trimer immunization often elicits non-neutralizing responses to the base of the trimer , a neo-epitope exposed on soluble trimers but inaccessible on viral membrane-bound Env ( Bianchi et al . , 2018; Cottrell et al . , 2020; Hu et al . , 2015; Kulp et al . , 2017 ) .",
"Ongoing clinical trials will evaluate the immunogenicity of BG505 SOSIP trimer variants in humans ( ClinicalTrials . gov Identifiers: NCT03699241 , NCT04177355 , and NCT03783130 ) .",
"Below , we use mutational antigenic profiling to directly map the dominant neutralizing antibody specificities present within a panel of polyclonal sera from rabbits immunized the BG505 SOSIP trimer variants .",
"In parallel , we map the binding specificities of these sera using EMPEM , providing a holistic view of both serum binding and neutralization ."
],
[
"We chose a small panel of rabbit sera to optimize mutational antigenic profiling of polyclonal sera .",
"We used sera from rabbits sequentially vaccinated with BG505 SOSIP trimer variants: either BG505 SOSIP . 664 , which contains the T332N mutation ( Sanders et al . , 2013 ) , or the further stabilized BG505 SOSIP . V4 . 1 ( de Taeye et al . , 2015 ) , each administered either three or four times .",
"Details of the immunization schemes and characterization of some of these rabbits’ sera responses at earlier time points have been reported previously ( Klasse et al . , 2018; Klasse et al . , 2016; Ringe et al . , 2019; Ringe et al . , 2017 ) .",
"We chose serum samples with various specificities , including sera that predominantly target the 241/289 GH or C3/V5 epitopes alone , both of these epitopes , or neither of these epitopes .",
"To identify such sera , we performed preliminary TZM-bl neutralization assay mapping using pseudoviruses bearing mutations that affect each of these epitopes , as well as the V1 epitope rarely targeted in rabbits .",
"The resulting sera panel and associated preliminary mapping data are shown in Figure 1A .",
"These sera do not represent an unbiased collection of rabbit immune responses , but rather a curated selection of potent responses with different specificities .",
"We performed mutational antigenic profiling ( Dingens et al . , 2017 ) of each serum using libraries of replication-competent HIV virions expressing all mutants of the BG505 . T332N Env ( Haddox et al . , 2018 ) , allowing us to map autologous responses to the strain-matched BG505 trimer immunogen .",
"Briefly , this approach ( Figure 1B ) first involves generating libraries of mutant viruses containing all single amino-acid mutations to Env compatible with viral replication .",
"Each mutant virus library is then incubated with a highly selective concentration of sera before infecting a T-cell line such that only viruses that escape neutralization can enter cells .",
"In our experiments , we chose serum concentrations to keep the average level of selection exerted by each serum relatively constant; across all replicates , between 0 . 02% and 9 . 27% of the library escaped neutralization , with the across-replicate averages for each serum ranging from 0 . 3% and 2 . 7% ( Figure 1—figure supplement 1 ) .",
"The frequency of each mutation among viruses that are able to escape neutralization is quantified by Illumina sequencing of the viral cDNA produced in infected cells .",
"Comparing the relative frequency of each mutation in the sera-selected condition to a non-selected control condition quantifies the effect of each mutation on resistance to sera neutralization ( Figure 1B ) .",
"As an additional control , we also incubated viral libraries with pre-vaccine sera for each rabbit .",
"Figure 1—figure supplement 1 details the serum dilutions , the number of replicates ( three to six per post-immunization sera; median values are reported throughout ) , and the level of neutralization achieved by each serum in each experiment .",
"Median mutation differential selection values across all experimental replicates for a given serum are presented throughout ( see Figure 1—figure supplement 1 for replicate-to-replicate correlations ) .",
"The Env mutations that affect neutralization by each serum are plotted in Figure 2 .",
"The results are largely concordant with prior knowledge on BG505 trimer immunogenicity in rabbits , with each serum targeting one or both of the C3/V5 or GH epitopes , which are indicated by blue and green , respectively , in Figure 2 .",
"Additionally , the antigenic profiling largely agrees with the crude epitope specificities mapped using a small panel of pseudovirus point mutants ( Figure 1A , with mutations tested in preliminary TZM-bl assays plotted in black in Figure 2B ) .",
"Notably , most sera select neutralization-escape mutations in the C3/V5 epitope to some extent , including sera from rabbit 2124 , which appeared to predominantly target the GH based on the preliminary point-mutant mapping ( Figure 1A ) .",
"Figure 2 is just one approach to visualizing these complex datasets; to facilitate more flexible data exploration , antigenic profiling data for each sera can be interactively explored in dms-view ( Hilton et al . , 2020 ) by visiting https://jbloomlab . github . io/Vacc_Rabbit_Sera_MAP/ .",
"This interactive visualization reduces potential interpretation and presentation biases by allowing users to examine the extent of neutralization-escape by mutations at any site and projecting the mutations onto interactive structures of the Env trimer or monomer .",
"We hypothesized that sera with a few dominant sites of escape ( e . g . , serum 5724 in Figure 2 ) had neutralizing activity that was strongly focused on one epitope of Env , whereas sera with smaller-effect escape mutations to multiple regions ( e . g . , serum 2214 in Figure 2 ) had neutralizing responses that targeted multiple distinct epitopes .",
"To explore this hypothesis , we tested if the small effect sizes observed in mutational antigenic profiling for some sera accurately reflect the effect of mutations in TZM-bl neutralization assays .",
"We identified the most selected mutation at the most selected site for each serum , generated pseudoviruses bearing these mutations , and tested them in serum neutralization assays .",
"The fold enrichment in mutational antigenic profiling was well correlated with the fold change in ID50 in the neutralization assays ( Figure 2—figure supplement 7 , Pearson’s r = 0 . 97 , p value = 0 . 0011 from a two-tailed Pearson correlation test ) .",
"For example , the 5724 escape profile is focused entirely on the C3/V5 epitope: T464H is enriched ~190-fold in this serum’s mutational antigenic profiling and shifts the ID50 64-fold in a TZM-bl neutralization assay ( Figure 2—figure supplement 7 ) .",
"In contrast , 2423 targets both the C3/V5 and GH epitopes ( Figure 2 ) , and the maximal effect mutant N356K has just a ≈2-fold effect in both mutational antigenic profiling and neutralization assays ( Figure 2—figure supplement 7 ) .",
"A caveat is that the extent of mutant enrichment upon serum selection is also influenced by the serum dilution used in experiments , as shown in Figure 2—figure supplement 1–6 .",
"However , the good correlation of the extent of focusing in the escape-mutation mapping and the TZM-bl neutralization assays suggests the mutational antigenic profiling data captures the amount of focusing in the neutralization response reasonably well .",
"To contrast the serum neutralization specificities described above with the serum binding specificities , we performed EMPEM on the same set of sera to directly visualize antibody binding to Env ( Figure 1C ) .",
"We reasoned that collecting both types of data would enable us to compare and contrast the sites where antibodies bind to the sites where mutations mediate escape from neutralization .",
"Figure 3 presents the refined 3D reconstructions from negative stain electron microscopy of serum Fabs bound to immunogen-matched BG505 SOSIP trimer alongside mutational antigenic profiling data .",
"Across all sera , it is immediately clear that the binding responses identified by EMPEM include many epitopes where mutations do not affect viral neutralization .",
"For five of six sera , binding responses to both the GH and C3/V5 epitopes are observed .",
"All sera contain additional binding responses to other epitopes .",
"For example , even 5724 , where we observe narrow , strongly focused viral escape in just the C3/V5 epitope , contains numerous additional binding responses , including the GH , N611 glycan , base-of-trimer , and V1/V3 epitopes .",
"Some of the differences in binding vs neutralization-escape are easily explained by antigenicity differences between stabilized Env trimers and replication-competent virus .",
"First , while the base-of-trimer epitope is presented on recombinant trimers and commonly elicited by trimer immunization ( Bianchi et al . , 2018; Cottrell et al . , 2020; Hu et al . , 2015 ) , it is inaccessible in the replication-competent virus and hence does not elicit neutralizing antibodies .",
"Therefore , base-binding responses are observed in all sera , but of course are not mapped as neutralizing epitopes .",
"Second , binding responses to the N611 glycan region are also observed in all sera ( Figure 3 ) , but we do not observe viral escape by disrupting this glycosylation motif ( Figure 2 ) .",
"It has been previously shown that site N611 is less glycosylated in BG505 SOSIP trimers than in virus , which enhances the immunogenicity of the exposed region when this glycan is missing ( Derking et al . , 2020 ) .",
"Accordingly , EMPEM identified binding responses to this region using the immunogen-matched trimer bait , while neutralizing responses are not apparent in mutational antigenic profiling because the virus libraries are likely to have higher glycan occupancy at N611 .",
"Third , gp120 interface responses are observed in two sera; both of these rabbits were immunized with BG505 SOSIP . v4 trimers .",
"This gp120 interface epitope includes the A316W stabilizing mutation added to the SOSIP . v4 to reduce the exposure of the V3 loop ( de Taeye et al . , 2015 ) .",
"We have recently found that the A316W mutation alters immunogenicity to this region , eliciting mutation-specific responses to this region that would not cross-react with the A316-bearing viral libraries ( manuscript in preparation ) .",
"Together , these trimer-binding but non-neutralizing responses to the base , N611 glycan , and gp120 interface reflect and expand on previously characterized antigenic differences between Env trimers to Env on the surface of virus ( Bianchi et al . , 2018; Cottrell et al . , 2020; Derking et al . , 2020; Hu et al . , 2015 ) .",
"The few remaining differences in binding and neutralization could reflect a number of factors .",
"Some binding responses may be non-neutralizing , although it is often assumed that antibodies that bind native Env present on the virus – mimicked by stabilized Env trimers – are neutralizing ( Burton et al . , 2000; Yang et al . , 2006 ) .",
"Alternatively , at the serum concentrations we tested , the antibody occupancy on the virions or the binding kinetics may disfavor neutralization .",
"Furthermore , some observed binding responses may be neutralizing but at such subdominant levels that they do not exert selective pressure in the mutational antigenic profiling at the serum concentrations tested .",
"Alternatively , some epitopes may require saturation in order to be neutralizing and did not reach these saturating levels at the serum concentrations tested in mutational antigenic profiling .",
"To investigate these hypotheses , we tested each sera’s ability to neutralize pseudovirus point mutants bearing mutations to one or both of the C3/V5 and GH epitopes ( Figure 3—figure supplement 1 and summarized in Figure 3C ) .",
"Comparing the effect of single epitope mutations to multiple epitope mutations can help define the relative dominance of different neutralizing specificities ( Klasse et al . , 2018 ) .",
"A caveat is that neutralization assays for different sets of mutants were performed in two different laboratories; we therefore only present general interpretations in Figure 3C .",
"For both 5724 and 2425 , single or multiple glycan knock in mutations to the GH epitope had negligible effects , while single or multiple glycan knock in mutations to the C3/V5 epitope had large effects ( Figure 3—figure supplement 1 ) .",
"For these two sera , knocking in a glycan to each epitope ( S241N + I358T ) did not have much larger effects than the single C3 glycan knock in ( I358T ) , suggesting these sera have limited GH neutralizing responses even after eliminating some of the C3/V5 directed neutralizing response .",
"While this matches the mutational antigenic profiling , binding GH responses are observed in these sera ( Figure 3A ) .",
"This suggests that these GH responses are either non-neutralizing or much less dominant than the C3/V5 neutralizing antibody responses .",
"Of note , while the resolution of the negative stain EMPEM in this present study limits fine epitope interpretations , GH binding responses ( specifically ‘GH2’-like responses ) have previously been identified in non-neutralizing sera using cryo-EMPEM ( Bianchi et al . , 2018 ) .",
"When the effect of a mutation to one ( e . g . , epitope A ) is apparent when another epitope ( e . g . , epitope B ) is knocked out , but not when testing the epitope A mutation alone , we can interpret the neutralizing antibody response to epitope A as being ‘subdominant’ relative to the epitope B response .",
"Here , 5727 , 2124 , 2214 , and 2423 all displayed a greater effect for the double epitope glycan knock in mutations ( S241N + I358T ) than either of the single epitope knock in mutations .",
"Comparing the effects of the single and double epitope mutations suggests sera 2423 had relatively equivalent neutralizing responses to both the GH and C3/V5 epitopes , sera 2124 and 2214 had a dominant response to GH and a subdominant response to C3/V5 , and sera 5727 had a dominant response to C3/V5 and a subdominant response to GH ( Figure 3 , Figure 3—figure supplement 1 ) .",
"EMPEM identified binding responses to both epitopes in three of four of these sera , while mutational antigenic profiling identified neutralizing responses to both epitopes in only two of four sera ( Figure 3 ) .",
"Since there were a number of instances in which subdominant neutralizing responses to the GH or C3/V5 epitope identified with TZM-bl point-mutant mapping did not appear in either EMPEM or mutational antigenic profiling ( e . g . , GH response for 5727 is absent in mutational antigenic profiling , and a C3/V5 response for 2214 is absent in EMPEM ) , we examined if there were additional unobserved subdominant responses .",
"We focused on the effect of insertion mutations to V1 , an epitope occasionally targeted in rabbits ( Klasse et al . , 2018 ) .",
"We tested sera for neutralization of V1 and V1 + GH epitope mutants and compared effects to GH mutations alone .",
"While V1 insertions alone did not affect neutralization of any sera , they had an additional effect when tested with GH mutations relative to GH mutations alone ( Figure 3—figure supplement 1 ) .",
"This suggests that there are subdominant neutralizing antibody responses to V1 in all sera – though we cannot rule out that the V1 + GH double mutants broadly affect antigenicity .",
"Mutational antigenic profiling did not clearly identify any V1 responses , while EMPEM identified a V1/V3 binding response – which overlaps the V1 insertion mutations – in one serum ( 5724 , Figure 3 ) .",
"The mutational antigenic profiling data also allows for residue-level refinement of the dominant targets of the neutralizing antibody response .",
"For example , it is immediately apparent that the clustered , surface-exposed sites 464 and 356 and/or 358 ‘anchor’ the C3/V5 epitope , with many mutations at these sites having large effects for nearly all sera that strongly target this epitope ( Figure 4 ) .",
"However , detailed epitope specificity at other C3/V5 sites varies across rabbits: some sera are more focused on various regions of C3 , including sites 350 and 351 ( e . g . , sera 5727 , 2423 , 2124 ) or 354–357 ( e . g . , sera 5727 , 2423 , 2124 , 2425 ) , whereas serum 5724 is more narrowly focused on the V5 region previously identified by knocking in a glycan at site 465 .",
"Notably , site 396 bridges these two epitope regions in both linear sequence and structural space and is a site of escape for most sera .",
"We validated this residue-level specificity using TZM-bl neutralization assays ( Figure 4 ) .",
"For example , mutations to sites 350 , 351 , 355 , and 356 have very little effect on 5724 , while mutations to 358 and 464 have large effects .",
"Similarly , 2425 is most focused on residues 354–358 in the C3 region , mutations to these sites gave larger effects , while those to 350 and 251 did not .",
"While the magnitude of effect size varied across sera ( note the differing y-axis in Figure 4 ) , the TZM-bl mapping generally reflected these effect sizes .",
"It is also clear that the mutational antigenic profiling better explores the effects of different possible escape mutations than testing smaller panels of pseudovirus mutants .",
"For example , while knocking in a glycosylation site at site 465 ( T465N ) escapes many of the sera as previously shown ( Klasse et al . , 2018 ) , other mutants at this site have similar or even larger effects ( Figures 2 and 3 ) .",
"This suggests that the immune response is directed at site 465 and neighboring V5 residues , as opposed to targeting this general epitope region that is obstructed by adding a bulky glycan to site 465 .",
"There is greater variation in both the residue-level specificity and magnitude of neutralizing antibody responses to the GH epitope .",
"However , overlaying data onto the trimer structures make it clear that a subset of the sera target the 241/289 GH .",
"Examining data for three sera with clear enrichment in this region reveal selective pressure in a large epitope region , spanning from site 85 near the fusion peptide , through the 241/289 GH region , and extending to site 347 in/near the C3/V5 epitope .",
"While we arbitrarily classify site 347 as part of the GH epitope because enrichment at this site more closely tracks with sera that target the GH epitope ( Figure 2 ) , the blending of these epitopes supports the notion that the C3/V5 and GH epitopes together constitute a BG505-specific immunodominant ‘super-epitope’ in rabbits ( Nogal et al . , 2020 ) .",
"Mutations at site 629 , particularly L629P , are also enriched strongly in two sera that target the GH epitope ( Figure 5 ) .",
"Site 629 is near the N-terminus of the HR-2 , located below and buried beneath the GH epitope in the pre-fusion structure; the proline mutation may disrupt the HR-2 alpha helix , altering its antigenicity or accessibility of the GH epitope .",
"Mutations in the GH epitope were oftentimes only moderately enriched compared to the C3/V5 epitope , and TZM-bl assays also showed only small shifts in the neutralization curve for these mutants .",
"Sera without clear enrichment of escape mutations in this epitope also were not affected by epitope mutations in TZM-bl neutralization assays ( Figure 5—figure supplement 1 ) ."
],
[
"We have used mutational antigenic profiling to map the dominant neutralizing antibody specificities in polyclonal rabbit sera elicited with Env trimer immunization .",
"In parallel , we used EMPEM to map the total serum binding specificities .",
"Contrasting the serum binding and neutralizing specificities suggests that the dominant neutralizing antibody responses are only a subset of binding responses – even when just examining bona fide neutralizing antibody epitopes .",
"Additional differences in binding and neutralization highlight antigenicity differences between soluble SOSIP Env trimers and Env on the surface of viruses , which is relevant to the use of trimers as immunogens .",
"This work also shows the utility of mutational antigenic profiling in mapping polyclonal serum responses to HIV Env .",
"We recapitulate and extend prior knowledge on rabbit antibody responses to BG505 trimer immunization ( Bianchi et al . , 2018; Klasse et al . , 2018; McCoy et al . , 2016 ) , with the C3/V5 and GH regions being the dominant immunogenic epitopes .",
"We map responses to multiple epitopes of polyclonal serum responses at once , a significant advance over more traditional mapping approaches .",
"Furthermore , we refine the residue-level specificity of these epitopes directly from sera , revealing fine-grain epitope differences .",
"For example , some C3/V5 responses were more focused on the C3 region than others .",
"But there were sites that appeared to ‘anchor’ this epitope – all sera targeting this epitope were strongly affected by mutations to sites 358 and 454 .",
"It remains to be determined if these different epitope specificities are the result of monoclonal responses with varying binding footprints or if they vary due to differing sets of multiple overlapping antibodies targeting the same epitope region .",
"Lastly , while rabbit antibody responses to BG505 SOSIP trimer immunization have been well-characterized , these unbiased sera mapping approaches have the potential to identify novel specificities .",
"Indeed , some sera do display minor enrichment of possible escape mutations outside of known epitopes ( e . g . , sites 507 and 509 for sera 5727 ) .",
"The GH epitope is not a desirable broadly neutralizing epitope that can be exploited for vaccine design ( Yang et al . , 2020 ) , and the C3/V5 epitope is similarly problematic due to its relatively low sequence conservation .",
"While the autologous neutralizing responses we mapped here are encouraging , we hypothesize it will be important to silence these immunodominant and autologous neutralizing responses to aid in redirecting response to more broadly conserved epitopes .",
"This is particularly important given that similar specificities have also been observed in non-human primates after BG505 SOSIP trimer immunization ( Cottrell et al . , 2020; Nogal et al . , 2020 ) .",
"Our mutational antigenic profiling provides a rich map of potential alterations that disrupt these epitopes , which could be of use in resurfacing trimer immunogens .",
"While this work has allowed us to compare total polyclonal serum neutralization and binding , there are important limitations to keep in mind .",
"One key point is that these methods have different sensitivities for dominant versus subdominant responses , making it impossible to directly quantify serum neutralization and binding on the same scale .",
"Indeed , TZM-bl neutralization assays using single and double epitope mutant pseudoviruses suggest that both approaches may have ‘missed’ subdominant neutralizing antibody responses to the V1 epitopes ( Figure 3 , Figure 3—figure supplement 1 ) .",
"The mutant libraries used to map neutralization have on average only ~1 Env mutation per virus; a given virion can therefore likely only escape one component of polyclonal responses , limiting the ability to detect subdominant responses .",
"Furthermore , rigorous quantification of both binding and neutralizing responses – and comparisons of responses across sera – are not yet possible with existing technology and analytical approaches .",
"Furthermore , while mutational antigenic profiling examines the entire serum neutralizing antibody response , EMPEM maps the binding of IgG Fabs after purification .",
"Lastly , easily visualizing and interpreting these complex datasets remains difficult , a challenge we have attempted to overcome here with the interactive visualizations available at https://jbloomlab . github . io/Vacc_Rabbit_Sera_MAP/ .",
"Nonetheless , this work describes the most detailed mapping yet of the specificities of polyclonal sera at both the binding and neutralizing level .",
"While both rational , structure-based vaccine design ( Alam et al . , 2017; Correia et al . , 2014; Dubrovskaya et al . , 2019; Haynes et al . , 2012; Kong et al . , 2019; Kwong and Mascola , 2018; Saunders et al . , 2017; Xu et al . , 2018 ) and broadly neutralizing antibody germline-targeting ( Briney et al . , 2016; Dosenovic et al . , 2015; Escolano et al . , 2019; Escolano et al . , 2016; Jardine et al . , 2013; Jardine et al . , 2016; Medina-Ramírez et al . , 2017; Steichen et al . , 2019; Steichen et al . , 2016; Tian et al . , 2016 ) approaches have begun to show exciting promise for HIV , continued progress will require iterative rounds of evaluating vaccine responses and redesigning immunogens and vaccine regimens ( Ward and Wilson , 2020 ) .",
"Determining the extent of immunofocusing to targeted epitopes , while also tracking – and subsequently eliminating – off-target responses to less conserved regions will be critical to these efforts .",
"Together , EMPEM and mutational antigenic profiling can aid in rational vaccine design by directly mapping polyclonal serum specificities for both binding and neutralization ."
],
[
"Mutational antigenic profiling was performed as previously described ( Dingens et al . , 2017 ) .",
"Briefly , 5 × 105 infectious units of BG505 . T332N mutant virus libraries ( Haddox et al . , 2018 ) were neutralized with serum dilutions for 1 hr at 37°C as specified in Figure 1—figure supplement 1 .",
"We performed additional experimental replicates for sera with smaller effect sizes in order to increase our signal relative to noise ( Figure 1—figure supplement 1 ) .",
"All replicates ( Figure 1—figure supplement 1 ) were included in the analysis , without any removal or quality filtering of whole replicates or individual mutation counts .",
"Different library numbers ( e . g . , 1 , 2 , 3 ) are viral libraries generated from independently generated DNA libraries ( Haddox et al . , 2018 ) , and letter labels ( e . g . , a , b , c ) correspond to experiments done on different days with matched non-selected control library selections .",
"After neutralization , viral libraries were then infected into 1 × 106 SupT1 . CCR5 cells in R10 containing 100 μg/mL DEAE-dextran .",
"Three hours post-infection , the cells were resuspended in 1 mL R10 ( RPMI [GE Healthcare Life Sciences; SH30255 . 01] , supplemented with 10% fetal bovine serum , 1% 200 mM l-glutamine containing a 1% of a solution of 10 , 000 units/mL penicillin and 10 , 000 mg/mL streptomycin ) .",
"At 12 hr post-infection , cells were washed once with phosphate-buffered saline , and non-integrated viral cDNA was isolated from cells using a miniprep .",
"Each mutant virus library was also subjected to a mock selection ( no serum ) , and duplicate four 10-fold serial dilutions of each mutant virus library were also infected into 1 × 106 cells to serve as an infectivity standard curve ( Figure 1—figure supplement 1 ) .",
"The proportion of the library that survived neutralization and entered cells was quantified using a qPCR and interpolation of the infectivity standard curve ( Dingens et al . , 2019 ) .",
"Sequencing libraries were generated using a barcoded subamplicon sequencing approach as previously described ( Haddox et al . , 2018; Haddox et al . , 2016 ) and detailed at https://jbloomlab . github . io/dms_tools2/bcsubamp . html .",
"Libraries were sequenced using 2 × 250 bp paired-end Illumina HiSeq runs .",
"Data was analyzed with dms_tools2 version 2 . 2 . 6 ( https://jbloomlab . github . io/dms_tools2/ ) ( Bloom , 2015 ) .",
"Differential selection is the log2-transformed enrichment of a given mutation relative to wild type in the sera-selected condition relative to the non-selected control condition .",
"This statistic is corrected for sequencing error , determined by sequencing wild-type plasmid , in a site- and mutation-specific manner .",
"To reduce noise associated with low sequencing counts for a given mutations , a pseudocount of 5 ( scaled up for the deeper-sequenced sample by the relative sequencing depth of the selected and non-selected libraries , to avoid biases introduced by differing sequencing depths across samples ) is added to sequencing counts .",
"See prior work ( Dingens et al . , 2017; Doud et al . , 2017 ) or https://jbloomlab . github . io/dms_tools2/diffsel . html for additional details .",
"Short tandem repeat profiling on our stock of SupT1 . CCR5 cells found that 11 of 14 alleles plus both amelogenin alleles matched the reference of the parental SupT1 cells ( ATCC #CRL-1942 ) reference profile .",
"These cells also tested negative for mycoplasma .",
"IgG from rabbit sera was affinity-purified with equal parts of Protein G Sepharose ( Sigma–Aldrich , P3296 ) and 150 ml Protein A Sepharose ( GE Healthcare , 17-5138-01 ) in a Poly-Prep Chromatography Column ( Bio-Rad , 731–1550 ) .",
"Fabs were generated by Papain cleavage and purified by the use of the Pierce Fab Preparation Kit according to the manufacturers’ instructions ( Thermo Scientific - #44985 ) .",
"The purity of the Fabs was confirmed by SDS–PAGE .",
"BG505 SOSIP . 664 or BG505 SOSIP v4 . 1/Fab complexes were made by mixing 15 μg SOSIP with 1 mg of polyclonal Fabs and allowed to incubate for 18–24 hr at room temperature .",
"Complex samples were SEC purified using a Superose 6 Increase 10/300 GL ( GE Healthcare ) column to remove excess Fab prior to electron microscopy grid preparation .",
"Fractions containing the SOSIP/Fab complexes were pooled and concentrated using 10 kDa Amicon spin concentrators ( Millipore ) .",
"Samples were diluted to 0 . 02 mg/mL in TBS ( 0 . 05 M Tris pH 7 . 4 , 0 . 15 M NaCl ) and adsorbed onto glow discharged carbon-coated Cu400 EM grids ( Electron Microscopy Sciences ) and blotted after 10 s .",
"The grids were then stained with 3 μL of 2% ( wt/vol ) uranyl formate , immediately blotted , waiting for 10 s before being stained again for 35 s followed by a final blot .",
"Image collection and data processing were performed on TFS Talos F200C microscope ( 1 . 98 Å/pixel; 73 , 000× magnification ) with an electron dose of ∼25 electrons/Å2 using Leginon ( Pugach et al . , 2015; Suloway et al . , 2005 ) .",
"2D classification , 3D sorting , and 3D refinement were conducted using Relion v3 . 0 ( Zivanov et al . , 2018 ) .",
"EM density maps were visualized using UCSF Chimera ( Pettersen et al . , 2004 ) and segmented using Segger ( Pintilie et al . , 2010 ) .",
"Figures were generated using UCSF Chimera ( Pettersen et al . , 2004 ) .",
"TZM-bl neutralization assays used to preliminarily map the specificity of our sera panel ( Figure 1A ) were performed in laboratories at Weill Cornell as previously described ( Klasse et al . , 2018 ) .",
"The remainder of the TZM-bl neutralization assays were performed in laboratories at the Fred Hutch as previously described ( Dingens et al . , 2017 ) .",
"These protocols are very similar and based on widely used protocols such as TZM-bl neutralization assay protocols ( Sarzotti-Kelsoe et al . , 2014 ) .",
"Validation assays were completed in technical duplicate two to four times .",
"In a small number of instances , the top of the neutralization plateau did not fit for single replicates; while fold change in ID50 values were always calculated using at minimum two replicates ( with standard deviations [SD] between biological replicates reported ) , the maximum neutralization plateau with SD reported as ‘ ( * ) ’ were from single replicates in which the naturalization plateau fits accurately .",
"The entire mutational antigenic profiling analysis pipeline , as well as processed data are available as https://github . com/jbloomlab/Vacc_Rabbit_Sera_MAP; Dingens , 2021; copy archived at swh:1:rev:b8d312d2bf5c2c117ce1d1601ea3738b58e62c20 .",
"Illumina sequencing reads were uploaded to the NCBI SRA as BioProject PRJNA656582 with sample identifiers SRR12431153-SRR12431189 .",
"EMPEM 3D maps are deposited into the EMDB with codes EMD-23366 to EMD-23371 ."
]
] | [
"Mapping polyclonal serum responses is critical to rational vaccine design .",
"However , most high-resolution mapping approaches involve isolating and characterizing individual antibodies , which incompletely defines the polyclonal response .",
"Here we use two complementary approaches to directly map the specificities of the neutralizing and binding antibodies of polyclonal anti-HIV-1 sera from rabbits immunized with BG505 Env SOSIP trimers .",
"We used mutational antigenic profiling to determine how all mutations in Env affected viral neutralization and electron microscopy polyclonal epitope mapping ( EMPEM ) to directly visualize serum Fabs bound to Env trimers .",
"The dominant neutralizing specificities were generally only a subset of the more diverse binding specificities .",
"Additional differences between binding and neutralization reflected antigenicity differences between virus and soluble Env trimer .",
"Furthermore , we refined residue-level epitope specificity directly from sera , revealing subtle differences across sera .",
"Together , mutational antigenic profiling and EMPEM yield a holistic view of the binding and neutralizing specificity of polyclonal sera ."
] | [
"Vaccines work by stimulating the immune system to produce proteins called antibodies .",
"These antibodies bind to the virus targeted by the vaccine and block the virus from infecting cells .",
"It has been difficult to develop a vaccine for HIV because frequent mutations allow it to evade antibodies .",
"Understanding exactly how these proteins bind to HIV and how various mutations enable the virus to escape them is crucial to designing a successful HIV vaccine .",
"Over the last decade , scientists have developed new techniques for studying individual antibodies and how they bind to viruses .",
"Now , they are using these insights to design vaccines .",
"Most vaccines result in the production of many antibodies that bind to different parts of the virus , making it harder for a virus to escape .",
"But studying many antibodies with different targets on the virus simultaneously remains challenging .",
"By combining two-cutting edge approaches , Dingens et al . catalogued the many antibodies that rabbits produce in response to an experimental vaccine for HIV .",
"In the experiments , they mapped how two types of rabbit antibodies target the virus: those that could bind to the virus , and those that could both bind and neutralize the virus ( i . e . , block it from infecting cells ) .",
"The experiments showed that small differences between the HIV virus and the vaccine explained why some rabbit antibodies created in response to the vaccine could bind but not neutralize the virus .",
"Moreover , the ability to stop HIV from infecting the cells appeared to be reserved to antibodies that could bind to several different locations at the virus .",
"Dingens et al . further documented all the virus mutations that would allow it to evade neutralizing antibodies .",
"The techniques used in the experiments may help scientists identify the best sites on the HIV virus to target with vaccines and to better understand the binding and neutralizing activity of antibodies .",
"The results of the experiments may also help to redesign the experimental HIV vaccine – which is currently being tested in humans – to be even more effective ."
] | 2021 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cancer biology"
] | Transient rapamycin treatment can increase lifespan and healthspan in middle-aged mice | elife-16351-v2 | [
[
"Successful interventions that increase healthy longevity in people could have profound benefits for quality of life , productivity , and reduced healthcare costs ( Goldman et al . , 2013; Kaeberlein et al . , 2015 ) .",
"The drug rapamycin is a promising candidate for such an intervention , as it has been shown to increase lifespan in numerous species ( Johnson et al . , 2013 ) and to delay or reverse multiple age-associated phenotypes in mice including cognitive decline ( Halloran et al . , 2012; Majumder et al . , 2012 ) , cardiac dysfunction ( Dai et al . , 2014; Flynn et al . , 2013 ) , immune senescence ( Chen et al . , 2009 ) , and cancer ( Anisimov et al . , 2011 ) .",
"Recently , a six week treatment with the rapamycin derivative RAD001 was reported to improve immune function in elderly people , as measured by response to influenza vaccine ( Mannick et al . , 2014 ) , suggesting that at least some of the effects on aging in mice are conserved in humans .",
"Despite these impressive results , the utility of rapamycin or other mTOR inhibitors to delay aging may be limited by side effects .",
"The high doses of rapamycin and its derivatives used clinically to prevent organ transplant rejection are associated with adverse events , including impaired wound healing , edema , elevated circulating triglycerides , impaired glucose homeostasis , gastrointestinal discomfort , and mouth ulcers ( Augustine et al . , 2007; de Oliveira et al . , 2011 ) .",
"While many of these side effects have not been observed in mice at the lower doses that extend lifespan , chronic treatment with encapsulated rapamycin ( eRapa ) in the diet at 14 ppm has been reported to cause gonadal degeneration in males , increased risk of cataracts , and impaired response to a glucose tolerance test ( Wilkinson et al . , 2012; Lamming et al . , 2012 ) ."
],
[
"Based on the premise that transient treatment with rapamycin during middle-age might be more suitable for clinical efforts to promote healthy aging than continuous treatment throughout life , we set out to investigate whether a single three-month treatment regimen can extend lifespan and healthspan in C57BL/6JNia mice starting at 20–21 months of age .",
"We initially used a treatment regimen consisting of intraperitoneal ( i . p . ) injections of 8 mg/kg rapamycin daily for 90 days .",
"This dose was selected because we have previously found that it increases survival and alleviates disease phenotypes in short-lived mouse models of dilated cardiomyopathy , muscular dystrophy , and the severe mitochondrial disease Leigh Syndrome ( Ramos et al . , 2012; Johnson et al . , 2013 ) .",
"Based on efficacy in the Leigh Syndrome mouse model and serum drug levels in wild type mice , we estimate that this treatment regimen is comparable to dietary delivery of eRapa at approximately 378 ppm ( Johnson et al . , 2015 ) , or 27-fold higher levels than initially shown to extend lifespan in mice when continuous treatment is initiated at either 9 months or 20 months of age ( Harrison et al . , 2009; Miller et al . , 2011 ) .",
"Because prior studies have noted differences in the magnitude of lifespan extension following continuous rapamycin treatment in male versus female animals ( Harrison et al . , 2009; Miller et al . , 2011 , 2014 ) , we examined the effect of this regimen in both sexes independently .",
"Serum rapamycin levels did not differ significantly between male and female animals in our study ( Figure 1—figure supplement 1 ) .",
"During the three-month treatment period , we noted a significant decline in body weight of male mice receiving rapamycin injections relative to vehicle treated controls ( Figure 1A ) , although food intake remained similar during the treatment ( Figure 1—figure supplement 2 ) .",
"Decreased body weight persisted for several weeks following cessation of treatment ( Figure 1A ) .",
"This was accompanied by a striking increase in median life expectancy from the end of treatment of 60% ( p=0 . 02 , Figure 1B , Table 1 ) and an increase in overall median lifespan from birth of 16% ( p=0 . 03 , Figure 1—figure supplement 3 , Table 2 ) .",
"This effect is larger than both the absolute and relative magnitude of lifespan extension resulting from continuous treatment to death with 14 ppm eRapa starting at around the same age in UMHET3 mice ( Harrison et al . , 2009 ) .",
"The longest-lived rapamycin-treated male in our cohort survived for 710 days post treatment to approximately 1400 days of age .",
"Based on a survey of the literature , this is likely one of the longest-lived wild type C57BL/6 animals ever reported . 10 . 7554/eLife . 16351 . 003Figure 1 . Rapamycin injection at 8 mg/kg/day for 3 months extends life expectancy of male mice .",
"( A ) Body weight of male mice measured weekly after starting rapamycin and vehicle treatment .",
"Data are indicated as mean ± s . e . m . *p<0 . 05 .",
"**p<0 . 01 .",
"( B ) Survival of control and rapamycin-treated male mice following the end of treatment .",
"p=0 . 02 .",
"N=18 vehicle injected , N=17 rapamycin . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 00310 . 7554/eLife . 16351 . 004Figure 1—figure supplement 1 . Rapamycin serum level does not differ between female and male mice . Blood levels of rapamycin in mice 24 hr after finishing daily i . p . injections for 3 months .",
"p=0 . 359 .",
"Females , N = 16 .",
"Males , N = 16 .",
"Data are indicated as mean ± s . e . m . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 00410 . 7554/eLife . 16351 . 005Figure 1—figure supplement 2 . Food intake of male mice receiving 8 mg/kg/day i . p . rapamycin or vehicle injections . Food intake measured weekly by weighing the food given to each cage and the food remaining on the wire rack in each cage . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 00510 . 7554/eLife . 16351 . 006Figure 1—figure supplement 3 . Survival plots of male mice treated with 8 mg/kg/day i . p . rapamycin for 90 days starting around 600 days of age . Gray box approximately indicates treatment period .",
"Note: although the curves start at age=0 to represent all of life , we have no data on animals that may have died in this cohort prior to receipt from NIA ( dashed lines ) .",
"N=18 vehicle injected , N=17 rapamycin . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 00610 . 7554/eLife . 16351 . 007Figure 1—figure supplement 4 . Inclusion of non-age-related deaths does not alter survival outcomes . Lifespan curve of male mice injected with either vehicle or 8 mg/kg/day rapamycin including deaths during the treatment period from non-age-related causes .",
"N=20 vehicle injected , N=20 rapamycin . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 00710 . 7554/eLife . 16351 . 008Table 1 . Sex-segregated comparison of median and mean post-treatment life expectancy for mice receiving rapamycin by injection ( 8 mg/kg/day ) or feeding ( 128 ppm ) .",
"M: males , F: females . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 008Median life expectancy ( days ) Percent life expectancy increaseMean life expectancy ( days ) Percent life expectancy increaseVehicle M231235Rapamycin ( 8mg/kg/day ) M3726135853Vehicle F161177Rapamycin ( 8mg/kg/day ) F1610171-3Eudragit M193199Rapamycin ( 126 ppm ) M27944 . 624221Eudragit F184175Rapamycin ( 126 ppm ) F256392403710 . 7554/eLife . 16351 . 009Table 2 . Sex-segregated comparison of median and mean lifespan for mice receiving rapamycin by injection ( 8 mg/kg/day ) or feeding ( 128 ppm ) .",
"M: males , F: females . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 009Median lifespan ( days ) Percent median lifespan increaseMean lifespan ( days ) Percent mean lifespan increaseVehicle M925929Rapamycin ( 8mg/kg/day ) M105414105013Vehicle F847858Rapamycin ( 8mg/kg/day ) F8470853-1Eudragit M914912Rapamycin ( 126 ppm ) M1037149848Eudragit F879883Rapamycin ( 126 ppm ) F96099518 This rapamycin regimen also resulted in a decline in body weight of female mice together with an increased trend of food intake in vehicle-treated animals with time ( Figure 2A , Figure 2—figure supplement 2 ) ; however , we failed to detect a similar increase in post-treatment survival and lifespan in females ( p=0 . 261 , Figure 2B , Figure 2—figure supplement 1 , Tables 1–2 ) .",
"Cox proportional hazards regression with robust standard errors and adjusting for cohort indicated significant evidence of a treatment difference between male and female mice ( p=0 . 023 ) .",
"We speculated that this lack of lifespan extension results from an increase in aggressive hematopoietic cancers in the rapamycin treated females .",
"Histopathological analysis showed that while 6/12 control females had round cell tumors ( lymphoma and histiocytic sarcoma of hematopoietic origins ) , comparable to previous studies in C57BL/6 mice ( Blackwell et al . , 1995; Treuting et al . , 2008 ) , all the rapamycin-treated females examined ( 16 out of 16 ) had round cell tumors ( Figure 3A , B and Figure 3—figure supplement 1 , p=0 . 002 ) .",
"Additionally , an uncommon variant of lymphoma with plasmacytoid morphology affected 4 out of 16 mice examined in the rapamycin group and no vehicle treated mice ( Figure 3A and Figure 3—figure supplement 1A , B ) .",
"Round cell tumors affected more organs in rapamycin treated females , with multiple ( ≥2 ) organs affected in all of the rapamycin treated females compared to only 3 vehicle treated females ( Figure 3C , p=0 . 01 ) .",
"Together , these data indicate a more aggressive phenotype of hematopoietic tumors in the high dose rapamycin treated females .",
"In contrast , the incidence of non-hematopoietic neoplasms was dramatically decreased in the rapamycin group ( Figure 3D ) .",
"Nine non-hematopoietic neoplasms ( 5 pituitary adenomas , and 2 pulmonary adenomas , and 2 thyroid adenomas ) were detected in 7 out of the 12 examined vehicle treated mice; whereas , only 1 out of 16 rapamycin treated females had non-hematopoietic neoplasia ( 1 pituitary adenoma ) ( Figure 3D , p=0 . 004 ) . 10 . 7554/eLife . 16351 . 010Figure 2 . Rapamycin injection at 8 mg/kg/day for 3 months does not increase life expectancy of female mice .",
"( A ) Body weight of female mice measured weekly after starting rapamycin and vehicle treatment .",
"Data are indicated as mean ± s . e . m . *p<0 . 05 , **p<0 . 01 ( B ) Survival of control and rapamycin-treated female mice following the end of treatment .",
"p=0 . 261 .",
"N=20 vehicle injected , N=20 rapamycin . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 01010 . 7554/eLife . 16351 . 011Figure 2—figure supplement 1 . Survival plots of female mice treated with 8 mg/kg/day i . p . rapamycin for 90 days starting around 600 days of age . Note: although the curves start at age=0 to represent all of life , we have no data on animals that may have died in this cohort prior to receipt from NIA ( dashed lines ) .",
"Gray box approximately indicates treatment period .",
"N=20 vehicle injected , N=20 rapamycin . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 01110 . 7554/eLife . 16351 . 012Figure 2—figure supplement 2 . Food intake of female mice receiving 8 mg/kg/day i . p . rapamycin or vehicle injections . Food intake measured weekly by weighing the food given to each cage and the food remaining on the wire rack in each cage . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 01210 . 7554/eLife . 16351 . 013Figure 3 . Rapamycin injection at 8 mg/kg/day for 3 months alters cancer incidence of female mice .",
"( A ) Hematoxylin and eosin ( H&E ) sections of multisystemic aggressive lymphoma ( top ) and atypical plasmacytoid lymphoma ( bottom ) from rapamycin-treated female mice .",
"Arrows indicate a bizarre mitotic figure ( top ) and round cells with strongly eosinophilic cytoplasm ( plasmacytoid morphology , bottom ) .",
"Original magnification 60x .",
"Bar = 10 µm .",
"( B ) Hematopoietic cancer incidence of rapamycin-treated ( 16 female ) and vehicle-treated ( 12 female ) mice .",
"( C ) Incidence of multiple organ invasion of hematopoietic tumors in rapamycin-treated ( 16 female ) and vehicle-treated ( 6 female ) hematopoietic tumor-bearing mice .",
"( D ) , Non-hematopoietic cancer incidence of rapamycin-treated ( 16 female ) and vehicle-treated ( 12 female ) mice .",
"*p<0 . 05 .",
"**p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 01310 . 7554/eLife . 16351 . 014Figure 3—figure supplement 1 . Morphologies of aggressive hematopoietic tumors observed in rapamycin injected females . H&E sections of 3 representative hematopoietic tumor types observed in rapamycin treated female mice .",
"Lower magnification ( A , C , E . Original magnification 4x ) represents an uncommon site of invasion for hematopoietic tumors in C57BL/6 mice .",
"Higher magnification ( B , D , F . Original magnification 40x . The region is approximately indicated by the box in A , C , E , respectively ) shows the morphology of each cancer cell type .",
"( A ) Sheets of neoplastic round cells infiltrate the soft tissues of the head including the Harderian gland ( * ) and retrobulbar musculature ( indicated by box ) .",
"( B ) Neoplastic round cells have a plasmacytoid morphology characterized by an eccentric nucleus , perinuclear halo , and occasionally strongly eosinophilic cytoplasm .",
"( C ) A round cell neoplasm morphologically consistent with histiocytic sarcoma in a high dose rapamycin treated female expands and infiltrates the meninges ( * ) and soft tissues of the head ( indicated by box ) .",
"( D ) A round cell neoplasm morphologically consistent with histiocytic sarcoma .",
"Neoplastic histiocytes with abundant eosinophilic cytoplasm and occasional multinucleated giant cells separate and surround nerves .",
"( E ) Subcutaneous round cell neoplasm in a high dose rapamycin treated female associated with severe necrosis ( * ) .",
"Box indicates neoplasm composed of sheets of neoplastic lymphocytes .",
"( F ) Neoplastic lymphocytes have scant to moderate cytoplasm , marked anisocytosis and anisokaryosis , and frequent mitotic cells . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 01410 . 7554/eLife . 16351 . 015Figure 3—figure supplement 2 . Cancer incidence of male mice with rapamycin injection at 8 mg/kg/day for 3 months .",
"( A ) Hematopoietic cancer incidence of rapamycin-treated ( 15 male ) and vehicle-treated ( 14 male ) mice .",
"( B ) Incidence of multiple organ invasion of hematopoietic tumors in rapamycin-treated ( 9 male ) and vehicle-treated ( 14 male ) hematopoietic tumor-bearing mice .",
"( C ) Non-systemic hematopoietic cancer incidence of rapamycin-treated ( 15 male ) and vehicle-treated ( 14 male ) mice .",
"**p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 015 Both control and rapamycin treated male cohorts had a high incidence of systemic round cell neoplasia which affected ≥2 organs , and had an intravascular component and a morphology most consistent with histiocytic sarcoma , although immunohistochemistry was not performed ( Figure 3—figure supplement 2 ) .",
"Several male mice also had eosinophilic ( hyaline ) protein droplets in the renal tubules consistent with lysozyme accumulation associated with histiocytic sarcoma ( Hard and Snowden , 1991 ) .",
"Of the examined vehicle treated males , all ( 14 out of 14 ) had hematopoietic neoplasia affecting at least one organ .",
"Additionally , 7 other types of neoplasia were detected on histopathology from the vehicle treated male group , including 1 gastric neoplasia , 1 thyroid adenoma , and 5 pulmonary adenomas .",
"One male mouse had a subcutaneous facial mass characterized by sheets of round cells , most consistent with an extramedullary plasma cell tumor or mast cell tumor .",
"Rapamycin treated males also had a high incidence of systemic round cell neoplasia affecting at least one organ ( 9 out of 13 ) .",
"In the rapamycin treated male group , 12 additional non-round cell tumors were detected on histopathology , including 1 hepatocellular carcinoma , 2 intestinal adenomas , 1 splenic hemangiosarcoma , 5 pulmonary adenomas , 1 pulmonary carcinoma , 1 thyroid adenoma , and 1 Zymbal’s gland adenoma ( Figure 3—figure supplement 2 ) .",
"In the male group , no cases of systemic lymphoma with plasmacytoid morphology were seen .",
"Although the number of mice examined is relatively small , based on these observations we speculate that this regimen of rapamycin treatment induced the detrimental side effect of aggressive hematopoietic cancers specifically in female mice , which occurred earlier in life and were sufficient to prevent lifespan extension in these animals despite other beneficial effects , such as reduced non-hematopoietic cancers .",
"When data from both sexes are pooled together , daily injection of 8 mg/kg rapamycin for three months resulted in a non-significant ( p=0 . 16 ) increase in life expectancy of 23% ( Table 3 ) .",
"Between randomization and the end of treatment , 5 male mice ( 3 vehicle , 2 rapamycin ) died of non-age related causes and were excluded from the analysis .",
"Their inclusion in the analysis of survival does not significantly affect the results as described above ( Figure 1—figure supplement 4 ) . 10 . 7554/eLife . 16351 . 016Table 3 . Sex-pooled comparison of median and mean post-treatment life expectancy for mice receiving rapamycin by injection ( 8 mg/kg/day ) or feeding ( 128 ppm ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 016Median life expectancy ( days ) Percent life expectancy increaseMean Life expectancy ( days ) Percent life expectancy increaseVehicle188204Rapamycin ( 8mg/kg/day ) 2312225726Eudragit190188Rapamycin ( 126 ppm ) 2704225737 In order to assess whether transient treatment with a lower dose and different delivery of rapamycin might reduce side effects in female mice and increase lifespan in both sexes , we utilized dietary eRapa at 126 ppm .",
"Mice were fed a diet containing eRapa or the encapsulation control eudragit diet for 90 days starting at 20–21 months of age and then returned to a standard chow diet .",
"In contrast with the effects of daily injection of 8 mg/kg rapamycin ( Figures 1A and 2A ) , body weight and food intake were largely unaffected by this dietary rapamycin regimen ( Figure 4 A , C , E , Figure 4—figure supplement 1 ) , except for a transient , small increase in body weight in female mice fed eRapa , as determined by Student’s t test ( Figure 4E ) .",
"Median post-treatment life expectancy was significantly increased by 42% when female and male animals were considered together ( p=0 . 002 , Figure 4B , Table 3 ) and overall lifespan was increased by 13% ( p=0 . 003 , Figure 4—figure supplement 2A , Table 4 ) .",
"Similar results were observed stratifying on sex; three months of 126 ppm eRapa administration significantly increased post-treatment survival in both males and females independently ( Figure 4D , F , Figure 4—figure supplement 2B , C and Tables 1–2 ) .",
"Cox proportional hazards regression with robust standard error estimates did not find evidence that sex modifies the treatment effect ( p=0 . 904 ) .",
"Between randomization and the end of treatment , 1 male mouse died of non-age related causes and was excluded from the analysis .",
"Its inclusion in the analysis does not significantly affect the results as described above ( Figure 4—figure supplement 3A , B ) . 10 . 7554/eLife . 16351 . 017Figure 4 . Rapamycin feeding at 126 ppm for 3 months extends life expectancy .",
"( A , C , E )",
"Body weight of ( A ) sex-pooled , ( C ) male , and ( E ) female mice measured weekly after starting rapamycin and eudragit treatment .",
"*p<0 . 05 .",
"Survival of ( B ) sex-pooled control and rapamycin-treated mice following the end of treatment .",
"p=0 . 002 .",
"N=38 vehicle injected , N=37 rapamycin .",
"( C ) .",
"Survival of ( D ) male , and ( F ) female control and rapamycin-treated mice following the end of treatment .",
"( D ) N=20 eudragit males , N=19 126 ppm eRapa males .",
"( F ) N=18 eudragit females , N=18 eRapa females .",
"( G ) Forelimb grip strength tests measured prior to treatment initiation ( baseline ) , upon cessation of treatment ( 3 months ) , and 3 months after the drug withdrawal ( 6 months ) .",
"( H ) Rotarod performance tested prior to treatment initiation ( baseline ) , upon cessation of treatment ( 3 months ) , and 3 months after the drug withdrawal ( 6 months ) .",
"Data are plotted with box-whisker plots , showing median , 25th and 75th percentile , as well as individual outliers .",
"Statistical significance was calculated with a linear mixed-effect model , using treatment group , measurement date , and measurement day as fixed effects and individual mice identifiers as random variables .",
"*p<0 . 05 rapamycin vs . control at 6 months .",
"**p<0 . 01 rapamycin at 6 months vs . rapamycin at baseline . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 01710 . 7554/eLife . 16351 . 018Figure 4—figure supplement 1 . Food intake of mice receiving 126 ppm eRapa or eudragit control . Food intake measured weekly by weighing the food given to each cage and the food remaining on the wire rack in each cage after 5–7 days . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 01810 . 7554/eLife . 16351 . 019Figure 4—figure supplement 2 . Effects of 126 ppm eRapa treatment on lifespan in male and female mice .",
"( A ) Survival plots of sex-pooled mice treated with 126 ppm eudragit or eRapa for 90 days starting around 600 days of age .",
"Note: although the curves start at age=0 to represent all of life , we have no data on animals that may have died in this cohort prior to receipt from NIA .",
"Gray box approximately indicates treatment period .",
"N=38 vehicle injected , N=370 eRapa .",
"( B , C )",
"Sex-segregated survival plots of male ( B ) and female ( C ) mice fed 126 ppm eudragit or eRapa for 90 days starting around 600 days of age .",
"Note: although the curves start at age=0 to represent all of life , we have no data on animals that may have died in this cohort prior to receipt from NIA .",
"Gray box approximately indicates treatment period .",
"( B ) N=20 eudragit males , N=19 126 ppm eRapa males .",
"( C ) N=18 eudragit females , N=18 eRapa females . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 01910 . 7554/eLife . 16351 . 020Figure 4—figure supplement 3 . Inclusion of non-age-related deaths does not alter survival outcomes .",
"( A , B ) .",
"Survival plots of ( A ) male and ( B ) sex-pooled mice fed 126 ppm eudragit or eRapa for 90 days including deaths during the treatment period from non-age-related causes .",
"( A ) N=38 vehicle injected , N=38 eRapa .",
"( B ) N=20 eudragit males , N=20 126 ppm eRapa males . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 02010 . 7554/eLife . 16351 . 021Table 4 . Sex-pooled comparison of median and mean lifespan for mice receiving rapamycin by injection ( 8 mg/kg/day ) or feeding ( 128 ppm ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 021Median lifespan ( days ) Percent median lifespan increaseMean lifespan ( days ) Percent mean lifespan increaseVehicle874892Rapamycin ( 8mg/kg/day ) 92259445Eudragit885899Rapamycin ( 126 ppm ) 996139688 In parallel with survival , we also examined healthspan parameters in these cohorts .",
"The effects of rapamycin on age-associated decline in muscle function and motor coordination were assessed by testing forelimb grip strength and Rotarod performance .",
"Assessments were performed in the same animals before onset of treatment , at the end of the 90 day treatment , and 90 days after the end of the treatment .",
"While eudragit-fed mice showed a steady decline in both grip strength and Rotarod performance , rapamycin-fed mice scored significantly better than control mice in both assays after treatment ( Figure 4G , H ) , suggesting that the healthspan promoting effect of rapamycin continues after treatment is discontinued .",
"Furthermore , only rapamycin-fed mice showed a progressive increase in Rotarod performance during the 3 days of training and over the whole study , with mice performing better at 6 months compared to baseline ( Figure 4H , large bracket ) .",
"In the course of routine animal husbandry , we noted that mice treated with either rapamycin regimen produced consistently smaller fecal pellets than age-matched controls ( Figure 5A–D ) .",
"This difference was present in both dry ( Figure 5C , D ) and freshly excreted feces ( Figure 5—figure supplement 1A ) , indicating that water content is not a major factor affecting feces size .",
"In addition , feces size was affected independently of the method of rapamycin delivery ( Figure 5C , D ) , and the effect was persistent after cessation of treatment ( Figure 5—figure supplement 1B , C ) .",
"We hypothesized that changes in the microbiome may underlie this phenotype and therefore analyzed the fecal microbiome for each of the cohorts used in this study by deep-sequencing of bacterial 16S rRNA .",
"Distance based permutation multivariate analysis of variance ( MANOVA ) ( Anderson , 2001 ) indicated that rapamycin treatment induced a significant change in the composition of fecal microbiome ( p=0 . 018 for injection cohorts . p=0 . 015 for feeding cohorts . p=0 . 005 for pooled cohorts ) , even after accounting for delivery method and batch effects ( Figure 6—figure supplements 1–3 ) . 10 . 7554/eLife . 16351 . 022Figure 5 . Rapamycin decreases fecal pellet size .",
"( A ) Photograph of feces collected from rapamycin-injected and vehicle-injected animals at 3 months of the treatment .",
"( B ) Photograph of feces collected from rapamycin-fed and eudragit-fed animals at 3 months of the treatment .",
"( C ) The weight of fecal pellets collected from rapamycin-injected and vehicle-injected animals at 3 months of the treatment .",
"N = 22–24 .",
"( D ) The weight of fecal pellets collected from rapamycin-fed and eudragit-fed female animals at 3 months of the treatment .",
"N = 11 .",
"Data are indicated as mean ± s . e . m . *p<0 . 05 .",
"**p<0 . 01 .",
"***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 02210 . 7554/eLife . 16351 . 023Figure 5—figure supplement 1 . Rapamycin decreases fecal pellet size persistently after cessation of treatment .",
"( A ) Weight of freshly excreted fecal pellets collected from rapamycin-injected and vehicle-injected male animals at 3 months of the treatment .",
"N = 13 Vehicle , N = 19 Rapamycin .",
"( B ) Weight of fecal pellets collected from rapamycin-injected and vehicle-injected animals 1 month after cessation of the treatment .",
"N = 71 vehicle female , N = 90 rapamycin female , N = 47 vehicle male , N = 36 rapamycin male .",
"( C ) Weight of fecal pellets collected from rapamycin-injected and vehicle-injected animals 4 months after cessation of the treatment .",
"N = 22 vehicle female , N = 21 rapamycin female , N = 23 vehicle male , N = 22 rapamycin male .",
"Data are indicated as mean ± s . e . m . *p<0 . 05 .",
"**p<0 . 01 .",
"***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 023 Among the most notable changes in fecal bacterial DNA content seen in the global microbiome analysis was a significant increase in prevalence of segmented filamentous bacteria ( SFB , Candidatus Arthromitus sp . ) in the rapamycin treated animals ( Figure 6A , Figure 6—figure supplement 2A ) .",
"SFB are intestinal Gram-positive bacteria with a segmented and filamentous morphology , and are not normally present at high levels in aged mice ( Ericsson et al . , 2014 ) .",
"The SFB genome lacks a majority of virulence factors and SFB are not invasive ( Prakash et al . , 2011 ) ; however , their tight adhesion to the intestinal epithelial cell induces differentiation of host immune cells ( Atarashi et al . , 2015 ) .",
"The increase in SFB following rapamycin treatment was confirmed by real-time PCR of DNA from fecal samples obtained from both mice receiving injections or encapsulated rapamycin ( Figure 6B , Figure 6—figure supplement 2B ) , as well as by semi-quantitative histological scoring of the small intestine in an independent cohort of mice obtained from the Harrison Lab at the Jackson Laboratory and injected with 8 mg/kg/day of for 3 months at the University of Washington ( Figure 6C , D , Figure 6—figure supplement 2C ) .",
"Since increased SFB DNA was observed both in mice injected with rapamycin and mice fed dietary eRapa , this effect is independent of mode of drug delivery .",
"To the best of our knowledge , this represents the first pharmacological intervention to increase SFB in any animal .",
"It will be of interest to determine whether these and other effects of rapamycin on the microbiome are shared across species and play any causal role in the beneficial or detrimental effects of this drug . 10 . 7554/eLife . 16351 . 024Figure 6 . Rapamycin changes the composition of gut microbiota and increases segmented filamentous bacteria .",
"( A ) Violin plots representing the Log2 16S rRNA gene abundance for operation taxonomy units ( OTUs ) in fecal samples that are significantly differentially prevalent at a false discovery rate of 0 . 05 using the R package metagenomeSeq , controlling for the delivery method , gender and mouse batch effects .",
"( C ) Arthromitus ( Candidatus Arthromitus sp . ) refers to segmented filamentous bacteria ( SFB ) .",
"CT and RP indicate control and rapamycin , respectively .",
"( B ) The ratio of SFB DNA to total bacterial DNA in fecal samples measured by real-time PCR .",
"N = 32 .",
"Data are indicated as mean ± s . e . m . ***p<0 . 001 .",
"( C ) Representative H&E section of the intestine of rapamycin-treated mouse .",
"Arrow indicates SFB attached to the intestinal epithelial cells .",
"Lower right quadrant: magnification of area enclosed in black rectangle .",
"( D ) Semi-quantitative grading of SFB amount in the intestinal tissue section .",
"0 indicates the absence of SFB .",
"1–4 indicates the grades of the SFB amount with 1 lowest and 4 highest .",
"Fisher’s exact test p=3 . 6 x 10–5 . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 02410 . 7554/eLife . 16351 . 025Figure 6—figure supplement 1 . Effect of rapamycin on the fecal microbiome .",
"( A ) Heatmap of the Log2 16S rRNA gene abundance by mouse ( column ) and operation taxonomy units ( OTU ) s ( row ) .",
"The clustering is done on a subset of the OTUs with total abundance of more than 10 and relatively large variability ( standard deviation of more than 1 ) .",
"Batch numbers indicates mice shipped and received into our facility at the same time .",
"Feed and Inject indicate drug delivery via encapsulated feed and via injection , respectively .",
"The hierarchical clustering shows the large effects of drug delivery method and batch effect on the microbial composition .",
"( B ) Table of P values from multivariate analysis for rapamycin vs . control ( treatment ) , female vs . male ( sex ) , mouse cages received into our facility at different times ( batch ) and injection vs . feeding regimen ( delivery ) .",
"*p<0 . 05 .",
"**p<0 . 01 .",
"***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 02510 . 7554/eLife . 16351 . 026Figure 6—figure supplement 2 . Bacterial DNA in fecal samples significantly different between control and rapamycin .",
"( A ) .",
"Prevalence distribution of operation taxonomy units ( OTUs ) ( Log2 of 16 rRNA gene abundance ) in fecal samples that have p-values less than 0 . 05 using the R package metagenomeSeq , contrasting control and rapamycin group after controlling for the delivery method , gender and mouse batch effects .",
"CT and RP indicate control and rapamycin , respectively .",
"( B ) The ratio of SFB DNA to total bacterial DNA in fecal samples measured by Real-time PCR divided by sex and treatment method .",
"N = 4–10 .",
"Data are indicated as mean ± s . e . m . *p<0 . 05 .",
"**p<0 . 01 .",
"( C ) Semi-quantitative grading of SFB amount in the intestinal tissue section .",
"0 indicates the absence .",
"1–4 indicates the grades of the SFB amount with 1 lowest and 4 highest . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 02610 . 7554/eLife . 16351 . 027Figure 6—figure supplement 3 . Bacterial composition in fecal samples at phylum level .",
"( A ) Stacked bar plot showing the proportion of phylum within each fecal sample .",
"b1-5 indicate mouse batches that were shipped at a different time .",
"F and M indicate female and male , respectively .",
"Feed and inject indicate drug delivery via encapsulated feed and via injection , respectively .",
"( B ) Violin plot of Firmicutes to Bacteroidetes ratio in fecal samples .",
"CT and RP indicate control and rapamycin , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 16351 . 027"
],
[
"Taken together , our data demonstrate that a single three-month regimen of rapamycin is sufficient to robustly increase life expectancy in middle-aged mice , comparable to the effects previously reported for life-long treatment , while also improving measures of healthspan and substantially altering the microbiome .",
"This work extends prior evidence indicating that short-term rapamycin treatment can improve health in mice , including one experiment suggesting that 4 mg/kg rapamycin every other day for 6 weeks enhanced survival until around 30 months of age in a small cohort ( Chen et al . , 2009 ) , and studies reporting improvements in cardiac ( Dai et al . , 2014; Flynn et al . , 2013 ) and immune ( Chen et al . , 2009 ) function following transient treatment with rapamycin .",
"In the animals treated with the 126 ppm eRapa diet in this study , the improvements in lifespan and health were achieved without overt detrimental side effects , although it is possible that some side effects were undetected , and we did not explicitly test for cataracts , gonadal degeneration , and other adverse outcomes .",
"In the case of the 8 mg/kg/day injection regimen , serious side effects were noted in female , but not male mice .",
"Intriguingly , a dramatic shift toward aggressive hematopoietic cancers and away from non-hematopoietic cancers was observed in these female mice .",
"This is consistent with a similar weak trend seen in kidney transplant patients receiving rapamycin to prevent organ rejection , suggesting a possible conservation of mechanism and clinical relevance ( Mathew et al . , 2004 ) .",
"Our data indicate a need to carefully consider sex effects when optimizing treatment regimens and mechanism of drug action .",
"They also illustrate the importance of better understanding the effects of mTOR inhibitors on differential cancer risk , particularly as mTOR inhibitors are being tested and used clinically for a variety of purposes including the treatment of some rare forms of cancer .",
"The importance of evaluating potential risks and adverse side effects when developing interventions to promote healthy aging should not be underestimated .",
"This study extends other recent work aimed at developing mid-life interventions to promote healthy aging .",
"Of particular note are two studies reporting improved healthspan from short-term treatments in older mice with a JAK pathway inhibitor ( Xu et al . , 2015 ) and increased lifespan after transient treatment with the NAD+ precursor nicotinamide riboside ( Zhang et al . , 2016 ) .",
"From a translational perspective , a healthy aging intervention that can be applied for a relatively short period of time during mid- or late-life is likely to have advantages in cost , practicability and quality of life of people , and we look forward to further developments in this area ."
],
[
"All lifespan and healthspan experiments were performed on 19–20 month old C57BL6/JNia obtained from the National Institute on Aging Aged Rodent Colony .",
"A separate cohort of C57BL6/J ranging from 17 weeks to 100 weeks of age was obtained from the Harrison lab at the Jackson Laboratory for histological analysis of SFB .",
"Animals were housed in individually ventilated cages ( Allentown , Allentown , NJ ) containing corncob bedding ( Andersons , Maumee , OH ) and nestlets .",
"Mice were fed irradiated Picolab Rodent Diet 20 #5053 ( Lab Diet , St . Louis , MO ) .",
"Animals were maintained in a specific pathogen free facility within a Helicobacter spp .",
"-free room .",
"Mice were housed in groups ( 5 per cage at a maximum ) and aggressive male mice were isolated to prevent fighting .",
"Mice were acclimatized at least two weeks before onset of experiments .",
"Mice were inspected daily , and medicated for non-life threatening conditions as directed by the veterinary staff .",
"Date of death was recorded when mice were found dead or unlikely to survive longer than 48 hr at the time of inspection .",
"Mice were euthanized according to the following criteria ( modified from the Intervention Testing Program protocol [Harrison et al . , 2009] ) when they showed one of these symptoms: ( 1 ) inability to eat or drink , ( 2 ) severe lethargy , as indicated by a lack of response such as a reluctance to move when gently prodded , ( 3 ) severe respiratory difficulty while at rest , indicated by a regular pattern of deep abdominal excursions or gasping , or showing any combination of the following features:",
"( a ) severe balance and gait disturbance ,",
"( b ) an ulcerated or bleeding tumor , visible to the naked eye and breaking through the skin of the animal , or rapid weight gain associated with visible or palpable masses , or",
"( c ) Body Condition Score equal to 1 or loss of 20% of body weight in the course of seven days .",
"Survival data is shown from the end of treatment ( 23–24 months of age ) .",
"Deaths before and during the three month treatment period by group were as follows: 2 vehicle-injected males , 3 rapamycin-injected males , 1 eudragit fed female , 1 eRapa fed male .",
"Sentinel mice ( Crl:CD1[ICR]; Charles River , Wilmington , MA ) were tested quarterly and were negative for endo/ectoparasites , mouse norovirus , mouse hepatitis virus , mouse parvovirus , and rotavirus .",
"Sentinel mice were tested annually for Mycoplasma pulmonis , pneumonia virus of mice , reovirus 3 , Sendai virus , and Theiler murine encephalomyelitis virus .",
"All care of experimental animals was in accordance with the University of Washington institutional guidelines and experiments were performed as approved by the Institutional Animal Care and Use Committee .",
"For the injection experiments , rapamycin ( LC Laboratories ) was dissolved in DMSO to 100 mg/ml , then further diluted in 5% PEG-400/5% Tween-80 to a final concentration of 1 . 2 mg/ml , sterile filtered , and stored at −80°C for long-term storage .",
"37 rapamycin treated mice ( 17 males , 20 females ) were i . p . injected daily for 3 months with 66 µl/10g body weight for a final dosage of 8 . 0 mg/kg starting at 20–21 months old .",
"38 Control mice ( 18 males , 20 females ) were i . p . injected with vehicle solution ( 5% PEG-400/5% Tween-20 ) for 3 months .",
"For the feeding model , encapsulated rapamycin was obtained from Rapamycin Holdings , Inc .",
"Irradiated PicoLab Diet 20 5053 pellets were ground and mixed with encapsulated rapamycin at 126 ppm .",
"300 ml of 1% agar melted in sterile water and 200 ml of sterile distilled water were added per kilogram of powdered chow , in order to make pellets .",
"Pellets were stored at −20°C until use .",
"Control food contained the same concentration of agar and encapsulation material ( eudragit ) without rapamycin at the concentration that matched the rapamycin chow .",
"37 mice ( 19 on eudragit , 18 on rapamycin ) received assigned diet treatments at 20–21 months of age , lasting for 90 days .",
"Body weight was measured at least weekly from the beginning of treatment until three months after the end of treatment .",
"For male mice receiving injections ( Figure 1A ) , at each time point from the start point , sample size was as follows: vehicle N = 19 , 18 , 18 , 18 , 18 , 18 , 18 , 18 , 18 , 18 , 18 , 18 , 18 , 17 , 17 , 17 , 17 , 17 , 17 , 17 , 17 , 17 , 16 , 16 , 16 , 16 , 14 , 13 , 13 , 13 , 13 , 13 , 13; rapamycin N = 20 , 19 , 19 , 19 , 19 , 19 , 19 , 19 , 19 , 19 , 18 , 18 , 18 , 17 , 17 , 17 , 17 , 17 , 17 , 17 , 17 , 17 , 17 , 17 , 16 , 16 , 16 , 16 , 16 , 16 , 16 , 15 , 15 .",
"For female mice receiving injections ( Figure 2A ) , at each time point from the start point , sample size was as follows: vehicle N = 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 19 , 19 , 19 , 19 , 18 , 18 , 18 , 18 , 17 , 17 , 17 , 16 , 15 , 15 , 13 , 12 , 11 vehicle; rapamycin N = 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 19 , 18 , 18 , 18 , 18 , 17 , 17 , 17 , 16 , 16 , 16 , 16 , 15 , 14 , 13 , 13 .",
"For mice receiving micro-encapsulated diets ( Figure 4 ) , at each time from the start point , sample size was as follows: eudragit males N= 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 20 , 19 , 18 , 18 , 18 , 18 , 18 , 18 , 18; eudragit females N= 19 , 19 , 19 , 19 , 19 , 19 , 19 , 19 , 19 , 19 , 19 , 19 , 19 , 19 , 18 , 18 , 18 , 18 , 18 , 17 , 17 , 17 , 17 , 16 , 16 , 15 , 15; eRapa males N= 19 , 19 , 19 , 19 , 19 , 19 , 19 , 19 , 19 , 18 , 18 , 18 , 18 , 18 , 17 , 17 , 17 , 17 , 17 , 17 , 17 , 17 , 17 , 17 , 17 , 17; eRapa females N= 18 , 18 , 18 , 18 , 18 , 18 , 18 , 18 , 18 , 18 , 18 , 18 , 18 , 17 , 17 , 17 , 17 , 17 , 17 , 16 , 16 , 16 , 16 , 15 , 15 , 15 .",
"Food intake was measured weekly or twice per week per cage by subtracting the amount of food remaining on the wire rack from the amount given 4–7 days before .",
"Average food intake per mouse was calculated by dividing this value by the number of mice in the cage , then averaging all the values from cages hosting mice under the same treatment and of the same sex .",
"Gross examination was performed following the natural death or euthanasia of animals .",
"Tissues from mice were fixed with 10% neutral buffered formalin , routinely processed and embedded in paraffin , and stained with haematoxylin and eosin ( H&E ) .",
"16 rapamycin injected female , 15 rapamycin injected male , 12 vehicle administered female , and 14 vehicle administered male counterparts in the lifespan study were analyzed for tumor incidence and extent by two board certified veterinary pathologists ( J . M . S . and P . M . T ) .",
"7/15 rapamycin injected male mice were autolyzed to varying degrees , which may have complicated histological detection of systemic neoplasia , although disease sufficient to result in death was determined for 3 of these mice and systemic neoplasia was detected on histological examination of 5 of these 7 mice .",
"Autolysis also complicated the histological assessment of 2 female vehicle treated mice , although systemic neoplasia was detected on histological examination of one of these two mice .",
"Major organs ( including decalcified cross section of the head , skin , lung , heart , liver , kidney , spleen , pancreas , lymph node , salivary gland , gastrointestinal tract and reproductive tract ) were examined histologically .",
"For semi-quantitative SFB analysis , 14 female and 16 male mice i . p . injected with rapamycin ( 8 mg/kg/day , daily for 3 months ) and 9 female and 17 male mice injected with vehicle ( daily for 3 months ) were sacrificed the day after the end of treatment , and H&E sections of the gastrointestinal tract were routinely prepared and examined .",
"The amount of SFB in the intestinal tissue was graded on a 0 to 4 scale , with 0 representing no SFB , 1 representing rare SFB , 2 representing mild colonization by SFB , 3 representing moderate colonization by SFB , and 4 representing severe colonization of the small intestine by SFB .",
"Images of representative lesions were acquired using NIS-Elements BR 3 . 2 64-bit and plated in Adobe Photoshop Elements .",
"Image brightness and contrast was adjusted using Auto Smart Fix and Auto White Balance manipulations applied to the entire image .",
"Original magnification is stated .",
"Fresh blood was collected upon euthanasia via cervical dislocation 24 hr after the last injection , and sera were isolated using serum separators tube ( BD , Franklin Lakes , NJ ) and immediately stored at −20 C . Rapamycin was extracted with 100 mM ZnSO4 at room temperature and analyzed using a Waters 2795 LC/QuattroMicro MS ( Waters , Milford MA ) .",
"Mice were tested on a Rotamex V rotarod ( Columbus Instruments , Columbus OH ) with a constant acceleration of 0 . 1 rpm/second over a period of four days .",
"On the first day , all animals were allowed to acclimate to the rotarod with a single round of testing .",
"Over the following three days ( labeled day 1 , 2 , and 3 ) all mice were tested three times , with a 30 min minimum resting period in between rounds .",
"All mice were subjected to rotarod testing prior to initiation of treatment , upon cessation of treatment , and 90 days after cessation of treatment .",
"Individuals running the rotarod test were not blinded to the treatment .",
"Forelimb grip strength was tested with a Chatillon DFE-050 force gauge ( AMETEK , Largo FL ) .",
"Animals were held by the base of the tail , allowed to grip the bar of the gauge , and slowly pulled away from the testing apparatus with a smooth horizontal movement .",
"Each mouse was tested 5 times per round , and maximum grip strength was recorded .",
"Animals were tested prior to initiation of treatment , upon cessation of treatment , and 90 days after cessation of treatment .",
"At least 72 hr rest was allowed between grip strength and rotarod testing .",
"Individuals running the grip strength test were not blinded to the treatment .",
"All fecal samples were collected per cage at 3 months of treatment immediately after excretion from the mice analyzed for lifespan analysis and frozen with liquid nitrogen .",
"Microbial DNA was extracted as previously described ( Ericsson et al . , 2015 ) .",
"Briefly , fecal samples were collected into 800 µL of lysis buffer ( 500 mM NaCl , 50 mM tris-HCl , 50 mM EDTA , and 4% SDS ) , homogenized in a Qiagen Tissuelyser II ( Valencia CA ) , and incubated at 70°C for 20 min .",
"The supernatant was mixed with 1 µL of 2 M ammonium acetate , incubated on ice , and then centrifuged at 16 , 000 × g for 10 min at room temperature .",
"The supernatant was then mixed with an equal volume of ice-cold isopropanol and incubated for 30 min on ice .",
"The contents of the tube were then centrifuged at 4°C for 15 min to pellet DNA .",
"The pellet was rinsed twice with 70% EtOH and re-suspended in 150 µL of tris-EDTA buffer .",
"DNA was further purified using DNeasy kit ( Qiagen , Valencia CA ) according to the manufacturer’s protocol .",
"For Metagenomic sequencing , sequencing of the V4 region of the 16S rRNA gene was performed on the Illumina MiSeq platform ( San Diego CA ) , as previously described ( Ericsson et al . , 2015 ) .",
"The raw metagenome data are publicly available at the European Nucleotide Archive ( ENA ) database ( ERP014805 ) .",
"Real-time PCR to measure SFB and total bacterial DNA was performed on a CFX384 Real-Time System with a C1000-Touch thermal cycler ( BioRad , Hercules , CA ) and a StepOnePlus ( Applied Biosystems , Foster City , CA ) with a Sybr Green method as previously described ( Ericsson et al . , 2015 ) .",
"10 ng of DNA collected from fecal samples was analyzed .",
"Primers for SFB are 5’ TGTGGGTTGTGAATAACAAT 3’ and 5’ GCGAGCTTCCCTCATTACAAGG 3’ .",
"Primers for the detection of total bacteria ( 16s rRNA gene ) are 5’ TCCTACGGGAGGCAGCAGT 3’ and 5’ GGACTACCAGGGTATCTAATCCTGTT 3’ .",
"An unpaired two-sample t test was used to compare two experimental groups unless otherwise mentioned .",
"Post-treatment survival data were analyzed using a one-sided Mann Whitney U test ( http://vassarstats . net/utest . html ) .",
"Fisher’s exact test was used for semi-quantitative analysis of SFB and the incidence of malignancies .",
"Rotarod p-values were calculated by applying the glht ( ) function for general linear hypotheses for mixed-effects models from the R multcomp package ( Hothorn et al . , 2008 ) to each of the outputs from the lmer ( ) function in the R lme4 package ( Bates et al . , 2014 ) .",
"For the differential abundance analysis of microbiome population , we used the fitZig ( ) function in the R package metagenomeSeq ( Paulson et al . , 2013 ) .",
"The Figure 6—figure supplement 1 heatmap of hierarchical clustering of 16S rRNA gene prevalence was generated using Euclidian Distance using the R package pheatmap .",
"The shift in the microbial population after controlling for the experimental effect was tested using distance matrix based permutation MANOVA implemented in R package vegan as the adonis ( ) function using the weighted Unifrac distance .",
"Violin plots and stacked barplot were created using R package ggplot2 .",
"In the above analysis we removed a library that exhibited signs of experimental failure ( total library size <10000 ) ."
]
] | [
"The FDA approved drug rapamycin increases lifespan in rodents and delays age-related dysfunction in rodents and humans .",
"Nevertheless , important questions remain regarding the optimal dose , duration , and mechanisms of action in the context of healthy aging .",
"Here we show that 3 months of rapamycin treatment is sufficient to increase life expectancy by up to 60% and improve measures of healthspan in middle-aged mice .",
"This transient treatment is also associated with a remodeling of the microbiome , including dramatically increased prevalence of segmented filamentous bacteria in the small intestine .",
"We also define a dose in female mice that does not extend lifespan , but is associated with a striking shift in cancer prevalence toward aggressive hematopoietic cancers and away from non-hematopoietic malignancies .",
"These data suggest that a short-term rapamycin treatment late in life has persistent effects that can robustly delay aging , influence cancer prevalence , and modulate the microbiome ."
] | [
"Old age is the single greatest risk factor for many diseases including heart disease , arthritis , cancer and dementia .",
"By delaying the biological aging process , it may be possible to reduce the impact of age-related diseases , which could have great benefits for society and the quality of life of individuals .",
"A drug called rapamycin , which is currently used to prevent organ rejection in transplant recipients , is a leading candidate for targeting aging .",
"Rapamycin increases lifespan in several types of animals and delays the onset of many age-related conditions in mice .",
"Nearly all of the aging-related studies in mice have used the same dose of rapamycin given throughout the lives of the animals .",
"Lifelong treatment with rapamycin wouldn’t be practical in humans and is likely to result in undesirable side effects .",
"For example , the high doses of rapamycin used in transplant patients cause side effects including poor wound healing , elevated blood cholesterol levels , and mouth ulcers .",
"Before rapamycin can be used to promote healthy aging in humans , researchers must better understand at what point in life the drug is most effective , and what dose to use to provide the biggest benefit while limiting the side effects .",
"Now , Bitto et al . show that treating mice with rapamycin for a short period during middle age increases the life expectancy of the mice by up to 60% .",
"In the experiments , mice were given two different doses of rapamycin for only three months starting at 20 months old ( equivalent to about 60-65 years old in humans ) .",
"After receiving the lower dose , both male and female mice lived about 50% longer than untreated mice , and showed improvements in their muscle strength and motor coordination .",
"When given the higher dose , male mice showed an even greater increase in life expectancy , but the female mice did not .",
"These female mice had an increased risk of developing rare and aggressive forms of blood cancer , but were protected from other types of cancer .",
"Both drug treatments also caused substantial changes in the gut bacteria of the male and female mice , which could be related to effects of rapamycin on metabolism , immunity and health .",
"More studies are needed to uncover precisely how such short-term treatments can yield long-term changes in the body , and how such changes are related to lifespan and healthy aging ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology",
"neuroscience"
] | FcγRIIb-SHIP2 axis links Aβ to tau pathology by disrupting phosphoinositide metabolism in Alzheimer's disease model | elife-18691-v1 | [
[
"Alzheimer's disease ( AD ) is characterized by the progressive loss of memory and the neuronal degeneration ( Mattson , 2004 ) .",
"The pathological hallmarks of AD are the presence of senile plaques consisting of Aβ peptide and neurofibrillary tangles ( NFT ) formed by abnormally hyperphosphorylated tau ( Walsh and Selkoe , 2004 ) .",
"Aβ species are generated from amyloid precursor protein ( APP ) by β- and γ-secretases , and accumulate extracellularly ( Haass and Selkoe , 2007 ) .",
"In general , Aβ contributes to AD pathology by exhibiting toxicity in susceptible neurons , facilitating tau hyperphosphorylation , disrupting proteasome activity , and triggering synaptic dysfunction ( LaFerla et al . , 2007; Kam et al . , 2014 ) .",
"Tau is a microtubule-binding protein but dissociates from the microtubules and accumulates in neurons as it becomes highly phosphorylated at multiple sites , resulting in the impairment of microtubule assembly and function ( Ballatore et al . , 2007 ) .",
"Accumulating evidence strongly indicate that these two hallmarks are strongly interrelated in AD .",
"In the amyloid cascade hypothesis , tau is believed to be one of the major downstream targets of Aβ to produce neurotoxicity ( Hardy and Selkoe , 2002 ) .",
"Aβ accelerates neurodegeneration in neuronal cells but not in tau-deficient neurons ( Rapoport et al . , 2002 ) .",
"Moreover , tau depletion in mutant APP transgenic mice prevented Aβ pathologies , including learning and memory impairment ( Roberson et al . , 2007 ) .",
"The role of Aβ in tau pathology was also shown in 3xTg-AD mice expressing APP , presenilin , and tau transgenes in which Aβ immunization reduced not only Aβ accumulation but also tau pathology ( Oddo et al . , 2004 ) .",
"In addition , higher levels of NFT have been observed in APPswe/P301L transgenic mice ( Lewis et al . , 2001 ) and in 3xTg-AD mice ( Oddo et al . , 2003 ) .",
"More importantly , tau hyperphosphorylation is frequently found in AD brains ( Grundke-Iqbal et al . , 1989 ) .",
"Apparently , tau kinases , such as glycogen synthase kinase-3β ( GSK-3β ) , are activated by Aβ for tau phosphorylation in vitro and in vivo ( Hoshi et al . , 2003; Ma et al . , 2006; Terwel et al . , 2008; Park et al . , 2012 ) .",
"All of these findings indicate the presence of a pathologic signal pathway starting with extracellular Aβ and ending in the phosphorylation of intracellular tau .",
"However , the mechanism connecting the two pathologic hallmarks of AD remains unknown .",
"Phosphoinositides , the phosphorylated derivatives of phosphatidylinositol ( PtdIns ) , such as PtdIns ( 3 , 4 , 5 ) P3 , PtdIns ( 4 , 5 ) P2 , and PtdIns ( 3 , 4 ) P2 , are known to play a major role in signal transduction upon cellular stimulation ( Di Paolo and De Camilli , 2006 ) .",
"Among them , the biological roles of PtdIns ( 3 , 4 , 5 ) P3 and PtdIns ( 4 , 5 ) P2 have been relatively well characterized in cell survival , proliferation , and synaptic function via their binding proteins ( Bunney and Katan , 2010; Khuong et al . , 2013 ) , but the function of PtdIns ( 3 , 4 ) P2 is largely unknown .",
"Unlike PtdIns ( 4 , 5 ) P2 , PtdIns ( 3 , 4 ) P2 and PtdIns ( 3 , 4 , 5 ) P3 are formed when cells respond to signals ( Zhang and Majerus , 1998; Lemmon , 2008 ) .",
"SH2 domain-containing phosphatidylinositol 5′-phosphatase ( SHIP ) removes 5′ phosphate from PtdIns ( 3 , 4 , 5 ) P3 to produce PtdIns ( 3 , 4 ) P2 ( Damen et al . , 1996 ) .",
"Increasing evidence has revealed that phosphoinositide metabolism is dysregulated in AD; specifically , the level of PtdIns ( 4 , 5 ) P2 is decreased in human and mouse AD brains , and in the primary cortical neurons exposed to oligomeric Aβ ( Stokes and Hawthorne , 1987; Jope et al . , 1994; Berman et al . , 2008 ) , and recovery of PtdIns ( 4 , 5 ) P2 deficiency prevents AD-related cognitive deficits in mouse models ( McIntire et al . , 2012; Zhu et al . , 2015 ) .",
"However , how phosphoinositide metabolism , including levels of PtdIns ( 3 , 4 ) P2 , is regulated by Aβ during AD pathogenesis and the consequences of its dysregulation in AD needs to be resolved .",
"Until now , Aβ was reported to bind to many receptors , including alpha7 nicotinic acetylcholine receptors ( α7 nAChR ) , NMDA receptor , receptors for advanced glycation end- products ( RAGE ) , Aβ-binding alcohol dehydrogenase ( ABAD ) , the Ephrin-type B2 receptor ( EphB2 ) , cellular prion protein ( PrPc ) , and paired immunoglobulin-like receptor B ( PirB ) ( Yan et al . , 1996; Wang et al . , 2000; Lustbader , 2004; Snyder et al . , 2005; Laurén et al . , 2009; Cissé et al . , 2011a; Kim et al . , 2013 ) .",
"Although these receptors were shown to be responsible for Aβ neurotoxicity , especially memory impairment in AD mice , their role as neuronal receptors in Aβ-induced tau pathologies was limitedly shown in α7 nAChR and NMDA receptor ( reviewed in Stancu et al . , 2014 ) .",
"Of particular note , while α7 nAChR was reported to mediate Aβ-induced tau phosphorylation , the finding was based on in vitro and ex vivo system ( Wang et al . , 2003 ) .",
"Furthermore , evidence showing a correlation of the proposed molecular mechanism with pathologic evidence was not much provided .",
"In particular , the CAMKK2-AMPK at down-stream of NMDA receptor was recently proposed to mediate the synaptotoxic effects of Aβ oligomers through tau phosphorylation and this event is very likely caused by NMDA receptor-induced increase of intracellular calcium , not by direct interaction of NMDA receptor with Aβ ( Mairet-Coello et al . , 2013 ) .",
"Therefore , a neuronal receptor that is important in Aβ-induced tau pathology needs to be elucidated .",
"Recently , we showed that Fc gamma receptor IIb ( FcγRIIb ) is also expressed in neurons and directly interacts with Aβ1-42 to mediate Aβ neurotoxicity , synaptic dysfunction , and memory impairment in AD pathogenesis ( Nimmerjahn and Ravetch , 2008; Kam et al . , 2013 ) .",
"Here , we show that FcγRIIb is phosphorylated at tyrosine 273 by Aβ1-42 in neurons and in AD brains , and that this phosphorylation recruits SH2 domain-containing phosphatidylinositol 5′-phosphatase 2 ( SHIP2 , INPPL1 ) to increase PtdIns ( 3 , 4 ) P2 levels for tau hyperphosphorylation .",
"Further , Fcgr2b or Inppl1 deficiency in 3xTg-AD mice or pharmacological inhibition of either protein abrogates all of these observations , highlighting the importance of the FcγRIIb-SHIP2 axis in the Aβ-induced tau pathology ."
],
[
"Given that FcγRIIb was previously identified as a receptor for Aβ1-42 and a mediator of memory impairment in hAPP-J20 mice expressing only familial mutant APP ( Kam et al . , 2013 ) , and that tau as well as Aβ is essential for memory impairment in AD mouse ( Ramsden et al . , 2005 ) , we hypothesized that FcγRIIb is responsible for tau hyperphosphorylation in response to Aβ1-42 .",
"We first assessed and confirmed that both FcγRIIb mRNAs and proteins were significantly expressed in neurons as well as non-neuronal cells in mouse brains ( Figure 1—figure supplement 1 ) .",
"Interestingly , incubation of primary cortical neurons with synthetic Aβ1-42 oligomers increased tau phosphorylation at several pathological epitopes , including Ser396/Ser404 ( PHF1 ) , Thr231/Ser235 ( AT180 ) , and Ser202 ( CP13 ) , but these phosphorylations were abrogated in Fcgr2b knockout ( KO ) neurons ( Figure 1A , B ) .",
"Immunocytochemical analysis also showed that immunoreactivity against the phosphorylated tau was increased by treatment with Aβ1-42 in the neuron-specific enolase ( NSE ) -positive cortical neurons , but not in the Fcgr2b KO neurons ( Figure 1C ) . 10 . 7554/eLife . 18691 . 003Figure 1 . Fcgr2b deficiency prevents tau hyperphosphorylation and memory deficits in 3xTg mice .",
"( A , B )",
"Fcgr2b KO neurons are resistant to Aβ-induced tau phosphorylation .",
"Mouse primary cortical neurons from wild-type ( WT ) or Fcgr2b KO embryos ( DIV 8 ) were incubated with oligomeric forms of 1 μM synthetic Aβ1-42 for 24 hr and cell extracts were subjected to western blotting ( A ) .",
"The levels of phosphorylated tau were quantified by densitometry and normalized by total tau ( TG5 ) .",
"Values are means ± s . e . m . ; *p<0 . 05 , **p<0 . 005 , one-way ANOVA ( n = 4 ) ( B ) .",
"( C ) Immunocytochemical analysis of Aβ-induced tau phosphorylation in WT and Fcgr2b KO neurons .",
"( D ) Fcgr2b deficiency prevents cell-derived Aβ-induced hyperphosphorylation of tau .",
"Mouse primary hippocampal neurons from WT or Fcgr2b KO embryos were cocultured with CHO or 7PA2 cells for 24 hr ( left ) and tau phosphorylation was analyzed by western blotting ( right ) .",
"( E–F )",
"Rescue of memory impairment in 3xTg-AD/Fcgr2b KO mice .",
"Y-maze ( E ) , novel object recognition ( F ) , and passive avoidance ( G ) tests were performed in 8–9 month-old WT , Fcgr2b KO , 3xTg-AD , and 3xTg-AD/Fcgr2b KO mice ( n = 9–14 mice per group; WT , 5 males and 4 females; KO , 5 males and 4 females; 3xTg-AD , 8 males and 5 females; 3x-Tg-AD/KO , 7 males and 7 females ) .",
"Data are means ± s . e . m . ; *p<0 . 05 , ***p<0 . 001 , unpaired t-test .",
"( H , I )",
"Reduced hyperphosphorylation of tau in 3xTg-AD/Fcgr2b KO mice .",
"The hippocampal lysates of 9 month-old mice were subjected to western blotting ( H ) .",
"The levels of phosphorylated tau were quantified as in ( A ) .",
"Values are means ± s . e . m . ; *p<0 . 05 , unpaired t-test ( n = 3 ) ( I ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 18691 . 00310 . 7554/eLife . 18691 . 004Figure 1—figure supplement 1 . Neuronal expression of FcγRIIb in the mouse brain .",
"( A ) Fcgr2b mRNA level in captured neurons .",
"NeuN- or GFAP-positive cells were isolated from wild-type mouse brains by laser capture microdissection ( top ) .",
"Relative mRNA levels of Fcgr2b , Neun , and Gfap were quantified by real-time PCR in total extract ( T ) , captured neurons ( N ) or astrocytes ( A ) ( n = 3 mice ) ( bottom ) .",
"( B ) FcγRIIb protein in neurons .",
"Neurons were isolated from total mouse brain cells ( input ) using iodixanol density gradient assay .",
"The purified fractions were subjected to western blotting using anti-FcγRIIb ( 2 . 4G2 ) , anti-NeuN , and anti-GFAP antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 18691 . 00410 . 7554/eLife . 18691 . 005Figure 1—figure supplement 2 . FcγRIIb is required for cell-derived Aβ oligomer-induced tau phosphorylation in the primary neurons .",
"( A , B )",
"Induction of tau phosphorylation in primary neurons by naturally secreted Aβ .",
"The cell-derived Aβ in 7PA2-CHO cells were detected by immunoprecipitation and western blot analysis using Aβ antibody .",
"Compound E ( CompE ) was used as a negative control and synthetic Aβ1-42 oligomers ( 200 ng ) were used as a loading control .",
"Monomer ( mono ) , dimer ( Di ) and trimers ( Tri ) of Aβ1-42 are shown ( A ) .",
"Primary neurons were co-cultured with donor CHO or 7PA2 cells w/wo compound E for 24 hr , and cell lysates were subjected to western blotting ( B ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 18691 . 00510 . 7554/eLife . 18691 . 006Figure 1—figure supplement 3 . FcγRIIb is also required for Aβ-induced tau phosphorylation in the hippocampus of aged 3xTg-AD and hAPP ( J20 ) mouse lines .",
"( A ) Aβ levels in the hippocampus of 3xTg-AD and 3xTg-AD/Fcgr2b KO mice .",
"Levels of Aβ1-40 and Aβ1-42 in the hippocampus of 9-month-old 3xTg-AD or 3xTg-AD/KO mice were determined by ELISA ( n = 3 ) .",
"Each group mutually did not differ significantly by Student’s t-test and values are means ± s . e . m . ( B , C ) Age-dependent increase of hyperphosphorylated tau in the hippocampus of 3x Tg-AD mice .",
"The hippocampal lysates of 6 and 20 month-old mice were subjected to western blotting ( B ) .",
"The level of hyperphosphorylated tau detected by PHF1 and CP13 were quantified and normalized by TG5-positive total tau ( C ) .",
"Values are means ± s . e . m . ; *p<0 . 05 , **p<0 . 01 , unpaired t-test ( n = 3 ) .",
"( D , E )",
"Decrease of hyperphosphorylated tau by Fcgr2b KO in aged 3x Tg-AD mice .",
"The hippocampal lysates of 15 month-old mice were subjected to western blotting ( D ) .",
"The levels of hyperphosphorylated tau ( PHF1 and CP13 ) were quantified and normalized by TG5-positive total tau ( E ) .",
"Values are means ± s . e . m . ; *p<0 . 05 , unpaired t-test ( n = 3 ) .",
"( F , G )",
"Reduced hyperphosphorylation of tau in hAPP/Fcgr2b KO mice .",
"Hippocampal extracts of 11-month-old WT , Fcgr2b KO , hAPP and hAPP/Fcgr2b KO ( hAPP/KO ) mice were analyzed by western blotting using the indicated antibodies ( D ) .",
"Levels of the phosphorylated tau were measured by densitometric analysis .",
"Values are means ± s . e . m . ; **p<0 . 005 , ***p<0 . 0005 , one-way ANOVA ( n = 3 ) ( E ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 18691 . 006 Instead of using a high dose of synthetic Aβ1-42 , we further utilized a low-dose of cell-derived , naturally secreted Aβ oligomers ( Walsh et al . , 2002 ) .",
"The presence of the secreted , soluble Aβ oligomers in the conditioned medium ( CM ) of 7PA2 cells was confirmed by western blot analysis , and its production was inhibited by treatment with a γ-secretase inhibitor , compound E ( Figure 1—figure supplement 2A ) .",
"We found that tau phosphorylation was increased in the primary hippocampal neurons that were cocultured with 7PA2 cells ( Figure 1D ) , but not in the neurons cocultured with compound E-treated cells ( Figure 1—figure supplement 2B ) .",
"Further , we found that there was no hyperphosphorylation of tau in the Fcgr2b KO neurons that were cocultured with 7PA2 cells ( Figure 1D ) .",
"These results suggest that tau hyperphosphorylation in the cultured neurons was induced by physiologically relevant levels of Aβ oligomers through FcγRIIb .",
"The 3xTg-AD mice develop age-dependent and progressive Aβ and tau pathologies , including memory impairment ( Oddo et al . , 2003 ) .",
"Thus , in order to uncover the role of FcγRIIb in tau pathology , we crossed 3xTg-AD mice with Fcgr2b KO mice to generate double transgenic mice ( 3xTg-AD/Fcgr2b KO ) .",
"Each group of mice was tested for spatial working memory in a Y-maze task .",
"Although the total number of arm entries was not significantly different between groups , Fcgr2b deficiency improved the spatial working memory of 3xTg-AD mice ( Figure 1E ) .",
"In the novel object recognition test , only 3xTg-AD/Fcgr2b KO mice , but not 3xTg-AD mice , discriminated between novel and familiar objects in the second and third trials ( Figure 1F ) , indicating that the recognition memory deficit of 3xTg-AD mice was prevented by Fcgr2b deficiency .",
"Further , 3xTg-AD/Fcgr2b KO mice performed well in the passive avoidance task , whereas 3xTg-AD mice showed a deficit in passive avoidance memory ( Figure 1G ) .",
"These data indicate that FcγRIIb is required for the learning and memory impairments in 3xTg-AD mice .",
"Because FcγRIIb itself did not affect Aβ levels in these mice ( Figure 1—figure supplement 3A ) and the hippocampus of 3xTg-AD mice at nine months of age was plaque-free ( Hirata-Fukae et al . , 2008 ) , we next examined the role of FcγRIIb in tau pathology in the mouse brains .",
"As reported ( Hirata-Fukae et al . , 2008 ) , the pathologic hyperphosphorylation of tau detected by CP13 , PHF1 , and AT180 antibodies was found to be increased in the 3xTg-AD mice showing memory impairment ( Figure 1H , I ) .",
"In contrast , genetic deletion of Fcgr2b in 3xTg-AD mice abolished the hyperphosphorylation of tau .",
"In the brains of 20 month-old 3xTg-AD mice harboring Aβ plaques , tau phosphorylation was markedly elevated as compared to 6 month-old mice ( Figure 1—figure supplement 3B , C ) .",
"FcγRIIb deficiency also prevented the hyperphosphorylation of tau in 15 month-old 3xTg-AD mice ( Figure 1—figure supplement 3D , E ) , indicating that tau phosphorylation mediated by FcγRIIb occurs at the onset of the disease and lasts to the late stage .",
"Similarly , this inhibitory effect of Fcgr2b deficiency on tau phosphorylation was observed in another AD model , hAPP-J20 mice ( Figure 1—figure supplement 3F , G ) .",
"These results indicate that FcγRIIb is crucial for tau hyperphosphorylation in AD model mice showing memory impairment .",
"Because Aβ transduces toxic signals into the neurons via direct interaction with FcγRIIb ( Kam et al . , 2013 ) , we examined whether this interaction is necessary for tau hyperphosphorylation .",
"Compared to Aβ only-treated neurons , addition of the purified hFcγRIIb ectodomain ( hFcγRIIb-ED ) to culture medium blocked Aβ-induced tau hyperphosphorylation in primary cortical neurons ( Figure 2A , B ) .",
"With the notion that both Aβ1-42 oligomers and immunoglobulin complexes share the same binding site on FcγRIIb ( Kam et al . , 2013 ) , we found that the incubation of cultured cells with anti-FcγRIIb antibody ( 2 . 4G2 ) blocked the interaction between Aβ1-42 and HA-tagged FcγRIIb ( Figure 2—figure supplement 1A , B ) .",
"This antibody selectively recognized FcγRIIb but did not crossreact with Aβ1-40 or Aβ1-42 ( Figure 2—figure supplement 1C ) .",
"Consistently , treatment with 2 . 4G2 antibody drastically decreased Aβ-induced tau hyperphosphorylation in mouse primary cortical neurons in a dose-dependent manner ( Figure 2C , D ) .",
"These observations suggest that the interaction between Aβ1-42 and FcγRIIb is an initial step for tau phosphorylation in the cultured neurons . 10 . 7554/eLife . 18691 . 007Figure 2 . Inhibition of Aβ1-42-FcγRIIb interaction blocks Aβ-induced tau phosphorylation and memory impairment .",
"( A , B )",
"Inhibition of Aβ1-42-induced tau phosphorylation by the addition of purified hFcγRIIb-ED protein .",
"Mouse primary cortical neurons were incubated for 24 hr with 1 μM synthetic Aβ1-42 w/wo 50 μg/ml purified hFcγRIIb-ED and cell extracts were subjected to western blotting ( A ) .",
"The levels of phosphorylated tau ( PHF1 , CP13 , and AT180 ) were normalized by total tau ( TG5 ) .",
"Values are means ± s . e . m . ; *p<0 . 05 , **p<0 . 005 , ***p<0 . 0005 , one-way ANOVA ( n = 3 ) ( B ) .",
"( C , D )",
"Prevention of Aβ1-42-induced tau phosphorylation by anti-FcγRIIb antibody ( 2 . 4G2 ) .",
"Mouse primary cortical neurons were pre-incubated with 2 . 4G2 antibody for 2 hr and treated w/wo 1 μM Aβ1-42 oligomers for 24 hr .",
"Cell extracts were analyzed with western blotting using indicated antibodies ( C ) .",
"Levels of phosphorylated tau were quantified by densitometric measurement .",
"Values are means ± s . e . m . ; *p<0 . 05 , **p<0 . 005 , ***p<0 . 0005 , one-way ANOVA ( n = 3 ) ( D ) .",
"( E–G )",
"Suppression of Aβ1-42-induced cognitive deficits by coinjection of anti-FcγRIIb antibody .",
"WT mice ( 8 weeks old ) were i . c . v . -injected with PBS or Aβ1-42 ( 410 pmol ) together w/wo either 2 μg IgG or 2 . 4G2 antibody .",
"The mice ( n = 10 for each group ) were analyzed by Y-maze ( E; **p<0 . 005 , ***p<0 . 0005 , unpaired t-test ) , novel object recognition ( F; **p<0 . 005 , one-way ANOVA ) , and passive avoidance ( G; *p<0 . 02 , **p<0 . 005 , unpaired t-test ) tests as described in the methods .",
"Bars represent means ± s . e . m . ( H ) Inhibition of i . c . v . Aβ1-42-induced tau phosphorylation by anti-FcγRIIb antibody .",
"Brain extracts of the Aβ- and/or antibody-injected mice were subjected to western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 18691 . 00710 . 7554/eLife . 18691 . 008Figure 2—figure supplement 1 . FcγRIIb-antagonizing antibody , 2 . 4G2 , inhibits the interaction of Aβ1-42 with FcγRIIb .",
"( A , B )",
"Inhibition of the interaction between Aβ and FcγRIIb by 2 . 4G2 antibody .",
"SH-SY5Y cells that stably express hFcγRIIb-HA were left untreated or incubated with 1 μM Aβ1-42 for 1 hr in the presence or absence of 2 . 4G2 antibody .",
"Cell lysates were subjected to immunoprecipitation ( IP ) assay using anti-HA antibody ( A ) or anti-Aβ ( Nu-1 ) antibody ( B ) .",
"Murine pre-immune ( Pre ) served as a negative control .",
"The immunoprecipitates were then analyzed with western blotting using anti-Aβ antibody ( A ) or anti-HA antibody ( B ) .",
"( C ) Selectivity of 2 . 4G2 antibody .",
"Synthetic Aβ1-40 , Aβ1-42 oligomers and mouse FcγRIIb-HA-overexpressed HEK293 cell lysates were subjected to western blotting using anti-FcγRIIb ( 2 . 4G2 ) and anti-Aβ antibodies .",
"( D ) Total arm entries of the mice i . c . v-injected with oligomeric Aβ alone or together with either immunoglobulin G ( IgG ) or 2 . 4G2 were analyzed in Y-maze test ( n = 10 mice per group ) .",
"No significant difference in total arm entries was observed among groups .",
"Data are means ± s . e . m . DOI: http://dx . doi . org/10 . 7554/eLife . 18691 . 008 Next , we addressed the effects of 2 . 4G2 antibody on Aβ-induced acute memory impairment in mice .",
"When Aβ1-42 was directly injected into the intracerebroventricular ( i . c . v . ) region of wild-type ( WT ) mice ( Kam et al . , 2013 ) , the mice showed impaired behaviors ( Figure 2E–G ) .",
"Interestingly , coinjection of Aβ1-42 and 2 . 4G2 antibodies significantly rescued the deficits of spontaneous alternation behavior ( Figure 2E ) , object recognition memory ( Figure 2F ) , and passive avoidance memory ( Figure 2G ) .",
"On the other hand , injection with normal immunoglobulin G ( IgG ) did not exhibit such inhibitory effects on memory impairment .",
"There was no difference in total movements between these groups of mice , as reflected by total arm entry in the Y-maze test ( Figure 2—figure supplement 1D ) .",
"From western blot analysis , we found that tau phosphorylation was highly increased in the hippocampus of Aβ1-42-injected mice ( Figure 2H ) .",
"In contrast , Aβ-induced tau phosphorylation was abolished in the hippocampus by coinjection with 2 . 4G2 antibody , but not with IgG ( Figure 2H ) .",
"Thus , we found that the interaction between Aβ1-42 and FcγRIIb is essential for tau hyperphosphorylation and memory impairment in mice .",
"As reported ( Plattner et al . , 2006 ) , we found that tau kinases , such as GSK3β and Cdk5 , were activated for tau phosphorylation in cultured neurons by treatment with Aβ1-42 ( Figure 3—figure supplement 1A , B ) .",
"Notably , Fcgr2b deficiency in primary cortical neurons abrogated the activation of GSK3β and tau phosphorylation triggered by Aβ1-42 , but not that of Cdk5 .",
"Conversely , ectopic expression of FcγRIIb induced tau hyperphosphorylation , detected by PHF1 , CP13 , and AT180 antibodies , in both SH-SY5Y cells and primary cortical neurons ( Figure 3—figure supplement 1C–F ) .",
"Consistently , treatment with SB-415286 , a GSK3β inhibitor , markedly prevented FcγRIIb-induced tau hyperphosphorylation at those epitopes ( Figure 3—figure supplement 1C , E ) , whereas roscovitine , a Cdk5 inhibitor , did not affect tau phosphorylation ( Figure 3—figure supplement 1D , F ) .",
"These observations suggest that FcγRIIb transduces Aβ signal into the neuronal cells to activate GSK3β for tau hyperphosphorylation .",
"We next investigated the mechanism for FcγRIIb transduction of Aβ signal into the neurons for tau phosphorylation , and found that treatment with Aβ1-42 induced phosphorylation at Tyr273 within an immunoreceptor tyrosine-based inhibitory motif ( ITIM ) in the cytosolic region of FcγRIIb in SH-SY5Y cells ( Figure 3A ) .",
"This phosphorylation of FcγRIIb was also detected in Aβ-treated primary cortical neurons , but not in Fcgr2b KO neurons ( Figure 3B ) .",
"Further , addition of purified hFcγRIIb-ED protein prevented Aβ-induced phosphorylation of FcγRIIb-Tyr273 in SH-SY5Y cells ( Figure 3C ) , indicating that FcγRIIb is phosphorylated on Tyr273 after its interaction with Aβ1-42 . 10 . 7554/eLife . 18691 . 009Figure 3 . FcγRIIb Tyr273 phosphorylation is found in AD brains and mediates Aβ-induced tau phosphorylation .",
"( A ) Human FcγRIIb is phosphorylated at Tyr273 by Aβ1-42 .",
"Wild-type and FCGR2B knockdown SH-SY5Y cells ( SH-SY5Y/shFCGR2B ) were incubated with 1 μM Aβ1-42 oligomers and cell extracts were subjected to western blotting using phospho-FcγRIIb and total FcγRIIb antibodies ( left ) .",
"Levels of the phosphorylated FcγRIIb were quantified by densitometric measurement .",
"Values are means ± s . d . ; *p<0 . 05 , **p<0 . 005 , two-tailed t-test ( n = 3 ) ( right ) .",
"( B ) FcγRIIb is phosphorylated by Aβ1-42 in primary cortical neurons .",
"WT and Fcgr2b KO cortical neurons were incubated with 1 μM Aβ oligomers for 24 hr .",
"Cell lysates were immunoprecipitated using FcγRIIb antibody ( 2 . 4G2 ) and analyzed with western blotting using phospho-tyrosine antibodies .",
"( C ) Inhibition of Aβ1-42-induced FcγRIIb phosphorylation by hFcγRIIb-ED protein .",
"SH-SY5Y cells were co-incubated with Aβ1-42 and hFcγRIIb-ED protein .",
"( D ) FcγRIIb , not FcγRIIb Y273F-GFP ( Y273F ) , is phosphorylated at Tyr273 .",
"( E ) FcγRIIb Tyr273 phosphorylation is required for Aβ-induced tau phosphorylation .",
"Fcgr2b KO neurons ( DIV 8 ) were transfected with the indicated constructs , followed by incubation with 1 μM Aβ1-42 .",
"Cell extracts were subjected to western blotting ( left ) .",
"The signals on the blot were quantified and bar graph represents phospho-tau levels normalized by TG5 ( right ) .",
"All data shown are means ± s . e . m . ; *p<0 . 05 , **p<0 . 005 , one-way ANOVA .",
"( F ) FcγRIIb is phosphorylated at Tyr273 in AD brains .",
"Hippocampal homogenates from normal , MCI ( Braak III ) , and AD patients ( Braak V/VI ) were analyzed with western blotting ( left ) .",
"Levels of phosphorylated FcγRIIb and total FcγRIIb were quantified and values are means ± s . e . m . ; **p<0 . 01 , two-tailed t-test ( right ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 18691 . 00910 . 7554/eLife . 18691 . 010Figure 3—figure supplement 1 . FcγRIIb-mediated tau phosphorylation is dependent on GSK3β , not CDK5 . ( A , B ) Inhibition of Aβ-induced GSK3β activation in Fcgr2b KO neurons .",
"Primary cortical neurons from WT or Fcgr2b KO embryos ( DIV 8 ) were incubated with 5 μM Aβ1-42 for 24 hr .",
"Cell extracts were subjected to western blotting using PHF1 , Tau5 , p-GSK3β , GSK3β , p35/p25 , FcγRIIb ( 2 . 4G2 ) and β-actin antibodies ( A ) .",
"The signals of p-GSK3β , GSK3β and p25 in ( A ) were measured by densitometric analysis .",
"Values are means ± s . e . m . ( n = 3 ) .",
"*p<0 . 05 , one-way ANOVA ( B ) .",
"( C-F )",
"Prevention of FcγRIIb-mediated tau hyperphosphorylation by GSK3b inhibitor .",
"SH-SY5Y cells ( C , D ) or primary cortical neurons ( E , F ) were transfected with vehicle or pFCGR2B-flag alone ( E , F ) or together with GFP-tau ( C , D ) for 36 hr and then exposed to the indicated doses of SB-415286 ( SB ) ( C , E ) or roscovitine ( Rosco ) ( D , F ) for 12 hr .",
"Cell extracts were prepared and subjected to western blot analysis using the indicated antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 18691 . 01010 . 7554/eLife . 18691 . 011Figure 3—figure supplement 2 . Phosphorylation of FcγRIIb Tyr273 is required for Aβ neurotoxicity .",
"( A ) Association of the neurotoxic activity of FcγRIIb with ITIM .",
"A schematic diagram showing the structure of FcγRIIb protein .",
"SS; signal sequence , EX; extracellular domain , TM; transmembrane domain , Cyto; cytosolic domain ( left ) .",
"HT22 cells were transiently transfected with pEGFP , FcγRIIb-GFP ( WT ) , FcγRIIb ITIM-deleted form ( 1-270; ΔITIM ) , FcγRIIb cytosolic domain-deleted form ( 1-240; ΔCyto ) or FcγRIIb phosphorylation-defective mutant in ITIM ( Y273F ) .",
"GFP-positive cells showing condensed and fragmented nuclei after staining with Hoechst 33258 were counted as apoptotic cells under a fluorescence microscope .",
"At least 900 cells in 3–4 random spots per sample per condition were counted .",
"Values are means ± s . d . ; n = 3 ( right ) .",
"( B ) A FcγRIIb Y273F mutant prevents Aβ neurotoxicity .",
"SH-SY5Y cells were transfected with WT or FcγRIIb Y273F mutant , and then incubated w/wo oligomeric Aβ1-42 .",
"Bars depict the incidence of cell death .",
"Values are means ± s . d; n = 3 .",
"* , #p<0 . 05 , ** , ##p<0 . 005 , unpaired t-test , compared to cells transfected with pEGFP ( Mock ) and treated with PBS ( * , ** ) or synthetic Aβ ( # , ## ) , respectively .",
"N . S , not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 18691 . 011 By using FcγRIIb mutants , we further characterized the role of FcγRIIb phosphorylation in neuronal cells .",
"We generated several FcγRIIb mutants lacking the ITIM ( ΔITIM ) or cytoplasmic region ( Δcyto ) , or substituting the tyrosine residue with phenylalanine ( Y273F ) , and examined their effects on Aβ1-42 neurotoxicity and tau phosphorylation .",
"Unlike FcγRIIb , ectopic expression of FcγRIIb mutants did not induce neuronal death in mouse hippocampal HT22 cells ( Figure 3—figure supplement 2A ) .",
"Moreover , overexpression of FcγRIIb Y273F mutant , which was not phosphorylated ( Figure 3D ) , blocked Aβ1-42 neurotoxicity to the control level in SH-SY5Y cells , whereas FcγRIIb WT potentiated it ( Figure 3—figure supplement 2B ) , indicating that FcγRIIb-Tyr273 phosphorylation is required for Aβ1-42 neurotoxicity .",
"Next , we examined the role of FcγRIIb phosphorylation in tau phosphorylation using a reconstitution analysis in Fcgr2b KO neurons .",
"Unlike that in Fcgr2b-deficient neurons , reconstitution of the Fcgr2b KO primary cortical neurons with FcγRIIb WT recovered Aβ-induced hyperphosphorylation of tau , detected by PHF1 , CP13 , AT180 , and 12E8 antibodies , as seen in WT neurons ( Figure 3E ) .",
"On the other hand , reconstitution with FcγRIIb-ΔCyto or FcγRIIb-Y273F failed to show tau phosphorylation at those epitopes in response to Aβ1-42 ( Figure 3E ) .",
"These results suggest that the phosphorylation of FcγRIIb-Tyr273 by Aβ1-42 is critical for tau phosphorylation .",
"Even more interestingly , when we analyzed the phosphorylation status of FcγRIIb in the brains of AD patients , we found FcγRIIb phosphorylation on Tyr273 in the hippocampal tissues of five out of six AD patients ( stage V and VI ) , but not in normal and mild cognitive impairment ( MCI ) patients ( stage III ) ( Figure 3F ) .",
"In addition , tau phosphorylation at PHF was observed in four out of the five AD patients with FcγRIIb-Tyr273 phosphorylation-positive brains .",
"As we previously reported , the expression level of FcγRIIb was increased in AD brains ( Figure 3F ) .",
"With the notion that FcγRIIb phosphorylation is required for tau phosphorylation and Aβ1-42 neurotoxicity , it is likely that the phosphorylation of FcγRIIb found in AD brains is associated with tau phosphorylation and neuronal loss during AD pathogenesis .",
"In B cells , the SHIP ( SHIP1 and",
"2 ) is known to bind to the phosphorylated ITIM region of FcγRIIb and inhibit downstream responses triggered by immune receptors ( Ono et al . , 1996; Muraille , 2000 ) .",
"Given that SHIP2 is highly expressed in the brain and SHIP1 is expressed predominantly in hematopoietic cells ( Astle et al . , 2007 ) , we focused on SHIP2 and examined its ability to bind to phosphorylated FcγRIIb in neuronal cells .",
"Immunoprecipitation assays revealed that overexpressed FcγRIIb-GFP bound to SHIP2-His in SH-SY5Y cells , whereas the phospho-defective FcγRIIb ( Y273F ) mutant failed to do so ( Figure 4A ) .",
"Similar results were observed in the reverse immunoprecipitation assay using GFP antibody ( Figure 4B ) .",
"Interestingly , compared to the lack of or weak interaction in untreated control cells , we found a drastic increase in the binding between endogenous SHIP2 and FcγRIIb in SH-SY5Y cells after exposure to Aβ1-42 ( Figure 4C , D ) , suggesting that SHIP2 binds to FcγRIIb in neuronal cells in response to Aβ1-42 . 10 . 7554/eLife . 18691 . 012Figure 4 . SHIP2 is recruited and binds to phosphorylated FcγRIIb in response to Aβ .",
"( A , B )",
"Interaction between phosphorylated FcγRIIb and SHIP2 .",
"SH-SY5Y cells were co-transfected with pSHIP2-His and either pFcγRIIb WT-GFP ( WT ) or pFcγRIIb Y273F-GFP ( YF ) for 36 hr and cell extracts were subjected to immunoprecipitation analysis using anti-His ( A ) and anti-GFP ( B ) antibody , respectively .",
"Whole cell lysates ( WCL ) and the immunoprecipitates were probed by western blotting using anti-GFP and anti-His antibodies .",
"( C , D )",
"Regulated interaction between SHIP2 and FcγRIIb2 in response to Aβ1-42 .",
"SH-SY5Y cells were left untreated or incubated with 1 μM Aβ1-42 oligomers for 24 hr and cell extracts were immunoprecipitated using anti-FcγRIIb antibody ( C ) or anti-SHIP2 antibody ( D ) .",
"( E ) Aβ induces colocalization of FcγRIIb and SHIP2 on the plasma membrane .",
"SH-SY5Y cells were incubated with 1 μM Aβ1-42 for 24 hr and then subjected to immunocytochemical analysis using anti-FcγRIIb and anti-SHIP2 antibodies .",
"Hoechst dye was used for nuclear staining .",
"( F ) Increased targeting of SHIP2 to the plasma membrane by Aβ1-42 .",
"SH-SY5Y cells were treated with 1 μM Aβ1-42 for 24 hr and then subjected to subcellular fractionation assay to separate the plasma membrane from the cytosol .",
"The fractions were analyzed by western blotting .",
"The α-tubulin antibody was used as a marker for the cytosolic fraction ( left ) .",
"The relative expression of SHIP2 at each fraction was quantified by densitometric analysis ( right ) .",
"Values are means ± s . d . ; *p<0 . 05 , two-tailed t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 18691 . 01210 . 7554/eLife . 18691 . 013Figure 4—figure supplement 1 . Expression of SHIP2 in human brain .",
"( A , B )",
"Hippocampal extracts from normal , MCI ( Braak III ) , and AD patients ( Braak V/VI ) were analyzed with western blotting ( A ) .",
"Levels of SHIP2 were quantified by densitometric analysis and bars indicate means ± s . e . m . ; N . S . not significant , one-way ANOVA ( B ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 18691 . 013 Because SHIP2 is known to be localized in the cytosol but to translocate to the cell membrane upon stimulation ( Dyson et al . , 2001; Wang et al . , 2004 ) , we further examined subcellular localization of SHIP2 in neuronal cells exposed to Aβ1-42 .",
"Treatment of SH-SY5Y cells with Aβ1-42 enhanced subcellular localization of SHIP2 to the plasma membrane and thus colocalization of SHIP2 with FcγRIIb ( Figure 4E ) .",
"In addition , subcellular fractionation assays revealed similar results , showing that SHIP2 was enriched in the membrane fraction following Aβ1-42 treatment , while it was detected in both the cytosol and membrane fractions in untreated control cells ( Figure 4F ) .",
"However , SHIP2 expression was not changed by Aβ1-42 treatment in SH-SY5Y cells and in the brains of MCI and AD patients ( Figure 4F andFigure 4—figure supplement 1 ) .",
"Overall , our data indicates that SHIP2 is recruited to the membrane to interact with FcγRIIb in neuronal cells following Aβ1-42 treatment .",
"Because SHIP2 is an inositol phosphatase that dephosphorylates PtdIns ( 3 , 4 , 5 ) P3 to produce PtdIns ( 3 , 4 ) P2 ( Damen et al . , 1996 ) , we examined whether Aβ1-42 affects phosphoinositide metabolism through FcγRIIb and SHIP2 .",
"We measured the levels of PtdIns ( 3 , 4 ) P2 with a protein lipid overlay ( PLO ) assay using the purified His-tagged pleckstrin homology ( PH ) domain of tandem PH domain-containing protein-1 ( TAPP1 ) as a probe ( Dowler et al . , 2002 ) .",
"From the PLO assays using total lipid extracts , we found that the amount of TAPP1-PH-interacting PtdIns ( 3 , 4 ) P2 was increased in SH-SY5Y cells following exposure to Aβ1-42 ( Figure 5—figure supplement 1A ) .",
"In contrast , there was no such increase by Aβ1-42 in SH-SY5Y/FCGR2B or SH-SY5Y/INPPL1 knockdown cells ( Figure 5—figure supplement 1B ) .",
"Conversely , the amount of the general receptor for phosphoinositides-1 ( GRP1 ) -PH-interacting lipids , mainly PtdIns ( 3 , 4 , 5 ) P3 ( Guillou et al . , 2007 ) , was reduced by Aβ1-42 in SH-SY5Y cells , but not in SH-SY5Y/FCGR2B or SH-SY5Y/INPPL1 knockdown cells ( Figure 5—figure supplement 1B ) .",
"Similar patterns of changes in the levels of TAPP1-PH-interacting or GRP1-PH-interacting lipids were also observed in WT primary cortical neurons after treatment with Aβ1-42 ( Figure 5A , B ) .",
"Consistently , these changes were not observed in Fcgr2b KO neurons .",
"These results indicate that phosphoinositide metabolism is affected by the FcγRIIb-SHIP2 axis .",
"When we directly measured the levels of phosphoinositides with an ELISA assay , we observed that the levels of PtdIns ( 3 , 4 ) P2 were increased by 30% in primary cortical neurons after exposure to Aβ1-42 , whereas PtdIns ( 3 , 4 , 5 ) P3 was decreased by 17% ( Figure 5C ) .",
"However , Fcgr2b-deficiency abrogated these changes in PtdIns ( 3 , 4 ) P2 and PtdIns ( 3 , 4 , 5 ) P2 levels .",
"On the other hand , the levels of PtdIns ( 4 , 5 ) P2 were lowered by Aβ1-42 in WT neurons , consistent with a previous report ( Berman et al . , 2008 ) , and also in FcgR2b KO neurons ( Figure 5C ) .",
"We further traced the changes in PtdIns ( 3 , 4 ) P2 using GFP-tagged TAPP1-PH under a fluorescence microscope .",
"While TAPP1-PH-GFP was found in a diffuse pattern in the cytosol of untreated SH-SY5Y cells , treatment with Aβ1-42 enhanced the fluorescence of TAPP1-PH-GFP and concentrated it at the plasma membrane ( Figure 5D ) .",
"In contrast , Aβ-induced accumulation of TAPP1-PH-GFP at the plasma membrane was impaired in SH-SY5Y/FCGR2B knockdown cells ( Figure 5D ) .",
"As reported ( Cheung et al . , 2007 ) , hydrogen peroxide also induced membrane localization of TAPP1-PH-GFP in SH-SY5Y cells .",
"However , this localization was not affected by Fcgr2b deficiency ( Figure 5—figure supplement 1C ) .",
"These observations further support the idea that Aβ1-42 selectively dysregulates PtdIns ( 3 , 4 ) P2 and PtdIns ( 3 , 4 , 5 ) P3 levels in neurons via FcγRIIb . 10 . 7554/eLife . 18691 . 014Figure 5 . FcγRIIb-SHIP2 axis deregulates PtdIns ( 3 , 4 ) P2 metabolism for tau phosphorylation .",
"( A–C )",
"Aβ1-42 increase PtdIns ( 3 , 4 ) P2 levels through FcγRIIb .",
"Primary cortical neurons from WT and Fcgr2b KO embryos were incubated w/wo 1 μM Aβ1-42 oligomers for 24 hr .",
"Total lipids were extracted and analyzed by PLO assay using purified TAPP1-PH and GRP1-PH proteins which bind to PtdIns ( 3 , 4 ) P2 and PtdIns ( 3 , 4 , 5 ) P3 , respectively ( A ) or analyzed by ELISA to quantify PtdIns levels ( C ) .",
"The signals on the blots in ( A ) were quantified by densitometric analysis ( B ) .",
"All data shown are means ± s . d . ; **p<0 . 005 , ***p<0 . 0005 , unpaired t-test .",
"( D ) Aβ1-42 increase PtdIns ( 3 , 4 ) P2 levels at the plasma membrane through FcγRIIb .",
"SH-SY5Y/pSuper and SH-SY5Y/shFCGR2B stable cells were transfected with the GFP-PHTAPP1 probe and stimulated with 1 μM Aβ1-42 oligomers for 24 hr .",
"The fluorescence of GFP-PHTAPP1 was observed by confocal microscopy ( top ) .",
"The fluorescence intensity of the GFP-PHTAPP1 probe in the plasma membrane ( two external peaks ) was quantified after Aβ1-42 treatment ( bottom ) .",
"( E ) Intracellular delivery of PtdIns ( 3 , 4 ) P2 induces tau hyperphosphorylation .",
"Primary cortical neurons were incubated with the indicated concentrations of PtdIns ( 3 , 4 ) P2 w/wo carriers for 24 hr and cell lysates were subjected to western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 18691 . 01410 . 7554/eLife . 18691 . 015Figure 5—figure supplement 1 . The FcγRIIb-SHIP2 axis is required for Aβ-induced PtdIns ( 3 , 4 ) P2 dysregulation for tau phosphorylation .",
"( A ) Aβ oligomers increase TAPP1-PH-bound PtdIns ( 3 , 4 ) P2 levels in primary cortical neurons .",
"Serial dilutions ( 500 , 250 , 125 , 62 . 5 , 32 , and 16 pmol ) of the indicated phosphoinositides were spotted onto nitrocellulose membranes and the membrane was then incubated with purified His-TAPP1-PH protein .",
"His-fusion proteins bound to the phosphoinositides were detected by western blotting using His antibody ( left ) .",
"Primary cortical neurons were incubated with PBS or Aβ oligomers for 24 hr and cell extracts were analyzed by a PLO assay using purified TAPP1-PH protein ( right ) .",
"( B ) Requirement of FcγRIIb and SHIP2 for Aβ-induced PtdIns ( 3 , 4 ) P2 dysregulation .",
"SH-SY5Y/pSuper , SH-SY5Y/shFCGR2B and SH-SY5Y/shINPPL1 knockdown cells were incubated with Aβ1-42 oligomers for 24 hr , and TAPP1-PH-bound PtdIns ( 3 , 4 ) P2 or GRP1-PH-bound PtdIns ( 3 , 4 , 5 ) P3 was then analyzed by a PLO assay .",
"( C ) No difference between SH-SY5Y/pSuper and SH-SY5Y/shFCGR2B cells in H2O2-induced localization of GFP-PHTAPP1 to the plasma membrane .",
"SH-SY5Y/pSuper and SH-SY5Y/shFCGR2B cells were transfected with GFP-PHTAPP1 for 24 hr and treated with 300 μM H2O2 for 1 hr . DOI: http://dx . doi . org/10 . 7554/eLife . 18691 . 01510 . 7554/eLife . 18691 . 016Figure 5—figure supplement 2 . ER stress links SHIP2 to GSK3β for tau phosphorylation .",
"( A ) Cellular delivery of PtdIns ( 3 , 4 ) P2 triggers ER stress and GSK3β activation .",
"Primary cortical neurons ( DIV 7 ) were incubated with 10 μM phosphoinositides with carriers for 24 hr .",
"Cell lysates were subjected to western blotting .",
"( B ) Alleviating ER stress reduces PtdIns ( 3 , 4 ) P2-evoked GSK3β activation and tau hyperphosphorylation .",
"Primary cortical neurons ( DIV 10 ) were preincubated with 3 mM 4-phenylbutyric acid ( 4-PBA ) or 75 μM Salubrinal for 2 hr and treated with 10 μM PtdIns ( 3 , 4 ) P2 for 24 hr .",
"Cell lysates were subjected to western blotting .",
"( C ) Suppression of Aβ-induced ER stress and GSK3β activation by SHIP2 inhibitor .",
"Primary cortical neurons ( DIV 10 ) were preincubated with 10 μM AS1949490 ( AS ) for 2 hr and treated with 1 μM Aβ1-42 oligomers for 24 hr .",
"Cell lysates were subjected to western blotting .",
"( D ) Suppression of Aβ-induced ER stress and GSK3β activation by Inppl1 knockdown .",
"Primary cortical neurons ( DIV 10 ) were infected with control or Inppl1 siRNA-containing lentivirus ( LV-siInppl1 ) for 3 days , and then incubated with 1 μM Aβ1-42 oligomers for 24 hr .",
"Cell lysates were subjected to western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 18691 . 016 To address the important question of whether the increase in PtdIns ( 3 , 4 ) P2 by Aβ1-42 can influence tau phosphorylation , we directly delivered phosphoinositide into living neurons using a carrier ( Ozaki et al . , 2000 ) .",
"Compared to untreated control cells , treatment with PtdIns ( 3 , 4 ) P2 increased tau phosphorylation ( AT180 , CP13 , PHF1 ) in primary cortical neurons in a dose-dependent manner ( Figure 5E ) .",
"Interestingly , the increase in tau hyperphosphorylation was specific to PtdIns ( 3 , 4 ) P2; other phosphoinositides , such as PtdIns ( 4 , 5 ) P2 , PtdIns ( 3 , 5 ) P2 , and PtdIns ( 3 , 4 , 5 ) P3 , failed to do so .",
"Consistent with the activation of GSK3β by Aβ1-42 , PtdIns ( 3 , 4 ) P2 treatment also reduced the inhibitory phosphorylation of GSK3β at Ser9 in neurons ( Figure 5—figure supplement 2A ) .",
"Moreover , PtdIns ( 3 , 4 ) P2 induced the expression of GRP78 , a typical marker of unfolded protein response ( UPR ) , as well ( Figure 5—figure supplement 2A ) .",
"We further found that PtdIns ( 3 , 4 ) P2-induced GSK3β activation and tau phosphorylation were attenuated by the treatment with ER stress inhibitors , such as 4-PBA and Salubrinal , a chemical chaperone and an eIF2α dephosphorylation inhibitor , respectively ( Figure 5—figure supplement 2B ) .",
"We confirmed that ER stress response and GSK3β activation triggered by Aβ were all declined by a SHIP2 inhibitor AS1949490 or lentiviral expression of Inppl1 siRNA ( Figure 5—figure supplement 2C , D ) .",
"Combined with the previous study showing that ER stress stimulates GSK3β activity ( Ren et al . , 2015 ) , these results suggest that an increase in the PtdIns ( 3 , 4 ) P2 level by Aβ1-42 activates GSK3β through ER stress for tau hyperphosphorylation in neuronal cells .",
"We further determined whether SHIP2 , a downstream signal mediator of FcγRIIb , is essential for tau phosphorylation by examining the effects of SHIP2 knockdown .",
"Unlike enhanced tau phosphorylation by Aβ1-42 in control cells , knockdown of SHIP2 expression in SH-SY5Y cells abrogated tau phosphorylation by Aβ1-42 ( Figure 6—figure supplement 1A ) .",
"Conversely , overexpression of SHIP2 alone was sufficient to increase tau hyperphosphorylation in SH-SY5Y cells ( Figure 6—figure supplement 1D ) .",
"On the other hand , these effects were not observed using an activity-dead SHIP2 mutant with Asp608 replaced by Ala ( Nakatsu et al . , 2010 ) ( Figure 6—figure supplement 1E ) .",
"In addition , neuronal cell death triggered by Aβ1-42 treatment or FcγRIIb overexpression was greatly reduced by knockdown of SHIP2 expression in SH-SY5Y cells ( Figure 6—figure supplement 1B , C ) .",
"Together , these data suggest that SHIP2 is a key signal mediator of FcγRIIb in Aβ-induced tau hyperphosphorylation and neurotoxicity .",
"Then , we examined the role of SHIP2 ( INPPL1 ) in memory impairment in vivo using Inppl1 siRNA-expressing lentivirus ( lenti-siInppl1 ) .",
"We confirmed that infection with lenti-siInppl1 reduced SHIP2 levels in both HT22 cells and primary cortical neurons ( Figure 6—figure supplement 2A , B ) .",
"Consistently , we observed that infection with lenti-siInppl1 abrogated Aβ-induced tau phosphorylation ( PHF1 , CP13 ) in primary cortical neurons ( Figure 6A ) .",
"When we stereotaxically injected lenti-siInppl1 into the dentate gyrus of WT and 3xTg-AD mice , we observed that compared to control 3xTg AD mice , lenti-siInppl1-injected 3xTg AD mice showed no significant memory deficits in Y-maze and novel object recognition tests at 20 days after viral injection ( Figure 6B , C ) .",
"When we also monitored tau phosphorylation and SHIP2 levels in brains by western blot analysis , we found that tau phosphorylation and SHIP2 levels were reduced in the hippocampi of lenti-siInppl1-injected 3xTg-AD mice ( Figure 6D ) .",
"The amelioration of memory impairment by lenti-siInppl1 was maintained for 30 days post-injection ( data not shown ) .",
"These results indicate that SHIP2 is critical to memory impairment and tau hyperphosphorylation in 3xTg-AD mice . 10 . 7554/eLife . 18691 . 017Figure 6 . Lentiviral or pharmacological inhibition of SHIP2 prevents Aβ-mediated memory impairments and tau phosphorylation in vivo .",
"( A ) Suppression of Aβ-induced tau phosphorylation by Inppl1 knockdown .",
"Primary cortical neurons were infected with control ( LV-Con ) or Inppl1 siRNA-containing lentivirus ( LV-si Inppl1 ) .",
"On day 3 , cells were incubated with 1 μM Aβ1-42 oligomers for 24 hr and Aβ-induced tau phosphorylation was analyzed by western blotting .",
"( B–D )",
"Prevention of memory impairments and tau hyperphosphorylation by Inppl1 knockdown in 3xTg-AD mice .",
"Time schedule of the memory experiments ( top ) .",
"LV-Com or LV-siInppl1 was injected into the dentate gyrus of 7–8 month-old mice .",
"Beginning 20 days after the injection , mice were subjected to Y-maze ( B ) and novel object recognition ( C ) tests ( n = 7–10 mice per group ) .",
"*p<0 . 05 , **p<0 . 005 , ***p<0 . 0005 , one-way ANOVA .",
"The hippocampal lysates were subjected to western blotting ( D ) .",
"( E ) Suppression of Aβ-induced tau phosphorylation by a SHIP2 inhibitor .",
"Primary cortical neurons were incubated with AS1949490 for 2 hr and then with 1 μM Aβ1-42 for 24 hr .",
"Cell extracts were analyzed with western blotting ( top ) .",
"The signals on the blots were quantified .",
"*p<0 . 01 , **p<0 . 005 , unpaired t-test ( bottom ) .",
"( F–H )",
"SHIP2 inhibitor prevents Aβ-induced memory deficits .",
"The 2-month-old WT mice were injected with Aβ1-42 alone or together with 10 μg AS1949490 ( n = 10 per groups ) .",
"One day later , mice were analyzed with Y-maze ( F ) , novel object recognition ( G ) , and passive avoidance ( H ) tests .",
"**p<0 . 005 , ***p<0 . 0005 , one-way ANOVA .",
"( I ) Suppression of Aβ-induced tau phosphorylation in vivo by SHIP2 inhibitor .",
"The hippocampal extracts were subjected to western blotting .",
"All data are means ± s . e . m . ( n = 3 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 18691 . 01710 . 7554/eLife . 18691 . 018Figure 6—figure supplement 1 . SHIP2 is required for Aβ-induced tau phosphorylation and neurotoxicity .",
"( A ) Inhibition of Aβ-induced tau phosphorylation by INPPL1 knockdown in SH-SY5Y cells .",
"SH-SY5Y/pSuper and SH-SY5Y/shINPPL1 cells were infected with GFP-tau adenovirus for 36 hr and then left untreated or incubated with 5 μM Aβ1-42 for 24 hr .",
"Cell extracts were subjected to western blotting .",
"( B , C )",
"Requirement of SHIP2 in Aβ- and FcγRIIb-induced cell death .",
"SH-SY5Y/pSuper and SH-SY5Y/shINPPL1 cells were treated with oligomeric Aβ1-42 for 48 hr ( B ) or transfected with pFcγRIIb for 36 hr ( C ) , and cell death was then examined .",
"Values are means ± s . d . ; n = 3 .",
"*p<0 . 05 , ***p<0 . 0005 , two-tailed t-test .",
"( D ) Increase of tau phosphorylation by ectopic expression of SHIP2 .",
"SH-SY5Y cells were transiently transfected with pGFP-tau alone or together with pSHIP2 for 36 hr .",
"Cell lysates were subjected to western blotting .",
"( E ) Requirement of inositol phosphatase activity of SHIP2 in the activation of AKT-GSK3β pathway .",
"SH-SY5Y cells were transfected with SHIP2 WT or D608A catalytically inactive mutant for 36 hr .",
"Cell extracts were analyzed by western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 18691 . 01810 . 7554/eLife . 18691 . 019Figure 6—figure supplement 2 . Knockdown of SHIP2 expression with siRNA-carrying lentivirus ( A ) HT22 cells were infected by lentivirus ( LV ) -empty or LV-siInppl1 with MOI 2 or 5 .",
"After 5 days , SHIP2 expression was analyzed by western blotting .",
"( B ) Primary cortical neurons ( DIV 8 ) were infected by LV-empty or LV-siInppl1 for 4 days and knockdown of SHIP2 expression was assessed by western blotting . DOI: http://dx . doi . org/10 . 7554/eLife . 18691 . 01910 . 7554/eLife . 18691 . 020Figure 6—figure supplement 3 . A SHIP2 inhibitor prevents Aβ-induced neurotoxicity .",
"( A ) Inhibition of Aβ-induced cell death in neurons by SHIP2 inhibitor .",
"Primary cortical neurons were preincubated with 10 μM AS1949490 for 2 hr and further treated with Aβ1-42 for 48 hr .",
"Cell viability was determined using Calcein-AM assay .",
"Data are means ± s . d . ( n = 3 ) ; **p<0 . 001 , One-way ANOVA .",
"( B ) Total arm entries of AS1949490-injected mice were analyzed in Y-maze test ( n = 10 per groups ) .",
"Data are means ± s . e . m . DOI: http://dx . doi . org/10 . 7554/eLife . 18691 . 020 In addition , we assessed the impact of pharmacological SHIP2 inhibition on Aβ-induced tau phosphorylation and memory impairment using a SHIP2-selective inhibitor , AS1949490 , which is a 30-fold more potent inhibitor against SHIP2 than SHIP1 ( Suwa et al . , 2009 ) .",
"Treatment of primary cortical neurons with AS1949490 inhibited Aβ-induced tau hyperphosphorylation and neuronal cell death ( Figure 6E and Figure 6—figure supplement 3A ) .",
"When we examined the effect of AS1949490 on the memory impairment triggered by i . c . v . -injected Aβ1-42 in mice , we directly delivered AS1949490 into mouse brains because of its poor bioavailability ( Suwa et al . , 2010 ) .",
"Behavioral tests following the i . c . v . injection of sub-lethal doses of Aβ1-42 and AS1949490 revealed that AS1949490 significantly rescued Aβ1-42-induced impairments of spatial working memory ( Figure 6F ) , object recognition memory ( Figure 6G ) , and passive avoidance memory ( Figure 6H ) .",
"Total movements , determined by arm entries in the Y-maze test , were not significantly different between the groups of mice ( Figure 6—figure supplement 3B ) .",
"Moreover , from western blot analysis of the mouse hippocampal tissues , we found that AS1949490 suppressed Aβ1-42-induced tau phosphorylation ( PHF1 , CP13 , AT180 ) ( Figure 6I ) .",
"Taken together , these observations support the view that pharmacological manipulation of SHIP2 is amenable to the development of AD therapeutics targeting Aβ1-42-induced tau phosphorylation and memory impairment ."
],
[
"Despite tremendous efforts showing that Aβ plays a central role in the pathogenesis of AD , including memory impairment , synaptic loss , and neuronal cell death ( Cleary et al . , 2005; LaFerla et al . , 2007 ) , mechanistic understanding of tau phosphorylation in Aβ-induced memory deficits remains poor ( Rapoport et al . , 2002; Roberson et al . , 2007; Shipton et al . , 2011 ) .",
"To our knowledge , this is the first study showing crucial mediator , the FcγRIIb-SHIP2 axis , which is responsible for Aβ-induced tau phosphorylation , memory impairment , and neuronal loss in AD models .",
"Because the interaction of Aβ species , especially oligomeric Aβ1-42 , with FcγRIIb accounts for such neuropathogenic defects of AD as an initiation step , the selective interaction of oligomeric Aβ1-42 with FcγRIIb may hint at why Aβ1-42 , but not Aβ1-40 , is important in tau pathology ( Oddo et al . , 2008; Kam et al . , 2013 ) .",
"Consequently , inhibition of the interaction between FcγRIIb and Aβ using anti-FcγRIIb antibody prevents Aβ-induced tau phosphorylation and memory deficits .",
"In general , Aβ oligomers play a key role in AD pathogenesis , while soluble Aβ oligomers are still heterogeneous , including low or high n oligomers , and the proposals on which species of Aβ oligomers are responsible for the pathogenesis are a little in debate ( reviewed in Benilova et al . , 2012 ) .",
"We have here used 3 different sources of Aβ oligomers; synthetic Aβ oligomers , naturally secreted Aβ oligomers ( 7PA2 cells ) , and Aβ of 3xTg-AD model mice .",
"Although synthetic Aβ oligomers are well-characterized and have been used widely for neurotoxicity , the acting concentration of synthetic Aβ ( μM range ) is relatively higher than that in AD brains .",
"Compared to synthetic Aβ oligomers , conditioned medium from 7PA2 cells mainly contains not only Aβ dimers and trimers but also the different pools of Aβ oligomers , including zeta peptide , and show more potent neurotoxic properties ( nM range ) ( Qi-Takahara , 2005; Haass and Selkoe , 2007 ) .",
"Moreover , the oligomers generated in 3xTg-AD mouse brain may be more complicated and needs to be identified .",
"Nonetheless , we propose here that FcγRIIb plays a crucial role in tau phosphorylation and neurotoxicity in vitro and in vivo in response to these species of Aβ , probably a certain common species among the different sources of Aβ oligomers .",
"Given that FcγRIIb was initially reported as a hematopoietic receptor which is mainly expressed in B cells , macrophages , and neutrophils ( Nimmerjahn and Ravetch , 2008 ) , our data using 3xTg-AD/FcγRIIb KO mice raised a possibility that FcγRIIb in non-neuronal cells might contribute to APP/Aβ-induced tau pathologies via neuroinflammatory function .",
"As expected , we observed that microglia was activated in 3xTg-AD mice and this activation was also reduced significantly in the cortex and marginally in the hippocampus by FcγRIIb deficiency ( data not shown ) .",
"Recently , however , we and other colleagues identified that both FcγRIIb mRNA and protein are also expressed in neurons ( Figure 1—figure supplement 1; Cahoy et al . , 2008; Suemitsu et al . , 2010; Kam et al . , 2013 ) , though FcγRIIb expression in neurons is low compared to astrocytes and immune tissues .",
"Notably , its expression is increased at least several folds in primary neurons after exposure to Aβ1-42 and in AD brain ( Kam et al . , 2013 ) .",
"In addition , transgenic expression of the FcγRIIb mutant lacking its cytoplasmic domain in the neurons blocked memory impairment in 3xTg AD mice ( data not shown ) .",
"Moreover , the results showing that Aβ-induced tau phosphorylation was prevented by FcγRIIb deficiency , antagonistic FcγRIIb antibody , or SHIP2 inhibitor in primary cultured neurons assure neuronal function of the FcγRIIb-SHIP2 axis .",
"Together , we believe that the inflammation mediated by FcγRIIb in 3xTg-AD mice is not likely a major cause of the memory impairment but contributes to the aggravation of memory impairment in the mice .",
"Unlike other FcγRs that have an immunoreceptor tyrosine-based activating motif ( ITAM ) , FcγRIIb has a unique ITIM in its cytoplasmic tail and thus acts as an inhibitory receptor in B cells ( Okun et al . , 2010 ) .",
"Excitingly , the binding of extracellular Aβ1-42 to FcγRIIb induces phosphorylation in the ITIM of FcγRIIb in neuronal cells .",
"We observed that Lyn kinase is expressed in neurons and that knockdown of LYN expression blocks Aβ-induced FcγRIIb phosphorylation and neurotoxicity ( data not shown ) .",
"Thus , it is likely that Lyn phosphorylates the ITIM of FcγRIIb in neuronal cells in response to Aβ1-42 .",
"Moreover , we also observed this phosphorylation of FcγRIIb in AD brains ( stage V and VI ) in which tau was highly phosphorylated .",
"Then , how is FcγRIIb different from other Aβ receptors ?",
"It is reasonable to propose that different mechanisms or even the same mechanism exerts multiple effects at different stages of disease progression ( De Strooper and Karran , 2016 ) .",
"For instance , RAGE is now believed to mainly function to transport Aβ in the blood brain barrier ( Deane et al . , 2003 ) and ABAD acts for mitochondrial toxicity as an intracellular binding partner of Aβ ( Lustbader , 2004 ) .",
"In case of PrPc , it's debatable whether it is involved in Aβ-induced memory impairments and thus needs to be further characterized ( Balducci et al . , 2010; Gimbel et al . , 2010; Cissé et al . , 2011b ) .",
"Further , compared to those receptors , our observations that the phosphorylation of FcγRIIb at tyrosine 273 is found in the brain of AD patients and is required for both oligomeric Aβ neurotoxicity and tau hyperphosphorylation can make it distinct from other Aβ-binding receptors .",
"In the case of PirB that shares structural similarity with FcγRIIb and also acts as an Aβ receptor for synaptic plasticity , the phosphorylation of PirB is not associated with Aβ signaling ( Kim et al . , 2013 ) .",
"Thus , we believe that FcγRIIb facilitates tau phosphorylation and neuronal loss in AD brains , consistent with the proposed role of tau in AD pathogenesis , such as severe memory impairment and neuronal loss ( Ballatore et al . , 2007 ) .",
"Interestingly , we show that SHIP2 is a key mediator in delivering the toxic signal of Aβ1-42 to tau by binding to the phosphorylated FcγRIIb .",
"Many studies on SHIP2 have focused on its inhibitory effect on insulin signaling ( Ishihara et al . , 1999; Wada et al . , 2001 ) .",
"Inppl1 transgenic mice show impaired insulin signaling and glucose intolerance , while Inppl1 KO mice are highly resistant to weight gain on a high-fat diet ( Sleeman et al . , 2005; Kagawa et al . , 2008 ) .",
"Thus , targeting SHIP2 is thought to be a promising approach for the treatment of other diseases , including type 2 diabetes ( Vanhanen et al . , 2006 ) .",
"Given that metabolic syndrome , including insulin resistance and glucose intolerance , is highly associated with AD ( Vanhanen et al . , 2006; Razay et al . , 2007 ) , we speculate that SHIP2 may play a dual role in AD and diabetes .",
"In support of this , a SHIP2 inhibitor exhibited an ameliorating effect on the impaired memory function of diabetic mice ( Soeda et al . , 2010 ) .",
"Several single-nucleotide polymorphisms ( SNPs ) of SHIP2 are involved in metabolic syndrome ( Kaisaki et al . , 2004; Kagawa et al . , 2005 ) ; thus , it will be very interesting to evaluate the effect of these SHIP2 SNPs on AD pathogenesis in our FcγRIIb-SHIP2 axis .",
"Dysregulation of phosphoinositide metabolism is increasingly recognized as important in various diseases , such as cancer , diabetes , and myopathy ( Wymann and Schneiter , 2008; Kok et al . , 2009 ) .",
"The phosphoinositide pool is also altered in AD ( Stokes and Hawthorne , 1987; Jope et al . , 1994 ) .",
"In particular , this work shows that a change in the PtdIns ( 3 , 4 ) P2 level is implicated in AD pathogenesis through tau hyperphosphorylation .",
"PtdIns ( 3 , 4 ) P2 is scarce under normal conditions and increases through signaling ( Lemmon , 2008 ) .",
"While PtdIns ( 3 , 4 ) P2 and PtdIns ( 3 , 4 , 5 ) P3 are known to overlap to some degree in their functions , they apparently have distinct roles in neurodegeneration .",
"Recently , it was shown that PtdIns ( 3 , 4 ) P2 , but not PtdIns ( 3 , 4 , 5 ) P3 and PtdIns ( 4 , 5 ) P2 , potentiates glutamate-induced cell death in neurons ( Sasaki et al . , 2010 ) .",
"In addition , PtdIns ( 3 , 4 ) P2 phosphatase INPP4A deficiency shows increased level of PtdIns ( 3 , 4 ) P2 and leads to neurodegeneration in brains ( Sasaki et al . , 2010 ) .",
"We also observed that PtdIns ( 3 , 4 ) P2 selectively induces tau hyperphosphorylation in neurons .",
"In addition , tensin homolog deletion on chromosome 10 ( PTEN ) also dephosphorylates PtdIns ( 3 , 4 , 5 ) P3 at position 3 and generates PtdIns ( 4 , 5 ) P2 ( Li et al . , 1997 ) .",
"The reduced level of PTEN in AD brains correlates with an increase in tau phosphorylation and , thus , dysregulation of PTEN also contributes to tau pathology ( Kerr et al . , 2006; Zhang et al . , 2006 ) .",
"More recently , genetic reduction of synaptojanin 1 ( Synj1 ) , the major PtdIns ( 4 , 5 ) P2 phosphatase in the brain , ameliorates behavioral and synaptic deficits and accelerates Aβ clearance through rescuing PtdIns ( 4 , 5 ) P2 deficiency ( McIntire et al . , 2012; Zhu et al . , 2015 ) .",
"Because oligomeric Aβ decreases PtdIns ( 4 , 5 ) P2 levels in AD ( Berman et al . , 2008 ) , as we also observed , there is a possibility that SHIP2 activation together with either Synj1 upregulation or PTEN downregulation induces the imbalance in the phosphoinositide pool between PtdIns ( 3 , 4 ) P2 and PtdIns ( 4 , 5 ) P2 , and leads to AD pathogenesis .",
"In conclusion , the FcγRIIb-SHIP2 signaling axis provides the missing link between Aβ and tau pathologies .",
"Notably , Aβ1-42 induces FcγRIIb phosphorylation to recruit SHIP2 , leading to disruption of phosphoinositide metabolism for tau hyperphosphorylation and memory impairment in neurons and AD model mice .",
"Together with the Aβ-lowering strategy , our results provide new ways for AD therapeutics to rescue Aβ and tau pathology:",
"i ) selective inhibition of the interaction between FcγRIIb and Aβ1-42 ,",
"ii ) inhibition of a kinase ( i . e . , Lyn ) which phosphorylates FcγRIIb upon Aβ1-42 stimulation , and",
"iii ) inhibition of SHIP2 which disrupts phosphoinositide metabolism ."
],
[
"WT ( C57BL/6 ) , Fcgr2b KO C57BL/6 ( Takai et al . , 1996 ) , 3xTg-AD and hAPP ( J20 , The Jackson Laboratory , Bar Harbor , ME ) mice were used .",
"All experiments involving animals were performed according to the protocols approved by the Seoul National University Institutional Animal Care and Use Committee ( SNU IACUC ) guidelines .",
"For biochemical assays , mice were anesthetized and the brains were rapidly dissected into subregions ( cortex and hippocampus ) and snap-frozen at –80°C .",
"The hemibrains were homogenized in lysis buffer [20 mM Tris-HCl ( pH 7 . 4 ) , 150 mM NaCl , 1% Triton X-100 , protease inhibitor cocktail] and centrifuged at 14 , 000 g for 20 min at 4°C .",
"Supernatant was then collected and the protein concentration was determined using the Bradford method ( GE Healthcare ) .",
"Hippocampal tissues from AD ( Braak V-VI ) ( mean age , 80 . 2 ± 10 . 6 years; mean post-mortem intervals , 16 . 3 ± 6 . 7 hr ) , MCI ( Braak III ) ( mean age , 84 . 2 ± 2 . 9 years; mean post-mortem intervals , 20 . 1 ± 8 . 0 hr ) and non-AD patients ( mean age , 67 . 8 ± 16 . 5 years; mean post-mortem intervals , 20 . 1 ± 5 . 8 hr ) were kindly provided by the Harvard Brain Tissue Resource Center ( McLean Hospital ) .",
"Hippocampal tissues of AD patients were homogenized in ice-cold Tris-buffered saline ( TBS ) buffer [20 mM Tris-HCl ( pH 7 . 4 ) , 150 mM NaCl and protease inhibitor cocktails] .",
"The homogenates were clarified by centrifugation at 14 , 000 g for 20 min at 4°C , aliquoted and stored at –80°C until use .",
"The supernatants were subjected to SDS-PAGE .",
"Seven to 8-month-old WT and 3xTg-AD mice were deeply anesthetized with a mixture of ketamine ( 100 mg/kg ) and xylazine ( 10 mg/kg ) .",
"Lentivirus expressing shRNA against mouse Inppl1 ( 5′-GAA GGG AGG GCA CGT TAA TTT-3′ ) ( Sigma-Aldrich ) was used for injection and pLKO . 1-Neo-CMV-tGFP non-target virus was used as a control .",
"The lentivirus ( 1 . 1 × 1099 TU/ml , TU; transduction unit ) was stereotaxically injected bilaterally into the dentate gyrus ( 2 μl per hemisphere at 0 . 4 μl/min ) with the following coordinates: anteroposterior = 2 . 1 mm from bregma , mediolateral = ± 1 . 8 mm , dorsoventral = 2 . 0 mm .",
"After the injection , the cannula was maintained for an additional 5 min for a complete absorption of the virus .",
"Behavior tests were performed 20 and 30 days after the injection .",
"For western blot analysis , brains were removed 25 days after viral injection , hippocampal region was dissected and its protein samples were prepared as described above .",
"Intracerebroventricular injection of PBS or Aβ1-42 ( Sigma-Aldrich , St . Louis , MO ) was performed as described previously ( Kam et al . , 2013 ) .",
"In the case of coinjection experiments , oligomeric Aβ1-42 was incubated alone or together with AS1949490 , IgG or 2 . 4G2 antibody before use .",
"Behavior tests for double transgenic or injected mice were performed as described previously ( Kam et al . , 2013 ) .",
"All apparatus and objects were cleaned with 70% ethanol before and after each trial .",
"In Y-maze test , the mice were placed in the end of one arm ( 32 . 5 cm length × 15 cm height ) of apparatus and allowed to move freely for 7 min .",
"When the all four paws were into the arm , the entry was counted .",
"The percentage of spontaneous alternations was calculated as the ratio of the number of successful alternations to the number of total alternations .",
"In novel object recognition test , the mice were habituated in a chamber ( 22 cm wide × 27 cm long × 30 cm high ) for 7 min with 24 hr intervals .",
"In training trial #1 ( 2 days after habituation ) , the mice were exposed to two objects and allowed to explore freely for 7 min .",
"In the testing trial #2 ( a day after training ) , one of the familiar objects was replaced to a novel object and recognition was counted for 7 min .",
"The same test was repeated after 24 hr , but with another novel object ( trial #3 ) .",
"The object recognition was defined as spending time with orienting toward the object in a distance of 1 cm or less , sniffing the object or touching with the nose .",
"The passive avoidance test was done in an apparatus consist of a light and dark compartment ( 20 × 20 × 20 cm each ) separated by a guillotine door .",
"The mice were allowed to explore the box for 5 min with the door open for habituation , and then returned to home cage .",
"After 24 hr , the mice were placed into the light compartment for conditioning .",
"The door was closed after entering into the dark room and then an electric foot shock ( 0 . 25 mA , 2 s ) was delivered by the floor grids .",
"The latency time for mice to enter the dark room was measured with a 5 min cut-off after 24 hr .",
"Synthetic Aβ1-42 oligomers were prepared from lyophilized monomers ( rPeptide , Bogart , GA ) .",
"The hydroxyfluroisopropanol ( HFIP ) -treated Aβ1-42 peptide was dissolved in dimethylsulfoxide ( DMSO ) and then diluted in phosphate-buffered saline ( PBS ) .",
"The stock solution was incubated at 4°C for 24 hr and stored at –80°C until use .",
"Before use , the solution was centrifuged at 12 , 000 g for 10 min and the supernatant was used as an oligomeric Aβ ( ADDLs ) .",
"The oligomeric status of Aβ1-42 was evaluated by western blot analysis and atomic force microscopy ( Kam et al . , 2013 ) .",
"The conditioned medium of 7PA2-CHO cells ( kindly provided by Dr . D . J . Selkoe , Harvard Medical School ) were collected and used as a naturally secreted Aβ oligomers .",
"Aβ levels in the 3xTg-AD and 3xTg-AD/Fcgr2b KO mice were analyzed using a sandwich enzyme-linked immunosorbent assay ( ELISA ) kit ( Invitrogen , Carlsbad , CA ) following the manufacturer’s instructions .",
"Briefly , the mice were anesthetized and the brains were microdissected .",
"The hippocampus was carefully isolated and homogenized in 10 volumes of ice-cold guanidine buffer ( 5 M guanidine-HCl/50 mM Tris-Cl , pH 8 . 0 ) and then mixed for 3 hr at room temperature .",
"The brain homogenates were further diluted 1:10 with cold reaction buffer ( 5% BSA , 0 . 03% Tween-20 , and 5 mM EDTA in PBS supplemented with protease inhibitor cocktail ) and then centrifuged at 16 , 000 g for 20 min at 4°C .",
"The diluents were mixed 1:1 with standard dilution buffer in the assay kit .",
"Primary cortical and hippocampal neurons were cultured from embryonic day 17 ( E17 ) mice .",
"The neurons were plated on poly-L-lysine ( 0 . 01% in 100 mM borate buffer , pH 8 . 5 ) -coated glass coverslips and maintained in neurobasal medium containing 2% B-27 supplement ( Invitrogen ) and 0 . 5 mM L-glutamine ( Invitrogen ) .",
"Half of the medium was exchanged every 3 days .",
"Primary neurons were not authenticated , and were not tested for mycoplasma .",
"SH-SY5Y , HEK293T and CHO cells ( ATCC , Manassas , VA ) , which were authenticated by ATCC and were negative for mycoplasma , were cultured in DMEM ( HyClone , Logan , UT ) supplemented with 10% fetal bovine serum ( FBS ) ( HyClone ) , penicillin and streptomycin ( Invitrogen ) .",
"Cells were grown at 37°C under an atmosphere of 5% CO2 .",
"Primary neurons were transfected using Lipofectamine 2000 reagent ( Invitrogen ) , whereas other cells were transfected using Polyfect reagent ( Qiagen , Germany ) according to the manufacturer’s instructions .",
"If required , cells were treated with SB-415286 , roscovitine ( Sigma-Aldrich ) or AS1949490 ( Tocris , United Kingdom ) as indicated .",
"All primers used in this study are listed in Supplementary file 1 .",
"Human FCGR2B cDNAs was amplified by PCR from a human brain cDNA library and subcloned into pEGFP-N1 vector .",
"The cDNAs of FCGR2B deletion mutants , ΔCyto , ΔITIM and ΔC-term was generated by PCR and subcloned into pEGFP-N1 vector .",
"A point mutant of FCGR2B ( Y273F ) was generated by site-directed mutagenesis .",
"All mutants were confirmed by DNA sequencing analysis .",
"Human FCGR2B and human INPPL1 shRNAs were synthesized , annealed and cloned into the pSUPER-neo vector .",
"Human tau ( 0N4R ) cDNAs were subcloned into pcDNA3-HA and pEGFP-C1 vector as described previously ( Park et al . , 2012 ) .",
"His-tagged mouse Ship2 cDNA is a generous gift from Dr . M . G . Tomlinson ( University of Birmingham , UK ) .",
"GFP-tagged mouse Ship2 and D608A mutant were kindly provided by Dr . P . De Camilli ( Yale University ) .",
"SH-SY5Y cell were transfected with pcDNA3-HA , pFCGR2B-HA , pSuper-neo , pFCGR2B shRNAs or pINPPL1 shRNAs for 36 hr and then cultivated in the selection medium containing 1 mg/ml G418 ( Invitrogen ) for at least two weeks .",
"A single cell was further cultivated to form stable cell colony and the expression of each cell lines was analyzed by western blotting and reverse transcriptase ( RT ) PCR .",
"Cells were lyzed in lysis buffer ( 50 mM Tris-HCl pH 7 . 4 , 30 mM NaCl , 1% Triton X-100 , 0 . 1% SDS , 1 mM EDTA , 1 mM PMSF , 1 mM Na3VO4 , 1 mM NaF , 1 μg/ml each of aprotinin , leupeptin and pepstatin A ) .",
"The lysates were centrifuged at 14 , 000 g for 10 min at 4°C and the supernatant was separated by SDS-PAGE and blotted onto PVDF membrane .",
"The blots were blocked for 1 hr at room temperature and incubated with following antibodies: anti-phospho-FcγRIIb , anti-FcγRIIb ( Epitomics , Burlingame , CA ) , anti-Aβ ( 4G8 , Signet , Dedham , MA ) , anti-phospho-tau ( AT180 and AT100 , Innogenetics , Alpharetta , GA ) , anti-NSE ( Zymed , South San Francisco , CA ) , anti-phospho-GSK3β , anti-p35/25 ( Cell signaling , Danvers , MA ) , anti-GSK3β ( BD Biosciences , San Jose , CA ) , anti-SHIP2 , anti-mFcγRIIb , anti-GFP , anti-His ( Santa Cruz Biotechnology Inc . , Dallas , TX ) , anti-α-tubulin and anti-β-actin ( Sigma-Aldrich ) , Nu-1 ( kindly provided by Dr . W . L . Klein , Northwestern University ) , PHF1 , CP13 and TG5 ( a generous gifts from Dr . P . Davies , Albert Einstein College of Medicine ) , and 12E8 ( a generous gift from Dr . P . Seubert ( Elan Pharmaceuticals ) .",
"Membranes were rinsed three times with TBS-T ( 10 mM Tris-Cl , pH 7 . 5 , 150 mM NaCl , 0 . 1% Tween-20 ) , further incubated for 1 hr with peroxidase-conjugated secondary antibodies and visualized using ECL detection system .",
"Mouse primary cortical or hippocampal neurons were fixed in 4% paraformaldehyde ( PFA ) ( Sigma-Aldrich ) for 10 min , rinsed three times with PBS and then permeabilized with 0 . 1% Triton X-100 in PBS .",
"After blocking with 5% BSA in PBS , neurons were incubated overnight at 4°C with the following antibodies: AT180 ( 1:200 ) , AT8 ( 1:200 ) , anti-NSE ( 1:200 ) , anti-FcγRIIb ( 1:500 ) and anti-SHIP2 ( 1:250 ) .",
"After rinsing three times with PBS , cells were incubated with FITC- or TRITC-conjugated secondary antibodies ( Jackson Laboratory Inc . ) at room temperature for 1 hr .",
"The coverslips were placed with mounting solution ( Sigma-Aldrich ) and observed on a confocal laser scanning microscope ( Carl Zeiss Inc . , Thornwood , NY ) .",
"For endogenous immunoprecipitation ( IP ) assay , Aβ1-42-treated SH-SY5Y cell extracts were incubated with anti-FcγRIIb or anti-SHIP2 antibodies in IP buffer [50 mM Tris-Cl ( pH7 . 4 ) , 150 mM NaCl , 1% Triton X-100 , 1 mM EDTA , a mixture of protease inhibitors] for 12 hr at 4°C , and then pulled-down by protein G-Sepharose beads ( GE Healthcare , United Kingdom ) .",
"For co-immunoprecipitation assay , HEK293T cells which transiently overexpressed His-tagged SHIP2 with either GFP-tagged FcγRIIb or FcγRIIb Y273F mutant were lyzed and incubated with anti-GFP or anti-His antibodies for 12 hr at 4°C , and then pulled-down by protein G-Sepharose beads .",
"After a short centrifugation , the beads were washed three times with IP buffer and subjected to western blotting .",
"SH-SY5Y cells treated with Aβ1-42 were harvested with the buffer ( 20 mM Tris-HCl pH7 . 5 , 150 mM NaCl , 0 . 1% Triton X-100 , 1 mM EDTA ) and then mechanically disrupted using a 26-gauge needle with passing it 20 times .",
"Cell lysates were centrifuged at 1000 g for 10 min at 4°C to remove the nuclei or unbroken cells .",
"The supernatant was collected into new tube and again centrifuged at 100 , 000 g for 1 hr at 4°C with Beckman SW41 rotor .",
"The pellet was resuspended in lysis buffer and used as a crude membrane fraction , whereas the supernatant used as a cytosolic fraction .",
"The separated fractions were confirmed with western blot analysis using anti-FcγRIIb and anti-α-tubulin antibodies for the membrane and cytosolic markers , respectively .",
"The lipid extraction from primary cortical neurons or SH-SY5Y cells was performed as described ( Gray et al . , 2003 ) .",
"After stimulation , cells were harvested with ice-cold 0 . 5 M trichloroacetic acid ( TCA ) solution , standing on ice for 5 min and centrifuged at 200 g for 5 min .",
"The pellet was washed with 5% TCA with 1 mM EDTA solution .",
"Neutral lipids were extracted from the pellet with a 2:1 solution of methanol and chloroform , followed by vigorous vortexing for 10 min at room temperature .",
"The extracts were centrifuged at 200 g for 5 min , and the acidic lipids were then extracted .",
"A 80:40:1 solution of methanol , chloroform and 12 M HCl was added to the pellet and vortexed for 15 min at room temperature , and then centrifuged at 200 g for 5 min .",
"The 750 μl of supernatant was transferred to a new tube and added with 250 μl of chloroform and 450 μl of 0 . 1 M HCl .",
"After mixing , the samples were centrifuged to separate the organic and aqueous phases and the lower organic phase was collected and dried under vacuum .",
"The lipids were then resuspended by sonication in a water bath with an appropriate buffer ( 1:2:0 . 8 solution of chloroform , methanol and water for PLO assay , or PBS-T buffer for ELISA ) .",
"For the purification of recombinant proteins , the PH domain of TAPP1 and GRP1 was inserted into pET-28a vector .",
"E . coli BL21 cells were transformed with the plasmids and cultured to reach an OD600 of 0 . 6 , before induction with 1 mM IPTG .",
"After incubation for 18 hr at 16°C , cells were harvested and lyzed by sonication .",
"His-fused TAPP1-PH and GRP1-PH were purified from cell lysates using Ni-NTA chelating agarose CL-6B ( Peptron , Korea ) .",
"PLO assays were performed as described previously with minor modification ( Dowler et al . , 2002 ) .",
"Lyophilized PtdIns ( 3 , 4 ) P2 , PtdIns ( 4 , 5 ) P2 and PtdIns ( 3 , 4 , 5 ) P3 diC16 ( Echelon , Salt Lake City , UT ) were reconstituted in a 1:2:0 . 8 solution of chloroform , methanol and water , and used as positive controls .",
"The lipid extracts from the cells were serially diluted and spotted on PVDF membranes which were pre-wetted in methanol , washed in TBS-T buffer and then air-dried .",
"The membranes were dried completely and blocked with 3% BSA in TBS-T buffer for 1 hr .",
"The blots were incubated overnight at 4°C with gentle rocking in the fresh blocking buffer containing purified 10 μM His-TAPP1-PH or His-GRP1-PH .",
"The membranes were washed 5 times over 50 min in TBS-T buffer and incubated with anti-His antibody for 1 hr at room temperature .",
"The membranes were further incubated for 1 hr with peroxidase-conjugated secondary antibodies and bound proteins were visualized using ECL detection system .",
"PtdIns ( 3 , 4 ) P2 , PtdIns ( 4 , 5 ) P2 or PtdIns ( 3 , 4 , 5 ) P3 levels were quantified by ELISA kit ( Echelon ) following the manufacturer’s instructions .",
"The synthetic phosphoinositides diC16 were incubated with histone carriers ( Echelon ) with a 0 . 5–3:1 molar ratio for 15 min with a vigorous vortexing .",
"The histone-phosphoinositides complex was diluted 1:10 with neurobasal media and added to primary cortical neurons .",
"The phosphoinositides without carriers and the only carriers without phosphoinositides were used as negative controls .",
"Statistical analyses were performed with GraphPad Prism software .",
"Differences between two means were assessed by paired or unpaired t-test .",
"Differences among multiple means were assessed by one-way ANOVA , followed by Tukey’s post-hoc test .",
"Error bars represent s . d . or s . e . m . as indicated ."
]
] | [
"Amyloid-β ( Aβ ) -containing extracellular plaques and hyperphosphorylated tau-loaded intracellular neurofibrillary tangles are neuropathological hallmarks of Alzheimer's disease ( AD ) .",
"Although Aβ exerts neuropathogenic activity through tau , the mechanistic link between Aβ and tau pathology remains unknown .",
"Here , we showed that the FcγRIIb-SHIP2 axis is critical in Aβ1-42-induced tau pathology .",
"Fcgr2b knockout or antagonistic FcγRIIb antibody inhibited Aβ1-42-induced tau hyperphosphorylation and rescued memory impairments in AD mouse models .",
"FcγRIIb phosphorylation at Tyr273 was found in AD brains , in neuronal cells exposed to Aβ1-42 , and recruited SHIP2 to form a protein complex .",
"Consequently , treatment with Aβ1-42 increased PtdIns ( 3 , 4 ) P2 levels from PtdIns ( 3 , 4 , 5 ) P3 to mediate tau hyperphosphorylation .",
"Further , we found that targeting SHIP2 expression by lentiviral siRNA in 3xTg-AD mice or pharmacological inhibition of SHIP2 potently rescued tau hyperphosphorylation and memory impairments .",
"Thus , we concluded that the FcγRIIb-SHIP2 axis links Aβ neurotoxicity to tau pathology by dysregulating PtdIns ( 3 , 4 ) P2 metabolism , providing insight into therapeutic potential against AD ."
] | [
"In Alzheimer’s disease , damage to neurons in the brain gradually causes memory loss and difficulties with thinking .",
"The main hallmarks of this damage are seen in the accumulation of proteins in and around neurons .",
"First , a protein called amyloid beta forms aggregates outside the cell .",
"This appears to lead to the build up of an abnormal form of a protein called tau inside the cell .",
"These abnormal tau proteins have excessive numbers of phosphate groups attached to them , and so are known as “hyperphosphorylated” .",
"The molecular mechanism underlying amyloid beta’s role in the hyperphosphorylation of tau proteins was not known .",
"Amyloid beta binds to many different receptor proteins – including one called Fc gamma receptor IIb – on the surface of neurons .",
"Kam , Park et al . investigated whether interactions between amyloid beta and Fc gamma receptor IIb might regulate the phosphorylation of tau within neurons .",
"Adding amyloid beta to mouse neurons caused tau proteins to become hyperphosphorylated .",
"However , removing Fc gamma receptor IIb from the neurons , or stopping it from binding to amyloid beta , abolished this effect .",
"When amyloid beta was bound to Fc gamma receptor IIb , the receptor became phosphorylated .",
"This in turn triggered a series of further phosphorylation events , culminating in an increase in the level of a molecule that relays signals from cell receptors – called SHIP2 – inside the neurons .",
"This molecule increases tau phosphorylation when added to neurons .",
"Reducing the activity or amount of SHIP2 in mice that present the symptoms of Alzheimer’s disease reduced the hyperphosphorylation of the tau protein in their neurons and restored their memory to normal levels .",
"Kam , Park et al . also looked at samples taken from the brains of human Alzheimer's disease patients .",
"Unlike samples taken from people without Alzheimer’s disease , neurons in these samples contain both phosphorylated Fc gamma receptor IIb and hyperphosphorylated tau proteins .",
"By uncovering the molecules that link amyloid beta with tau hyperphosphorylation , Kam , Park et al . ’s results suggest new targets for therapies to treat the symptoms of Alzheimer’s disease .",
"More research is now needed to investigate whether this could lead to the design of new drugs ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | NG2 glia are required for vessel network formation during embryonic development | elife-09102-v3 | [
[
"The vascular system consists of two highly organized , branched , and stereotypic circuits of blood vessels and lymphatic vessels ( Adams and Alitalo , 2007; Larrivee et al . , 2009 ) .",
"Blood vessels develop by two unique mechanisms: vasculogenesis , de novo synthesis involving differentiation of cells from endothelial precursor cells ( angioblasts ) , and angiogenesis , formation of vessels from pre-existing vessels by either",
"( a ) sprouting , proliferation , and migration of endothelial cells ( ECs ) or",
"( b ) bifurcation of a preexisting blood vessel into two ( Adams and Alitalo , 2007; Jain , 2003; Larrivee et al . , 2009 ) .",
"The selection of sprouting ECs is controlled positively by the pro-angiogenic vascular endothelial growth factor ( VEGF ) and negatively by pericytes , extracellular matrix molecules or VEGF inhibitors .",
"Thereafter , the endothelial tip cells have the capacity to extend filopodia in order to respond to attractive or repulsive signals of the environment .",
"Guidepost cells have been shown to direct tip cells pathfinding by cell contact adhesion or by secreting soluble guidance cues .",
"Therefore , endothelial tip cells share numerous similarities with axonal growth cones and it is admitted that axons and blood vessels use similar mechanisms in order to form complex networks ( Carmeliet and Tessier-Lavigne , 2005; Eichmann et al . , 2005 ) .",
"Additionally , pathfinding of blood vessels has been shown to require Sema3E/PlexinD1 , Netrin-1/Unc5B , and Slit/Robo4 signaling ( Adams and Alitalo , 2007; Carmeliet and Tessier-Lavigne , 2005; Lu et al . , 2004; Larrivee et al . , 2007; Eichmann et al . , 2005 ) .",
"Sprout extension is made by the migration of the stalk ECs behind the tip cell or by local proliferation of stalk ECs .",
"The fusion of adjacent sprouts and vessels that occurs after tip cells encounter each other is regulated by adhesive or repulsive interactions .",
"Blood vessels in the telencephalon of embryonic mice are divided into pial or periventricular vessels based on their anatomical location , their growth patterns and development .",
"The periventricular vessels are believed to give birth to the arterial networks , while the pial vessels may generate the venous sinuses ( Hiruma et al . , 2002 ) .",
"As soon as E9 , developing pial vessels forming a vascular plexus surround the entire brain without following any obvious spatial or temporal gradient .",
"By contrast , the periventricular vessels develop in a ventral-to-dorsal gradient within the telencephalon .",
"Angiogenesis in the embryonic telencephalon , in addition to the extrinsic factors listed above , is also controlled by a set of region-specific transcription factors , from the ventral telencephalon such as Nkx2 . 1 and Dlx2 , and from the dorsal telencephalon like Pax6 that are expressed in a subset of ECs ( Vasudevan and Bhide , 2008; Vasudevan et al . , 2008 ) .",
"The distinct spatial and temporal expression of these transcription factors has been shown to direct telencephalic vascular development .",
"Until now , macrophages that lie in close proximity to the blood vessels have been known to act as angiogenic agents and have been implicated in blood vessel development during growth and repair ( Fantin et al . , 2010; Newman and Hughes , 2012; Nucera et al . , 2011; Outtz et al . , 2011; Pollard , 2009 ) .",
"In brain , early embryonic macrophages travel from the yolk sac , express Tie2 and the neuropilin 1 receptor , and are present at the time of brain vascularization .",
"They were observed to localize at vessels junctions and interact with the endothelial tip cells , by forming bridges to align them and to prepare them for the later fusion ( Fantin et al . , 2010; Nucera et al . , 2011; Outtz et al . , 2011 ) .",
"In this paper , we discovered that another class of glial cells , NG2+ glia , are also involved in the vascular network formation in the early mouse telencephalon .",
"NG2+ glia or polydendrocytes constitute a population of cells that are different from neurons , mature oligodendrocytes , astrocytes , and microglia ( Nishiyama et al . , 2002; Nishiyama et al . , 2009 ) .",
"They express NG2 ( Nerve/glial antigen 2 or CSPG for chondroitin sulfate proteoglycan ) and Olig2 ( Oligodendrocyte precursor bHLH transcription factor 2 ) but lack astrocytic markers namely GFAP ( glial fibrillary acidic protein ) and GLAST ( Astrocyte-specific glutamate and aspartate transporter ) .",
"They display complex highly branched morphology and are uniformly distributed within the grey and white matter throughout all layers .",
"They generate oligodendrocytes in vitro and have been considered since a long time as oligodendrocyte progenitor cells ( Polito and Reynolds , 2005; Nishiyama et al . , 1996a , 1996b , 2009; Zhu et al . , 2008b ) .",
"However , recently , NG2+ glia have also been shown to differentiate into neurons and protoplasmic astrocytes in the grey matter as well as in the white matter ( Rivers et al . , 2008; Zhu et al . , 2008b; Zhu et al . , 2008a ) .",
"Furthermore , electrophysiological studies indicate that NG2+ glia receive synaptic input from neurons , and like astroglia , probably participate to the neuronal network ( Paukert and Bergles , 2006; Butt et al . , 2005 ) .",
"NG2+ glia have also been implicated in synaptic reorganization after cortical and spinal cord injury , but their precise role is still unknown ( Nishiyama , 2007; Nishiyama et al . , 2009 ) .",
"This study displays a novel function for this rather recently classified glial cell population .",
"The embryonic NG2+ glia of the dorsal telencephalon originate from the subpallial Nkx2 . 1+ progenitors .",
"Interestingly , they occupy the telencephalon at the same time as the establishment of the blood vessel network .",
"Additionally , they not only originate at the same time but also reside very closely to the developing vessel network .",
"Hence , the strategic temporal occurrence is conjugated with spatial proximity to the vessel network .",
"They are closely juxtaposed to the blood vessels , either at sprouting tip cells , branching points or along the vessel walls .",
"Remarkably , in their absence , the embryonic vasculature was poorly developed and exhibited reduced connectivity .",
"Our results bring forth a new function for this class of glia .",
"In summary , our findings propose the participation of Nkx2 . 1-derived NG2+ glia toward blood vessel network formation and stabilization during late embryonic ages .",
"Therefore , this study gives new insights into the mechanisms involved in brain angiogenesis and implies that transient NG2+ glia work together with macrophages in guiding vessels ."
],
[
"Here , we identified early NG2+ glia within the embryonic telencephalon and investigated their function during development .",
"To this purpose , we first characterized the molecular identity and origin of the embryonic NG2+ glia that populated the telencephalon .",
"To selectively fate map the NG2+ glia , we used NG2 immunostaining in wild-type ( WT ) mice , Cspg4-cre+/Rosa-EYFP ( CSPG or chondroitin sulfate proteoglycan also known as NG2 ) and Nkx2 . 1-cre+/Rosa-EYFP mice .",
"Between E12 . 5-to-E16 . 5 , we observed that the NG2+ glia were positioned within the marginal zone ( MZ ) , the subplate , the intermediate zone ( IZ ) and the sub-ventricular zone ( SVZ ) of the lateral cortical area , and in the septum ( SEP ) of the WT mice ( Figure 1 ) .",
"They populated the midline corticoseptal boundary ( CSB ) region at E12 . 5 ( n=3 ) ( Figure 1A ) , and the cingulate bundle ( CI ) , the cingulate ( CCi ) and frontal ( CFr ) cortices at E14 . 5 ( n=3 ) ( Figure 1B and 1C ) .",
"By E16 . 5 , NG2+ glia were ubiquitously dispersed within the WT dorsal telencephalon ( n=3 ) ( Figure 1D ) . 10 . 7554/eLife . 09102 . 003Figure 1 . NG2+ glia are in close contact with blood vessels .",
"( A–D )",
"Double immunohistochemistry for NG2 and Isolectin ( A1–A3 , B1–B3 ) and for NG2 and PECAM ( C1–C3 , D1–D2 ) on coronal cingulate cortex ( CCi ) and cingulate bundle ( CI ) sections of wild-type mice ( n=3 each ) at E12 . 5 ( A1–A3 ) , at E14 . 5 ( B1–B3 and C1–C3 ) , and at E16 . 5 ( D1–D2 ) .",
"A3 , A2 , B2 , B3 , C2 , C3 , and D2 are higher power views of the region in A1 , B1 , C1 , and D1 , respectively ( white arrowheads ) .",
"D3 is an isosurface reconstruction of the labeling seen in D2 .",
"The processes of the NG2+ glia are in close contact with adjacent blood vessels ( open arrowheads in A3 , B3 , and C3 ) .",
"Bar = 675 μm in A1 , B1 , and D1; 50 μm in A2 , B2 , C1 , and D2; 40 μm in A3 , B3 , C2 , and C3 .",
"CSB , corticoseptal boundary at the midline where the corpus callosum will form . DOI: http://dx . doi . org/10 . 7554/eLife . 09102 . 003 We then analyzed in detail the Cspg4-cre+/Rosa-EYFP mice wherein the NG2 promoter dictated specific Cre recombinase expression which then lead to permanent YFP expression from the constitutively active Rosa promoter .",
"In Cspg4-cre+/Rosa-EYFP mice , the YFP signal was detected in a majority of embryonic NG2+ glia of the dorsal telencephalon ( at E18 . 5: 71 . 7 ± 14 . 6% in the corpus callosum ( CC ) , 57 ± 3 . 8% in the CI and 69 ± 5 . 3% in the CCi; n=3 ) ( Figure 2—figure supplement 1A ) .",
"The entire cell population visualized by the YFP signal at E16 . 5–E18 . 5 co-expressed NG2 ( n=3 ) and Olig2 ( n=3 ) , two well-known markers for NG2 glia , and also S100β ( n=3 ) , considered as a marker for astrocytes and NG2+ glia ( Cahoy et al . , 2008; Honsa et al . , 2012; Rivers et al . , 2008 ) ( Figure 2A–C and Figure 2—figure supplement 1E ) .",
"As expected , at same ages they did not express the specific astrocytic markers GLAST ( n=3 ) and GFAP ( n=3 ) ( Figure 2D–E and Figure 2—figure supplement 1E ) .",
"Although immunostaining showed that in WT mice , PDGFR-β+ pericytes adjacent to the vessels were NG2+ ( Figure 3D ) , Cre-mediated recombination in Cspg4-cre+/Rosa-EYFP mice did not occur properly in the pericytes .",
"As a result , although NG2 is expressed by pericytes ( Levine and Nishiyama , 1996; Stallcup and Huang , 2008; Virgintino et al . , 2007 ) , we found only very few PDGFR-β+ pericytes labeled for the YFP in Cspg4-cre+/Rosa-EYFP telencephalon ( Figure 3B , n=3 ) .",
"A substantial proportion of the PDGFR-β+ pericytes population was YFP- .",
"Quantifications of the two populations: PDGFR-β+/YFP- pericytes and PDGFR-β+/YFP+ pericytes showed that only 4 . 95 ± 1 . 54% of total PDGFR-β+ pericyte-population was co-labeled with YFP ( Figure 3G , n=10 ) .",
"Thus , vast majority of the YFP signal in Cspg4-cre+/Rosa-EYFP brains was present in NG2+ glia alone . 10 . 7554/eLife . 09102 . 004Figure 2 . NG2+ glia of the dorsal telencephalon are derived from Nkx2 . 1+ progenitors of the subpallium .",
"( A–E )",
"Double immunohistochemistry for the YFP and NG2 ( A1–A2 ) ( n=3 ) , the YFP and Olig2 ( B ) ( n=3 ) , the YFP and S100β ( C ) ( n=3 ) , the YFP and GLAST ( D ) ( n=3 ) , and the YFP and GFAP ( E ) ( n=3 ) on telencephalic coronal slices of Cspg4-cre+/Rosa-EYFP mice at E16 . 5 ( B and D ) and E18 . 5 ( A1 , A2 , C , and E ) .",
"( F–J )",
"Double immunohistochemistry for the YFP and NG2 ( F1–F2 ) ( n=5 ) , the YFP and Olig2 ( G ) ( n=5 ) , the YFP and S100β ( H ) ( n=4 ) , the YFP and GLAST ( I ) ( n=4 ) , and the YFP and GFAP ( J ) ( n=3 ) on telencephalic coronal slices of Nkx2 . 1-cre+/Rosa-EYFP mice at E16 . 5 ( F1 , F2 , and H ) and E18 . 5 ( G , I , and J ) .",
"A2 and F2 are higher power views of the cingulate region in A1 and F1 , respectively .",
"The NG2-derived and the Nkx2 . 1-derived YFP+ cells co-express polydendroglial markers , NG2 and Olig2 , together with S100β ( white arrowheads ) but lack expression of GLAST and GFAP ( open arrowheads in D and I ) .",
"Bar=675 μm in A1 , F1; 100 μm in E , J; 50 μm in A2 , B , C , D , F2 , G , H , I . DOI: http://dx . doi . org/10 . 7554/eLife . 09102 . 00410 . 7554/eLife . 09102 . 005Figure 2—figure supplement 1 . YFP signal in Nkx2 . 1-cre+/Rosa-EYFP and Cspg4-cre+/Rosa-EYFP mice is present in NG2 glia .",
"( A ) Bars ( means ± SEM ) represent the percentage of YFP-labeled NG2 glia in corpus callosum ( CC ) , cingulate bundle ( CI ) , and cingulate cortex ( CCi ) sections of E18 . 5 Cspg4-cre+/Rosa-EYFP mice ( n=3 ) .",
"The YFP signal in Cspg4-cre+/Rosa-EYFP mice was detected in a majority of NG2+ embryonic glia of the dorsal telencephalon ( 71 . 7 ± 14 . 6% in the CC , 57 ± 3 . 8% in the CI and 69 ± 5 . 3% in the CCi at E16 . 5 , n=3 ) .",
"( B–C )",
"Double immunohistochemistry for NG2 and PECAM in CCi coronal sections of Cspg4-cre-/Rosa-DTA ( n=3 ) ( B ) and Cspg4-cre+/Rosa-DTA ( n=3 ) ( C ) mutant mice at E18 . 5 .",
"Scale bar = 50 µm in B and C . ( D ) Bars ( means ± SEM; unpaired Student’s t-test ) represent the percentage of remaining NG2 glia in the telencephalon of E18 . 5 Cspg4-cre+/Rosa-DTA mice compared to control mice .",
"A drastic loss of NG2+ glia followed by severe vascular defects was observed in Cspg4-cre+/Rosa-DTA mice compared to Cspg4-cre-/Rosa-DTA .",
"( E ) Bars ( means ) represent the percentage of glial markers expression by YFP-labeled NG2 glia in the CC , Cl , and CCi of E18 . 5 Cspg4-cre+/Rosa-EYFP mice .",
"In Cspg4-cre+/Rosa-EYFP mice , all YFP-labeled NG2 glia of the dorsal telencephalon expressed NG2+/Olig2+ ( n=2 ) , many S100β+ ( n=2 ) but no GLAST+ ( n=3 ) or GFAP+ ( n=2 ) .",
"( F ) Bars ( means ) represent the percentage of NG2 markers expression by YFP-labeled cells in the telencephalon of E18 . 5 Nkx2 . 1-cre+/Rosa-EYFP mice ( n=4 ) ( 47 . 24% in the CC and 27 . 2% in the Cl ) .",
"CC , corpus callosum; CCi , cingulate cortex; Cl , cingulate bundle . DOI: http://dx . doi . org/10 . 7554/eLife . 09102 . 00510 . 7554/eLife . 09102 . 006Figure 2—figure supplement 2 . Nkx2 . 1-derived NG2 and Olig2 glia are transient and gradually disappear from the dorsal pallium at postnatal ages .",
"( A–C )",
"Double immunohistochemistry for GFP and NG2 on coronal telencephalicsections from Nkx2 . 1-cre+/Rosa-EYFP mice at P0 ( n=3 ) ( A1–A2 ) , P2 ( n=3 ) ( B1–B2 ) , and P8 ( n=2 ) ( C1–C2 ) .",
"( D ) Double immunohistochemistry for GFP and Olig2 on coronal telencephalicsections from Nkx2 . 1-cre+/Rosa-EYFP mice at P8 ( n=2 ) ( D1–D3 ) .",
"Cell nuclei were counterstained in blue with Hoechst ( A1 , B1 , and C1 ) .",
"A2 , B2 , C2 , and D3 are high-power views of the cingulate bundle ( Cl ) seen in A1 , B1 , C1 , and D1 , respectively .",
"In the Cl of Nkx2 . 1-cre+/Rosa-EYFP mice brains at P2 , only very few Nkx2 . 1-derived NG2 glia remained ( arrowheads B2 ) .",
"At P8 , Nkx2 . 1-derived NG2 and Olig2 glia disappeared completely and were replaced by NG2 and Olig2 glia that did not expressed the YFP and were not derived from Nkx2 . 1 germinal domains ( open arrowheads in A2 and C2 ) .",
"At all postnatal ages many Nkx2 . 1-derived GABAergic interneurons found in this region were not labeled for NG2 ( arrows in B2 and C2 ) .",
"Scale bar = 675 µm in A1 , B1 , C1 and D1; 40 µm in A2 , B2 and C2; 100 µm in D2 and D3 .",
"CC , corpus callosum; CCi , cingulate cortex; LV , lateral ventricles; SEP , septum . DOI: http://dx . doi . org/10 . 7554/eLife . 09102 . 00610 . 7554/eLife . 09102 . 007Figure 3 . NG2+ glia , but not pericytes , control blood vessels formation .",
"( A–C )",
"Double immunohistochemistry for the YFP and Isolectin ( A1–A2 ) or PDGFRβ ( B1–B2 , C1–C2 ) on coronal cingulate cortex ( CCi ) and cingulate bundle ( CI ) sections of Nkx2 . 1-cre+/Rosa-EYFP ( A1 , C1–C2 ) and Cspg4-cre+/Rosa-EYFP ( A2 , B1–B2 ) mice ( n=3 ) at E16 . 5 ( A1–A2 ) and E18 . 5 ( B1–B2 and C1–C2 ) .",
"( A–C )",
"From E16 . 5 to E18 . 5 , numerous YFP+ NG2 glia are surrounding cortical blood vessels .",
"( B–C )",
"The Cre-mediated recombination , visualized by the YFP signal , can be observed in only very few pericytes surrounding blood vessels in Cspg4-cre+/Rosa-EYFP mice ( B2 , boxed region showing high magnification of region marked with white arrowhead ) , but not in Nkx2 . 1-cre+/Rosa-EYFP mice ( C2 , boxed region showing high magnification of region marked with arrow ) .",
"( D–F )",
"Double immunohistochemistry for NG2 and PDGFRβ on coronal CI sections in wild-type ( D1–D2 ) , Nkx2 . 1-cre+/Rosa-DTA ( E1–E2 ) ( n=3 ) and Cspg4-cre+/Rosa-DTA ( F1–F2 ) ( n=3 ) mice at E18 . 5 .",
"( D–F )",
"The NG2+ glia form a complex cellular network around the cortico-cerebral blood vessels outlined by NG2 and PDGFRβ staining .",
"The DTA under the control of Nkx2 . 1 ( E ) and Cspg or NG2 ( F ) promoters selectively depletes NG2+ glia but not pericytes .",
"D2 , E2 , and F2 are higher power views of the cingulate region in D1 , E1 , and F1 , respectively ( white arrowheads ) .",
"( G ) Bars ( means ± SEM ) represent the percentage of YFP-negative and YFP-positive PDGFRβ labeled pericytes in dorsal telencephalon sections of E18 . 5 Cspg4-cre+/Rosa-EYFP mice ( n=10 ) .",
"The YFP signal in Cspg4-cre+/Rosa-EYFP mice was not detected in PDGFRβ+ embryonic pericytes of the dorsal telencephalon ( 95 . 05 ± 1 . 54% of pericytes are YFP-negative in the CI at E18 . 5 , n=10 ) .",
"( H ) Bars ( means ± SEM; unpaired Student’s t-test ) represent the percentage of remaining PDGFRβ+ pericytes in cingulate bundle ( Cl ) sections of E18 . 5 Cspg4-cre+/Rosa-DTA mice ( n=11 ) compared to control mice ( n=11 ) .",
"No loss of PDGFRβ+ pericytes was observed in Cspg4-cre+/Rosa-DTA mice compared to Cspg4-cre-/Rosa-DTA .",
"Bar = 100 μm in B1 , C1; 50 μm in A1 , A2 , D1 , E1 , F1; 40 μmin B2 , C2 , D2 , E2 , F2 . DOI: http://dx . doi . org/10 . 7554/eLife . 09102 . 00710 . 7554/eLife . 09102 . 008Figure 3—figure supplement 1 . Drastic depletion of GAD67-GFP+ neurons in Nkx2 . 1-/- , Nkx2 . 1-cre+/Rosa-DTA and Nkx2 . 1-cre+/Eno2-DTA cortices .",
"( A–D )",
"Double immunohistochemistry for the GFP and L1 on coronal CI sections in Gad1-EGFP ( n=8 ) ( A1–A2 ) , Nkx2 . 1-cre+/Rosa-DTA:Gad1-EGFP ( n=5 ) ( B1–B2 ) and Nkx2 . 1-cre+/Eno2-DTA:Gad1-EGFP ( n= 6 ) ( C1–C2 ) mice at E18 . 5 .",
"A2 , B2 , and C2 are higher power views showing the quantification of the GAD67-GFP+ interneurons ( white spots ) within the CI region pointed with a white arrowhead in A1 , B1 , and C1 , respectively .",
"( D ) Bars ( means ± SEM; unpaired Student’s t-test ) represent the percentage of remaining GAD67-GFP+ interneurons in the CI of E18 . 5 Nkx2 . 1-cre+/Rosa-DTA:Gad1-EGFP and Nkx2 . 1-cre+/Eno2-DTA:Gad1-EGFP mice compared to Gad1-EGFP mice .",
"Both the mutant mice exhibited equivalent loss of neurons when compared to wild-type mice .",
"( E ) Table of the corresponding cell density values ( cell number*103/mm3 ) ( unpaired Student’s t-test ) .",
"Scale bar = 675 µm in A1 , B1 , and C1; 100 µm in A2 , B2 , and C2 .",
"CC , corpus callosum; CCi , cingulate cortex; Cl , cingulate bundle; SEP , septum . DOI: http://dx . doi . org/10 . 7554/eLife . 09102 . 00810 . 7554/eLife . 09102 . 009Figure 3—figure supplement 2 . GLAST+ astrocytes are not affected in Nkx2 . 1-cre+/Rosa-DTA cingulate cortex at E18 . 5 . ( A–B ) Double immunohistochemistry for Olig2 and GLAST in cingulate cortex ( CCi ) coronal sections of Nkx2 . 1-cre-/Rosa-DTA mutant mice ( n=4 ) ( A ) and Nkx2 . 1-cre+/Rosa-DTA control mice ( n=4 ) ( B ) at E18 . 5 .",
"Scale bar = 50 µm in A and B . ( C ) Bars ( means ± SEM; unpaired Student’s t-test ) represent the density number of remaining GLAST+ astroglia in the CCi of E18 . 5 Nkx2 . 1-cre+/Rosa-DTA mice compared to control mice .",
"No significant loss of GLAST+ astroglia was observed in Nkx2 . 1-cre+/Rosa-DTA CCi compared to control mice . DOI: http://dx . doi . org/10 . 7554/eLife . 09102 . 009 As Nkx2 . 1-regulated precursors have been previously shown in embryos to produce transient oligodendrocyte precursor cells ( OPCs ) in addition to giving rise to GABAergic interneurons and astrocytes ( Nery et al . , 2001; Kessaris et al . , 2006 , Minocha et al . , 2015 ) , we decided to make use of the Nkx2 . 1-cre+/Rosa-EYFP mice to look further at the subpallial origin , time of appearance and spatial arrangement of the embryonic NG2+ glia that ubiquitously occupied the dorsal telencephalon toward the end of embryonic development .",
"Using our Nkx2 . 1-cre+/Rosa-EYFP mice , our findings confirmed that Nkx2 . 1-derived precursors produced YFP+/NG2+ glia that colonized the cingulate cortical area and the midline ( Figure 2F , Figure 2—figure supplement 1F ) ( Kessaris et al . , 2006 ) .",
"The YFP signal was detected in all embryonic NG2+ glia of the dorsal telencephalon from E16 . 5 to E18 . 5 ( 100% colocalization in the CC , and the CCi at E16 . 5 and E18 . 5; n=5 ) ( Figure 2F ) .",
"Nkx2 . 1-derived NG2+ glia expressed Olig2 ( n=5 ) and S100β ( n=4 ) ( Figure 2G–H ) , and lacked GLAST ( n=4 ) or GFAP ( n=3 ) expression ( Figure 2I–J ) .",
"This further emphasized that subpallial domains are sites for early NG2+ glia genesis .",
"Furthermore , we followed the presence of these YFP+ /NG2+ glia after birth .",
"We observed that the YFP+/Olig2+/NG2+ glia originating from Nkx2 . 1 domains were transient in nature and disappeared abruptly from the cortex around P8 ( n=3 ) ( Figure 2—figure supplement 2 ) .",
"These results are coherent with previous studies , wherein the OPCs generated from the Nkx2 . 1-expressing precursors were shown to disappear around P10 ( Kessaris et al . , 2006 ) .",
"Interestingly , we never detected any YFP signal in the ECs and the pericytes of the cerebral vasculature ( Figure 3A1 and 3C ; n=5 ) .",
"Thus , based on the specificity of the Cre- reporter strains ( both Cspg4-cre and Nkx2 . 1-cre ) for NG2+ glia , these mice can be further utilized to determine the function of the embryonic NG2+ glia independent of ECs and pericytes .",
"These results altogether show that in embryos , NG2+ glia of the dorsal telencephalon are derived from Nkx2 . 1+ progenitors of the subpallium and transiently occupy the telencephalon .",
"The disappearance of the Nkx2 . 1-derived NG2+ glia a few days after birth underlines their functional requirement for events occurring during embryonic and early postnatal period .",
"We further studied the precise localization of the embryonic NG2+ glial population to elucidate their functional relevance .",
"At E14 . 5 ( n=3 ) , the first pioneer NG2+ glia were observed in the CCi cortical plate ( CP ) ( Figure 1 ) .",
"As such , they occupied the CP at the same time as establishment of the blood vessel network and were seen to accompany it while growing and invading the dorsal telencephalon .",
"The NG2+ glia were present from E14 . 5-to-E16 . 5 throughout the dorsal telencephalon during the early phase of angiogenesis when vessels begin to extend , sprout , and form new branches , as well as during the later phase from E16 . 5-to-E18 . 5 when branches fuse together or retract ( Figures 1 and 2 ) .",
"Interestingly , we observed that NG2+ glia formed a complex cellular network around the cerebral vessels outlined by NG2 , PECAM , Isolectin , or PDGFR-β labeling ( Figures 1 and 2 ) .",
"They were localized at sprouting tip cells or at branch fusion points , and also all along the vessel walls ( Figure 1 ) .",
"Several long and slender processes of the NG2+ glia appeared to wrap or tether to the vascular walls , possibly reflective of a functional interaction ( Figure 1A3 , 1B3 , 1C3; open arrowheads ) .",
"The interactions between blood vessel network and NG2+ glia enhanced as embryonic age progressed ( compare Figure 1A to 1D and 3D ) .",
"A careful analysis using highly magnified three-dimensional ( 3D ) confocal pictures combined with iso-surface representations revealed that NG2+ glia make multiple close contacts with different cortical blood vessels and created bridges between neighboring vessels ( Figure 1D3 ) .",
"The bridges are composed of several NG2+ glia that make connections via their entangled processes , and connect the neighbor vessels ( Figure 1B–D ) .",
"The strategic position of NG2+ glia between the blood vessels created an elaborate mesh-like structure , and reflected the possibility that NG2+ glia participated toward the vasculature connectivity ( Figure 1B–D ) .",
"Therefore , the location and timing of appearance of the transient NG2+ glia raise the possibility that they actively participate in the branching and refinement of the cerebral vasculature .",
"The concurrence of angiogenic development , NG2+ gliogenesis together with the elaborate connectivity between NG2+ glia and vessels made us wonder if the NG2+ glia were involved in regulating brain angiogenesis .",
"To test this hypothesis , we used mice expressing a 'floxed' diphtheria toxin gene that allows selective ablation of cells in the whole brain ( Rosa-DTA ) ( Brockschnieder et al . , 2006 ) .",
"By crossing Nkx2 . 1-cre+ mice with Rosa26-DTA mice that expressed the diphtheria toxin under the control of the Nkx2 . 1 promoter , only Nkx2 . 1-expressing cells were selectively depleted ( Minocha et al . , 2015 ) .",
"Nkx2 . 1-cre+/Rosa-DTA mice allowed a selective ablation of Nkx2 . 1-derived post-mitotic cells without affecting Nkx2 . 1+ precursors because the diphtheria toxin expression under the control of Nkx2 . 1 promoter took several days ( Minocha et al . , 2015 ) .",
"As the Nkx2 . 1-regulated precursors are known to give rise to GABAergic interneurons ( Anderson et al . , 2001; Corbin et al . , 2001; Marin and Rubenstein , 2001; Sussel et al . , 1999; Xu et al . , 2008 ) and astrocytes ( Minocha et al . , 2015 ) , we aimed to identify the contribution of GABAergic interneurons and astrocytes in our assays , as they can also get ablated in Nkx2 . 1-cre+/Rosa-DTA mice .",
"To do so , we incorporated the reporter Gad1-EGFP ( Gad1 corresponds to the Gad67 gene ) into the Nkx2 . 1-cre+/Rosa-DTA mice to observe the GFP+ interneurons .",
"In the dorsal telencephalon of Nkx2 . 1-cre+/Rosa-DTA:Gad1-EGFP ( n=5 ) , Nkx2 . 1-derived post-mitotic GABAergic neurons were depleted severely by ~50% at E18 . 5 ( Figure 3—figure supplement 1B and 1D ) .",
"Next , we aimed to confirm that the GLAST+ astrocytes are not affected in Nkx2 . 1-cre+/Rosa-DTA mice brains at E18 . 5 .",
"In Nkx2 . 1-cre+/Rosa-EYFP and Nkx2 . 1-cre+/Rosa-tdTomato mice , recombination can only been seen in GLAST+ astrocytes of the ventral telencephalon , whereas no recombination can be seen in those that occupy the dorsal telencephalon ( Figure 2I and not shown ) .",
"Thus , we did not expect to see an ablation in GLAST+ astrocytes of the dorsal telencephalon with Nkx2 . 1-cre+/Rosa-DTA mice brains .",
"Indeed , upon co-immunostaining with anti-GLAST , we found that there was no ablation of GLAST+ cell population in Nkx2 . 1-cre+/Rosa-DTA mice ( n=4 ) when compared to control Nkx2 . 1-cre-/Rosa-DTA mice ( n=4 ) in the cingulate cortex ( Figure 3—figure supplement 2 ) .",
"Interestingly , staining for NG2 revealed a nearly complete loss of NG2+ glia in all medio-dorsal cortical areas in all rostrocaudal sections of Nkx2 . 1-cre+/Rosa-DTA mice brains compared to control mice brains at E16 . 5 ( n=3 ) and E18 . 5 ( n=6 ) ( Figure 4B and Figure 4—figure supplement 1 ) .",
"Thus , this result underlined the presence of only Nkx2 . 1-derived NG2+ glia in the cortical midline regions specifically .",
"No evident morphological changes were seen in lateral cortical regions where Nkx2 . 1-derived NG2+ glia intermixed with other NG2+ glia having different identities and origins ( not shown ) .",
"Also , the ECs and pericytes were not ablated in Nkx2 . 1-cre+/Rosa-DTA , due to the lack of Cre-mediated recombination in these cells ( Figure 3C and 3E; n=3 ) .",
"The lack of effective Cre-mediated recombination in ECs and pericytes provided us with a useful tool since the other cells that are involved in vessel network formation were not ablated .",
"Thus , we could specifically assess the effects of a narrower population ( i . e . NG2+ glia ) . 10 . 7554/eLife . 09102 . 010Figure 4 . Drastic depletion of embryonic NG2+ glia in Nkx2 . 1-cre+/Rosa-DTA and Cspg4-cre+/Rosa-DTA midline dorsal telencephalon . DAB staining for NG2 in wild-type ( A1–A3 ) ( n=6 ) , Nkx2 . 1-Cre+/Rosa-DTA ( B1–B3 ) ( n=3 ) , Cspg4-cre+/Rosa-DTA ( C1–C3 ) ( n=3 ) , and Nkx2 . 1-cre+/Eno2-DTA ( D1–D3 ) ( n=3 ) telencephalic coronal slices at E18 . 5 .",
"NG2+ glia were completely depleted from the corpus callosum ( CC ) , the cingulate cortex ( CCi ) , and cingulate bundle ( Cl ) of Nkx2 . 1-cre+/Rosa-DTA ( B1–B3 ) mutant mice compared to wild-type mice ( A1–A3 ) .",
"In Cspg4-cre+/Rosa-DTA mutant mice ( C1–C3 ) , there was also a drastic loss of NG2+ cells in medial cortical areas of the dorsal telencephalon with only few remaining cells .",
"In Nkx2 . 1-cre+/Eno2-DTA ( D1–D3 ) mutant mice , there was no loss of NG2+ glia in all the observed regions .",
"A2–A3 , B2–B3 , C2–C3 , and D2–D3 are higher power views of the Cl regions in A1 , B1 , C1 , and D1 , respectively ( white arrowheads ) .",
"Bar = 500 μm in A1 , B1 , C1 , and D1; 250 μm in A2 , B2 , C2 , and D2; 125 μm in A3 , B3 , C3 , and D3 . DOI: http://dx . doi . org/10 . 7554/eLife . 09102 . 01010 . 7554/eLife . 09102 . 011Figure 4—figure supplement 1 . Drastic depletion of embryonic NG2+ glia in Nkx2 . 1-cre+/Rosa-DTA midline dorsal telencephalon starts at E16 . 5 . ( A–D ) Immunostaining for NG2 in Nkx2 . 1-cre-/Rosa-DTA ( A1–A2 to C1–C2 ) and Nkx2 . 1-cre+/Rosa-DTA ( D1–D2 to F1–F2 ) telencephalic coronal slices at E14 . 5 ( A1–A2 , D1–D2 ) , E16 . 5 ( B1–B2 , E1–E2 ) and E18 . 5 ( C1–C2 , F1–F2 ) .",
"A2 , B2 , C2 , D2 , E2 , and D2 are higher power views of the region in A1 , B1 , C1 , D1 , E1 , and F1 , respectively ( white arrowheads ) .",
"At E14 . 5 , there were no significant differences in NG2+ glia observed in the cingulate regionof Nkx2 . 1-cre+/Rosa-DTA mutant mice ( n=3 ) compared to wild-type mice ( n=3 ) ( A1–A2 , D1–D2 ) .",
"By contrast , later at E16 . 5 , there was already a drastic loss of NG2+ glia populating the medio-dorsal telencephalon regionsin the Nkx2 . 1-cre+/Rosa-DTA ( n=3 ) mutant mice compared to control mice ( n=3 ) ( B1–B2 , E1–E2 ) .",
"This loss was maintained at E18 . 5 in the Nkx2 . 1-cre+/Rosa-DTA ( n=3 ) mutant mice compared to control mice ( n=3 ) ( C1–C2 , F1–F2 ) .",
"Scale bar = 675 µm in A1 , B1 , C1 , D1 , E1 , and F1; 40 μmin A2 , B2 , C2 , D2 , E2 , and F2 . DOI: http://dx . doi . org/10 . 7554/eLife . 09102 . 011 Remarkably , we observed that vessels stained for vessel markers ( NG2 , PECAM or Isolectin ) formed a poorly developed vascular network in multiple telencephalic regions , such as the septum and the cerebral cortices of Nkx2 . 1-cre+/Rosa-DTA mice ( n=4 for PECAM at E14 . 5 and E16 . 5; n=3 for NG2; n=4 for PECAM; n=5 for isolectin at E18 . 5 ) compared to WT mice ( n=4 for PECAM at E14 . 5 and E16 . 5 , n=6 for NG2; n=8 for PECAM; n= 5 for isolectin at E18 . 5 ) as soon as E16 . 5 and becoming fully evident by E18 . 5 in all the rostrocaudal levels ( Figure 4B , Figure 5B , Figure 6B , Figure 5—figure supplement 1D and Figure 5—figure supplement 2B ) .",
"No defects were visualized prior to E16 . 5 ( Figure 5—figure supplement 1B ) .",
"Cortical vessels of Nkx2 . 1-cre+/Rosa-DTA mice exhibited a drastic and significant reduction of intersections ( nodes ) , connections and of the density of the vascular network ( reduction of nodes: 14 . 1 ± 4 . 8% , p<0 . 05; reduction of branches: 14 . 7 ± 4 . 9% , p<0 . 05; reduction of the total volume of the vascular network: 17 . 78 ± 4 . 33% , p<0 . 05; Figure 5B , G and Table 1; n=4; unpaired Student’s t-test ) .",
"Due to the defects observed in branching pattern and connectivity , the regular vascular pattern was not observed any more in mutants , and vessels formed only isolated units ( Figure 5B and Figure 5—figure supplement 1D ) .",
"After co-staining for Isolectin and lymphocyte antigen LY-76 marker ( Ter119 ) ( Figure 6 ) and DAB staining allowing the visualization of erythrocytes ( Figure 5 ) , our analyses revealed that , in addition , cortical vessels lost their regular diameter and erythrocytes accumulated at the level of enlarged vessel segments ( Figure 5E and 5H , Figure 6B and Table 2; n=5; unpaired Student’s t-test ) .",
"These results are strongly suggestive of brain vessel dysfunction in the mutant mice .",
"Finally , observations made at high magnification after Isolectin and F4/80 staining in both mutant mice did not reveal any defect in tip cell induction and in macrophage recruitment , two processes required for vessels anastomosis ( Adams and Alitalo , 2007; Fantin et al . , 2010 ) ( Figure 6B and not shown ) .",
"Quantifications revealed that the number of macrophages was not altered in the Nkx2 . 1-cre+/Rosa-DTA mice compared to control mice , excluding the direct involvement of macrophages in generating the observed defects ( not shown ) . 10 . 7554/eLife . 09102 . 012Figure 5 . Blood vessel branching is similarly impaired in Nkx2 . 1Cre+/Rosa-DTA and Cspg4-cre+/Rosa-DTA mice .",
"( A–C )",
"DAB staining for PECAM and reconstitution of the vascular network using the Neurolucida tracing tool in wild-type ( A1–A3 ) ( n=8 ) , Nkx2 . 1-cre+/Rosa-DTA ( B1–B3 ) ( n=4 ) and Cspg4-cre+/Rosa-DTA ( C1–C3 ) ( n=4 ) cortical coronal sections at E18 . 5 .",
"A2 , B2 , and C2 are higher magnified views of the boxed regions seen in A1 , B1 , and C1 , respectively .",
"( D–F )",
"DAB staining for erythrocytes in wild-type ( D1–D2 ) ( n=10 ) , Nkx2 . 1-cre+/Rosa-DTA ( E1–E2 ) ( n=5 ) , and Cspg4-cre+/Rosa-DTA ( F1–F2 ) ( n=5 ) cortical coronal sections at E18 . 5 .",
"( G ) Bars ( means ± SEM ) represent the percentages of vessel nodes , vessel branches and volume of the vascular network in mutants compared to wild-type ( n=4; unpaired Student’s t-test ) .",
"The respective absolute values per CCi section are given in Table 1 .",
"All the quantified parameters were significantly decreased in mutant mice .",
"( H ) Bars ( means ± SEM ) represent the percentage of erythrocyte clusters and individual erythrocytes in mutants compared to wild-type .",
"The respective absolute values per CCi section are given in Table 2 ( n=5; unpaired Student’s t-test ) .",
"The number of erythrocyte clusters showed a significant increase in all mutants .",
"The total number of individual erythrocytes remained unchanged .",
"Bar = A1 , B1 , C1: 250 μm; D1 , E1 , F1: 125 μm; A2 , A3 , B2 , B3 , C2 , C3: 62 . 5 μm; D2 , E2 , F2: 50 μm .",
"CCi , cingulate cortex . DOI: http://dx . doi . org/10 . 7554/eLife . 09102 . 01210 . 7554/eLife . 09102 . 013Figure 5—figure supplement 1 . Embryonic Nkx2 . 1-derived NG2 glia do not control cortical blood vessels outgrowth before E16 . 5 . ( A–D ) DAB staining for PECAM in Nkx2 . 1-cre-/Rosa-DTA ( n=4 ) ( A1-A2 , C1–C2 ) and Nkx2 . 1-cre+/Rosa-DTA ( n=4 ) ( B1–B2 , D1–D2 ) telencephalic coronal slices at E14 . 5 ( A1–A2 , B1–B2 ) and E16 . 5 ( C1–C2 , D1–D2 ) .",
"At E14 . 5 , there was no significant blood vessel network defect observed in the cingulate region of Nkx2 . 1-cre+/Rosa-DTA ( B1–B2 ) mutant mice compared to wild-type mice ( A1–A2 ) .",
"By contrast , later at E16 . 5 , the blood vessel network is less developed in the Nkx2 . 1-cre+/Rosa-DTA ( D1–D2 ) mutant mice ( D1–D2 ) compared to control mice ( C1–C2 ) .",
"Scale bar = 250 µm in A1 , B1 , C1 , and D1; 125 µm in A2 , B2 , C2 , and D2 . DOI: http://dx . doi . org/10 . 7554/eLife . 09102 . 01310 . 7554/eLife . 09102 . 014Figure 5—figure supplement 2 . Blood vessel structure is impaired in the septum of Nkx2 . 1-cre+/Rosa-DTA and Cspg4-cre+/Rosa-DTA mice at E18 . 5 . ( A–D ) DAB staining for PECAM in wild-type ( n=12 ) ( A1–A2 ) , Nkx2 . 1Cre+/Rosa-DTA ( n=4 ) ( B1–B2 ) , Cspg4-cre+/Rosa-DTA ( n=4 ) ( C1–C2 ) and Nkx2 . 1-cre+/Eno2-DTA ( n=4 ) ( D1–D2 ) telencephalic coronal slices at E18 . 5 .",
"The blood vessel network was significantly impaired in the septal region ( SEP ) of Nkx2 . 1-cre+/Rosa-DTA ( B1–B2 ) and Cspg4-cre+/Rosa-DTA ( C1–C2 ) mutant mice compared to wild-type mice ( A1–A2 ) .",
"In Nkx2 . 1-cre+/Eno2-DTA ( D1–D2 ) mutant mice , there was no significant blood vessel network defect observed in the septal region .",
"Scale bar = 500 µm in A1 , B1 , C1 , and D1; 125 µm in A2 , B2 , C2 , and D2 . DOI: http://dx . doi . org/10 . 7554/eLife . 09102 . 01410 . 7554/eLife . 09102 . 015Figure 5—figure supplement 3 . Embryonic Nkx2 . 1-derived GABAergic neurons do not control the development and function of cortical blood vessels . DAB staining for PECAM ( A1–A2 , B1–B2 ) and for erythrocytes ( C1–C2 , D1–D2 ) in coronal slices of the dorsal telencephalon of E18 . 5 wild-type ( n=3 ) ( A1–A2 , C1–C2 ) and Nkx2 . 1-cre+/Eno2-DTA ( n=3 ) ( B1–B2 , D1–D2 ) mutant mice .",
"( A2 , B2 , C2 , and D2 ) are higher magnified views of the cingulate bundle ( CI ) region pointed with a black arrowhead in ( A1 , B1 , C1 , and D1 ) , respectively .",
"PECAM and erythrocyte staining revealed that the loss of GABAergic neurons in Nkx2 . 1-cre+/Eno2-DTA mutant mice did not cause any vascular anomalies ( B2 and D2 ) compared to control mice ( A2 and C2 ) .",
"Scale bar = 500 µm in A1 , B1 , C1 , and D1; 125 µm in A2 , B2 , C2 , and D2 .",
"CC , corpus callosum; CCi , cingulate cortex . DOI: http://dx . doi . org/10 . 7554/eLife . 09102 . 01510 . 7554/eLife . 09102 . 016Figure 5—figure supplement 4 . No significant blood vessel network defects are observed in Cspg4-cre+/Rosa-DTA postnatal brains .",
"( A–D )",
"DAB staining for PECAM in Cspg4-cre-/Rosa-DTA ( n=3 ) ( A1–A2 ) and Cspg4-cre+/Rosa-DTA ( n=3 ) ( B1–B2 ) telencephalic coronal slices at P17-P19 ( A1–A2 , B1–B2 ) .",
"At all postnatal ages , there was no significant blood vessel network defect observed in the cingulate region and corpus callosum of Cspg4-cre+/Rosa-DTA ( B1–B2 ) mutant mice compared to control Cspg4-cre-/Rosa-DTA ( A1–A2 ) mice .",
"Scale bar = 500 μm in A1–A2 and B1–B2 . DOI: http://dx . doi . org/10 . 7554/eLife . 09102 . 01610 . 7554/eLife . 09102 . 017Figure 6 . Macrophages and tip cells are not affected in Nkx2-1Cre+/Rosa-DTA mice .",
"( A–B )",
"Double immunohistochemistry for Isolectin and Ter119 , to visualize erythrocytes , on 250-μm-thick coronal of corpus callosum ( CC ) and cingulate bundle ( CI ) sections in wild-type ( A1–A4 ) ( n=5 ) and Nkx2 . 1-cre+/Rosa-DTA ( B1–B4 ) ( n=5 ) mice at E18 . 5 .",
"In the CC and the CI ( B1–B2 ) of Nkx2 . 1-cre+/Rosa-DTA mice , the blood vessels have a twisted shape and the erythrocytes are clustered ( white arrowheads in B1 and B2 ) compared to the wild-type vessels that formed a regular network ( white arrowheads in A1 and A2 ) .",
"In the CC and the CI of both wild-type ( A3–A4 ) and Nkx2 . 1-cre+/Rosa-DTA mice ( B3–B4 ) , guidepost macrophages labeled by the isolectin ( white arrowheads in A3–A4 and B3–B4 ) are found in the close vicinity of the tip cells ( white arrows in A3–A4 and B3–B4 ) .",
"The tip cells exhibit the same morphology and the same number of filopodia with similar length in both circumstances .",
"Bar = 60 μm in A1 , B1; 40 μm in A2 , B2 , A3 , A4 , B3 , B4 .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 09102 . 01710 . 7554/eLife . 09102 . 018Table 1 . Nodes , branches and volume of the vascular network visualized per CCi section in control and transgenic mice used to ablate Nkx2 . 1-derived or only NG2+ cells .",
"The values ( mean ± SEM ) corresponding to the number of nodes , the number of branches and the volume of the vascular network per CCi section are given for: ( 1 ) Nkx2 . 1-cre+/Rosa-DTA mice and their corresponding control Nkx2 . 1-cre-/Rosa-DTA mice , and ( 2 ) Cspg4-cre+/Rosa-DTA mice and their corresponding control Cspg4-cre-/Rosa-DTA mice ( n=4 each; unpaired Student’s t-test ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 09102 . 018Values ( mean ± SEM ) for the vascular network per CCi sectionGenotypeNodes numberBranches numberVascular volume * 103 ( µm³ ) Nkx2 . 1-cre-/Rosa-DTA 103 . 44 ± 7 . 60241 . 77 ± 14 . 6520302 . 06 ± 1290 . 76Nkx2 . 1-cre+/Rosa-DTA 84 . 63 ± 6 . 65207 . 75 ± 11 . 5616692 . 46 ± 879 . 03Cspg4-cre-/Rosa-DTA 135 . 50 ± 8 . 60299 . 50 ± 16 . 8920361 . 28 ± 1429 . 69Cspg4-cre+/Rosa-DTA 109 . 25 ± 6 . 20250 . 87 ± 1315921 . 88 ± 403 . 4410 . 7554/eLife . 09102 . 019Table 2 . Number of erythrocyte clusters and of individual erythrocytes visualized per CCi section in control and transgenic mice used to ablate Nkx2 . 1-derived or only NG2+ cells .",
"The values ( mean ± SEM ) corresponding to the number of erythrocyte clusters and the number of individual erythrocytes per CCi are given for: ( 1 ) Nkx2 . 1-cre+/Rosa-DTA mice and their corresponding control Nkx2 . 1-cre-/Rosa-DTA mice , and ( 2 ) Cspg4-cre+/Rosa-DTA mice and their corresponding control Cspg4-cre-/Rosa-DTA mice ( n=5 each; unpaired Student’s t-test ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 09102 . 019Values ( mean ± SEM ) per CCi sectionGenotypeNumber of erythrocyte clusters per CCi sectionNumber of individual erythrocytes per CCi sectionNkx2 . 1-cre-/Rosa-DTA 6 . 60 ± 0 . 88302 . 33 ± 19 . 12Nkx2 . 1-cre+/RosaDTA 13 . 00 ± 2 . 62273 . 42 ± 41 . 44Cspg4-cre-/Rosa-DTA 5 . 83 ± 0 . 62397 . 80 ± 18 . 96Cspg4-cre+/Rosa-DTA 14 . 95 ± 1 . 77447 . 30 ± 22 . 91 Next , we tested whether the observed loss of cortical GABAergic interneurons might participate toward the observed vascular defects .",
"In order to address this issue , we selectively ablated neuronal cells without affecting NG2+ glia , by using Nkx2 . 1-cre+/Eno2-DTA:Gad1-EGFP mice .",
"In these mice , the diphtheria toxin ( DTA ) is expressed under the control of a neuron-specific promoter ( enolase 2 , Eno2 ) whose action is dictated by the Cre-mediated recombination ( Nkx2 . 1-cre+ ) .",
"In Nkx2 . 1-cre+/Eno2-DTA:Gad1-EGFP mice , we noticed a significant depletion of GAD67-GFP+ GABAergic neurons in the medial cortex ( Figure 3—figure supplement 1C–E; n=6; unpaired Student’s t-test ) closely equivalent to the loss seen in Nkx2 . 1-cre+/Rosa-DTA:Gad1-EGFP mice .",
"In this mice strain , NG2+ glia were not affected and immunostaining analysis with vessel markers ( NG2; n=3 and PECAM; n=4 ) and erythrocytes ( n=3 ) did not reveal any developmental vessel defects that accompanied the substantial loss of GABAergic neurons in the Nkx2 . 1-cre+/Eno2-DTA cortex ( Figure 4D , Figure 5—figure supplement 2 and 3 ) .",
"Thus , these findings exclude the possibility that the poorly developed vascular network in Nkx2 . 1-cre+/Rosa-DTA mice could be a direct result of GABAergic interneuron loss .",
"Altogether , these results strongly suggest that the loss of Nkx2 . 1-derived cells , most probably NG2+ glia , was responsible for brain angiogenesis defects .",
"To confirm that NG2+ glia play a role in the establishment of the vascular network , we used Cspg4-cre+/Rosa-DTA mice in which NG2+ glia were specifically depleted with a drastic loss of 55% ( 18 . 96 ± 0 . 98 x 103/mm3 NG2+ glia in CCi sections of Cspg4-cre-/Rosa-DTA mice versus 8 . 52 ± 1 . 06 x 103/mm3 NG2+ glia in CCi sections of Cspg4-cre+/Rosa-DTA mice; n=3 , unpaired Student’s t-test; p<0 . 001 ) ( Figure 2—figure supplement 1B–D , Figure 3F , 4C ) .",
"Consistent with the very low level of Cre-mediated recombination in the brain vessel pericytes of Cspg4-cre+/Rosa-EYFP mice , we did not notice any obvious loss of PDGFR-β+ pericytes in the Cspg4-cre+/Rosa-DTA cortex as well ( compare Figure 3F and Figure 3D; n=3 ) .",
"The number of PDGFR-β+ pericytes observed in Cspg4-cre+/Rosa-DTA mice ( n=11 ) was similar to those seen in Cspg4-cre-/Rosa-DTA mice ( n=11 ) control cingulate bundle , as proved by the quantification analyses ( Figure 3H ) .",
"Thus , these results additional confirm that due to the lack of sufficient Cre-mediated recombination in the pericytes , the blood vessel network seen with Cspg4-cre+/Rosa-DTA mice brains can be majorly attributed to the NG2+ glia .",
"The vessel defects in the cerebral cortex and septum of Cspg4-cre+/Rosa-DTA mice were seen at all rostrocaudal levels and were similar to those that were aforementioned for Nkx2 . 1-cre+/Rosa-DTA mice ( Figure 5C and Figure 5—figure supplement 2C; n=5 ) .",
"The vessels displayed a significant reduction in the number of nodes and branches and of the density of the vascular network ( reduction of nodes 16 . 23 ± 4 . 33% , p<0 . 05; reduction of branches 12 . 93 ± 2 . 37% , p<0 . 05; reduction of the total volume of the vascular network: 21 . 8 ± 1 . 98% , p<0 . 05; n=4 , unpaired Student’s t-test , Figure 5C , G and Table 1 ) .",
"Moreover , the number of erythrocyte aggregates was significantly increased too ( Figure 5F , H and Table 2; n=5 , unpaired Student’s t-test ) .",
"These secondary defects may reflect brain vessel dysfunction as a result of disturbed brain vessel network development .",
"As in Nkx2 . 1-cre+/Rosa-DTA mice , the involvement of pericytes can be directly excluded as a causative factor of the observed vasculature defects in Cspg4-cre+/Rosa-DTA due to lack of recombination in this cell population ( Figure 3B and 3F ) .",
"The only population that is centrally affected in both transgenic mice strains , Cspg4-cre+/Rosa-DTA and Nkx2 . 1-cre+/Rosa-DTA mice , are NG2+ glia .",
"Hence , the poor development of the vascular network and exactly similar vascular defects in both transgenic mice strains , Cspg4-cre+/Rosa-DTA and Nkx2 . 1-cre+/Rosa-DTA mice , is triggered by the absence of NG2+ glia .",
"Thus , confirming the involvement of Nkx2 . 1-derived NG2+ glia in controlling brain angiogenesis .",
"We also noticed some axonal guidance defects in the telencephalic commissures of Nkx2 . 1-cre+/Rosa-DTA mice ( Minocha et al . , 2015 ) .",
"As these defects were not at all visible in Cspg4-cre+/Rosa-DTA mice , the involvement of NG2+ glia could be excluded .",
"Analyses of postnatal Cspg4-cre+/Rosa-DTA brains at P17-P21 ( n=3 ) , however , did not reveal any significant blood vessel network defects when compared to littermate Cspg4-cre-/Rosa-DTA brains ( n=3 ) ( Figure 5—figure supplement 4 ) .",
"Thus , it appears that the contribution of the NG2+ glia is important to keep up with the fast pace of embryonic brain development; however , their loss in postnatal brain is may be compensated by either later appearing waves of NG2+ glia like shown previously ( Kessaris et al . , 2006 ) or through other cells .",
"Taken together , this study shows for the first time that NG2+ glia play a major role in embryonic brain angiogenesis .",
"The temporal and spatial presence of these NG2+ glia is specifically articulated to aid proper vessel network formation during embryogenesis .",
"The poor development of the blood vessel network in absence of NG2+ glia underlines the importance of understanding the point-to-point connectivity that is generated by the contact between vessels and polydendrocyte processes .",
"Our results , altogether , reveal that brain angiogenesis in embryos requires the presence of Nkx2 . 1-derived NG2+ glia .",
"The Nkx2 . 1-derived NG2+ glia begin to populate the entire telencephalon by E14 . 5 but are transient in nature and disappear by the early postnatal stages .",
"The astrocytes and macrophages have already been reported to play divergent roles during angiogenesis ( Fantin et al . , 2010; Larrivee et al . , 2009; He et al . , 2013 ) .",
"This study , however , shows for the first time that Nkx2 . 1-derived NG2+ glia play a major role in brain angiogenesis .",
"This unravels the important interplay between the ventral and the dorsal telencephalon in multiple physiological functions .",
"We show here that the ventral telencephalon participates not only to the establishment of the cortical neuronal circuitry by generating tangentially migrating GABAergic interneurons , but also to embryonic angiogenesis .",
"By revealing a novel and essential role for NG2+ glia originating from the ventral telencephalon in cortical angiogenesis , our study brings new perspectives to pathophysiological consequences linked to tangential migration defects ."
],
[
"The generation of brain’s vascular network starts very early during embryogenesis , and it occurs concomitantly with the development of neuronal network in the brain ( Patan , 2000; Vasudevan and Bhide , 2008 ) .",
"Efficient growth of the vasculature is essential to meet the metabolic needs of the developing brain .",
"However , the mechanisms regulating vessel network’s establishment are not yet fully understood .",
"Several studies , so far , have shown that angiogenesis involves participation of ECs along with perivascular cells , referred to as pericytes , and macrophages ( Fantin et al . , 2010; Newman and Hughes , 2012; Nucera et al . , 2011; Outtz et al . , 2011; Pollard , 2009; Bergers and Song , 2005 ) .",
"This angiogenic process is completed by a complex repertoire of spatially and temporally orchestrated events that engage several transcription factors , growth factors with their receptors , adhesion molecules , proteases , chemokines , cytokines , and extracellular matrix ( Adams and Alitalo , 2007; Carmeliet and Tessier-Lavigne , 2005; Lu et al . , 2004; Larrivee et al . , 2007; Eichmann et al . , 2005 ) .",
"In our model to study embryonic angiogenesis , we have used cell-specific ablation strategy with the help of Cre reporter strains , namely Nkx2 . 1-cre and Cspg4-cre mice , together with 'floxed' diphtheria toxin ( Rosa-DTA ) to selectively kill cells under Nkx2 . 1 or NG2 promoter , respectively .",
"With Nkx2 . 1-cre+/Rosa-DTA mice , we observed the formation of a poorly developed vessel network in several telencephalic regions .",
"A significant reduction of intersections , connectivity and vasculature density was observed .",
"The cortical vessels were seen to loose their regular diameter and harbored erythrocyte accumulations at the level of enlarged vessel segments .",
"Since , the Nkx2 . 1-derived cells comprise of GABAergic interneurons ( Marin and Rubenstein , 2001; Xu et al . , 2008; Sussel et al . , 1999; Du et al . , 2008; Xu et al . , 2004 ) and NG2+ glia , we investigated the two populations separately to dissect the responsible cell type .",
"With Nkx2 . 1-cre+/Rosa-DTA mice , a drastic decrease of 50% GABAergic interneurons and complete loss of NG2+ glia was seen at midline regions .",
"Complete loss of NG2+ glia with Nkx2 . 1-cre+/Rosa-DTA mice displayed the subpallial origin of the polydendrocyte population at the midline specifically .",
"Further on , to look into the role of GABAergic interneurons in the observed vessel network phenotype , we made use of Nkx2 . 1-cre+/Eno2-DTA:Gad1-EGFP mice .",
"These mice did not show any loss of NG2+ glia nor any vessel network formation defects , although the loss of GABAergic neurons was comparable to that seen with Nkx2 . 1-cre+/Rosa-DTA mice .",
"Thus , we could exclude the GABAergic interneurons from being the cause of the observed phenotype .",
"We could also exclude the role of astrocytes because Nkx2 . 1-cre+mice recombination can only been seen in GLAST+ astrocytes of the ventral telencephalon , and dorsal telencephalon astrocytes remain unaffected in Nkx2 . 1-cre+/Rosa-DTA mice .",
"Next , we aimed to target the NG2+ population .",
"To this purpose , we additionally used Cspg4-cre+/Rosa-DTA mice where NG2+ glia were depleted by more than 50% .",
"Interestingly , we visualized similar vessel network defects as Nkx2 . 1-cre+/Rosa-DTA mice .",
"Notably , NG2 chondroitin sulfate proteoglycan is expressed not only in glia but also on surface of pericytes in the brain ( Nishiyama et al . , 2009; Ozerdem et al . , 2001 ) .",
"The loss of pericytes can also lead to aberrations in fetal brain vasculature and maintenance of blood–brain barrier leading to vascular instability ( Stallcup and Huang , 2008; Armulik et al . , 2010; Daneman et al . , 2010 ) .",
"Since the role of both perivascular cells has been well established in angiogenesis , we investigated if our Nkx2 . 1-cre+/Rosa-DTA and Cspg4-cre+/Rosa-DTA mice lack pericytes or ECs .",
"The Nkx2 . 1-cre+/Rosa-DTA mice directly excluded the involvement of pericytes and ECs due to the absence of Cre expression in both cell types .",
"As a result , we can exclude that the defects can be due to the intrinsic action of Nkx2 . 1 in ECs as observed in Nkx2 . 1 KO mice ( Vasudevan and Bhide , 2008; Vasudevan , et al . , 2008 ) .",
"Also , the Cspg4-cre+/Rosa-DTA mice did not show any loss of pericytes due to very low levels for Cre recombination in these cells .",
"Therefore , our cell-specific ablation analysis directly points toward involvement of NG2+ glia , the only common affected cell population , in embryonic brain angiogenesis .",
"The origin of these NG2+ glia coincides not only temporally with the brain vessel network formation but also spatially due to their close proximity to the developing vessel network .",
"Another identified key player in brain vessel network are macrophages that have been implicated in both physiological and pathological angiogenesis ( Newman and Hughes , 2012; Nucera et al . , 2011; Pollard , 2009 ) .",
"The macrophages colonize the embryonic brain even before vascularization is built ( Fantin et al . , 2010 ) and have been observed to localize at vessels junctions and to interact with the endothelial tip cells , by forming sort of bridges in order to align them and to prepare them for the fusion ( Fantin et al . , 2010 ) .",
"Similar to macrophages , the NG2+ glia were localized at sprouting tip cells or at branch fusion points , but , in addition , were also present all along the vessel walls .",
"They made several contacts with mural cells with their extended processes .",
"Although our selective cell ablation strategy did not affect the macrophage population , it is probable that the two populations may influence the vessel network through a concerted action .",
"The strategic positioning of NG2+ glia raises the possibility that they interact functionally by making synapses and generating action potentials ( De Biase et al . , 2010; Clarke et al . , 2012 ) or by sending signals to mural cells .",
"Their positioning along and around the developing networks might provide them with the proximity required to monitor the firing patterns of surrounding cells ( De Biase et al . , 2010; Clarke et al . , 2012 ) .",
"Additionally , the NG2+ glia may act as guidepost cells by secreting growing factors or guidance molecules required for the proper development and function of brain vessels .",
"During development , the formation of the vessel network is dependent on several growth factors like vascular endothelial growth factor A ( VEGF-A ) , VEGF-B , VEGF-C , VEGF-D , and placental growth factor ( PlGF ) that bind to different tyrosine kinase receptors ( VEGFR ) expressed on ECs ( Adams and Alitalo , 2007; Coultas et al . , 2005; Fantin et al . , 2010; Ferrara et al . , 2003; Gerhardt et al . , 2003; Grunewald et al . , 2006; Laakkonen et al . , 2007; Lee et al . , 2005; Neufeld et al . , 2002; Pan et al . , 2007; Plate , 1999; Ruhrberg et al . , 2002; Shibuya , 2009 ) .",
"In addition to these growth factors , other signaling pathways also aid in the development of the vascular circuitry like angiopoietin1/Tie2; Notch/Delta-like-4/JAG1; Wnt signaling and guidance cues with their associated receptors such as ephrin-B2 and EphB4 , Slit and Robo4 , Netrins and Unc5/Dcc , class 3 semaphorins and Neuropilins/Plexins ( Adams and Alitalo , 2007; Adams and Eichmann , 2010; Bedell et al . , 2005; Bray , 2006; Eichmann et al . , 2005; Gerhardt et al . , 2004; Gitler et al . , 2004; Gu et al . , 2005; Hellstrom et al . , 2007; Klagsbrun et al . , 2002; Kruger et al . , 2005; Larrivee et al . , 2009; Leslie et al . , 2007; Lobov et al . , 2007; Lu et al . , 2004; Neufeld et al . , 2005; Park et al . , 2003; Sainson et al . , 2005; Suchting et al . , 2007;Torres-Vazquez et al . , 2004; Wilson et al . , 2006 ) .",
"Recently , an elegant study performed in postnatal mice showed that the OPCs-intrinsic Hypoxia-inducible factors 1/2 ( HIF1/2 ) signaling stimulates EC proliferation in vitro and increases CC white matter angiogenesis in vivo ( Yuen et al . , 2014 ) .",
"Conversely , OPCs-encoded HIF1/2a inactivation causes insufficient CC angiogenesis and commissural axon’s degeneration .",
"This study leads us to question if the NG2+ embryonic glia we have identified also regulate vessels outgrowth through HIF1a and/or HIF2a action .",
"Notably , the population addressed by Yuen et al . , 2014 primarily focuses on the effects in postnatal brains , whereas the NG2+ glia identified by us disappear by P8 .",
"Nonetheless , it will be interesting to perform in vivo brain analyses of angiogenesis in mouse embryos from Cspg4-cre mice crossed with HIF1a floxed , HIF2a floxed or HIF1/2a floxed mice , allowing selective gene inactivation of HIFs in NG2+ glia only .",
"Several studies have shown that growth factors can regulate the NG2+ glial cell proliferation and migration – of which some belong to PI3K/mTOR and Wnt/β-catenin signaling pathways ( Hill et al . , 2013; Frost et al . , 2009 ) .",
"In particular , Wnt/β-catenin signaling pathway has been suggested to be the important player required for molecular and cellular steps necessary for proper brain vascularization .",
"The broad expression of Wnt7a/7b ligands in the developing early embryonic CNS and induction of Wnt/ β-catenin signaling is required for formation and differentiation of the embryonic brain vasculature ( Stenman et al . , 2008 ) .",
"Additionally , the Wnt/ β-catenin signaling is also important for the blood–brain barrier , a structure formed by the brain blood vessels .",
"The effects of the absence of Wnt7a/7b ligands are evident already in early embryonic ages , that is E12 . 5 , and are crucial for ventral CNS vasculature .",
"Notably , the NG2+glia are not present in the dorsal telencephalon as early as E12 . 5 .",
"Hence , a more closer dissection of these and other Wnts would be required to ascertain if the NG2+glia are mediating their roles in vascularization through Wnts at later stages , that is , when we observed the defects ( E16 . 5–E18 . 5 ) .",
"Since the repertoire of factors expressed by NG2+ glia has not been explored much yet , a complete dissection of the different angiogenic factors will be required for a better understanding .",
"It would be also necessary to analyze the coordinated and oriented cell movements that are necessary for the development of the vascular network especially during sprouting and subsequent maturation phases .",
"The complete understanding of molecular mechanisms undertaken by NG2+ glia to guide and aid angiogenesis will provide significant insight into angiogenesis .",
"This study highlights the importance of spatially and temporally synchronized generation of NG2+ glia that act as guidepost cells for embryonic vasculature establishment .",
"The migratory profile of the NG2+ glia has been explored in several contexts before , but there is no such available information if they directly use the vasculature .",
"Like the other major population of Nkx2 . 1-derived GABAergic interneurons ( Marin and Rubenstein , 2001; Guo and Anton , 2014; Elias et al . , 2008; Chedotal and Rijli , 2009 ) , we believe that the NG2+ glia also use tangential migration to colonize the cortex .",
"Clues for this are evident from our immunostaining of the NG2+ glia at embryonic and early postnatal stages in the control brains .",
"Previous reports have discussed the migration of sub-populations of the NG2+ cells during embryonic and early postnatal development ( Sugimoto et al . , 2001; Cayre et al . , 2009; Aguirre and Gallo , 2004 ) .",
"Additionally , in adult tissues , the migration of NG2+ cells has also been observed where these cells migrate in relation to each other ( Hughes et al . , 2013 ) .",
"Also , some cell–cell interactions between axonal tracts and OPCs has been believed to be important for migration , and molecules such as N-cadherins , neuregulins , ephrins , and integrins have been implicated ( de Castro and Bribian , 2005 ) .",
"A recent study showed that the proteoglycan NG2 itself could play a role in the directed localization of NG2+ cells ( Biname et al . , 2013 ) .",
"Fittingly , the NG2+ cells exhibit maximal migration upon encountering a region deficient of other NG2+ cells ( Cayre et al . , 2009 ) or when the region has been induced to lack NG2+ cells either through experimental intervention or as a result of injury ( Blakemore and Irvine , 2008 ) .",
"Our work depicting the elucidation of the role of NG2+ glia in early phases of brain vessel network provides new insights into the mechanisms that take place during embryonic brain angiogenesis .",
"The requirement of a properly developed vessel network is immense during embryonic ages to meet the growing nutritional needs of the embryo .",
"Several human neuropathologies related to neonatal encephalopathy are induced by incidents involving hypoxia to the brain ( e . g . in cerebral palsy ) .",
"We suspect that one possible reason for cerebral palsy could be the presence of dysfunctional NG2+ glia , which then leads to decrease in vascularization followed by neuronal injury generating the pathological condition .",
"OPCs have been shown to be involved in glioblastoma genesis and evolution ( Barrett et al . , 2012; Lindberg et al . , 2009; Persson et al . , 2010; Sugiarto et al . , 2011; Svendsen et al . , 2011 ) .",
"Therefore , our research may help in the understanding of the pathological cellular and vessel processes that take place during brain tumor evolution .",
"Strategies based on NG2+ cell targeting may significantly improve long-term outcome of patients with glioblastoma ( GBM ) .",
"It will be interesting to further characterize the role of NG2+ glia in GBMs tumour angiogenesis and to investigate whether NG2+ glia-encoded HIFs are involved in tumour-associated NG2+ glial differentiation and angiogenesis .",
"Altogether , the combined role play of ECs , pericytes , macrophages , and glia and the molecular mechanisms defining their respective mode of actions can aid in better understanding of the blood vessel network formation , and help alleviate many disease pathologies ."
],
[
"All studies on mice of either sex have been performed in compliance with the national and international guidelines and with the approval of the Federation of Swiss cantonal Veterinary Officers ( 2164 ) .",
"For staging of embryos , midday of the day of vaginal plug formation was considered as embryonic day 0 . 5 ( E0 . 5 ) .",
"WT mice maintained on a C57Bl/6 genetic background were used for developmental analysis .",
"We used heterozygous Gad1-EGFP knock-in mice , described in this work as Gad1-EGFP mice ( Tamamaki et al . , 2003 ) .",
"We used Nkx2 . 1-cre ( Xu et al . , 2008 ) and Cspg4-cre ( Jackson Laboratory: B6;FVB-Tg ( Cspg4-cre ) 1Akik/J ) ( Zhu et al . , 2008a ) transgenic mice that have been described previously .",
"The reporter mouse Rosa26R–Yellow fluorescent protein ( YFP ) ( Srinivas et al . , 2001 ) was used to reliably express YFP under the control of the Rosa26 promoter upon Cre-mediated recombination .",
"Embryos were recognized by their YFP fluorescence .",
"The reporter Rosa26:lacZ/DTA ( Rosa-DTA ) mouse line ( Brockschnieder et al . , 2006 ) was used to express conditionally the cytototoxic diphtheria toxin polypeptide toxic fragment A ( DTA ) allele under the control of ubiquitously active Rosa26 promoter .",
"By crossing Nkx2 . 1-cre mice with Rosa26-DTA mice that express the diphtheria toxin under the control of the Nkx2 . 1 promoter , only Nkx2 . 1-expressing post-mitotic cells were depleted in the whole brain without affecting the Nkx2 . 1+ precursors in the progenitor zones .",
"The neuron-specific enolase ( Eno2 ) -stop-DTA ( Eno2-DTA ) mice ( Kobayakawa , et al . , 2007 ) were used to induce the expression of highly potent diphtheria toxin fragment A ( DTA ) from neuron-specific enolase locus , and resulted in specific ablation of neurons only .",
"Embryos were collected after caesarean section and quickly killed by decapitation .",
"Their brains were dissected out and fixed by immersion overnight in 4% paraformaldehyde solution in 0 . 1 M phosphate buffer ( pH 7 . 4 ) at 4°C .",
"Postnatal mice were profoundly anesthetized and perfused with the same fixative , and their brains post-fixed for 4 hr .",
"Brains were cryoprotected in a solution of 30% sucrose in 0 . 1 M phosphate buffer ( pH 7 . 4 ) , frozen and cut in 50-µm-thick coronal sections for immunostaining .",
"Rat monoclonal antibodies used were: Lymphocyte antigen76 ( Ter119 ) ( Lifespan Bioscience , Switzerland ) ; PDGFRα ( BD Bioscience , Switzerland ) ; PECAM ( CD31 ) ( BD Pharmingen , Switzerland ) .",
"Rabbit polyclonal antibodies used were: GFAP ( DAKO , Carpinteria , CA ) ; GFP ( Molecular Probes , Zug , Switzerland ) ; NG2 ( Chemicon , Temecula , CA ) ; Nkx2 . 1 ( Biopat , Caserta , Italy ) ; Olig2 ( Millipore , Switzerland ) ; S100β ( Swant , Bellinzona , Switzerland ) .",
"Goat polyclonal antibodies used were: PDGFRβ ( R&D Systems , Switzerland ) .",
"Guinea pig antibody was: GLAST ( Chemicon , Temecula , CA ) .",
"Chicken antibody was: GFP ( Aves , UK ) .",
"Biotin-conjugated antibodies were: Isolectin Ig-IB4 ( Molecular probes , Zug , Switzerland ) .",
"Fluorescence immunostaining: Non-specific bindings were blocked during pre-incubation and incubations after adding 2% normal horse serum in PBS 1X solution supplemented with 0 . 3% Triton X-100 .",
"The primary antibodies were detected with Cy3-conjugated ( Jackson ImmunoResearch laboratories , West Grove , PA ) and Alexa 488- , Alexa 594- or Alexa 647-conjugated antibodies or streptavidin ( Molecular Probes , Eugene , OR ) .",
"Sections were counterstained with Hoechst 33 , 258 ( Molecular Probes , Switzerland ) , mounted on glassslides and covered in Mowiol 4–88 mounting medium ( Calbiochem , Bad Soden , Germany ) .",
"DAB immunostaining: Endogenous peroxidase reaction was quenched with 0 . 5% hydrogen peroxide in methanol , and unspecific binding was blocked by adding 2% normal horse serum during incubations in the Tris-buffered solutions containing 0 . 3% Triton X-100 .",
"The primary antibodies were detected with biotinylated secondary antibodies ( Jackson ImmunoResearch , West Grove , PA ) and the Vector-Elite ABC kit ( Vector Laboratories , Burlingame , CA ) .",
"The slices were mounted on glassslides , dried , dehydrated , and covered with Eukitt mounting medium .",
"DAB immunostaining was adapted for erythrocytes staining: Endogenous peroxidase reaction was directly used to visualize the erythrocytes .",
"Therefore , no hydrogen peroxide was added .",
"Sections were pre-incubated for 10 min in 0 . 05% DAB diluted in 0 . 1 M Tris buffer .",
"The reaction was started with the same solution supplemented with 0 . 5% H2O2 and stopped with 0 . 1 M Tris buffer after 10 min .",
"The slices were mounted as per standard protocol .",
"DAB-stained sections were imaged with a Zeiss Axioplan2 microscope equipped with 10× , 20× , 40× , or 100× Plan neofluar objectives and coupled to a CCD camera ( Axiocam MRc 1388x1040 pixels ) .",
"Fluorescent-immunostained sections were imaged using confocal microscopes ( Leica SP5 or Zeiss LSM 710 Quasar ) equipped with 10× , 20× , 40× oil Plan neofluar and 63× oil Plan apochromat objectives .",
"Fluorophore excitation and scanning were done with an Argon laser 458 , 488 , 514 nm ( blue excitation for GFP and Alexa 488 ) , with a HeNe laser 543 nm ( green excitation for Alexa 594 , CY3 and DiI ) , with a HeNe laser 633 nm ( excitation for Alexa 647 and CY5 ) , and with a Diode laser 405 nm ( for Hoechst-stained sections ) .",
"Z-stacks of 10–25 planes were acquired for each coronal section in a multitrack mode avoiding crosstalk , and the creation of isosurfaces was done with Imaris 7 . 2 . 1 software ( Bitplane Inc . ) .",
"All 3D Z-stack reconstructions and image processing’s were performed with Imaris 7 . 2 . 1 software .",
"The generation of iso-surfaces ( object defining a surface surrounding voxels located between two threshold values ) allowed us to visualize the contours of cells , of blood vessels and of growth cones .",
"The colocalization between two fluorochromes was calculated and visualized by creating a yellow channel .",
"Figures were processed in Adobe Photoshop CS5 .",
"Loss of GABAergic neurons: In 50-μm-thick coronal sections of Nkx2 . 1-cre+/Rosa-DTA:GAD67-GFP , Nkx2 . 1-cre-/Rosa-DTA:GAD67-GFP , Nkx2 . 1-cre+/Eno2-DTA:Gad1-EGFP and Nkx2 . 1-cre-/Eno2-DTA:Gad1-EGFP embryos , GABAergic neurons of the cortical region ( CCi and CI ) were counted at E18 . 5 as the number of cells labeled for the GFP .",
"Quantification were done on a sample of n=8 sections in Nkx2 . 1-cre+/Rosa-DTA:Gad1-EGFP and corresponding controls and on a sample of n=6 sections in Nkx2 . 1-cre+/Eno2-DTA:Gad1-EGFP and corresponding controls .",
"To study the density of GABAergic neurons , the values were quantified in all the scanned planes of each stack and were reported per volume unit ( number of cells/mm3 ) .",
"The quantifications were done using the Imaris 7 . 2 . 1 software .",
"Preservation of GLAST+ glia in the cingulate cortex: In 50-μm-thick brain sections , the GLAST+ glia in the CCi labeled for GLAST were counted from a sample of n=4 sections of E18 . 5 Nkx2 . 1-cre-/Rosa-DTA and of Nkx2 . 1-cre+/Rosa-DTA embryos .",
"To study the density of GABAergic neurons and GLAST+ glia , the values were quantified in all the scanned planes of each stack and were reported per volume unit ( number of cells/mm3 ) .",
"The quantifications were done using the Imaris 7 . 3 . 1 software .",
"Cspg4-cre recombination level analysis and percentage of NG2 , Olig2 , S100β , GFAP and GLAST markers expression by YFP-labeled NG2+ glia in Cspg4-cre+/Rosa-EYFP mice: In 50-μm-thick brain sections of Cspg4-cre+/Rosa-EYFP embryos at E18 . 5 , glia of the CC ( midline ) , CI and CCi labeled for YFP and NG2 were counted ( n=3 ) .",
"For each condition , at least 3 different Z-stacks were obtained at 63× magnification by using a Leica SP5 microscope .",
"To study the density of NG2+ glia , the values were quantified in all the scanned planes of each stack and were reported per volume unit ( number of cells/mm3 ) .",
"Cspg4-cre recombination level analysis and percentage of PDGFRβ pericyte markers expression by YFP-labeled NG2+ cells in Cspg4-cre+/Rosa-EYFP mice: In 50-μm-thick brain sections of Cspg4-cre+/Rosa-EYFP embryos at E18 . 5 ( n=10 ) , YFP+ pericytes and YFP+ pericytes of the dorsal telencephalic regions labeled for NG2 were counted .",
"The percentage of Cre-mediated recombination within the NG2+ cells was calculated as follows: YFP+ cells/NG2+ cells X100 .",
"The percentage of YFP+/PDGFRβ+ , YFP+/NG2+ , Olig2+/NG2+ , YFP+/S100β+ , YFP+/GFAP+ and YFP+/GLAST+ glia within YFP+ cells was calculated .",
"The quantification was done using Imaris 7 . 2 . 1 software .",
"Percentage of NG2 marker expression by YFP-labeled cells in Nkx2 . 1-cre+/Rosa-EYFP mice: In 50-μm-thick brain sections of Nkx2 . 1-cre+/Rosa-EYFP embryos at E18 . 5 , YFP+ cells of the CI labeled for the NG2 were counted ( n=3 ) .",
"For each condition , at least 3 different Z-stacks were obtained at 63x magnification by using a Leica SP5 microscope .",
"To study the density of YFP+/NG2+ glia , the values were quantified in all the scanned planes of each stack and were reported per volume unit ( number of cells/mm3 ) .",
"The percentage of YFP+/NG2+ glia within YFP+ cells was calculated .",
"The quantification was done using Imaris 7 . 2 . 1 software .",
"Loss of NG2+ glia and PDGFRβ+ pericytes:In 50-μm-thick brain sections , the NG2+ glia in the CI labeled for NG2 were counted from a sample of n=11 sections of E18 . 5 Cspg4-cre-/Rosa-DTA and of Cspg4-cre+/Rosa-DTA embryos .",
"Similarly , the pericytes in the dorsal telencephalon labeled for PDGFRβ were counted from a sample of n=11 sections of Cspg4-cre-/Rosa-DTA and of Cspg4-cre+/Rosa-DTA mice .",
"To study the density of NG2+ glia , PDGFRβ+ pericytes in each condition , the values were quantified in all the scanned planes of each stack and were reported per volume unit ( number of cells/mm3 ) .",
"The quantification was done using Imaris 7 . 2 . 1 software .",
"Blood vessels morphology and erythrocytes distribution analysis:In 50-μm-thick brain sections of Nkx2 . 1-cre+/Rosa-DTA , Nkx2 . 1-cre-/Rosa-DTA , Cspg4-cre+/Rosa-DTA and Cspg4-cre-/Rosa-DTA embryos at E18 . 5 , the vessels network morphology and erythrocytes were analyzed from a sample of more that n=10 sections for each condition .",
"DAB staining for PECAM was used to label blood vessels .",
"The blood vessels morphology was analyzed in a predetermined region comprising of the CC , CI , and CCi ( surface area/section=6*106μm2 ) .",
"The 3D blood vessels morphology was reconstructed using Neurolucida 9 . 0 software ( MicroBrightField , Williston , VT ) .",
"The axonal tracing tool allows to trace and to calculate for each vessels network the number of individual trees , the number of nodes , the number of branches , the length , the surface , and the volume of the blood vessels network for each condition .",
"DAB-stained erythrocytes were counted in a predetermined region comprising of the CC , CI , and CCi ( surface area/section=20*106μm2 ) .",
"The erythrocytes were counted as a cluster when more than five consecutive erythrocytes were closely apposed to each other , while the well separated individual erythrocytes were counted as individual cells .",
"The quantification was done using Neurolucida 9 . 0 software .",
"The results from all quantifications were analyzed with the aid of Statview software ( SAS Institute ) .",
"For all analysis , values from at least three independent experiments were first tested for normality .",
"Values that followed a normal distribution were compared using Student's t-test ( *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 ) .",
"The neuroanatomical nomenclature is based on the 'Atlas of the prenatal mouse brain' ( Schambra et al . , 1991 ) ."
]
] | [
"The NG2+ glia , also known as polydendrocytes or oligodendrocyte precursor cells , represent a new entity among glial cell populations in the central nervous system .",
"However , the complete repertoire of their roles is not yet identified .",
"The embryonic NG2+ glia originate from the Nkx2 . 1+ progenitors of the ventral telencephalon .",
"Our analysis unravels that , beginning from E12 . 5 until E16 . 5 , the NG2+ glia populate the entire dorsal telencephalon .",
"Interestingly , their appearance temporally coincides with the establishment of blood vessel network in the embryonic brain .",
"NG2+ glia are closely apposed to developing cerebral vessels by being either positioned at the sprouting tip cells or tethered along the vessel walls .",
"Absence of NG2+ glia drastically affects the vascular development leading to severe reduction of ramifications and connections by E18 . 5 .",
"By revealing a novel and fundamental role for NG2+ glia , our study brings new perspectives to mechanisms underlying proper vessels network formation in embryonic brains ."
] | [
"In the brain , nerve cells and blood vessels form complex networks that are interconnected .",
"The networks of blood vessels form as the main regions of the brain develop in the embryo .",
"During this time , glial cells provide physical support to the developing nerve cells and produce signals that guide them to the correct location .",
"NG2+ glial cells are different to other types of glial cells .",
"They spread throughout the developing brain and form complex , highly branched shapes .",
"They also appear to be involved in several other processes in the developing brain as they give rise to other glial cells and to neurons .",
"However , the full scope of their roles during brain development was not clear .",
"Here , Minocha , Valloton et al . investigate the role of NG2+ glia in the developing mouse brain by first marking the cells that form NG2+ glia with a fluorescent protein .",
"This approach allowed Minocha , Valloton et al . to track where and when NG2+ glia form .",
"The experiments revealed that midway through the development of mouse embryos , these glia populate an entire region called the telencephalon , which later forms a region of the brain called the cerebral cortex .",
"The appearance of these cells coincides with the formation of the brain’s blood vessel networks .",
"These early NG2+ glia cells then disappear a few days after birth .",
"Minocha , Valloton et al . also found that NG2+ glia form many close contacts with blood vessels in the cortex .",
"The cells are found at the tip of newly branching vessels , or are tethered along the walls of the vessels .",
"Furthermore , the NG2+ glial cells also create bridges between neighboring vessels .",
"When these glia are missing from the brains of mouse embryos , blood vessel networks in the telencephalon fail to branch normally and connect with each other .",
"Thus , NG2+ glial cells are important for the branching , connection and refinement of blood vessels in the cerebral cortex during development .",
"Future studies will aim to identify which of the molecules produced by glial cells influence the formation of the blood vessel network ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Entrainment and maintenance of an internal metronome in supplementary motor area | elife-38983-v2 | [
[
"Adaptive behavior benefits from the ability to discern temporal regularities in the environment .",
"To exploit these regularities , the brain must be able to measure time intervals between repetitive events ( Buhusi and Meck , 2005; de Lafuente et al . , 2015; Confais et al . , 2012; Leon and Shadlen , 2003; Grahn and Brett , 2007; Merchant and Lafuente , 2014; Merchant et al . , 2015 ) , and use this timing information to anticipate future events ( Goel and Buonomano , 2014; Jazayeri and Shadlen , 2010; Uematsu et al . , 2017 ) .",
"This behavior is evident when we dance to music , which requires perceiving rhythms and generating movements in sync with them ( Levitin et al . , 2018 ) .",
"Nonhuman primates and other vertebrates are capable synchronizing their movements to periodic rhythms ( Merchant et al . , 2013; Takeya et al . , 2017; Gámez et al . , 2018 ) , and we recently showed that monkeys can internally maintain rhythms of different tempos in the absence of overt motor actions ( García-Garibay et al . , 2016 ) .",
"Ample evidence indicates that cortical and subcortical motor circuits participate in behavioral tasks that require time perception and temporally precise behavioral responses ( Mita et al . , 2009; Crowe et al . , 2014; Bartolo et al . , 2014; Merchant and Averbeck , 2017; Grahn and Brett , 2007; Ivry and Spencer , 2004; Murray et al . , 2014 ) .",
"Nonetheless , the neuronal mechanisms that allow motor structures to encode rhythms of different tempos , in the absence of motor commands , are not yet completely understood .",
"We developed a novel visual metronome task in which nonhuman primates had to observe , and then internally maintain , a temporal rhythm defined by a left-right alternating visual stimulus .",
"Crucially , subjects had to track the rhythm in the absence of overt movements ( García-Garibay et al . , 2016 ) .",
"By uncoupling rhythm encoding and maintenance from motor actions , we aimed to identify the mechanism that allows the brain to internally maintain rhythms of different tempos .",
"While monkeys performed the task , we recorded the local field potentials ( LFPs ) and spiking activity of single neurons in the supplementary motor area ( SMA ) that has been implicated in timing and rhythm perception ( Buzsáki et al . , 2012; Pesaran et al . , 2002 ) .",
"Our results show that bursts of lower gamma band activity ( 30–40 Hz ) reflect the internally maintained tempos by a simple mechanism: the intervals defining the rhythm are encoded by the periodic onset of gamma bursts .",
"Moreover , increasing amplitudes of gamma bursts reflected an estimate of total elapsed time ( i . e . the total time since the rhythm began ) .",
"Importantly , gamma bursts encoded both rhythm and the total elapsed time in the absence of sensory stimulation and overt motor activity ."
],
[
"We trained two rhesus monkeys ( M . mulatta ) to perform a visual metronome task ( Figure 1A ) .",
"While maintaining eye and hand fixation over the screen , monkeys saw a visual stimulus that appeared on one side , switched to the other , and the back to the initial location .",
"This alternating stimulus defined three entrainment intervals of an isochronous rhythm .",
"On each trial , the interval duration was pseudo-randomly chosen to be 500 , 750 , or 1000 ms . In this manner , animals were presented with a visual metronome whose tempo was changed on a trial-by-trial basis ( Figure 1A ) .",
"After the third entrainment interval , the visual stimulus disappeared , and subjects had to maintain the rhythm internally by keeping track of the virtual position ( left or right ) of the stimulus as a function of elapsed time .",
"To test the ability of subjects to maintain the rhythms , a go-cue at the middle of any one of up to four maintenance intervals instructed the subjects to reach towards the stimulus location ( the go-cue consisted of removing the hand fixation point; the number of maintenance intervals was pseudo-randomly chosen; Figure 1A ) .",
"Thus , the key parameters in the visual metronome task were ( 1 ) interval duration ( 500 , 750 , or 1000 ms ) , and ( 2 ) the number of maintenance intervals that subjects had to wait after the visual stimulus was gone .",
"We characterized monkeys’ ability to maintain the rhythms by plotting the proportion of correct responses as a function of the elapsed time since the initiation of the first maintenance interval ( Figure 1B ) .",
"The behavioral results show that monkeys satisfactorily performed the task and were able to correctly estimate the location of the stimulus in more than 80% of trials ( 94 ± 0 . 2% monkey 1; 86 ± 0 . 3% monkey 2; mean ± s . e . over sessions , n = 131 sessions ) .",
"Importantly , performance as a function of time displays the hallmark of a timing task: the proportion of correct responses declines as a function of the number of maintenance intervals ( or equivalently , elapsed time ) .",
"The proportion of correct responses started close to 100% and declined to approximately 75% for the last maintenance intervals ( last two data points for each curve ) .",
"This behavior is consistent with the internal rhythm gradually drifting away from the true tempo of the stimulus ( Gibbon et al . , 1997; Grondin , 2001 ) .",
"As we described in previous work ( García-Garibay et al . , 2016 ) , this pattern is well captured by a model in which the subject’s time estimates arise from increasingly noisy ( wider ) distributions , described by Weber’s Law of time ( also called the scalar property of timing ) ( Laje et al . , 2011 ) .",
"The increase in timing variability causes the subjects to eventually fall out of synchrony with the true stimulus position ( getting ahead , or behind the true tempo ) , thus explaining the decrease in correct responses as a function of elapsed time ( Figure 1B , the colored curves are fits of this model to the data; pooled data across monkeys; see also Figure 1—figure supplement 1 ) .",
"Behavioral performance for the 4th maintenance interval of the 750 and 1000 ms tempos is higher than would be expected , that is it is higher than the performance on the previous 3rd interval .",
"This is likely due to the fact that our experiment only included up to four maintenance intervals .",
"We speculate that monkeys exploited this information and halted the maintenance at the 4th interval , so that they could avoid errors due to moving onto the 5th interval .",
"In the future , we plan to mitigate this bias by choosing the number of entrainment and maintenance intervals from an exponential distribution with a flat hazard rate .",
"Reaction times to the go-cue increases significantly in proportion to elapsed time within a narrow window ranging between 350 ms after the first maintenance interval of the fastest tempo ( 500 ms intervals ) , to 400 ms after the last interval of the slowest tempo ( 1000 ms intervals ) ( Figure 1C; R2 = 0 . 72 , slope = 11 ms/s , p<0 . 001; monkey 1 = 10 . 2 ms/s±0 . 8; monkey 2 = 11 . 3 ms/s ± 0 . 5 ) .",
"This increase in reaction times could be a result of the increasing difficulty in estimating the true stimulus position .",
"As expected by scalar variability , the subject’s estimate of the stimulus position becomes noisier with time , thus increasing uncertainty and the reaction time necessary to make a decision .",
"In Figure 1—figure supplement 2 , we provide the LFP spectrogram aligned to movement onset , demonstrating that gamma band activity decreases , and it is replaced by low-frequency oscillations at movement onset .",
"We also demonstrate that larger gamma band amplitudes are correlated with increased reaction times .",
"Overall , behavioral results show that monkeys were able to entrain to a rhythm , and maintain it in the absence of sensory stimuli , and importantly , in the absence of overt motor commands .",
"While the monkeys performed the visual metronome task , we recorded neural activity in 131 experimental sessions ( 84 and 47 for monkeys 1 and 2 , respectively; Figure 1E ) , and analyzed the local field potentials ( LFPs ) within 5–80 Hz band .",
"As a first step , we calculated the mean spectrogram for both monkeys , across all recording sessions ( Figure 1D; 500 ms interval shown; combined data across monkeys ) .",
"Modulations of LFP amplitude were especially salient in the 30–40 Hz frequencies , which we will refer to as gamma band .",
"In this band , LFP power was up to two-fold larger than the baseline activity recorded 500 ms before trial initiation ( p<0 . 001; permutation test of the time-frequency bins , 1000 permutations ) .",
"The LFP amplitude in the gamma band had a rhythmic structure .",
"It increased markedly with the presentation of the last visible stimulus ( 3rd entrainment interval , Figure 1D ) , as well as near the time when the non-visible stimulus would be switching its position from one side of the screen to the other during maintenance intervals ( Figure 1D; broken red lines ) .",
"To test this observation quantitatively , we verified that the average gamma amplitude at the time of switches was significantly higher than halfway between them ( t-test , p<0 . 01 for the three tempos; window sizes 1/4th of interval length; see Materials and methods ) .",
"In addition to the rhythmic modulation , gamma oscillations increased in amplitude as a function of total elapsed time ( Figures 1D and 3C; note that the last maintenance interval displays the largest amplitude ) .",
"The analyses so far focused on mean LFP activity across sessions .",
"To gain further insight into the LFP dynamics supporting the maintenance of internal rhythms , we analyzed LFP amplitude modulations within single trials .",
"The LFP recordings from single trials ( band-passed at 30–40 Hz ) revealed short-duration bursts during which the oscillations transiently increase in amplitude ( Figure 2B ) , consistent with recent findings in the putamen ( Bartolo et al . , 2014 ) and the prefrontal cortex ( Lundqvist et al . , 2016 ) .",
"Importantly , we observed that during the maintenance epoch , these bursts tended to coincide with the times at which the stimulus would have changed position , as is shown by the peaks in the spectrogram of the example single trials ( Figure 2A ) .",
"This trend is readily visualized by color-coding the amplitude of gamma oscillations and plotting all recorded trials on a single panel ( Figure 2C ) .",
"It is readily apparent that gamma bursts during maintenance tend to appear around the times at which the stimulus should be switching from one side of the screen to the other .",
"This pattern is captured by the mean gamma amplitude , across trials , as a function of elapsed time ( Figure 2D; p<0 . 01 , t-test that compared amplitudes at the times of switch [0 . 5 and 1 s] versus amplitudes at the middle of the interval [0 . 75 and 1 . 25 s]; 125 ms windows ) .",
"These salient temporal features of the gamma LFP were consistent across the three interval durations ( Figure 3A; 500 , 750 , and 1000 ms intervals ) .",
"To better illustrate the time distribution of gamma bursts , trials were sorted according to burst-onset time in each maintenance interval .",
"It is important to emphasize that there are no motor actions during the maintenance intervals , and no periodic stimuli is shown on the screen .",
"The only difference between the three groups of trials ( 500 , 750 , 1000 ms ) is the tempo of the internal rhythm that subjects are maintaining .",
"In other words , the rapid succession of the gamma bursts in the 500 ms intervals , and the more temporally distant bursts in the 1000 ms intervals , are a reflection of the subject’s internal maintenance of a visuo-spatial rhythm for the fast and slow tempos , respectively .",
"This finding reveals a neural signature of rhythms , of different tempos , that are maintained internally .",
"Alignment of the gamma bursts to their onset time revealed that bursts have a similar temporal profile across tempos and elapsed intervals ( Figure 3B ) .",
"Importantly , we found that the amplitude of these bursts increased in proportion to the time elapsed since the initiation of the internal rhythm ( Figure 3C; R2 = 0 . 86 , exponential model ) .",
"The results presented so far indicate that ( 1 ) the LFPs in SMA encode internal rhythms by means of gamma bursts that occur in sync with the beats ( i . e . location switch ) of a visual metronome presented earlier; and that ( 2 ) these bursts increase in amplitude , providing a neural correlate for total elapsed time .",
"In a previous study , we demonstrated that human subjects tend to lag behind fast tempos and get ahead of slow ones ( García-Garibay et al . , 2016 ) .",
"This predicts that animals might systematically overestimate the 500 ms rhythms , and underestimate the 1000 ms rhythms .",
"However , since animals only had two response options ( left or right ) , it was not possible to use behavioral responses to disambiguate errors in which the animals were ahead or behind the true tempo .",
"Nonetheless , we hypothesized that systematic over- and under-estimations of the intervals should be reflected in the patters of gamma activity in SMA .",
"We therefore compared the profile of gamma activity on correct and error trials ( Figure 4A ) .",
"The results showed that , on fast tempo trials ( 500 ms interval ) , the dynamics of gamma on error trials was right-shifted with respect to correct trials .",
"That is , error trials displayed slower dynamics compared to correct trials ( Figure 4A , upper panel ) .",
"This trend was captured by the power spectrums of error and correct trials , which showed that error trials indeed oscillated at lower frequencies ( Figure 4A , inset on upper panel ) .",
"Conversely , the dynamics of errors on slow tempo trials ( 1000 ms ) resemble a left-shifted version of the correct trials , that is errors displayed faster dynamics as compared to the correct trials ( Figure 4A; bottom panel ) .",
"This pattern is captured by the power spectrums of correct and error trials , which show that error trials oscillated at higher frequencies compared to correct trials ( Figure 4A , inset on the bottom panel ) .",
"These results suggest that monkeys were lagging behind fast tempos and getting ahead of slow ones .",
"The internal rhythm increasingly getting out of synchrony was also demonstrated by the ability of a logistic classifier to differentiate between correct and error trials ( Figure 4B; see Materials and methods ) .",
"This analysis shows that correct and error trials are increasingly easier to classify as a function of elapsed time , just as it would be expected from a rhythm that increasingly falls out of sync with the correct tempo .",
"This pattern holds true for a classifier that cumulatively uses gamma amplitude information as the trial develops , and also for a classifier using the information from a sliding window of constant length ( Figure 4B ) .",
"On average , monkeys tend lag behind fast rhythms and get ahead of slow ones .",
"However , we must note that mean error activity comes from a mixture of lagging and leading tempos ( see Figure 4—figure supplement 1 ) .",
"Thus , mean error activity does not necessarily reflect the half a cycle de-synchronization that must underlie incorrect responses on single trials .",
"Since SMA participates in the preparation of impending motor actions , it is possible that the rhythmic gamma bursts that we observed arise because this premotor area rhythmically prepare reach movements alternatively to the left and right locations of the screen .",
"To test this possibility , we recorded the LFPs in a delayed-reach control task ( Hwang and Andersen , 2011 ) in which subjects were required to reach to the left or the right after being cued by a briefly presented visual stimulus ( Figure 5A ) .",
"In this task , monkeys waited a pseudo-randomly chosen time ( 1100 to 3000 ms , exponential distribution ) before a go-cue prompted a reach towards the location specified by the cue ( Figure 5B ) .",
"The results of this control task show that , as monkeys prepare an impending reach movement , the LFPs in SMA generate bursts of gamma band activity that occur more frequently , and with increasing amplitude , as a function of total elapsed time ( Figure 5C , delay period ) .",
"These findings are consistent with the idea that gamma bursts in SMA encode impending motor commands .",
"Moreover , the results of this control task are consistent with the idea that the SMA circuits reflect internal rhythms by means of rhythmically alternating motor plans to make a reach movement to the left and right locations of the screen .",
"According to the previous delayed-reach experiment , gamma bursts might be reflecting an internal rhythm by periodically alternating ‘reach-left’ and ‘reach-right’ motor plans .",
"However , our task is designed such that a motor response was never required during the three entrainment intervals .",
"For this reason , we next analyzed the gamma band activity during the entrainment intervals in which the presentation of the alternating visuo-spatial stimuli defined the different tempos of the visual metronome task ( 500 , 750 , and 1000 ms intervals; Figure 6A–B ) .",
"The results showed that even during entrainment intervals , which did not involve any motor planning , bursts of gamma oscillations were present in each interval , and their amplitude progressively increased after the presentation of each visual stimulus ( Figure 6A–C ) .",
"It is important to note that gamma activity in entrainment intervals peaked after each stimulus presentation .",
"This is in contrast to what was observed during maintenance intervals , in which the peaks of gamma occurred when the stimulus switched sides .",
"We speculate that this phase offset could be related to the process of estimating interval duration , a process that necessarily happens during entrainment intervals .",
"A potential concern is that the gamma bursts in entrainment intervals are merely sensory responses to visual stimuli .",
"However , a pure sensory response should produce similar gamma dynamics after each stimulus presentation , both across consecutive entrainment intervals ( 1st , 2nd , 3rd ) , and also similar across tempos ( 500 , 750 , 1000 ms ) , which was clearly not the case in our results ( Figure 6A ) .",
"In particular , two observations suggest that gamma bursts during entrainment cannot be explained solely in terms of a sensory response .",
"First , gamma bursts increased in amplitude as a function of elapsed time , but the amplitude dropped sharply 500 ms after the onset of the third entrainment interval ( Figures 6A , 750 and 1000 ms panels ) .",
"Therefore , gamma bursts carry information about the animals’ knowledge that the third entrainment interval was the last visible interval , that is the last interval that could be used for estimating the tempo .",
"Thus , gamma dynamics likely incorporate aspects of higher cognitive processing .",
"Second , the times of burst onset do not have a fixed temporal profile with respect to stimulus presentation ( Figure 6B ) .",
"To demonstrate this , we measured the distribution of burst onset time across each consecutive interval ( 1st , 2nd , and 3rd entrainment intervals ) and across metronome tempos ( 500 , 750 , and 1000 ms ) , and then performed Chi-squared tests between these distributions ( by using burst onset time we removed the effect of burst amplitude ) .",
"The tests demonstrated that the temporal profiles of gamma onset times significantly differ , both across consecutive intervals and across metronome tempos ( p<0 . 01; corrected for multiple comparisons ) .",
"In fact , gamma responses to stimulus onset are similar only during the first 500 ms of the first entrainment interval , which is the only epoch in which monkeys have no information about the metronome tempo ( Figure 6C ) .",
"These results indicate that gamma bursts reflect cognitive processes related to estimating the rhythm of the visual metronome .",
"To quantify the extent to which gamma burst amplitude encodes total elapsed time , we measured burst amplitudes in each of the three entrainment intervals ( Figure 6D ) .",
"We found that burst amplitude increased linearly in proportion to total elapsed time ( R2 = 0 . 94 ) .",
"In this manner , in addition to periodically generating bursts in each entrainment interval , the SMA circuit reflected the total elapsed time since the beginning of the entrainment epoch .",
"Simultaneously with LFPs , we recorded the extracellular spike potentials of 113 neurons ( 78 monkey 1; 35 monkey 2 ) .",
"The temporal profile of the mean firing rates largely resembled the modulations of gamma-band activity in the LFP in the sense that firing rates ( 1 ) display oscillatory amplitude modulations , both during entrainment and maintenance intervals ( Figure 7A , B ) ; ( 2 ) firing rates increase as a function of total elapsed time; and ( 3 ) the activity of neurons in the delayed-reach task increase during the period preceding reach movements to a target signaled by a brief visual cue ( Figure 7C ) .",
"We found that SMA neurons had a preferred spatial location , that is they were more active when the stimulus was presented ( entrainment intervals ) , or was estimated to be ( maintenance intervals ) , on one side of the screen .",
"Of the 113 recorded neurons , 74 preferred the right side of the screen , and 39 preferred the left side ( Materials and methods ) .",
"This side preference allowed us to detrend the firing rates by subtracting the mean firing rate across sides ( mean between preferred and non-preferred sides of the screen; Figure 7D ) .",
"The detrended firing rates demonstrated the cyclic oscillations in the activity and , importantly , allowed to calculate the cross-correlation function between correct and incorrect trials .",
"The cross-correlation analysis between firing rates provided an independent corroboration of the hypothesis that that errors are mostly due to the internal metronome lagging behind fast rhythms , and getting ahead of slow ones ( Figure 7E ) .",
"The cross-correlogram for the 500 and 750 ms intervals peak at negative lags , demonstrating that incorrect trials lag behind the correct tempo .",
"The opposite pattern was observed for the slow tempo ( 1000 ms tempo ) .",
"To explore the relationship between single-neuron spiking and the simultaneously recorded LFP , we calculated the spike-triggered average ( STA ) LFP , and its spectral density , within a window of −100 to 100 ms surrounding each spike ( Figure 8A–B , see Materials and methods ) ( Denker et al . , 2011; Fries et al . , 2001 ) .",
"We found that the LFP activity simultaneously recorded with each spike has a power peak at 30 Hz , and this peak is especially salient during maintenance intervals ( Figure 8A , bottom panel; factorial ANOVA: interaction band/condition F = 12 . 11 p<0 . 05 , Bonferroni tests of Gamma power in maintenance and entrainment vs baseline: p<0 . 05 ) .",
"Moreover , the association between spikes and the 25–40 Hz frequency band is stronger at the times of stimulus transitions , that is . around the times at which the stimulus switches from one side of the screen to the other ( Figure 8C; window length around switch: half an interval , t-test p<0 . 005 ) .",
"To demonstrate that gamma is closely associated with the timing of spikes we performed a control analysis in which we jittered the spike times by ±15 ms with the resulting loss of the observed peak at the gamma band ( random uniform distribution; grey traces Figure 8c; t test between jittered data in switch and non-switch conditions p=0 . 21 ) .",
"These analyses demonstrate that the performance of the metronome task is accompanied by a tighter temporal relationship between the gamma bursts and the firing of single neurons , and this association is more prominent at the times of stimulus switching during the maintenance intervals .",
"These results are consistent with previous investigations proposing that LFP oscillations near the gamma frequencies could help single neurons synchronize their firing , and thus have a larger and more temporally precise influence on downstream target structures ( Siegle et al . , 2014; Veit et al . , 2017; Fries , 2015 ) .",
"Finally , we demonstrate that the LFP signals we recorded reflect local interaction and were not the result of signals being volume-conducted from other brain regions .",
"We measured the coherence between LFPs of simultaneously recorded electrodes and plotted this measure as a function of the distance between them .",
"The results show that coherence decayed as a function of electrode distance ( Figure 8D , R2 = 0 . 72 ) , as expected by an LFP signal that is generated in the neuronal circuits within the vicinity of the recording electrode ."
],
[
"Our results show that ( 1 ) monkeys can maintain rhythms in the absence of sensory stimuli and in the absence of overt motor commands .",
"( 2 ) Those internal rhythms are encoded by bursts of low gamma-band LFP oscillations in SMA whose timing and amplitude indicate rhythm intervals and total elapsed time , respectively .",
"( 3 ) The spikes of single neurons are associated with the low gamma band frequency of the LFP , which is consistent with the idea that gamma oscillations might help to synchronize populations of neurons whose temporally coincident firing would have a larger impact on its postsynaptic targets ( Buzsáki and Schomburg , 2015; Cardin et al . , 2009; Fries , 2015; Siegle et al . , 2014; Veit et al . , 2017; Womelsdorf et al . , 2007; Wong et al . , 2016 ) .",
"In our metronome task , the go-cue can arrive at the middle of any of the four maintenance intervals .",
"So , there is a rhythmic modulation in the likelihood of motor response initiation .",
"This could be related the periodic modulation of the gamma bursts and the firing rates of SMA neurons .",
"However , we must emphasize that the rhythmicity in the gamma bursts and firing rates is also observed in entrainment intervals , where no movement is ever required .",
"In addition to rhythmic modulations , the probability of the go-cue appearing , given that it has not appeared yet , increases as a function of total elapsed time ( hazard rate ) .",
"Thus , the increase in gamma amplitude and in the firing rates that is observed as total time elapses , could be related to the increasing likelihood of a motor response .",
"We must note , however , that in the delayed-reach task we used an exponential distribution of delay times , resulting in a flat hazard rate .",
"Even with a flat hazard rate , we observed increases in gamma amplitude , and in the firing rates , that are related to total elapsed time .",
"Overall , we favor the interpretation that SMA participates in the metronome task by generating a motor plan that dynamically matches the spatio-temporal tempo defined by the rhythmic visual stimulus .",
"SMA plays a central role in learning , imaging , planning and executing complex motor actions ( Nachev et al . , 2008; Romo and Schultz , 1992; Kurata and Wise , 1988; Shima and Tanji , 2000; Murakami et al . , 2014 ) .",
"It is densely and reciprocally connected to M1 and to the parietal and frontal lobes , and has direct projections to motor nuclei in the brain stem ( Jürgens , 1984 ) .",
"Thanks to this diverse input and output relationships its activity been found to correlate not only with motor actions but also with cognitive , emotional , and perceptual functions ( Narayana et al . , 2012; Vergara et al . , 2016; de Lafuente and Romo , 2005 ) .",
"SMA is active before the actual movement begins , participating in action selection , and importantly , determining the time at which actions are performed ( Merchant and Averbeck , 2017; Mita et al . , 2009; Chen et al . , 2010; Ohara et al . , 2001; Yokoyama et al . , 2016; Shima and Tanji , 2000 ) .",
"Preparatory activity can be observed even when monkeys are required to rapidly produce a movement in response to a sensory cue ( Lara et al . , 2018 ) , and it has been proposed that this preparatory activity constitutes the initial step of the temporal evolution of a dynamical system for the control of movement ( Churchland et al . , 2010; Remington et al . , 2018 ) .",
"Consistent with this view , our results show that LFP and single neuron activity in SMA starts during the entrainment epoch of the metronome task , seconds before an actual movement will be required .",
"Thus , the internal metronome is encoded as a dynamic motor plan that is initiated by the presentation of entrainment intervals .",
"That behavioral performance decreases as a function of time while gamma amplitude increases with elapsed time might seem counterintuitive .",
"However , we must note that the tempo of the internal metronome is encoded by the timing , not the amplitude of the gamma bursts .",
"On Figure 4—figure supplement 1 we show a six-choice variation of the metronome task that allowed determining that error trials are not explained by random behavioral responses .",
"Instead , the behavioral responses on the six-choice version of the task demonstrate that error trials are due to the internal metronome lagging or getting ahead of the true tempo .",
"Even on error trials , gamma activity , and the firing pattern of neurons , show rhythmic dynamics and a mean amplitude that increases with total elapsed time .",
"Thus , there are three independent lines of evidence supporting the notion that error trials arise from the internal metronome falling out of sync with the intended tempo .",
"First , the periodograms of gamma activity indicate that errors on fast trials ( 500 ms interval ) oscillate at slower frequencies as compared to correct trials .",
"Conversely , errors on slow tempos ( 1000 ms ) oscillate faster than correct responses .",
"Second , these same patterns were demonstrated by the cross-correlograms of the firing rates in correct and incorrect trials ( Figure 7E ) .",
"Finally , the behavioral results on the six-choice version of the metronome task showed that errors are not uniformly distributed across choices ( as would be expected from lapses of attention ) , but distribute around the correct stimulus position , with increasing variability for longer elapsed times , as would be expected from the scalar property of timing ( Figure 4—figure supplement 1 ) .",
"Moreover , the distributions show that errors tend to be behind the true stimulus position on fast trials , and ahead on slow trials .",
"It has been debated whether subjects performing a rhythmic task measure individual intervals separately , or instead rely on an estimate of total elapsed time ( Laje et al . , 2011 ) .",
"Our results now reveal that rhythms of different tempos are supported by the presence of rhythmic neuronal activity outlining each individual interval .",
"In addition to this , the increases in gamma burst amplitude , and mean firing rates , provide information about total elapsed time .",
"Gamma synchronization might be useful to the formation of local ensembles of neurons that increase the temporal coordination of presynaptic spikes on postsynaptic targets , allowing brief windows of effective communication ( Wong et al . , 2016; Womelsdorf et al . , 2007; Buzsáki and Schomburg , 2015 ) .",
"Previous results show that gamma oscillations increase before the execution of a motor action , and then shut down at the time of movement onset ( Yokoyama et al . , 2016 ) a result replicated by our data .",
"Previous work by Merchant and colleagues found that LFP gamma band activity in the basal ganglia was associated with the presentation of sensory stimuli defining the intervals within a hand tapping task ( Bartolo et al . , 2014 ) .",
"They found that bursts of gamma were selective for intervals of different durations , and thus different cell populations were selective for different time intervals .",
"We found no such duration selectivity in the SMA cortex , instead observing that gamma bursts encoded intervals of different durations .",
"Signals associated with timing tasks can be found across multiple brain areas , including parietal , motor , and premotor cortices , as well as dopaminergic midbrain neuron in the primate ( Ghose and Maunsell , 2002; Genovesio et al . , 2006; Lebedev and Wise , 2000; Mita et al . , 2009; Harrington et al . , 2010 ) .",
"For example , Jazayeri and Shadlen , 2015 have shown that activity of single neurons in the lateral intraparietal area encodes the time elapsed from a previous sensory stimuli , as well as the time remaining to initiate a saccadic eye movement ( Jazayeri and Shadlen , 2015 ) .",
"Importantly , they showed that these signals calibrate themselves according to the underlying probability to make an eye movement within a given temporal window .",
"A recent important result by Jazayeri and colleagues demonstrated that encoding intervals of different lengths is achieved by means of speeding up or slowing down the temporal dynamics of populations of neurons that , individually , display widely different firing patterns ( Wang et al . , 2018 ) .",
"Our results extend this finding to the dynamics of the LFP oscillations by demonstrating that they also show temporal scaling ( Figures 3A and 6C ) .",
"A coherent picture is thus emerging , indicating that time-estimation and time-production signals are present as dynamic motor plans that are distributed across the motor structures that participate in executing timely motor actions ."
],
[
"Two adult male Rhesus monkeys ( Macaca mulatta ) participated in the study ( weight: 5–7 kg , age: 5 , 7 years ) .",
"Experimental procedures were approved by the Ethics in Research Committee of the Institute of Neurobiology and were in agreement with the principles outlined in the Guide for Care and Use of Laboratory Animals ( National Institutes of Health ) .",
"Each monkey was surgically implanted with titanium head bolts and a titanium recording chamber over the left supplementary motor area ( SMA ) .",
"Placement of the chambers over the SMA was guided by structural MRI for both monkeys ( Figure 1E ) .",
"Monkeys were trained in a visual metronome task described in detail in a previous report ( García-Garibay et al . , 2016 ) .",
"Briefly , while maintain eye and hand fixation over a touch screen ( ELO Touch Solutions , model 1939L; ASL Eye-Track 6 ) , subjects observed a visual stimulus ( gray circle , 10° diameter , 25° eccentricity ) that periodically changed position from one side of the screen to the other , at regular intervals ( entrainment epoch; 500 , 750 , or 1000 ms interval; pseudo-randomly selected on each trial; Figure 1A ) .",
"After three entrainment intervals the visual stimulus disappeared , and subjects had to continue estimating its position ( left or right ) as a function of elapsed time ( maintenance intervals; Figure 1A ) .",
"This visuo-spatial rhythm task is similar to a visual metronome that paces a rhythm which subjects have to keep internally during the maintenance epoch .",
"To quantify the ability of the subjects to maintain rhythms of different tempos a go-cue ( disappearance of the hand fixation area ) was presented at the middle of any of the four maintenance intervals ( randomly selected , uniform distribution; Figure 1A ) .",
"This go-cue instructed the subjects to make a reach movement towards the estimated target position ( left or right ) .",
"It is important to note that this was not an interception task , that is once the go-cue was presented the non-visible stimulus no longer changed position .",
"Performance was measured as the proportion of correct responses plotted as a function of the elapsed time since the initiation of the maintenance epoch ( Figure 1B ) .",
"Visual stimuli and task control was achieved with the Expo software ( designed by Peter Lennie , maintained by Robert Dotson; available at https://sites . google . com/a/nyu . edu/expo/ ) .",
"In this task , monkeys were required wait a variable delay period before making a reach movement to one side of the screen signaled by a brief visual stimulus ( Figure 5 ) .",
"The stimulus appeared for 500 ms on either the left or side of the screen ( randomly selected ) , and the delay period was randomly selected from a truncated exponential distribution with a minimum delay duration of 1 . 1 s and a maximum of 3 s .",
"For analyzing the activity during the delay period ( Figures 5 and 7 ) , we used every trial up to the time before the go-cue .",
"This ‘attrition’ method allows to use all available information up a given point in time ( without the go-cue , or movement related- activity ) .",
"In this manner , before 1 . 1 s all trials contribute to the mean activity .",
"Then , there is a progressive attrition of trials so that for the 3 s time point ~300 trials contribute to the mean .",
"Figure 5B shows every trial in which the visual cue appeared on the left .",
"Neural recordings were performed with seven independent movable microelectrodes ( 2–3 MΩ , Thomas Recordings , Giessen , Germany ) .",
"Electrodes were advanced in the coronal plane into the supplementary motor area until single unit activity was obtained in at least one of the electrodes .",
"At each recording site , spikes were isolated online ( Cerebus acquisition system , Blackrock Microsystems , Salt Lake City , UT ) and sampled at 30 KHz .",
"The local field potentials ( LFPs ) were obtained by filtering the electrode signal at 0 . 5 to 500 Hz , at a 2 KHz rate .",
"Offline , the signal was down sampled to 1 KHz , and band-pass filtered to the 2–50 Hz band .",
"Analyses were performed with MATLAB 2013b ( The Mathworks , Natick , MA ) , making use of the Chronux Toolbox for the time- frequency maps ( Mitra and Bokil , 2007 ) .",
"Spectral estimation was performed using multitaper methods ( Pesaran et al . , 2002; Mitra and Pesaran , 1999; Cohen , 2014 ) .",
"A 200 ms windows sliding at 5 ms steps was used for the time-frequency maps ( one taper was used , 5 Hz bandwidth ) .",
"Spectrogram power was normalized by dividing each frequency and time bin by the average power in a 500 ms baseline window before trial initiation .",
"For the entrainment intervals , we compared the mean gamma amplitude ( across the trials of one session ) at the time of switches ( dotted lines in Figure 1D ) , with the gamma amplitude in between switches ( at the middle of each interval ) .",
"We used window lengths of 25% the interval duration .",
"For each tempo , each recording session contributed three pairs of switch/non-switch windows .",
"Thus , the degrees of freedom of the t-test were ( 131 sessions ) x ( 3 pairs per session ) = 393–1 degrees of freedom .",
"We performed three such tests , one for each metronome tempo ( 500 , 750 , 1000 ms ) .",
"The three tests had p<0 . 01 .",
"To characterize how the amplitude of the gamma oscillations is modulated over time , we averaged the normalized spectrograms over the low gamma band frequencies ( 30–40 Hz ) .",
"Narrow-band filtering with analytic envelopes and complex Morlet wavelet convolution yielded similar results .",
"Gamma bursts were defined as the period of time in which gamma amplitude exceeded the 90th percentile of overall activity for at least 100 ms ( i . e . for at least four cycles of the gamma oscillations ) .",
"On panel 2C trials were sorted by the burst-onset time on the last maintenance interval , thus , the previous gamma bursts are not aligned .",
"On panel 3A gamma bursts were aligned independently on each interval , that is the bursts were aligned for the first transition , then re-aligned for the second transition and so on .",
"This was done for display purposes only; the mean gamma activity is not affected by how trials are sorted .",
"A logistic function was used to identify correct and error trials:pcorrect=11+e- ( β0+t1β1+t2β2+…tnβn ) where t1 correspond to the gamma amplitude in the first time-bin , t2 to the amplitude on second time bin , and so on ( 10 time bins per interval , 35 time-bins for each trial ) .",
"Thus , the predicted behavior arises from a linear combination of the gamma activity used to fit the logistic function .",
"The classifier accuracy was measured on 100 trials ( 50 correct and 50 error trials; randomly selected ) not used in fitting the logistic function .",
"Fitting and testing was repeated 100 times , randomly selecting the test trials .",
"For the cumulative window classifier ( Figure 4B , green line ) , we used the gamma amplitude on the first time-bin and then tested the accuracy of decoding , then we added the data of the second time-bin and recalculated accuracy , and so on until the last time-bin .",
"In a second approach that we called ‘sliding window’ , a window of 5 time-bins were used to fit the classifier and calculate accuracy .",
"This window moved across the trial to calculate accuracy as a function of elapsed time ( Figure 4B , blue line ) .",
"To estimate each neuron’s spatial preference , we computed the cross-correlation between the stimulus position ( left and right ) and the mean firing rate .",
"For this analysis , we concatenated the mean firing rate of trials starting on the left with those starting the right , and generated the stimulus position signal accordingly .",
"The sign at the peak of the cross-correlogram tells us if increasing firing rates are significantly correlated , or anti-correlated , with the stimulus position being on the left .",
"With each neuron’s spatial preference , we were able to generate the mean firing rate of trials starting on the neuron’s preferred location , and the mean firing rate of trials starting in the opposite location ( Figure 7B ) .",
"The detrended firing rates were obtained by subtracting the mean activity across all trials , from the mean firing rates of each trial type ( starting on the preferred and non-preferred location; Figure 7D ) .",
"To estimate the synchronization between the spikes and the simultaneously recorded LFP , 200 ms windows centered on each spike were analyzed ( Fries et al . , 2001; Denker et al . , 2011 ) .",
"The average LFP in these windows were computed and normalized peak-to-valley to values between 0 and 1 .",
"This procedure was applied before spectral decomposition of the STA ( Figure 8A , power spectrum ) , allowing the comparison of spectral density maintaining the same maximum amplitude across conditions ( baseline , entrainment and maintenance epochs ) .",
"To assess statistical significance , we performed a factorial ANOVA with the factors condition ( baseline , entrainment , maintenance ) , and frequency ( alpha , beta , gamma ) , where the dependent variable was the average amplitude between 6 and 10 Hz for alpha , 15 to 24 Hz for beta and 30 to 40 Hz for gamma .",
"This analysis demonstrated that the average power of the STA over the gamma band was significantly larger during entrainment and maintenance , as compared to the baseline period ( p<0 . 01; Figure 8B ) .",
"We normalized the amplitude of the LFP traces surrounding each spike to account for the increase in gamma amplitude with total elapsed time .",
"To assess the locality of the observed LFP oscillations we estimated the phase clustering between the LFPs in pairs of simultaneously recorded electrodes .",
"We used the time series of all trials recorded while the monkeys performed the task .",
"For each electrode pair , we band-pass filtered the signal ( 30–40 Hz ) and estimated the analytic envelope to obtain the instantaneous phase .",
"Then , for each time point we estimated the difference angles between signals in the complex plane .",
"The coherence was defined as the length of the average vector of all difference angles , a procedure that results in magnitudes between 1 ( all difference angles are aligned to the same direction ) and zero ( random distribution ) ( Cohen , 2014 ) .",
"To quantify how coherence decreased as a function of electrode separation we grouped the distance variable into 50 bins containing the same number of observations per bin .",
"A linear regression was then applied to these data ( Figure 8D ) ."
]
] | [
"To prepare timely motor actions , we constantly predict future events .",
"Regularly repeating events are often perceived as a rhythm to which we can readily synchronize our movements , just as in dancing to music .",
"However , the neuronal mechanisms underlying the capacity to encode and maintain rhythms are not understood .",
"We trained nonhuman primates to maintain the rhythm of a visual metronome of diverse tempos and recorded neural activity in the supplementary motor area ( SMA ) .",
"SMA exhibited rhythmic bursts of gamma band ( 30–40 Hz ) reflecting an internal tempo that matched the extinguished visual metronome .",
"Moreover , gamma amplitude increased throughout the trial , providing an estimate of total elapsed time .",
"Notably , the timing of gamma bursts and firing rate modulations allowed predicting whether monkeys were ahead or behind the correct tempo .",
"Our results indicate that SMA uses dynamic motor plans to encode a metronome for rhythms and a stopwatch for total elapsed time ."
] | [
"A catchy tune on the radio , and suddenly we are tapping our foot and moving our bodies to the rhythm of the music .",
"We can follow a beat because our motor neurons , the nerve cells that control movements , work together in circuits .",
"During actions that require precise timing – such as dancing to a rhythm – the motor neurons within these circuits increase and decrease their activity in complex patterns .",
"But recent evidence shows that these motor neuron circuits also ‘switch on’ simply when we perceive a rhythm , even if we do not move to it .",
"In fact , just imagining a rhythm triggers the same symphony of electrical activity in the brain .",
"How do motor neurons generate coordinated patterns of activity without movement or even an external stimulus ?",
"Cadena-Valencia et al . set out to answer this question by training monkeys to follow a rhythm .",
"The animals learned to track a dot that appeared alternately on the left and right sides of a touchscreen with a regular tempo .",
"After a few repeats , the dot disappeared .",
"The monkeys then had to continue mentally tracking where the dot would have been .",
"A group of neurons in a brain region called the supplementary motor area synchronized their activity with the dot .",
"Whenever the dot was due to appear , the neurons in the area showed a burst of rapid firing .",
"These spikes of activity , called gamma bursts , helped the motor neurons to communicate with one another within their circuits .",
"The gamma bursts thus acted as an internal metronome , making it easier for the monkeys to follow the rhythm .",
"These results should be a starting point for other studies to pinpoint exactly where and how this rhythmic activity arises , and how the brain uses gamma bursts to synchronize our movements to a tempo ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"plant biology",
"developmental biology"
] | Coordination of tissue cell polarity by auxin transport and signaling | elife-51061-v1 | [
[
"How the polarity of cells in a tissue is coordinated is a central question in biology .",
"In animals , the coordination of this tissue cell polarity requires direct cell-cell communication and often cell movements ( Goodrich and Strutt , 2011 ) , both of which are precluded in plants by a wall that holds cells apart and in place; therefore , tissue cell polarity is coordinated differently in plants .",
"The formation of plant veins is an expression of such coordination of tissue cell polarity; this is most evident in developing leaves .",
"Consider , for example , the formation of the midvein at the center of the cylindrical leaf primordium .",
"Initially , the plasma-membrane ( PM ) -localized PIN-FORMED1 ( PIN1 ) protein of Arabidopsis ( Gälweiler et al . , 1998 ) , which catalyzes cellular efflux of the plant signal auxin ( Petrásek et al . , 2006 ) , is expressed in all the inner cells of the leaf primordium ( Benková et al . , 2003; Reinhardt et al . , 2003; Heisler et al . , 2005; Scarpella et al . , 2006; Wenzel et al . , 2007; Bayer et al . , 2009; Verna et al . , 2015 ) ; over time , however , PIN1 expression becomes gradually restricted to the file of cells that will form the midvein .",
"PIN1 localization at the PM of the inner cells is initially isotropic , but as PIN1 expression becomes restricted to the site of midvein formation , PIN1 localization becomes polarized: in the cells surrounding the developing midvein , PIN1 localization gradually changes from isotropic to medial , that is toward the developing midvein , to mediobasal; in the cells of the developing midvein , PIN1 becomes uniformly localized toward the base of the leaf primordium , where the midvein will connect to the pre-existing vasculature .",
"The correlation between coordination of tissue cell polarity , as expressed by the coordination of PIN1 polar localization between cells; polar auxin transport , as expressed by the auxin-transport-polarity-defining localization of PIN1 ( Wisniewska et al . , 2006 ) ; and vein formation does not seem to be coincidental .",
"Auxin application to developing leaves induces the formation of broad expression domains of isotropically localized PIN1; such domains become restricted to the sites of auxin-induced vein formation , and PIN1 localization becomes polarized toward the pre-existing vasculature ( Scarpella et al . , 2006 ) .",
"Both the restriction of PIN1 expression domains and the polarization of PIN1 localization are delayed by chemical inhibition of auxin transport ( Scarpella et al . , 2006; Wenzel et al . , 2007 ) , which induces vein pattern defects similar to , though stronger than , those of pin1 mutants ( Mattsson et al . , 1999; Sieburth , 1999; Sawchuk et al . , 2013 ) .",
"Therefore , available evidence suggests that auxin coordinates tissue cell polarity to induce vein formation , and that the coordinative and inductive property of auxin depends on the function of PIN1 and possibly other PIN genes .",
"How auxin coordinates tissue cell polarity to induce vein formation is unclear , but the current hypothesis is that the GNOM ( GN ) guanine-nucleotide exchange factor for ADP-ribosylation-factor GTPases , which regulates vesicle formation in membrane trafficking , controls the cellular localization of PIN1 and possibly other auxin transporters; the resulting cell-to-cell , polar transport of auxin would coordinate tissue cell polarity and control developmental processes such as vein formation ( reviewed in , e . g . , Berleth et al . , 2000; Richter et al . , 2010; Nakamura et al . , 2012; Linh et al . , 2018 ) .",
"Here we tested this hypothesis by a combination of cellular imaging , molecular genetic analysis , and chemical induction and inhibition .",
"Contrary to predictions of the hypothesis , we found that auxin-induced vein formation occurs in the absence of PIN proteins or any other intercellular auxin transporter; that the residual auxin-transport-independent vein-patterning activity relies on auxin signaling; and that a GN-dependent tissue-cell-polarizing signal acts upstream of both auxin transport and signaling ."
],
[
"The current hypothesis of how auxin coordinates tissue cell polarity to induce vein formation proposes that GN controls the cellular localization of PIN1 and possibly other auxin transporters; the resulting cell‐to‐cell , polar transport of auxin would coordinate tissue cell polarity and control developmental processes such as vein formation ( reviewed in , e . g . , Berleth et al . , 2000; Richter et al . , 2010; Nakamura et al . , 2012; Linh et al . , 2018 ) .",
"The hypothesis makes three testable predictions: Here we tested these predictions .",
"We tested this prediction by imaging expression domains of PIN1::PIN1:YFP ( PIN1:YFP fusion protein expressed by the PIN1 promoter [Xu et al . , 2006] ) and cellular localization of expression of PIN1::PIN1:GFP ( Benková et al . , 2003 ) during leaf development in WT and in the new strong allele gn‐13 ( Supplementary file 1 ) .",
"Consistent with previous reports ( Benková et al . , 2003; Reinhardt et al . , 2003; Heisler et al . , 2005; Scarpella et al . , 2006; Wenzel et al . , 2007; Bayer et al . , 2009; Sawchuk et al . , 2013; Marcos and Berleth , 2014; Verna et al . , 2015 ) , in WT leaves PIN1::PIN1:YFP was expressed in all the cells at early stages of tissue development .",
"Over time , epidermal expression became restricted to the basalmost cells , and inner tissue expression became restricted to developing veins ( Figure 1A–J ) .",
"In gn leaves too , PIN1::PIN1:YFP was expressed in all the cells at early stages of tissue development , and over time epidermal expression became restricted to the basalmost cells; however , inner tissue expression failed to become restricted to developing veins and remained nearly ubiquitous even at very late stages of leaf development ( Figure 1K–O ) .",
"Consistent with previous reports ( Benková et al . , 2003; Reinhardt et al . , 2003; Heisler et al . , 2005; Scarpella et al . , 2006; Wenzel et al . , 2007; Bayer et al . , 2009; Sawchuk et al . , 2013; Marcos and Berleth , 2014; Verna et al . , 2015 ) , in the cells of the second pair of vein loops ( ‘second loop’ hereafter ) at early stages of its development in WT leaves , PIN1::PIN1:GFP expression was mainly localized to the side of the plasma membrane ( PM ) facing the midvein; in the inner cells flanking the developing loop , PIN1::PIN1:GFP expression was mainly localized to the side of the PM facing the developing loop; and in the inner cells further away from the developing loop , PIN1::PIN1:GFP expression was localized isotropically at the PM ( Figure 1C , P ) .",
"At later stages of second‐loop development , by which time PIN1::PIN1:GFP expression had become restricted to the cells of the developing loop , PIN1::PIN1:GFP expression was localized to the side of the PM facing the midvein ( Figure 1D , T ) .",
"At early stages of development of the tissue that in gn leaves corresponds to that from which the second loop forms in WT leaves , PIN1::PIN1:GFP was expressed uniformly in the outermost inner tissue , and expression was localized isotropically at the PM ( Figure 1Q , R ) .",
"PIN1::PIN1:GFP was expressed more heterogeneously in the innermost inner tissue , but expression remained localized isotropically at the PM , except in cells near the edge of higher-expression domains: in those cells , localization of PIN1::PIN1:GFP expression at the PM was weakly polar , but such weak cell polarities pointed in seemingly random directions ( Figure 1Q , S ) .",
"At late stages of gn leaf development , heterogeneity of PIN1::PIN1:GFP expression had spread to the outermost inner tissue , but expression remained localized isotropically at the PM , except in cells near the edge of higher-expression domains: in those cells , localization of PIN1::PIN1:GFP expression at the PM was weakly polar , but such weak cell polarities pointed in seemingly random directions ( Figure 1U , V ) .",
"Heterogeneity of PIN1::PIN1:GFP expression in the innermost inner tissue had become more pronounced at late stages of gn leaf development , and the weakly polar localization of PIN1::PIN1:GFP expression at the PM had spread to the center of the higher-expression domains ( Figure 1U , W ) ; nevertheless , such weak cell polarities still pointed in seemingly random directions ( Figure 1U , W ) .",
"In conclusion , both restriction of PIN1 expression domains and coordination of PIN1 polar localization occur only to a very limited extent or fail to occur altogether during gn leaf development , which is consistent with the current hypothesis of how auxin coordinates tissue cell polarity to induce vein formation .",
"To test this prediction , we first asked what the phenotype were of the quintuple mutant between the strong allele gn‐13 ( Figure 2 ) and mutation in PIN1 , PIN3 , PIN4 , and PIN7 — that is the PM‐PIN genes with vein patterning function ( Figure 3 ) .",
"gn;pin1 , 3;4;7 embryos were viable ( Supplementary file 2J ) and developed into seedlings ( Supplementary file 2K ) whose cotyledon and leaf vascular defects were no different from those of gn ( Figure 6A , B , E; Figure 6—figure supplement 1; Figure 3—figure supplement 2A , B; Figure 3—figure supplement 3; Figure 6—figure supplement 2A-D; Figure 6—figure supplement 3A-F; Figure 6—figure supplement 4; Figure 6—figure supplement 5A-D , F-H , K ) , suggesting that the vascular phenotype of gn is epistatic to that of pin1 , 3;4;7 .",
"We next asked what the phenotype were of the septuple mutant between the strong allele gn‐13 ( Figure 2 ) and mutation in all the PIN genes with vein patterning function ( Figure 3 ) .",
"gn;pin1 , 3 , 6;4;7;8 embryos were viable ( Supplementary file 2J ) and developed into seedlings ( Supplementary file 2K ) whose cotyledon and leaf vascular defects were no different from those of gn ( Figure 6A , C , E; Figure 6—figure supplement 1; Figure 3—figure supplement 2A , D; Figure 3—figure supplement 3; Figure 6—figure supplement 2A , C , E , F; Figure 6—figure supplement 3A , B , F-I; Figure 6—figure supplement 4; Figure 6—figure supplement 5A-G , I-K ) , suggesting that the vascular phenotype of gn is epistatic to that of pin1 , 3 , 6;4;7;8 .",
"Finally , NPA failed to induce additional vein pattern defects in gn leaves ( Figure 6D , E; Figure 6—figure supplement 1 ) .",
"In conclusion , auxin transport inhibition fails to induce defects in gn that approximate those which it induces in GN .",
"Therefore , our results also fail to support Prediction 3 of the current hypothesis of how auxin coordinates tissue cell polarity to induce vein formation .",
"Consequently , the hypothesis must be revised ."
],
[
"Overwhelming experimental evidence suggests that the patterned formation of veins depends on polar auxin transport ( reviewed in Sachs , 1981; Sachs , 1991; Berleth et al . , 2000; Sachs , 2000; Sawchuk and Scarpella , 2013 ) .",
"The polarity of auxin transport is determined by the asymmetric localization of efflux carriers of the PIN family at the PM of auxin-transporting cells ( Wisniewska et al . , 2006 ) .",
"Therefore , loss of function of all the PM-PIN proteins should lead to loss of reproducible vein-pattern features or even , in the most extreme case , to the inability to form veins .",
"Neither prediction is , however , supported by evidence: mutants in all the PM-PIN genes with vein patterning function — PIN1 , PIN3 , PIN4 and PIN7 — or in all the PM-PIN genes — PIN1–PIN4 and PIN7 — form veins , and these veins are arranged in reproducible , albeit abnormal , patterns .",
"We conclude that vein patterning is controlled by additional , PM-PIN-independent auxin-transport pathways .",
"The existence of PM-PIN-independent auxin-transport pathways with vein patterning function can also be inferred from the discrepancy between the vein pattern defects of pin1 , 3;4;7 or pin1 , 3;2;4;7 and those induced by NPA , which is thought to be a specific inhibitor of cellular auxin efflux ( Cande and Ray , 1976; Sussman and Goldsmith , 1981; Petrásek et al . , 2003; Dhonukshe et al . , 2008 ) .",
"The vein pattern defects of WT grown in the presence of NPA are more severe than those of pin1 , 3;4;7 or pin1 , 3;2;4;7 , suggesting the existence of an NPA-sensitive auxin-transport pathway with vein patterning function besides that controlled by PM-PIN proteins , a suggestion that is supported by the ability of NPA to enhance the vein pattern defects of pin1 , 3;4;7 to match those induced in WT by NPA .",
"Such PM-PIN-independent NPA-sensitive auxin-transport pathway with vein patterning function depends on the activity of the ER-PIN proteins PIN6 and PIN8 , as inferred from the identity of the vein pattern defects induced in WT by NPA and those of pin1 , 3 , 6;4;7;8 , and from the inability of NPA to induce further defects in pin1 , 3 , 6;4;7;8 .",
"Moreover , that NPA-grown WT phenocopies pin1 , 3 , 6;4;7;8; that no further defects can be induced in pin1 , 3 , 6;4;7;8 by NPA; and that the vein patterns of pin1 , 3 , 6;4;7;8 and NPA-grown WT fall into the same single phenotype-class suggest no NPA-sensitive vein-patterning activity beyond that provided by PIN1 , PIN3 , PIN4 , PIN6 , PIN7 , and PIN8 , and hence the existence of NPA-insensitive vein-patterning pathways .",
"These NPA-insensitive vein-patterning pathways unlikely depend on the function of other intercellular auxin transporters — the AUX1/LAX influx carriers ( Yang et al . , 2006; Swarup et al . , 2008; Péret et al . , 2012 ) and the ABCB efflux carriers ( Geisler et al . , 2005; Bouchard et al . , 2006; Petrásek et al . , 2006 ) — as their mutation fails to enhance the vein pattern defects of pin1 , 3 , 6 and of the NPA-induced phenocopy of pin1 , 3 , 6;4;7;8 .",
"The NPA-insensitive vein-patterning pathways also unlikely depend on NPA-insensitive auxin transport because as little as 10 µM NPA ( a fraction of the concentration we used ) is sufficient to inhibit polar auxin transport completely in tissue segments ( Okada et al . , 1991; Kaneda et al . , 2011 ) .",
"Whatever the molecular nature of the NPA-insensitive vein-patterning pathways , they do contribute to the polar propagation of the inductive auxin signal: application of auxin to pin1 , 3 , 6;4;7;8 leaves , just as to WT leaves , induces the formation of veins that connect the applied auxin to the pre-existing vasculature basal to the site of auxin application .",
"The residual NPA-insensitive auxin-dependent vein-patterning activity of pin1 , 3 , 6;4;7;8 relies , at least in part , on the signal transduction mediated by the TIR1/AFB auxin receptors and their post-translational regulator AXR1 .",
"Loss of AXR1; loss of TIR1 and AFB2 , the two auxin receptors that most contribute to auxin signaling ( Dharmasiri et al . , 2005 ) ; or growth in the presence of the auxin signaling inhibitor PBA ( Matthes and Torres-Ruiz , 2016 ) induces entirely new vein-pattern defects in pin1 , 3 , 6;4;7;8 or in its NPA-induced phenocopy .",
"In the more-severely affected leaves of axr1;pin1 , 3 , 6;4;7;8 , tir1;afb2;pin1 , 3 , 6;4;7;8 , NPA-grown axr1 , NPA-grown tir1;afb2 , and NPA- and PBA-grown WT , the end-to-end alignment of vascular elements oriented with their axis along the axis of the vein is often replaced by the clustered differentiation of abnormally oriented vascular elements .",
"Not only are these defects never observed in pin1 , 3 , 6;4;7;8 or NPA-grown WT , but they are more severe than the predicted sum of the defects of pin1 , 3 , 6;4;7;8 or NPA-grown WT , on the one hand , and of axr1 , tir1;afb2 , or PBA-grown WT , on the other .",
"This synthetic enhancement between the vein pattern defects caused by reduced auxin signaling and those caused by reduced auxin transport suggests non-homologous redundancy of auxin signaling and auxin transport in vein patterning , a conclusion which is consistent with observations in the shoot apical meristem ( Schuetz et al . , 2008 ) .",
"Unlike in the shoot apical meristem , however , in the leaf such redundancy is unequal: whereas auxin transport is required for vein patterning even in the presence of normal auxin signaling , the vein patterning activity of auxin signaling is only exposed in conditions of compromised auxin transport .",
"How auxin signaling , inherently non-directional ( Leyser , 2018 ) , could contribute to the polar propagation of the inductive auxin signal in the absence of polar auxin transport is unclear .",
"One possibility is that auxin signaling promotes the passive diffusion of auxin through the tissue by controlling , for example , the proton gradient across the PM ( Fendrych et al . , 2016 ) .",
"However , it is difficult to conceive how auxin diffusion through a specific side of the PM could positively feed back on the ability of auxin to diffuse through that specific side of the PM — a positive feedback that would be required to drain neighboring cells from auxin and thereby form veins , that is channels of preferential auxin movement ( Sachs , 1969 ) .",
"One other possibility is that auxin signaling promotes the facilitated diffusion of auxin through the plasmodesmata intercellular channels , a possibility that had previously been suggested ( Mitchison , 1980 ) and that has received some experimental support ( Han et al . , 2014 ) .",
"Here , it is conceivable how auxin movement through a specific side of the PM could positively feed back on the ability of the cell to move auxin through that specific side of the PM ( e . g . , Cieslak et al . , 2015 ) , but no experimental evidence exists of such feedback or that auxin movement through plasmodesmata controls vein patterning .",
"Yet another possibility is that auxin signaling activates an unknown mobile signal .",
"Such signal need not be chemical: alternatives , for example a mechanical signal , have been suggested ( Couder et al . , 2002; Laguna et al . , 2008; Corson et al . , 2009; Lee et al . , 2014 ) and have been implicated in other auxin-driven processes ( e . g . , Hamant et al . , 2008; Heisler et al . , 2010; Peaucelle et al . , 2011; Nakayama et al . , 2012; Braybrook and Peaucelle , 2013 ) .",
"However , whether a mechanical signal controls vein patterning remains to be tested .",
"The vein pattern defects of leaves in which both auxin transport and signaling are compromised are never observed in leaves in which either process is; yet those defects are not unprecedented: they are observed — though in more extreme form — in leaves of gn mutants , suggesting that GN controls both auxin transport and signaling during vein patterning .",
"That GN controls PM-PIN-mediated auxin transport during vein patterning is also suggested by the very limited or altogether missing restriction of PIN1 expression domains and coordination of PIN1 polar localization during gn leaf development , which is consistent with observations in embryos and roots ( Steinmann et al . , 1999; Kleine-Vehn et al . , 2008 ) .",
"However , if failure to coordinate the polar localization of PIN1 — and possibly other PM-PIN proteins — were the sole cause of the vein pattern defects of gn , these defects would depend on PM-PIN function and would therefore be masked by those of pin1 , 3;4;7 in the gn;pin1 , 3;4;7 mutant .",
"The epistasis of the vein pattern defects of gn to those of pin1 , 3;4;7 instead suggests that the vein pattern defects of gn are independent of PM-PIN function; that the vein pattern defects of gn are not the sole result of loss or abnormal polarity of PM-PIN-mediated auxin transport; and that GN acts upstream of PM-PIN genes in vein patterning .",
"Moreover , the epistasis of the vein pattern defects of gn to those of pin1 , 3 , 6;4;7;8 , and the inability of NPA , which phenocopies the vein pattern defects of pin1 , 3 , 6;4;7;8 , to induce additional defects in gn suggest that the vein pattern defects of gn are independent of all the PIN genes with vein patterning function; that the vein pattern defects of gn are not the sole result of loss or abnormal polarity of PIN-mediated auxin transport; and that GN acts upstream of all the PIN genes in vein patterning .",
"Mechanisms by which GN controls PM-PIN-mediated auxin transport have been suggested ( e . g . , Richter et al . , 2010; Luschnig and Vert , 2014; Naramoto et al . , 2014 ) ; it is instead unclear how GN could control auxin transport mediated by the ER-localized PIN6 and PIN8 .",
"One possibility is that such control depends on GN function in ER-Golgi trafficking ( Richter et al . , 2007; Teh and Moore , 2007; Nakano et al . , 2009 ) .",
"Irrespective of the mechanism by which GN controls PIN-mediated auxin transport , however , our results suggest that the function of GN in coordination of tissue cell polarity and vein patterning entails more than such control , a conclusion which is consistent with functions of GN that seem to be unrelated to auxin transport or independent of PIN function ( Shevell et al . , 2000; Fischer et al . , 2006; Irani et al . , 2012; Nielsen et al . , 2012; Moriwaki et al . , 2014 ) .",
"The auxin-transport- , PIN-independent functions of GN in coordination of tissue cell polarity and vein patterning are , at least in part , mediated by TIR1/AFB2- and AXR1-mediated auxin signaling .",
"This conclusion is suggested by the ability of simultaneous reduction in auxin transport and signaling to phenocopy defects in coordination of tissue cell polarity , auxin response , and vein patterning of gn; it is also supported by the epistasis of the vein pattern defects of gn to those of axr1 , an observation which is consistent with genetic analysis placing GN upstream of auxin signaling in the formation of apical-basal polarity in the embryo ( Mayer et al . , 1993 ) .",
"Though it is unclear how GN controls auxin signaling during vein patterning , the most parsimonious account is that GN controls the coordinated localization of proteins produced in response to auxin signaling .",
"Auxin signaling indeed controls the production of proteins that are polarly localized at the plasma membrane of root cells ( e . g . , Scacchi et al . , 2009; Scacchi et al . , 2010; Yoshida et al . , 2019 ) , and at least some of these proteins act synergistically with PIN-mediated auxin transport in the root ( e . g . , Marhava et al . , 2018 ) ; however , it remains to be tested whether such proteins have vein patterning activity , whether their localization is controlled by GN , and whether they mediate GN function in auxin signaling during vein patterning .",
"Alternatively , because cell wall composition and properties are abnormal in gn ( Shevell et al . , 2000 ) , GN may control the production , propagation , or interpretation of a mechanical signal that has been proposed to be upstream of both auxin signaling and transport in the shoot apical meristem ( Heisler et al . , 2010; Nakayama et al . , 2012 ) ; however , whether a mechanical signal controls vein patterning and whether such signal acts downstream of GN remains to be tested .",
"Irrespective of the mechanism of action , our results reveal synergism between auxin transport and signaling , and their unsuspected control by GN in the coordination of tissue cell polarity during vein patterning , a control whose logic is unprecedented in multicellular organisms ."
],
[
"In agreement with Crittenden et al . ( 1996 ) , linked genes or mutations ( <2 , 500 kb apart , which in Arabidopsis corresponds , on average , to ~10 cM [Lukowitz et al . , 2000] ) are separated by a comma , unlinked ones by a semicolon , and homologous chromosomes by a slash .",
"Origin and nature of lines , and oligonucleotide sequences are in Supplementary file 1; genotyping strategies are in Supplementary file 2Q .",
"Seeds were sterilized and sown as in Sawchuk et al . ( 2008 ) .",
"Stratified seeds were germinated and seedlings were grown at 22°C under continuous fluorescent light ( ~80 µmol m‐2 s‐1 ) .",
"Plants were grown at 25°C under fluorescent light ( ~110 μmol m‐2 s‐1 ) in a 16‐h‐light/8‐h‐dark cycle .",
"Plants were transformed and representative lines were selected as in Sawchuk et al . ( 2008 ) .",
"NPA and PBA were dissolved in dimethyl sulfoxide and water , respectively; dissolved chemicals were added ( 100 μM final NPA concentration , unless otherwise noted ) to growth medium just before sowing .",
"IAA was dissolved in melted ( 55°C ) lanolin; the IAA‐lanolin paste ( 1% final IAA concentration ) was applied to first leaves 4 days after germination and was reapplied weekly .",
"Total RNA was extracted as in Chomczynski and Sacchi ( 1987 ) from 4-day-old seedlings grown as in Odat et al . ( 2014 ) .",
"RT-PCR was performed as in Odat et al . ( 2014 ) with the following oligonucleotides: ‘GN_qFb’ and ‘GN_qRb’ , and ‘ROC1 F’ and ‘ROC1 R’ ( Beeckman et al . , 2002 ) ; ‘Aux_F380’ and ‘Aux_R380’ , and ‘ROC1 F’ and ‘ROC1 R’; and ‘Lax_F513’ and ‘Lax_R513’ , and ‘‘ROC1 F’ and ‘ROC1 R’ ( Supplementary file 1 ) .",
"Developing leaves were mounted and imaged as in Sawchuk et al . ( 2013 ) , except that emission was collected from ~2 . 5 μm-thick optical slices .",
"Light paths are in Supplementary file 2R .",
"Mature leaves were fixed in 3 : 1 or 6 : 1 ethanol : acetic acid , rehydrated in 70% ethanol and water , cleared briefly ( few seconds to few minutes ) — when necessary — in 0 . 4 M sodium hydroxide , washed in water , mounted in 80% glycerol or in 1 : 2 : 8 or 1 : 3 : 8 water : glycerol : chloral hydrate , and imaged as in Odat et al . ( 2014 ) .",
"Grayscaled RGB color images were turned into 8-bit images , look-up-tables were applied , and brightness and contrast were adjusted by linear stretching of the histogram in the Fiji distribution ( Schindelin et al . , 2012 ) of ImageJ ( Schneider et al . , 2012; Schindelin et al . , 2015; Rueden et al . , 2017 ) ."
]
] | [
"Plants coordinate the polarity of hundreds of cells during vein formation , but how they do so is unclear .",
"The prevailing hypothesis proposes that GNOM , a regulator of membrane trafficking , positions PIN-FORMED auxin transporters to the correct side of the plasma membrane; the resulting cell-to-cell , polar transport of auxin would coordinate tissue cell polarity and induce vein formation .",
"Contrary to predictions of the hypothesis , we find that vein formation occurs in the absence of PIN-FORMED or any other intercellular auxin-transporter; that the residual auxin-transport-independent vein-patterning activity relies on auxin signaling; and that a GNOM-dependent signal acts upstream of both auxin transport and signaling to coordinate tissue cell polarity and induce vein formation .",
"Our results reveal synergism between auxin transport and signaling , and their unsuspected control by GNOM in the coordination of tissue cell polarity during vein patterning , one of the most informative expressions of tissue cell polarization in plants ."
] | [
"Plants , animals and other living things grow and develop over their lifetimes: for example , oak trees come from acorns and chickens begin their lives as eggs .",
"To achieve these transformations , the cells in those living things must grow , divide and change their shape and other features .",
"Plants and animals specify the directions in which their cells will grow and develop by gathering specific proteins to one side of the cells .",
"This makes one side different from all the other sides , which the cells use as an internal compass that points in one direction .",
"To align their internal compasses , animal cells touch one another and often move around inside the body .",
"Plant cells , on the other hand , are surrounded by a wall that keeps them apart and prevents them from moving around .",
"So how do plant cells align their internal compasses ?",
"Scientists have long thought that a protein called GNOM aligns the internal compasses of plant cells .",
"The hypothesis proposes that GNOM gathers another protein , called PIN1 , to one side of a cell .",
"PIN1 would then pump a plant hormone known as auxin out of this first cell and , in doing so , would also drain auxin away from the cell on the opposite side .",
"In this second cell , GNOM would then gather PIN1 to the side facing the first cell , and this process would repeat until all the cells' compasses were aligned .",
"To test this hypothesis , Verna et al . combined microscopy with genetic approaches to study how cells' compasses are aligned in the leaves of a plant called Arabidopsis thaliana .",
"The experiments revealed that auxin needs to move from cell-to-cell to align the cells’ compasses .",
"However , contrary to the above hypothesis , this movement of auxin was not sufficient: the cells also needed to be able to detect and respond to the auxin that entered them .",
"Along with controlling how auxin moved between the cells , GNOM also regulated how the cells responded to the auxin .",
"These findings reveal how plants specify which directions their cells grow and develop .",
"In the future , this knowledge may eventually aid efforts to improve crop yields by controlling the growth and development of crop plants ."
] | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Social structure learning in human anterior insula | elife-53162-v2 | [
[
"Being able to distinguish ‘us’ from ‘them’ is a core social capacity ( Brown , 1991 ) .",
"In an increasingly interconnected world where people have multiple intersecting identities that guide their thoughts , feelings , and behaviors , being able to differentiate friend from foe is of paramount importance .",
"Yet we know surprisingly little about how social group boundaries are learned and represented in the brain—particularly in the absence of overt cues to individuals’ group membership .",
"One dominant account is that people use judgments of similarity to one’s self on some contextually relevant feature ( e . g . , skin tone ) .",
"Accordingly , neuroimaging studies have attempted to identify an overlap between brain regions associated with self-referential processes and categorization of others as in-group members ( Molenberghs and Morrison , 2014; Morrison et al . , 2012 ) A ventral region of medial prefrontal cortex ( vmPFC ) , including pregenual anterior cingulate cortex ( pgACC ) , is reliably associated with thinking about one’s own and similar others’ traits , mental states , and characteristics ( Cikara et al . , 2014; Heleven and Van Overwalle , 2016; Jenkins et al . , 2008 ) .",
"But are similarity-based estimates sufficient for categorizing others as in-group versus out-group members and informing subsequent behavior ?",
"Classic social psychological theories of intergroup relations indicate that there are other dimensions by which groups are defined ( Sherif , 1966 ) .",
"Rather than prioritizing similarity to oneself , people may rely on functional relations between one’s self and a target ( e . g . , 'Are you with me or against me ? '; Cikara and Fiske , 2013 ) .",
"Given that social categorization is such a flexible , dynamic process , how do people accumulate group structure information ( especially in the absence of overt cues to group membership ) ?",
"On one hand , they might try to characterize their ties with each individual ( e . g . , how well do I get along with Sue , with Dan , etc . ) .",
"However , social group dynamics may be better captured by a model that integrates information about how agents relate to one another in addition to oneself ( e . g . , how do Sue and Dan get along with each other , and how do I get along with either of them ? ) , which would allow perceivers to infer social latent group structure .",
"If people represent social latent group structure ( Figure 1A ) in addition to dyadic similarities , then even when two agents’ choices ( Agents A's and B's ) are equally similar to their own , the presence of a third agent ( Agent C ) altering the group structure should influence their decisions ( Figure 1B ) .",
"Importantly , dyadic similarity accounts would not predict differential ally-choice behavior in these cases ( because similarity is equated for the two agents in question ) .",
"In other words , the key difference between the two models is whether or not the presence of the third agent can affect how the first two agents are perceived .",
"To determine whether people possess different neural mechanisms for dyadic similarity and latent group structure learning , we created a structure-learning task in which participants reported their own position on a political issue and then guessed and learned via feedback the positions of three other agents , Agents A , B , and C , on the same issue ( Figure 2A ) .",
"After repeating this for eight political issues , participants were shown pictures of two of the three agents and asked to indicate with which of the two agents , Agent A or Agent B , they would align on an unknown political issue ( Figure 2B; Lau et al . , 2018 ) .",
"We focused on political issues because recent evidence suggests that implicit bias and behavioral discrimination along political party lines is now as potent as bias against racial out-groups ( Iyengar et al . , 2012; Iyengar and Westwood , 2015 ) .",
"This design allowed us to",
"i ) investigate participants’ trial-by-trial alignment signals based on dyadic similarity ( with each respective agent ) , feature similarity-over-agents , and social latent structures , and",
"ii ) identify which brain regions tracked each of these representations .",
"We then tested whether variability in the neural signal associated with these representations improved prediction of variability in participants’ ally-choice behavior ."
],
[
"To model the probability of choosing Agent B’s choice in the ally-choice trial as a function of social latent structure , we used a logistic regression predicting whether our participants in the scanner ( N = 42 ) chose Agent B’s choice during the ally-choice trial as a function of Agent B’s agreement and Agent C’s agreement with the participant .",
"( See Materials and methods for analysis of the full N = 333 sample . )",
"Because Agent A’s preferences were always the inverse of Agent B’s , including Agent A’s agreement would have created a multicollinearity problem ( recall also that participants could only choose either Agent B or Agent A ) .",
"Including random slopes to account for subject-level effects resulted in a singular fit of the model ( i . e . , overfitting ) , so we removed them .",
"We compared the full model including both main effects and the interaction with simpler models ( including only main effects or including only Agent B’s agreement with the participant ) .",
"Likelihood-ratio tests indicated that the fully saturated model with both main effects and the interaction term fit the data better than without the interaction term ( χ2 ( 1 ) = 7 . 246 , p=0 . 007 ) .",
"Replicating previous behavioral results ( Lau et al . , 2018 ) , we found that increasing Agent C’s alignment with the participant made respondents more likely to choose Agent B on the ally-choice trial , above and beyond the participant’s similarity with Agents A and B ( Figure 3 ) .",
"As a simple dyadic similarity account would predict , the model indicated a significant positive effect of Agent B’s agreement in predicting the likelihood of choosing Agent B in the ally-choice trial , b = 2 . 325 , Wald’s z = 4 . 099 , 95% CI [1 . 284 , 3 . 519] , p<0 . 001 .",
"However , as predicted by the latent structure learning account , the model also indicated a significant positive effect of Agent C’s agreement in predicting the likelihood of choosing Agent B in the ally-choice trial , b = 1 . 322 , Wald’s z = 2 . 633 , 95% CI [0 . 371 , 2 . 349] , p=0 . 008 ( Figure 3 ) .",
"This was qualified by a significant negative interaction between the agreements of Agent B and Agent C , b = −0 . 307 , Wald’s z = −2 . 588 , 95% CI [−0 . 550 , –0 . 082] , p=0 . 010 .",
"In other words , even when adjusting for Agent B’s agreement with the participant , increasing Agent C’s alignment with the participant made respondents more likely to choose Agent B on the ally-choice trial .",
"However , this result was expectably qualified by a weak interaction: when Agent B agreed with the participant a majority of the time , the additional variance explained by Agent C's agreement decreased .",
"While a dyadic similarity model would not predict that the level of agreement with Agent C should matter for choosing on the ally-choice trial , the latent structure learning model does predict that Agent C’s level of agreement with the participant should matter in whether or not participants choose Agent B on the ally-choice trial .",
"Indeed , any difference in choice behavior as a result of Agent C’s level of agreement is already inconsistent with the dyadic similarity account .",
"Disambiguation between these two accounts of ally-choice have been demonstrated previously with model simulations and behavioral studies ( Gershman et al . , 2017; Lau et al . , 2018 ) .",
"We developed three models to capture participants’ trial-by-trial estimates of",
"i ) dyadic similarity with each agent ,",
"ii ) similarity-over-agents , and",
"iii ) social latent structure .",
"We calculated dyadic similarity Sd as a function of the number of previous agreement instances between the agent under consideration and the participant divided by the number of trials elapsed , initialized at 0 . 50 for each agent ( see Materials and methods for model details ) .",
"For example , if an agent agreed with the participant on the first political issue , Sd would be calculated as 0 . 66 for the second issue; if the agent did not agree with the participant on the first political issue , Sd would be calculated as 0 . 33 for the second issue .",
"Unlike the latent structure learning model described below , the dyadic similarity model did not account for the feedback of other agents when calculating Sd .",
"In other words , for the third trial , the dyadic similarity model would calculate a similarity for an agent who had agreed twice with the participant on the previous two trials as 0 . 75 , regardless of how the other two agents had responded .",
"On each new run , Sd for each agent was set to 0 . 50 given that participants had no information about those three new agents .",
"As such , this value reflected how likely each agent was to agree with the participant on a new issue given that agent’s agreement with the participant on previous issues .",
"The person-as-feature similarity model calculated Sf as the correlation between rows of a similarity matrix constructed from all possible dyadic similarities ( i . e . , Sd between the participant and each agent as well as Sd between each pair of agents ) .",
"In other words , Sf could be considered a second-order dyadic similarity in that it captures similarity over people rather than over choices .",
"As such , this value reflected how likely each agent was to agree with the participant given that the agent's and the participant’s political preferences similarly resembled those of other agents .",
"In contrast , the latent structure learning model ( Gershman et al . , 2017 ) assumes that participants infer latent group assignments ( a partition of agents into groups ) on the basis of the agents’ choice data ( see Materials and methods for model details ) .",
"This model uses the Chinese restaurant process ( Aldous , 1985 ) as a prior over group assignments , which effectively ‘infers’ the most probable number of clusters in the environment given the existing data , therefore bypassing the need to a priori set an expected number of clusters ( e . g . , one rarely walks into a room expecting there to be n number of groups ) .",
"Through the observations of agents’ choices , the posterior is inferred using Bayes’ rule , and the likelihood , derived from analytically marginalizing the latent parameters under a Dirichlet-Multinomial model , will favor groupings where individuals in the same group exhibit similar choice patterns .",
"Parametric modulator values for the latent structure learning model were calculated as the marginal posterior probabilities of relevant partitions ( i . e . , partitions wherein the participant and the respective agent were grouped together ) .",
"To generate values , we did not fit any free parameters to participant behavior .",
"Unlike our other two models , information about all three agents contributed on each trial to the prior for guessing about each particular agent .",
"In other words , the prior for the second agent during the third trial took into account the feedback for the first agent in the third trial as well as the feedback for all three agents during the first and second trials .",
"As such , this value reflected how likely each agent was to belong to the same social group as the participant based on how all of the agents and the participant related to one another on previous issues .",
"We examined which voxels’ signal correlated with the dyadic similarity , feature similarity-over-agents , and latent structure learning parametric modulator contrasts ( Table 1; no other regions other than the ones reported here exceeded our corrected threshold . ) .",
"As predicted , trial-by-trial dyadic similarity correlated with activity in the ventral medial prefrontal cortex/pregenual anterior cingulate ( pgACC; Figure 4 , green ) .",
"The similarity-over-agents modulator identified clusters in the pgACC , bilateral temporoparietal junction , right superior temporal sulcus , and left supplementary motor area ( Figure 4 , yellow ) .",
"Perhaps unsurprisingly , the pgACC cluster from this parametric modulator encompassed the pgACC cluster found by the dyadic similarity modulator .",
"In contrast , the latent structure learning model parametric modulator identified only a cluster in right anterior insula ( rAI ) that extended into the inferior frontal gyrus ( IFG pars orbitalis; Figure 4 , red ) .",
"This rAI cluster did not overlap with any of the clusters identified by the previous two models .",
"Of note , this rAI cluster overlapped with a separately identified rAI cluster associated with non-social cluster assignment updating ( Tomov et al . , 2018 ) .",
"To provide a description of the similarity: our rAI overlapped with 44 . 7% of that rAI result ( i . e . , number of common voxels across both ROIs divided by total number of rAI voxels in Tomov et al . , 2018; Figure 4—figure supplement 1 ) .",
"Activity in this independently defined ROI correlated significantly with our latent group model , cluster-level FDR-corrected q = 0 . 014 .",
"We conducted a k-fold cross validation ( leave one out procedure ) and tested whether the latent structure learning model ( compared to the other models ) explained more variance in the rAI .",
"To do this , we iteratively generated rAI and pgACC clusters by conducting second-level analyses using all participants but one and tested the fit of the models in each cluster on the left-out participant .",
"For each iteration , we computed a Bayesian information criterion for each model as that particular model score and multiplied it by −0 . 5 to convert it into log model evidence .",
"We used this calculated log model evidence ( one for each model for each fold ) for Bayesian model selection and calculated protected exceedance probabilities ( PXP ) and Bayesian omnibus risk ( BOR; Rigoux et al . , 2014 ) .",
"A PXP reflects the probability that a particular model is more frequent in the population compared to the other models considered ( beyond what would be expected by chance ) , while BORs reflect the probability that all model frequencies are equal to one another .",
"To put these results into context , a previous ROC analysis found the disambiguation threshold , or the point at which we can best discriminate between H0 ( that both models are equally represented ) and H1 ( that one model is represented more so than another ) , to exist somewhere around 50% for PXPs and around 0 . 25 for BORs ( Rigoux et al . , 2014 ) .",
"The PXP in the rAI was 82 . 34% for the latent structure model , but only 6 . 44% for the dyadic similarity model and 11 . 23% for the similarity-over-agents model .",
"The BOR was 0 . 190 .",
"In sum , the latent structure model explained significant variance in the rAI .",
"On the other hand , when using the same method to test the specificity of the dyadic similarity model in the pgACC , we found that the PXPs were 51 . 58% for the latent structure model , 23 . 31% for the dyadic similarity model , and 25 . 11% for the similarity-over-agents model .",
"The BOR was 0 . 675 .",
"In other words , for the pgACC , no single model was especially frequent over the other two .",
"We also tested whether variability in the brain signal from our resulting ROIs would help predict variability in participants’ behavior during the ally-choice trial .",
"In other words , we asked whether the neural ‘noise’ from our clusters improved prediction of participant choice above and beyond mere model predictions .",
"We first decoded the neural signal in each ROI—pgACC and rAI—corresponding to the parametric modulator by removing the variance corresponding to other regressors in our model ( see Materials and methods ) .",
"We then isolated the signal corresponding to the temporal onsets of the photos of Agents A and B , which appeared right before each ally-choice trial .",
"For each agent , we averaged the signal of interest across voxels within each ROI , respectively ( i . e . , we calculated the signals corresponding to Agent A and Agent B for the pgACC and the signals corresponding to Agent A and Agent B for the rAI ) .",
"For each ROI , we then calculated the log difference between the signals corresponding to each agent .",
"We tested whether this signal would improve the fit of a logistic regression that modeled ally-choice behavior against model predictions from each of our models .",
"Model predictions were calculated as the log difference between either the similarity with A and the similarity with B ( e . g . , in the case of the dyadic similarity model ) at the end of the eight learning trials or the probability of the participant being grouped with A and the probability of the participant being grouped with B ( in the case of the latent structure model ) at the end of the eight learning trials .",
"Log difference of the signals was orthogonalized with respect to the log difference of corresponding model predictions .",
"Note that our parametric modulator ROIs were identified by fitting the signal during the learning phase of each run , whereas in the behavior prediction analyses here , we used signal from the ally-choice phase .",
"As such , there is no circularity in this analysis .",
"While a likelihood ratio test showed that adding the signal from the rAI cluster helped to better predict variability in choice behavior for the latent structure model ( χ2 ( 1 ) = 5 . 312 , p=0 . 021 ) , neither the addition of the signal from the pgACC to the dyadic similarity model ( χ2 ( 1 ) = 1 . 526 , p=0 . 217 ) , nor the addition of signal from any of the clusters associated with the similarity-over-agents model helped improve predictions of choice variability ( χ2 ( 1 ) s < 2 . 112 , ps >0 . 250 ) ."
],
[
"Here , we used a model-based analysis to compare different accounts by which people may differentiate ‘us’ from ‘them’ and found evidence for separable neural areas tracking each concurrently .",
"While social alignment estimates based on dyadic similarity and feature similarity-over-agents recruited the pgACC , allyship estimates via latent structure learning recruited the rAI .",
"Additionally , signal variability in the rAI cluster , but not any other cluster identified by the two other models , during the ally-choice trials helped predict ally-choice above and beyond model predictions .",
"Furthermore , a cross-validation demonstrated that the variability explained in the rAI by our latent structure learning model was much higher than the competing models .",
"Several aspects of these results merit further discussion .",
"First , this is the only evidence of which we are aware that pgACC supports incremental revisions of representations of similarity between oneself and others , both directly and across third agents .",
"In contrast to research that relies on preexisting knowledge about specific groups or individuals , using novel agents allowed us to examine how participants’ degree of alignment changed as they learned agents’ preferences over time .",
"Second , our rAI result is consistent with previous work on updating of non-social latent structure ( Tomov et al . , 2018 ) .",
"Note , though , that social categorization is distinct from other forms of categorization because it requires participants to categorize themselves ( Turner et al . , 1987 ) .",
"Thus , our results demonstrate that rAI is capable of learning egocentrically defined latent structures .",
"Anterior insula is topographically well-situated to relay social information related to coalition structure .",
"Connectivity of the anterior insula with the anterior cingulate cortex , amygdala , and ventral tegmental area ( a ‘salience detection’ network; Seeley et al . , 2007 ) and the dorsolateral prefrontal cortex ( associated with cognitive control; Chang et al . , 2013 ) likely affords the flexibility required to represent context-specific coalition members—someone who may be a coalition member at a debate may not be a fellow coalition member at a sports event .",
"AI is also heavily involved in socially-relevant computations , including but not limited to self-awareness tasks such as awareness of emotions and subjective pain , and exhibits hypoactivity in persons with autism spectrum disorder on tasks such as face processing and theory of mind ( Uddin and Menon , 2009 ) .",
"The rAI region we identified included a part of the IFG ( specifically , pars orbitalis ) .",
"In studies of hierarchical processing in music and language , this area has been associated with sentence comprehension ( Silbert et al . , 2014 ) and found to exhibit sensitivity to violations of hierarchical regularities ( Cheung et al . , 2018 ) .",
"This area is also involved in building up sentence structure and meaning as semantic information is processed ( Jeon and Friederici , 2013 ) .",
"Additionally , the IFG has been hypothesized to represent individual episodes for later recombination ( Preston and Eichenbaum , 2013 ) .",
"Just as this area may be recruited to build up the structures of sentences and tonal patterns , it may also be building up inferences of social latent structures as participants learn more about other agents’ preferences .",
"Finally , it is worth noting that while the brain tracks both of these alignment signals , variability in the signal from only one of the regions , rAI , helped improve model predictions of behavioral choices .",
"It is possible that had participants been asked to make a different choice ( e . g . , identify which agent better represented a particular trait ) , the pgACC signal may have been more relevant .",
"Nonetheless , this result underscores the need to further understand how social latent structures and coalitions feature in shaping people’s social choices .",
"Accurately distinguishing ‘us’ from ‘them’ is crucial to navigating our social lives .",
"To do so , we could rely on computing dyadic similarity with each individual agent or across agents; however , a more sophisticated approach would be to incorporate information about how agents relate to one another in order to infer latent groups in the environment through Bayesian inference .",
"Our approach moves beyond explicit category labels and mere similarity as the sole inputs to social group representations , appealing instead to a domain-general latent structure learning mechanism , which we demonstrate predicts ally-choice .",
"Furthermore , we provide evidence for separable neural areas tracking each of these processes; not only do we demonstrate that the rAI tracks estimates of any agent being a fellow coalition member , but we also show that the pgACC can track the fluctuations in similarity between oneself and agents ( both with individual agents and over agents ) in the environment .",
"These findings advance our understanding of the complex processes underlying social group inference and ally selection in humans and potentially other species ."
],
[
"We first recruited participants ( N = 333 ) under the pretense of playing a game in lab in which they would tell us about their political issue preferences and learn about others’ preferences .",
"All participants first completed this behavioral version of the task to familiarize themselves with the task and the political issues prior to scanning .",
"Participants completed all six runs of the task as described in the scanner procedure in the following section .",
"The main differences between the behavioral version and the scanner version were that",
"( i ) the behavioral version allowed participants to spend as much time as needed to read the prompt before proceeding , while the scanner version limited the reading time to 6 s ,",
"( ii ) the behavioral version did not allow for participants to acquaint themselves with the political issues before beginning the main task , and",
"( iii ) the response buttons differed ( i . e . , the behavioral version involved pushing ‘E’ and ‘I’ on a keyboard rather than ‘1’ and ‘2’ on a button box ) .",
"We could then ensure that we were only recruiting participants who could successfully make responses within the time limit .",
"Analysis of behavioral data from this phase can be found below .",
"Upon completion of the behavioral task , participants were asked if they were interested in completing a similar fMRI study for pay and asked to confirm or disconfirm a series of statements relating to qualifications for participating in an fMRI study ( e . g . , whether they had metal in their body , being able to lie still for over an hour , etc . ) .",
"Interested participants who reported no contra-indicators were invited to participate in the scanner task .",
"In the task for the participant selection phase , we fixed Agent A’s and Agent B’s agreement with the participant such that each agent agreed with the participant on only four of the eight trials .",
"Additionally , Agent C always agreed with Agent B and Agent A on five trials and three trials , respectively .",
"To create our conditions , we varied Agent C’s agreement with the participant such that Agent C agreed with the participant on either seven trials ( high-C ) or only one trial ( low-C ) .",
"Given that participants had to respond within 6 s , some participants missed ally-choice trials , and only trials where data was recorded were analyzed ( high-C: 906 trials , low-C: 918 trials ) .",
"We used a logistic regression to model the probability of choosing Agent B’s choice in the ally-choice trial as a function of condition ( high-C or low-C ) .",
"Including random slopes to account for subject-level effects resulted in a singular fit of the model ( i . e . , overfitting ) , so we removed them .",
"We did not find a significant difference between our two conditions in predicting the probability for choosing Agent B , b = 0 . 023 , Wald’s z = 0 . 246 , 95% CI = [−0 . 161 , 0 . 207] , p>0 . 250 .",
"Nonetheless , we have stated before this is a small behavioral effect when previously demonstrating it across another set of experiments ( Lau et al . , 2018 ) .",
"Our sample size here may have been too small to detect a difference .",
"Additionally , because participants were inexperienced and had only 6 s to respond ( whereas our previous participants had an unlimited time to respond ) , they may have responded differently .",
"When we limit the analysis to only the final , sixth ally-choice trial , we see a small effect of condition on the probability of choosing Agent B , b = 0 . 454 , Wald’s z = 2 . 00 , 95% CI = [0 . 010 , 0 . 901] , p=0 . 046 .",
"Finally , our two conditions , which drastically varied Agent C’s level of agreement ( i . e . , Agent C either mostly agreed or mostly disagreed with the participant ) , may have been too obvious for a version of the task that was not self-paced .",
"One participant reported as much in the debriefing .",
"In the actual scanner version of the study , we varied the degree to which Agents A , B , and C agreed with the participant while maintaining the same agreement relationships between the agents as found in the behavioral portion to avoid this limitation .",
"From our 333 participants , we recruited 61 right-handed participants ( 48 female , Mage = 21 . 74 years , SD = 4 . 17 ) in order to achieve a sample size of at least 40 participants after exclusions .",
"This sample size was determined based on sample sizes used in other fMRI experiments ( e . g . , Tomov et al . , 2018; Cheung et al . , 2018 ) .",
"Two participants requested to be removed from the scanner prior to the end of the study , and two participants were excluded due to a computer crash , resulting in unrecorded responses .",
"Prior to any data analysis , we excluded five participants who fell asleep in the scanner , eight participants for excessive head movements of 4 mm or more , one participant who correctly deduced the hypothesis of the study , and one participant for missing at least one self-response per run .",
"This left us with a sample size of 42 participants ( 32 female , Mage = 22 . 07 years , SD = 4 . 82 ) .",
"Participants provided informed consent to participate and consent to publish; all procedures complied with Harvard University’s Committee on the Use of Human Subjects’ guidelines ( Protocol #IRB15-2048 ) .",
"To develop stimuli , we used ISideWith . com , a website that helps people determine the political party and/or candidate with which their positions best align based on yes and no responses to nationally relevant , political issues ( e . g . , 'Do you support the death penalty ? ' ) .",
"The website also aggregates survey responses and makes this data publicly available ( https://isidewith . com/polls ) .",
"We selected issues that had accumulated at least 500 , 000 votes and had the greatest agreement/disagreement discrepancies , as described in Experiment 2 in Lau et al . ( 2018 ) .",
"We included the 48 issues with the lowest yes-no differences as of May 2017 in the main task ( see OSF for complete materials ) .",
"On each trial , we displayed the issue as text at the top of the screen .",
"Underneath , we signified a ‘yes’ or ‘no’ response to the issue by superimposing a green check mark or a red ‘X , ’ respectively , atop an image representing the issue .",
"To avoid confusion , we also displayed the words , ‘YES’ and ‘NO’ , underneath the corresponding images .",
"The order of presentation of the 48 issues as well as the sides on which the agreement positions appeared on the screen were randomized for each participant .",
"For agent pictures , we selected a total of 36 photos from the Chicago Face Database ( CFD; Ma et al . , 2015 ) and gender-matched agents to the participant .",
"We extracted the pool of ‘White’ faces ( based on CFD designations ) and eliminated faces based on the norming data provided by the CFD until 18 female and 18 male faces were left .",
"Given that the pool of faces varies for male and female faces , face selection processes varied slightly .",
"For female faces , at least half of the respondents had to rate the face as looking ‘white’ , and then we eliminated any face that respondents rated as unusual compared to other white females ( i . e . , above the midpoint of a 7-point Likert scale ranging from 1 , not at all unusual , to 7 , very unusual ) .",
"Using the ratings of how prototypical the faces looked compared to other white females ( 5-point Likert scale ranging from 1 , not at all prototypical , to 5 , very prototypical ) , we then removed the faces scoring less than one standard deviation from the average of this pool to remove non-prototypical faces .",
"We also then removed the faces that scored one standard deviation below the mean in terms of femininity ( 1 , not at all feminine , to 7 , extremely feminine ) .",
"To norm for attractiveness , we eliminated anyone who was rated as one standard deviation above and below the mean of attractiveness ratings , leaving us with 38 faces .",
"Any face two standard deviations above or below the mean age was then removed , leaving us with 37 faces , and the youngest face was then removed to generate a set of 36 faces with an average age of 27 . 01 years ( SD = 3 . 81 ) .",
"For male faces , we followed the same initial five steps , except we eliminated any face that scored one standard deviation above the mean in femininity ratings in order to retain the more masculine-looking faces .",
"Of the remaining 43 faces , we then eliminated anyone who was one and a half standard deviations above or below the mean age , which left us with 38 faces .",
"For the final step , we removed the two youngest faces in the set of faces to generate a set of 36 male faces with an average age of 28 . 58 years ( SD = 5 . 23 ) .",
"After being consented , participants first completed a round of instructions guiding them through a trial .",
"They expressed their own opinion on a topic ( ‘Should cartoons include plotlines involving duck-hunting ? ’ ) by selecting ‘Yes’ or ‘No’ and then guessed and received feedback on the opinions of Bugs and Daffy .",
"Participants were then guided through an ally-choice trial .",
"We told participants that for these trials , gray boxes with question marks on them would represent two different positions on a political issue .",
"The only information participants had about the boxes was the choices of other agents—the same ones whose preferences they had just learned .",
"We told participants to select the box they would prefer based on the other agents’ choices .",
"Participants were then introduced to the timing of the task ( see below ) and completed four practice trials ( ‘Should cartoon characters conquer Mars ? ' , ‘Should cartoon characters be allowed to pilot planes ? ' , ‘Should cartoon plotlines feature day jobs ? ' , ‘Should cartoon characters be allowed to do yoga ? ' ) with these timings in place with Bugs and Daffy again . An ally-choice trial followed these four practice trials in which participants were asked to choose between Bugs' and Daffy’s mystery positions .",
"After this , the computer displayed how many responses they successfully made within the time limits , and participants were prompted to notify the experimenter .",
"The experimenter checked that all participants made at least 11 of the 13 responses within the time limits and probed for any remaining questions about the task .",
"Given that participants had limited time to think about the political issue at hand and because their responses were important for generating the feedback they received from the other agents , we then gave participants a list of all of the political issues they would be seeing in the scanner prior to being scanned and asked them to review all the issues .",
"Each run consisted of two phases:",
"( i ) eight learning trials during which participants expressed their own preferences on a political issue and learned about three others’ preferences;",
"( ii ) an ally-choice trial .",
"Each learning trial began with the participant seeing a political issue for 6 s .",
"They then had 4 . 5 s to choose whether they would support that issue; a gray rectangle appeared around their selection when they had made their choice .",
"A gray arrow pointing to their choice appeared for 3 s to confirm their choice .",
"Each participant then learned about the preferences of other three individuals ( Agents A , B , and C ) via feedback .",
"To avoid participants weighting information from agents who looked more similar to them , we gender-matched the agents to participants’ self-reported gender and only used White faces .",
"Participants first saw a picture of one of the agents alongside his/her name and were asked to predict the preference of that agent with regards to the issue ( e . g . , ‘Which do you think Annie chose ? ' ) ; each participant had 4 . 5 s to guess , and a gray rectangle appeared around their selection after they made a choice . They then learned of the agent’s choice when a gray arrow pointing to one of the preferences ( ‘Yes’ or ‘No’ ) appeared for 3 s ( Figure 2A ) .",
"Participants then repeated this guess and feedback process for the other two individuals .",
"Between each screen , a fixation cross appeared for 3 s-16 s ( jittered ) .",
"Jitter was generated using the optseq algorithm ( http://www . surfer . nmr . mgh . harvard . edu/optseq ) .",
"Once this process ( political issue prompt , self-choice , confirmation of self-choice , guess and feedback for A , guess and feedback for B , and guess and feedback for C ) was completed for one issue , participants started a new regular trial by repeating the process for a new issue with the same three Agents .",
"A table consisting of eight rows and four columns displayed on the right-hand side of the screen recorded the participant’s and each Agent’s responses on each trial .",
"The order of the policy positions and agents was randomized; every participant saw each of the 48 policies and 18 agents only once during the experiment .",
"The order of the agents was also randomized across runs .",
"Agents were randomly assigned names from a preset list of names of five-letter length .",
"Following the eight learning trials , participants saw one ‘mystery’ ally-choice trial .",
"On these trials , participants saw pictures of Agents A and B sequentially presented in a random order , each of which was followed by a fixation cross that appeared for 3 s-16 s ( jittered ) , prior to reaching the decision screen .",
"On the decision screen , participants saw two boxes with question marks representing two unknown positions on a political issue .",
"Underneath the two boxes were photos of Agents A and B and gray arrows pointing to their respective choices ( Figure 2B ) .",
"We told participants that the mystery boxes contained Agent A’s and Agent B’s preferred positions .",
"Participants had to indicate which one of the two unknown positions they would rather choose ( e . g . , 'Which would you choose ? Remember , Annie and Betsy know what’s inside the boxes . ' ) .",
"A gray rectangle appeared around the selection after they indicated their choice .",
"Thus , participants had to align themselves with one of the two agents .",
"The response table summarizing participants’ and all agents’ preferences during the block was visible during the ally-choice trial .",
"After the ally-choice trial , participants started a new run with a new set of three agents and a new set of eight policy positions .",
"Participants completed six runs in total .",
"For each run , Agents B and C agreed on five issues; Agents A and C agreed on three issues , and Agents A and B never agreed with each other .",
"This agreement structure made it more likely that participants would cluster Agents B and C together and that Agent C’s agreement with the participant would increase the selection of Agent B on the ally-choice trial .",
"However , agreement counts between the participant and each agent varied across blocks and participants .",
"All agent positions were entirely randomly assigned ( within the constraints of the agreement/disagreement structure ) .",
"Thus , across all runs and participants , some agents expressed a mixture of highly partisan left and right beliefs .",
"In our previous behavioral studies ( Lau et al . , 2018 ) , Experiment 2 ) , we found that participants still exhibited the behavior predicted by the latent structure learning model despite learning about agents with less ideologically coherent profiles ( i . e . , agents with preference profiles constructed from a mix of left and right partisan topics ) .",
"We collected data using a 32-channel head coil in a 3 . 0-tesla Prisma MRI scanner ( Siemens ) located at the University .",
"At the beginning of each scan session , we acquired a high-resolution T-1 weighted anatomical image ( T1-MPRAGE , 1 × 1 × 1 mm , parallel to the anterior commissure-posterior commissure plane ) for use in registering activity to each participant’s anatomy and spatially normalizing data across participants .",
"Functional images were then acquired through six echo-planar imaging ( EPI ) sessions each lasting 12 min .",
"For whole brain coverage , we acquired 69 interleaved 2 . 0 mm slices ( repetition time = 1 . 5 s; echo time = 30 ms; flip angle = 75 degrees; field of view = 208 mm; matrix = 104 × 104; in-plane acceleration ( GRAPPA ) = 2; multi-band acceleration factor = 3 ) .",
"The multi-band EPI sequence was provided by the University of Minnesota Center for Magnetic Resonance Research ( Moeller et al . , 2010; Feinberg et al . , 2010; Setsompop et al . , 2012; Xu et al . , 2013 ) .",
"We conducted preprocessing and statistical analyses using SPM12 ( Wellcome Trust Centre for Neuroimaging , London , UK , http://www . fil . ion . ucl . ac . uk/spm ) .",
"We realigned functional images to the first volume , unwarped the functional images , segmented the structural image into its respective tissue types , and normalized the gray matter of the structural to the gray matter of a standard Montreal Neurological Institute ( MNI ) reference brain .",
"The mean functional images were co-registered to the structural image , and functional images were normalized to the MNI template , resliced to 2 mm × 2 mm × 2 mm voxels , and smoothed using an 8 mm FWHM Gaussian kernel .",
"We modeled data with an event-related design using a general linear model .",
"For each of the six runs , one regressor modeling the onset of self-choice ( eight onsets ) , a second regressor modeling the onset of guesses for each of the three agents across the eight issues ( 24 onsets ) and a parametric modulator for the second regressor , were convolved with the canonical hemodynamic response function .",
"The parametric modulator values indexed the specific model output ( the dyadic similarity , feature similarity-over-agents , or the latent structure learning model; see Computational models below ) of either the similarity between the participant and the target or the prior probability of the participant belonging to the same group as the target of the guess ( i . e . , Agents A , B , or C ) .",
"For example , when the participant was making a guess for Agent A on the seventh trial in a block , the latent structure parametric modulator took on the value of the probability that Agent A was in the same group as the participant given previous feedback .",
"No clusters survived correction when all three models were included as parametric modulators .",
"We did not orthogonalize parametric modulators with respect to the second regressor ( the onsets of guesses ) given that we were interested in which voxels tracked our parametric modulator values rather than the mean-centered values ( Mumford et al . , 2015 ) .",
"In addition , we included six nuisance regressors containing the temporal and spatial derivatives for the main regressor and six run regressors .",
"Alignment values from the latent structure learning model and the dyadic similarity model were modeled in separate models given that each represented a different hypothesis about processes in the brain .",
"To determine which areas of the brain tracked each these models , we then entered the images resulting from contrasting the parametric modulator against baseline into a second-level analysis treating participants as a random effect .",
"We used a voxelwise threshold of p<0 . 001 and corrected for multiple comparisons using whole-brain cluster-wise family-wise error ( FWE ) correction from bspmview ( http://www . bobspunt . com/bspmview ) at the α = 0 . 05 level .",
"Given that all three model outputs were derived from similar inputs , the correlations among the different parametric modulators were moderate to large .",
"The average correlation between dyadic similarity and latent structure modulators was 0 . 8627 , with values ranging from 0 . 5592 to 0 . 9704 .",
"The average correlation between the latent structure and feature similarity-over-agents parametric modulators was 0 . 7889 ( ranging from 0 . 3778 to 0 . 9496 ) .",
"Finally , the average correlation between the dyadic similarity and feature similarity-over-agents parametric modulators was 0 . 9614 ( ranging from 0 . 9023 to 0 . 9874 ) .",
"To measure collinearity between the dyadic similarity , feature similarity-over-agents and latent structure modulators , we calculated the VIF ( 1/ ( 1-R2 ) , where R2 is the r-squared from regressing one parametric modulator on the other ) .",
"The VIF between dyadic similarity and latent structure modulators was 5 . 063 ( generally regarded as low collinearity ) , while the VIF between dyadic similarity and feature similarity-over-agents parametric modulators was 13 . 786 .",
"Dyadic similarity , Sd , is calculated as a function of the number of previous agreement instances between the agent and the participant divided by the number of trials elapsed , where priors for the first trial are 0 . 50 for each agent: ( 1 ) Sd ( agent , participant ) =∑i=0n−1Agreementagent+1n+1 Feature similarity-over-agents , Sf , uses output from the dyadic similarity model to construct a similarity matrix whose values are Sd between the participant and each agent as well as Sd between agents .",
"Feature similarity between a participant and a particular agent is computed as the correlation between the row of the similarity matrix representing the dyadic similarity between the participant and each agent and the row of the similarity matrix representing the dyadic similarity between that particular agent and everyone else ( i . e . , the participant and the other two agents; Equation 2 ) .",
"To transform these correlations to interpretable probabilities , output values were rescaled to a 0 to 1 range , and a log odds transformation ( i . e . , log ( Sf ) – log ( 1- Sf ) ) was applied . Sdsim=[1Sd ( A , p ) Sd ( B , p ) Sd ( C , p ) Sd ( A , p ) 1Sd ( A , B ) Sd ( A , C ) Sd ( B , p ) Sd ( B , A ) 1Sd ( B , C ) Sd ( C , p ) Sd ( C , A ) Sd ( C , B ) 1] ( 2 ) Sf ( agent , participant ) =corr ( Sdsim ) agent , participant The latent structure learning model assumes that participants infer latent group assignments ( a partition of agents ) based on agents’ choice data .",
"The prior distribution over group assignments is a Chinese restaurant process ( Aldous , 1985 ) , where the probability of partition z = [z1 , … , zM] given M individuals is our prior: ( 3 ) P ( z|α ) =αKΓ ( α ) Πk ( Tk ) Γ ( M+α ) where α ≥ 0 serves as the dispersion parameter ( as α approaches infinity , each individual is assigned to a unique group ) , Tk is the number of individuals assigned to group k and Γ ( ∙ ) is the gamma function .",
"In our modeling , we used α = 2 , though the results are relatively robust to variation in this parameter .",
"An infinite number of groups can be generated , but a ‘rich get richer’ dynamic favoring more popular clusters will produce more parsimonious groupings ( see Gershman and Blei , 2012 ) .",
"We can derive the posterior using Bayes’ rule with observed choices C = [c1 , … , cM]: ( 4 ) P ( z|C ) =P ( C|z ) P ( z ) ∑z′P ( C|z′ ) P ( z′ ) The likelihood is obtained by analytically marginalizing the latent parameters under a Dirichlet-Multinomial model: ( 5 ) P ( C|z ) =∫θP ( C|θ , z ) P ( θ ) dθ=∏n∏kΓ ( |χn|γ ) Γ ( Tk+|χn|γ ) ∏cΓ ( Lknc+γ ) Γ ( γ ) Where θ is a set of multinomial parameters , |χn| is the number of options on problem n and Lknc is the number of individuals assigned to group k who choose stance c on issue n .",
"The likelihood favors group assignments for which choice patterns are similar between individuals assigned to the same group .",
"Parametric modulator values use the probability that the agent under consideration is in the same group as the participant and are derived as the marginal posterior probability of the relevant partitions: ( 6 ) P ( za=zp|C ) =∑kP ( za=k|C ) P ( zp=k|C ) The neural signal of interest can be algebraically derived from the standard GLM with L2-norm regularization: ( 7 ) Signal^interest= ( Y−∑i≠interestβ^iXi ) β^interestβ^interest2+λIwhere Y is the overall signal from the voxel and X is the corresponding vector from the original design matrix .",
"For these analyses , we set our regularization parameter , λ , to a value of 1 following Tomov et al . ( 2018 ) ."
]
] | [
"Humans form social coalitions in every society , yet we know little about how we learn and represent social group boundaries .",
"Here we derive predictions from a computational model of latent structure learning to move beyond explicit category labels and interpersonal , or dyadic , similarity as the sole inputs to social group representations .",
"Using a model-based analysis of functional neuroimaging data , we find that separate areas correlate with dyadic similarity and latent structure learning .",
"Trial-by-trial estimates of ‘allyship’ based on dyadic similarity between participants and each agent recruited medial prefrontal cortex/pregenual anterior cingulate ( pgACC ) .",
"Latent social group structure-based allyship estimates , in contrast , recruited right anterior insula ( rAI ) .",
"Variability in the brain signal from rAI improved prediction of variability in ally-choice behavior , whereas variability from the pgACC did not .",
"These results provide novel insights into the psychological and neural mechanisms by which people learn to distinguish ‘us’ from ‘them . ’"
] | [
"In every society , people form social coalitions — we draw boundaries between 'us' and 'them' .",
"But how do we decide who is one of 'us' and who is one of 'them' ?",
"One way is to use arbitrary categories .",
"For example , we say that those living 49 degrees north of the Earth’s equator are Canadian , whereas those living south of it are American .",
"Another possibility is to use physical characteristics .",
"But what about when neither of these options are available ?",
"By monitoring brain activity in healthy volunteers learning about other people’s political values , Lau et al . obtained insights into how people make these decisions .",
"Participants lying in a brain scanner were asked to report their position on a political issue .",
"They then learned the positions of three other hypothetical participants – A , B and C – on the same issue .",
"After repeating this procedure for eight different issues , the volunteers had to decide whether they would align with A or with B on a 'mystery' political issue .",
"So how do participants choose between A and B ?",
"One possibility is that they simply choose whichever one has views most similar to their own .",
"If this is the case , the views of hypothetical person C should not affect their decision .",
"But in practice , C's views – specifically how much they resemble the volunteer's own – do influence whether the volunteer chooses A or B . This suggests that we choose our allies based on more than just their similarity to ourselves .",
"Using a mathematical model , Lau et al . show that volunteers also take into account how similar the views of the other ‘participants’ are to each other .",
"In other words , they consider the structure of the social group as a whole .",
"Moreover , the results from brain imaging show that different regions of the brain are active when volunteers track the structure of the entire group , as opposed to their own similarity with each individual .",
"Notably though , the activity of the group-tracking region explains people's alignment choices better than the activity of the similarity-tracking region .",
"This suggests that we base our judgments of 'us' versus 'them' more on the structure of the group as a whole than on our own similarity with individual group members .",
"Understanding how we determine whether others are on the same ‘team’ as ourselves could ultimately help us find ways to reduce bias and discrimination between groups ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"computational and systems biology"
] | Rapid cell-free forward engineering of novel genetic ring oscillators | elife-09771-v3 | [
[
"A central tenet of engineering involves characterizing and verifying complex systems in a simplified environment ( Lu et al . , 2009 ) .",
"Electronic circuits are tested on a breadboard to verify circuit design and aircraft prototypes are tested in a wind tunnel to characterize their aerodynamics .",
"A simplified environment does not exist for characterizing and engineering complex biological networks , requiring system analysis to be conducted primarily in cells .",
"Performing extensive , quantitative and rapid network characterization in cells is limited due to difficulties associated with measuring parts , components , and systems in complex and ill-defined cellular hosts ( Kwok , 2010 ) .",
"Particular problems include: I ) lack of precise control over network component concentrations , II ) unpredictable interactions and integration with host cell processes , III ) cumbersome molecular cloning , and IV ) technical challenges and limited throughput associated with single cell measurements .",
"Cell-free systems promise to be efficient and effective tools to rapidly and precisely characterize native and engineered biological systems to understand their operating regimes .",
"Reconstituted biochemical systems have allowed the study of complex dynamic and self-organizing behaviors outside of cells such as switches , oscillators and pattern-forming regulatory networks ( Schwille and Diez , 2009; Genot et al . , 2013; van Roekel et al . , 2015 ) .",
"Networks assembled from simplified biochemistries such as oligonucleotide polymerization and degradation reactions can produce complex behaviors such as oscillations and provide insights into the working principles of biological regulatory systems ( Genot et al . , 2013; van Roekel et al . , 2015 ) .",
"While a high degree of abstraction and simplification makes it easier to analyze the underlying principles of biological networks , it becomes challenging to implement more complex networks and to directly transfer results and networks between the cell-free and the cellular environment .",
"Implementation of genetic networks in transcription-translation reactions has gained considerable traction because they rely on the cellular biosynthesis machinery and are compatible with a broad range of regulatory mechanisms .",
"A growing number of synthetic gene networks with increasing complexity have been implemented in cell-free transcription-translation systems ( Noireaux et al . , 2003; Shin and Noireaux , 2012; Takahashi et al . , 2015; Pardee , 2014 ) .",
"We and others have recently shown that oscillating genetic networks can be implemented in vitro outside of cells using microfluidic devices ( Niederholtmeyer et al . , 2013; Karzbrun et al . , 2014 ) .",
"However , whether these cell-free systems reflect the cellular environment sufficiently well to be of significance to biological systems engineering and analysis remains an open question .",
"A few studies investigated whether individual components such as promoters and ribosomal binding sites express at comparable strengths in cell-free systems and in cells ( Sun et al . , 2014; Chappell et al . , 2013 ) .",
"Comparisons of the behavior of genetic networks in cell-free systems and in cells are still limited to a few examples such as repressor-promoter pairs ( Chappell et al . , 2013; Karig et al . , 2012 ) and a RNA transcriptional repressor cascade ( Takahashi et al . , 2015 ) .",
"Thus far , however , it has not been shown whether genetic networks with complex dynamic behavior function similarly in cell-free and cellular environments .",
"Here , we demonstrate that cell-free systems can be used to characterize and engineer complex dynamic behaviors of genetic networks by implementing and characterizing novel 3-node , 4-node , and 5-node negative feedback architectures in vitro .",
"We go on to show that our 3- and 5-node oscillator networks were functional in cells and that their periods were comparable to those observed in the cell-free system , indicating that the cell-free system accurately emulated the cellular environment for the complex dynamic networks developed and tested in this study .",
"Cells carrying our 3-node networks oscillated on a population level , which was previously only observed in actively synchronized oscillators using quorum-based coupling ( Danino et al . , 2010 ) .",
"The in vitro and in vivo oscillations of our 5-node negative feedback networks confirm theoretical predictions on biomolecular ring oscillators and represent the largest synthetic negative feedback networks implemented simultaneously in cell-free systems and in cells ."
],
[
"Cell-free expression systems prepared from an E . coli extract ( ‘TX-TL’ ) can preserve the endogenous E . coli transcription machinery as well as native mRNA and protein degradation mechanisms ( Shin and Noireaux , 2010a; 2010b; 2012; Sun et al . , 2013 ) .",
"We have recently described a microfluidic nano-reactor device capable of emulating cellular growth and division .",
"A discontinuous flow of TX-TL reagents through miniaturized reactors at rates matching natural E . coli dilution rates keeps transcription and translation rates at constant steady state levels and removes reaction products , which is critical for the implementation of dynamic genetic networks ( Niederholtmeyer et al . , 2013 ) .",
"Using a TX-TL expression system in the microfluidic nano-reactor device thus provides a simplified and controlled environment , which can be applied to rapid prototyping ( Sun et al . , 2014 ) and characterization of genetic networks ( Figure 1 , top , Figure 1—figure supplement 1 ) .",
"In addition , TX-TL provides significant time savings over traditional prototyping in cells ( Figure 1 , bottom , Figure 1—source data 1 ) .",
"Linear DNA can be used in lieu of plasmid DNA , and the DNA source does not require specific compatible origins of replication or antibiotic markers .",
"Therefore , DNA can be assembled completely in vitro based on premade modules within 3 hr ( Sun et al . , 2014 ) .",
"Importantly , the time needed does not scale with circuit complexity , as each circuit component can be added as a separate linear template , which can be easily replaced or varied in concentration in following design-build-test cycles .",
"This is particularly advantageous for large circuits , where dosage or part changes require extensive re-cloning of plasmids for cellular testing , but no additional time for in vitro testing .",
"We estimate that prototyping a genetic circuit in TX-TL requires 4 to 78 hr , as opposed to prototyping in E . coli , which requires a minimum of 52 hr to several weeks . 10 . 7554/eLife . 09771 . 003Figure 1 . Cell-free systems allow rapid and extensive characterization of biological systems . Schematic representation of the design-build-test cycle using the cell-free system ( top ) .",
"A design is first modeled to obtain intuition about the architecture .",
"Parts are then assembled on linear DNA without cloning , and tested in vitro .",
"With functional parts , circuit variants can then be tested and working circuits can be extensively characterized .",
"Final circuits are cloned onto plasmids and implemented in vivo .",
"For a specific example of the cell-free system applied to engineering a 5-node oscillator network see Figure",
"1 . Bottom shows a comparison of the time required for testing a genetic circuit by the cell-free approach versus traditional engineering in cells . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 00310 . 7554/eLife . 09771 . 004Figure 1—source data 1 . Comparison of a Test Cycle in TX-TL vs . a Test Cycle in traditional prototyping . Shown are time estimates per step for each process .",
"Assumption is that modular PCR fragments exist that can be assembled into Linear DNA .",
"Parts used from Sun et al . , ( 2014 ) Supplementary Table S2 .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 00410 . 7554/eLife . 09771 . 005Figure 1—figure supplement 1 . Engineering a 5-node negative feedback oscillator using the cell-free framework .",
"A novel network architecture , which shows the intended behavior in silico is first assembled on linear DNA using in vitro characterized parts .",
"Initial circuit testing on linear DNA is advantageous because: ( I ) linear DNA can be synthesized in a few hours , ( II ) it allows rapid testing of multiple circuit variants , ( III ) and allows expression strengths of network components to be easily tuned by varying their relative concentrations .",
"A functional circuit can then be further characterized to identify parameter ranges that support the desired behavior and to experimentally test hypotheses .",
"If an in vivo implementation is intended , the cloned plasmids are verified for correct function in vitro before in vivo implementation . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 005 We asked whether this cell-free system could be used to run and characterize an existing synthetic in vivo circuit and chose to test the repressilator ( Elowitz and Leibler , 2000 ) as a model circuit .",
"We successfully implemented the original repressilator network in our cell-free system and observed long-term sustained oscillations with periods matching the in vivo study ( Figure 2 , Video 1 ) .",
"We compared the original repressilator to a modified version containing a point mutation in one of the CI repressor binding sites in the promoter regulating LacI ( Figure 2A ) .",
"This mutation increases the repressor concentration necessary for half-maximal repression ( K ) , and reduces cooperativity ( Rosenfeld et al . , 2005 ) .",
"At long dilution times ( td ) both circuits oscillated , but with shifted absolute reporter protein concentrations ( Figure 2B ) .",
"At decreasing dilution times amplitudes decreased and periods became faster with a linear dependence on td .",
"Faster dilution times , however , did not support oscillations for the modified network ( Figure 2B , C ) .",
"Experimentally , the range of dilution times supporting oscillations can serve as a measure for robust oscillator function , which generally diminishes with decreasing synthesis rates or when binding of one repressor to its promoter is weakened as in the OR2* mutant ( Figure 2—figure supplements 1–3 ) .",
"Initial conditions can influence the dynamic behavior of nonlinear systems but are difficult to control in cells .",
"In order to explore the dynamics of the repressilator in response to different initial conditions we varied the starting concentrations of TetR and CI repressor .",
"For all conditions tested the system quickly approached limit cycle oscillations and was invariant to initial conditions ( Figure 2D ) .",
"This analysis of the repressilator network in phase space provides an example for an experimental characterization that would be challenging or impossible to perform in a cellular environment . 10 . 7554/eLife . 09771 . 006Figure 2 . Cell-free repressilator characterization .",
"( A ) Application of cell-free systems to characterize the original repressilator ( Elowitz and Leibler , 2000 ) and a modified version with a point mutation in the CI promoter ( OR2* ) located in one of the binding sites of the CI repressor .",
"( B ) Expression from the three promoters of the repressilator and the OR2* version at different dilution times .",
"( C ) Oscillation periods of the repressilator as a function of dilution time .",
"In the OR2* version sustained oscillations were supported in a narrower range of dilution times as compared to the original repressilator network .",
"( D ) Phase portrait of repressilator oscillations starting from different initial TetR and CI repressor concentrations . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 00610 . 7554/eLife . 09771 . 007Figure 2—figure supplement 1 . Oscillation parameter regime for a 3-node repressilator network in terms of dilution time and synthesis rates . Transcription ( TX ) and translation ( TL ) rates supporting oscillations at different dilution times for a 3-node repressilator network .",
"Synthesis rates resulting from the plasmid concentrations used in Figure 2—figure supplement 2 are indicated by broken lines . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 00710 . 7554/eLife . 09771 . 008Figure 2—figure supplement 2 . Existence of oscillations and periods for a 3-node repressilator network in terms of dilution time . We experimentally studied the effect of varying transcription rates on the repressilator network by measuring the range of dilution times that supported sustained oscillations .",
"Transcription rates could be rapidly adjusted by varying DNA template concentrations of the repressilator plasmid .",
"For different DNA template concentrations , oscillations occurred in different ranges of dilution times .",
"Markers at a period of 0 hr indicate a stable steady state , and shaded regions highlight dilution times that did not support oscillations for a specific DNA template concentration .",
"A simulation of the repressilator network produced similar results but did not capture loading effects on the biosynthetic machinery for high DNA template concentrations . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 00810 . 7554/eLife . 09771 . 009Figure 2—figure supplement 3 . Oscillation parameter regime for a 3-node repressilator network in terms of dilution time and repressor binding affinityIncreasing the KM value , the repressor concentration needed for half-maximal repression , of one repressor , as for CI repressor in the OR2* repressilator version , reduces the range of dilution rates that support oscillations as indicated by our experimental results ( Figure 2C ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 00910 . 7554/eLife . 09771 . 010Video 1 . 3-color in vitro run of repressilator at td = 47 min . Shown on the right are individual channels of pCI-Citrine-ssrA , pTetR-Cerulean-ssrA , and pLacI-mCherry-ssrA .",
"These are combined in the composite at the left . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 010 The cell-free system also allows rapid characterization of individual network components .",
"We measured the transfer functions of repressor-promoter pairs in the repressilator network ( Figure 3A , Figure 3—figure supplements 1–3 , Figure 3—source data",
"1 ) and found that the network is symmetric in terms of transfer functions .",
"In the CI promoter OR2* mutant we observed the expected shift in K value and decreased steepness of the transfer function .",
"We also characterized TetR repressor homologs as building blocks for novel negative feedback circuits ( Figure 3A ) and with the exception of QacR observed similar transfer functions as observed in vivo ( Stanton et al . , 2014 ) ( Figure 3—figure supplement 3 ) . 10 . 7554/eLife . 09771 . 011Figure 3 . Cell-free prototyping and characterization of novel negative feedback circuits .",
"( A ) Transfer functions of the repressilator repressor-promoter pairs ( top ) and TetR homologs ( bottom ) .",
"The TetR repressor was tested against two different promoters: the promoter used in the repressilator ( top panel ) and the J23119-TetR promoter ( Stanton et al . , 2014 ) ( bottom panel ) .",
"Lines are Hill function fits .",
"( B ) Oscillations of a novel 3-node ring oscillator ( 3n1 ) constructed on plasmid DNA .",
"( C ) Two versions of a second 3-node ring oscillator ( 3n2 ) on linear DNA were used to study the effect of ClpXP degradation on oscillator function .",
"One version was ssrA-tagged on all repressor genes while the other version did not carry degradation tags on the repressors .",
"The same reporter with a medium-strength degradation tag was used in both versions .",
"( D ) A 4-node cyclic negative feedback network on linear DNA has two stable steady states that depend on the initial conditions .",
"IPTG switched the network into the state where pPhlF was on and pTetR off .",
"An initial pulse of aTc resulted in the opposite stable steady state .",
"( E ) Oscillations of two novel 5-node ring oscillators ( 5n1 , 5n2 ) constructed on linear DNA .",
"( F ) 5-node ring oscillators oscillate with longer periods than 3-node ring oscillators , as predicted by simulations ( Materials and methods ) and shown by experimental data . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 01110 . 7554/eLife . 09771 . 012Figure 3—source data 1 . Transfer function parameters . Parameter values of repressor – promoter pairs were determined by fitting to the Hill equation ( Materials and methods ) .",
"Promoter sequences were taken from the references cited .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 01210 . 7554/eLife . 09771 . 013Figure 3—figure supplement 1 . Measurement of transfer functions . Transfer functions of the repressor – promoter pairs were determined using the cell-free framework ( Materials and methods ) .",
"Shown are experimental results and analysis using LacI - pLacI ( r ) as an example .",
"Synthesis rates from the promoter of interest could be followed by Citrine fluorescence .",
"Varying repressor template DNA concentration over time allowed us to determine synthesis rates at different repressor concentrations .",
"Cerulean was co-expressed with the repressor and served as reporter for repressor concentration .",
"Transfer functions were obtained by plotting Citrine synthesis rates from highest to lowest repressor concentration ( grey shaded area ) against total Cerulean concentration and were identical for different dilution times set in the nano-reactor device . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 01310 . 7554/eLife . 09771 . 014Figure 3—figure supplement 2 . Comparison of relative promoter strengths in vitro and in vivo . Comparison of relative promoter strengths ( vmax ) , determined in vitro and in vivo .",
"pCI ( r ) , pTetR ( r ) , and pLacI ( r ) are from ( Elowitz and Leibler , 2000 ) ; pTetR is from ( Stanton et al . , 2014 ) and pLacI from ( Lutz and Bujard , 1997 ) .",
"Error bars indicate standard deviations of three replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 01410 . 7554/eLife . 09771 . 015Figure 3—figure supplement 3 . Comparison of half-maximal repressor concentrations needed for repression in vitro and in vivo . Comparison of KM values measured in vitro in this study with KM values determined in vivo by Stanton et al . ( Stanton et al . , 2014 ) .",
"KM values were normalized to the KM of TetR . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 015 Using three new repressors , BetI , PhlF and SrpR , we constructed a novel 3-node ( 3n ) circuit , 3n1 , and observed high-amplitude oscillations over a broad range of dilution times with the same dependence of amplitude and period on td as for the repressilator ( Figure 3B ) .",
"In our characterization of the repressilator network and the 3n1 oscillator we found dilution rates to be critical for the existence , period and amplitude of oscillations .",
"Protein degradation is similar to dilution in that it results in removal of repressor proteins .",
"In order to study the effect of degradation we constructed a second 3n network ( 3n2 ) using TetR , PhlF and SrpR repressors on linear DNA .",
"One version of the circuit used strong ssrA ClpXP degradation tags , while the second used untagged repressors .",
"We observed oscillations for both circuits ( Figure 3C ) .",
"However , the circuit without ssrA-tag mediated protein degradation exhibited slower oscillations , which extended to lower dilution times , showing that protein degradation , just like dilution , affects oscillator function and period .",
"Effects of ClpXP-mediated protein degradation , which have been shown to be important for existence and frequency of oscillations in vivo ( Cookson et al . , 2011; Prindle et al . , 2014 ) , can thus be emulated in a cell-free environment .",
"We characterized the repressilator ( Figure",
"2 ) and the novel 3n1 network ( Figure 3B ) on plasmid DNA , reasoning that this is the closest approximation to the situation in a cell and because promoter strengths compare better when measured on a plasmid ( Sun et al . , 2014; Chappell et al . , 2013 ) .",
"However , for a more rapid analysis of novel networks and network variants it is advantageous if laborious cloning steps are not required to obtain initial results on circuit performance .",
"Construction and comparison of two 3n2 network variants , which only required a few PCR reactions to synthesize the linear DNA templates , showed that it is possible to go from theoretical design of a circuit to first experimental results in a very short timeframe ( Figure 3C ) .",
"Next , we went on to test this concept for novel and more complex network architectures .",
"Theory predicts that ring architectures built from an odd number of repressors oscillate , while even-numbered architectures have stable steady states ( Smith , 1987; Hori et al . , 2013 ) .",
"We experimentally built and tested a 4-node circuit from LacI , TetR , PhlF and SrpR on linear DNA .",
"Initial pulses of LacI inducer IPTG or TetR inducer aTc allowed us to switch expression into either one of the two stable steady states ( Figure 3D ) .",
"Stable steady states were reached after an initial adjustment phase of 5 to 10 hr and remained stable until the experiment was terminated after 22 hr .",
"These results show that non-oscillating networks also function in the cell-free environment and that the oscillations we observe for the 3-node networks are determined by network architecture and not established by particular reaction conditions in the microfluidic reactor .",
"Encouraged by the robust oscillations observed in the 3n networks and the expected behavior of the 4-node bistable switch , we built two 5-node ring networks ( 5n ) to test our prototyping environment on another novel synthetic network architecture ( Figure 3E ) .",
"We expected these circuits to oscillate , as they were built from an odd number of repressors .",
"Despite their considerable complexity both circuits indeed oscillated over a broad range of dilution times .",
"The period of the 5n networks ( up to 19 hr ) was significantly longer than that of the 3n networks ( up to 8 hr ) .",
"Comparing all ssrA-tagged 3n and 5n ring architectures , we show that the observed periods could be accurately predicted for all four networks by computational simulations ( Figure 3F ) .",
"Our cell-free system allows characterization of complex networks from rapid testing on linear DNA to verifying networks cloned onto a single plasmid , which is the closest approximation to cellular implementation ( Figure 1—figure supplement 1 ) .",
"To validate our cell-free approach we cloned the 3n1 and 3n2 networks onto low-copy plasmids and co-transformed each with a medium-copy reporter plasmid into lacI-JS006 E . coli ( Stricker et al . , 2008 ) .",
"When tested on a microfluidic device ( mother machine ( Wang et al . , 2010 ) ) , both 3n oscillators showed regular oscillations with periods of 6 ± 1 hr for at least 30 hr ( Figure 4A , Videos 2 , 3 ) .",
"Both oscillators were surprisingly robust as all cells undergoing healthy cellular division oscillated ( n=71 ) ( Figure 4—figure supplement 1 , Video 4 ) . 10 . 7554/eLife . 09771 . 016Figure 4 . Novel 3-node and 5-node ring oscillators in cells .",
"( A ) Time series traces of 3-node ring oscillators running in E . coli ( mother machine ) .",
"Single trap traces of 3n1 and 3n2 observed for 36 hr in vivo using a strong pPhlF sfGFP-ssrA reporter and a representative image from an ‘on’ and ‘off’ state of oscillation .",
"Scale bar: 5 µm .",
"( B ) Time series traces of 5-node ring oscillators running in E . coli ( mother machine ) .",
"Single trap traces of 5n1 and 5n2 observed for 72 hr in vivo using a weak pPhlF sfGFP-ssrA reporter .",
"( C ) 3n1 displays population-wide oscillation pulses in vivo ( CellASIC ) .",
"Time series micrographs of 3n1 under a strong pPhlF sfGFP-ssrA reporter every 160 min; inset shows individual cells of the initial microcolony .",
"Scale bar: 10 µm and 5 µm ( inset ) .",
"( D ) Relationship between period and division time in vivo .",
"Left , 3n1 in vivo under a strong pPhlF sfGFP-ssrA reporter .",
"The in vitro data is shown for comparison .",
"Each point in the in vivo data corresponds to the period and division time from aCellASIC experiment run under different media type and flow rates .",
"Right , 5n2 in vivo under aweak pPhlF sfGFP-ssrA reporter .",
"In vivo periods determined at 29ºC and 21ºC growth temperature in mother machine experiments .",
"Boxes represent the inner quartile range with the median .",
"( E ) Influence of reporter concentration on oscillation periods by competing for ClpXP degradation .",
"Left , with constant amounts of ClpXP the reporter concentration affects repressor degradation and thus oscillation period .",
"Histograms of the periods observed with a weak and a strong pPhlF sfGFP-ssrA reporter for both 3n1 and 5n2 run in the mother machine .",
"Dashed lines indicate the medians . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 01610 . 7554/eLife . 09771 . 017Figure 4—figure supplement 1 . Robust oscillations of 3-node and 5-node oscillators in vivo . 3-node ( top ) and 5-node networks ( bottom ) oscillate with periods that depend on the network size in vivo .",
"Shown are the distributions of observed period lengths with medians indicated by dashed lines .",
"Both 3-node and 5-node networks exhibited robust oscillation with all growing cells oscillating for the 3-node networks and more than 95% of growing cells oscillating for the 5-node networks ( defined as at least two distinct oscillation peaks per trace ) .",
"Shown are four example traces for all oscillators in addition to the ones shown in Figure 3A , B .",
"Both 3-node networks were analyzed using a strong pPhlF sfGFP-ssrA reporter and the two 5-node networks were analyzed using a weak pPhlF sfGFP-ssrA reporter . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 01710 . 7554/eLife . 09771 . 018Figure 4—figure supplement 2 . Population-level oscillations of 3n2 oscillator in vivo . 3n2 oscillator displays phase synchrony in vivo .",
"3n2 is run under a strong pPhlF sfGFP-ssrA reporter in the CellASIC microfluidic device .",
"Scale bar: 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 01810 . 7554/eLife . 09771 . 019Figure 4—figure supplement 3 . Three-color oscillations and population-level oscillations of 3n2 oscillator in vivo . 3n2 displays phase synchrony observing 3 reporters simultaneously .",
"Reporters are a strong pPhlF Citrine-ssrA , pTetR mCherry-ssrA , and pSrpR Cerulean-ssrA .",
"Shown is one oscillation cycle .",
"Scale bars: 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 01910 . 7554/eLife . 09771 . 020Figure 4—figure supplement 4 . Original repressilator and OR2* mutant repressilator do not show population-level oscillations . Original repressilator and OR2* repressilator do not show phase synchrony .",
"These are run under pTetR ( r ) -eGFP ( ASV ) in M9 minimal media; oscillations were not supported in LB .",
"Scale bars: 10 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 02010 . 7554/eLife . 09771 . 021Video 2 . 3n1 in mother machine , single trap . 3n1 using a pPhlF-BCD20-sfGFP-ssrA ( strong ) reporter is run in the mother machine at 29ºC in LB . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 02110 . 7554/eLife . 09771 . 022Video 3 . 3n2 in mother machine , single trap . 3n2 using a pPhlF-BCD20-sfGFP-ssrA ( strong ) reporter is run in the mother machine at 29ºC in LB . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 02210 . 7554/eLife . 09771 . 023Video 4 . Run of a panel of 3n2 oscillators in mother machine , using pPhlF-BCD20-sfGFP-ssrA ( strong ) reporter . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 023 We next turned to testing our 5n oscillators in vivo .",
"Like the 3n networks we cloned both 5n oscillators onto low-copy number plasmids to transfer the two networks we had initially prototyped on linear DNA ( Figure 3E ) into E . coli .",
"Figure 1—figure supplement 1 shows the complete cell-free prototyping cycle for the 5n1 oscillator from design by testing on linear DNA to validation of the final cloned oscillator plasmid .",
"In E . coli , 5n1 was not viable when co-transformed with a high expression-strength reporter , but was viable with a low expression-strength reporter .",
"Specifically , 5n1 with a high expression-strength reporter caused slow growth and high cell death rates when run on the mother machine – we hypothesize that this is due to loading effects from high protein production , as decreasing the reporter expression strength resolved cell viability issues ( Ceroni et al . , 2015 ) .",
"When tested with a low expression-strength reporter both 5n oscillators showed robust oscillations in E . coli that were maintained for at least 70 hr , and over 95% of all analyzed traps containing healthy cells oscillated ( n=104 ) .",
"In addition , both 5n networks oscillated with similar periods: 8 hr for 5n1 , and 9 hr for 5n2 ( Figure 4B , Figure 4—figure supplement 1 , Videos 5–7 ) . 10 . 7554/eLife . 09771 . 024Video 5 . 5n1 in mother machine . 5n1 using a pPhlF-BCD22-sfGFP-ssrA ( weak ) reporter is run in the mother machine at 29ºC in LB . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 02410 . 7554/eLife . 09771 . 025Video 6 . 5n2 in mother machine . 5n2 using a pPhlF-BCD22-sfGFP-ssrA ( weak ) reporter is run in the mother machine at 29ºC in LB . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 02510 . 7554/eLife . 09771 . 026Video 7 . Run of a panel of 5n2 oscillators in mother machine , using pPhlF-BCD22-sfGFP-ssrA ( weak ) reporter . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 026 We also tested both 3n oscillators on a CellASIC system , which allows planar single-layer colony formation .",
"Starting from a single cell we observed striking oscillation pulses of the entire growing microcolony ( Figure 4C , Figure 4—figure supplement 2 , Videos 8 , 9 ) .",
"These population level pulses were also apparent when using three different fluorescent reporters simultaneously ( Figure 4—figure supplement 3 , Video 10 ) .",
"We did not observe population level oscillations in either the original repressilator , the OR2* mutant ( Figure 4—figure supplement 4 ) or the 5n networks .",
"Synchronized oscillations were not reported with the original repressilator ( Elowitz and Leibler , 2000 ) , and have only been observed in oscillators using intercellular communication ( Danino et al . , 2010; Prindle et al . , 2012 ) .",
"In contrast to these quorum-sensing mechanisms that can actively couple and synchronize oscillator states ( Danino et al . , 2010; Prindle et al . , 2012 ) we do not believe the population-wide in-phase oscillations we observed are due to an active coupling mechanism .",
"We hypothesize that the population-level oscillations of the 3n1 and 3n2 networks are due to increased repressor concentrations as compared to the original repressilator network , which increases the inheritance of the period phenotype and minimizes the rapid de-phasing expected from stochastic cellular protein fluctuations ( Kiviet et al . , 2014 ) .",
"However , a quantitative characterization of this slow de-phasing phenotype requires more in depth understanding of stochastic effects in vivo .",
"Because cells stayed synchronized , we were able to analyze the population as a whole to make general conclusions of oscillator behavior .",
"We varied dilution time by using different media conditions and media flow rates , and found a direct relationship between division times and period , consistent with the cell-free data collected .",
"Oscillation periods of the 5n oscillators were also consistent with our cell-free results and showed a similar dependence on doubling time ( Figure 4D ) . 10 . 7554/eLife . 09771 . 027Video 8 . 3n1 in CellASIC .",
"3n1 using a pPhlF-BCD20-sfGFP-ssrA ( strong ) reporter is run in CellASIC . This video corresponds to Figure 4C .",
"Conditions: 29º°C , LB media , 12 . 5% lamp intensity , 200 ms exposure , 2 psi flow rate . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 02710 . 7554/eLife . 09771 . 028Video 9 . 3n2 in CellASIC .",
"3n2 using a pPhlF-BCD20-sfGFP-ssrA ( strong ) reporter is run in CellASIC . This video corresponds to Figure 4—figure supplement",
"2 . Conditions: 29ºC , LB media , 12 . 5% lamp intensity , 200 ms exposure , 2 psi flow rate . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 02810 . 7554/eLife . 09771 . 029Video 10 . 3n2 with 3-color output run in CellASIC . 3n2 using a pPhlF-BCD20-Citrine-ssrA , pTetR-BCD20-mCherry-ssrA , pSrpR-Cerulean-ssrA ( strong ) reporter is run in CellASIC .",
"This video corresponds to Figure S5B .",
"Conditions: 29 C , LB media , 12 . 5% lamp intensity , 200 ms exposure for Citrine and Cerulean ( 500 ms for mCherry ) , 2 psi flow rate . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 029 Finally , we compared 3n1 and 5n2 with weak and strong reporters in vivo to analyze the effect of protein degradation on the oscillator period .",
"We theorized that given a constant concentration of ClpXP , stronger reporters would result in more ClpXP loading , thereby slowing the period of oscillation .",
"ClpXP is thought to influence oscillation dynamics in vivo in this manner ( Cookson et al . , 2011 ) .",
"We found that in the mother machine , both the period distributions of 3n1 and 5n2 showed this characteristic ( Figure 4E ) , which reflects our cell-free findings of differential –ssrA tag dependent period length ( Figure 3C ) ."
],
[
"We showed that synthetic dynamic networks can be readily implemented , characterized , and engineered in a cell-free system and subsequently transferred to cellular hosts ( Figure 1 ) .",
"Our results demonstrate the utility of this approach for biological systems engineering and component characterization .",
"The cell-free system resulted in the experimental validation of previous theoretical predictions on even- and odd-numbered cyclic negative feedback circuits ( Smith , 1987; Hori et al . , 2013 ) and enabled the in vivo implementation of robust 5-node genetic oscillators .",
"The cell-free environment can thus fill the gap between theoretical network design and in vivo implementation of biological systems and provide a simplified and controlled environment that drastically reduces the design-build-test cycle ( Sun et al . , 2014 ) .",
"In order to match the cell-free and cellular environments as closely as possible we prepared an extract-based TX-TL expression system from the same E . coli strain we used for in vivo experiments by a method that preserves the endogenous biosynthetic and protein degradation machinery ( Shin and Noireaux , 2010a; 2010b; 2012; Sun et al . , 2013 ) .",
"Similar expression systems had previously been applied to the prototyping of promoters and ribosomal binding sites ( Sun et al . , 2014; Chappell et al . , 2013 ) .",
"Here we showed that they can also be used for the prototyping of entire genetic networks when they are analyzed in a microfluidic system that emulates cellular growth .",
"Oscillation periods in negative feedback ring oscillators are mainly determined by degradation and dilution rates ( Hori et al . , 2013 ) , which explains the good correspondence between oscillation periods in our cell-free environment and in E . coli .",
"By constructing and testing novel network architectures we show that almost all prototyping can be done on linear DNA , which requires less than 8 hr to assemble and test .",
"This allows rapid screening of different network topologies and rapid screening of parameters important for the desired function of a network .",
"We observed some differences between the cell-free and cellular environment , particularly in the difficulty of predicting cellular toxicity and loading effects of the 5n oscillators in vivo , and some differences between promoter and repressor strengths .",
"With a better understanding of loading effects cell-free prototyping environments may predict when cells will be overloaded .",
"We also add that the complete cell-free prototyping cycle that worked for 5n1 ( Figure 1—figure supplement 1 ) was not entirely successful for the 5n2 network .",
"5n2 showed similar oscillations as compared to 5n1 when analyzed on linear DNA in the cell-free environment ( Figure 3E ) and on plasmid DNA in E . coli ( Figure 4 ) , but we did not observe oscillations for 5n2 when run from the same plasmid DNA in the cell-free environment .",
"We hypothesize this is due to differences in expression efficiencies from linear and plasmid DNA in cell-free prototyping environments ( Sun et al . , 2014; Chappell et al . , 2013 ) , and/or differences between repressor strengths in the cell-free and the cellular environment ( particularly for QacR , Figure 3—figure supplement 3 ) .",
"While more work is necessary describing and explaining differences between in vitro and in vivo environments , the observed behavior of complex networks in our cell-free environment reflected network behavior in vivo well .",
"Cell-free systems are thus a powerful emulator of the cellular environment allowing precise control over experimental conditions and enabling studies that are difficult or time consuming to perform in cells .",
"We envision that it will not only be useful for prototyping and characterizing novel synthetic systems but also facilitate in-depth analyses of native biological networks in a simplified setting .",
"With further developments in cell-free lysate systems and supporting technologies , the cell-free approach is posed to play an increasing role in biological systems engineering and provides a unique opportunity to design , build , and analyze biological systems ."
],
[
"DNA was constructed using either Golden Gate Assembly or Isothermal Assembly .",
"For linear DNA , all DNA was constructed using previously published Rapid Assembly protocols on a ‘v1-1’ vector ( Sun et al . , 2014 ) .",
"Linear DNA constructs are summarized in Supplementary file 1A .",
"The original repressilator plasmid , pZS1 ( Elowitz and Leibler , 2000 ) was used as a template for initial characterization and for construction of the OR2* mutant .",
"Transfer function plasmids were constructed by Transcriptic , Inc .",
"For other plasmids , partial sequences were either obtained from Addgene ( Stanton et al . , 2014 ) or synthesized on gBlocks or ssDNA annealed oligonucleotides ( Integrated DNA Technologies ) .",
"Specific plasmids required secondary-structure free segments , which were designed by R2oDNA ( Casini et al . , 2014 ) .",
"JS006 ( Stricker et al . , 2008 ) was co-transformed with origin-of-replication compatible plasmids to create engineered strains .",
"Specifically , negative-feedback oscillator units were cloned onto pSC101* low copy plasmids ( ampR or kanR ) , while reporters were cloned onto colE1 medium copy plasmids ( kanR or cmR ) ( Supplementary files 1B , C ) .",
"To modulate the reporter copy number , all experiments were conducted below 37°C ( Fitzwater et al . , 1988 ) .",
"Strain passage was minimized to avoid plasmid deletions due to the recA + nature of JS006 and the high complexity of oscillator plasmids or triple-reporter plasmid .",
"Based on the in vitro and in silico results , we used strong transcriptional and translational ( Mutalik et al . , 2013 ) units to maximize gain .",
"Preparation of TX-TL was conducted as described previously ( Sun et al . , 2013 ) , but using strain 'JS006' co-transformed with Rosetta2 plasmid and performing a 1:2:1 extract:DNA:buffer ratio .",
"This resulted in extract ‘eZS4’ with: 8 . 7 mg/mL protein , 10 . 5 mM Mg-glutamate , 100 mM K-glutamate , 0 . 25 mM DTT , 0 . 75 mM each amino acid except leucine , 0 . 63 mM leucine , 50 mM HEPES , 1 . 5 mM ATP and GTP , 0 . 9 mM CTP and UTP , 0 . 2 mg/mL tRNA , 0 . 26 mM CoA , 0 . 33 mM NAD , 0 . 75 mM cAMP , 0 . 068 mM folinic acid , 1 mM spermidine , 30 mM 3-PGA , 2% PEG-8000 .",
"For experiments utilizing linear DNA GamS was added to a final concentration of 3 . 5 µM ( Sun et al . , 2014 ) .",
"Experiments were performed in a microfluidic nano-reactor device as described previously ( Niederholtmeyer et al . , 2013; Sun et al . , 2013 ) with some modifications to optimize the conditions for the lysate-based TX-TL mix .",
"Reaction temperature was 33°C .",
"Lysate was diluted to 2x of the final concentration in 5 mM HEPES 5 mM NaCl buffer ( pH 7 . 2 ) .",
"The reaction buffer mix was combined with template DNA and brought to a final concentration of 2x .",
"For a 24 hr experiment 30 µl of these stocks were prepared .",
"During the experiment , lysate and buffer/DNA solutions were kept in separate tubing feeding onto the chip , cooled to approximately 6ºC , and combined on-chip .",
"We ran experiments with dilution rates ( µ ) between approximately 2 . 8 and 0 . 5 hr-1 , which corresponds to dilution times , td = ln ( 2 ) µ-1 , between 15 and 85 min .",
"These were achieved with dilution steps exchanging between 7 and 25% of the reactor volume with time intervals of 7 to 10 min , which alternately added fresh lysate stock or fresh buffer/DNA solution into the reactors .",
"Dilution rates were calibrated before each experiment .",
"Initial conditions for the limit cycle analysis of the repressilator network were set by adding pre-synthesized repressor protein at the beginning of each experiment .",
"For this , CI repressor ( together with Citrine reporter ) and TetR repressor ( together with Cerulean reporter ) were expressed for 2 . 5 hr in batch .",
"On chip the initial reaction was mixed to be composed of 25% pre-synthesis reaction and 75% fresh TX-TL mix and repressilator template DNA .",
"Then , the experiment was performed at a td of 19 . 2 ± 0 . 3 min .",
"Initial conditions for the 4-node experiment were 2 . 5 µM aTc or 250 µM IPTG , and the experiment was performed at a td of 44 . 5 ± 0 . 9 min .",
"DNA template concentrations used in steady-state reactions are listed in Supplementary file 1D .",
"Arbitrary fluorescence values were converted to absolute concentrations from a calibration using purified Citrine , Cerulean , and mCherry , which were prepared using previously published protocols utilizing a His6 purification method followed by size-exclusion chromatography and a Bradford assay to determine protein concentration ( Sun et al . , 2014 ) .",
"Transfer functions of the repressor – promoter pairs were determined in the nano-reactor device at a minimum of two different dilution times ( Figure 3—figure supplement 1 ) .",
"All tested promoters were cloned into a plasmid in front of a BCD7 ribosomal binding site and the Citrine open reading frame .",
"A non-saturating concentration of 1 nM plasmid was used in the experiment .",
"The repressors were expressed from linear templates carrying the J23151 promoter and the BCD7 ribosomal binding site with time-varying concentrations , which were increased from 0 to 2 . 5 nM and decreased back to 0 during the course of the experiment ( Niederholtmeyer et al . , 2013 ) .",
"Simultaneously we expressed Cerulean as a reporter for the repressor concentration from a linear template at an identical concentration as the repressor template .",
"From the concentration of the Citrine reporter we calculated the synthesis rate of the fluorescent protein over time using a model of steady state protein synthesis in the nano-reactor device ( Niederholtmeyer et al . , 2013 ) , ( 1 ) Pd ( t+∆t ) = Pd ( t ) +syn ( t ) ·∆t-mat·Pd ( t ) ·∆t-dil·Pd ( t ) ( 2 ) Pf ( t+∆t ) =Pf ( t ) +mat·Pd ( t ) ·∆t-dil·Pf ( t ) where Pd and Pf are dark and fluorescent reporter concentration respectively , t is time , Δt is the time interval between dilution steps , dil is the volume fraction replaced per dilution step , which was determined during the calibration of the device , and mat is maturation rate of the fluorescent protein .",
"Maturation times of Citrine and Cerulean were determined as described previously ( Niederholtmeyer et al . , 2013 ) and were 15 ± 4 min for Cerulean and 29 ± 3 min for Citrine .",
"Dark fluorescent protein was calculated from ( Equation 2 ) : ( 3 ) Pd ( t ) =Pf ( t+∆t ) -Pf ( t ) +dil·Pf ( t ) mat·∆t and the synthesis rate was calculated from Equation 1: ( 4 ) syn ( t ) =Pd ( t+∆t ) -Pd ( t ) +mat·Pd ( t ) ·∆t+dil·Pd ( t ) We used the sum of measured fluorescent Cerulean concentration and ( Equation 3 ) for dark Cerulean as a measure of the total repressor protein present at any time during the experiment .",
"The synthesis rates were normalized to their respective maximal values ( vmax ) and plotted against the concentration of the repressor reporter using only repressor concentrations higher than 1nM .",
"The transfer curves were then fit to a Hill function ( 5 ) y=f ( x ) =ymin+ ( 1-ymin ) KMnKMn+xn where y is the synthesis rate , ymin is the minimum synthesis rate , n is the Hill coefficient and KM is the Michaelis Menten constant for half maximal promoter activity .",
"The fitting was performed in Igor Pro using orthogonal distance regression with ODRPACK95 assuming a 9% error in the measurements of Citrine and Cerulean fluorescence .",
"Relative promoter strengths ( vmax values ) were determined using the transfer function promoter plasmids .",
"In vitro strengths were determined in 5 µl TX-TL reactions at a DNA template concentration of 1 nM .",
"Reactions were assembled in 384-well plates , overlaid with 35 µl Chill-Out Liquid wax ( BioRad ) and analyzed using a Biotek SynergyMx plate reader set to 33ºC reaction temperature , and reading Citrine fluorescence with Exc: 510 ± 9 nm and Em: 540 ± 9 nm .",
"For comparison , Citrine fluorescence at 6 hr was normalized to the value of pLacI .",
"In vivo strengths were determined using E . coli JS006 transformed with the same plasmids .",
"Cells were grown at 29ºC in MOPS medium supplemented with 0 . 4% glycerol and 0 . 2% casaminoacids .",
"For each strain , three independent overnight cultures were diluted 1:50 and grown to mid-log phase .",
"They were then diluted to a starting OD600 of 0 . 15 into 100 µl growth medium in a 96-well plate and grown in the plate reader at 29ºC with periodic shaking measuring Citrine fluorescence .",
"Fluorescence values were normalized to OD resulting in steady state values after 2 hr .",
"Average steady state values were normalized to pLacI for comparison with the in vitro measurement .",
"Mother machine ( Wang et al . , 2010 ) experiments were conducted with custom-made microfluidic chips ( mold courtesy of M . Delincé and J . McKinney , EPFL ) .",
"E . coli cells were trapped in channels of 30 µm length , 2 µm width and 1 . 2 µm height .",
"Before loading onto the device , cells were grown from a frozen stock to stationery phase .",
"Cells were then concentrated tenfold and loaded onto the chip .",
"Experiments were performed using LB medium supplemented with 0 . 075% Tween-20 at a flow rate of 400 µl/hr .",
"Oscillation traces were collected from single mother machine traps using the background subtracted average fluorescence intensity of the entire trap .",
"CellASIC experiments were conducted using B04A plates ( Merck Millipore , Darmstadt Germany ) .",
"Flow rates were varied between 0 . 25 psi – 2 psi .",
"Cells were grown from frozen stock in media at running temperature to stationery phase .",
"Cells were then diluted 1:100 for 2 hr , and loaded on a equilibrated plate at 1:1000 or less to achieve single-cell loading efficiencies per chamber .",
"To vary cellular doubling times , different growth media were used: LB ( BD Biosciences ) , M9CA ( Sigma Aldrich ) with 0 . 2% glucose , 2xYT ( MP Bio ) , MOPS EZ Rich ( Teknova ) .",
"Cells were imaged in time series every 10-–20 min using a 100x phase objective minimizing both lamp intensity ( 12% Xcite 120 , Excelitas Inc . Waltam MA or 1–2% CoolLED pE-2 , Custom interconnected Ltd . , UK ) and exposure times ( <500 ms ) to limit photo-toxicity .",
"Images were processed and stitched ( Preibisch et al . , 2009 ) , if necessary , using Fiji/ImageJ ( Schindelin et al . , 2012 ) .",
"Fluorescence traces of cell populations with synchronized oscillations were extracted from CellASIC movies using background corrected mean fluorescence intensity from the entire field of view .",
"For cells that were not synchronized over the complete field of view , we tracked regions of oscillating sister cells at the edge of the microcolony .",
"We used ImageJ to define polygonal regions around those cells and manually shifted the polygonal region to track the front of growing cells .",
"Periods were determined from fluorescence traces derived from mother machine and CellASIC movies by measuring the time from one oscillation peak to the next peak .",
"Doubling times were estimated by averaging over the doubling times of at least ten individual cells .",
"We consider an n-node negative cyclic feedback biocircuit and denote the genes , mRNAs and proteins by G1 , G2 , … , Gn , and M1 , M2 , … , Mn and P1 , P2 , … , Pn , respectively .",
"Let ri ( t ) and pi ( t ) denote the concentrations of mRNA Mi and protein Pi , respectively .",
"For example , the novel 3-node ring oscillator in Figure 3B is defined by n=3 , r1 ( t ) = [BetI mRNA] , r2 ( t ) = [PhlF mRNA] , r3 ( t ) = [SrpR mRNA] , p1 ( t ) = [BetI protein] , p2 ( t ) = [PhlF protein] , p3 ( t ) = [SrpR protein] .",
"Our mathematical model considers transcription , translation and degradation of mRNA and protein molecules as summarized in Table 1 , where ai and bi represent the degradation rates of Mi and Pi , respectively , and ci and βi are the translation and transcription rates .",
"The constants Ki-1 and νi are the Michaelis-Menten constant and the Hill coefficient associated with the protein Pi-1 and the corresponding promoter on gene Gi .",
"We hereafter use subscripts 0 and n + 1 as the substitutes of n and 1 , respectively , to avoid notational clutter . 10 . 7554/eLife . 09771 . 030Table 1 . Stoichiometry and reaction rates . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 030DescriptionReactionReaction rateTranscription of Mi Gi + Pi−1→Gi + Pi−1 + MiβiKi−1viKi−1vi + pi−1viTranslation of Mi Mi→Mi + Pici ri Degradation of Mi Mi→∅ai ri Degradation of Pi Pi→∅bi pi Using the law of mass action and the quasi-steady state approximation , the dynamics of the mRNA and protein concentrations can be modeled by the following ordinary differential equations ( ODE ) ( 6 ) rι ( t ) =− ( ai+μ ) ri ( t ) +βigKi−1viKi−1vi+pi−1vi ( t ) , pι ( t ) =− ( bi+μ ) pi ( t ) +ciri ( t ) , where i = 1 , 2 , … , n , and g is the concentration of the circuit plasmid .",
"The constant µ is the dilution rate of mRNA and proteins by the microfluidic device .",
"The dilution time of the microfluidic device is defined by ( 7 ) Td :=ln ( 2 ) μ The ODE model ( Equation 6 ) was numerically simulated using ode45 solver of MATLAB R2013b to obtain qualitative insight into the period as well as the oscillatory parameter regime ( Figure 3F and Figure 2—figure supplement 2 ) .",
"The parameters summarized in Table 2 were used for the simulations . 10 . 7554/eLife . 09771 . 031Table 2 . Parameters used for simulations . DOI: http://dx . doi . org/10 . 7554/eLife . 09771 . 031DescriptionParameter valueai Degradation rate of mRNAs ( min-1 ) ln ( 2 ) /8 ( half-life time: 8 min ) bi Degradation rate of proteins ( min-1 ) ln ( 2 ) /90 ( half-life time: 90 min ) βi Transcription rate ( nM ⋅· min-1 ⋅· plasmid concentration-1 ) 0 . 4ci Translation rate ( nM ·min-1 · mRNA concentration-1 ) 0 . 5Ki Michaelis-Menten constant ( nM ) 5 . 0νi Hill-coefficient2 . 0 The plasmid concentration g was set as g = 5 . 0 nM for Figure 3F .",
"The initial concentrations for the simulations were r1 ( 0 ) =30 , p1 ( 0 ) =0 and ri ( 0 ) = pi ( 0 ) = 0 for i = 2 , 3 , . . . , n .",
"The period of oscillations was calculated based on the autocorrelation of the simulated protein concentration p1 ( t ) .",
"More specifically , let ( 8 ) R ( τ ) :=∫T1T2 p1 ( t+τ ) p1 ( t ) dt where T1 is a positive constant such that p1 ( t ) is steady state at t = T1 , and T2 is a sufficiently large constant compared to the period of oscillations .",
"The period of oscillations Tperiod was determined by Tperiod = minτ>0 argmaxτR ( τ ) .",
"The simulation result is also consistent with the analytic estimation of the oscillation period in Hori et al . ( Hori et al . , 2013 ) in that the period increases monotonically with the dilution time Td .",
"The parameter region for oscillations ( Figure 2—figure supplement 1 ) was obtained based on the analysis result ( Theorem 3 ) by Hori et al . ( Hori et al . , 2011 ) .",
"Since parameter values do not depend on the subscript i as shown in the parameters table above , we remove the subscript i and define a : = a1 ( = a2 = … an ) .",
"In the same way , we define b , c , β , K and ν .",
"It was shown that the protein concentrations pi ( i = 1 , 2 , … , n ) oscillate if both of the following inequalities are satisfied ( Hori et al . , 2011 ) .",
"( 9 ) ν>W ( n , Q ) , ( 10 ) cβ> ( W ( n , Q ) ν−W ( n , Q ) ) 1ν ( νν−W ( n , Q ) ) K ( a + d ) ( b + d ) , where W ( n , Q ) :=2 ( −cos ( πn ) +cos2 ( πn ) +Q2sin2 ( πn ) ) Q2sin2 ( πn ) and Q := ( a+d ) ( b+d ) ( a+b+2d ) /2 .",
"To obtain the parameter region in Figure 2—figure supplement 1 , we substituted n =3 and the parameters shown in the table above into the right-hand side of the inequality condition ( Equation 10 ) , then we varied Td ( = ln ( 2 ) /µ ) between 5 to 80 .",
"The inequality ( Equation 9 ) was always satisfied for these parameters .",
"The parameter region of Figure 2—figure supplement 3 was obtained by the local stability analysis of the model ( Equation 6 ) .",
"The previous theoretical result ( Hori et al . , 2011 ) showed that the model ( Equation 6 ) has a unique equilibrium point and the protein concentrations pi ( i = 1 , 2 , … , n ) show stable oscillations if the Jacobian matrix evaluated at the equilibrium point has an eigenvalue in the open right-half complex plane .",
"Based on this result , we computed the Jacobian eigenvalues with varying K3 , which we denote by KcI , and Td .",
"The values in the parameters table above were used for the other parameters .",
"The plasmid concentration was set as g = 5 . 0 nM in the computation ."
]
] | [
"While complex dynamic biological networks control gene expression in all living organisms , the forward engineering of comparable synthetic networks remains challenging .",
"The current paradigm of characterizing synthetic networks in cells results in lengthy design-build-test cycles , minimal data collection , and poor quantitative characterization .",
"Cell-free systems are appealing alternative environments , but it remains questionable whether biological networks behave similarly in cell-free systems and in cells .",
"We characterized in a cell-free system the ‘repressilator’ , a three-node synthetic oscillator .",
"We then engineered novel three , four , and five-gene ring architectures , from characterization of circuit components to rapid analysis of complete networks .",
"When implemented in cells , our novel 3-node networks produced population-wide oscillations and 95% of 5-node oscillator cells oscillated for up to 72 hr .",
"Oscillation periods in cells matched the cell-free system results for all networks tested .",
"An alternate forward engineering paradigm using cell-free systems can thus accurately capture cellular behavior ."
] | [
"Engineers often use simplified models to test their ideas .",
"For example , engineers test small-scale models of new airplane designs in wind tunnels to see how easily air flows by them .",
"This saves the engineers the time and expense of building a full-sized aircraft only to learn it has serious design flaws .",
"The interactions of genes and proteins within living cells can be incredibly complex , and working out how a particular network works can take months or years in living cells .",
"To try to speed up and simplify the process , scientists are developing models that do not involve cells .",
"These models replicate the chemistry inside of the cells and allow scientists to observe complex interactions between genes , proteins and other cellular components .",
"Some scientists have recreated complex patterns of gene expression in these cell-free models , but these systems still take a long time to make .",
"It is also not yet clear whether these models accurately depict what happens in living cells .",
"Now , Niederholtmeyer , Sun et al . have created a cell-free system that allows the interactions of a large network of genes to be examined in a single day – a process that would previously have taken weeks or months .",
"To test the model , Niederholtmeyer , Sun et al . recreated how networks of genes in the bacterium Escherichia coli interact to form “oscillations” , which produce a regular rhythm of gene expression .",
"When the cell-free oscillator networks were inserted into live E . coli cells , the oscillators continued to produce the same patterns of gene expression as they did outside the cells .",
"Overall , the experiments show that cell-free models can accurately reproduce , or emulate , the behavior of cellular networks .",
"This work now opens the door for engineering ever more complex genetic networks in a cell-free system , which in turn will enable rapid prototyping and detailed characterization of complex biological reaction networks ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"structural biology and molecular biophysics"
] | Crystal structure and dynamics of a lipid-induced potential desensitized-state of a pentameric ligand-gated channel | elife-23886-v3 | [
[
"Fast synaptic transmission throughout the central and peripheral nervous system is mediated by pentameric ligand-gated ion channels ( pLGICs ) , also referred to as Cys-loop receptors .",
"The vertebrate channels of this superfamily include both inhibitory anion-selective channels ( γ-aminobutyric acid receptors- GABAAR and glycine receptors- GlyR ) and excitatory cation-selective channels ( nicotinic acetylcholine receptors- nAChR and serotonin receptors- 5HT3AR ) .",
"The binding of a neurotransmitter initiates a cascade of protein motions that lead to transitions between resting , open , and desensitized conformations ( Unwin and Fujiyoshi , 2012; Du et al . , 2015; Sauguet et al . , 2014; Althoff et al . , 2014 ) ( Figure 1A ) .",
"Ionic fluxes , and hence the post-synaptic responses , are critically governed by the transition rates and equilibrium populations of these functional states .",
"Aberration of these molecular events underlies many neurological disorders and therefore , pLGICs are therapeutic targets for treating these conditions .",
"To develop a molecular understanding of the pLGIC gating mechanism and its modulation requires high-resolution structures of the channel in multiple functional states .",
"While there has been ground-breaking progress in determining the structures of several members of the pLGIC family , from both prokaryotic and eukaryotic origin , an unequivocal assignment of functional states to these conformations has not been achieved ( Unwin and Fujiyoshi , 2012; Du et al . , 2015; Hilf and Dutzler , 2009 , 2008; Sauguet et al . , 2013; Bocquet et al . , 2009; Miller and Aricescu , 2014; Hassaine et al . , 2014; Hibbs and Gouaux , 2011 ) .",
"Structural mechanisms underlying channel opening ( Du et al . , 2015; Althoff et al . , 2014; Sauguet et al . , 2013; Velisetty et al . , 2012 ) and desensitization have been areas of extensive investigation ( Du et al . , 2015; Miller and Aricescu , 2014; Morales-Perez et al . , 2016 ) . 10 . 7554/eLife . 23886 . 003Figure 1 . DHA modulation of GLIC function .",
"( A ) A minimal gating scheme showing three fundamental conformational states that constitute pLGIC function: a resting state [C] , a transient open-state [O] , and a desensitized state [D] .",
"Agonist-binding shifts the equilibrium towards the high-affinity D state such that under steady-state conditions , the channels are predominantly in the desensitized state .",
"Allosteric modulators exert their effect by altering the transition , and hence the equilibrium , between the three states .",
"( B ) The trace shows a continuous recording of GLIC currents in oocytes measured by two electrode voltage-clamp ( TEVC ) in response to multiple pH-4 . 5 pulses .",
"The pH-pulses were interspaced by perfusion with the pH 7 . 4 solution ( for deactivation and recovery ) .",
"Currents were measured in the absence ( marked by red lines ) or presence of 50 μM DHA ( marked by red and blue lines ) .",
"The baseline is marked as a dotted black line .",
"DHA inhibits GLIC currents by increasing desensitization ( faster current decay and lower steady-state currents; highlighted by the vertical blue arrow and dotted blue/red lines ) .",
"The effect of DHA on the current decay was fully reversible , as seen in the second and fourth pH-pulses .",
"( C ) A plot of the ratio of steady-state ( measured at t = 2 . 2 min ) over the peak current amplitude for the two conditions ( n = 12 ) with s . d shown as error bars . DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 00310 . 7554/eLife . 23886 . 004Figure 1—figure supplement 1 . Effect of DHA on GLIC desensitization .",
"( A ) The effect of DHA at various concentrations ( left ) on currents at pH 4 . 5 and the effect of 50 μM DHA on currents elicited by various extracellular pH ( right ) .",
"The currents were recorded by TEVC at a holding potential of −60 mV .",
"In each case , a ratio of the steady-state current ( measured at t = 4 . 5 min ) to the peak amplitude was plotted .",
"The error bars denote s . d ( n = 6 ) ( B ) Normalized peak amplitudes in the presence ( blue ) and absence ( red ) of 50 μM DHA plotted as a function of pH , and the data were fitted with the Hill equation to yield pH504 . 87 ± 0 . 04 and nH1 . 6 ± 0 . 2 in the absence of DHA; pH505 . 02 ± 0 . 03 and nH1 . 9 ± 0 . 3 in the presence of DHA .",
"The error bars denote s . d ( n = 3 ) ( C ) Outward currents recorded at pH 4 . 5 and +60 mV holding potential in the presence and absence of 50 μM DHA .",
"The dashed lines and arrow mark the level of steady-state currents under the two conditions . DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 00410 . 7554/eLife . 23886 . 005Figure 1—figure supplement 2 . Effect of DHA-pre-application on GLIC currents . Representative pH-elicited GLIC-currents recorded by TEVC at −60 mV membrane potential in response to pre-application of 50 μM DHA at pH 7 . 4 ( 2 . 2 min duration ) ( middle trace ) and compared with currents recorded without DHA pre-application ( first and third pulses ) .",
"All the three pH 4 . 5-pulses had 50 μM DHA .",
"The ratio of the peak-current amplitudes measured with and without pre-application of DHA was 0 . 64 ± 0 . 11 ( n = 5 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 005 Desensitization regulates the frequency and amplitude of synaptic response during fast , repetitive stimulation and is implicated to play a role in the synaptic plasticity of neural networks associated with learning , memory , and attention ( Giniatullin et al . , 2005; Elenes et al . , 2006; Jones and Westbrook , 1996 ) .",
"Mutations in nAChR , GABAAR , and GlyR that lead to altered desensitization kinetics have been associated with congenital myasthenic syndrome , frontal lobe epilepsy , and human startle disease ( Bertrand et al . , 2002; Matsushima et al . , 2002; De Fusco et al . , 2000; Sine et al . , 2002; Saul et al . , 1999; Bowser et al . , 2002 ) .",
"Desensitization is modulated by a range of endogenous and exogenous factors , including Ca2+ , membrane lipids ( cholesterol , anionic lipids , and polyunsaturated fatty acids ) , neurosteroids , alcohols , and anesthetics ( Arias , 1998; Fenster et al . , 1999; Huganir and Greengard , 1990 ) .",
"In particular , positive allosteric modulators of nAChR , which slow desensitization , are being developed for the treatment of Alzheimer’s disease , schizophrenia , depression , and pain ( Gopalakrishnan et al . , 2007; Donnelly-Roberts et al . , 2011; Hurst et al . , 2005; Young et al . , 2008 ) .",
"Strategies targeting desensitization and allosteric modulation are being considered as a means to design safer therapeutics that have fewer side effects ( Buccafusco et al . , 2009 ) .",
"It is now well established that membrane-lipid constituents modulate gating transitions in many pLGIC members ( Borroni et al . , 2016; Baenziger and Corringer , 2011; Rankin et al . , 1997; Sunshine and McNamee , 1992; Heidmann et al . , 1980 ) .",
"In addition , they regulate the allosteric effects of alcohols , anesthetics , neurosteroids , and free fatty acids .",
"These molecules partition into the lipid bilayer and affect channel properties by either interacting directly with the channel or by altering the interaction of the channel with surrounding lipids ( Howard et al . , 2014; Changeux , 2012; Campagna et al . , 2003; Mihic et al . , 1997 ) .",
"In particular , docosahexaenoic acid ( DHA , an ω−3 polyunsaturated fatty acid with 22 carbons and six double bonds ) has previously been reported to modulate nAChR and GABAAR function by increasing the rate and extent of desensitization ( Hamano et al . , 1996; Nabekura et al . , 1998; Bouzat and Barrantes , 1993; Witt et al . , 1999 ) .",
"DHA is a major polyunsaturated fatty acid ( PUFA ) in the brain and is found in high concentrations ( up to 50 mol% of the total acyl chains of phospholipids ) in synaptic plasma membranes .",
"Reduced levels are linked with impaired learning ability ( Hashimoto et al . , 2016; Janssen and Kiliaan , 2014 ) .",
"DHA is readily esterified and incorporated into membrane phospholipids , altering both the physical properties of the membrane and the expression and function of associated membrane proteins .",
"Free DHA released by intracellular enzymes such as phospholipase A2 and diacylglycerol lipase ( Piomelli and Greengard , 1990 ) also influences membrane protein function ( Moreno et al . , 2012; Boland and Drzewiecki , 2008 ) .",
"While there are numerous reports describing the effects of DHA on several channel types , the mechanism of its action remains elusive ( Bruno et al . , 2007 ) .",
"Mutational analysis and subunit/isoform specific effects in a number of channels have implicated direct interaction with DHA ( Ottosson et al . , 2014; Hoshi et al . , 2013a , 2013b ) , however evidence for a DHA binding site on these channels is still lacking .",
"Although the prokaryotic pLGIC are significantly less stringent in their need for specific membrane constituents , their function is modulated by lipids in an analogous way ( Labriola et al . , 2013; Velisetty and Chakrapani , 2012 ) .",
"Here , we show in electrophysiological recordings that desensitization in GLIC , a prokaryotic pH-gated pLGIC , is enhanced in the presence of DHA .",
"GLIC has previously been crystallized in its resting ( closed ) and putative open states ( Sauguet et al . , 2014; Hilf and Dutzler , 2009; Bocquet et al . , 2009 ) , however , the desensitized state of the channel has been structurally elusive .",
"Co-crystals of GLIC in the presence of DHA allowed us to successfully stabilize the channel in a novel state that is likely to be a desensitized conformation .",
"The structure reveals a DHA molecule bound at the channel periphery , close to the M4 segment , and interacting with Arg118 in the Cys-loop ( β6-β7 loop ) through a salt-bridge .",
"Both the M4 segment ( also referred to as the ‘lipid-sensor’ in nAChR ) and the Cys-loop are implicated in transducing agonist-induced conformational changes from across the extracellular domain ( ECD ) to the transmembrane domain ( TMD ) .",
"Mutations in the ECD-TMD interfacial region have been shown to affect desensitization ( Bouzat et al . , 2008 ) .",
"The most striking feature of our structure is the new pore conformation , which provides a molecular view of how ion permeation could potentially be occluded in a desensitized state ."
],
[
"To test the effect of DHA on GLIC function , we expressed GLIC in Xenopus laevis oocytes and measured currents by two-electrode voltage-clamp ( TEVC ) techniques ( Materials and methods ) .",
"As previously shown , GLIC is activated by extracellular protons ( pH 4 . 5 ) ( Hilf and Dutzler , 2009; Bocquet et al . , 2007 ) and the currents display a slow decay as the channels desensitize ( Figure 1B ) .",
"When DHA ( 50 μM ) was co-applied with pH 4 . 5 , the macroscopic decay from the peak was accelerated , leading to much smaller steady-state currents ( Figure 1B , blue arrow ) .",
"Upon deactivation at pH 7 . 0 , subsequent pH change to 4 . 5 resulted in currents with peak amplitudes and decay phases indistinguishable from the first pulse , revealing that the effect of DHA was fully reversible .",
"In addition to the effect on current decay , DHA decreases the amount of steady-state current ( measured at 2 . 2 min from the start of application ) as shown in the plot of the steady-state-to-peak ratio , suggesting that both the rate and the extent of desensitization are increased ( Figure 1C ) .",
"The Figure 1—figure supplement 1A shows a detailed analysis of the effect of DHA at different concentrations and at various activating pH . The effect on desensitization was observed at DHA concentrations above 5 μM , and was more pronounced at higher proton concentrations , suggesting that channel activation promotes the effect of DHA .",
"Additionally , in the presence of DHA , a small left-shift in pH-response is observed for GLIC ( Figure 1—figure supplement 1B ) .",
"These findings are in fact expected for a modulator that promotes desensitization ( the conformational state with the highest agonist-affinity ) .",
"Further , outward current decay was also accelerated in the presence of DHA ( Figure 1—figure supplement 1C ) , similar to the effect on inward currents .",
"Upon pre-application of DHA ( at pH 7 . 4 ) prior to co-application at pH 4 . 5 , additional decrease in peak amplitudes is observed , suggesting that enhanced availability of DHA could result in larger effects on GLIC currents ( Figure 1—figure supplement 2 ) .",
"The effects of DHA were fully reversible in all of the measured conditions .",
"To further confirm that DHA indeed promotes an agonist-induced desensitized state rather than a pre-open resting state , we studied the effect of DHA on an alanine mutation at the Ile9’ position in M2 .",
"Mutation at the equivalent position in several pLGIC has been shown to increase agonist sensitivity and slow desensitization ( Bocquet et al . , 2007; Labarca et al . , 1995; Filatov and White , 1995; Akabas et al . , 1992; Yakel et al . , 1993; Chang et al . , 1996; Revah et al . , 1991 ) .",
"The prediction is that perturbations that destabilize the desensitized state should lower the effect of DHA .",
"The I9′A mutant exhibits a gain-of-function phenotype ( Bocquet et al . , 2007; Parikh et al . , 2011; Gonzalez-Gutierrez et al . , 2013 ) resulting in leaky oocytes , an effect that can be offset with a background mutation ( H11′F [Wang et al . , 2012; Rienzo et al . , 2014] ) that reduces pH-sensitivity ( Schmandt et al . , 2015 ) .",
"We found that the double-mutant ( I9′A/H11′F ) shows robust non-desensitizing currents with a pH50 5 . 17 ± 0 . 19 ( Figure 2A ) .",
"Quite remarkably , DHA had no effect on this mutant over a range of pH conditions , and even up to a 100 μM concentration ( Figure 2B and C , and Figure 2—figure supplement 1 ) .",
"Since the mutant’s pH response is close to wt , the DHA effect cannot be explained by stabilization of the resting or pre-open states .",
"We would like to point out that the technical limitations of TEVC , which include slower perfusion rates , preclude us from resolving fast kinetic components of desensitization .",
"We therefore , at this point , cannot ascertain which of the multiple desensitized states that DHA stabilizes .",
"Nevertheless , above findings demonstrate that transitions to the desensitized state are necessary for the DHA effect , and that DHA may stabilize a desensitized conformation induced by the agonist during gating . 10 . 7554/eLife . 23886 . 006Figure 2 . DHA has no effect on the non-desensitizing GLIC I9′A/H11′F mutant .",
"( A ) Normalized pH-response for GLIC-wt ( red ) and GLIC I9′A/H11′F double mutant ( orange ) at −60 mV .",
"The error bars denote s . d and the curve is a fit to the Hill equation .",
"GLIC-wt ( pH50 4 . 87 ± 0 . 04 and nH 1 . 6 ± 0 . 2; n = 3 ) and GLIC I9′A/H11′F ( pH50 5 . 17 ± 0 . 19 and nH 0 . 88 ± 0 . 29; n = 7 ) .",
"( B ) Macroscopic currents measured by TEVC for GLIC I9′A/H11′F in response to pH jumps ( from 7 . 4 to 4 . 5 ) , at −60 mV holding potential , in the presence or absence of 50 μM DHA .",
"( C ) Currents recorded at pH 4 . 5 in the presence of either 50 μM DHA or 100 μM DHA . DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 00610 . 7554/eLife . 23886 . 007Figure 2—figure supplement 1 . Lack of DHA effect on the non-desensitizing GLIC I9′A/H11′F mutant . Macroscopic currents measured by TEVC for GLIC I9′A/H11′F in response to pH jumps ( from 7 . 4 to the indicated pH value ) , at −60 mV holding potential , in the presence of either 50 μM DHA or 100 μM DHA . DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 007 To better understand the mechanism of DHA action and to attempt trapping GLIC in a desensitized conformation , co-crystals of GLIC were grown in the presence of 50 μM of DHA under acidic conditions , similar to those previously reported for GLIC wt ( Hilf and Dutzler , 2009 ) .",
"The crystals diffracted up to 3 . 25 Å and the structure was solved using GLIC-pH4 ( PDB ID: 4HFI ) as the starting model ( Sauguet et al . , 2013 ) .",
"Statistics for data collection and refinement are summarized in Table 1 .",
"Well-defined electron density was found for DHA in all the subunits in a pocket at the channel periphery; lined by M4 , the M2-M3 linker , and the β6-β7 loop ( Figure 3A , also see Figure 3—figure supplement 1 ) .",
"The DHA molecule appears bent with a twisted/curled orientation , and makes a salt-bridge interaction with an arginine sidechain ( Arg118 ) in the β6-β7 loop .",
"Besides this interaction , DHA does not appear to engage with the rest of the protein .",
"However , it must be noted that the density for the DHA tail is not resolved beyond C13 , and therefore was not built in the model .",
"Considering the high degree of conformational flexibility in the polyunsaturated aliphatic tail of DHA , it is not surprising that this region is less well defined .",
"We therefore cannot exclude additional interactions of the protein with the flexible fatty acid tail .",
"Several residues in the vicinity of the DHA binding site ( in the β6−β7 loop , the M2-M3 linker , M1 , M3 , and M4 ) show small changes in rotameric orientation ( Figure 3—figure supplement 2 ) , although the overall conformation of these regions is similar to the GLIC-pH4 structure .",
"In the GLIC-pH4 structure , the Arg118 side-chain lines a phospholipid-binding pocket within the intra-subunit cavity formed by M1 , M3 , and M4 ( Sauguet et al . , 2013 ) .",
"In comparison , the density for the lipid molecule ( PLC ) in GLIC-pH4-DHA is well-defined in only one subunit and the head-group of this lipid appears to be reoriented in comparison to its position in GLIC-pH4 structure ( Figure 3B and Figure 3—figure supplement 2 ) . 10 . 7554/eLife . 23886 . 008Figure 3 . DHA binding site in GLIC .",
"( A ) A side-view of the GLIC-pH4-DHA structure at pH 4 . 0 solved to 3 . 25 Å resolution with a bound DHA molecule shown in stick representation .",
"Only one subunit is colored for clarity ( The TM helices are colored as: M1-blue , M2-green , M3-cyan , and M4-wheat ) .",
"The 2Fo-Fc electron density map for DHA , contoured at 1 . 0 σ-level , is shown as a blue mesh .",
"The phospholipid molecule ( PLC ) , shown in sticks , was also present in previously reported GLIC structures at acidic pH . ( B ) The chemical structure of the DHA molecule ( top ) and zoomed-in views of the region marked by the inset in panel A ( bottom ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 00810 . 7554/eLife . 23886 . 009Figure 3—figure supplement 1 . The DHA binding site . The GLIC-pentamer viewed from the extracellular side , overlaid with the Fo-Fc ‘omit’ electron density map generated by excluding DHA molecules from structure factor calculations .",
"The green mesh is contoured at 2 . 0 σ and DHA molecules are drawn as ball-and-sticks .",
"Prominent electron density is visible for all the five subunits although the continuity was variable among subunits .",
"The lipid molecule ( PLC ) is also shown in a ball-and-stick presentation .",
"Clear density for the bound PLC was observed at only one subunit . DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 00910 . 7554/eLife . 23886 . 010Figure 3—figure supplement 2 . Reorientation of lipid molecule . An alignment of GLIC-pH4 ( PDB ID: 4HFI ) ( Sauguet et al . , 2013 ) and GLIC-pH4-DHA structures shows that the lipid molecule ( PLC ) bound close to M4 is reoriented .",
"( A ) A side view of the DHA binding site is shown .",
"( B ) A top view of the DHA binding pocket .",
"Small changes in side-chain orientation are seen for residues in the β6−β7 loop ( Arg117 , Arg118 , and Phe121 ) , the M2-M3 linker ( Met252 ) , M3 ( Phe260 ) , M4 ( Phe315 ) , and M1 from the adjacent subunit ( Phe195 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 01010 . 7554/eLife . 23886 . 011Figure 3—figure supplement 3 . R118A mutation reduces the effect of DHA on desensitization . Sequence for the β6−β7 loop is shown , and the position Arg118 is highlighted in red .",
"Typical GLIC-R118A currents measured by TEVC in response to pH jumps ( from 7 . 4 to 4 . 5 ) , at −60 mV holding potential , in the presence or absence of 50 μM DHA ( left ) .",
"Ratio of the steady-state current ( measured at 2 . 2 min ) to the peak amplitude was plotted ( n = 12 for GLIC wt and n = 7 for R118A ) with s . d shown as error bars ( right ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 01110 . 7554/eLife . 23886 . 012Table 1 . Data collection and refinement and statistics of GLIC . DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 012Data collectionBeamlineNE-CAT 24-ID-C/EWavelength0 . 97870Space groupC121Cell dimensions a , b , c , ( Å ) ; β ( ° ) 181 . 87 , 133 . 32 , 159 . 92; 102 . 36No .",
"of observations198167No .",
"of unique observations64340Resolution range ( Å ) 61 . 31–3 . 25 ( 3 . 34–3 . 25 ) CC1/2 = 0 . 3 ( Å ) *3 . 25Mean I/σ ( I ) 6 . 7 ( 1 . 4 ) Rpim†0 . 057 ( 0 . 439 ) Completeness ( % ) 96 . 7 ( 98 . 6 ) Multiplicity2 . 8 ( 2 . 8 ) Average Mosaicity0 . 49RefinementResolution ( Å ) 30 . 0–3 . 25Rwork ( % ) 23 . 15Rfree‡ ( % ) 26 . 11B-factor ( Å2 ) Protein102 . 55R . M .",
"S deviations:Bond lengths ( Å ) 0 . 006Bond angles ( ° ) 1 . 45Molprobity Score98th percentileRamachandran Analysis§Favored88 . 15%Allowed11 . 32%Generously Allowed all Allowed80 . 53% ( 8 residues ) *CC1/2 is the Pearson correlation coefficient of two-half data sets ( Karplus et al . , 2012 ) .",
"†Rpim ( all I+/I- ) .",
"‡5 . 0% of reflections were excluded from refinement for calculation of Rfree .",
"§Calculated using PROCHECK ( Laskowski et al . , 1993 ) .",
"To determine if the interaction of DHA with Arg118 is necessary for the observed effect on channel gating , we probed the functional consequence of mutating Arg118 to Ala , and studied the effect of DHA on R118A desensitization .",
"The R118A mutant showed robust pH-induced currents in oocytes , however in comparison to GLIC wt , the current decay was much less affected by 50 μM DHA ( Figure 3—figure supplement 3 ) .",
"This finding thereby validates the crystallographically-captured DHA-binding site , and further implicates a novel role for the Arg118 in lipid-channel interactions .",
"The most prominent difference noted in the GLIC-pH4-DHA structure in comparison to other GLIC structures ( at pH 7 . 0 and pH 4 . 0 ) , is at the level of M2 lining the channel pore ( Figure 4 ) .",
"The conformation of the GLIC-pH4-DHA pore does not align with either the resting ( GLIC-pH7 ) ( Sauguet et al . , 2014 ) or the putative open state ( GLIC-pH4 ) ( Sauguet et al . , 2013 ) GLIC structures ( Figure 4—figure supplements 1 and 2 ) .",
"In this new M2 conformation , the pore is funnel-shaped with the hydrophobic extracellular-end ( Ala13′ to Thr20′ ) wide-open to a pore radius greater than 5 Å , reminiscent of the GLIC-pH4 structure , while the polar intracellular-half ( between Ile9′ and Thr2′ ) is constricted to 2 . 5 Å , resembling the GLIC-pH7 closed structure ( Figure 4A and Figure 4—figure supplement 4A ) .",
"The pore radius at the Glu-2’ position was essentially the same as seen in GLIC-pH7 and GLIC-pH4 .",
"The Fo-Fc omit map for the M2/M2-M3 linker indicates that the GLIC-pH4-DHA structure is not a mixture of closed and open conformations in the crystal ( Sauguet et al . , 2014 ) ( Figure 4—figure supplement 3 ) .",
"Further , the observed M2 conformation in GLIC-pH4-DHA is also different from the previously reported locally-closed GLIC structures at pH 4 . 0 ( Prevost et al . , 2012 ) ( Figure 4—figure supplement 4B ) , and therefore represents a unique pore conformation . 10 . 7554/eLife . 23886 . 013Figure 4 . Conformational changes in the GLIC pore .",
"( A ) The ion-permeation pathway through the channel pore , as determined by the MOLE PyMOL plugin ( Petrek et al . , 2007 ) .",
"The M2 from two subunits are shown using a ribbon representation , with residues lining the pore represented as sticks ( left ) .",
"Pore radius along the channel axis in GLIC structures at pH 7 . 0 ( PDB ID: 4NPQ ) , pH 4 . 0 ( PDB ID: 4HFI ) , and at pH 4 . 0 in the presence of DHA calculated using HOLE software ( Petrek et al . , 2007; Smart et al . , 1996 ) ( right ) .",
"The constricted region from Ile9′ to Thr2′ is highlighted by a grey box .",
"( B ) Fo-Fc omit electron density of six dodecyl-maltoside molecules ( shown in stick representation ) and water pentagon ( shown as red spheres ) at 3 . 0 σ-level in M2 for different GLIC structures .",
"The PDB ID for the structures are: GLIC-pH7: 4NPQ; GLIC-pH4: 4HFI; GLIC-pH4 ( lower resolution ) : 3UU8 .",
"The resolution for each of the structures is indicated below . DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 01310 . 7554/eLife . 23886 . 014Figure 4—figure supplement 1 . M2 conformation in the GLIC-pH4-DHA structure . Fo-Fc ‘omit’ electron density map ( green mesh , contoured at 2 . 3 σ ) for the M2 and M2-M3 linker ( 228-255 ) of the GLIC-pH4-DHA structure ( shown in stick representation ) .",
"Two non-adjacent opposed subunits are shown for clarity from the side and top views . DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 01410 . 7554/eLife . 23886 . 015Figure 4—figure supplement 2 . Conformational changes in the GLIC-pH4-DHA structure . The 2Fo-Fc electron density map for the GLIC-pH4-DHA structure contoured at 1 . 5 σ-level and shown for two non-adjacent subunits .",
"( A ) Views of M2 and the M2-M3 linker parallel to the membrane ( left ) and from the extracellular end of the membrane ( right ) for the GLIC-pH4-DHA structure .",
"( B ) The GLIC-pH4-DHA structure aligned with the GLIC-pH4 structure ( PDB ID: 4HFI ) ( Sauguet et al . , 2013 ) .",
"( C ) The GLIC-pH4-DHA structure aligned with the GLIC-pH7 structure ( PDB ID: 4NPQ ) ( Sauguet et al . , 2014 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 01510 . 7554/eLife . 23886 . 016Figure 4—figure supplement 3 . Comparison of Fo-Fc omit maps for the GLIC-pH4-DHA with that of the GLIC-His10-pH4 structure . Fo-Fc ‘omit’ electron density map ( green mesh , contoured at 2 . 4 σ ) for the M2 and M2-M3 linker ( 228-255 ) of the GLIC-pH4-DHA structure ( blue , ribbon representation ) overlaid with the Fo-Fc map ( grey mesh , contoured at 2 . 4 σ ) for the M2 and M2-M3 linker ( 228-255 ) of the GLIC-His10-pH4 structure ( PDB ID: 4NPP ) ( Sauguet et al . , 2014 ) containing a mixture of locally-closed and open conformations .",
"Magenta , ribbon representation shows the locally-closed conformation in 4NPP .",
"Two non-adjacent subunits are shown for clarity from the side and top views . DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 01610 . 7554/eLife . 23886 . 017Figure 4—figure supplement 4 . Pore conformations in GLIC structures .",
"( A ) Cα-Cα distance for the residues lining the narrow constrictions in the GLIC-pH7 , GLIC-pH4 , and GLIC-pH4-DHA structures ( B ) Pore radius calculated using HOLE ( Smart et al . , 1996 ) for GLIC-pH7 ( PDB ID: 4NPQ , green ) , GLIC-pH4 ( PDB ID: 4HFI , orange ) , GLIC-pH4-DHA ( blue ) , and various locally-closed GLIC structures ( PDB ID: 3TLS , purple; PDB ID: 3TLT , wine; PDB ID: 3TLV , pink ) ( Prevost et al . , 2012 ) .",
"Residues Leu22′- Ala13′ form narrow constrictions at the extracellular activation gate .",
"The Ile9′ position and the residues below , through the intracellular end , contribute to the desensitization gate ( s ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 017 The reduction in the GLIC-pH4-DHA pore radius is accompanied by notable changes in the occupancy of detergent molecules , water , and ions within the pore .",
"While the electron density was observed for the bundle of six dodecyl-maltoside molecules in the upper M2 , they were more disordered in comparison to the GLIC-pH4 structures ( Figure 4B ) .",
"The reduced detergent occupancy is expected , due to the effect of pore constriction at Ile9′ .",
"Additionally , the channel pore in the GLIC-pH4 structure ( PDB ID: 4HFI ) reveals a cation binding site ( at the level of Thr2′ coordinated by water molecules beneath it ) and an ordered pentagonal-ring of water molecules that are in-plane with the γ-O atoms of Ser6’ ( Sauguet et al . , 2013 ) ( Figure 4B ) .",
"These densities were also noted in lower-resolution structures ( comparable in resolution to GLIC-pH4-DHA ) , although they appeared more diffuse ( Hilf and Dutzler , 2009; Bocquet et al . , 2009 ) .",
"In contrast , the GLIC-pH4-DHA structure shows a distinctly different hydration profile at the intracellular half of the pore , with loss of densities for water and ions .",
"Interestingly , the orientation of Ser6 sidechains is implicated in directly influencing the organization of the water ring at this position ( Sauguet et al . , 2013 ) .",
"A small change at this position in GLIC-pH4-DHA brought about by the compression at Ile9’ may thus be responsible for the loss of ions and water .",
"With the exception of changes in M2 , GLIC-pH4-DHA adopts a conformation almost identical to GLIC-pH4 ( RMSD for alignment of ECD pentamer ( residues 5–191 ) with 4HFI is 0 . 34 Å and that for the TMD ( residues 192–315 ) is 0 . 49 Å ) .",
"Surprisingly , DHA had minimal effect on the protein conformation in and around M4 .",
"Similarly , crystal structures of GLIC at neutral and acidic pH also reveal minimal positional differences in M4 ( Figure 5—figure supplement 1 ) .",
"This is somewhat unexpected considering that M4 plays a role in relaying modulatory effects of lipid-protein interactions on to the channel pore , and that M4 perturbations have functional consequences ( Bouzat et al . , 1998; Lasalde et al . , 1996; Lee et al . , 1994; Mitra et al . , 2004 ) .",
"The lack of a structural change could potentially result from the absence of a membrane environment in crystallographic conditions , with the effect being most pronounced on the lipid-exposed M4 segment .",
"Additionally , crystal packing and lattice forces may overwhelm the energetics of conformational equilibrium , thereby masking the effect induced by ligands and mutations ( Gonzalez-Gutierrez et al . , 2012 ) .",
"To better understand the role of M4 in regulating lipid-sensitive gating , it is important to first know how M4 moves during channel activation and desensitization .",
"To probe the conformational changes in M4 during the transition to the desensitized state , we used site-directed spin labeling ( SDSL ) and Continuous-Wave ( CW ) EPR spectroscopic methods in membrane-reconstituted GLIC .",
"Single-cysteine mutations were made along the length of the segment ( ~31 positions ) on a cysteine-less background template .",
"Previous studies have also shown that M4 mutations are well-behaved , with the exception of Pro300 , where no pH-activated currents were observed ( Carswell et al . , 2015; Hénault et al . , 2015 ) Individual cys-mutants were purified , labeled with MTSL , and reconstituted into asolectin membranes for EPR studies .",
"As with any study involving side-chain perturbations , attachment of spin-probes could lead to functional alterations of the channel .",
"We therefore tested the functionality of representative purified cysteine mutants upon spin-labeling and reconstitution by patch-clamp recordings of excised membranes ( Figure 5—figure supplement 2 ) .",
"Although the spin-labeled M4 mutants exhibit pH-activated currents , the current traces show qualitative differences in decay profiles .",
"These differences in desensitization kinetics may arise from cys mutagenesis and/or SDSL .",
"Functional perturbation accompanying SDSL remains a caveat of this approach .",
"However , since EPR measurements are made at steady-state conditions , the changes in ‘faster’ components of kinetics may not significantly impact the overall interpretation .",
"The CW spectral analysis included the determination of two parameters: ( 1 ) ΔH0−1 , which is measured as the inverse of the central linewidth .",
"This lineshape parameter has been routinely utilized to assess changes in mobility .",
"However , in some cases changes in ΔH0−1 may arise from alterations in oxygen accessibility that result in line broadening and may not reflect a change in mobility .",
"Nevertheless , this lineshape parameter is useful to reflect changes occurring among the states .",
"( 2 ) Π , accessibility to either membrane or water measured in the presence of lipid-soluble oxygen ( O2 ) and water-soluble Ni ( II ) ethylenediaminediacetic acid ( NiEDDA ) , respectively .",
"Each of these measurements was made for samples at pH 7 . 0 and at pH 3 . 0 .",
"Since in membranes , exposure to acidic pH activates and subsequently desensitizes GLIC , under steady-state EPR conditions , the channels are expected to be predominantly in their closed conformation at pH 7 . 0 and in their desensitized conformation at pH 3 . 0 ( Velisetty et al . , 2012 ) .",
"An overlay of the EPR line-shapes in the two states for all of the positions studied are shown in Figure 5 and Figure 5—figure supplement 3 . 10 . 7554/eLife . 23886 . 018Figure 5 . pH-dependent conformational changes in M4 for membrane-reconstituted GLIC . Spin-normalized CW-EPR spectra for representative positions along M4 in the closed ( black , pH 7 . 0 ) and desensitized ( red , pH 3 . 0 ) states .",
"Location of spin-labels are shown on the GLIC-pH4 structure ( PDB ID: 4HFI ) .",
"Only two subunits are shown for clarity .",
"The position of Pro300 , which introduces a kink in the helix , is marked by a green star .",
"Positions close to and above Pro300 ( 295-298 ) show an increase in ΔHo−1 in the desensitized state ( marked by magenta balls ) , while positions further below are essentially unchanged in the two conformations ( marked by blue balls ) .",
"Dotted lines marked as ‘i’ ( cyan ) and ‘m’ ( dark blue ) represent the immobile and mobile components of the spectra , respectively .",
"The two components may arise from two different rotameric orientations of the spin-labels and/or from two conformational states of the protein in equilibrium . DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 01810 . 7554/eLife . 23886 . 019Figure 5—figure supplement 1 . Alignment of GLIC transmembrane domains ( TMD ) .",
"A superposition of GLIC-TMDs ( GLIC-pH7 , PDB ID: 4NPQ; GLIC-pH4 , PDB ID: 4HFI; and GLIC-pH4-DHA ) shows minimal conformational differences in the M4 segment . DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 01910 . 7554/eLife . 23886 . 020Figure 5—figure supplement 2 . Functional characterization of spin-labeled M4 mutants by patch-clamp recordings in reconstituted proteoliposomes . Macroscopic current traces from ‘inside-out’ patches of representative spin-labeled M4 mutants reconstituted into asolectin membranes .",
"Currents were recorded in response to fast application of pH-jumps from 7 . 0 to 3 . 0 .",
"Currents were recorded under symmetrical 150 mM Na+ at −50 mV holding membrane potential . DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 02010 . 7554/eLife . 23886 . 021Figure 5—figure supplement 3 . Conformational changes in M4 reported by EPR lineshapes . CW-EPR spectra for the M4 residues in the closed ( pH 7 . 0 , black ) and desensitized ( pH 3 . 0 , red ) states .",
"The residues facing the intrasubunit cavity are highlighted by green asterisks and those facing the membrane are indicated by black circles .",
"Dotted lines marked as ‘i’ and ‘m’ represent the immobile and mobile components of the spectra , respectively .",
"Grey box highlights residues at positions ( i-5 ) to ( i-1 ) with respect to Pro300 ( i = 0 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 021 In the closed state , the spectra at positions lining the TMD-facing interface of M4 ( Figure 5 , and marked by green asterisks in Figure 5—figure supplement 3 ) are broad in comparison to the spectra at positions along the membrane-facing M4 interface ( marked by black circles in Figure 5—figure supplement 3 ) , suggesting that residues on the TMD-facing interface are packed in a sterically constrained environment .",
"In the desensitized conformation , spectra at the intracellular end ( Pro285-Ala294 ) show minimal changes in lineshape .",
"However , there are dramatic changes in lineshapes for residues above Ala294 that are reflective of an increase in mobility ( particularly Ser295 , Arg296 , Ile297 and Ala298 , grey box in Figure 5—figure supplement 3; compare the dark blue and cyan dotted lines that indicate the mobile and immobile components of the spectra , respectively ) .",
"The flexibility in this region is likely due to the hinge/kink introduced by the conserved Pro300 ( Figure 5 , marked by a green star ) , arising from steric hindrance of the sidechain and the loss of the backbone hydrogen bonds between Pro300 and Arg296 .",
"Proline-induced local distortion of the helix and increases in flexibility are predicted to have global effects on protein conformational changes ( Cordes et al . , 2002 ) .",
"Consistent with this idea , mutational perturbation of the conserved proline side-chain leads to non-functional channels ( Hénault et al . , 2015 ) , suggesting that the helix-bending at this position may play a role in channel gating .",
"Above this region ( towards the extracellular end ) , for the TMD-facing side of M4 , a large change in lineshape ( observed as a decrease in spectral broadening and an increase in amplitude; also see the changes in mobile and immobile spectral components marked by the dotted lines ) is noted at several positions ( Phe299 , Val302 , Phe303 , Phe314 , and Phe315 ) and modest changes are observed at others ( Ala306 , Asn307 , and Leu310 ) .",
"In contrast , for the membrane-facing side of M4 , the spectral changes are small ( Figure 5—figure supplement 3 , indicated by black circles ) , indicating that channel gating is accompanied by rearrangement of the packing-interface of the intra-subunit cavity .",
"A complete plot of ΔH0−1 and accessibility parameters for M4 residues is presented for the closed and desensitized conformations ( see Materials and methods for details on the estimation of these parameters ) , and the difference in values of individual parameters between the two states are mapped on the GLIC-pH4 structure ( Figure 6 ) .",
"In the closed conformation , positions with low values of ΔH0−1 appear to be three to four residues apart and line up on the inward-face of M4 ( marked by green asterisk , Figure 6A ) .",
"Further , high ΠO2 values for the membrane-exposed residues and low values for the tertiary contacts show that M4 is closely associated with the rest of the TM helices , such that one face of M4 is protected from lipids in this state .",
"In the desensitized conformation , there is an overall increase in ΠO2 , which indicates an increase in the membrane exposure of M4 .",
"Although , potential differences in membrane permeability of O2 in the two pH conditions could affect this measurement .",
"Nevertheless , in comparison to the membrane-facing residues , a larger increase in ΠO2 is observed for the TMD-facing residues ( Ala289 , Ile291 , Ser295 , Phe299 , Val302 , Phe303 , Ala306 , Asn307 , Leu310 , Phe314 , and Phe315 ) ( Figure 6B ) , suggesting that the intimate association of M4 with M3 and M1 helices is disrupted in the desensitized conformation .",
"Consistent with the observed lineshape changes of residues in the vicinity of the Pro300 kink ( 295-299 ) , these positions also show high ΠO2 values in the desensitized state .",
"Overall , the C-terminal half of M4 shows the most dramatic changes in the CW lineshapes , which are correlated with increases in ΠO2 .",
"Further , the membrane boundaries of M4 are clearly defined by high ΠNiEDDA values at the extracellular and intracellular end with little water penetration observed in the M4 vicinity .",
"Besides a decrease in water exposure at the C-terminal tip of M4 , no major differences are observed in the ΠNiEDDA pattern in the two conformations .",
"A lack of NiEDDA accessibility for the residues facing the intra-subunit cavity is inline with the hydrophobic nature of this region . 10 . 7554/eLife . 23886 . 022Figure 6 . Solvent accessibility changes in M4 during desensitization .",
"( A ) A plot of residue environmental parameters for the closed ( shown in grey ) and desensitized ( shown in color ) states .",
"ΔHo−1 parameter ( top ) ; O2 accessibility ΠO2 ( middle ) ; water accessibility ΠNiEDDA ( bottom ) .",
"Positions along the protein-facing side of M4 are marked by green asterisks .",
"Regions of most prominent change are highlighted within grey boxes .",
"( B ) Difference in individual parameters between the desensitized and closed states are mapped on the GLIC-pH4 structure ( PDB ID: 4HFI ) and color-coded with red denoting an increase and blue representing a decrease in the environmental parameter .",
"The direction of putative M4 motion is indicated by the arrows .",
"The putative membrane boundaries as reflected by NiEDDA accessibility are marked by solid black lines in the bottom panel . DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 02210 . 7554/eLife . 23886 . 023Figure 6—figure supplement 1 . Changes in EPR lineshapes due to relaxation broadening vs mobility changes . CW-EPR spectra for positions along M4 in the closed ( pH 7 . 0 ) and desensitized ( pH 3 . 0 ) states before ( left ) and after purging N2 ( for 15 min ) ( right ) .",
"Positions of notable change are marked by black arrows .",
"Positions on the TMD-facing side of M4 are marked green asterisks ( these residues are highlighted in the GLIC-pH4 structure as a stick representation ) .",
"The grey box highlights positions in the vicinity of Pro300 that show large changes in linehapes under the two conditions . DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 023 Overall , residues facing the intra-subunit cavity show a change in nitroxide lineshapes and increased accessibility to O2 reflective of an outward motion of M4 that is accompanied by increased lipid exposure ( indicated by the arrows in Figure 6B ) .",
"Notably , a decrease in water exposure accompanied by an increase in membrane accessibility at the M4 tip is also consistent with an outward M4 motion that would bury the tip further within the membrane .",
"To quantify the extent of M4 movement , we measured average interspin distances in the closed and desensitized conformations for several positions in M4 ( Ser295 , Arg296 , Phe303 , and Leu304 ) with double electron-electron resonance ( DEER ) experiments .",
"Although CW spectra suggest a smaller conformational change compared to the C-terminal end , these positions were chosen because they were closer to the distance range that can be reliably measured by DEER ( 20–60 Å ) .",
"Since DEER measurements of longer distances are particularly challenging to determine in liposomes , we reconstituted labeled GLIC in nanodiscs using the membrane scaffolding protein , MSP1E3D1 ( Ritchie et al . , 2009 ) .",
"Previous work has shown that the use of nanodisc technology in combination with Q-band for data collection improved DEER sensitivity by lowering background contributions ( Zou and McHaourab , 2010 ) .",
"As expected for a pentameric system with five labels , the distance distribution for all the positions showed at least two main components , one corresponding to the short adjacent distance and the other to the long non-adjacent distance ( Figure 7 , Figure 7—figure supplement 1 ) .",
"The DEER distances in the closed state were broadly consistent with the GLIC-pH7 structure ( PDB ID: 4NPQ ) ( Table 2 ) .",
"Positions S295R1 and L304R1 also reveal multiple components for the adjacent distances indicative of conformational heterogeneity of the M4 helix .",
"However , the DEER distances in the desensitized state were consistently longer ( for three of four positions ) than those predicted from the GLIC-pH4 structure ( PDB ID: 4HFI ) and the GLIC-pH4-DHA structure .",
"For positions Arg296 , Phe303 , and Leu304 , both distributions move toward longer distances in the desensitized state compared to the closed state .",
"Increases in DEER distances are a consequence of M4 moving away from the five-fold axis , up to 4 Å at the mid-M4 region and likely to be greater at the tip of M4 .",
"Based on CW and DEER measurements , we conclude that M4 undergoes major conformational changes during its transition to the ligand-activated desensitized state , with an increase in lipid exposure of this segment , particularly at positions along the protein-facing side of M4 . 10 . 7554/eLife . 23886 . 024Figure 7 . Changes in M4 distance measured by DEER for GLIC in nanodiscs . GLIC structure showing the positions investigated by DEER and the two expected distance distributions ( from the adjacent and non-adjacent subunits ) .",
"Background subtracted DEER- echo intensity is plotted against evolution time and fit using model-free Tikhonov regularization .",
"The corresponding inter-spin distance distribution ( right ) for the closed ( black , pH 7 . 0 ) and desensitized ( red , pH 4 . 0 ) states for different spin-labeled positions .",
"The arrows highlight the direction of change . DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 02410 . 7554/eLife . 23886 . 025Figure 7—figure supplement 1 . Analysis of DEER data .",
"( A ) Background corrected Q-band dipolar evolution data for spin-labeled GLIC mutants reconstituted in nanodisc .",
"( B ) Corresponding Tikhonov L-curve .",
"The highlighted blue circle in the L-curve represents the regularization parameter ( α = 100 ) corresponding to the distance distribution in panel D . ( C ) The FT spectra of the traces shown in panel A . The black and red traces are fits based on the distance distribution shown in panel D for samples in the closed ( pH 7 . 0 ) and desensitized ( pH 4 . 0 ) states , respectively .",
"( D ) Distance distribution obtained by Tikhonov regularization . DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 02510 . 7554/eLife . 23886 . 026Table 2 . DEER distances measured in nanodiscs compared with ( cβ-cβ ) distances from crystal structures . DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 026pH 7 . 0pH 4 . 0ResidueShort ( Å ) Long ( Å ) Short ( Å ) Long ( Å ) S295R1GLIC Crystal Structure26 . 342 . 626 . 542 . 9GLIC Nanodisc DEER27 . 8/34 . 346 . 631 . 647 . 2R296R1GLIC Crystal Structure28 . 045 . 328 . 245 . 7GLIC Nanodisc DEER27 . 846 . 131 . 146 . 7F303R1GLIC Crystal Structure29 . 247 . 329 . 547 . 8GLIC Nanodisc DEER28 . 148 . 332 . 249 . 4L304R1GLIC Crystal Structure34 . 555 . 935 . 357GLIC Nanodisc DEER21 . 4/33 . 847 . 822 . 6/35 . 649 . 4GLIC crystal structure at pH 7 . 0 ( PDB ID: 4NPQ ) ( Sauguet et al . , 2014 ) .",
"GLIC crystal structure at pH 4 . 0 ( PDB ID: 4HFI ) ( Sauguet et al . , 2013 ) .",
"Outside of the canonical gating scheme as described in Figure 1A , pLGICs are shown to also exist in a lipid-induced , non-activatable conformation , referred to as the ‘uncoupled’ state ( daCosta and Baenziger , 2009; daCosta et al . , 2013 ) .",
"This conformational state , which is distinct from the desensitized state , is refractory to agonist-induced transitions and is characterized by lower agonist-affinity ( similar to the closed state ) .",
"To determine if the DHA-stabilized state is similar to the ‘uncoupled’ state , we measured CW-spectra at representative positions on M4 under steady-state conditions of neutral and acidic pH ( that favor the closed and desensitized conformations , respectively ) , each in the presence and absence of DHA ( Figure 8 ) .",
"If the observed effect of DHA was through an increase in the rate of agonist-induced desensitization , under conditions of prolonged agonist exposure , the equilibrium population , both in the presence and absence of DHA , is likely to be shifted towards the desensitized state .",
"However , if DHA were to stabilize the uncoupled conformation , then a mixture of closed and uncoupled states , with very little population of the desensitized states , is expected in the acidic pH condition .",
"A comparison of the spectra ( Figure 8 ) shows that there is indeed no effect of DHA on the lineshapes of the two end-states ( closed and desensitized ) .",
"These results further confirm that DHA stabilizes an agonist-induced desensitized state . 10 . 7554/eLife . 23886 . 027Figure 8 . EPR spectral analysis of M4 positions in the absence or presence of DHA . Spin-normalized CW-spectra for representative positions along M4 in the closed ( pH 7 . 0 ) and desensitized ( pH 3 . 0 ) states and measured in the presence or absence of 50 μM DHA . DOI: http://dx . doi . org/10 . 7554/eLife . 23886 . 027"
],
[
"The GLIC-pH4-DHA structure presented here reveals a novel lipid-induced conformation in the crystal that is physically distinct from previously observed pLGIC conformations ( Du et al . , 2015; Sauguet et al . , 2014; Hilf and Dutzler , 2009 , 2008; Sauguet et al . , 2013; Miller and Aricescu , 2014; Hassaine et al . , 2014; Hibbs and Gouaux , 2011; Morales-Perez et al . , 2016 ) .",
"A comparison of GLIC-pH7 and GLIC-pH4 structures suggest that activation involves an outward tilting of M2 from the pore-axis leading to channel opening with a radius >5 Å at the extracellular end ( Hilf and Dutzler , 2009; Sauguet et al . , 2013 ) .",
"In the GLIC-pH7 structure , the M2 helices come together to form a tightly packed bundle with the pore constricted to less than ~2 . 5 Å at Ile16′ , Ile9′ , Ser6′ , and Thr2 , effectively occluding ion permeation ( the metal-oxygen distance of hydrated monovalent cations is ~2 . 1–3 . 1 Å ( Hille , 2001; Marcus , 1988 ) .",
"In the GLIC-pH4 structure , the narrowest region of the pore is formed by the selectivity filter region ( between Thr2′ to Glu-2′ ) with a radius ~2 . 5 Å at Thr2′ .",
"Although the pore in this region is too narrow to allow fully-hydrated cations to pass , polar side-chains at Ser6′ , Thr2′ and Glu-2′ could potentially coordinate partially hydrated ions .",
"Since it is not known whether GLIC can permeate partially hydrated ions , the conformational state of GLIC-pH4 ( open , pre-open , or desensitized ) is still debatable .",
"In contrast , in the GLIC-pH4-DHA structure , while the extracellular end remains open , the intracellular-half of M2 ( lined by Ile9′ , Ser 6′ , and Thr2′ ) undergoes constriction , with the largest effect seen at Ile9′ ( pore radii ~2 . 5 Å ) .",
"In this conformation , occupancy of the ions and water molecules in the selectivity filter area is greatly reduced , which could be induced by the closure at Ile9′ .",
"The pore radii at Glu-2′ is unchanged .",
"However , lower resolution of our structure limits us from making significant interpretations about the side-chain conformation at the −2′ position .",
"We therefore suggest that the GLIC-pH4-DHA structure represents a desensitized conformation ( perhaps a lipid-induced , deeper state ) , where ion permeation is occluded in the intracellular-end of M2 below the 9′ position .",
"Consistent with this idea , reducing the side-chain volume or hydrophobicity at the conserved 9′ position increases the open state stability and decreases the apparent desensitization rate in many members of the pLGIC family ( Bocquet et al . , 2007; Labarca et al . , 1995; Filatov and White , 1995; Akabas et al . , 1992; Yakel et al . , 1993; Chang et al . , 1996; Revah et al . , 1991 ) .",
"Further , constriction of the inner half of M2 in the desensitized state was previously predicted based on our EPR spectral broadening information ( Velisetty et al . , 2012 ) .",
"This mechanism is also consistent with lidocaine slowing desensitization by a ‘foot-in-the-door mechanism’ ( Velisetty and Chakrapani , 2012 ) .",
"Other studies have suggested that lidocaine accelerates GLIC desensitization ( although these were measured at lower lidocaine concentrations ) ( Gonzalez-Gutierrez and Grosman , 2015 ) .",
"Pore dehydration and loss of ion occupancy at the selectivity filter region have been implicated in C-type inactivation in voltage-gated channels ( Cuello et al . , 2010a ) .",
"Our structure lends strong support to the original ‘two-gate’ hypothesis that suggests the presence of two structurally distinct activation and desensitization gates ( Auerbach and Akk , 1998 ) .",
"This model is also consistent with accessibility measurements of the pore lining residues in the three conformations ( Wilson and Karlin , 2001 ) .",
"At the functional level , pLGICs are known to display multiple desensitized states with dwell times ranging from milliseconds to minutes ( Elenes and Auerbach , 2002 ) .",
"Although , an unequivocal assignment to one of these states is not discernable , prolonged agonist exposure favors a long-lived desensitized state , and we therefore hypothesize that this state may be favored in the crystal form .",
"Recently , structures of GABAAR-β3 , GlyR , and nAChR-α4β2 were solved in the presence of agonists and are likely to represent desensitized conformations ( Du et al . , 2015; Miller and Aricescu , 2014; Morales-Perez et al . , 2016 ) .",
"In these structures , a local constriction was noted in the M2 intracellular end , closer to the −2′ position , which is consistent with findings that picrotoxin binding at −2′ slows desensitization ( Gielen et al . , 2015 ) .",
"Interestingly , the extent of pore closure at −2′ appears to bear some correlation with the desensitization properties of the these channels: a stronger desensitization was observed for GABAAR-β3 and nAChR-α4β2 currents ( Miller and Aricescu , 2014; Morales-Perez et al . , 2016 ) in comparison to ivermectin/glycine evoked currents for GlyR ( Du et al . , 2015 ) .",
"In terms of the structures , the pore is narrower in GABAAR-β3 and nAChR-α4β2 ( ~1 . 5–2 Å radius ) compared to GlyR ( ~2 . 5 Å radius ) .",
"Furthermore , in ELIC ( a GABA-gated cation channel ) , the intracellular compression is suggested to also involve residues further up ( 6′ to −2′ positions ) ( Kinde et al . , 2015 ) .",
"Overall , these mechanistic differences may underlie the broad range of desensitization kinetics observed within the family , encompassing time constants that span several orders of magnitude ( <1 ms for α7-nAChR , tens of seconds for GLIC and ELIC , and very little current decay observed for ρ1 GABAAR and α GlyR , 5-HT3AR ) ( Keramidas and Lynch , 2013 ) .",
"A notable aspect of the new conformation reported here is that it is stabilized by a physiologically relevant lipid molecule .",
"The question still remains as to how DHA induces the observed conformational state .",
"The DHA binding-site in the M4 vicinity was not surprising , considering the extensive functional and biophysical studies that have implicated M4 as the ‘lipid-sensor’ in pLGIC .",
"Further , earlier studies predicted that the effect of free PUFA on nAChR inhibition occurs through allosteric mechanisms that alter lipid-protein interactions ( Villar et al . , 1988; Andreasen and McNamee , 1980; Fernández Nievas et al . , 2008 ) rather than through changes in bulk membrane fluidity .",
"Although the M4 segment is least conserved in sequence , mutations ( including those at the lipid-facing side of the helix ) have been shown to alter gating , presumably by perturbing the protein structure or the lipid-protein interactions ( Bouzat et al . , 1998; Lasalde et al . , 1996; Li et al . , 1992 ) .",
"Similarly , DHA appears to alter the interaction of the channel with an annular lipid molecule ( PLC ) in the M4 groove and , perhaps as a consequence , exerts a long-range allosteric effect on the pore conformation .",
"Consistent with this idea , the occupancy of PLC in this site appears to be influenced by the conformational state of the channel .",
"Particularly , the GLIC-pH7 and locally-closed GLIC structures show no electron density at this PLC site , suggesting that the open conformation may stabilize the binding of this lipid molecule ( Sauguet et al . , 2014; Prevost et al . , 2012 ) .",
"Further , the most notable consequence of propofol binding in the GLIC structure was the change in the orientation of PLC , suggesting that alterations in channel properties may arise from changes in lipid-protein interactions ( Nury et al . , 2011 ) .",
"In remarkable agreement , our EPR data show that residues lining the PLC pocket in the crystal structure ( 302–316 in M4 and the 118–121 in the β6-β7 loop [Velisetty et al . , 2014] ) show an increase in O2 accessibility in the desensitized state , indicating that the lipid-accessibility of this pocket is increased upon activation .",
"However , the pathway for allosteric coupling between the DHA binding site and the pore is not clear , since the conformations of M4 , the M2-M3 linker , and the β6−β7 loop are nearly identical to those observed in the GLIC-pH4 structure .",
"One possibility is that this region undergoes minimal structural change between the open and desensitized states .",
"Further studies to stabilize the open GLIC conformation in membranes are needed to fully address this question .",
"Based on our EPR data , which show that the lipidic environment of M4 changes quite dramatically between the closed and desensitized states accompanied by an increase in intra-subunit distances , we propose that the M4 segment undergoes an outward tilt upon activation , mediated by the hinge/bend at the conserved proline residue in the middle of M4 .",
"The structural changes observed in EPR are consistent with the study in GLIC M4 that shows that mutational perturbations in the extracellular half of M4 have a greater effect on pH50 in comparison to the intracellular end ( Hénault et al . , 2015 ) .",
"The proposed M4 conformational change is also consistent with the lipid-mediated M4 tilt observed in isolated peptides ( Antollini et al . , 2005 ) .",
"Since this proline residue is also present in other members of the family including GABAARs and GlyRs , the proposed M4 movement could be a conserved mechanism .",
"The outward M4 motion is associated with changes in both the polarity and the volume of the intra-subunit cavity and such a conformational change may underlie state-dependent accessibility of several lipophilic modulators ( such as alcohols and anesthetics ) of the pLGIC which are shown to bind in these cavities ( Mihic et al . , 1997; Nury et al . , 2011; Howard et al . , 2011 ) .",
"Importantly , several residues on the TMD-facing side of M4lining this hydrophobic cavity , are proposed to bind allosteric modulators such as PNU-120596 ( Young et al . , 2008 ) and endogenous steroid ( THDOC ) ( Hosie et al . , 2006 ) , that modulate desensitization in nAChR and GABAAR , respectively .",
"From a structural point of view , it is intriguing that the M4 position appears to be fixed with respect to the rest of the TM helices in the pLGIC structures , even though these structures may represent different conformational states .",
"In contrast , functional analysis of M4 mutations and our EPR data seem to suggest that M4 is dynamic and experiences extensive change in environment during gating .",
"Although we cannot explain this disparity with certainty , it is conceivable that M4 movements could be masked in a detergent environment .",
"It is also possible that the lipidic environment around M4 changes without a significant change in the M4 backbone .",
"We believe that high-resolution structural studies of pLGIC in a membrane environment , such as nanodiscs , are needed to address these differences .",
"In conclusion , we show that distinct regions of the pore control activation and desensitization in pLGICs , in contrast to the mechanisms proposed in tetrameric ligand-gated channels ( Sobolevsky , 2015 ) .",
"Pore dehydration and collapse of the selectivity region during desensitization/inactivation appears to be a conserved mechanism across many channel types ( Cuello et al . , 2010b ) .",
"The present work provides a structural view of the long-studied allosteric modulation of lipids on pLGIC desensitization and opens up new avenues for the investigation of more complex regulatory mechanisms ."
],
[
"The gene encoding GLIC was inserted into the pTLN vector for oocyte expression and confirmed by DNA sequencing .",
"The DNA was then linearized with the Mlu1 restriction enzyme overnight at 37°C .",
"The mRNA was synthesized using the mMessage mMachine kit ( Ambion , Life Technologies , Carlsbad , CA ) , purified with RNAeasy ( Qiagen , Germantown , MD ) , and injected ( 5–15 ng ) into Xenopus laevis oocytes ( stages V-VI ) .",
"Control ooctyes were injected with the same volume of water to verify endogenous currents were not present .",
"Oocytes were maintained at 18°C in OR3 media ( Leibovitz media , GIBCO BRL: Life Technologies , Carlsbad , CA ) containing glutamate , 500 units each of penicillin and streptomycin , pH adjusted to 7 . 5 , osmolarity adjusted to 197 mOsm ) .",
"Two electrode voltage-clamp experiments were then performed at room temperature 2–5 days after injection .",
"A Warner Instruments ( Hamden , CT ) Oocyte clamp OC-725 was used for the measurements , and the current was sampled and digitized at 500 Hz with a Digidata 1440A ( Molecular Devices , Sunnyvale , CA ) .",
"Oocytes were clamped at a holding potential of −60 mV , and current traces were recorded in response to ligand application .",
"Solutions were changed using a syringe pump perfusion system flowing at a rate of 2 ml/min .",
"The electrophysiological solutions contain 96 mM NaCl , 2 mM KCl , 1 . 8 mM CaCl2 , 1 mM MgCl2 , and 5 mM HEPES ( pH 7 . 4 , osmolarity adjusted to 195 mOsm ) or 5 mM Sodium Citrate ( Hilf and Dutzler , 2009; Parikh et al . , 2011; Hilf et al . , 2010; Goyal et al . , 2011 ) ( at acidic pH buffer , pH adjusted to indicated value ( 4 . 0–6 ) ; osmolarity adjusted to 195 mOsm ) .",
"All chemical reagents were purchased from Sigma-Aldrich .",
"DHA stock solutions were freshly prepared in DMSO prior to each experiment and diluted to the final concentration in the electrophysiology solutions ( mentioned above ) .",
"The highest DMSO concentration used was ~0 . 016% and comparison with control experiments were made using the highest concentration of DMSO in the test conditions .",
"The traces were analyzed by Clampfit 10 . 2 ( Molecular Devices , Sunnyvale , CA ) .",
"Dose response curves were fit in Origin ( OriginLab , Northampton , MA ) to determine the pH50 and Hill coefficient ( nH ) .",
"The values for ‘n’ in the figure legends referto the number of oocytes .",
"The GLIC gene cloned into a modified pET26b vector was expressed as a fusion construct with N-terminal maltose binding protein ( MBP ) as previously described ( Hilf and Dutzler , 2009; Bocquet et al . , 2009 ) .",
"The protein was expressed and purified as previously described ( Hilf and Dutzler , 2008; Bocquet et al . , 2009; Velisetty and Chakrapani , 2012 ) .",
"Briefly , C43 E . coli cells ( Lucigen Corporation , Middleton , WI ) transformed with the construct were grown in terrific broth media containing 50 μg/ml kanamycin at 37°C to O . D600 of 1 . 0 .",
"Cells were induced with 0 . 2 mM isopropyl 1-thio-β-d-galactopyranoside ( Gold Biotechnology , Olivette , MO ) overnight at 18°C .",
"Membranes were prepared by homogenizing the cells in Buffer A ( 100 mM NaCl , 20 mM Tris-HCl ( pH 7 . 4 ) ) with protease inhibitors and centrifuged at 100 , 000 x g for 45 min .",
"Membranes were solubilized in Buffer A using 40 mM DDM ( n-dodecyl-β-d-maltopyranoside , Anatrace Inc , Maumee , OH ) at 4°C .",
"The protein was purified by binding to amylose resin and eluting with 20 mM maltose .",
"The maltose binding protein tag was cleaved with human rhinovirus 3C protease ( GE Healthcare , Wauwatosa , WI ) , and the pentameric protein was separated from MBP using size exclusion chromatography on a Superdex 20/200 column ( GE Healthcare , Wauwatosa , WI ) with Buffer A and 0 . 5 mM DDM .",
"The native Cys ( C27 ) was mutated to Ser and single Cys mutants in M4 were generated using the Cys-free construct ( C27S ) as the template .",
"Mutant proteins were expressed and purified similar to the wild type channels .",
"Purified mutants were labeled with a methanethiosulfonate spin probe MTSL ( 1-Oxyl-2 , 2 , 5 , 5-tetramethylpyrrolidin-3-yl ) methyl methanethiosulfonate ) ( Toronto Research Chemicals Inc , North York , ON , Canada ) at a 10:1 label:protein molar ratio and incubated on ice for 30 min , after which a 5-fold molar excess of the MTSL was added and further incubated for 2 hr for better labeling efficiency ( Velisetty et al . , 2012 ) .",
"The labeled protein was then purified by size exclusion chromatography on a Superdex 20/200 column ( GE healthcare , Wauwatosa , WI ) in Buffer A supplemented with 0 . 5 mM DDM .",
"Spin-labeled samples were reconstituted at a 1:3000 protein:lipid ( molar ratio ) in asolectin , incubated with biobeads to remove solubilizing detergent , and centrifuged to obtain a pellet of the proteoliposomes .",
"Continuous Wave-Electron Paramagnetic Resonance ( CW-EPR ) spectroscopy measurements were performed at room temperature on an EMX X-band spectrometer ( Bruker , Billerica , MA ) equipped with a dielectric resonator and a gas permeable TPX plastic capillary .",
"First derivative absorption spectra were recorded at an incident microwave power of 2 . 0 mW , modulation frequency of 100 kHz , and modulation amplitude of 1 . 0 G . The EPR signal is normalized to the total number of spins in the sample by dividing the spectra by the peak-to-peak value of the double integral ( which is proportional to the total number of spins ) .",
"Our analyses were centered on two types of dynamic EPR structural information ( Farahbakhsh et al . , 1992; Altenbach et al . , 2005 ) : the first is lineshape parameter ΔHo−1 , calculated as the inverse of the central line width of the first derivative absorption spectra , which often times correlates with the mobility .",
"ΔHo−1 is governed both by the local steric contacts in the immediate vicinity of the probe and by the flexibility of the backbone to which it is attached ( Mchaourab et al . , 1996 ) .",
"Lineshape changes at times may not arise from changes in mobility but occur because of relaxation broadening due to accessibility to paramagnetic O2 .",
"This scenario can be distinguished by measuring lineshapes upon purging N2 ( See Figure 6—figure supplement 1 ) .",
"For positions where relaxation is observed , we will refer to variations in ΔHo−1 as lineshape changes rather than differences in mobility .",
"The second is spin-probe solvent accessibility evaluated by collisional relaxation methods .",
"Here , polar Ni ( II ) ethylenediaminediacetic acid ( ΠNiEDDA ) and nonpolar molecular O2 serve to evaluate the extent of water and membrane exposure , respectively ( Farahbakhsh et al . , 1992; Gross and Hubbell , 2002 ) .",
"The accessibility parameter ( П ) is estimated from power saturation experiments in which the vertical peak-to-peak amplitude of the central line of the first derivative EPR spectra is measured as a function of increasing incident microwave power ( Farahbakhsh et al . , 1992 ) .",
"Conformational changes were measured by equilibrating the sample with appropriate buffers ( pH 7 . 0 and 3 . 0 ) in a 42°C water-bath .",
"The samples were centrifuged and the process was repeated three times to ensure complete buffer exchange .",
"These conditions ensured saturation of pH-induced changes in EPR line-shape ( Velisetty et al . , 2012 ) .",
"Reversibility of structural changes was ensured by switching back to pH 7 . 0 .",
"Membrane scaffold protein ( MSP1E3D1 ) was expressed and purified as previously described ( Boldog et al . , 2007; Mishra et al . , 2014 ) with some modifications .",
"The MSP1E3D1 gene in pET-28a ( a gift from Stephen Sligar: Addgene plasmid # 20066 ) ( Denisov et al . , 2007 ) was transformed in E . coli BL21 ( DE3 ) cells ( Agilent Technologies , Santa Clara , CA ) and plated on LB-agar plates supplemented with kanamycin ( 25 μg mL−1 ) .",
"An overnight culture from a single colony was set up with LB supplemented with kanamycin ( 25 μg mL−1 ) and 1% glucose .",
"The overnight culture was used to inoculate a 1L culture of Terrific broth supplemented with kanamycin ( 25 μg mL−1 ) and 0 . 2% glucose .",
"The culture was grown at 37°C with shaking to an OD600 of ~1 . 0 , and induced by with 1 mM IPTG for 4 hr at 37°C .",
"Cells were harvested by centrifugation and the cell pellet resuspended in Buffer A containing 1 mM PMSF and Complete EDTA-free protease inhibitor cocktail tablet ( Roche ) and lysed by homogenization .",
"The lysate was centrifuged at 30 , 000 x g for for 30 min and the supernatant was bound to Ni-NTA equilibrated with Buffer A . The resin was washed with four bed volumes of Buffer B ( 300 mM NaCl , 40 mM Tris-HCl , and pH 8 . 0 ) containing 1% Triton X-100 , four bed volumes of Buffer B containing 50 mM sodium cholate , four bed volumes of Buffer B , four bed volumes of Buffer B containing 20 mM imidazole , and eluted with Buffer B containing 300 mM imidazole .",
"The eluted MSP1E3D1 was passed through a desalting column equilibrated with Buffer C ( 100 mM NaCl , 50 mM Tris-HCl , 0 . 5 mM EDTA , and pH 7 . 5 ) , and the concentration was determined by absorbance at 280 nm ( extinction coefficient = 29 , 910 M−1 cm−1 ) .",
"The purity was assessed by SDS–PAGE and size-exclusion chromatography .",
"Detergent-solubilized spin-labeled GLIC mutants passed through gel-filtration columns were incorporated into lipid nanodiscs as previously described ( Mishra et al . , 2014 ) with some modifications .",
"Briefly , asolectin dissolved in chloroform was dried using nitrogen stream and rehydrated in Buffer A supplemented with 2 mM DDM .",
"Each spin labeled mutant was mixed with rehydrated lipids and MSP1E3D1 in the GLIC:MSP:lipid = 1:3:360 molar ratio .",
"The mixture was incubated at 4°C for 30 min with gentle rotation .",
"Bio-beads SM-2 ( Bio-Rad Laboratories , Hercules , CA ) were added to initiate reconstitution overnight at 4°C with gentle rotation .",
"Bio-beads were then removed and the reconstitution mixture assessed by size-exclusion chromatography and SDS–PAGE .",
"Samples were equilibrated in pH 7 . 0 ( closed state ) or pH 4 . 0 ( desensitized state ) .",
"We used pH 4 . 0 for DEER measurements due to potential instability issues of membrane scaffolding proteins in nanodisc at more acidic pH conditions .",
"The peak corresponding to pentameric GLIC reconstituted into nanodiscs was collected and flash-frozen in 20% glycerol for DEER measurements .",
"Inter-subunit distances ( <50 Å ) were measured using Double Electron-Electron Resonance ( DEER ) methods ( Zou and McHaourab , 2010; Jeschke et al . , 2002 ) for spin-labeled samples reconstituted in nanodiscs .",
"Four-pulse DEER experiments were performed using a Bruker ELEXSYS E580 spectrometer equipped with a SuperQ-FT pulse Q-band system with a 10 W amplifier and EN5107D2 resonator .",
"The sample was loaded into a 1 . 1 mm inner diameter quartz capillary ( Wilmad LabGlass , Buena , NJ ) and mounted into the sample holder ( plastic rod ) inserted into the resonator .",
"Dipolar time evolution data were obtained at 80K using a standard DEER four-pulse sequence ( π/2 ) mw1–τ1– ( π ) mw1–τ1– ( π ) mw2–τ2– ( π ) mw1–τ2–echo ( Pannier et al . , 2000 ) at Q-band frequency ( ~33 . 9 GHz ) .",
"The experimental conditions were: pulse lengths for ( π/2 ) mw1 and ( π ) mw1 were 10 and 20 ns , respectively , and 24 ns for ( π ) mw2 , 80 MHz of frequency difference between probe and pump pulse , shot repetition time determined by spin-lattice relaxation rate ( T1 ) , 100 echo/point , and 2-step phase cycling .",
"Data were collected out to ~2 . 0 µs for overnight data acquisition time .",
"DEER signals were background-corrected assuming a 3D homogeneous background and analyzed by the Tikhonov regularization in the DEER Analysis 2014 software ( Jeschke et al . , 2006; Chiang et al . , 2005 ) to determine average distances and distributions in distance .",
"The regularization parameter in the L curve was optimized by examining the fit of the time domain .",
"Electrophysiological measurements were made by patch clamp recordings in channel-reconstituted liposomes prepared as described earlier ( Velisetty and Chakrapani , 2012; Delcour et al . , 1989; Cortes et al . , 2001; Chakrapani et al . , 2007 ) .",
"Purified and spin-labelled GLIC mutants were reconstituted into preformed asolectin vesicles by diluting in 150 mM NaCl , 10 mM MOPS , pH 7 . 0 ( reconstitution buffer ) .",
"The detergent was removed by incubating the proteoliposome suspension with biobeads overnight at 4°C .",
"The suspension was centrifuged at 100 , 000g for 1 hr and the pellet re-suspended in reconstitution buffer .",
"A drop of the proteoliposome was placed on a glass slide and dried overnight in a desiccator at 4°C .",
"The sample was then re-hydrated with 20 μl of buffer , which yielded giant liposomes .",
"Channels were reconstituted in 1:10000 protein:lipid ( molar ratio ) for macroscopic currents .",
"All currents measurements were made at room temperature by inside-out patch-clamping of proteoliposome under symmetrical NaCl concentrations .",
"Recording pipettes were pulled from thin walled borosilicate glass , heat polished to a resistance of 1 . 5–2 MΩ , and filled with 150 mM NaCl , 10 mM MOPS , pH 7 . 0 .",
"Currents were elicited in response to pH jumps ( to pH 3 . 0 , 150 mM NaCl and 10 mM sodium citrate buffer ) using an RCS-200 fast solution exchanger ( switch time 2 ms ) fed by gravity ( BioLogic , Knoxville , TN ) .",
"Currents were measured using Axopatch 200B , digitized at 10 kHz sampling frequency and were analysed using Clampfit 10 . 2 .",
"GLIC wt in 100 mM NaCl , 10 mM Tris-HCl ( pH 7 . 4 ) , and 0 . 5 mM DDM was concentrated to between 9–10 mg/ml with an Amicon Ultra 50 KDa cutoff concentrator ( EMD Millipore , Billerica , MA ) .",
"Prior to crystallization setup , the protein was supplemented with 50 μM DHA ( from 300 mM DHA stock in ethanol ) and 0 . 5 mg/ml E . coli polar extract ( Avanti Polar Lipids ) and incubated on ice for 1 hr .",
"The protein was crystallized at 4°C by sitting drop vapor diffusion in Cryschem plates ( Hampton Research , Aliso Viejo , CA ) with a 1:1 mixture ( 1 μl each ) of protein and reservoir solution ( 225 mM ammonium sulfate , 50 mM sodium acetate , pH 3 . 9–4 . 2 and 9–12% PEG4000 ) .",
"Crystals typically formed within one week and typically took 2–3 weeks to reach full size .",
"The crystals were cryoprotected by adding 6 μL reservoir solution supplemented with 30% ethyleneglycol to the drop , and directly frozen in liquid nitrogen using appropriately sized microloops ( MiTeGen , Ithaca , NY ) or cryoloops ( Hampton Research ) .",
"X-ray diffraction data were acquired on NE/CAT beamlines 24ID-C at the Advanced Photon Source at Argonne National Laboratory .",
"The data was indexed using iMosflm ( Battye et al . , 2011 ) and further processed using programs within the CCP4 suite ( Collaborative Computational Project , 1994 ) .",
"The crystals belong to space group C121 with one pentamer in the asymmetric unit .",
"Initial phases were obtained by molecular replacement using PHASER ( McCoy , 2007 ) with GLIC ( PDB ID: 4HFI ) ( Sauguet et al . , 2013 ) crystal structures as a search model .",
"The initial model was refined with REFMAC5 ( Murshudov et al . , 1997 ) and was followed by manual model building/fitting in COOT ( Emsley et al . , 2010 ) .",
"On each successive cycle of model building and refinement , we analyzed the structure with Molprobity software ( Chen et al . , 2010 ) .",
"MOLE ( Petrek et al . , 2007 ) and HOLE ( Smart et al . , 1996 ) software were used to compute pore radius profiles ( Absalom et al . , 2003 ) ."
]
] | [
"Desensitization in pentameric ligand-gated ion channels plays an important role in regulating neuronal excitability .",
"Here , we show that docosahexaenoic acid ( DHA ) , a key ω−3 polyunsaturated fatty acid in synaptic membranes , enhances the agonist-induced transition to the desensitized state in the prokaryotic channel GLIC .",
"We determined a 3 . 25 Å crystal structure of the GLIC-DHA complex in a potentially desensitized conformation .",
"The DHA molecule is bound at the channel-periphery near the M4 helix and exerts a long-range allosteric effect on the pore across domain-interfaces .",
"In this previously unobserved conformation , the extracellular-half of the pore-lining M2 is splayed open , reminiscent of the open conformation , while the intracellular-half is constricted , leading to a loss of both water and permeant ions .",
"These findings , in combination with spin-labeling/EPR spectroscopic measurements in reconstituted-membranes , provide novel mechanistic details of desensitization in pentameric channels ."
] | [
"The nerve cells ( or neurons ) in the brain communicate with each other by releasing chemicals called neurotransmitters that bind to ion channels on neighboring neurons .",
"This ultimately causes ions to flow in or out of the receiving neuron through these ion channels; this ion flow determines how the neuron responds .",
"One family of ion channels that is found at the junction between neurons , and between neurons and muscle fibers , is known as the pentameric ligand-gated ion channels ( or pLGICs ) .",
"These channels act as ‘gates’ that open to allow ions through them when a neurotransmitter binds to the channel .",
"In addition to the open ‘active’ state , the channels can take on two different ‘inactive’ states that do not allow ions to pass through the channel: a closed ( resting ) state and a desensitized state ( that is still bound to the neurotransmitter ) .",
"Understanding how channels switch between these states is important for designing drugs that correct problems that cause the channels to work incorrectly .",
"Problems that affect the desensitized state have been linked to neurological disorders such as epilepsy .",
"Medically important molecules such as anesthetics and alcohols are thought to affect desensitization , and drugs that target desensitized ion channels may present ways of treating neurological disorders with fewer side effects .",
"Docosahexaenoic acid ( DHA ) is an abundant lipid molecule that is present in the membranes of neurons .",
"It is one of the key ingredients in fish oil supplements and is thought to enhance learning and memory .",
"DHA affects the desensitization of pLGICs but it is not clear exactly how it does so .",
"Basak et al . now show that DHA affects a bacterial pLGIC in the same way as it affects human channels – by enhancing desensitization .",
"Using a technique called X-ray crystallography to analyze the channel while bound to DHA revealed a previously unobserved channel structure .",
"The DHA molecule binds to a site at the edge of the channel and causes a change in its structure that leaves the upper part of the channel open while the lower part is constricted .",
"Basak et al . predict that molecules such as anesthetics target this desensitized state .",
"The next step will be to obtain the structures of bacterial and human pLGIC channels in a natural membrane environment .",
"This will allow us to better understand the changes in structure that the channels go through as they transmit signals between neurons , and so help in the development of new treatments for neurological disorders ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"structural biology and molecular biophysics",
"immunology and inflammation"
] | Structural basis for the prion-like MAVS filaments in antiviral innate immunity | elife-01489-v1 | [
[
"Viral infection of host cells triggers innate and adaptive immune responses that are essential for the survival of the host ( Iwasaki and Medzhitov , 2010; Ronald and Beutler , 2010; Takeuchi and Akira , 2010 ) .",
"Initiation of innate immune response relies on a group of pattern recognition receptors , which recognize specific pathogen-associated molecular patterns ( PAMPs ) , including microbial nucleic acids , bacterial cell wall components and certain highly conserved proteins .",
"Two groups of pattern recognition receptors are responsible for detecting viral RNAs .",
"The first group is the membrane-anchored Toll-like receptors ( TLRs ) , located on the cell surface or in endosomal membranes and acting as sensors of viral RNAs encountered in extracellular environment ( Kawai and Akira , 2006 ) .",
"The second group of sensors resides in the cytosol .",
"They belong to a family of cytosolic RNA helicases called RIG-I-like receptors ( RLRs ) , including retinoic acid inducible gene-I ( RIG-I ) , melanoma differentiation-association gene 5 ( MDA5 ) and laboratory of genetics and physiology 2 ( LGP2 ) ( Yoneyama and Fujita , 2009 ) .",
"Recognition of viral RNAs by RLRs leads to the activation of mitochondrial antiviral signaling protein ( MAVS; also known as IPS1 , VISA and CARDIF ) .",
"Activated MAVS triggers rapid production of type I interferons and proinflammatory cytokines ( Kawai et al . , 2005; Meylan et al . , 2005; Seth et al . , 2005; Xu et al . , 2005 ) .",
"RIG-I and MDA5 are two DExD/H-box helicases that belong to the superfamily 2 of RNA helicases ( Yoneyama et al . , 2004; Fairman-Williams et al . , 2010 ) .",
"Both proteins contain N-terminal tandem caspase activation and recruitment domains ( CARDs ) , a central RNA helicase , and a C-terminal regulatory domain ( CTD ) .",
"Despite sharing a similar domain structure , RIG-I and MDA5 are activated by complementary sets of viral RNA ligands through distinct mechanisms ( Kato et al . , 2008; Loo et al . , 2008; Iwasaki , 2012 ) .",
"RIG-I recognizes short blunt ends of dsRNA with 5′-triphosphate caps ( Hornung et al . , 2006; Schlee et al . , 2009 ) , as well as long dsRNAs ( Kohlway et al . , 2013; Patel et al . , 2013; Peisley et al . , 2013 ) .",
"Ligand binding to the helicase domain and CTD induces a conformational change that liberates RIG-I from an autoinhibited state and exposes its N-terminal tandem CARDs ( Hou et al . , 2011; Kowalinski et al . , 2011; Luo et al . , 2011 ) .",
"In contrast , MDA5 detects long dsRNAs made of hundreds to thousands of base pairs ( Kato et al . , 2008 ) .",
"The helicase domain and CTD of MDA5 cooperatively assemble into helical filaments along the dsRNA , leaving the tandem CARDs of MDA5 flexibly exposed on the periphery of the filament ( Peisley et al . , 2011; Berke and Modis , 2012; Wu et al . , 2013 ) .",
"The exposed CARDs of RIG-I and MDA5 subsequently bind to unanchored lysine-63 ( K63 ) polyubiquitin chains and form oligomers .",
"The latter gain a high capacity of activating MAVS on mitochondria , presumably through CARD–CARD interactions ( Zeng et al . , 2010; Jiang et al . , 2012 ) .",
"The exact mechanism for the polyubiquitin-dependent interaction between active RIG-I and inactive MAVS is not well defined .",
"MAVS is ubiquitously expressed on the outer membrane of mitochondria .",
"It consists of an N-terminal CARD domain , a proline-rich region ( PRR ) preceding a poorly structured middle segment , and a monotopic transmembrane ( TM ) domain at the very C-terminus ( Figure 1A ) .",
"Recent studies have shown that upon activation , MAVS molecules polymerize themselves into functional aggregates ( Hou et al . , 2011 ) .",
"These high molecular weight aggregates behave like prion fibers , because they are detergent-resistant , protease-resistant and self-perpetuating by inducing inactive MAVS to form functional aggregates .",
"The N-terminal CARD domain of MAVS is necessary and sufficient for forming active MAVS aggregates .",
"It shares some homology with the first CARD domains in both MDA5 and RIG-I ( 25% and 20% sequence identity respectively ) .",
"A crystal structure of the MAVS CARD , fused to a maltose-binding protein ( MBP ) , exhibits a typical helical bundle of six antiparallel α-helices ( Potter et al . , 2008 ) .",
"However , the isolated MAVS CARD without the MBP self-assembles into filamentous structures , which can promote endogenous , inactive MAVS to form highly active aggregates ( Hou et al . , 2011 ) .",
"How the CARD domain triggers MAVS aggregation remains poorly understood . 10 . 7554/eLife . 01489 . 003Figure 1 . CryoEM reconstruction of MAVS CARD filaments .",
"( A ) Diagrams of the domain organization in MAVS and deletion mutants used in this study .",
"( B ) Flag-MAVS CARD purified from HEK293T cells analyzed by silver stained SDS-PAGE and cryoEM imaging .",
"The cryoEM image is displayed in reversed contrast ( protein in black ) for better visualization .",
"( C ) Left: a side view of the final 3D reconstruction of MAVS CARD filament .",
"The X-ray crystal structure model of the human MAVS CARD ( PDB: 2VGQ ) was docked into the cryoEM map .",
"Three strands are colored differently .",
"Right: the rod-like densities at the periphery of the cryoEM density map allowed positioning of H1 , H4 , H3 and H6 without modification ( top; see Video 2 ) .",
"When a front part of the density map was sectioned off with a clipping plane ( red mesh ) , the H1 helix ( blue ) fitted into a rod-like density very well ( bottom ) .",
"( D ) Pseudoatomic model of MAVS CARD filament .",
"Dashed lines indicate the inter- ( red ) and intra-strand ( yellow ) interaction interfaces . DOI: http://dx . doi . org/10 . 7554/eLife . 01489 . 00310 . 7554/eLife . 01489 . 004Figure 1—figure supplement 1 . Processing of cryoEM images of the MAVS CARD filaments .",
"( A ) Left , summed power spectrum from a set of horizontally centered particles; right , summed power spectrum from 360 projections of the final cryoEM map of MAVS CARD filament .",
"To better exhibit the weak signal within the resolution range of 16 . 8 to 8 . 2 Å ( red circle ) , the power spectrum from the cryoEM map ( right side ) was presented after the application of a square root function .",
"The red arrows point to the four characteristic layer lines that were seen in the raw data ( left panel ) .",
"( B ) The first 12 Eigen images after the multivariate statistical analysis of the boxed segments from individual MAVS CARD filaments .",
"( C and D )",
"Convergence of the two helical symmetry parameters: the azimuthal rotation ( ΔΦ in C ) and the axial rise ( Δz in D ) .",
"In the middle of the refinement , changes in axial rise were manually imposed twice to check the robustness of the convergence .",
"( E ) The Fourier shell correlation ( FSC ) between two 3D reconstructions that were calculated independently from the top and bottom halves of the dataset ( ‘Materials and methods’ ) .",
"The estimated resolution at FSC = 0 . 5 is 9 . 6 Å .",
"( F ) Representative class averages of MAVS CARD filaments after multivariate statistical analysis and hierarchical classification .",
"( G ) When the four-start helical symmetry ( n=4; −4 was the mirrored symmetry ) was used in the IHRSR , the refinement did not converge to the correct axial rise ( 17 . 1 instead of 16 . 7 Å as in 1A ) .",
"Full refinements with n=−4 led to maps with densities averaged out , and no features corresponding to the secondary structures showed up either . DOI: http://dx . doi . org/10 . 7554/eLife . 01489 . 00410 . 7554/eLife . 01489 . 005Figure 1—figure supplement 2 . Structural model of MAVS CARD filaments and its chirality determination by cryo electron tomography ( cryo-ET ) .",
"( A ) A cross-eye stereo view of the cryoEM map of the MAVS CARD filament with multiple copies of the MAVS CARD X-ray models ( PDB: 2VGQ ) docked in position , showing the left-handed three-stranded helix .",
"Colored arrows mark the three strands .",
"( B ) The projection image calculated from a 2 . 7 nm slice out of the cryo-ET reconstruction as viewed from the outer surface of the filaments .",
"Yellow arrows point to portions of multiple filaments where apparent helical stripes were resolved .",
"Three zoomed views from filaments of different orientations display a well-matched left-handedness and groove dimension with the cryo-EM map .",
"The measured distance spanning four stripes ( ∼7 layers ) is ∼118 angstroms and the measured angle between the helical strips and the helical axis is ∼50° , both consistent with the cryoEM map . DOI: http://dx . doi . org/10 . 7554/eLife . 01489 . 005 The unique filamentous structure of MAVS CARD probably results from its distinct chemical properties not shared by other CARDs .",
"To understand the structural basis underlying the filament formation , we solved the 3D structure of MAVS CARD filaments at 9 . 6 Å resolution by cryo-electron microscopy ( cryoEM ) and iterative helical real space refinement ( IHRSR ) ( Frank , 2006; Egelman , 2007 ) .",
"Based on the cryoEM map and the crystal structure of the individual MAVS CARD , we built a pseudoatomic model of the filament and identified two new CARD–CARD interfaces that are important for filament formation .",
"Mutations of residues found at the two interfaces disrupted MAVS self-association and abrogated the activation of the signaling pathway in cells .",
"In order to understand the domain arrangement of the native MAVS aggregates , we obtained a 16 . 4 Å cryoEM map of a nearly full-length MAVS protein without part of the proline-rich region ( PRR ) and the C-terminal transmembrane domain ( MAVSΔProTM ) .",
"The cryoEM map of the MAVSΔProTM filament has the same CARD filament in the center , which is surrounded by extra fragmented densities in the periphery .",
"This arrangement makes the CARD filament the organization center of the MAVS aggregates , and suggests a novel mechanism to expose the central segments of MAVS for downstream signaling effector recognition and signal amplification .",
"To visualize the full-length MAVS filaments in virus-infected cells , we obtained three-dimensional Structured Illumination Microscopic ( 3D-SIM ) images that achieved a sufficiently high resolution for us to discern the rod-shaped ultrathin MAVS filaments .",
"These native thin filaments are on average ∼400 nm long and usually seen among mitochondrial membranes .",
"In accordance with the cryoEM studies , point mutations that disrupted MAVS filament formation abrogated the redistribution and aggregation of MAVS on mitochondrial membrane and blocked the induction of interferon-β ( IFNβ ) in response to RNA virus infection .",
"These results elucidate the structural mechanism for the formation of functional MAVS filaments ."
],
[
"Our previous electron microscopic ( EM ) images of negatively stained specimens suggested that the MAVS CARD assembles into a filament-like structure in vitro ( Hou et al . , 2011 ) .",
"To further uncover the molecular mechanism governing the MAVS CARD self-association , we utilized cryoEM to determine the molecular structure of the CARD filament .",
"Flag-tagged MAVS CARD ( residues 1–100 ) was expressed in HEK293T cells and purified to apparent homogeneity ( Figure 1B ) .",
"The purified protein formed filaments that eluted from gel filtration column in the void volume .",
"CryoEM images of the purified filaments showed helical diffraction ( Figure 1—figure supplement 1A , left ) .",
"We selected good EM micrographs and high-quality Falcon Direct Detector images , and built a large dataset for IHRSR analysis .",
"IHRSR describes the helical symmetry with a general definition of azimuthal rotation ( ΔΦ ) and axial displacement ( Δz ) per subunit relative to the helical axis .",
"Individual datasets from different sessions of data collection were first analyzed separately to confirm that the total power of centered particles in each dataset showed the typical layer lines ( Figure 1—figure supplement 1A , left ) .",
"Good datasets showed a clear meridional line at ∼16 . 7 Å ( layer line #9 ) , which is a good estimate of Δz .",
"Different datasets were scaled and merged into a large set by their symmetry parameters ( Δz; details in the ‘Materials and methods’ ) .",
"The final dataset contained 48 , 884 particles .",
"Multivariate statistical analysis of the images revealed obvious helical properties and imperfection in some of the Eigen images ( Figure 1—figure supplement 1B ) .",
"The IHRSR analysis of the dataset started with a featureless cylinder as the initial reference and converged to a stable solution ( Figure 1—figure supplement 1C , D ) .",
"After sorting the filaments to consider variations in helical symmetry and accounting for the filaments that may be tilted out of the horizontal plane by up to 15° , we calculated a final map from 15 , 366 boxed segments of filaments .",
"The resolution of the map was estimated to be 9 . 6 Å from Fourier Shell Correlation ( FSC ) between two independently calculated 3D reconstructions ( FSC = 0 . 5; Figure 1C , Figure 1—figure supplement 1E , F; and Video 1 ) .",
"The cryoEM map shows a three-stranded helical assembly with a central pore that is about 18 Å in diameter .",
"Neighboring subunits in each strand are related by an azimuthal rotation angle ( ΔΦ ) of 53 . 6° and an axial rise ( Δz ) of 16 . 8 Å along the helical axis ( Figure 1C ) . 10 . 7554/eLife . 01489 . 006Video 1 . 3D reconstruction of the MAVS CARD filament . DOI: http://dx . doi . org/10 . 7554/eLife . 01489 . 006 In order to determine the handedness of the helical assembly of MAVS CARD , we obtained cryo-electron tomograms ( cryo-ET ) of the CARD filaments ( ‘Materials and methods’ ) .",
"Figure1—figure supplement 2B shows a 2 . 7 nm-thick slice of the tomogram as viewed from the outer surface of the filaments .",
"Apparent helical stripes observed in multiple filaments of different orientations suggest that the actual helical structure is left-handed .",
"The measured distance between the helical strands from the cryo-ET reconstruction matches well with the cryo-EM reconstruction of MAVS CARD filament ( Figure 1—figure supplement 2A ) .",
"Although the MAVS filaments share some features with prion fibers , such as self-perpetuation , they could not be stained with Congo red , a dye commonly used to label β sheet-rich insoluble amyloid aggregates formed by most prions ( Hou et al . , 2011 ) .",
"MAVS CARD lacks the glutamine/asparagine-rich regions that are responsible for forming the parallel β-sheet structures of most amyloid fibers ( Michelitsch and Weissman , 2000; Nelson et al . , 2005 ) .",
"There is no evidence that during filament formation MAVS CARD undergoes a helix-to-beta-sheet transition in its secondary structure .",
"We therefore built a model of MAVS oligomer by directly fitting the crystal structure of individual MAVS CARD into the cryoEM density map ( Video 2; Potter et al . , 2008 ) . 10 . 7554/eLife . 01489 . 007Video 2 . Building a pseudoatomic model from the cryoEM map . The cryoEM map of the MAVS CARD filament is shown at two different density levels together with the pseudoatomic model , and individual units are assembled into a long filament . DOI: http://dx . doi . org/10 . 7554/eLife . 01489 . 007 At the peripheral surface of the cryoEM map , three rod-shaped EM densities are clearly discernable ( Figure 1C; Video 2 ) , which are likely contributed by three of the six α-helixes of each CARD molecule .",
"These features enabled us to determine a unique orientation of the crystal structure of the MAVS CARD ( residues 1–93 ) in the cryoEM map .",
"Recognition of these structural features also allowed for independent confirmation of the chirality to the cryoEM map because only the left-handed map allowed the three helices ( H1 , H4 and H3 ) to be positioned well into the density map without modification ( Video 2 ) .",
"After the docking of these three helices , the 6th helix ( H6 ) was naturally fit into a rod-shaped feature next to the H1 .",
"Rigid body refinement using SITUS ( Wriggers , 2010 ) then locally optimized the agreement between the X-ray model and the cryoEM map ( insets in Figure 1C; Video 2 ) .",
"Even though both H2 and H5 are contained in the density map , there is a small discrepancy between their orientations and the density features lining the inner pore of the filament ( Video 2 ) , suggesting a possible local rearrangement of the CARD domain upon filament formation ( Figure 1C ) .",
"Because the rearrangement of H2 and H5 is not well defined by the density features and there was a small density next to H5 that is not fully accounted for due to probably inexact segmentation of the helical density map into individual units , we did not use flexible fitting to optimize the local positions of these two helices .",
"Helical symmetry operations and threefold symmetry were subsequently applied to generate a pseudo-atomic model for the CARD filament ( Figure 1C; Video 2 ) .",
"Overall the MAVS CARD filament adopts a densely packed structure containing three intertwined helical strands .",
"Each turn of one helical strand contains 6 . 7 ( 360/53 . 6 ) CARD monomers .",
"The first ( H1 ) and last ( H6 ) helices of the CARD are positioned at the outer surface of the filament ( Figure 1C; Video 2 ) .",
"This arrangement provides the structural basis for other domains to be connected to the filaments in the center , in keeping with previous observations that a N-terminal small ubiquitin-like modifier ( SUMO ) tag and other domains added to the C-terminus of CARD did not prevent filament formation ( Hou et al . , 2011 ) .",
"There is some unoccupied density at the surface of the EM map next to the N- and C-termini of the docked CARD model .",
"This may be attributed to the residual density from the N-terminal Flag tag and the additional seven residues at the C-terminus in the protein used for preparing the cryoEM specimens , but not in the crystal structure ( Figure 1C ) .",
"In the structural model of the filament , each MAVS CARD monomer directly interacts with four nearby monomers: two from the same strand and the other two respectively from two adjacent strands ( Figure 1D ) .",
"Within one layer perpendicular to the helical axis , the three subunits do not make direct contact with each other .",
"Two types of interfaces are involved in the packing interactions .",
"The intra-strand interface makes contacts between adjacent CARDs within the same strand ( Figure 1D , Figure 1—figure supplement 2A ) while the inter-strand interface holds the three strands together .",
"At the inter-strand interface , the positive and negative charges are alternately distributed at two opposite ends of each CARD subunit , indicating that the inter-strand interaction is mainly electrostatic ( Figure 2A , B ) .",
"The positively charged residues R37 in the 3rd helix ( H3 ) , R64 and R65 in the loop between the 4th and 5th helices ( H4 and H5 ) from the CARD in a lower layer ( purple in Figure 2B ) are in close proximity to the negatively charged residues D23 in the loop between the 1st and 2nd helices ( H1b and H2 ) and E26 in the 2nd helix ( H2 ) of another CARD molecule ( cyan in Figure 2B ) in the upper layer .",
"E26 , R64 and R65 are highly conserved among MAVS molecules from different species , but D23 and R37 are less so ( Figure 2A ) .",
"Replacement of D23 with a histidine residue and R37 with either an asparagine or a serine residue in the MAVS orthologs introduces good H-bonders to these less conserved positions , and may compensate for the lost electrostatic pairs . 10 . 7554/eLife . 01489 . 008Figure 2 . Charged residues are conserved at the inter-strand interface of MAVS CARD filament .",
"( A ) Sequence alignment of the MAVS CARD from 10 different species with the secondary structures based on the X-ray model of human MAVS CARD .",
"The alignment was generated with ClustalW ( http://www . ebi . ac . uk/clustalw/ ) and formatted using ESPript ( http://espript . ibcp . fr/ESPript/ESPript/ ) .",
"The colored arrowheads mark the residues mutated in this study: pink , key negatively charged residues at the inter-strand interface; blue , key positively charged residues at the inter-strand interface; cyan , key residues at the intra-strand interface; black , residues showing normal activity when substituted with alanine .",
"( B ) MAVS CARD hexamer model with key residues at the inter-strand interface between two subunits ( purple and cyan ) shown as yellow sticks .",
"The inset on the right side is an expanded view of these residues .",
"The side chains of these residues ( D23 , E26 , R37 , R64 and R65 ) in the model were optimized by testing different rotamers in Coot .",
"( C ) Effects of mutating the residues at the inter-strand interface and other conserved charged residues on MAVS activity .",
"Wild-type MAVS and CARD mutants at different positions were transiently expressed in HEK293T-IFNβ-luciferase reporter cells .",
"Cells were lysed 24 hr later , and the MAVS signaling was tested by luciferase reporter assay in a dose-dependent manner .",
"Western blot was done to monitor the expression level of the transfected MAVS proteins with α-tubulin as loading controls .",
"( D ) A stereo view of MAVS CARD model with the surface-exposed , conserved charged residues shown as sticks ( blue , positively charged residues; red , negatively charged residues ) .",
"Mutations of these surface residues , which are not involved in the inter-strand interactions , do not impair MAVS signaling . DOI: http://dx . doi . org/10 . 7554/eLife . 01489 . 00810 . 7554/eLife . 01489 . 009Figure 2—figure supplement 1 . Mutations at the inter-strand interface disrupt MAVS CARD polymerization .",
"( A ) Size-exclusion chromatography of recombinant wild-type human MAVS CARD and the E26R mutant .",
"Aliquots of the fractions were analyzed by silver staining ( left ) or Coomassie blue staining ( right ) .",
"Wild-type MAVS CARD eluted at the void volume ( ∼0 . 8 ml ) , while the soluble mutant E26R eluted at ∼1 . 9 ml .",
"( B ) Negative-stained EM images of human MAVS CARD E26R .",
"No filaments were observed .",
"Scale bar , 100 nm .",
"( C ) Superimposition of the crystal structure of wild-type human MAVS CARD ( PDB: 2VGQ , yellow ) with those of the horse MAVS CARD E26R ( PDB: 4O9L , magenta ) and R64C mutants ( PDB: 4O9F , blue ) .",
"The Cα atoms of E26R and R64C in the crystal structures are shown in sphere representations ( red and blue , respectively ) .",
"( D ) Mapping of the important residues for MAVS polymerization obtained from solubility screen to the crystal structure .",
"Most of them are located at the two interfaces of the MAVS CARD filament . DOI: http://dx . doi . org/10 . 7554/eLife . 01489 . 009 To test the energetic contributions of these charged residues to the stability of the filament and the in vivo MAVS signaling activity , we mutated them individually to an alanine residue or residues with reversed charges .",
"The cDNAs encoding these MAVS mutants were transiently expressed in HEK293T cells together with an IFNβ luciferase reporter plasmid .",
"IFNβ induction was measured by a luciferase assay .",
"Compared with the wild-type MAVS , which potently induced IFNβ in a dose-dependent manner , point mutations at multiple locations of the inter-strand interface abolished MAVS activity ( Figure 2C ) .",
"The gel-filtration profiles ( E26R as an example in Figure 2—figure supplement 1A ) and the EM images of the negatively stained proteins ( Figure 2—figure supplement 1B ) verified that these mutations impaired the filament formation of the MAVS CARD .",
"X-ray crystallographic studies of several horse MAVS CARD mutants , which were monomeric in solution and yielded well diffracting crystals , showed that they adopt almost exactly the same structure as the wild-type protein ( e . g . , Figure 2—figure supplement 1C for the horse E26R and R64C structures; Table 1 ) , suggesting that these mutations did not disrupt the filament by changing the structural fold of each subunit , but instead by altering the interface chemistry . 10 . 7554/eLife . 01489 . 010Table 1 . Summary of MAVS CARD mutantsDOI: http://dx . doi . org/10 . 7554/eLife . 01489 . 010HumanHorseResiduesMutantsScreenSolubilityActivityMutantsScreenSolubilityCrystalF16F16ANF16HYF16I✓++D23D23APD23N✓E26E26A++NE26R+++NE26R++✓E26R/R64ENY30Y30ANY30FYY30HYY30C✓+R37R37ANR37K✓A44A44D✓A44T++A44T✓++✓L48L48AYL48DNL48KNR52R52APD53D53ANW56W56A++NW56FYW56YYW56DNW56E+++W56R✓++R64R64ANR64E+++NR64Q✓++R64C++R64C✓+++✓R64S✓+++✓R65R65ANR65ENR65Q✓R65S✓++R65H✓*Solubility is based on the estimated final yield of the purified protein per liter of bacterial culture .",
"+++: >5 mg/L; ++: 1–5mg/L; +: <1 mg/L .",
"†Activity: N , no activity; P , partial activity; Y , activity close to wild-type .",
"To test the specificity of the electrostatic interactions at the MAVS CARD assembly interface , we mutated several charged residues outside the interface and assessed their effects on MAVS activity .",
"E70 and R77 in the H5 , E80 in the H5-H6 loop , as well as D86 and E87 in the H6 are outside the interfaces ( Figure 2D ) .",
"Their replacement with alanine or reversely charged residues still allowed potent induction of IFNβ production in HEK293T cells ( Figure 2C ) .",
"In contrast , D40 , R41 and R43 are located in the H3 and very close to the inter-strand interface .",
"Their mutations did exert strong negative effects on MAVS signaling ( Figure 2C ) .",
"The exact packing of these three residues and their contribution to the inter-strand interface require better resolution of the cryoEM map .",
"The cryoEM model predicts that the intra-strand interface is stabilized mainly by hydrophobic interaction or hydrogen bonds among residues F16 , W56 , Y30 , D53 and L48 ( Figure 3A ) .",
"W56 is well conserved ( leucine residue in horse ) .",
"In the crystal structure of MAVS CARD , the side chain of W56 adopts different conformations , with a major conformer ( 60% occupancy ) exposed to solvent and stacked against the ring structure of residue F16 in immediate vicinity ( magnified view on the right side of Figure 3A ) ( Potter et al . , 2008 ) .",
"Y30 is a fairly conserved residue ( histidine in cattle ) that protrudes to the close proximity of W56 in the other CARD molecule at the interface .",
"Even though the atomic details of the side chain packing are not fully resolved , the three ring structures from these three residues appear to be important for the intra-strand stability .",
"Single alanine substitution of F16 , W56 or Y30 almost completely abolished MAVS activity ( Figure 3B ) , but mutations of some of the residues next to them ( R52 and S49 as examples ) had little effect ( Figure 2C , Figure 3B ) .",
"Interestingly , when we introduced different ring-containing residues to these positions , such as F16H , W56F , W56Y , Y30F and Y30H , we were able to largely rescue MAVS activity ( Figure 3B ) .",
"These results support that the hydrophobic packing among the three ring-containing residues is important for the intra-strand interface .",
"D53 is one helical turn below W56 , and its side chain sits at the boundary between the aqueous phase and the hydrophobic core packing of F16 , W56 and Y30 .",
"When we introduced an alanine to this position , it completely abolished the MAVS activity ( Figure 3B ) , suggesting that the charged D53 may enforce the hydrophobic interaction above the aqueous surface .",
"Close to the central pore of the helical filament , residue L48 appears in close contact with a group of short chain residues and contributes to a separate hydrophobic patch .",
"Even though atomic details in side chain packing are unclear , substitution of L48 with charged residues ( D or K ) , but not alanine , completely abolished MAVS activity ( Figure 3B ) , suggesting that this second hydrophobic patch is also important for intra-strand interaction . 10 . 7554/eLife . 01489 . 011Figure 3 . The intra-strand interface is mediated by hydrophobic interactions and hydrogen bonding .",
"( A ) MAVS CARD hexamer model with interacting residues at the intra-strand interface shown as yellow sticks .",
"Residues that showed normal activity when substituted with alanine are displayed in brown .",
"( B ) MAVS proteins with point mutations at the intra-strand interface were tested for IFNβ induction and protein expression as in Figure 2C . DOI: http://dx . doi . org/10 . 7554/eLife . 01489 . 011 To further test whether these mutations impaired the MAVS signaling activity because they interfered with CARD polymerization , CARD mutants were purified from Escherichia coli and their oligomerization states were examined .",
"Unlike the wild-type MAVS CARD , which eluted at the void volume in gel filtration chromatography , W56A , W56E and W56R mutants eluted as monomers ( Table 1; Figure 3A ) .",
"These results indicate that mutations that abrogate hydrophobic interactions at the intra-strand interface prevent MAVS CARD oligomerization and abolish MAVS activity .",
"According to our pseudoatomic model , residues in the first helix ( H1 ) are not in direct contact with any adjacent molecule ( Figure 1C; Video 2 ) .",
"This agrees with our previous finding that deletion of the first ten residues in MAVS CARD did not impair its filament formation ( Hou et al . , 2011 ) .",
"In addition , no cysteine pairs are at the interface , which explains the prior observation that MAVS filaments were not disrupted by a high concentration of reducing agent DTT ( Hou et al . , 2011 ) .",
"As an alternative verification of the key residues for MAVS CARD self-association , we searched for mutants that disrupt the CARD polymerization in E . coli ( Table 1 ) .",
"We performed random mutagenesis and screened for soluble CARD mutants by using a recently developed solubility screen ( Harada et al . , 2008 ) .",
"Mutated CARDs fused to murine dihydrofolate reductase ( mDHFR ) were expressed .",
"While mDHFR fused to wild-type CARD appeared in inclusion bodies due to CARD oligomerization , it remained soluble when it was fused a CARD mutant that failed to oligomerize .",
"Expression of soluble mDHFR-CARD was selected by trimethoprim , which specifically inhibits bacterial DHFR , but not mDHFR .",
"Our screen recognized multiple sites that are important for CARD oligomerization ( Table 1; Figure 2—figure supplement 1D ) .",
"Biochemical analysis confirmed that most mutants from the screen were expressed as soluble proteins , and eluted as homogenous monomers in gel-filtration chromatography ( e . g . , Figure 2—figure supplement 1A ) .",
"Almost all the mutations out of the solubility screen are conserved and at the protein surface .",
"They can be mapped to the two interfaces identified in the cryoEM model ( Figures 2 and 3 ) .",
"Previously we have shown that a deletion mutant of MAVS ( MAVSΔProTM; Figure 1A ) lacking part of the proline-rich region ( PRR , residues 103–153 ) and the C-terminal transmembrane domain ( TM; residues 461–540 ) allowed the production of a large amount of protein from E . coli , and remained capable of inducing wild-type MAVS to form fibers and potently activate IRF3 dimerization in cytosolic extracts ( Hou et al . , 2011 ) .",
"Compared to full-length MAVS , MAVSΔProTM is soluble when fused with SUMO , and can form functional polymers after removal of SUMO .",
"Because MAVSΔProTM resembles the soluble part of the wild-type MAVS , its filament structure probably closely represents the polymerization of endogenous MAVS on mitochondria .",
"When we compared the EM images of the MAVSΔProTM filaments with those of the CARD filaments , the former apparently had extra mass and were larger in diameter than the latter ( Figure 4A , Figure 4—figure supplement 1 ) .",
"We built a cryo dataset of 8909 boxed segments ( particles ) out of individual MAVSΔProTM filaments .",
"The summed power spectrum of the raw particles exhibited a similar pattern of layer lines as the CARD filaments ( Figure 4—figure supplement 1B vs Figure 1—figure supplement 1A ) .",
"We calculated a cryoEM map of MAVSΔProTM at a nominal 16 . 4 Å resolution ( FSC0 . 5; Figure 4—figure supplement 1C ) .",
"Despite the extra mass , the MAVSΔProTM map is surprisingly similar to that of the CARD filament ( Figure 4B ) .",
"When a 90 Å-thick portion in the middle of the MAVSΔProTM map was compared with the map of the CARD filament ( gray surface vs purple mesh in Figure 4B ) , the two overlapped very well at various threshold levels , suggesting that the core of the MAVSΔProTM fiber takes the same left-handed three-stranded helical structure .",
"The symmetry parameters , a rotational angle of 52 . 9° and an axial rise of 16 . 9 Å , are almost the same as those of the CARD filament ( Figure 1C ) . 10 . 7554/eLife . 01489 . 012Figure 4 . CARD filament is the organization center of the MAVS Filament .",
"( A ) Segments from negative-stain EM images of ( 1 ) Flag-MAVS CARD and ( 2 ) MAVSΔProTM and those from cryoEM images of ( 3 ) Flag-MAVS CARD and ( 4 ) MAVSΔProTM .",
"For better visualization , protein is black in the cryoEM images .",
"Yellow arrows point to the extra mass that made the MAVSΔProTM filaments larger in diameter .",
"( B ) Side and top views of the cryoEM reconstruction of MAVSΔProTM filament ( gray , surface ) , which has the same helical symmetry as the MAVS CARD filament ( purple , mesh ) .",
"Only the middle 90 Å portion of the MAVSΔProTM map is shown .",
"( C ) Side and top views of the full MAVSΔProTM map ( gray , surface ) whose threshold was set at a proper level to overlap well with the CARD-only map ( purple , mesh ) .",
"A cylindrical sheet of extra density appeared at ∼15 Å away from the central CARD filament .",
"( D ) Schematic view of the helical packing of MAVSΔProTM with the sequences C-terminal to the CARD shown as dashed coils . DOI: http://dx . doi . org/10 . 7554/eLife . 01489 . 01210 . 7554/eLife . 01489 . 013Figure 4—figure supplement 1 . Image processing for the MAVSΔProTM filaments .",
"( A ) A typical cryoEM image of MAVSΔProTM filaments ( protein is black ) .",
"Scale bar , 100 nm .",
"( B ) Summed power spectrum of boxed segments of MAVSΔProTM filaments after central alignment along X-axis ( left ) is compared with the modulus image ( square root of power spectrum ) of the averaged power spectrum calculated from 360 projections of the final 3D map ( right ) .",
"Similar to Figure 1–figure supplement 1A , the square root function enhanced the signal to noise ratio for the resolution range beyond 16 . 8 Å .",
"The red arrows point to the characteristic layer lines seen in the power spectrum of the raw data .",
"( C ) Fourier Shell Correlation ( FSC ) between two independently calculated maps from two halves of the data .",
"The estimated resolution is 16 . 4 Å at FSC = 0 . 5 .",
"( D ) 10 class averages after 5-round hierarchical classification of the dataset .",
"Outside the central CARD filament density , there were clear densities at the neighboring region ( red arrow heads ) , which were probably contributed by the intervening segment between the CARD and the TM domain .",
"( E ) Two views of the 3D map of MAVSΔProTM before symmetry imposition .",
"The helical symmetry is relatively robust , and the extra density surrounding the CARD filament is fairly strong ( bottom panel ) .",
"The scale bars in both panels = 12 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 01489 . 013 When the whole MAVSΔProTM reconstruction is presented at a proper contour level so that the middle CARD filament has the same volume as the CARD-only filament , a cylindrical sheet of periodic densities appears at a distance of ∼15 Å away from the perimeter of the central filament ( Figure 4C , Figure 4—figure supplement 1D , E ) .",
"MAVSΔProTM has ∼300 extra residues C-terminal to its CARD .",
"Some of these extra residues most likely contribute to the peripheral densities in the cryoEM map .",
"These extra densities were clearly visible in the class averages calculated from the aligned filament images ( red arrowheads in Figure 4—figure supplement 1D ) .",
"Because of the intrinsic disorder in the segment between the CARD and the TM domain of MAVS , the majority of the extra residues did not produce significant density in the cryoEM map .",
"A key conclusion from this comparison is that the CARD filament is in the center of the MAVS aggregates and the rest of the molecule is connected to the peripheral surface of the filament .",
"Because the C-terminal TM domain is integrated in the mitochondrial membrane , the CARD filament and the membrane anchor the two ends of each MAVS molecule so that the intervening region is exposed to recruit cytosolic signaling effector molecules such as the TRAF ( tumor necrosis factor receptor-associated factor ) proteins ( Liu et al . , 2013; Figure 4D ) .",
"To visualize the filament formation of full-length MAVS , we stably reconstituted Mavs-null murine embryonic fibroblasts ( MEFs ) with Flag-tagged wild-type MAVS or its mutants .",
"Although transient expression of wild-type MAVS resulted in constitutive signaling ( Kawai et al . , 2005; Meylan et al . , 2005; Seth et al . , 2005; Xu et al . , 2005 ) , the low expression level in the stable cell lines did not lead to constitutive activation of downstream target genes .",
"Like endogenous protein , Flag-tagged MAVS was properly localized to the mitochondrial membranes , as demonstrated by its co-localization with either MitoTracker or TOM20 , a 20 kDa subunit of the translocase in the outer mitochondrial membrane ( Figure 5—figure supplement 1 , 3A , Mock ) .",
"Infection by Sendai virus induced the redistribution of MAVS protein and the formation of densely packed , speckled MAVS puncta on the surface of mitochondria , along with the nuclear translocation of NF-κB subunit p65 and induction of interferon-β ( IFNβ; Figure 5—figure supplement 1 , 3 ) .",
"In addition , Sendai virus also induced MAVS aggregation , which was detected by semi-denaturing detergent agarose electrophoresis ( SDD-AGE; Figure 5—figure supplement 3D; Hou et al . , 2011 ) .",
"Based on the nuclear translocation of p65 after viral infection , these bright MAVS puncta were observed in a majority of virus-infected cells ( Figure 5—figure supplement 3B ) .",
"In contrast , the bright puncta did not form in cells expressing MAVS mutants that failed to form filaments ( Figure 5—figure supplement 1 , 2 and 3A ) .",
"As negative controls , mutants ( E80A and F16H ) that do not affect the MAVS filament formation were found to have normal puncta formation ( Figure 5—figure supplement 1 , 2 and 3C ) .",
"Together these results support that the CARD-mediated aggregate formation is the key structural element for activating MAVS signaling in cells ( Figure 5—figure supplement 3C , D ) .",
"Because the confocal fluorescent images of the MAVS aggregates did not have enough resolution to reveal the shape of the puncta in virus-infected cells , we next performed the experiments by Super-Resolution Structured Illumination Microscopy ( SR-SIM ) ( Gustafsson et al . , 2008 ) .",
"The resolution of conventional fluorescence microscopy is limited to ∼200 nm in lateral ( x , y ) dimensions , and ∼500 nm along the optical axis .",
"SR-SIM increases both the lateral and axial resolutions by a factor of two ( Gustafsson et al . , 2008 ) .",
"SR-SIM images of Flag-tagged wild-type MAVS showed a fairly uniform distribution on the surface of mitochondria as it appeared in concentric rings around almost every MitoTracker-stained mitochondrion ( Figure 5A , the last image in the top row ) .",
"In cells infected with Sendai virus , the SR-SIM images revealed a clear redistribution of MAVS into rod-shaped clusters that interfaced with only a small fraction of mitochondria ( e . g . , white arrowheads in the rightmost image of the bottom row in Figure 5A ) .",
"Because the expression level of MAVS in cells remained the same before and after viral infection ( Figure 5—figure supplement 3D and references ) ( Hou et al . , 2011; Liu et al . , 2013 ) , the redistribution of MAVS from one mitochondrion to another during the puncta formation may result from mitochondrial fusion ( Yasukawa et al . , 2009; Castanier et al . , 2010; Koshiba et al . , 2011 ) .",
"Alternatively , MAVS aggregates on some mitochondria may be degraded through an unknown mechanism , which is less likely because of the unchanged level of MAVS protein .",
"The diameter of the rod-shaped MAVS clusters is probably less than 100 nm , the lateral resolution limit of SR-SIM . 10 . 7554/eLife . 01489 . 014Figure 5 . In virus-infected cells MAVS redistributes and forms rod-shaped puncta on the surface of mitochondria .",
"( A ) Mavs−/− MEF cells stably expressing Flag-tagged wild-type MAVS were mock-treated or infected with Sendai virus ( SeV ) for 12 hr and stained with MitoTracker ( Mitochondria; red ) and anti-Flag antibody ( Flag-MAVS; green ) .",
"Redistribution of MAVS among mitochondria was examined using SR-SIM .",
"Expanded views of the areas within the yellow windows in the merged images were shown on the right .",
"The SeV-infected cells contain bright foci of Flag-MAVS .",
"The white arrowheads in the rightmost image of the bottom row highlight a few bright rod-shaped MAVS clusters .",
"Scale bars , 5 . 0 μm .",
"( B ) 3D reconstruction of MAVS clusters ( green ) on the surface of mitochondria ( red ) .",
"Scale bar , 1 . 0 μm .",
"The areas within the yellow windows in the merged image ( right most ) were expanded to show a few clusters that appear to bridge between mitochondrial membranes .",
"( C ) Histogram and Gaussian fit ( black curve ) of the length distribution that was measured from the SIM images of individual MAVS clusters as in panel A ( SeV; N = 74 ) in virus-infected cells . DOI: http://dx . doi . org/10 . 7554/eLife . 01489 . 01410 . 7554/eLife . 01489 . 015Figure 5—figure supplement 1 . Mutations at the inter-strand interface that disrupt CARD polymerization abolish the SeV-induced redistribution of MAVS on mitochondria . Mavs−/− MEF cells stably expressing Flag-tagged wild-type MAVS or its mutants were mock-treated or infected with SeV for 12 hr and stained with MitoTracker ( Mitochondrial matrix; red ) , anti-Flag antibody ( Flag-MAVS; green ) , anti-p65 antibody ( grey ) and DAPI ( blue ) .",
"MAVS redistribution was examined using confocal microscopy .",
"Areas within the yellow windows in the merged images are expanded and shown in the rightmost images .",
"MAVS puncta are highlighted by white arrowheads .",
"WT and E80A MAVS showed strong puncta formation and clear p65 nuclear translocation , whereas E26A , a mutation at the inter-strand interface , did not .",
"E80A is a conserved charged residue as a control .",
"Scale bar , 5 . 0 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01489 . 01510 . 7554/eLife . 01489 . 016Figure 5—figure supplement 2 . Mutations at the intra-strand interface that disrupt CARD polymerization abolish the SeV-induced redistribution of MAVS on mitochondria . Similar to the Figure 5—figure supplement 1 , mutations of two important residues ( F16 and Y30 ) at the intra-strand interface were tested in a similar fashion .",
"Scale bar , 5 . 0 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01489 . 01610 . 7554/eLife . 01489 . 017Figure 5—figure supplement 3 . Strong correlations among MAVS puncta formation , MAVS signaling and MAVS CARD polymerization . Mavs −/− MEF cells stably expressing Flag-tagged wild-type MAVS or its mutants were mock-treated or infected with SeV for 12 hr . ( A ) As in Figure 5—figure supplement 1 , MAVS puncta formation and redistribution were examined using confocal microscopy .",
"Mitochondria were stained by TOM20 antibody ( mitochondrial outer membrane ) .",
"Areas within the yellow windows in the merged images are expanded and shown in the rightmost images .",
"MAVS puncta are highlighted by white arrowheads .",
"Scale bars , 5 . 0 μm .",
"( B ) Statistical analysis of the correlation between nuclear translocation of p65 and SeV-induced MAVS puncta formation in Mavs−/− MEF cells expressing wild-type MAVS .",
"No p65 nuclear translocation was seen in uninfected cells .",
"Among cells showing clear p65 translocation , only those showing large MAVS puncta ( probably > 0 . 4 μm ) were classified as positive .",
"Even with such strict constraints , more than 70% of the cells were positive .",
"( C ) IFNβ production was rescued by expressing MAVS wild-type and mutants ( E80A and F16H ) that retain the capability of forming MAVS puncta , but not those ( E26A , F16A and Y30A ) that failed to form puncta ( Figure 5—figure supplement 1 ) .",
"The IFNβ mRNA level was quantified by q-RT-PCR .",
"( D ) MAVS proteins in the mitochondrial extracts from different cells were separated by SDD-AGE ( top ) or SDS-PAGE ( bottom ) followed by western blotting .",
"Cells expressing wild-type MAVS and mutants ( E80A , L48A , F16H , W56Y , Y30F and R77A ) that rescued IFNβ production gave rise to strong aggregate signal in the SDD-AGE assay , whereas those expressing MAVS mutants defective in forming puncta or inducing IFNβ ( E26A , R64A , R65A , F16A or Y30A ) did not form aggregates .",
"Y30A showed slightly higher basal aggregation signal , independent of viral infection . DOI: http://dx . doi . org/10 . 7554/eLife . 01489 . 017 When a series of SR-SIM images were combined in IMARIS to reconstruct a 3D volume of MAVS and mitochondria in virus-infected cells ( Figure 5B ) , it became clear that the MAVS aggregates were not evenly distributed around individual mitochondria , but were clustered into narrow regions on the surface of mitochondria ( the rightmost image in Figure 5B ) .",
"Many of these rod-shaped clusters bridge between two or more mitochondrial membranes , suggesting that MAVS molecules from multiple mitochondria may contribute to the formation of one MAVS filament ( magnified insets of Figure 5B ) .",
"To quantify the average number of MAVS molecules for the rod-shaped clusters , we measured the length distribution of many observed clusters in the 2D SR-SIM images ( Figure 5A , C ) .",
"The mean filament length in the observed Poisson distribution is approximately 400 nm ( full width at half maximum , or FWHM , n = 74 ) and the longer ones are ∼800 nm .",
"Based on the cryoEM map , each 400 nm filament contains approximately 720 MAVS molecules ."
],
[
"The pseudoatomic model for the MAVS CARD filaments from our cryoEM study suggests that after viral infection , activated MAVS molecules on the mitochondrial surface interact with each other at both the intra- and inter-strand interfaces between their CARD domains ( Figure 6 ) .",
"The MAVS aggregates in cells are indeed rod-shaped clusters that may contain MAVS molecules from multiple mitochondria .",
"The CARD filaments form the central elements of the MAVS aggregates , and can promote their own growth by attracting new CARDs into the pre-poised interaction interfaces .",
"The filaments are localized on the mitochondrial surface and the MAVS TM domains are embedded inside the outer mitochondrial membranes .",
"These two ends of each MAVS molecule provide important spatial constraints that may force the intervening coiled sequence to be well extended and exposed for recruiting down-stream signaling molecules ( Liu et al . , 2013; Figure 6 ) .",
"Bioinformatic analysis suggests that the middle segment of MAVS forms random coils , which , if present by themselves in aqueous phase , would not likely be fully extended and thus may deter efficient binding of multiple positive or negative regulators of MAVS .",
"The spatial arrangement between the CARD filament and the mitochondrial membrane provides a good solution to this problem of intrinsic disorder .",
"The prion-like filament formation of MAVS thus uses very different chemistry than other prion proteins , and orchestrates the signaling domains of MAVS into a high-affinity platform for rapid and efficient signaling . 10 . 7554/eLife . 01489 . 018Figure 6 . A working model for MAVS activation by RIG-I/RNA complexes . Detection of 5’-pppRNA by RIG-I or dsRNA by MDA5 or RIG-I triggers the formation of RIG-I ( or MDA5 ) /RNA/polyUb complex .",
"The CARD domains of individual complexes are poised properly to attract three MAVS CARD domains and support the nucleation of the filament .",
"In the resting state , MAVS CARD is sequestered and has a low probability of forming polymers .",
"The RIG-I ( or MDA5 ) /RNA/polyUb complexes stabilize the MAVS CARDs in the exposed state and bring three copies together to initiate the filament formation .",
"Once started , a short MAVS CARD filament promotes its own elongation by attracting more MAVS CARDs into the assembly .",
"The filament can form on the surface of one mitochondrion or between two or more mitochondrial membranes .",
"Inset , one or more mitochondria might be involved in MAVS filament formation . DOI: http://dx . doi . org/10 . 7554/eLife . 01489 . 018 The filament formation of MAVS CARDs is based on collective interactions at the four interfaces of each subunit ( Figures 1D , 2B and 3A ) .",
"These interfaces maintain the tight and dense packing of individual CARDs in the filaments , which , together with the strong inter-strand electrostatic interactions , probably make the filaments detergent-resistant .",
"During filament formation , there are counter-acting forces , such as decrease in entropy and possible structural adjustment at the pore-lining surface of the CARD filament ( Figure 1C; Video 2 ) , which would increase the energy level of the filaments .",
"The net decrease in free energy supporting the filament formation is due to the four interaction interfaces for every CARD .",
"But the counter-balance of these positive and negative energy terms likely makes the filaments fairly sensitive to mutations .",
"Indeed , the four interfaces appear to be dominated by hot spots because point mutations in multiple positions are capable of destabilizing the filaments .",
"The open ends of the CARD filaments provide three binding sites for each incoming CARD molecule .",
"Because of the net gain in free energy for each CARD , the longer filaments are expected to be more stable than the shorter ones , leading to the filaments that are up to 10 microns long under cryoEM conditions .",
"Inside virus-infected cells , the average length of the rod-shaped MAVS clusters is merely ∼400 nm .",
"The physical length of MAVS filaments in cells may be limited by the number of MAVS molecules in each mitochondrion and by the dynamics of mitochondrial fusion and fission ( Onoguchi et al . , 2010; Figure 5C ) .",
"The three-stranded helical filaments at the center of the MAVS aggregates and the complete separation of three CARD units in each layer suggest that a successful initiation of filament formation needs at least two independent molecules ( Figure 6 ) .",
"These independent molecules need to be juxtaposed in order to poise three CARDs to form the first layer of the filament .",
"The tandem CARDs in one RIG-I molecule are restrained by a short α-helix running between them , and are less likely to serve this role .",
"There are two possible scenarios for two RIG-I/RNA complexes to join for initiation: ( 1 ) Two individual RIG-I/RNA monomers can come together; or ( 2 ) RIG-I molecules may form dimers or oligomers along short duplex RNAs .",
"Crystallographic studies of RIG-I ( full-length or truncated ) have led to the proposal that the RIG-I/RNA complex might act as monomers in conjunction with polyubiquitin to activate MAVS ( Civril et al . , 2011; Hild et al . , 2011; Jiang et al . , 2011; Kowalinski et al . , 2011; Lee et al . , 2011; Luo et al . , 2012 , 2013 ) .",
"Should two or more RIG-I/short RNA monomeric complexes be able to nucleate a MAVS filament , we would expect the short RNAs ( 10–17 bp ) used in the crystallographic studies to activate the pathway .",
"But the published data did not lead to a consensus on the activities of these short RNAs ( Fujita , 2009; Schlee et al . , 2009; Marq et al . , 2011; Kohlway et al . , 2013 ) .",
"Recent biochemical data showed that RIG-I tandem CARDs and K63 polyubiquitin form relatively stable 4:4 oligomers , which may nucleate the helical filamentous structure formed by the MAVS CARDs ( Figure 6; Jiang et al . , 2012 ) .",
"In a similar fashion , the RIG-I ( or MDA5 ) /RNA helical complex may present multiple CARD domains on its periphery where multiple MAVS filaments may be initiated ( Berke and Modis , 2012; Wu et al . , 2013 ) .",
"Moreover , negative-stain EM studies have suggested either parallel or anti-parallel dimers to be the active form of RLRs ( Murali et al . , 2008; Ranjith-Kumar et al . , 2009 ) .",
"Structural studies of active RIG-I-RNA and RIG-I-MAVS complexes are needed to provide a detailed mechanism by which RIG-I initiates MAVS polymerization .",
"Protein oligomerization mediated by the death domain ( DD ) superfamily , which include CARDs , has been studied in multiple signaling proteins .",
"For example , the helical assembly of MyDDosome was proposed to use three different types of DD–DD interfaces ( Lin et al . , 2010; Gay et al . , 2011; Kersse et al . , 2011 ) , none of which share any similarity to the intra- or inter-strand interfaces in the MAVS CARD filament ( Figure 1D ) .",
"The MyDDosome formation is thought to bring multiple kinase domains of the IRAKs together for efficient activation .",
"This affinity-enhancing scheme for recruiting downstream effectors is essentially the same as in the MAVS filaments .",
"The much longer MAVS filaments would thus entail a significant amplification of the signal that nucleates their formation and enhance the RLR sensitivity to viral RNAs .",
"Because TRAF2 and TRAF3 both form trimers ( Park et al . , 1999; Ni et al . , 2000; Zheng et al . , 2010; Napetschnig and Wu , 2013 ) , MAVS filaments may enhance the TRAF-MAVS interaction through increased avidity , and boost the signaling efficacy significantly .",
"The redistribution of MAVS molecules on the surface of mitochondria and the possible bridging of multiple mitochondria by the MAVS filaments ( Figures 5 , 6 ) were apparently a result of the filament formation .",
"The filament growth needs to bring randomly distributed MAVS into close vicinity .",
"The limited number of MAVS molecules per mitochondrion suggests that the long MAVS filaments may be contributed by multiple mitochondria .",
"The clustered MAVS molecules become sequestered during the dynamic mitochondrial fission and fusion cycles .",
"The fusion of sequestered clusters leads to the redistribution of MAVS from being ubiquitously present in almost all mitochondria to being segregated on a small number of them .",
"The self-promoting nature of in vivo MAVS filaments may be the driving force that leads to almost all MAVS being incorporated into filaments ( Figure 5C , Figure 6 ) .",
"Further analyses of MAVS redistribution and its correlation with mitochondrial fusion/fission dynamics by live-cell imaging and computational analysis may provide more detailed insights into this process .",
"In summary , our structural and functional studies of the MAVS polymerization revealed the unique three-stranded filaments and identified the interaction interfaces that drive the formation of self-perpetuating prion-like fibers .",
"The structural organization of the MAVS filaments on mitochondrial membranes is distinct from other oligomeric assemblies such as the MyDDosome and β-amyloid prions .",
"This virtually irreversible polymerization of MAVS provides a highly sensitive and robust mechanism of immune response .",
"Such digital ( i . e . , all or none ) response allows the organisms to defend against noxious agents such as lethal infections ."
],
[
"Mouse antibody against Flag-tag ( M2 ) and M2-conjugated agarose were purchased from Sigma-Aldrich ( St . Louis , MO ) ; rabbit antibodies against TOM20 and the p65 subunit of NF-κB were from Santa Cruz Biotechnology ( Dallas , TX ) ; Alexa Fluor 488 conjugated goat anti-mouse and anti-rabbit antibodies , Alexa Fluor 568 conjugated goat anti-mouse antibody , and Alexa Fluor 633 conjugated goat anti-rabbit antibody were from Invitrogen ( Carlsbad , CA ) .",
"Sendai virus ( SeV , Cantell strain , Charles River Laboratories ) was used at 100 hemagglutination ( HA ) units/ml culture media .",
"HEK293T , HEK293T-IFNβ-luciferase , Mavs−/− MEF cells and derivatives were cultured in Dulbecco’s modified Eagle’s medium ( DMEM ) supplemented with 10% ( v/v ) cosmic calf serum ( Hyclone , Thermo Fisher Scientific , Waltham , MA ) with penicillin ( 100 U/ml ) and streptomycin ( 100 μg/ml ) .",
"Other chemicals and reagents were from Sigma-Aldrich unless otherwise specified .",
"cDNA encoding Flag-tagged MAVS CARD ( 1–100 ) has been described previously ( Hou et al . , 2011 ) .",
"For protein expression , pcDNA3-Flag-MAVS CARD was transiently transfected into HEK293T cells .",
"Cells were harvested 36 hr after transfection and lysed in a buffer containing 20 mM Tris–HCl ( pH 8 . 0 ) , 150 mM NaCl , 10% glycerol , 0 . 10% Triton X-100 , 1 . 0 mM DTT , and EDTA-free protease inhibitor cocktail ( Roche , Basel , Switzerland ) .",
"After centrifugation at 10 , 000×g for 10 min , Flag-MAVS CARD was selectively bound to M2-antibody-conjugated agarose beads and eluted by Flag peptide .",
"The eluate was fractionated on a Superdex 200 PC 3 . 2/30 column ( GE Healthcare , Uppsala , Sweden ) equilibrated in a buffer containing 20 mM Tris–HCl ( pH 7 . 5 ) , 50 mM NaCl and 1 . 0 mM DTT .",
"Fractions were analyzed by SDS-PAGE and silver staining .",
"cDNA encoding the MAVSΔProTM mutant lacking the proline-rich region ( 103–153 ) and the C terminal transmembrane domain ( 461–540 ) has been described previously ( Hou et al . , 2011 ) .",
"The bacterial expression vector pET-28a-His6-Sumo-MAVSΔProTM was transformed into BL21 ( pLys ) .",
"Protein expression was induced with 0 . 20 mM IPTG at 18°C for four hours .",
"After sonication in a lysis buffer containing 10 mM Tris–HCl ( pH 8 . 0 ) , 500 mM NaCl , 0 . 50 mM DTT , 5 . 0% glycerol , 0 . 50 mM PMSF and 10 mM imidazole , cell lysates were centrifuged at 50 , 000×g for 30 min .",
"His6-Sumo-MAVSΔProTM in the supernatant was purified using Ni-NTA affinity resin ( QIAGEN , Limburg , Netherlands ) .",
"Subsequently , the protein was loaded onto HiTrap Q HP column ( GE Healthcare ) , and then eluted with a gradient of NaCl varying from 0 . 10 M to 0 . 50 M in a buffer made of 10 mM Tris–HCl ( pH 7 . 5 ) , 5 . 0% glycerol , 2 . 0 mM DTT , 1 . 0 mM EDTA and 0 . 50 mM PMSF .",
"The fractions containing His6-Sumo-MAVSΔProTM , which were eluted with 300 mM NaCl , were pooled together and applied to a Superdex 200 HR 10/30 column ( GE Healthcare ) equilibrated with a buffer made of 10 mM Tris–HCl ( pH 8 . 0 ) , 150 mM NaCl , 1 . 0 mM DTT , 1 . 0 mM EDTA and 0 . 50 mM PMSF .",
"His6-Sumo-MAVSΔProTM was then digested with SUMO protease at 4 . 0°C overnight .",
"The His6-SUMO tag was removed by running the reaction mixture in a Superdex 200 PC 3 . 2/30 column ( GE Healthcare ) , which was equilibrated in a buffer containing 10 mM Tris–HCl ( pH 8 . 0 ) , 150 mM NaCl and 1 . 0 mM DTT .",
"The peak fraction of the protein was collected for EM studies .",
"Copper grids ( Ted Pella Inc . , Redding , CA ) coated with a layer of thin carbon film ( 3–5 nm ) were rendered hydrophilic by negative glow discharge in air .",
"A 2-4 μl aliquot of the purified MAVS sample was loaded onto the grids .",
"After 30 s of incubation on the grid at room temperature , the sample was stained with 2 . 0% phosphotungstic acid ( PTA ) at pH 8 . 0 and blotted dry .",
"Samples were imaged in a JEOL 2200FS FEG electron microscope operated at 200 kV with a nominal magnification of 50 , 000 × ( 2 . 84 Å/pixel at the detector level ) using a defocus range of −0 . 7 to −1 . 5 μm .",
"Images were recorded with an electron dose of 20 e−/Å2 on a 2K × 2K Tietz slowscan Charge Coupled Device ( CCD ) camera .",
"Quantifoil R2/2 grids ( Quantifoil Micro Tools GmbH , Jena , Germany ) were coated with a thin carbon film ( 1–3 nm ) in order to retain more filaments for imaging .",
"Right before use , the grids were negatively glow-discharged in air .",
"2 . 5 μl purified MAVS was loaded onto the grids .",
"Grids were blotted in 100% humidity at 4°C for 5 s before being plunge-frozen into liquid ethane bathed in liquid nitrogen inside a Vitrobot ( FEI , Hillsboro , OR ) .",
"After the specimens were transferred into and kept frozen inside the JEOL 2200FS FEG electron microscope , images were recorded on SO163 films ( Eastman Kodak , Rochester , NY ) with a nominal magnification of 60 , 000 × under low-dose conditions ( ∼20 e−/Å2 ) .",
"A Gatan K2 Summit Direct Detector ( Gatan , Pleasanton , CA ) was used for testing specimens in the later phase of the project .",
"A total of 358 films were developed using full-strength D19 ( Kodak ) solution .",
"Micrographs were digitized with a PhotoScan film Scanner ( Z/I Imaging GmbH , Germany ) at a step size of 7 . 0 μm .",
"After 2 × 2 binning , the pixel size was 2 . 33 Å on the specimen .",
"The magnification calibration at the nominal 60 , 000 × was 61 , 950 × and the actual pixel size was 2 . 26 Å .",
"Datasets from other imaging conditions were scaled to this condition after the axial rise of each dataset was independently determined through IHRSR .",
"A total of 30 , 384 segments were boxed from film data using the EMAN2 program HelixBoxer ( Ludtke et al . , 1999 ) .",
"To accelerate data collection , we sent the grids of the MAVS CARD filaments to the HHMI Janelia Farm Research Campus , and collected ∼2 , 100 images with a 4K × 4K Falcon Direct Detector in a Titan Krios microscope .",
"The microscope was operated at 300 kV and was equipped with a Cs corrector .",
"Automatic data collection was run by proprietary software , EPU ( FEI , Hillsboro , OR ) .",
"Images were taken under −2 . 5 to −4 . 0 microns of defocus at 29 , 000 × , which gave rise to a calibrated pixel size of 2 . 30 Å at the specimen level .",
"The density of filaments in the cryo specimens was fairly low so that we had to collect a large number of images .",
"After the evaluation of their power spectra , 1 , 088 Falcon images were selected , and a total of 18 , 500 short filaments were boxed out for analysis .",
"The analysis of the datasets of both the MAVS CARD filaments and the MAVSΔProTM filaments followed the IHRSR method developed by Dr Edward Egelman’s group at University of Virginia .",
"The method was implemented in SPIDER .",
"Dr Egelman kindly provided the programs in SPIDER , and the HSEARCH_LORENTZ/HIMPOSE programs .",
"All SPIDER programs were rewritten in order to run with SPIDER Version 19 . 08 in a Linux cluster that runs the Redhat Enterprise 5 . 0 .",
"Besides , new SPIDER programs were written to refine the map for out-of-plane tilting of the filaments and for local optimization in angle assignment .",
"Extensive technical details could be found in reference ( Mukherjee et al . , 2014 ) .",
"Briefly , from all images obtained in one session of data collection , the filaments were boxed out into 200 pixel segments ( ∼45 . 2 nm long at the specimen level ) with 90% overlapping between neighboring ones from the same long filament .",
"Filaments shorter than 200 pixels were discarded .",
"The filament helical axis was always positioned roughly along the Y-axis .",
"The defocus and astigmatism information for each image were determined by CTFFIND2 in the MRC package ( IMAGE2010 ) .",
"The filament particles ( boxed segments out of the raw filaments ) were phase-flipped and band-pass filtered .",
"We also tested the Wiener-filtering with a constant of 0 . 2 ( 1/SNR ) , and did not find significant difference from the phase-flipped dataset .",
"Afterwards the filaments from different images out of one cryo session were pooled together as a subdataset .",
"To start the analysis , we made sure that the summed power spectrum of each subdataset showed a faint ( usually fuzzy ) layer line at the position of the first meridional line ( layer line 9; Figure 1—figure supplement 1A , Figure 4—figure supplement 1B ) .",
"The diameter of individual filaments was estimated from the raw images .",
"A cylindrical volume that had the estimated diameter was generated in SPIDER and projected along Y-axis to be used as the first reference .",
"The individual filaments were compared with the projection from the cylindrical volume .",
"The shift in X-direction for each boxed segment ( particle ) was rounded to the nearest integer and applied to center the particle horizontally .",
"The total power spectrum was calculated from all X-centered particles in the dataset .",
"The four layer lines ( LL = 1 , 4 , 5 , and",
"9 ) were measured to prepare a ( LL , Z* ) table .",
"The peak positions were indexed to make a table of ( n , R ) .",
"The n was estimated from a numeric table of Bessel functions from 0 to 14th orders .",
"The helical indexing led to the initial guess of a 3-start helix ( n = 3 or −3 ) , with some uncertainty of a 4-start one ( n = 4 or −4 ) .",
"We then tested the refinement for both a 3-start helix and 4-start one separately , and found that the model with a 3-start helical symmetry was able to converge to a 3D map with the right axial rise , but not the one with a 4-start symmetry ( Figure 1—figure supplement 1C , D , G ) .",
"Full refinement with n = −4 was done to confirm this point .",
"We therefore concluded that n = 3 was probably right ( n = −3 is the mirror and equally possible ) .",
"With this information , we did the initial analysis of all subdatasets from different cryo sessions independently in order to obtain the rotation angle ( ΔΦ ) and the axial rise ( Δz ) for each subdataset .",
"The axial rises for individual datasets collected at different magnifications or from different microscopes were brought together and compared to find the right parameters for interpolating the images in the different datasets and scaled them all together into one large dataset that contained 48 , 884 particles .",
"The refinement of the large dataset against a cylindrical volume of a diameter of 90 Å quickly led to a stable solution ( Figure 1—figure supplement 1C , D ) with the symmetry parameters of ΔΦ = 53 . 6° and Δz = 16 . 5 Å .",
"To test the robustness of the parameters , we also introduced 1 . 0 Å deviation to the axial rise in the middle of the refinement , and observed the quick recovery of the refinement to the stable solution ( Figure 1—figure supplement 1D ) .",
"This test suggested that the solution was quite stable .",
"When we performed multivariate statistical analysis and hierarchical classification of the large dataset in IMAGIC , the Eigen images clearly showed that local bending and symmetry breakdown ( Figure 1—figure supplement 1B ) appeared to be the major defects in the dataset , which might have been a limiting factor to a better resolution .",
"We tried to eliminate a small fraction of images ( ∼20% ) that corresponded to those class-averaged images showing clearly weak symmetry or distortions .",
"But after that , we still saw distortions in the Eigen images calculated from the rest of the images .",
"We therefore did not eliminate ∼50% or more of the data to examine the resolution .",
"After an initial refinement , we sorted the data into nine bins by aligning them against models whose symmetry parameters are centered around ( ΔΦ = 53 . 6° , Δz = 16 . 5 Å ) with ΔΔΦ = 2 . 0° and ΔΔz = 1 . 5 Å .",
"The distribution of the particles into these 9 bins followed a Gaussian distribution , and the central bin had 20 , 825 particles and was selected for further refinement where the out-of-plane tilting was refined in 2° steps to a maximum of 15° .",
"After 140 runs the final map was stable and the symmetry well converged to ΔΦ = 53 . 6° and Δz = 16 . 8 Å .",
"The final map was calculated from 15 , 366 boxed segments .",
"The map after IHRSR was corrected by an average CTF envelope calculated from the CTF parameters for all images used in the final map calculation .",
"After the FSC calculation ( see next paragraph ) , a negative B-factor of −1 , 100 Å2 was applied to sharpen the final map to 9 . 6 Å .",
"To estimate the resolution by FSC , the sorted dataset ( 20 , 825 particles ) was separated into top and bottom two halves .",
"Two completely independent volumes were obtained by refining the two half datasets against a cylinder .",
"The two refinements starting with different initial symmetry parameters converged to almost exactly the same symmetry parameters: ΔΦ = 53 . 61° and Δz = 16 . 68 Å for the first , and ΔΦ = 53 . 66° and Δz = 16 . 66 Å for the second .",
"The two volumes without symmetry imposition were aligned in UCSF Chimera ( Pettersen et al . , 2004 ) .",
"The second volume was shifted by 0 . 225 pixels and rotated by −28 . 96° around Y-axis before the FSC calculation ( Figure 1—figure supplement 1E ) .",
"The estimated resolution at FSC = 0 . 5 was 9 . 6 Å .",
"Because of almost exactly the same symmetry in two volumes , we worried about noise correlation and did not use the 0 . 143 threshold for our resolution estimate .",
"As a control , we calculated the FSC before the two volumes were aligned , and found that the FSC quickly fell down to below 0 . 1 at around 40 Å .",
"To confirm that the final map recapitulates the characteristic of the original dataset , we calculated the summed power spectrum of projections from the map at 360 different orientations around its helical axis ( Figure 1—figure supplement 1A , right ) .",
"The power spectrum showed the same patterns as in the total power of the raw dataset ( Figure 1—figure supplement 1A , left ) , with layer lines extending to higher resolution range .",
"Analysis of the MAVSΔProTM dataset followed the same procedure .",
"The dataset had only 8 , 909 particles ( Figure 4—figure supplement 1A , D , E ) .",
"We therefore did not sort them against models with varying symmetry parameters .",
"The FSC calculation gave rise to the resolution estimate of 16 . 4 Å ( Figure 4—figure supplement 1C ) .",
"Energy-filtered electron cryo-tomography on the MAVS CARD filaments was carried out from a dataset collected with an FEI Titan Krios cryo-EM at HHMI Janelia Farm Research Campus .",
"The tomography tilt series were collected in a Gatan K2 Summit direct electron detector installed behind a GIF Quantum energy filter .",
"A narrow energy slit of 5 eV was used together with a small objective aperture ( 50 microns ) to enhance image contrast .",
"The data were collected at a nominal magnification of 42 , 000 × , corresponding to 2 . 7 Å per pixel on the K2 camera .",
"The tilt range spans from −60 to +60° with a step size of 3° .",
"The defocus level was set at −6 . 0 microns .",
"Tomographic reconstruction was carried out using the standard weighted back projection procedure implemented in IMOD ( Yu et al . , 2013 ) .",
"The tilt series were aligned using patch tracking due to the absence of fiducial gold particles .",
"A non-linear anisotropic diffusion filter was applied to the reconstruction .",
"For better resolution of the helical stripes , five slices that cover the top surfaces of multiple filaments in the tomogram were used to calculate a projection image ( Figure 1—figure supplement 2B ) , and the view was from above the filaments .",
"The boxed segments were cropped out and the distance between stripes was measured in the image .",
"The angle between the stripes and the helical axis was estimated manually .",
"The crystal structure of MAVS CARD ( PDB Code: 2VGQ ) was first docked into the cryoEM density map manually in UCSF Chimera .",
"We tested the docking in the original map and its mirror , and found that the docking to the mirrored map , which has left-handed symmetry , allowed us to position three α-helices of the X-ray model directly into the density features in the map ( Figure 1C ) .",
"Recognition of these surface features led to a fairly unique position for the X-ray model and resolved the ambiguity in chirality , consistent with the cryoET results .",
"After one CARD was docked into the map , a hexamer model was built by applying symmetry operations .",
"We segmented the cryoEM density and extracted a portion that represents a hexamer formed by two layers of protein subunits using the segmentation tools in UCSF Chimera .",
"Subsequently , the hexamer model was optimized using SITUS ( Wriggers , 2010 ) to find a local best position with the symmetry constraints .",
"The side chains of a few charged residues ( D23 , E26 , R37 , R64 and R65 ) at the inter-strand interface in the model were optimized by testing different rotamers in Coot to produce the model in Figure 2B ( Emsley et al . , 2010 ) .",
"The two videos were made with Chimera .",
"Creation and solubility screen of mutant libraries of the human and horse CARDs were performed as described previously ( Harada et al . , 2008 ) .",
"Briefly , the coding regions of the MAVS CARD ( residues 2–98 ) were randomly mutagenized by using error-prone PCR , and sub-cloned into the pBAD/DHFR vector ( kindly provided by Dr James Bowie at UCLA ) between the NcoI/SalI restriction sites .",
"Plasmids were transformed into Top10 cells ( Invitrogen ) to express CARD mutants fused to the C-terminus of DHFR .",
"Cells were grown on M9 minimal medium plates containing 100 μg/ml ampicillin , 0 . 20% arabinose and with or without 1 μg/ml trimethoprim at 37°C for 72 hr .",
"Colonies that grew in the presence of trimethoprim were candidate clones expressing soluble CARD mutants .",
"Plasmids from these clones were sequenced to identify their mutations in the CARD domain .",
"The mutants were subsequently subcloned into a modified pET28a vector that encodes an N-terminal His6-tag and a cleavage site of human rhinovirus C3 protease ( He et al . , 2009 ) , and expressed in the bacterial strain BL21 DE3 using a standard procedure .",
"Proteins were purified using Ni-NTA and gel filtration chromatography .",
"The N-terminal tag was removed by treatment of human rhinovirus C3 protease .",
"Mutants were identified as soluble if the proteins could be purified and behaved as a homogenous monomeric peak in gel filtration chromatography .",
"Well-behaving CARD mutants were concentrated and subjected to crystallization trials .",
"Many mutants of horse CARD crystallized in multiple conditions , many of which belonged to the same crystal form as shown by preliminary diffraction experiments .",
"The E26R and R64C mutants , both mapped to the electrostatic interface for mediating MAVS oligomerization , were chosen for structure determination .",
"Crystals of E26R at 1 . 1 mg/ml grew in 0 . 10 M MES ( pH6 . 5 ) , 30%PEG 5000 MME , 0 . 20 M ammonium sulfate at 20°C .",
"Crystals of R64C at 1 . 5 mg/ml grew in Bis-Tris ( pH6 . 5 ) , 25% PEG 3350 , 0 . 20 M ammonium acetate at 20°C .",
"Crystals were flash-cooled in liquid nitrogen in their crystallization buffers supplemented with 25% glycerol .",
"Diffraction data for E26R were collected at 100 K in Beamline 19ID of the Advanced Photon Source ( Argonne National Laboratory ) .",
"Data for R64C were collected at 100 K with a Rigaku X-ray source using a Raxis IV detector .",
"Diffraction data were indexed , integrated and scaled by using HKL2000 ( Otwinowski and Minor , 1997 ) .",
"The structure of the human MAVS CARD ( PDB ID: 2VGQ ) was used as the search model for molecular replacement using Phaser in the Phenix package ( Adams et al . , 2002; McCoy et al . , 2007 ) .",
"Iterative model building and refinement were performed in Phenix and Coot , respectively ( Emsley and Cowtan , 2004 ) .",
"The data collection and refinement statistics were summarized in Supplementary file 1 .",
"HEK293T cells stably expressing both Renilla luciferase ( as an internal control ) and IFNβ promoter driving firefly luciferase were transfected with the indicated amounts of cDNAs for Flag-tagged wide-type MAVS or its mutants .",
"24 hr after transfection , cells were harvested to measure the expression of luciferase using a dual luciferase assay kit ( Promega , Madison , WI ) .",
"Mavs−/− MEFs stably expressing wide-type MAVS or its mutants were grown on sterile glass coverslips in 12-well plates .",
"12 hr after Sendai virus infection , cells were first stained with MitoTracker Red according to the manufacturer’s instructions ( Invitrogen ) .",
"Cells were then fixed with 4 . 0% paraformaldehyde in PBS for 15 min , permeabilized in PBS containing 0 . 10% Triton X-100 for 5 min , and blocked in PBS containing 0 . 10% Triton X-100 and 10% BSA for 30 min at room temperature .",
"After blocking , the cells were incubated with specific primary antibodies for 1 hr , washed and then incubated with suitable Alexa Fluor 488 ( or Alexa Fluor 568 or Alexa Fluor 633 ) -conjugated secondary antibodies for another hour .",
"After careful wash , the slides were mounted with the VECTASHIELD mounting medium with DAPI ( Vector Laboratories ) .",
"Imaging of the cells was carried out using a Zeiss LSM510 META laser scanning Confocal Microscope or a Zeiss ELYRA PS . 1",
"Super-Resolution Structured Illumination Microscope ( SR-SIM ) .",
"Z-stacks with an interval of 110 nm were used to section the whole cell for 3D-SR-SIM .",
"Images were analyzed using the Zen2011 software ( Zeiss ) or ImageJ .",
"Alignment and reconstruction of 3D-SIM images were performed using IMARIS ( Bitplane ) .",
"Total RNA was isolated using TRIzol ( Invitrogen ) .",
"0 . 1 μg of total RNA was reverse-transcribed into cDNA with iScript cDNA synthesis kit ( Bio-Rad , Hercules , CA ) .",
"The resulting cDNAs served as the templates for Quantitative-PCR analysis using iTaq Universal SYBR Green Supermix ( Bio-Rad ) and ViiTM7 Real-Time PCR System ( Applied Biosystems Inc . , Foster City , CA ) .",
"Primers for specific genes are: Mouse β-actin , 5′-TGACGTTGACATCCGTAAAGACC-3′ and 5′-AAGGGTGTAAAACGCAGCTCA-3′; Mouse IFNβ , 5′-CCCTATGGAGATGACGGAGA-3′ and 5′-CTGTCTGCTGGTGGAGTTCA-3′ .",
"The formation of prion-like aggregates of MAVS and its mutants was analyzed by SDD-AGE as previously described ( Hou et al . , 2011 ) ."
]
] | [
"Mitochondrial antiviral signaling ( MAVS ) protein is required for innate immune responses against RNA viruses .",
"In virus-infected cells MAVS forms prion-like aggregates to activate antiviral signaling cascades , but the underlying structural mechanism is unknown .",
"Here we report cryo-electron microscopic structures of the helical filaments formed by both the N-terminal caspase activation and recruitment domain ( CARD ) of MAVS and a truncated MAVS lacking part of the proline-rich region and the C-terminal transmembrane domain .",
"Both structures are left-handed three-stranded helical filaments , revealing specific interfaces between individual CARD subunits that are dictated by electrostatic interactions between neighboring strands and hydrophobic interactions within each strand .",
"Point mutations at multiple locations of these two interfaces impaired filament formation and antiviral signaling .",
"Super-resolution imaging of virus-infected cells revealed rod-shaped MAVS clusters on mitochondria .",
"These results elucidate the structural mechanism of MAVS polymerization , and explain how an α-helical domain uses distinct chemical interactions to form self-perpetuating filaments ."
] | [
"When infected by a virus , the body will generally launch an immune response to eliminate the infectious agent .",
"Activation of the innate immune system–the first line of defense against infection—requires the host cells to recognize the presence of a pathogen and to sound the alarm once the invader is detected .",
"Viruses can contain DNA or RNA , and when a virus containing double stranded RNA enters a cell , or starts replicating within the cytoplasm , proteins called RIG-I-like receptors ( RLRs ) will detect these RNA molecules .",
"This will trigger a signaling cascade that results in the production of type I interferons , the proteins that activate cells of the innate immune system .",
"Members of the RLR family of receptors , including RIG-I and MDA5 , initiate the signaling cascade by interacting with the mitochondrial antiviral-signaling ( MAVS ) protein .",
"Recent work revealed that upon activation by RIG-I or MDA5 , MAVS proteins aggregate on the surface of mitochondria and form protein filaments .",
"These filaments then activate inactive MAVS proteins , leading to the formation of more filaments .",
"While a region of the MAVS protein called caspase activation and recruitment domain ( CARD ) is known to be involved in the formation of the filaments , the chemical interactions that govern the formation process have yet to be described .",
"Now , using cryo-electron microscopy , Xu et al . have shown that these filaments are comprised of three-stranded helixes .",
"This came as something of a surprise because other similar filaments known as prions are made of tightly packed beta sheets .",
"Xu et al . went on to visualize full-length MAVS filaments in virus-infected cells , and to verify that mutations that impair the assembly of MAVS filaments also prevent RNA viruses from triggering the production of interferon .",
"These results have the potential to inform future studies of the innate immune response , as well as investigations into the assembly of proteins to form prion-like filaments ."
] | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"structural biology and molecular biophysics",
"neuroscience"
] | Knockout of Slo2.2 enhances itch, abolishes KNa current, and increases action potential firing frequency in DRG neurons | elife-10013-v1 | [
[
"Potassium channels regulated by cytosolic Na+ ( KNa ) are encoded by two homologous mammalian genes , Kcnt1 ( encoding the Slo2 . 2 or Slack channel ) ( Yuan et al . , 2003 ) and Kcnt2 ( encoding the Slo2 . 1 or Slick channel ) ( Bhattacharjee et al . , 2003 ) .",
"Recent work has revealed a critical role of KNa channels in neuronal function , through demonstration that several mutations in Kcnt1 are associated with intellectual disability and childhood epilepsy ( Barcia et al . , 2012; Heron et al . , 2012; Martin et al . , 2014 ) .",
"Yet , despite apparently wide-spread expression both in neurons ( Bhattacharjee et al . , 2002 , 2005 ) and other cells ( Kameyama et al . , 1984; Niu and Meech , 2000 ) , the physiological roles of KNa currents during normal patterns of neuronal activity remain poorly understood in part because of the absence of suitably selective pharmacological tools and also the complexities than can arise from manipulations of Na+ .",
"Because of potential coupling of KNa activation to Na+ influx through voltage-dependent Na+ ( Nav ) channels , KNa currents have been proposed to influence repetitive firing ( Yang et al . , 2007; Gribkoff and Kaczmarek , 2009 ) and postexcitatory afterhyperpolarizations ( Franceschetti et al . , 2003; Gao et al . , 2008 ) .",
"Recently , it has been suggested that KNa currents may be selectively activated by Na+ influx through Nav channel openings that persist at steady state following inactivation ( Hage and Salkoff , 2012 ) .",
"To further probe the role of KNa currents , we have genetically disrupted Kcnt1 and Kcnt2 genes to generate mouse strains in which Slo2 . 1 , Slo2 . 2 , or both subunits together ( Slo2 dKO ) have been deleted .",
"Because previous work has suggested an important role of Slo2 channels in sensory neurons ( Gao et al . , 2008; Nuwer et al . , 2010; Biton et al . , 2012 ) , we examined the consequences of KNa KO on sensory function and dorsal root ganglion ( DRG ) neuron excitability .",
"The results reveal a role of Slo2 . 2 channels in acute itch sensation .",
"Pruritic stimuli trigger an immediate increase in itch response in Slo2 . 2 KO mice , with later time points indistinguishable from WT animals .",
"Furthermore , KO of Slo2 . 2 , but not Slo2 . 1 , removes a KNa current from all small-diameter DRG neurons examined .",
"To examine effects of Slo2 KO on DRG excitability , we focused on small diameter neurons , immunoreactive for isolectin Β4 ( IB4+ ) , which are known to be enriched in neurons responsive to itch and pain stimuli ( Lallemend and Ernfors , 2012 ) .",
"Slo2 KO increases firing frequency at any level of current injection , while decreasing both rheobase and action potential ( AP ) threshold .",
"Contrary to the view that KNa current functions primarily during AP repolarization and afterhyperpolarization ( Schwindt et al . , 1989; Franceschetti et al . , 2003; Wallen et al . , 2007 ) , we propose that in DRG neurons activation of KNa current precedes AP initiation thereby acting as a brake to AP firing .",
"During completion of this work , another paper describing a Slo2 . 2 KO mouse ( Lu et al . , 2015 ) importantly identified a potential role of Slo2 . 2 in DRG in a neuropathic pain model .",
"Here we reveal a role of Slo2 . 2 in acute sensory responses and provide a new explanation for how cell firing is altered by Slo2 . 2 channels ."
],
[
"Slo2 . 1 ( gene: Kcnt2 ) and Slo2 . 2 ( gene: Kcnt1 ) KO mice were generated via homologous recombination of specific targeting DNA fragments ( Figure 1A , D ) into the genome of mouse embryonic stem ( ES ) cells with confirmation by Southern blot ( Figure 1B , E ) , generation of chimeric mice following injection of recombinant ES cells into C57BL/6 blastocysts , and then ultimately Cre/loxP mediated deletion of the targeted exons .",
"Successful incorporation of the mutant allele into mice was confirmed by PCR genotyping of genomic DNA extracted from mouse tails ( Figure 1C , F ) .",
"The absence of specific native Slo2 protein was confirmed by western blots of total brain membrane proteins ( Figure 2; See ‘Materials and methods’ for discussion of Slo2 epitopes identified by antibodies ) .",
"Enrichment of brain Slo2 protein via sequential co-immunoprecipitation ( co-IP ) and western blot further validated the successful KO of Slo2 proteins and also established that Slo2 . 1 and Slo2 . 2 coassemble in WT brain ( Figure 2B–E ) , as indicated in earlier work ( Chen et al . , 2009 ) .",
"As a guide to tissues of interest for future study , quantitative RT-PCR was employed on various tissues to define the relative abundance of message for Kcnt1 and Kcnt2 message ( Figure 2F ) .",
"mRNAs encoding either Slo2 . 1 and Slo2 . 2 are broadly present in the central nervous system , with message for Slo2 . 1 notably more abundant in heart and aorta and message for Slo2 . 2 relatively enriched in other tissues including DRG and cerebellum .",
"The selective expression of transcript for Slo2 . 1 in rat heart has been previously reported ( Bhattacharjee et al . , 2003 ) .",
"Based on the RT-PCR results , we examined DRG , spinal cord , cortex , cerebellum and heart for the presence of Slo2 . 1 and Slo2 . 2 subunits using sequential IP and western blot ( Figure 2G–J ) .",
"Slo2 . 1 protein was detected in DRG , spinal cord , cortex and heart , but only a very weak band was seen from cerebellum ( Figure 2G ) .",
"Slo2 . 2 was observed in DRG , spinal cord , cortex , and cerebellum , but not detectable in heart ( Figure 2J ) .",
"Co-IP between Slo2 . 1 and Slo2 . 2 was observed in those tissues for which both subunits were detectable: DRG , spinal cord , and cortex ( Figure 2H , I ) .",
"Because KNa currents have been described in sensory neurons ( Gao et al . , 2008; Tamsett et al . , 2009; Nuwer et al . , 2010 ) , we chose DRG as a convenient system for investigation of potential physiological roles . 10 . 7554/eLife . 10013 . 003Figure 1 . Construction and validation of Slo2 . 1 and Slo2 . 2 KO mice .",
"( A ) Upper row: map of WT mouse Kcnt2 ( encoding Slo2 . 1 ) gene locus within genomic DNA bracketing the targeted exon 22 .",
"Second row: map of the targeting vector , showing M1uI site for vector linearization , targeted exon 22 with a 1 . 8 kb neomycin gene cassette flanked by LoxP and FRT sites , and a 2 . 8 kb thymidine kinase ( TK ) gene cassette .",
"The overall size of the Kcnt2 genomic DNA for homologous recombination ( left arm + right arm ) is 16 . 3 kb .",
"Third row: map of the recombinant allele in targeted embryonic stem ( ES ) clones following homologous recombination of the Kcnt2 KO region into the targeted locus .",
"The neo gene cassette is eliminated by Flp-FRT mediated deletion .",
"Fourth row: map of the mutant kcnt allele following Cre-loxP mediated deletion of the targeted exon .",
"Shown are the elements and restriction enzyme sites used in generation and verification of the targeted mutant allele .",
"Location of the probe used in genomic Southerns for the selection of recombinant ES clones is indicated .",
"After enzyme digestion treatments , the WT allele fragments detected by the probe are 10 kb ( by EcoRV ) and 4 . 3 kb ( by PvuII ) , while the recombinant allele fragments detected by the probe are 4 . 2 kb ( by EcoRV ) and 3 kb ( by PvuII ) , respectively .",
"( B ) Genotype analysis of ES cell lines by Southern blot analysis .",
"After enzyme digestion with either EcoRV ( left ) or PvuII ( right ) , genome DNA obtained from recombinant ES colonies , containing both wild type allele and targeted recombinant allele , shows two corresponding fragments identified by the probe .",
"( C ) PCR verification of animal genotypes .",
"The target exon is removed by mating heterozygous ( HET ) F1 mice with early embryonic expression Cre-mice ( EIIa-Cre , Jackson ) .",
"The predicted amplicons are 579 bp for WT and 269 bp for the exon 22 deleted mutant .",
"( D ) Upper row: map of WT mouse Kcnt1 ( encoding Slo2 . 2 ) gene locus bracketing the targeted exon 11 .",
"Second row: map of the targeting vector , showing SpeI site for vector linearization , targeted exon 11 and a 1 . 8 kb neomycin gene cassette flanked by LoxP and FRT sites , and a TK gene cassette .",
"The overall size of Kcnt1 genomic DNA for homologous recombination is 16 . 6 kb .",
"Third row: map of the recombinant allele in targeted ES clones following homologous recombination of the kcnt1 region into the targeted locus .",
"The neo gene cassette is then eliminated by Flp-FRT mediated deletion .",
"Fourth row: map of the mutant Kcnt1 allele following Cre-loxP mediated deletion of the targeted exon .",
"The location of the probes used in genomic Southerns are also indicated .",
"After enzyme digestion treatments , the WT allele fragments detected by the probe are 6 . 4 kb ( by BclI ) and 3 . 4 kb ( by BglII ) , while the recombinant allele fragments detected by the probe are 4 . 3 kb ( by BclI ) and 5 . 3 kb ( by BglII ) respectively .",
"( E ) Genotype analysis of ES cell lines by Southern blot analysis .",
"Expected fragment sizes for either BclI ( left ) or BglII ( right ) restriction enzyme digestion are shown for both wild type and targeted homologous recombinant .",
"( F ) PCR verification of Kcnt1 exon 11 deletion .",
"The target exon is removed by mating HET F1 mice with early embryonic expression Cre-mice ( EIIa-Cre , Jackson ) .",
"The predicted amplicons for WT and the exon 11 deleted mutant were 607 bp and 200 bp , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 00310 . 7554/eLife . 10013 . 004Figure 2 . Slo2 . 1 and Slo2 . 2 subunits are absent in Kcnt2 and Kcnt1 KO mice , respectively , exhibit differential tissue distribution , and coassemble in some tissue .",
"( A ) Top , brain membrane proteins from WT , Slo2 . 1 KO , and Slo2 . 2 KO mice were probed with N11/33 anti-Slo2 . 1 antibody ( Antibodies Inc . ) .",
"Middle , brain membrane proteins were separated and probed with N3/26 anti-Slo2 . 2 mAb ( Antibodies , Inc ) .",
"Slo2 . 2 protein is absent in Slo2 . 2 KO mice .",
"No native Slo2 . 2 protein is present in the Slo2 KO mice , but is found in Slo2 . 1 KO mice .",
"Bottom , α-tubulin loaded in each lane was probed with anti-α-tubulin Ab .",
"15 μg of whole brain membrane proteins were loaded in each lane .",
"( B ) Slo2 . 1 Ab pulls down Slo2 . 1 protein from brain membrane proteins in WT and Slo2 KO mice , but not from Slo2 . 1 KO mice .",
"Anti-Slo2 . 1 Ab also pulls down Slo2 . 1 from proteins following mixing of separate Slo2 . 2 KO and Slo2 . 1 KO membrane preparations ( mix ) .",
"25 μg of whole brain proteins were subjected to IP procedures and the IP products were loaded in each lane .",
"( C ) IP with anti-Slo2 . 1 Ab pulls down Slo2 . 2 only in WT membrane proteins , but not in mixed proteins , or membrane proteins from Slo2 . 1 KO or Slo2 . 2 KO mice .",
"62 . 5 μg of whole brain proteins were subjected to IP procedures with the IP products loaded in each lane .",
"( D ) Following IP with anti-Slo2 . 2 , Slo2 . 2 is detected in proteins from WT , mixed , and Slo2 . 1 KO membranes .",
"25 μg of whole brain proteins were subjected to IP procedures and the products loaded in each lane .",
"( E ) IP with anti-Slo2 . 2 Ab pulls down Slo2 . 1 only from WT membrane proteins .",
"62 . 5 μg of whole brain proteins was subjected to IP procedures and the products loaded in each lane .",
"( F1 )",
"Abundance of message for Slo2 . 1 relative to β-actin message is plotted for various tissues .",
"Here and in ( F2 ) , message was measured in triplicate from each of three mice .",
"( F2 )",
"Slo2 . 2 message abundance is plotted .",
"( F3 )",
"The ratio of message for Slo2 . 1 to Slo2 . 2 measured by quantitative rt-PCR is shown for various tissues .",
"Dotted line indicates approximately equimolar RNA amounts .",
"Red arrows highlight enrichment of Slo2 . 2 message .",
"Horizontal blue bar and arrow highlight relative enrichment of message for Slo2 . 2 in heart tissues .",
"( G ) IP with anti-Slo2 . 1 shows presence of Slo2 . 1 protein in DRG , spinal cord , cortex and heart , but not cerebellum .",
"Protein amounts used in IPs were: DRG , 3 mg; spinal cord , 1 mg; cortex , 0 . 3 mg; cerebellum; 2 mg; heart , 30 mg .",
"( H ) IP with anti-Slo2 . 2 pulls down Slo2 . 1 in DRG , spinal cord and cortex , but not in cerebellum and heart .",
"Protein amounts used in IPs were: DRG , 3 mg; spinal cord , 1 mg; cortex , 0 . 5 mg; cerebellum; 0 . 25 mg; heart , 30 mg .",
"( I ) IP with anti-Slo2 . 1 pulls down Slo2 . 2 in spinal cord and cortex .",
"( J ) IP with anti-Slo2 . 2 shows presence of Slo2 . 2 in all tested tissues except heart .",
"Western blots were repeated three times in all cases , except twice for DRG . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 004 WT and Slo2 KO mouse strains were evaluated with various tests of sensory function .",
"In a 55°C hotplate test , single KO of either Slo2 . 1 or Slo2 . 2 did not influence the response latency , although Slo2 dKO mice exhibited a briefer latency than WT mice ( Figure 3A ) .",
"In a formalin test , no differences were observed between WT and Slo2 dKO mice ( Figure 3B ) .",
"The absence of a difference in hotplate or formalin response in Slo2 . 2 KO mice agrees with recent observations on another Slo2 . 2 KO mouse ( Lu et al . , 2015 ) . 10 . 7554/eLife . 10013 . 005Figure 3 . Slo2 dKO shortens hotplate response latency , increases responses to hindpaw injections of capsaicin , but does not influence formalin responses .",
"( A ) Latencies to aversive response following placement on a 55°C hotplate are plotted for the indicated genotypes , showing means , sem , and individual latencies .",
"From left to right , n = 19 , 19 , 24 , 11 , 13 , 9 , 17 , and 24 .",
"Only in the WT vs Slo2 dKO comparison was a difference noted ( p = 0 . 002; KS test ) .",
"( B ) Following formalin injection , time spent in licking the hindpaw was determined for 5 min intervals for WT ( n = 10 ) and Slo2 dKO ( n = 9 ) mice .",
"Here and below , behavioral tests over time display measurements centered in each 5 min interval .",
"( C ) Time course of licking response to hindpaw injection of 0 . 1 μg capsaicin .",
"Small symbols , individual mice .",
"p = 0 . 012 ( KS test ) .",
"Vehicle: 10 μl volume with 0 . 35% EtOH .",
"( D ) Time spent licking was determined over 10 min following hindpaw injections of the indicated capsaicin quantities in 10 μl vehicle for WT ( n = 9 , 9 , 20 , 20 , 18 , 20 , 20 , 20 , 20 , and 9 from low to high capsaicin ) and Slo2 dKO ( n = 10 , 10 , 11 , 9 , 14 , 26 , 13 , 18 , 10 , and",
"10 ) genotypes .",
"Vehicle alone was without effect ( n = 10 for both WT and Slo2 dKO ) .",
"For filled black , open black , and filled red stars , p values correspond to KS statistic with p = 0 . 000 ( filled black stars ) , p = 0 . 007 ( filled red stars ) , and p = 0 . 012 for open black star .",
"For open red stars , a t-test statistic was used with p < 0 . 01 .",
"Highest capsaicin concentrations showed no difference between WT and Slo2 dKO mice . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 005 Hindpaw injection of capsaicin elicits a characteristic licking behavior which was somewhat enhanced in Slo2 dKO mice ( Figure 3C ) .",
"Because the intensity of a sensory stimulus may affect whether KNa currents influence sensory function , we compared responses to a series of capsaicin doses ( Figure 3D ) .",
"Consistent with this idea , pronounced differences between WT and Slo2 dKO mice were present at doses in excess of 0 . 0001 μg up through 0 . 01 μg , with weaker differences at 0 . 03 , 0 . 1 μg , and higher concentrations .",
"These results indicate that mice lacking both Slo2 . 1 and Slo2 . 2 channels exhibit an enhanced aversion to moderate doses of capsaicin and that Slo2 dKO can influence the acute response to sensory stimuli .",
"We next tested several pruritic compounds in a standard itch assay ( Sun and Chen , 2007 ) .",
"Chloroquine ( CQ , Figure 4A–F ) , histamine ( HA , Figure 4G–L , Figure 4—figure supplement 1A–E ) , and compound 48–80 ( Figure 4—figure supplement 1F ) elicited robust enhancement of scratching behavior in Slo2 dKO mice ( Video 1 for the case of CQ ) , but not WT mice ( Video 2 ) , during the first 5 min following injection .",
"No difference in itch behavior was observed between WT and Slo2 dKO mice after the first 5 min .",
"KO of only Slo2 . 2 also revealed a similar alteration in the itch phenotype during the first 5 min after injection ( CQ: Figure 4C , D; HA: Figure 4I , J ) .",
"The enhanced itch was also observed in heterozygous Slo2 . 2 mice .",
"In contrast , WT and Slo2 . 1 mice exhibited no difference in response to either CQ ( Figure 4E , F ) or HA ( Figure 4K , L ) . 10 . 7554/eLife . 10013 . 006Figure 4 . The absence of Slo2 . 2 , but not Slo2 . 1 , results in enhancement of chloroquine ( CQ ) and histamine ( HA ) -induced itch .",
"( A ) Each point shows mean number of scratching bouts per 5 min bins for WT mice ( n = 15 , black circles ) and Slo2 dKO mice ( n = 19 , red circles ) after injection of 200 μg CQ .",
"( B ) Mean scratching bouts during first 5 min are summarized for WT and Slo2 dKO mice from ( A ) , along with determinations from individual mice ( circles ) .",
"Over the first 5 min , WT and Slo2 dKO mice differ at p = 0 . 000 ( KS-test ) .",
"( C ) Slo2 . 2 KO mice exhibit enhanced responsiveness to CQ injection .",
"( D ) Mean scratching bouts during the first 5 min after CQ injection for WT ( n = 12 ) , Slo2 HET mice ( n = 12 ) and Slo2 . 2 KO mice ( n = 16 ) .",
"KS-test comparisons: WT vs Slo2 . 2 HET , p = 0 . 005; WT vs Slo2 . 2 KO , p = 0 . 000; Slo2 . 2 HET vs Slo2 . 2 KO , p = 0 . 003 .",
"( E ) Slo2 . 1 KO mice exhibit CQ responsiveness identical to WT mice .",
"( F ) Mean scratching during the first 5 min after CQ injection for WT ( n = 16 ) , Slo2 . 1 HET ( n = 11 ) and Slo2 . 1 KO ( n = 16 ) mice .",
"( G ) Responses of WT ( n = 15 ) and Slo2 dKO ( n = 18 ) mice following injection of 1 mg HA .",
"( H ) Scratching during first 5 min following HA injection for WT and Slo2 dKO mice .",
"Over the first 5 min , WT and Slo2 dKO mice differ at p = 0 . 000 .",
"( I ) HA-induced scratching behavior for WT and Slo2 . 2 KO mice .",
"( J ) Mean and individual values of scratching during first 5 min for WT ( n = 11 ) , Slo2 . 2 HET ( n = 11 ) , and Slo2 . 2 KO ( n = 12 ) mice .",
"KS-test comparisons: WT vs Slo2 . 2 HET , p = 0 . 003; WT vs Slo2 . 2 KO , p = 0 . 000; Slo2 . 2 HET vs Slo2 . 2 KO , p = 0 . 121 .",
"( K ) HA-induced scratching behavior for WT and Slo2 . 1 KO mice .",
"( L ) Mean and individual values of scratching during first 5 min for WT ( n = 19 ) , Slo2 . 1 HET ( n = 13 ) , and Slo2 . 1 KO ( n = 30 ) mice . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 00610 . 7554/eLife . 10013 . 007Figure 4—figure supplement 1 . Concentration-dependence of itch response to HA and compound 48–80 . ( A ) Scratching behavior in WT and Slo2 dKO mice is plotted following injection of 1 mg HA .",
"( B ) Scratching behavior after 0 . 3 mg HA is compared .",
"( C ) Scratching behavior after 0 . 1 mg HA is displayed .",
"( D ) Scratching behavior after 0 . 03 mg HA is shown .",
"( E ) Dose-response relationship for HA injections is displayed showing individual estimates and mean responses for WT and Slo2 dKO mice during first 5 min after injection .",
"p values are the KS statistic .",
"Numbers of animals in each case are given in A–D .",
"( F ) Dose-response relationship for compound 48–80 injection is compared .",
"p values reflect the KS statistic .",
"At 3 μg , 10 WT mice were compared to 8 dKO mice; at 10 μg , 10 WT were compared to 10 dKO; at 100 μg , 10 WT were compared to 14 dKO mice . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 00710 . 7554/eLife . 10013 . 008Video 1 . Response of a Slo2 dKO mouse to CQ injection ( related to Figure 4A ) .",
"The nape of the neck of a Slo2 dKO mouse was injected with 10 μl 200 μM choroquine .",
"Video recording was begun about 10 s after injection . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 00810 . 7554/eLife . 10013 . 009Video 2 . Response of a WT mouse to CQ injection ( related to Figure 4A ) .",
"The nape of the neck of a WT mouse was injected with 10 μl 200 μM choroquine .",
"Acquisition of video was begun about 10 s after injection . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 009 Because the time course of the early itch response was similar to capsaicin responses ( Figure 3C ) , it seemed possible that pruritic stimuli in the Slo2 KO mice were perceived as something distinct from itch .",
"A cheek injection assay has been proposed to distinguish itch from pain ( Shimada and LaMotte , 2008 ) .",
"In the cheek , injection of HA elicits hindlimb scratching , while capsaicin injection elicits forepaw wiping ( Shimada and LaMotte , 2008 ) , suggesting that they are being perceived differently .",
"We wondered whether a pruritic stimulus injected into the cheek of a Slo2 dKO mouse might elicit a capsaicin-like forepaw wiping response .",
"In our hands , cheek injection of CQ in WT animals was associated with two types of behaviors , hindlimb scratching of the injected site , but also some forepaw wiping presumably reflecting grooming ( Figure 5 ) .",
"In the dKO animals , forepaw wiping was no different than in WT ( Figure 5B ) , but the hindlimb scratching was markedly increased only during the first 5 min ( Figure 5A ) .",
"Whatever the basis of the enhanced response to cheek injection of CQ in Slo2 dKO mice , the response is characteristic of pruritic stimuli and not of capsaicin . 10 . 7554/eLife . 10013 . 010Figure 5 . CQ enhances itch-type behavior following cheek injection , but not pain-type behavior .",
"( A ) Total scratching bouts using the hindpaw to scratch the cheek was monitored following cheek injection of 200 μg CQ in WT and Slo2 dKO mice .",
"During the first 5 min interval , distributions differed at p < 0 . 001 ( Student's t-test ) .",
"( B ) Bouts of forepaw grooming were monitored following CQ cheek injection for WT and Slo2 dKO mice .",
"There was no difference in the first 5 min . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 010 Sensory neurons contain a rich variety of K+ currents ( Vydyanathan et al . , 2005; Dobler et al . , 2007; Li et al . , 2007; Cho et al . , 2009; Zhang et al . , 2010b; Liu et al . , 2013 ) that complicate unambiguous definition of KNa current , for which selective pharmacological tools are lacking .",
"We have not had reliable success with subtractive methods involving Na+ current inhibition or Na+ replacement .",
"To test for the presence of KNa current in small diameter DRG neurons , we used a method previously applied to rat DRG neurons ( Bischoff et al . , 1998 ) : a K+ background current arising from defined pipette Na+ is measured using hyperpolarizing voltage-steps during the first 5 min following formation of the whole-cell recording configuration .",
"With 0 mM pipette Na+ , little background current is observed with voltage-steps from −80 to −120 mV ( Figure 6A , C ) .",
"With 70 mM pipette Na+ , net current elicited by the same voltage-step gradually increases over 3 min reaching a plateau near 1 nA ( Figure 6A , C ) .",
"At longer times following whole-cell access , current activated by 70 mM pipette Na+ gradually diminishes ( Figure 6—figure supplement",
"1 ) despite no change in voltage-dependent Na+ current .",
"As in rat DRG neurons ( Bischoff et al . , 1998 ) , the KNa current is blocked by extracellular 20 mM Cs+ , with stronger inhibition at −120 mV than −80 mV reflecting the voltage-dependence of Cs+ inhibition ( Figure 6A , Figure 6—figure supplement 2 ) .",
"The average amplitude of KNa current was similar for WT and Slo2 . 1 KO DRG neurons ( Figure 6B , C ) , while there was no KNa current in Slo2 . 2 KO or Slo2 dKO neurons ( Figure 6B , C ) .",
"Despite considerable variability in total KNa current among neurons from either WT or Slo2 . 1 KO animals ( Figure 6D ) , the total current always exceeds that observed in WT cells with 0 Na+ , or in Slo2 dKO or Slo2 . 2 KO cells with 70 mM Na+ ( Figure 6D ) .",
"Excised inside-out patches confirmed that Slo2 dKO removed a Na–dependent K+ channel ( Figure 6E ) which exhibited little voltage-dependence over the range of −80 through −20 mV ( Figure 6E , Figure 6—figure supplement 3A ) with a single channel conductance of about 127 pS ( Figure 6E , Figure 6—figure supplement 3B ) .",
"Finally , we compared the whole-cell steady-state current–voltage ( I–V ) relationship between WT and Slo2 dKO cells over the range of −125 to −25 mV , with 70 mM pipette Na+ along with the steady-state IV relationship persisting in WT cells after 30 min with 70 mM pipette Na+ ( Figure 6F ) .",
"This shows the relatively voltage-independent nature of the background KNa conductance ( reversal at EK ) when the cytosolic Na+ concentration is constant . 10 . 7554/eLife . 10013 . 011Figure 6 . The absence of Slo2 . 2 reduces Na+-dependent leak current in acutely dissociated mouse DRG neurons .",
"( A ) Traces on the top show currents ( evoked by indicated voltage protocol ) for four time points following formation of a whole-cell recording with 70 mM pipette Na+ .",
"Green: immediately following whole-cell access; black: 3 min following access; blue: following application of 20 mM Cs+; red: washout of Cs+ .",
"On the bottom , traces are from another WT neuron examined with the same procedure , but with 0 mM pipette Na+ .",
"( B ) Panels correspond to the same sequence as shown in ( A ) for a Slo2 . 2 KO neuron ( top left ) , a Slo2 . 1 KO neuron ( top right ) , and a Slo2 dKO neuron ( bottom ) .",
"( C ) The time courses of increases in net current evoked by steps from −80 to −120 mV are shown for WT and the three indicated Slo2 genotypes .",
"( D ) Mean estimates of leak current and standard errors measured 3 min following whole-cell access are plotted for different test conditions .",
"Circles correspond to individual cells .",
"t-test comparisons yielded: for 0 mM Na+ WT vs 70 mM Na+ WT , p = 0 . 0015; for 70 mM WT vs Slo2 dKO , p < 0 . 001; for 70 WT vs Slo2 . 2 KO , p < 0 . 001; for Slo2 . 1 KO vs Slo2 . 2 KO , p = 0 . 0038 .",
"All other comparisons were p > 0 . 1 .",
"( E ) Traces on the top show channel activity in a patch excised from a WT DRG neuron bathed either with 0 mM Na+ or 70 mM Na+ .",
"Bottom: a similar patch from a Slo2 dKO neuron reveals no channels activated by Na+ .",
"( F ) Voltage-step protocols over the range of −125 mV to −25 mV were used to compare steady-state conductance ( measured at the end of a 20 ms command step ) ( Figure 6—figure supplement",
"1 ) in WT and dKO neurons with 70 mM pipette Na+ , along with WT neurons with 70 mM Na+ after 30 min of recording . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 01110 . 7554/eLife . 10013 . 012Figure 6—figure supplement 1 . KNa current runs down during constant cytosolic 70 mM Na+ .",
"( A ) The indicated voltage protocol was used to elicit currents in a WT DRG neuron with 70 mM pipette Na+ .",
"Red trace indicates current activated by step to −5 mV .",
"Bottom record in each case shows current resulting from the step to −125 mV to highlight the loss of the conductance over negative voltages .",
"Application of loxapine following the example trace at 30 min resulted in partial restoration of the background conductance ( not shown ) .",
"For recordings in this figure , the extracellular solution contained 10 mM TEA to minimize any contribution from Ca2+-dependent K+ currents .",
"( B ) Current at the end of the 20 ms voltage steps in A is plotted for the different recording times .",
"( C ) The difference between peak inactivating current and steady-state current at 20 ms is plotted as a function of command voltage for the different recording times , revealing little change in Na+ current over the 30 min of recording . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 01210 . 7554/eLife . 10013 . 013Figure 6—figure supplement 2 . Cs+ inhibition of KNa current exhibits voltage-dependence .",
"( A ) Steady-state current was measured over the indicated voltages with 70 mM pipette Na+ .",
"Application of 20 mM extracellular Cs+ markedly inhibits the KNa current , with stronger inhibition at more negative potentials .",
"Symbols are means ( ±sem ) of current from four neurons .",
"( B ) Fractional inhibition is plotted as a function of membrane voltage .",
"Solid line is the best fit of the following equation:f ( V ) =1−A1+exp−zF ( V−Vh ) RT , where A ( = 0 . 39 ) is the amplitude of the voltage-dependent component of inhibition by 20 mM Cs+ , Vh ( = −82 . 2 mV ) reflects the voltage of half inhibition of the voltage-dependent component , and z ( = 0 . 68e ) reflects the voltage-dependence of that inhibition .",
"The function implies that at more positive potentials 20 mM Cs+ inhibits 61% of the current in a voltage-independent fashion . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 01310 . 7554/eLife . 10013 . 014Figure 6—figure supplement 3 . Confirmation of properties of single KNa channels that are deleted by Slo2 dKO .",
"( A ) The cytosolic face of an excised inside-out patch from a DRG neuron was exposed to 70 mM Na+ solution and channel activity was monitored over a range of voltages .",
"Average activity exhibited only weak voltage-dependence .",
"( B ) Single channel amplitude was measured at four voltages for a set of four patches , yielding a single channel conductance of 127 pS . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 01410 . 7554/eLife . 10013 . 015Figure 6—figure supplement 4 . Na+-dependent leak current is present in both IB4+ and IB4− neurons and runs down with time in culture .",
"( A ) Whole-cell current was activated by voltage-steps from −80 to −120 mV , with 70 mM Na+ in the pipette solution .",
"Both IB4+ and IB4− neurons exhibit substantial KNa current .",
"( B ) Dissociated DRG neurons were maintained in culture for up to about 2 days .",
"Following formation of whole-cell recordings , a step from −80 to −120 mV was used to determine leak current in the presence of 70 mM pipette Na+ .",
"p values at 20–32 hr and 46–52 hr of culture are T-test comparisons to the measurements from 2 to 10 hr in culture . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 01510 . 7554/eLife . 10013 . 016Figure 6—figure supplement 5 . The absence of Slo2 . 2 and , to a lesser extent , Slo2 . 1 , reduces Na+-dependent leak current in mouse DRG neurons in DRG tissue slices .",
"( A ) Traces on the left show currents evoked by the indicated voltage steps at three time points following formation of a whole-cell recording with 70 mM pipette Na+ , an initial trace immediately following whole-cell access ( green ) , ∼3 min following access ( black ) , and then following application of 20 mM Cs+ .",
"On the right , traces are from another WT neuron examined with the same procedure on the left , but with 0 mM pipette Na+ .",
"( B ) Panels correspond to the same sequence as shown in ( A ) for a Slo2 . 1 KO neuron ( left ) , a Slo2 . 2 KO neuron , and a Slo2 dKO neuron .",
"( C ) The time course of increases in net current evoked by steps from −80 to −120 mV is shown for the five indicated conditions .",
"( D ) Mean current amplitude ( ±sem ) of background current observed 2–3 min following formation of whole-cell recording is plotted for five conditions . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 016 In WT DRG neurons , the average KNa background current with 70 mM pipette Na+ in IB4+ neurons did not differ significantly from that in IB4− neurons ( Figure 6—figure supplement 4A ) .",
"Our results suggest that essentially all dissociated small diameter DRG neurons express KNa current which can be attributed exclusively to Slo2 . 2 subunits .",
"The magnitude of the KNa current decreased with time in culture , being ∼1013 ± 95 pA ( n = 44 cells ) after 2–10 hr of culture , but only 277 ± 70 pA ( n = 9 cells ) after 2–3 days in culture ( Figure 6—figure supplement 4B ) .",
"Measurement of KNa current in a set of small diameter neurons in DRG slices yielded similar results ( Figure 6—figure supplement 5 ) .",
"However , one difference was that , although Slo2 . 2 accounted for most of the KNa current in DRG neurons in slices , after 2–3 min of dialysis of the pipette solution into the Slo2 . 2 KO neurons , some residual Na+-dependent current was observed .",
"Although neurons in slices may be uniquely affected by dialysis of 70 mM cytosolic Na+ , this observation raises the possibility that channels containing Slo2 . 1 subunits may be present at more peripheral locations in the DRG neurons , perhaps consistent with the presence of message encoding Slo2 . 1 and some Slo2 . 1 protein in DRG samples , as described above .",
"Small diameter DRG neurons exhibit a complex range of electrical properties reflecting a rich variety of Nav ( Vijayaragavan et al . , 2001; Ho and O'Leary , 2011 ) and Kv channels ( Zhang et al . , 2010b ) .",
"Such neurons are also heterogeneous ( Petruska et al . , 2000; Dirajlal et al . , 2003 ) in regards to sensitivity to various chemical signals .",
"Given the presence of Slo2 . 2-dependent KNa current in all DRG neurons we sampled , KNa currents may influence excitability in several different classes of neurons .",
"Since tests for phenotypic consequences of Slo2 . 2 KO pointed to neurons involved in itch and , to a lesser extent , pain , we limited our analysis to small-diameter IB4+ neurons , likely to be enriched in neurons involved in itch and polymodal pain sensation ( Lallemend and Ernfors , 2012 ) .",
"Neurons were selected for recordings based on size defined from membrane capacitance ( WT: 16 . 1 ± 0 . 3 pF [±sem; n = 64]; Slo2 dKO: 15 . 9 ± 0 . 5; [n = 41] ) and the presence of IB4 reactivity ( Dirajlal et al . , 2003 ) .",
"Furthermore , neurons were prepared from 3 to 5 week old mice to help ensure relative numbers of IB4+ and Ret-expressing neurons ( Molliver et al . , 1997 ) more consistent with acquisition of adult itch and polymodal pain-sensing ( Lallemend and Ernfors , 2012 ) .",
"We used Slo2 dKO neurons to guarantee complete absence of any Slo2-dependent KNa current .",
"A 1 s current step to different amplitudes was used to compare numbers of evoked APs in both WT and Slo2 dKO neurons with either 10 mM ( Figure 7A ) or 0 mM pipette Na+ ( Figure 7B ) .",
"Average resting potential ( Vm ) was adjusted to −60 mV , prior to the depolarizing current pulses .",
"Despite considerable variability in the maximum firing rates among both WT and Slo2 dKO neurons , AP firing was , on average , more robustly elevated in Slo2 dKO neurons than in WT neurons for identical amounts of injected current ( Figure 7C , D ) .",
"The increase in firing in Slo2 dKO neurons was observed at all levels of current injection , both with 10 and 0 mM pipette Na+ ( Figure 7E–G ) .",
"AP firing did not differ between 10 and 0 mM pipette Na+ within WT neurons or within Slo2 dKO neurons .",
"The increase in AP firing associated with KNa loss is consistent with increased AP firing of embryonic ( E15 ) rat DRG neurons following protein kinase A-mediated internalization of KNa channels ( Nuwer et al . , 2010 ) . 10 . 7554/eLife . 10013 . 017Figure 7 . Evoked action potential ( AP ) firing is increased in IB4+ DRG neurons from Slo2 dKO mice .",
"( A ) 40 , 60 , 100 , and 200 pA current injections ( 1 s ) were used to elicit firing in WT ( left ) and dKO IB4+ DRG neurons from a holding potential of −60 mV .",
"The pipette solution contained 10 mM Na+ .",
"( B ) Similar injected currents were used to elicit firing in WT and Slo2 dKO neurons , but with 0 mM pipette Na+ .",
"( C ) Mean number of APs for each 1 s step is plotted as a function of injected current amplitude for WT and dKO neurons for 10 mM pipette Na+ .",
"WT and dKO AP firing was significantly different at all injected current levels .",
"( D ) Mean firing is compared for WT and dKO neurons recorded with 0 mM pipette Na+ .",
"( E ) Mean ( black circle ) and individual estimates ( red circles ) of AP firing for 1 s 60 pA current injections are summarized for 10 and 0 mM pipette Na+ .",
"p values , KS statistic .",
"For comparisons between 0 and 10 mM Na+ , for WT cells , p = 0 . 909; for dKO cells , p = 0 . 545 .",
"( F ) AP firing for 100 pA current injections .",
"Between 0 and 10 mM Na+ , for WT cells , p = 0 . 585; for dKO cells , p = 0 . 245 .",
"( G ) AP firing for 200 pA current injections .",
"Between 0 and 10 mM Na+ , for WT cells , p = 0 . 09; for dKO cells , p = 0 . 23 . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 017 Standard protocols were used to compare basic electrical properties of WT and Slo2 dKO neurons either with 10 mM ( Table 1 , top ) or 0 mM ( Table 1 , bottom ) pipette Na+ .",
"To measure rheobase , 20 ms depolarizing current injections were applied from a −60 mV holding potential either with 10 ( Figure 8A ) or 0 mM pipette Na+ ( Figure 8B ) .",
"This defines a minimal amount of injected current necessary to elicit an AP .",
"Despite considerable variance within both WT and dKO cells , both at 10 and 0 mM Na+ less current was required to elicit an AP in the dKO neurons ( Figure 8C; Table 1 ) .",
"This difference between WT and dKO neurons suggests that KNa is activated prior to or during the weak depolarizations that begin to elicit Nav activation and is not influenced by pipette Na+ over the range of 0–10 mM . 10 . 7554/eLife . 10013 . 018Table 1 . Properties of IB4+ WT and Slo2 dKO DRG neurons ( 10 and 0 mM pipette Na+ ) DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 018Pipette Na+IB4+ WTIB4+ dKOp-values10 mM Na+meansemnmeansemnK-S statisticCm ( pF ) 16 . 10 . 36415 . 90 . 5410 . 574m . p .",
"( mV ) −54 . 20 . 657−50 . 80 . 9410 . 001Rin ( MΩ ) 1251 . 1130 . 8131212 . 9148 . 8130 . 828rheobase ( pA ) 86 . 64 . 64458 . 13 . 4310 . 000dV/dt AP threshold ( mV ) −25 . 310 . 6414−27 . 890 . 65100 . 032AP peak ( mV ) 39 . 22 . 21441 . 71 . 6100 . 877AP half-width ( ms ) 5 . 70 . 3145 . 60 . 3100 . 771AHP ( mV ) −74 . 00 . 414−72 . 60 . 5100 . 12460 pA AP count2 . 30 . 5649 . 71 . 9410 . 000100 pA AP count5 . 50 . 86417 . 72 . 7410 . 000200 pA AP count11 . 41 . 56428 . 95 . 1410 . 000Pipette Na+IB4− WTIB4− dKOp-values0 Na+meansemnmeansemnK-S statisticCm ( pF ) 16 . 70 . 91216 . 70 . 8120 . 991m . p .",
"( mV ) −54 . 01 . 411−47 . 01 . 4120 . 007Rin ( MΩ ) 1381 . 0194 . 5121136 . 495 . 0110 . 459rheobase ( pA ) 92 . 58 . 71260 . 85 . 1120 . 0048dV/dt AP threshold ( mV ) −22 . 70 . 710−25 . 80 . 5100 . 001AP peak ( mV ) 45 . 32 . 91051 . 71 . 6100 . 313AP half-width ( ms ) 5 . 10 . 3104 . 50 . 2100 . 313AHP ( mV ) −72 . 50 . 710−73 . 70 . 4100 . 31360 pA AP count1 . 20 . 5125 . 81 . 2120 . 005100 pA AP count3 . 51 . 11212 . 32 . 1120 . 019200 pA AP count6 . 82 . 41119 . 93 . 0100 . 005Cm , cell capacitance; m . p . , resting potential; Rin , input resistance measured by current deflection arising from a 10 mV pulse from −60 to −70 mV; AP half-width , measured at half peak amplitude; AHP , measured following a single evoked AP; AP count , number of APs in 1 s of specified injected current .",
"Rheobase , defined as smallest injected current which elicited an action potential during a 20 ms current injection . AP , action potential . 10 . 7554/eLife . 10013 . 019Figure 8 . Slo2 dKO results in reduced AP threshold .",
"( A ) A 20 ms current injection of different amplitudes applied with membrane potential adjusted to −60 mV was used to examine AP threshold for a WT ( left ) and a Slo2 dKO ( right ) DRG neuron with 10 mM pipette Na+ .",
"Dotted red lines indicate 0 and −60 mV voltage levels .",
"Current injection amount that first elicited an AP is indicated on each panel .",
"( B ) A similar comparison of AP threshold for a WT ( left ) and dKO ( right ) neuron is shown with 0 mM pipette Na+ .",
"( C ) Mean and individual determinations of effective rheobase as determined in panels ( A ) and ( B ) are plotted for WT and dKO cells both for 10 and 0 mM pipette Na+ .",
"p values are the KS statistic for the indicated pairs .",
"There was no difference for comparisons of 0 and 10 mM Na+ within a given genotype .",
"( D ) Example single APs elicited by a 100 pA current injection for WT and Slo2 dKO neurons are shown ( 0 mM pipette Na+ ) .",
"( E ) dV/dt is plotted as function of membrane voltage for the APs in panel ( D ) ( dKO , red; WT , black ) .",
"Horizontal dotted lines correspond to the dV/dt value that is 10% of peak dV/dt for a given cell .",
"( F ) The dV/dt plot is shown for a more limited range of membrane voltage , with crossover with horizontal dotted lines of same color showing effective AP threshold .",
"( G ) Thresholds determined from dV/dt analysis are plotted for WT and dKO neurons ( p = 0 . 001 , KS statistic ) .",
"( H ) Currents activated by a 40 ms voltage-ramp from −60 to −20 mV from a holding potential of −60 mV were averaged for 10 WT and 11 dKO neurons ( 0 mM pipette Na+ ) .",
"The voltage at which the current becomes net inward is indicated by the arrows .",
"( I ) The membrane potential at which net current becomes inward during the voltage-ramp protocol shown in ( H ) is plotted for WT and dKO neurons with p = 0 . 007 ( KS statistic ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 019 We next compared the properties of single APs in WT and Slo2 dKO neurons elicited by a single 20 ms 100 pA current injection with 0 mM pipette Na+ ( Figure 8D ) , a stimulus usually sufficient to evoke an AP in both WT and dKO cells .",
"AP waveforms were then transformed into phase plots ( dV/dt vs V ) for each cell ( Figure 8E ) .",
"Since peak dV/dt can vary substantially among cells , we have defined the threshold in a given cell as the Vm value at which dV/dt reaches 10% of its peak value ( dotted lines on Figure 8E , F ) .",
"This comparison shows that APs are initiated from a more negative Vm in dKO cells than in WT cells ( Figure 8G ) .",
"In contrast to the effects of Slo2 dKO on AP initiation , a number of other properties of single APs , including peak AP amplitude , AP half-width , and AP after-hyperpolarization , showed no obvious differences ( Table 1 ) .",
"However , with both 10 and 0 mM pipette Na+ , dKO cells exhibited a somewhat more depolarized Vm , although no obvious difference in input resistance ( Rin ) measured from a 10 mV step from −60 to −70 mV was noted .",
"Potential reasons for the apparent discrepancy between Vm and Rin will be considered below .",
"If the difference in apparent AP threshold between WT and dKO cells arises from outward KNa current present in the WT cells that delays the activation of Nav current , a voltage-clamp ramp protocol that better approximates the slow depolarization preceding an AP might also reveal a difference between WT and dKO neurons .",
"From a holding potential of −60 mV , cells were therefore stimulated with a 40 ms voltage-ramp up to −20 mV ( Figure 8H ) .",
"We observed that the Vm at which the overall current became net inward was more negative in dKO cells compared to WT cells ( Figure 8H , I ) .",
"Prior to the surge of Nav current activation , the ramp reveals a modest outward current , which is larger on average in WT cells and which in WT cells slightly shifts rightward the voltage at which net current becomes inward , relative to dKO cells .",
"Although the properties of the ramp-activated outward current and shift in 0 current potential are generally consistent with the loss of outward current activated at the onset of depolarization , a concern in regards to the above experiments is that the comparisons are being made between cell populations from genotypically distinct animals .",
"For example , a shift in Nav channel activation to more negative potentials in dKO neurons might produce qualitatively similar effects .",
"We therefore tested several inhibitors and activators of KNa current as tools to examine the properties of ramp-activated current in WT cells , but slow onset of action and non-specific effects on other ion channels precluded their use .",
"As an alternative , having shown that extracellular Cs+ inhibits KNa current , we examined the ability of 20 mM Cs+ to influence excitability and ramp-activated currents in both WT and dKO DRG neurons with 0 mM pipette Na+ ( Figure 9 ) .",
"As shown above , the voltage-ramp activated a much more pronounced low-voltage outward current in WT cells than in dKO cells , with a marked shift in the 0 current potential ( Figure 9A ) .",
"Application of 20 mM extracellular Cs+ to WT neurons also resulted in a reduction in ramp-activated outward current and a shift in the 0 current voltage ( p = 0 . 000; Figure 9B ) , quite comparable to the current observed in the dKO neurons ( Figure 9C ) .",
"In contrast , application of 20 mM Cs+ to the dKO cells produced only small shifts in the 0 current voltage ( p = 0 . 675; Figure 9D ) .",
"Overall , 20 mM Cs+ mimicked the effect of Slo2 dKO on the 0 current voltage ( Figure 9E ) , while also producing essentially identical effects on measurement of rheobase in the same set of neurons ( Figure 9F ) .",
"That an apparent shift in the voltages over which the surge of inward current is observed can occur from K+ channel inhibition is also highlighted in comparisons of the normalized ramp activated currents ( Figure 9—figure supplement 1 ) , which clearly shows the ability of Cs+ to produce a shift in apparent inward current activation in WT cells which is much more reduced in the dKO cells .",
"Inhibition by Cs+ is likely to differ from Slo2 dKO in two primary ways: first , KNa will not be inhibited completely by Cs+ at these voltages and second , Cs+ is likely to inhibit other K+ currents in addition to KNa .",
"However , the results clearly support the view that an apparent shift in inward current activation occurs with inhibition of subthreshold K+ currents , likely to include KNa . 10 . 7554/eLife . 10013 . 020Figure 9 . Cs+ inhibition of outward current in WT , but not dKO , neurons recapitulates properties of Slo2 dKO .",
"( A ) Traces show averaged currents activated by the indicated voltage-ramp protocol ( top ) for 10 WT and 10 dKO neurons .",
"Number shows the voltage at which net current crosses the 0-current level ( indicated approximately by arrow heads ) .",
"( B ) Ramp-activated currents are shown for WT cells before and after application of 20 mM extracellular Cs+ .",
"( C ) Ramp-activated currents are compared for WT cells in the presence of 20 mM Cs+ and dKO cells .",
"( D ) Currents are shown for dKO neurons without and with 20 mM extracellular Cs+ .",
"( E ) The mean 0-current potential for sets of WT and dKO neurons without and with Cs+ are plotted , along with the individual estimates from each cell .",
"p values are KS statistics .",
"Other comparisons had p-value estimates >0 . 1 .",
"( F ) Mean rheobase for the same set of WT and dKO cells are plotted , along with individual estimates . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 02010 . 7554/eLife . 10013 . 021Figure 9—figure supplement 1 . Normalized ramp-activated currents reveals that application of Cs+ shifts the apparent range of inward current activation in a fashion similar to dKO of Slo2 currents . Ramp-activated currents as in Figure 9 were normalized in each cell to the largest inward current and then averaged .",
"The most negative inward current values of the averaged traces differ among each average because of temporal displacement of the exact minimum .",
"( A ) Normalized currents are shown for WT and dKO neurons ( 10 each ) for ramps up to −5 mV .",
"( B ) Application of 20 mM Cs+ to WT cells elicits a shift in the apparent range of inward current onset similar to that seen in dKO neurons .",
"( C ) The normalized ramp-activated currents compare dKO cells to WT cells in the presence of Cs+ .",
"( D ) Application of Cs+ to dKO cells produces only minor shifts in the normalized ramp-activated current properties . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 02110 . 7554/eLife . 10013 . 022Figure 9—figure supplement 2 . Comparison of step-activated inward and steady-state currents activated in WT and dKO cells for comparison of ramp-activated outward current .",
"( A ) 20 ms voltage steps from −120 mV to +40 mV were applied from a holding potential of −60 mV , with pipette and extracellular solutions appropriate to allow normal cell firing .",
"Figure plots peak inward current as a function of command voltage for 10 WT DRG neurons without and with application of 20 mM Cs+ .",
"( B ) Inward current as a function of command voltage is displayed for 10 dKO DRG neurons without and with Cs+ .",
"( C ) As an approximation of the voltage-dependence of inward current activation , currents at each command potential from A were converted to chord conductance estimates assuming ENa = 75 mV .",
"Boltzmann fits yielded , for control solution , Vh = −10 . 1 ± 0 . 4 mV with z = 4 . 1 ± 0 . 4e; with 20 mM Cs+ , Vh = −15 . 9 ± 0 . 2 mV with z = 3 . 7 ± 0 . 1e .",
"( D ) Mean Nav conductance was plotted as a function of command potential for 10 dKO DRG neurons and fit with a Boltzmann function .",
"For control solution , Vh = −13 . 9 ± 0 . 4 mV with z = 3 . 6 ± 0 . 2e; for 20 mM Cs+ , Vh = −16 . 4 ± 0 . 1 mV with z = 3 . 5 ± 0 . 1e .",
"( E ) From the same family of traces used to generate panels A–D and same cells used for Figure 9 , sustained current at the end of a 20 ms command step was measured and plotted as a function of command potential , for the 10 WT cells , without and with 20 mM Cs+ .",
"( F ) Sustained current as a function of command potential is plotted for 10 dKO cells .",
"Although maximum sustained current is similar between these sets of WT and DRG neurons , at voltages of −20 mV and more positive the activation of sustained current is shifted to more negative potentials in the dKO cells .",
"Note that in both WT and dKO cells the net blocking effect of Cs+ is stronger over potentials from −40 to −20 mV than at the more positive voltages .",
"A linear fit to the current values between −120 mV and −60 mV yielded a net conductance of 1 . 4 ± 0 . 04 nS for WT neurons , and 1 . 1 ± 0 . 1 nS for the dKO neurons .",
"However , in neither WT nor dKO cells , did Cs+ reduce this resting conductance , suggesting there is no basal KNa activation with 0 pipette Na+ .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 02210 . 7554/eLife . 10013 . 023Figure 9—figure supplement 3 . Evaluating the potential impact of a small K+ conductance near resting potential .",
"( A ) A modified GHK conductance equation ( top ) was used to calculate membrane potential .",
"In the absence of explicit estimates of specific ion conductances at rest , we assume EK = −80 mV , ENa = 60 mV , ECl = −50 mV with gK , gNa , and gCl set to 0 . 7 nS , 0 . 165 nS , and 0 . 3 nS for a total membrane conductance of 1 . 165 nS and a basal membrane potential of −52 . 4 mV .",
"A hypothetical gKNa was then added in increments of 0 . 02 nS to generate the relationship between net added gKNa and membrane potential .",
"Red dots correspond to the complete absence of KNa and with added 0 . 16 nS gKNa , ( B ) The relationship between Vm and total cell conductance is plotted .",
"We would propose that the ability of KNa to influence Vm will also be influenced by any factors affecting basal Na flux , such that at more negative voltages the contribution of KNa would diminish .",
"( C ) Assuming a maximal DRG gKNa of ∼69 nS ( Figure 6F ) , the relationship between effective Po of the KNa channels and gKNa is plotted .",
"( D ) The expected current amplitude of KNa at −50 mV for the given increments of KNa conductance is plotted . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 023 The same set of cells was also examined with standard voltage-step protocols to ascertain the properties of peak inward and steady-state outward current ( Figure 9—figure supplement 2 ) .",
"Step-activated inactivating current and the voltage of half-activation of the inward current was roughly similar in both WT and dKO neurons , with similar reductions produced by 20 mM Cs+ ( Figure 9—figure supplement 2A–D ) .",
"The absence of obvious differences between the Nav currents in WT and dKO neurons make it highly unlikely that a difference in Nav current between WT and dKO neurons accounts for the differences in rheobase and ramp-activated 0 current potential .",
"Furthermore , if the effect of Cs on ramp-activated current were to arise from an effect on Nav current , an inhibition of Nav current would be expected to shift the 0 current voltage rightward .",
"This is not observed .",
"Together , these results support the view that the Cs+ induced inhibition of ramp-activated outward current and the shift in the 0 current voltage arise solely from inhibition of a K+ current .",
"Whatever this current is , it is apparently absent in the dKO neurons .",
"Although it is perhaps possible that some other low voltage activated K+ current other than KNa is also absent in the dKO neurons , the simpler view is that the difference in excitability between the WT and dKO neurons arises from the absence of the KNa current itself .",
"To ascertain whether there might be changes in other components of current between this particular set of WT and dKO neurons , we also compared steady-state current at the end of a 20 ms voltage-step in the same set of cells ( Figure 9—figure supplement 2E , F ) .",
"Although net outward current was generally similar in both groups , the dKO cells exhibited a larger outward at command potentials from −10 mV and more positive ( Figure 9—figure supplement 2E ) .",
"However , over the range of −120 to almost −20 mV , there was no obvious difference in this steady-state current ( Figure 9—figure supplement 2F ) .",
"With 0 mM pipette Na+ , no significant difference was observed in the resting conductance measured from a fit of the I–Vs between −120 and −60 mV , suggesting that there is little obvious basal activation of KNa current with 0 mM pipette Na+ .",
"Although these results also indicate that , in the voltage range of −50 to −20 mV , there are other Cs+-sensitive K+ conductances besides KNa active at the end of 20 ms steps , these do not appear to differ significantly between WT and dKO neurons , again supporting the idea that the observed differences in excitability are likely to arise from changes in KNa alone .",
"Given that in many other cells KNa may play a role in slow AHPs , we also specifically addressed this question in WT DRG neurons .",
"For example , in cells of the thalamic paraventricular neurons ( Zhang et al . , 2010a ) , it has been shown that trains of APs produce a slow development of Na+-dependent AHPs dependent on the number and frequency of APs in the trains .",
"We therefore examined the consequences of an increasing number of APs on AHPs in IB4+ small diameter neurons .",
"Trains of 5 or 10 APs applied at 7 Hz were unable to elicit any slow AHP in IB4+ small diameter neurons different from that elicited by a single AP ( Figure 10 ) .",
"This further suggests that KNa current in DRG neurons , at least with physiological ionic solutions , contributes negligibly to membrane potential regulation following APs . 10 . 7554/eLife . 10013 . 024Figure 10 . AP trains in IB4+ small diameter DRG neurons do not develop slow AHPs .",
"( A ) A cell was maintained at a resting potential of −50 mV and stimulated with either 1 , 5 , of 10 pulses of 10 ms duration and 200 pA amplitude , with a pulse frequency of 7 . 1 Hz .",
"A brief afterhyperpolarization is associated with the last AP in each test , with no indication of any additional slow afterhyperpolarization persisting for 100 s of milliseconds .",
"( B ) In another cell maintained at a resting potential of −60 mV , the identical current injection sequence also failed to elicit any slow afterhyperpolarization .",
"Identical results were observed in three additional neurons . DOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 024 Overall , these results suggest that the contribution of KNa to DRG firing behavior is relatively insensitive to resting cytosolic Na+ levels up to 10 mM .",
"The absence of a difference in resting conductance between WT and dKO neurons at potentials between −60 and −120 mV suggests that basal KNa activation is minimal .",
"Therefore , an influence of KNa current at the foot of AP generation presumably requires a source of Na+ and perhaps a requirement for coupling to local Na+ influx .",
"Although it has been proposed that KNa channels may be coupled to influx through particular subtypes of Nav channels ( Hage and Salkoff , 2012 ) , the results here would require that such coupling must be very tight and occur immediately upon Nav activation .",
"Future work will be required to address these issues , but the results here suggest new considerations on the conditions under which KNa activation may occur ."
],
[
"Direct evidence supporting the existence of KNa channels in native cells first appeared about 25–30 years ago ( Kameyama et al . , 1984; Bader et al . , 1985; Dryer et al . , 1989 ) .",
"Yet , even with identification of the two mammalian Slo2 genes that encode KNa channels of the type observed in the early studies ( Bhattacharjee et al . , 2003; Yuan et al . , 2003 ) , full definition of functional properties and physiological roles of KNa currents have proven somewhat elusive , despite the apparently widespread distribution of Slo2 subunits in excitable tissue ( Bhattacharjee et al . , 2002 , 2005 ) .",
"Recent demonstration of neurological disorders linked to Slo2 . 2 ( Barcia et al . , 2012; Martin et al . , 2014 ) further highlights the potential importance of such channels .",
"The availability of Slo2 . 1 and Slo2 . 2 KO mice will now provide an additional tool to probe the roles of KNa currents .",
"The present results demonstrating the loci of expression of message for Slo2 . 1 and Slo2 . 2 subunits , the presence of Slo2 . 1 and Slo2 . 2 protein in various tissues , and the natural occurrence of Slo2 . 1/Slo2 . 2 heteromultimers confirms and extends earlier work regarding the loci of expression of Slo2 subunits ( Bhattacharjee et al . , 2002 , 2003; Chen et al . , 2009; Tamsett et al . , 2009 ) , while also providing KO controls confirming the utility of antibodies in western blots .",
"Here , focusing on the role of Slo2 . 2 in sensory function , we observed that Slo2 . 2 , but not Slo2 . 1 , KO results in enhanced itch and , to a lesser extent , pain responses .",
"Furthermore , KO of Slo2 . 2 , but not Slo2 . 1 , results in complete absence of the DRG KNa current .",
"This loss of Slo2 current results in increased excitability in response to depolarizing stimuli , likely accounting for the observed phenotypes .",
"An important point of the present results is that the primary effect of KNa removal is to reduce AP threshold , with little or no clear effect on AHPs following an AP .",
"The effect on AP threshold was revealed in multiple kinds of tests , a decrease in rheobase , a negative shift in AP threshold determined from the rate of AP rise ( dV/dt ) , and also from a similar negative shift in the voltage at which current becomes net inward during a ramp protocol .",
"Similar changes in AP threshold were observed with both 0 and 10 mM pipette Na+ , excluding a key role for differences in pipette Na+ as a determinant of basal KNa activation .",
"Overall , the results require that KNa acts as a mild brake to the onset of AP initiation .",
"Furthermore , we observed no difference in AHP amplitude measured following single APs between WT and KO cells , either with 0 or 10 mM pipette Na+ .",
"Trains of APs also did not evoke the development of slower AHPs .",
"A role of KNa activity preceding the AP upswing in DRG neurons differs fundamentally from other explanations of the proposed role of KNa current in other cells .",
"In different cases , a primary role for KNa current has been proposed either in fast ( Hess et al . , 2007; Yang et al . , 2007; Gribkoff and Kaczmarek , 2009; Markham et al . , 2013 ) or slower afterhyperpolarizations that may require trains of several APs to develop ( Schwindt et al . , 1989; Kim and McCormick , 1998; Franceschetti et al . , 2003; Wallen et al . , 2007; Zhang et al . , 2010a ) .",
"In the case of rapid coupling of KNa activation to single APs , this potentially provides a mechanism to facilitate high frequency firing ( Yang et al . , 2007 ) , while slow AHP development serves to support AP accommodation and termination of burst activity .",
"The present results do not preclude such roles for KNa either in rapid repolarization or slow AHPs in other cells .",
"However , the specific role of KNa channels in any cell would depend intimately on the balance of other repolarizing conductances , along with magnitude , spatial , and temporal properties of any cytosolic [Na+] elevation .",
"In this regards , it is worth mentioning that some of the best support for the presence of KNa currents in cortical neurons has required conditions under which Ca2+-dependent outward currents are inhibited ( Schwindt et al . , 1989 ) .",
"Given the abundance of other repolarizing conductances in DRG neurons , it is perhaps not unexpected that modest KNa activation during AP repolarization in DRG neurons may have negligible effects on Vm .",
"Our observations are , in fact , consistent with earlier results in rat DRG neurons , in which both AP duration and AHPs were unchanged when extracellular Na+ was replaced by Li+ ( Bischoff et al . , 1998 ) , despite the fact that Li+ does not substitute for Na+ in KNa activation ( Safronov and Vogel , 1996 ) .",
"If KNa activity acts as a brake to AP initiation , how is KNa activation elicited ?",
"Despite the well-established existence of KNa channels , the circumstances under which cytosolic Na+ elevation arising from physiological stimuli is sufficient to produce KNa activation remain unclear .",
"In fact , consideration of basic properties of Na+ diffusion and the expected Na+ flux through single channels have raised some doubt whether average [Na+]i can ever be sufficient to activate KNa ( Dryer , 1991 ) .",
"Some aspects of our data partially address these issues , but there are complexities in our observations that are not readily explained .",
"The differences in firing properties of WT and dKO cells , both with 0 and 10 mM pipette Na+ , suggest that KNa activation is unaffected over the range of 0–10 mM pipette Na+ .",
"This is not surprising given that the threshold for KNa activation may be higher than 10 mM ( Bischoff et al . , 1998; Tamsett et al . , 2009 ) .",
"Furthermore , when Rin was measured with a step from −60 to −70 mV , no difference between WT and dKO neurons was observed either with 0 and 10 mM pipette Na+ .",
"Similarly , with Rin measured by a fit to the I–V relationship over voltages from −120 to −60 mV with 0 Na+ pipette solution , no Cs+ dependent inhibition of conductance was observed in either WT or dKO neurons .",
"Although it has been suggested that some Slo2 . 2 channel activation may occur in 0 Na+ ( Huang et al . , 2013 ) , the present results suggest that in DRG neurons basal KNa activity at potentials negative to −60 mV does not occur .",
"However , both with 0 and 10 mM pipette Na+ , WT cells exhibited a slightly more negative Vm than dKO cells .",
"If pipette Na+ itself does not influence the differences in WT cells from dKO cells , how might these differences arise ?",
"The largely linear behavior of both the WT and dKO neuron steady-state I–V relationship from −120 mV to −60 mV begins to exhibit distinct upward curvature in the range of −60 to −50 mV , bracketing the range of measured membrane potentials .",
"This would suggest that conductances are active at rest that are apparently not active negative to −60 mV .",
"Based on the differences in WT and dKO resting potentials , KNa current is clearly a candidate for one of these conductances .",
"In addition , that Vm is close to −50 mV with EK ∼ −80 mV suggests that there may be appreciable inward Na current at potentials above −60 mV .",
"Future work will be required to assess the identity of any components of Na+ current active at such potentials .",
"Voltage-step protocols used here from a holding potential of −70 mV reveal little obvious inward current until at least −30 mV .",
"Both Nav1 . 8 and Nav1 . 9 channels are known to be expressed in some small diameter IB4+ neurons ( Fang et al . , 2006; Strickland et al . , 2008 ) and Nav1 . 9 , in particular , may begin to activate at potentials close to resting potentials we have observed ( Rugiero et al . , 2003; Coste et al . , 2004; Zhao et al . , 2011b ) .",
"Another possibility reflects the proposal that KNa currents in some neurons may be selectively activated by persistent TTX-sensitive Na+ currents ( Hage and Salkoff , 2012 ) .",
"TTX-sensitive Nav1 . 7 channels can be found in small diameter DRG neurons ( Nassar et al . , 2004 ) and , although such channels are largely inactivated near DRG resting potentials ( Vijayaragavan et al . , 2001 ) , it is possible that even under steady-state inactivated conditions some persistent openings occur .",
"Perhaps as Slo1 Ca2+-dependent K+ channels are coupled to specific Ca2+ channels ( Berkefeld et al . , 2006; Berkefeld and Fakler , 2008 ) , molecular coupling of KNa channels to specific sources of Na+ influx may occur .",
"Although the changes in rheobase , AP threshold , and increase in excitability observed in the dKO animals can largely consistent with what one would expect from the simple demonstrated removal of KNa current , there is also the possibility that genetic deletion of Slo2 protein may result in compensatory changes that account for some of the observed effects .",
"This issue might be of particular concern in regards to DRG neurons , since it is well-known that a variety of manipulations can readily induce changes in various DRG current properties , including Nav channels , resulting in altered excitability ( Chahine and O'Leary , 2014 ) .",
"Furthermore , in the particular case of Slo2 . 2 , it has been proposed that severe human pathologies associated with Slo2 . 2 mutation arise from extensive alterations in gene and protein expression throughout the nervous system ( Kaczmarek , 2013 ) .",
"In the present case , two possible alternative mechanisms by which rheobase , AP threshold , and excitability might be altered as observed in the Slo2 dKO neurons would be , first , a shift in Nav current activation to more negative potentials and , second , a loss of some other K+ current active in the subthreshold range of voltages .",
"Although we cannot fully exclude that there have been no changes other than loss of KNa current in the Slo2 dKO DRG neurons , the demonstration that inhibition of subthreshold K+ current by Cs+ mimicked the behavior of the dKO neurons strongly argues that a change in Nav channel expression does not underlie the observed phenotypes and , furthermore , that removal of a sub-threshold K+ conductance can produce the particular constellation of changes we have observed .",
"Finally , we did not observe any indication of a loss in a near threshold K+ conductance other than KNa , although any such change might be difficult to resolve .",
"It is instructive to consider how much KNa current activation might be required to account for changes in resting potential .",
"Assuming a simple modified GHK conductance equation and a relative balance of GK , GNa , and GCl ( net Rin = 858 MΩ; Gin = 1 . 165 nS ) to yield a resting potential near −50 mV , increasing the background GK of 0 . 7 nS with an additional activation of 0 . 16 nS GKNa results in additional hyperpolarization of ∼3 . 3 mV ( Figure 9 , Figure 9—figure supplement 3 ) .",
"From Figure 6F , we observed an average KNa conductance of 69 nS activated by 70 mM pipette Na+ around −50 mV .",
"Although 70 mM Na+ produces a less than maximal activation , if one assumes a maximal conductance of 69 nS , the fractional activation of KNa required to produce a 3–4 mV hyperpolarization corresponds to a Po of about 0 . 002 , which corresponds to 8 pA at −50 mV .",
"If these calculations are generally correct , it is not surprising that it would be difficult to identify procedures to directly examine such a current .",
"The severity of the human patients with apparent Slo2 . 2 mutations ( Barcia et al . , 2012; Heron et al . , 2012 ) naturally raises a question regarding whether suitable phenotype tests may uncover cognitive impairments in the Slo2 dKO mice .",
"Any such deficits , if they exist , apparently spare the basic ability of the dKO animals to eat , mate , and function in a generally normal way .",
"Perhaps relevant to the possibility that Slo2 . 2 KO may have apparently benign functional consequences , one set of the human Slo2 . 2 mutations corresponds to gain-of-function changes ( Barcia et al . , 2012 ) , resulting in larger KNa currents .",
"Perhaps the presence of Slo2 . 2 subunits of altered function results in more deleterious consequences than the complete absence of such subunits .",
"Recent work on another Slo2 . 2 KO model ( Lu et al . , 2015 ) also focused on sensory function with some complementary results .",
"In both cases , exon 11 of the gene encoding Slo2 . 2 was deleted .",
"Both groups observe similar absence of effects of Slo2 . 2 KO on hotplate and formalin tests .",
"Given the absence of effects on acute pain responses , Lu et al . ( 2015 ) focused on neuropathic pain responses , observing that Slo2 . 2 activation reduces neuropathic pain and does not acutely influence sensory responses .",
"However , our results clearly show that Slo2 . 2 KO influences the immediate response to sensory stimuli , in particular , itch .",
"Furthermore , an enhancement of the acute responses to capsaicin also occurs at lower doses .",
"Both groups also observed increased excitability in neurons lacking KNa current , although the basis for the enhanced excitability was not examined in detail by the other group ( Lu et al . , 2015 ) .",
"However , we suggest that the increase in DRG neuron excitability observed in both studies is consistent with enhancement of the immediate response to a sensory stimulus .",
"Why do some aversive tests , for example , hotplate , formalin , tail flick , and cold plate ( Lu et al . , 2015 ) , show no difference between WT and Slo2 . 2 KO ?",
"We envision three possible explanations .",
"First , as suggested by the dose-dependence of capsaicin responses , perhaps regulation of KNa current has more impact on weaker stimuli or relatively weak depolarizing drive , whereas , with stimuli that elicit strong initial depolarization , modest KNa activation will be less likely to influence AP generation .",
"Second , even if KNa is present in most small diameter DRG neurons , different categories of such neurons may have other conductances that diminish the impact of loss of KNa current .",
"Third , perhaps there are small diameter neurons , or certainly DRG neurons of other sizes , that may not have KNa current .",
"In sum , we propose that , in small diameter IB4+ DRG neurons , KNa currents influence AP onset , with greatest effect during low frequency firing .",
"The particular properties of KNa currents , specifically modest intrinsic voltage-dependence but voltage-dependence perhaps acquired through coupling to its cytosolic ligand , Na+ , may be well-suited to influence the initial upswing of AP generation , at a time when other K+ conductances are largely quiescent ."
],
[
"Animals were handled and housed according to the National Institutes of Health Committee on Laboratory Animal Resources guidelines .",
"All experimental protocols ( protocol #20130256 ) were approved by the Washington University in St Louis Institutional Animal Care and Use Committee .",
"Every effort was made to minimize pain and discomfort .",
"To generate the Slo2 . 1 KO ( deletion of Kcnt2 exon ) mouse , exon 22 ( 110 bp , encoding amino acids 829–865 of the Slo2 . 1 protein ) was targeted for deletion .",
"The deletion of exon 22 in Kcnt2 causes a frame-shift and the predicted residual protein is Slo2 . 1 ( 1–828 ) .",
"To generate the Slo2 . 2 KO ( deletion of Kcnt1 exon ) mouse , exon 11 ( 181 bp , encoding amino acids 253–313 of Slo2 . 2 , which includes part of the pore-forming region ) was targeted for deletion .",
"The deletion of exon 11 in Kcnt1 causes a frame-shift and the predicted residual protein is Slo2 . 2 ( 1–252 ) with an appended 26 amino acid peptide before the first stop codon .",
"Following germline transmission via recombinant ES cells , the F1 mice with targeted loci were bred with FLP delete mice ( B6 . 129S4-Gt ( ROSA ) 26Sortm1 ( FLP1 ) Dym/RainJ , Jackson Labs , Bar Harbor , ME , United States ) to generate floxed mouse lines and with early embryonic expression Cre-mice ( EIIa-Cre , Jackson ) for deletion of the targeted exons .",
"The Kcnt1 floxed mice and Kcnt2 floxed mice are available at The Jackson Laboratory as Stock No . 028418 and Stock No . 028419 , respectively .",
"Slo2 . 1 KO and Slo2 . 2 KO strains of mice have been maintained in a C57BL/6 background out to N = 12 .",
"Additional details of the generation of Slo2 . 1 and Slo2 . 2 KO mice are provided in the legend to Figure 1 .",
"All procedures related to animal care and treatment conformed to institutional and NIH guidelines .",
"Mice were maintained in a 12 hr light/dark cycle with free access to food and water .",
"Behavioral experiments were done on male mice of 10–12 weeks of age and mice were only used once for any of the tests .",
"Littermate mice were used in all behavioral studies , except those involving double KO of Slo2 . 1 and Slo2 . 2 .",
"For dKO mice , each allele had initially been breed within a C57BL/6 background out to N = 12 generations , so comparisons were made to the Jackson Labs WT C57BL/6 stock .",
"Animals were habituated to the experimental room with background white noise used to mask random noise ( SKI 000148 , San Diego Instruments , San Diego , CA , United States ) and monitored by an observer naïve to genotype .",
"RNA extraction and RT-PCR followed procedures previously used in this laboratory ( Yang et al . , 2011; Martinez-Espinosa et al . , 2014 ) .",
"Total RNA from different mouse tissues was isolated using the RNeasy Plus Mini Kit ( Qiagen , Valencia , CA , United States ) following the manufacturer's recommendations .",
"Before the reverse transcription , total RNA was treated to remove genome DNA with the DNA-free Kit ( AM1906 , Applied Biosystems , Waltham , MA , United States ) .",
"cDNA was synthesized using the Retroscript Kit ( AM1710 , Applied Biosystems ) .",
"For the negative control groups , all components except the reverse transcriptase MMLV-RT were included in the reaction mixtures .",
"Real-Time PCR with specific primers ( Table 2 ) was performed using Power SYBR Green PCR Master Mix ( Applied Biosystems ) .",
"Mouse β-actin gene was utilized here as the homogenous standard .",
"The running protocol extended to 40 cycles consisting of 95°C for 15 s and 60°C for 1 min using an Applied Biosystems 7500 Fast Real-time PCR system .",
"PCR specificity was checked by dissociation curve analysis and DNA electrophoresis .",
"Primer efficiency was validated as previously reported ( Yang et al . , 2009 ) .",
"Abundance was calculated from 2−dCt , with dCt = Ct ( target ) − Ct ( β-actin ) .",
"Each reported estimate is the average from three separately prepared mouse tissue RNA samples , with each sample run in triplicate . 10 . 7554/eLife . 10013 . 025Table 2 . Primers used for Real-Time PCRDOI: http://dx . doi . org/10 . 7554/eLife . 10013 . 025GenePrimerAmplicon lengthKcnt2Forward: 5′-TCTATTTGAAACAATACTCCTTGG-3′149 bpReverse: 5′-GAACAAATAGATTTCTTAAGGTGG-3′Kcnt1Forward: 5′-CTCACACACCCTTCCAACATGCGG-3′161 bpReverse: 5′-ATGCTGATACTAAATACTCGACCA-3′Β-actinForward: 5′-TGGAGAAGAGCTATGAGCTGCCTG-3′127 bpReverse: 5′-GTAGTTTCATGGATGCCACAGGAT-3′ Preparation and analysis of proteins from mouse tissues followed procedures recently used in this laboratory ( Yang et al . , 2011; Martinez-Espinosa et al . , 2014 ) .",
"Mature male mice were sacrificed for preparation of membrane proteins from whole brain , cerebellum , cortex and spinal cord , respectively .",
"1 g of mouse whole brain , cortex , cerebellum , or spinal was homogenized with Teflon-glass pestle in 10 ml ice-cold 0 . 32 sucrose in PBS , including 100 μl 1 . 5 M PMSF in acetone and 100 μl Protease Inhibitor Cocktail ( Sigma-Aldrich ) .",
"After spinning at 300×g for 10 min at 4°C , the supernatant was collected , followed by ultra-speed centrifugation in a 4°C Ti70 rotor at 150 , 000×g for 1 hr .",
"The membrane pellet was resuspended in 10 ml lysis buffer ( 50 mM Na phosphate , 150 mM NaCl , 10 mM KCl , 2% Triton X-100 , pH 7 . 2 ) , including 100 μl 1 . 5 M PMSF in acetone and 100 μl Protease Inhibitor Cocktail , and rocked at 4°C for 1 hr , followed by centrifugation at 14 , 000×g for 10 min . 10 ml supernatant was saved as the membrane protein preparation in the −80°C freezer .",
"Hearts from four mature male mice were dissected , washed with PBS and quickly frozen in liquid nitrogen .",
"The frozen hearts were pulverized with liquid nitrogen pulverizer and then homogenized on ice with Teflon-glass pestle in 3 ml TE ( pH 7 . 6 ) buffer containing 2% Triton X-100 , 20 μl PMSF ( 1 . 5 M in acetone ) and 20 μl Protease Inhibitor Cocktail .",
"The suspension was rocked at 4°C cold room for 1 hr , followed by spinning at 14 , 000Χg for 15 min .",
"Pellet was discarded and the 3 ml supernatant was saved as the heart total protein preparation .",
"DRGs collected from 10 mature male mice were homogenized on ice with a Teflon-glass pestle in 1 ml lysis buffer ( 50 mM Na phosphate , 150 mM NaCl , 10 mM KCl , 2% Triton X-100 , pH 7 . 2 ) , including 10 μl 1 . 5 M PMSF in acetone and 10 μl Protease Inhibitor Cocktail .",
"The suspension was rocked at 4°C cold room for 1 hr , followed by spinning at 14 , 000×g rpm for 15 min .",
"The 1 ml supernatant was saved as the DRG total protein preparation .",
"Samples of total protein preparations or membrane protein preparations appropriate for a given tissue were applied in the immunoprecipitation experiment .",
"70 μl Protein A/G Plus agarose beads ( Santa Cruz Biotechnology , Dallas , TX , United States ) were added to the preparation and mixed at 4°C cold room for 1 hr .",
"The beads were removed by a brief spin at 14 , 000×g .",
"The supernatant was carefully collected and mixed with 8 μg monoclonal anti-mSlo2 . 1 ( N11/33 ) or anti-mSlo2 . 2 ( N3/26 ) antibody ( Antibodies Inc . , Davis , CA , United States ) at 4°C cold room for 2 hr , followed by the addition of 80 μl Protein A/G Plus agarose beads .",
"The mixture was rocked overnight and then centrifuged briefly to collect the beads .",
"The beads were washed three times with 1 ml 1% Triton X-100 in PBS and the bound proteins were eluted from the beads with 100 μl SDS loading buffer containing 100 mM DTT .",
"For western blotting , aliquots of total protein preparations or membrane protein preparations was mixed well with an equal volume of 2× SDS loading buffer containing 100 mM DTT , maintained at room temperature for 30 min before loading onto 8% Precise Protein Gels ( Pierce , Life Technologies , Grand Island , NY , United States ) .",
"Protein markers were EZ-Run Prestained Rec Protein Ladder ( Fisher , Waltham , MA , United States ) .",
"Proteins were transferred to Immobilon-P PVDF membranes with the Trans-Blot Semi-Dry Transfer System ( Bio-Rad , Hercules , CA , United States ) .",
"Membranes were blocked with 5% nonfat milk in Tris-buffered saline-Tween 20 solution ( pH 7 . 3 ) at room temperature for 1 hr , followed by overnight incubation at 4°C in 5 ml blocking solution containing monoclonal anti-mSlo2 . 1 or anti-mSlo2 . 2 antibody ( 10 μg/ml , Antibodies Inc ) .",
"After washing with 5 ml blocking solution × 5 min for four times , membranes were incubated with 5 ml blocking solution containing Mouse Trueblot Ultra HRP-conjugated anti-mouse IgG ( at 1:2000 dilution , eBioscience , San Diego , CA , United States ) at room temperature for 1 hr .",
"After four-time washing with 5 ml blocking solution , HRP-labeling was developed using Amersham ECL Plus Western Blotting Detection System ( GE Healthcare , Pittsburgh , PA , United States ) .",
"A specific Slo2 . 1 band in WT heart Slo2 . 1-IP sample was not detected in the first round of western blot with the monoclonal anti-Slo2 . 1 antibody ( 10 μg/ml , Antibodies Inc . ) .",
"To visualize a Slo2 . 1-specific band , the initial western blot was stripped with Re-Blot Plus Mild Solution ( Millipore; Billerica , MA , United States ) and then the PVDF membrane was reblotted with the same antibody .",
"For western blotting , NeuroMab anti-Slo2 . 1 antibody ( #75-055 ) targets amino acids 564–624 of Slo2 . 1; NeuroMab anti-Slo2 . 2 antibody ( #75-051 ) is against amino acids 1168–1237 of Slo2 . 2 .",
"Slo2 . 1 KO predicts a residual protein Slo2 . 1 ( 1–828 ) with a predicted MW of 91 kDa , which should be recognized by the NeuroMab antibody , but is not observed in the Slo2 . 1 KO brain ( Figure 2 ) .",
"This indicates that , following deletion of exon 22 , no residual Slo2 . 1 protein remains in the knockout mouse , probably due to the instability of the truncated mRNA or protein .",
"In the case of Slo2 . 2 KO , deletion of exon 11 ( encoding amino acids 285–354 of Slo2 . 2 ) causes a frame-shift such that the predicted residual Slo2 . 2 ( 1–252 ) protein does not contain the sequence recognized by the NeuroMab anti-Slo2 . 2 antibody .",
"Therefore , no residual Slo2 . 2 protein was detected in Slo2 . 2 KO membrane samples .",
"Since the residual Slo2 . 2 ( 1–252 ) is terminated in the middle of the S6 segment of the inner helix , it seems unlikely that any residual Slo2 . 2 protein fragments in the knockout mouse would assemble into functional channels .",
"From Figure 2A , B , D , it is clear that knocking out the gene for Slo2 . 1 has no obvious effect on the presence of Slo2 . 2 protein , and vice versa .",
"After removal of DRG from 3 to 5 week old mice , ganglia were desheathed and then incubated in 15 U/ml papain/L-cysteine in HBSS without calcium and magnesium ( Life Technologies ) for 20 min at 37°C .",
"Ganglia were washed three times in HBSS , replaced with 1 . 5 mg/ml collagenase ( Sigma–Aldrich ) in HBSS and incubated for 20 min at 37°C .",
"After washing three times with Neurobasal-A medium supplemented with 10% FBS , B27 supplement , 100 U/ml penicillin/streptomycin , and Glutmax ( 2 mM L-alanyl-L-glutamine ) ( all from Life Technologies ) , ganglia were gently triturated with a flame-polished Pasteur pipette until the solution turned cloudy .",
"The dispersed cells were diluted with growth medium containing supplemented Neurobasal medium .",
"The cells were plated at a density of ∼2000 cells per well on 12 mm glass coverslips coated with Matrigel ( BD Biosciences , San Jose , CA , United States ) , and maintained at 37°C in humidified air with 5% CO2 for 1 hr before onset of recording .",
"Most experiments were done within 8 hr after dissociation and changes of the culture medium were not necessary .",
"For 2–3 days in culture , half the medium was replaced with fresh growth medium on the second day .",
"100 μm thin slices were prepared from dorsal root ganglia of 7–14 day old of mice using a previously described method ( Safronov et al . , 1996 ) .",
"In brief , mice were killed by CO2 inhalation , rapidly decapitated , and six ganglia from lower thoracic and lumber regions were carefully removed in ice-cold Hank's Balanced Salt Solution ( HBSS , Invitrogen; Carlsbad , CA , United States ) .",
"Ganglia were desheathed using fine forceps , placed in the center of a 35 mm petri dish , then filled with with 40°C 4% low melting agar ( wt/vol in HBSS ) .",
"The dish was then immediately submerged in ice-cold artificial CSF cutting solution , which contained the following ( in mM ) : 125 NaCl , 3 . 5 KCl , 0 . 5 CaCl2 , 3 . 5 MgCl2 , 26 NaHCO3 , and 10 D-glucose .",
"The solution was bubbled with 95%O2/5%CO2 to maintain pH at ∼7 . 4 .",
"After solidification of the agar , small blocks containing ganglia were cut out and glued onto the cutting platform of a vibratome ( VT100 , Leica , Buffalo Grove , IL , United States ) for cutting .",
"Slices were stored for 45 min at 35°C and kept at room temperature until recording .",
"The oxygenated storage solution contained the following ( in mM ) : 125 NaCl , 3 . 5 KCl , 26 NaHCO3 , 10 D-glucose , 2 . 5 CaCl2 , and 1 . 3 MgCl2 .",
"Individual slices were subsequently transferred to a recording chamber continuously perfused ( 3 ml/min ) with oxygenated saline at room temperature .",
"A Slicescope Pro 3000 ( Scientifica Ltd , East Sussex , United Kingdom ) microscope equipped with Nomarski optics , a 40× water-immersion lens , and infrared illumination was used to view DRG neurons in the slices .",
"Small diameter DRG neurons responsive to itch and pain stimuli ( Stucky and Lewin , 1999; Lallemend and Ernfors , 2012 ) express a cell surface antigen that binds a plant lectin , isolectin B4 ( Silverman and Kruger , 1990 ) .",
"To categorize neurons as either IB4+ or IB4− , prior to recording , DRG neurons , whether dissociated or in slices , were exposed to media containing either 5 μg/ml isolectin Β4 ( FITC ) or 1 μg/ml isolectin B4 ( Texas Red ) .",
"After 5 min incubation , cells were returned to normal extracellular solution and viewed with standard fluorescence microscopy .",
"Standard whole-cell recording methods were used for both voltage-clamp and current clamp using a Multiclamp Amplifier ( Molecular Dynamics , Sunnyvale , CA , United States ) , for both dissociated cells and for cells in slices .",
"Voltage- and current stimulation protocols and acquisition of voltage and current records were accomplished by Clampex 9 . 2 ( Molecular Dynamics ) with analysis of waveforms done via Clampfit .",
"Patch-clamp pipettes typically were of 1 . 5–2 . 5 MΩ .",
"Following whole-cell access , cells were used if the series resistance ( Rs ) was less than 10 GΩ .",
"Rs was compensated 85% .",
"For excised patch experiments , pipettes of similar size were used to form GΩ seals on dissociated DRG neurons before excision .",
"The standard internal solution contained ( in mM ) : 10 NaCl , 135 KCl , 1 MgCl , 5 EGTA , 10 HEPES , 3 Mg-ATP , 0 . 3 Na-GTP , pH 7 . 3 adjusted with KOH , OSM ∼300 .",
"In the nominally zero internal Na+ pipette solution , internal KCl was 145 mM , but contained 0 . 3 mM Na from Na-GTP .",
"The standard external solution contained the following ( in mM ) : 136 . 4 NaCl , 5 . 6 KCl , 2 . 2 CaCl , 1 MgCl2 , 11 D-Glucose , 10 HEPES , pH 7 . 4 adjusted with NaOH .",
"For inside-out patches , the pipette ( external ) solution contained ( in mM ) : 5 NaCl , 152 . 5 KCl , 1 MgCl2 , 5 HEPES , pH 7 . 4 adjusted with KOH; the internal solution contained ( in mM ) 73 . 6 KCl , 1 MgCl2 , 3 EGTA , 10 HEPES and 70 NaCl ( for 70 Na+ ) or 70 Choline-Cl ( for 0 Na ) , adjusted to pH 7 . 3 with KOH ( EK = 18 . 35 mV ) .",
"Both Tetraethylammonium and tetrodotoxin were added to the external solution at final concentrations of 1 mM and 100 nM , respectively , just before the start of experiments .",
"When Cs+ was used as a non-specific blocker of KNa current ( Bischoff et al . , 1998 ) , Cs+ replaced an equal molar concentration of NaCl .",
"For recording of ‘leak’ current , after whole-cell formation , to assess ‘leak current’ , the net difference in current observed from voltage-steps from −80 mV to −120 mV ( Figure 6C , D and Figure 6—figure supplement 4 ) was monitored ( Bischoff et al . , 1998 ) , either with 0 Na+ in the internal pipette solution to define Na+-independent ‘leak’ current , or with 70 mM Na+ .",
"The 70 and 0 mM sodium pipette solutions contained the following ( in mM , with 0 Na+ solutions in parenthesis ) : 70 ( 0 ) NaCl , 73 . 3 ( 140 ) KCl , 1 MgCl , 5 EGTA , 10 HEPES , 3 Mg-ATP , 0 . 3 Na-GTP , pH 7 . 3 adjusted with KOH , OsM 290–300 .",
"For leak current measurements in slices , the external solution contained the following ( in mM ) : 115 NaCl , 5 . 6 KCl , 1 MgCl2 , 1 . 8 CaCl2 , 11 D-Glucose , 1 NaH2PO4 , 25 NaHCO3 , bubbled with 95%O2/5%CO2 to maintain pH at ∼7 . 4 .",
"For acutely dissociated DRG , the solution contained ( in mM ) : 136 . 4 NaCl , 5 . 6 KCl , 1 MgCl2 , 1 . 8 CaCl2 , 11 D-Glucose , 10 Hepes , pH 7 . 4 adjusted with NaOH solution .",
"The Kolgoromov–Smirnov test was used to generate the KS statistic , P . For cases in which the number of entries in one or both sample populations was less than 10 , a two-tailed , unpaired Student's t-test was employed .",
"Data are presented as mean ± sem ."
]
] | [
"Two mammalian genes , Kcnt1 and Kcnt2 , encode pore-forming subunits of Na+-dependent K+ ( KNa ) channels .",
"Progress in understanding KNa channels has been hampered by the absence of specific tools and methods for rigorous KNa identification in native cells .",
"Here , we report the genetic disruption of both Kcnt1 and Kcnt2 , confirm the loss of Slo2 . 2 and Slo2 . 1 protein , respectively , in KO animals , and define tissues enriched in Slo2 expression .",
"Noting the prevalence of Slo2 . 2 in dorsal root ganglion , we find that KO of Slo2 . 2 , but not Slo2 . 1 , results in enhanced itch and pain responses .",
"In dissociated small diameter DRG neurons , KO of Slo2 . 2 , but not Slo2 . 1 , abolishes KNa current .",
"Utilizing isolectin B4+ neurons , the absence of KNa current results in an increase in action potential ( AP ) firing and a decrease in AP threshold .",
"Activation of KNa acts as a brake to initiation of the first depolarization-elicited AP with no discernible effect on afterhyperpolarizations ."
] | [
"The billions of neurons in the brain send information along their lengths in the form of electrical signals called action potentials .",
"These signals are produced by charged ions , such as sodium and potassium ions , moving into and out of the neuron .",
"To ‘fire’ an action potential , sodium ions rapidly enter the neuron .",
"This produces an electrical spike .",
"Potassium ions then exit the neuron , which causes the electrical activity to subside and allows the neuron to return to a resting state .",
"The sodium and potassium ions move in and out of the neuron through structures called ion channels .",
"The sodium-activated potassium channels are one type of ion channel; whether these ion channels let potassium ions out of a cell depends on the concentration of sodium ions inside the cell .",
"Slo2 . 1 and Slo2 . 2 are two such potassium channels that are present in many different cells , including neurons .",
"Nevertheless , and in spite of how common they are , the exact roles of these channels remain unclear .",
"Martinez-Espinosa et al . created mice that lack the genes encoding one or both of the Slo2 . 1 and Slo2 . 2 ion channels , and compared them with normal mice .",
"Mice that lacked Slo2 . 2 but not Slo2 . 1 initially scratched more intensely than normal mice when made to feel an itch , though this increased scratching only occurred briefly .",
"To some extent , the mice that lacked both Slo2 channels also had increased pain sensations .",
"Martinez-Espinosa et al . observed that in sensory neurons lacking the Slo2 . 2 sodium-dependent potassium channels , the neurons fired more action potentials .",
"The increase in firing is thought to underlie the enhanced itching and pain sensations .",
"Taken together , the results suggest that the activity of sodium-activated potassium ion channels makes it less likely for a neuron to fire an action potential .",
"Future work will need to address whether the activity of sodium-activated potassium channels is linked to specific kinds of sodium channels , and why the absence of the sodium-activated potassium current only enhances the immediate response to itch stimuli .",
"The availability of these mice that lack Slo2 subunits provides an important new tool for evaluating the role of sodium-activated channels in other neuronal systems ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"structural biology and molecular biophysics"
] | Mechanism of B-box 2 domain-mediated higher-order assembly of the retroviral restriction factor TRIM5α | elife-16309-v2 | [
[
"TRIM5α is a restriction factor that intercepts the incoming capsids of diverse retroviruses , including HIV-1 , and inhibits viral replication .",
"The mechanism of restriction is not yet fully understood , but is primarily associated with premature termination of reverse transcription and accelerated dissociation of the viral core ( Sayah et al . , 2004; Stremlau et al . , 2004; 2006 ) .",
"TRIM5α is also proposed to be a pattern recognition receptor for retroviral capsids , in that it initiates a signaling cascade to induce type I interferon upon capsid binding ( Pertel et al . , 2011 ) .",
"Ubiquitin ( Ub ) is implicated in both the antiviral ( restriction ) and signaling activities of TRIM5α .",
"In particular , TRIM5α’s E3 ligase activity creates K63-linked polyUb chains .",
"Although the functional target or targets of ubiquitination have not been established definitively , TRIM5α self-ligation correlates with the block in reverse transcription ( Campbell et al . , 2015; Fletcher et al . , 2015; Roa et al . , 2012 ) , whereas unanchored chains have been proposed to mediate interferon signaling ( Pertel et al . , 2011 ) .",
"Like all TRIM proteins , TRIM5α consists of an N-terminal tripartite or RBCC motif ( RING , B-box 2 , and coiled-coil domains ) , followed by a C-terminal domain ( Figure 1A ) ( Meroni and Diez-Roux , 2005 ) .",
"The B-box 2 and coiled-coil domains make an integrated antiparallel dimer fold ( Goldstone et al . , 2014; Sanchez et al . , 2014; Weinert et al . , 2015 ) , which acts as a scaffold that organizes the upstream and downstream domains ( Figure 1B ) .",
"In TRIM5α , the C-terminal domain is a β-sandwich fold called SPRY ( or PRYSPRY/B30 . 2 ) , which mediates direct binding to retroviral capsids ( Biris et al . , 2012; 2013; Diaz-Griffero et al . , 2006b; Kovalskyy and Ivanov , 2014; Sawyer et al . , 2005; Sayah et al . , 2004; Sebastian and Luban , 2005; Stremlau et al . , 2006; Yang et al . , 2012 ) .",
"The L2 linker that connects the SPRY domain to the coiled-coil packs against the coiled-coil scaffold , and so in the TRIM5α dimer , two SPRY domains are oriented to bind the capsid simultaneously ( Figure 1B ) ( Goldstone et al . , 2014; Li et al . , 2014; Sanchez et al . , 2014; Weinert et al . , 2015 ) .",
"Capsid recognition by TRIM5α is an avidity-driven interaction; that is , productive binding only occurs in context of assembled capsid and assembled TRIM5α ( Sebastian and Luban , 2005; Stremlau et al . , 2006 ) .",
"Higher-order assembly of TRIM5α requires the B-box 2 domain , which is thought to mediate three-fold symmetric interactions that connect coiled-coil mediated TRIM5α dimers into a hexagonal net ( Diaz-Griffero et al . , 2009; Ganser-Pornillos et al . , 2011; Javanbakht et al . , 2005; Li and Sodroski , 2008; Li et al . , 2016 ) .",
"This hexagonal scaffold is proposed to position the SPRY domains to match the orientations – both translational and rotational – of their corresponding binding epitopes on retroviral capsids , and thereby generate powerful avidity effects that amplify very weak ( millimolar level ( Biris et al . , 2013 ) ) intrinsic affinities between the SPRY and capsid subunits . 10 . 7554/eLife . 16309 . 003Figure 1 . Design and oligomeric behavior of miniTRIM proteins .",
"( A ) Schematic of the TRIM5α primary sequence .",
"( B ) Schematic of the antiparallel full-length dimer .",
"( C–D )",
"Schematic of the ( C ) RBcc miniTRIM and ( D ) Bcc miniTRIM .",
"( E ) SDS-PAGE profiles of purified miniTRIMs .",
"( F–G )",
"Size exclusion elution profiles of ( F ) RBcc and ( G ) Bcc miniTRIMs .",
"Wildtype ( WT ) constructs eluted early ( blue traces ) , whereas R121E mutants eluted late ( red traces ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 00310 . 7554/eLife . 16309 . 004Figure 1—figure supplement 1 . Primary sequence of the miniTRIMs . Residues are color-coded as in Figure 1B–D .",
"Zinc-coordinating residues in the RING and B-box 2 domains are colored in red and blue for reference .",
"The inverted triangle indicates the start position of Bcc miniTRIM . DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 004 Higher-order TRIM5α assembly is also reported to promote the E3 ligase activity of the upstream RING domain ( Pertel et al . , 2011; Yudina et al . , 2015 ) , which is connected to the B-box 2 domain by the L1 linker ( Figure 1A and B ) .",
"A segment of the L1 linker forms a 4-helix bundle that mediates dimerization of the RING domain , which is required for productive interactions with Ub-conjugated E2 enzymes and formation of the catalytically active Ub ligation complex ( Yudina et al . , 2015 ) .",
"Although the B-box 2 domain does not appear to have a direct role in catalysis , B-box/B-box interactions are expected to cluster their associated RING domains and promote RING dimerization .",
"In context of the TRIM hexagonal lattice , the apparent juxtaposition of three-way head-to-head interactions between the B-boxes and two-way interactions between the catalytic RING domains is suggested to facilitate TRIM5α self-ubiquitination ( Yudina et al . , 2015 ) .",
"The structural basis by which the B-box 2 domain promotes higher-order assembly of TRIM5α dimers has been challenging to decipher , largely because the bivalent nature of the TRIM5α dimer and the flexible architectures of both the TRIM dimer and hexagonal lattice have impeded crystallographic characterization .",
"Analysis of the isolated B-box 2 domain has been likewise problematic , because separating the B-box from the coiled-coil exposes a 'backside' hydrophobic surface that makes it prone to aggregation .",
"We therefore engineered artificial constructs – which we call miniTRIMs – that retain the integrated B-box/coiled-coil fold of the full-length dimer but are more amenable to biochemical and structural analyses .",
"These novel reagents allowed us to define the molecular details of how the B-box 2 domain facilitates higher-order assembly of TRIM5α , and how pattern recognition of retroviral capsids is coupled to ubiquitin-dependent downstream processes ."
],
[
"Our miniTRIM constructs were designed to be monovalent with respect to the RING and B-box 2 domains ( to uncouple interactions mediated by these domains from the coiled-coil dimer ) , and yet preserve the native , quaternary B-box/coiled-coil interface ( to prevent exposure of the backside hydrophobic B-box surface and non-specific aggregation ) .",
"The 'RBcc' miniTRIM contained residues 1–159 from rhesus TRIM5α ( which includes the RING , B-box 2 , and the first 26 residues of the coiled-coil ) ( Figure 1C and Figure 1—figure supplement 1 , colored in orange ) , followed by an antiparallel coiled-coil hairpin derived from a bacterial seryl-tRNA synthetase ( residues 49–78 of PDB 1SRY , gray ) , and then residues 225–265 of the TRIM5α coiled-coil ( green ) .",
"To further uncouple the B-box from potential dimeric interactions of the upstream RING domain , we also designed a second construct denoted 'Bcc' that lacks the RING ( residues 1–88 ) ( Figure 1D ) .",
"Both the RBcc and Bcc miniTRIMs proved well behaved in solution , and could be purified to homogeneity ( Figure 1E ) .",
"Higher-order assembly of full-length TRIM5α protein dimers requires interactions mediated by a surface patch on the B-box 2 domain that includes Arg121 ( rhesus TRIM5α numbering ) .",
"Biochemical and cell-based assays show that R121A and R121E mutants are deficient in capsid binding and restriction activities , and this correlates with defects in higher-order assembly ( Diaz-Griffero et al . , 2009; Ganser-Pornillos et al . , 2011; Li and Sodroski , 2008 ) .",
"The same patch also mediates self-association of the isolated B-box 2 domain in solution ( Diaz-Griffero et al . , 2009 ) .",
"We therefore expected the miniTRIMs to exhibit Arg121-dependent oligomeric behavior , and we tested this by using size exclusion chromatography .",
"Consistent with expectation , both the RBcc miniTRIM ( Figure 1F ) and Bcc miniTRIM ( Figure 1G ) eluted early from a Superdex 75 size exclusion column as asymmetric peaks with sharp leading edges and pronounced tails ( blue traces ) .",
"In contrast , miniTRIMs harboring the R121E mutation eluted late as more symmetrical peaks ( Figure 1F and G , red traces ) , with elution volumes consistent with monomeric species .",
"These results indicate that the protein-protein interactions required for higher-order assembly of full-length TRIM5α are also essential for miniTRIM oligomerization .",
"Despite the polydisperse nature of the miniTRIMs , we obtained numerous crystal hits .",
"We obtained high quality synchrotron diffraction data from three crystal forms of Bcc miniTRIM .",
"A P212121 crystal contained two trimers in the asymmetric unit ( 3 . 26 Å resolution , R/Rfree = 0 . 26/0 . 30 ) , a C2 form contained one dimer ( 2 . 1 Å , R/Rfree = 0 . 18/0 . 22 ) , and a P1 form contained two dimers ( 2 . 3 Å , R/Rfree = 0 . 22/0 . 26 ) ( Table 1 and Supplementary file 1A ) .",
"Altogether , these yielded 12 crystallographically independent views of Bcc miniTRIM .",
"All 12 structures were very similar to each other ( Figure 2—figure supplement 1 ) , and a complete structure is shown in Figure 2A .",
"In general , electron densities for the B-box 2 domains and proximal regions of the coiled-coil domains were well defined ( Figure 2B ) , whereas densities for the hairpin linker were of poorer quality or , in some cases , missing ( Figure 2C ) .",
"( Densities in Figure 2B and C are illustrated with a trimer subunit . )",
"Thus , our structures are of high quality at the functionally relevant regions , even though the artificial linker displayed significant disorder in some cases and may not be optimally designed .",
"Our 12 Bcc miniTRIM structures superimpose very well with the crystal structure of the TRIM5α B-box 2/coiled-coil dimer ( PDB 4TN3 ) ( Goldstone et al . , 2014 ) ( Figure 2D–F and Supplementary file 1B ) .",
"This indicates that our miniTRIM constructs are excellent structural surrogates for the B-box/coiled-coil core of the full RBCC motif . 10 . 7554/eLife . 16309 . 005Table 1 . Crystallographic statistics . DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 005DimerDimerTrimerDiffraction DataBeamlineAPS 22IDAPS 22IDAPS 22IDWavelength ( Å ) 1 . 01 . 01 . 0Processing programHKL2000HKL2000HKL2000Space groupC2P1P212121Cell dimensionsa = 72 . 7 Åa = 45 . 8 Åa = 71 . 2 Åb = 41 . 5 Åb = 52 . 3 Åb = 71 . 5 Åc = 111 . 3 Åc = 69 . 7 Åc = 213 . 8 Åα = 90° , β = 110° , γ = 90°α = 94 . 8° , β = 105 . 5° , γ = 103°α = 90° , β = 90° , γ = 90°Resolution range , Å50-1 . 90 ( 1 . 97-1 . 90 ) 50-2 . 30 ( 2 . 38-2 . 30 ) 50-3 . 25 ( 3 . 37-3 . 25 ) Rsym/Rmeas /Rpim0 . 18 ( 0 . 43 ) /0 . 12 ( 0 . 90 ) /0 . 06 ( 0 . 50 ) 0 . 07 ( 0 . 16 ) /0 . 10 ( 0 . 23 ) /0 . 07 ( 0 . 16 ) 0 . 08 ( 1 . 0 ) /0 . 05 ( 1 . 0 ) /0 . 08 ( 1 . 0 ) Mean I/σ<I>14 . 0 ( 1 . 2 ) 9 . 4 ( 4 . 0 ) 26 . 8 ( 1 . 6 ) Completeness , %98 . 6 ( 90 . 4 ) 93 . 9 ( 80 . 0 ) 100 ( 100 ) Average redundancy3 . 5 ( 2 . 7 ) 1 . 9 ( 1 . 7 ) 13 . 6 ( 9 . 4 ) Wilson B-factor , Å240 . 536 . 035 . 1Refinement StatisticsRefinement programPHENIXPHENIXPHENIXResolution range32 . 5-1 . 91 ( 1 . 98-1 . 91 ) 35 . 05-2 . 29 ( 2 . 38-2 . 29 ) 36 . 70-3 . 26 ( 3 . 37-3 . 26 ) No .",
"of unique reflections25 , 300 ( 2 , 301 ) 25 , 156 ( 2 , 171 ) 14 , 789 ( 181 ) Reflections in free set1254 ( 117 ) 1301 ( 113 ) 1431 ( 30 ) Rwork0 . 18 ( 0 . 30 ) 0 . 22 ( 0 . 26 ) 0 . 26 ( 0 . 30 ) Rfree0 . 22 ( 0 . 31 ) 0 . 26 ( 0 . 31 ) 0 . 30 ( 0 . 39 ) NCS copies246No .",
"of nonhydrogen atomsprotein and zinc2 , 2404 , 3495 , 052solvent112520Average B-factor ( Å2 ) protein and zinc6358 . 972 . 77solvent5353 . 1Coordinate deviationsbond lengths , Å0 . 0190 . 0040 . 005bond angles , °1 . 6440 . 7240 . 375Validation and DepositionRamachandran plotfavored , %999997 . 4outliers , %000MolProbity clashscore3 . 732 . 002 . 31PDB ID5EIU5F7T5IEAValues in parenthesis are for the highest resolution shell . 10 . 7554/eLife . 16309 . 006Figure 2 . Structure of the Bcc miniTRIM .",
"( A ) Complete structure of the Bcc miniTRIM , from a dimer subunit .",
"Residues derived from TRIM5α are colored in magenta , and the artificial hairpin linker is in gray .",
"Charcoal gray spheres indicate zinc atoms .",
"Residue numbers are indicated .",
"( B–C )",
"Electron density maps at two contour levels for ( B ) the B-box 2 domain and ( C ) the coiled-coil region of a trimeric Bcc subunit .",
"The model is colored according to B-factor , which indicates that the B-box and proximal coiled-coil regions are well defined .",
"B-box 2 sidechains within oligomerization interfaces are labeled to illustrate that these residues are well defined by the density .",
"( D–F )",
"Superposition of Bcc miniTRIM ( magenta ) with the corresponding B-box 2 and coiled-coil regions in the crystal structure of the rhesus TRIM5α B-box/coiled-coil fragment ( PDB 4TN3 ) ( Goldstone et al . , 2014 ) : ( D ) B-box and coiled-coil regions , ( E ) B-box alone , ( F ) coiled-coil alone .",
"Residue ranges used in the superposition are indicated , as are the average mean square deviations ± s . d . from pair-wise superpositions of each of the 12 monomer structures .",
"Deviations from each individual superposition are in Supplementary file 1B . DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 00610 . 7554/eLife . 16309 . 007Figure 2—figure supplement 1 . Ribbon representations of the 12 crystallographically independent Bcc miniTRIM structures solved in this study . Residues are colored according to B-factor . DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 007 The Bcc miniTRIM trimer is organized as a triskelion: three B-box 2 domains make a central three-fold symmetric vertex from which the coiled-coil domains emanate as spokes ( Figure 3A ) .",
"As expected , the trimerization interactions are principally mediated by the B-box domain , burying 578 Å2 of the available surface area from each subunit .",
"This result indicates that the three-fold vertexes of the TRIM5α hexagonal lattice are made by B-box trimers .",
"The B-box packs against the N-terminal end of the coiled-coil helix through a hydrophobic interface ( Figure 3A , asterisks ) , suggesting that trimer formation requires the presence of the coiled-coil .",
"This observation is consistent with the idea that higher-order assembly into a hexagonal lattice is a function of the integrated tripartite motif of TRIM5α , and does not simply arise from combining otherwise independent self-association motifs . 10 . 7554/eLife . 16309 . 008Figure 3 . Oligomeric structures of Bcc miniTRIM .",
"( A ) Trimer crystal structure and ( G ) dimer crystal structure , viewed from the 'top' ( closest to the N-termini ) .",
"( B , H )",
"Side views .",
"Three layers of interactions are boxed and expanded in the central panels .",
"Asterisks in A indicate a site of close packing between the B-box and the N-terminal end of the coiled-coil helix .",
"( C , I )",
"Interactions between layers .",
"( D–F , J–L )",
"Expanded views of three layers of interactions , in the same orientations as A and G . Relevant sidechains are shown as sticks and labeled .",
"Hydrogen bonds and salt bridges are indicated by black dashed lines .",
"Zinc atoms are shown as gray spheres . DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 00810 . 7554/eLife . 16309 . 009Figure 3—figure supplement 1 . Comparison of the B-box trimer ( A ) and dimer ( B ) .",
"Hydrophobic residues discussed in the text are shown as spheres .",
"Dashed circles highlight Leu118 and Leu132 , which are more close-packed in the trimer ( asterisks in Figure 3A ) compared to the dimer . DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 00910 . 7554/eLife . 16309 . 010Figure 3—figure supplement 2 . Superposition of representative subunits from the trimer ( cyan ) and dimer ( green ) indicate local bending of the coiled-coil helix spanning residues 133–139 . DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 01010 . 7554/eLife . 16309 . 011Figure 3—figure supplement 3 . Sedimentation equilibrium analytical ultracentrifugation profiles of Bcc miniTRIM . Upper panels show absorbance measurements at 280 nm ( symbols ) and best-fit curves ( solid lines ) .",
"Lower panels show residual differences .",
"Measurements were made at the indicated centrifugation speeds and loading protein concentrations .",
"All nine distributions were globally fit to an ideal monomer-dimer equilibrium model .",
"Fixing the molecular weight to the theoretical value for a monomer ( 16 . 4 kDa ) returned a dissociation constant of 0 . 94 μM . DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 01110 . 7554/eLife . 16309 . 012Figure 3—figure supplement 4 . SEC-MALS analysis of Bcc miniTRIM . Protein concentrations were monitored using absorbance ( not shown ) and refractive index ( black trace ) .",
"The masses of eluting species ( blue trace ) indicate that the injected sample ( 0 . 9 mM ) is polydisperse and consists primarily of dimers . DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 01210 . 7554/eLife . 16309 . 013Figure 3—figure supplement 5 . Slight clam shell-like opening of the B-box dimer interface . Ribbon representations of two dimer structures , superimposed on the right subunits ( C2 form , orange; P1 form , pink ) .",
"Equivalent atoms on the left subunits move by up to 4 Å ( indicated for one of the zinc atoms , gray spheres ) .",
"Sidechains for Trp117 and Arg121 are shown for reference .",
"Arrow indicates direction of movement for the subunit on the left . DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 013 Interestingly , the majority of Bcc miniTRIM crystals we obtained ( including three other forms that we did not refine ) were composed of dimers ( Figure 3G and Supplementary file 1A ) .",
"The dimer is quasi-equivalent to the trimer: the same sets of residues make the same interactions but with valence of two instead of three .",
"The dimer buries a smaller surface area per subunit ( 450 Å2 ) compared to the trimer .",
"Both the trimer and dimer are stabilized by three layers of interactions , with a hydrophobic layer sandwiched between two hydrophilic layers ( Figure 3B , H ) .",
"The top layers ( closest to the N-termini of the B-boxes ) make a ring of salt bridges mediated by Glu102 and Lys103 from each subunit ( Figure 3D , J ) .",
"The middle , hydrophobic layers center on Trp117 ( Figure 3E , K ) .",
"In both oligomers , these indole sidechains make a hydrophobic core surrounded by a collar of close packed leucine sidechains ( Leu105 , Leu106 , Leu118 , and Leu132 ) .",
"The third , bottom layers also consist of a ring of salt bridges , this time with Glu120 and Arg121 ( Figure 3F , L ) .",
"At the periphery of this layer , the sidechain hydroxyl of Thr30 donates a hydrogen bond to Glu120 .",
"Interactions between the layers consist of a hydrogen bond between Glu102 ( top ) and the indole amide of Trp117 ( middle ) , and close packing between the Trp117 sidechain ( middle ) and the Arg121 sidechain guanidinium group ( bottom ) ( Figure 3C , I ) .",
"The Trp117 indoles in the trimer form a channel that would be expected to have a highly negatively charged hole at the center ( Figure 3E ) , which is likely to be partially stabilized by the positively charged Arg121 sidechains .",
"Comparison of the trimer and dimer bonding interactions revealed that the two oligomers are distinguished primarily by intermolecular packing between the Trp117 , Leu118 , and Leu132 sidechains in the hydrophobic layer .",
"In particular , Leu118 and Leu132 , which are located at the outer edges of the binding surface , are in van der Waals contact in the trimer , but more separated in the dimer ( Figure 3—figure supplement 1 ) .",
"Another distinction is that the first two turns of the coiled-coil helix are overwound and bent in the dimer form ( Figure 3—figure supplement 2 ) , which might indicate some structural communication between the interacting B-boxes and the coiled-coil domain .",
"The apparent propensity of the B-box 2 domain to dimerize in our crystals prompted us to more closely analyze the oligomerization behavior of the miniTRIMs in solution .",
"In order to avoid possible contributions from proximity-induced RING/RING interactions , we focused on the Bcc miniTRIM construct .",
"We first analyzed Bcc miniTRIM by using sedimentation equilibrium analytical ultracentrifugation ( AUC ) at three different loading concentrations and three rotor speeds , and the combined data set was fit globally .",
"In control experiments , both the W117E and R121E Bcc miniTRIM mutants were monomeric , in agreement with the structures ( not shown ) , whereas the wildtype ( WT ) Bcc distributions indicated self-association ( Figure 3—figure supplement 3 ) .",
"Global fitting of the WT equilibrium distributions to a monomer-dimer-trimer model only returned stable values for the monomer-dimer dissociation constant ( 1 μM ) .",
"Indeed , the AUC data gave a satisfactory fit to a monomer-dimer equilibrium model , indicating that trimers ( or higher-order oligomers than dimers ) were disfavored in solution .",
"To determine whether higher-order oligomers would form at higher protein concentrations , we also analyzed the Bcc miniTRIM at 0 . 9 mM by using analytical size exclusion chromatography with multi-angle light scattering ( SEC-MALS ) .",
"The mass trace ( blue curve in Figure 3—figure supplement 4 ) had a sharp peak at the leading edge of the protein absorbance peak ( black curve ) , a curved plateau in the central region above the expected mass for a dimer , and tapered towards the expected monomer mass at the tail .",
"This again indicated dynamic equilibrium between monomer and dimer states , and that higher-order species were also present in the main peak , but at a much smaller fraction than dimers .",
"Thus , the Bcc miniTRIM was predominantly dimeric in solution .",
"The solution behavior of Bcc miniTRIM is in sharp contrast to that of full-length protein .",
"Published AUC analysis of freshly purified full-length TRIM5 did not show evidence of B-box mediated self-association , even at a loading concentration that is six-fold higher than our measured miniTRIM dimerization affinity ( Langelier et al . , 2008 ) .",
"Uncoupling of the B-box from the full coiled-coil and RING domains therefore appears to have amplified its propensity for oligomerization .",
"Another difference is that , upon incubation , full-length TRIM5α has a clear propensity to assemble into hexagonal arrays – and by inference , three-way B-box/B-box interactions – even at low μM concentrations ( Ganser-Pornillos et al . , 2011; Li et al . , 2016; and this study ) .",
"Since the miniTRIMs lack an intact coiled-coil , extended incubation did not result in lattice formation but only produced higher molecular weight aggregates ( not shown ) .",
"Our interpretation of all of these observations is that artificial uncoupling of the B-box 2 domain from the full coiled-coil and RING domains has stabilized the B-box dimer form .",
"In context of the full-length protein , assembly cooperativity drives the B-box into the trimer form , and so the B-box dimer might represent an intermediate state that can incorporate an additional B-box domain to form the trimer .",
"Consistent with this interpretation , our structures indicate that the Bcc miniTRIM dimers can open slightly like a clam shell , which we imagine can lead to a wider opening and provide access to a third subunit ( Figure 3—figure supplement 5 ) .",
"We note that this interpretation does not preclude a functional role for a B-box dimer .",
"For example , it is possible that B-box dimerization might be a mechanism to promote RING dimerization and E3 ligase activation .",
"Nevertheless , it seems clear that the B-box trimer is what facilitates hexagonal lattice assembly , at least in vitro .",
"To test the functional relevance of the B-box mediated interactions in our structures , we generated structure-based mutations in full-length rhesus TRIM5α and tested the mutant proteins for their ability to inhibit transduction of GFP-labeled HIV-1 in HeLa cells ( Figure 4 , Table 2 ) .",
"Charge reversal mutations in the top layer of interactions ( E102K , K103E , E102K/K103E ) diminished restriction activity ( Figure 4A ) , confirming that this ring of salt bridges has an appreciable contribution to higher-order TRIM5α assembly .",
"Individual charge reversals in the bottom layer ( E120R , R121E ) severely abrogated restriction activity ( Figure 4B ) , in agreement with previous work ( Diaz-Griffero et al . , 2009; Li and Sodroski , 2008 ) .",
"However , the E120R/R121E double mutation did not have a compensatory effect .",
"This is in contrast with previous studies ( Diaz-Griffero et al . , 2009; Li and Sodroski , 2008 ) but is consistent with the structures since Arg121 is involved in both a salt bridge and a hydrophobic packing interaction with Trp117 .",
"The repositioned guanidinium group in E120R seems unlikely to generate the optimal geometry for these interactions . 10 . 7554/eLife . 16309 . 014Figure 4 . Restriction activities of rhesus TRIM5α containing structure-based B-box 2 domain mutations .",
"For clarity , the data are presented in multiple panels .",
"( A–F )",
"GFP-labeled HIV-1 efficiently transduced HeLa cells that did not express exogenous TRIM5α ( no TRIM , open spheres ) .",
"Transduction was significantly inhibited in cells that expressed WT TRIM5α ( WT , filled spheres ) .",
"The same results were obtained in two independent experiments .",
"( G ) Immunoblots ( IB ) of whole cell lysates with anti-FLAG antibody to determine expression levels of rhesus TRIM5α mutants ( upper ) .",
"Anti-tubulin blots indicate that equivalent samples were loaded into each lane ( lower ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 01410 . 7554/eLife . 16309 . 015Table 2 . Summary of structure-based mutagenesis . DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 015MutationStructure contextRestriction activityCapsid bindingSpontaneous assemblyCo-assemblyTemplated assemblyNone ( WT ) ++++hexagonal1++++E102Atop layer+n . d . n . d . n . d . n . d . K103Atop layer+n . d . n . d . n . d . n . d . E102A , K103Atop layer+n . d . n . d . n . d . n . d . L105Amiddle layer++++macramé1++–L106Amiddle layer–+–1 , 2–n . d . W117Amiddle layer++–1 , 2 , striated3+–W117Emiddle layer–+–1 , 2–n . d . L118Amiddle layer+++–1 , hexagonal2n . d . n . d . L118Dmiddle layer–––1 , 2–n . d . L132Amiddle layer++–1 , hexagonal2n . d . n . d . L132Dmiddle layer+/–+–1 , 2–n . d . E120Rbottom layer–n . d . n . d . n . d . n . d . R121Ebottom layer–+*n . d . n . d . n . d . E120R , R121Ebottom layer–n . d . n . d . n . d . n . d . T130Abottom layer++n . d . n . d . n . d . n . d . 1At 1 mg/mL in standard low salt buffer2At >5 mg/mL in standard low salt buffer3At >5 mg/mL in high salt buffer*from previous study ( Ganser-Pornillos et al . , 2011 ) n . d . – not determined In the middle , hydrophobic layer , the W117E mutation was previously shown to abolish restriction activity ( Diaz-Griffero et al . , 2009 ) , which we have confirmed here ( Figure 4C ) .",
"Interestingly , however , the W117A mutant still harbored some restriction activity , most likely because the hydrophobic layer remains stabilized by an outer ring of leucines .",
"As with Trp117 , alanine substitution mutants for Leu118 ( Figure 4D ) and Leu132 ( Figure 4E ) had measurable restriction activity , whereas aspartate substitutions were more significantly disruptive .",
"Of the remaining two leucines , L105A had full activity and L106A did not restrict HIV-1 ( Figure 4F ) .",
"These results are again consistent with the structures since Leu105 is only partially buried , whereas Leu106 is completely buried within both the dimer and trimer interfaces .",
"Interestingly , we found that steady state expression levels of the TRIM5α B-box 2 domain mutants varied considerably , as noted previously ( Diaz-Griffero et al . , 2007 ) , and that furthermore there was an inverse correlation between the steady state expression levels of the mutant proteins and restriction activity ( Figure 4G ) .",
"The best expressing mutants ( e . g . , W117E and R121E ) did not restrict HIV , whereas the lowest expressing mutant ( L105A ) had WT-like restriction activity .",
"The expression levels also inversely correlated with in vitro assembly efficiency – W117E and R121E did not assemble , whereas L105A assembled efficiently ( Ganser-Pornillos et al . , 2011; also see below ) .",
"We therefore speculate that the ability to assemble reduces steady state protein levels because TRIM5α proteins that assemble are turned over more rapidly in cells .",
"An extension of this argument is that the ability of TRIM5α to assemble correlates with its ability to restrict HIV-1 .",
"We also tested the effect of the hydrophobic B-box 2 domain mutations on capsid binding activity in vitro .",
"For these experiments , we used TRIM5-21R , a chimeric construct described in previous studies as a functional and structural surrogate for TRIM5α due to its more favorable biochemical properties ( Diaz-Griffero et al . , 2006a; Ganser-Pornillos et al . , 2011; Kar et al . , 2008; Langelier et al . , 2008 ) .",
"We incubated crosslinked HIV-1 CA tubes ( biochemical surrogates for the HIV-1 capsid ) with WT and mutant TRIM5-21R proteins , and then measured binding in a co-pelleting assay ( Fribourgh et al . , 2014; Ganser-Pornillos et al . , 2011; Stremlau et al . , 2006 ) .",
"In control experiments , about 50% of WT TRIM5-21R was consistently found in the pellet ( Figure 5A ) .",
"Mutants were analyzed in parallel with a WT control , and results in Figure 5B are representative of mutant binding efficiencies relative to WT from at least two experiments with independent protein preparations .",
"Essentially all of the B-box mutants tested had detectable capsid binding activity , which was expected because all of these constructs contained an intact SPRY domain and because the R121E mutation that abolished restriction still supported capsid binding in a similar assay ( Ganser-Pornillos et al . , 2011 ) .",
"Nevertheless , our results showed that weaker or no capsid binding correlated with weaker or no restriction activity ( Table 2 ) , confirming that B-box 2 domain mediated interactions promote efficient capsid recognition . 10 . 7554/eLife . 16309 . 016Figure 5 . CA tube pull-down assay .",
"( A ) WT control .",
"( B ) Indicated mutants .",
"Purified TRIM5-21R proteins were incubated with disulfide-stabilized CA tubes and pelleted in a microcentrifuge .",
"Bound ( pellet ) and unbound ( supernatant ) proteins were visualized by SDS-PAGE with Coomassie staining and quantified .",
"Percentage values indicate the fraction of protein in the pellet .",
"Results are representative of at least two experiments per mutant , each done with an independent protein preparation .",
"L , load; S , supernatant; P , pellet .",
"CA and TRIM bands are indicated .",
"The asterisk indicates an apparent proteolytic fragment of TRIM5-21R . DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 016 We then directly determined the effects of the B-box mutations on TRIM5α assembly activity in vitro ( Figures 6–8 , and Table 2 ) .",
"For this analysis , we focused on the hydrophobic ( layer",
"2 ) mutations .",
"Previously , purified TRIM5-21R was shown to assemble spontaneously into hexagonal arrays when incubated in low salt buffer at about 1 mg/mL ( Ganser-Pornillos et al . , 2011 ) .",
"These micron-sized arrays can be readily visualized by negative stain electron microscopy ( Figure 6A ) .",
"In contrast , the R121E mutant did not assemble spontaneously even at concentrations up to 30 mg/mL ( Ganser-Pornillos et al . , 2011 ) .",
"We therefore tested our TRIM5-21R mutants for spontaneous assembly at both low ( 1 mg/mL ) and high ( >5 mg/mL ) protein concentrations .",
"We found that mutations that gave the most significant reductions in restriction activity ( L106A , W117E , L118D , and L132D ) also prevented assembly at all protein concentrations tested ( up to 18 mg/mL ) ( Table 2 and data not shown ) .",
"Two of the mutations that supported intermediate restriction activity ( L118A and L132A ) also supported hexagonal lattice assembly , but only at high protein concentrations and to a more limited extent compared to WT ( Figure 6B , C ) .",
"A third intermediate mutation , W117A , altered the assembly phenotype of TRIM5-21R .",
"This mutant failed to assemble in standard low salt buffer at both 1 and 5 mg/mL , but at high protein concentrations and higher salt ( >250 mM NaCl ) , it assembled into striated arrays ( Figure 6D ) .",
"Interestingly , the L105A mutation ( full restriction activity ) also altered the spontaneous assembly behavior of TRIM5-21R .",
"Under the same conditions as WT ( low salt buffer and 1 mg/mL protein ) , L105A formed macramé-like networks that appeared distinct from either the hexagonal or striated arrays ( Figure 6E ) . 10 . 7554/eLife . 16309 . 017Figure 6 . Spontaneous assembly of TRIM5-21R .",
"( A ) WT TRIM5-21R spontaneously assembled into hexagonal arrays at a concentration of 1 mg/mL in 25 mM Tris , pH 8 , 25 mM NaCl , 1 mM TCEP ( standard conditions ) ( Ganser-Pornillos et al . , 2011 ) .",
"Main panel shows a representative negatively stained image of the arrays; inset shows a Fourier transform of the image .",
"The unit cell spacing ( symmetry unimposed ) calculated from the diffraction pattern is indicated .",
"( B ) L118A and ( C ) L132A , which gave intermediate restriction phenotypes in context of rhesus TRIM5α , also assembled into hexagonal nets , but at higher protein concentrations ( 2 and 9 mg/mL , respectively ) .",
"( D ) W117A aggregated under standard conditions but at >5 mg/mL and 250 mM NaCl assembled into a striated array .",
"( E ) L105A , which was fully restriction competent , assembled spontaneously under standard conditions into networks that were neither hexagonal not striated .",
"Results are representative of two or three experiments per construct , each done with an independent protein preparation .",
"Scale bars = 100 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 01710 . 7554/eLife . 16309 . 018Figure 7 . Co-assembly of TRIM5-21R with HIV-1 CA .",
"( A ) Incubation of WT TRIM5-21R with soluble HIV-1 CA protein induced assembly of TRIM-coated capsid tubes .",
"A similar phenotype was observed when co-assembly is performed with African green monkey TRIM5α ( Li et al . , 2016 ) .",
"( B ) TRIM5-21R with the L105A mutation made similar decorated tubes as WT in this assay .",
"( C ) W117A also made similar decorations , but to a more limited extent .",
"Partially decorated and undecorated tubes were more prevalent ( arrows ) .",
"Results are representative of two or three experiments per construct , each done with an independent protein preparation .",
"( D ) Undecorated CA tubes shown for comparison .",
"Scale bars = 100 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 01810 . 7554/eLife . 16309 . 019Figure 8 . Slices of tomographic reconstructions of ( A ) WT and ( B ) L105A TRIM5-21R coated CA tubes . Left panels show peripheral slices , and right panels show central slices of the same tube .",
"( C ) Some of the L105A-coated tubes had ladder-like TRIM overlays that do not seem hexagonal .",
"These could be due to overlapping lattices or an alternative arrangement of TRIM dimers .",
"Scale bars = 50 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 019 Two-dimensional crystals of HIV-1 CA-NC , which mimic the hexagonal HIV capsid lattice at its planar limit , can promote assembly of TRIM5-21R and native TRIM5α proteins into flat hexagonal arrays ( Ganser-Pornillos et al . , 2011; Li et al . , 2016 ) .",
"We therefore tested whether the CA-NC arrays can 'rescue' the altered spontaneous assembly phenotype of L105A and W117A .",
"However , results were inconclusive; although the TRIM proteins appeared to associate with the CA-NC arrays , diffraction patterns that would indicate overlaying lattices were not observed ( Table 2 and data not shown ) .",
"We therefore used an alternative assay wherein soluble TRIM5α and HIV-1 CA are mixed and incubated under basic conditions and moderate salt concentrations , which results in formation of CA tubes that are almost uniformly decorated with TRIM ( Li et al . , 2016 ) .",
"The overlaying TRIM lattice on these tubes is related to the flat hexagonal arrays , except that it now follows the basal curvature of the CA tubes .",
"Both WT TRIM5-21R ( Figure 7A ) and the L105A mutant ( Figure 7B ) showed similar behavior in this assay; that is , virtually all the tubes were uniformly decorated , and the decorations looked similar to each other and to those made by WT TRIM5α ( Li et al . , 2016 ) .",
"In contrast to the flat arrays , hexagon shapes were more difficult to discern in projection images of the curved arrays , and so we also analyzed negatively stained samples by electron tomography .",
"As observed with vitrified samples ( Li et al . , 2016 ) , densities surrounding the CA tubes were readily discernible in the tomograms of our negatively-stained samples ( Figure 8 ) .",
"As expected , the peripheral TRIM5α layers exhibited a high degree of disorder , but for both WT ( Figure 8A ) and L105A ( Figure 8B ) , we could clearly observe regions of local order with hexagonal rings having the expected dimensions for a TRIM hexamer .",
"Interestingly , a proportion of the L105A decorations appeared more ladder-like ( Figure 8C ) , but again the distances between the repeating units were similar to the TRIM hexagon dimensions .",
"We therefore conclude that , although there is no clear structural explanation for the spontaneous assembly behavior of the L105A mutant , it can nevertheless form hexagonal arrays when it binds CA tubes in vitro .",
"The inability of this mutant to form flat lattices ( the endpoints of both the spontaneous assembly and template driven methods ) suggests that B-box/B-box interactions might have some link to lattice curvature .",
"We also used the co-assembly assay to characterize the W117A mutant that assembled into striated arrays ( Figure 6D ) .",
"This mutant also decorated the CA tubes , but the decorations were more limited in extent compared to either WT or L105A , and regions of undecorated CA were more readily apparent in the projection images ( Figure 7C ) .",
"Similarly , the tomograms revealed considerable disorder in the overlaying TRIM lattice , and hexagon-shaped decorations were not easily discerned ( not shown ) .",
"In summary , our analysis of structure-based B-box 2 domain mutations support a general correlation between TRIM5α restriction activity in cells , capsid binding efficiency in vitro , and hexagonal lattice assembly in vitro .",
"Our results therefore fit the model wherein both capsid recognition and antiviral restriction are facilitated by B-box mediated assembly of TRIM5α into a hexagonal lattice that wraps around the viral capsid .",
"The low-resolution structure of the TRIM5α hexagonal lattice consists of large lobes of density at the two-fold and three-fold symmetric positions connected by thin linkers of density ( Ganser-Pornillos et al . , 2011; Li et al . , 2016 ) .",
"The thin linkers are made by the antiparallel coiled-coil dimer scaffold and the two-fold lobes are made by the SPRY domain ( Ganser-Pornillos et al . , 2011; Goldstone et al . , 2014; Li et al . , 2016; Sanchez et al . , 2014; Weinert et al . , 2015; Li et al . , 2016 ) .",
"Our structural and biochemical analyses indicate that the three-fold vertexes are made by B-box 2 domain trimers .",
"By combining our miniTRIM trimer crystal structure with that of the B-box/coiled-coil dimer ( PDB 4TN3 ) ( Goldstone et al . , 2014 ) , we built a molecular model of a flat TRIM5α hexagonal lattice ( Figure 9A ) .",
"The unit cell spacing of this model is 338 . 5 Å , which is an almost exact match to the observed value for rhesus TRIM5α ( 340 Å ) ( Li et al . , 2016 ) .",
"All of the SPRY domains are located on one side of the lattice plane ( Figure 9B , blue spheres ) , where they could bind the capsid simultaneously .",
"At the three-fold vertexes , the N-terminal ends of the B-boxes point toward the other side of the lattice plane .",
"This suggests that the RING domains are oriented away from the capsid ( Figure 9B , red spheres ) .",
"This molecular architecture indicates that the higher-order TRIM scaffold also compartmentalizes the biochemical activities of the RING and SPRY domains . 10 . 7554/eLife . 16309 . 020Figure 9 . Molecular model of a flat TRIM5α hexagonal lattice .",
"( A ) Top view , with the B-box 2 domains colored in orange and the coiled-coil domains in green .",
"( B ) Side view , with the expected positions of the SPRY domains ( blue ) and RING domains ( red ) indicated by spheres .",
"The flat capsid lattice is shown for reference . DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 020 When TRIM5α binds to a retroviral capsid , it must accommodate the variable surface curvature of the capsid .",
"Capsid lattice curvature is generated by rigid body hinge motions between subunits , and we have previously identified such hinges by comparing crystallographically independent structures of the hexameric and pentameric capsid building blocks ( Pornillos et al . , 2009; 2011 ) .",
"The availability of 12 independent miniTRIM structures allowed us to perform a similar analysis here .",
"As illustrated in Figure 10A , superposition of the miniTRIM structures revealed that the coiled-coil can swing relative to the B-box 2 domain .",
"Flexion occurs about the B-box/coiled-coil interface , which is lined almost exclusively by aliphatic sidechains ( Figure 10B ) .",
"Note that in context of full-length TRIM5 , this 'greasy' interface is a quaternary contact between the subunits of the native dimer .",
"Bending of the first two turns of the coiled-coil helix in the dimer subunits produced the greatest change in orientation of the B-box relative to the coiled-coil , but the rigid body motions were also evident in comparing dimer subunits or trimer subunits alone ( not shown ) .",
"In context of the trimer , the three coiled-coils do not contact each other and can therefore move independently ( Figure 10C , D ) .",
"We conclude that TRIM5α uses the same general mechanism as retroviral capsids – flexion across quaternary interfaces and local conformational variations – to generate variable lattice curvature . 10 . 7554/eLife . 16309 . 021Figure 10 . Flexible architecture of the miniTRIMs .",
"( A ) Orthogonal views of 12 crystallographically independent structures of Bcc miniTRIM .",
"Superpositions of the structures on the B-box 2 domains ( orange ) reveals rigid body movements of the coiled-coil domains ( green ) .",
"( B ) Close-up view of the B-box/coiled-coil interface boxed in A . Relevant sidechains are shown explicitly and labeled .",
"( C , D )",
"Superpositions of multiple full-length triskelion models on the B-boxes illustrate that the coiled-coil arms can swing flexibly relative to the B-box trimer vertex .",
"( E , F )",
"Speculative illustrations of how flexible triskelion arms can simultaneously allow the assembling TRIM lattice ( green – coiled-coil; orange – B-box ) to follow the curvature of the capsid ( yellow orange ) while scanning for optimal binding positions of the SPRY domains ( blue ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 021 In addition to promoting avid capsid binding , B-box mediated interactions are expected to promote clustering of the upstream RING domain .",
"Indeed , TRIM5α assembly on retroviral capsids is reported to enhance E3 ligase activity ( Pertel et al . , 2011 ) .",
"The RING domain dimerizes to bind E2-Ub conjugates and catalyze Ub transfer ( Yudina et al . , 2015 ) .",
"So far , we have been unable to solve a crystal structure of the RBcc miniTRIM , but structures of monomeric ( inactive ) and dimeric ( active ) forms of the TRIM5α RING domain are both known ( Lienlaf et al . , 2011; Yudina et al . , 2015 ) , and so we used molecular modeling to determine possible configurations of the RINGs relative to the B-box trimer .",
"An important consideration here is the structure of the L1 linker that connects the RING and B-box domains , which we define as the 23 amino acids ( residues 72–94 ) that link the globular zinc-coordinating folds .",
"Residues 72–82 are disordered in the monomeric RING structure , but are folded into a 4-helix bundle in the dimeric RING ( Lienlaf et al . , 2011; Yudina et al . , 2015 ) .",
"Thus , a RING monomer has a longer ( and presumably more flexible ) linker to the B-box than a RING dimer subunit .",
"We used the program RANCH ( Bernadó et al . , 2007 ) to calculate an ensemble of 10 , 000 models wherein the monomeric RING is flexibly tethered to the B-box trimer .",
"Linker residues were modeled as impenetrable spheres , and the entire linker was assumed to be an 'intrinsically disordered' segment ( 'native' setting in RANCH ) .",
"This treatment seemed appropriate since the linker adopts alternative secondary structures ( Lienlaf et al . , 2011; Yudina et al . , 2015 ) .",
"In the resulting ensemble , the vast majority of the RING domains are located above the plane of the trimer ( Figure 11A , right ) .",
"Thus , even flexibly tethered monomeric RINGs are predominantly located on one side of the lattice and away from the capsid .",
"In a subset of our models , the RING/B-box linker folds down , in a configuration that brings the RING domain against the trimer vertex ( Figure 11A , left ) .",
"In principle , close packing of the RING domain and/or the RING/B-box linker against the trimer interface might explain the observation that the RING domain contributes to the efficiency of TRIM5α higher-order assembly ( Li et al . , 2011 ) . 10 . 7554/eLife . 16309 . 022Figure 11 . Models of RING domain configurations in context of the B-box trimer .",
"( A ) RING domains were modeled as monomers attached to their respective B-boxes by a flexible 23- residue linker .",
"Left panel shows three representative configurations of the RING monomer relative to the trimer plane; above , within , and below .",
"Right panel shows 500 of the 10 , 000 models calculated .",
"( B ) RING domains were modeled as dimers with a shorter , 12- residue linker .",
"This resulted in only 13 configurations that were sterochemically plausible .",
"( C ) Computational model of a self-ubiquitination complex .",
"Domains and proteins are color-coded as follows: RING , red; L1 linker , red ( N-term ) and white ( C-term ) ; B-box 2 , orange; coiled-coil , green; E2 , dark blue; ubiquitin , cyan .",
"Positions of the thioester ( yellow ) and Lys51 amine ( blue ) are indicated by spheres , and are about 7 Å apart in this model . DOI: http://dx . doi . org/10 . 7554/eLife . 16309 . 022 In the RING dimer , residues 72–82 fold into a 4-helix bundle , and destabilizing mutations in this region abrogate E3 ligase activity ( Yudina et al . , 2015 ) .",
"A RING dimer subunit is therefore separated from its B-box by a shorter linker of 12 residues ( 83EVKLSPEEGQKV94 ) .",
"Analysis of this sequence using the PEP-FOLD server ( Shen et al . , 2014 ) predicts that residues 87–94 have some propensity to fold into a short helix , and we therefore speculate that the dimeric RING/B-box linker might actually be a hinge rather than a flexible tether .",
"Since E2-Ub binding imposes additional spatial constraints , stereochemical clashes can only be avoided if the RING dimer is positioned above the B-box trimer ( Figure 11B ) .",
"We therefore conclude that in context of the TRIM hexagonal lattice , the B-box trimer spatially restricts the RING domain , such that an active E3 ligase can only form on the side of the lattice that faces the cytoplasm .",
"We also performed the same analysis on the B-box dimer; as expected , it also restricted RING positions , but to a lesser extent than the trimer ( not shown ) .",
"The functional target of TRIM5α ubiquitination has not been determined definitively , but self-ubiquitination correlates with inhibition of retroviral reverse transcription ( Campbell et al . , 2015; Fletcher et al . , 2015 ) .",
"The principal Ub attachment sites are in the RING domain ( Lys45 and Lys51 ) ( Fletcher et al . , 2015 ) .",
"Although a B-box dimer is more naturally compatible with a RING dimer , a B-box trimer suggests an intuitively appealing mechanism for self-ubiquitination because it clusters three RING domains .",
"In this model , two of the RING domains would dimerize and orient an E2-conjugated Ub for nucleophilic attack by the third RING ( Yudina et al . , 2015 ) .",
"To determine if such a mechanism of self-ubiquitination is stereochemically plausible , we constructed a model for a ubiquitination complex on a TRIM5α trimer vertex , by adding a third RING and an E2-Ub conjugate to one of our RING dimer/B-box trimer models .",
"The two RING subunits that form the dimer were connected to their corresponding B-boxes by 12-residue linkers , whereas the third RING was connected by a longer and more flexible 23-residue linker .",
"As illustrated in Figure 11C , we were able to identify stereochemically acceptable conformations that simultaneously allow trimerization of the B-box , dimerization of two RINGs , binding of the RING dimer to a Ub-conjugated E2 , and positioning of the appropriate lysine in the third RING for nucleophilic attack of the E2-Ub thioester bond .",
"Thus , although our modeling approach is somewhat crude , the results indicate that B-box trimerization is compatible with RING activation and TRIM5α self-ubiquitination ."
],
[
"The 'pattern recognition' model of capsid binding postulates that higher-order assembly of TRIM5α into a hexagonal lattice positions multiple SPRY domains to match both the orientations and spacing of their binding epitopes on the capsid surface ( Ganser-Pornillos et al . , 2011; Li et al . , 2016 ) .",
"Our studies are consistent with this model , and further indicate that three-fold symmetric interactions at the vertexes of the hexagonal net are directly mediated by the B-box 2 domain .",
"Indeed , our analysis suggests a 'rank order' of avidity promoting interactions , beginning with the 'minimal' coiled-coil dimer unit that positions ( or clusters ) two SPRY domains to bind the capsid simultaneously ( Goldstone et al . , 2014; Javanbakht et al . , 2007; Yap et al . , 2007 ) .",
"In principle , higher-order assembly of any geometry can amplify the recognition and restriction activities of the minimal dimer unit , but complete avidity and full restriction seem to occur when the TRIM5 dimers are arranged to match the lattice symmetry of the capsid .",
"This principle appears exemplified by the L105A and W117A B-box mutants ( Table 2 ) .",
"Like WT , the L105A mutant was fully restriction competent , bound to CA tubes efficiently in vitro , and formed observable hexagonal decorations on the tubes .",
"In contrast , the W117A mutant was impaired in all three activities , even though it can clearly assemble spontaneously into higher-order but non-hexagonal arrays in vitro .",
"Previous studies have analyzed how idealized ( flat ) capsid and TRIM lattices align in projection ( Ganser-Pornillos et al . , 2011; Goldstone et al . , 2014; Weinert et al . , 2015 ) .",
"This is reasonable because facets of the capsid surface are approximately flat , and because flat TRIM5α lattices assemble on two-dimensional crystals of the HIV-1 capsid protein .",
"However , retroviral capsids present highly curved surfaces for TRIM5 binding , and the TRIM lattice must accommodate this curvature .",
"Our structures demonstrate that the B-box/coiled-coil interface acts as a ball-and-socket joint , which imparts considerable flexibility in the way the coiled-coil arms emanate from the three-fold symmetric vertexes .",
"Interestingly , our models define two major trajectories along which the coiled-coil can swing relative to the trimer plane ( approximately parallel and approximately perpendicular ) .",
"Motions parallel to the trimer plane change the angle between adjacent coiled-coils emanating from a vertex .",
"This is consistent with the observation by Li et al . ( 2016 ) that the vertex angles in TRIM hexagonal lattices can deviate significantly from the ideal value of 120° , even in flat lattices .",
"In our models , the coiled-coil can make an arc of about 12° along this trajectory .",
"Angles between coiled-coil arms would therefore range from 120° ± 24° , which accounts for the full range of observed values ( 100–144° ) ( Li et al . , 2016 ) .",
"Perpendicular motions , on the other hand , modulate the concavity of the triskelion and can generate lattice curvature .",
"Figure 10E and F illustrate how the structural flexibility described above can allow the assembling TRIM lattice to scan for the most favorable SPRY domain positions and optimize local binding interactions , while simultaneously following the curvature of the bound capsid .",
"Flexibility in the triskelion arms also suggests a straightforward mechanism for generating multilayered TRIM assemblies , because a coiled-coil can extend a B-box 2 domain above or below its current lattice plane to nucleate a new lattice .",
"TRIM5α lattices in vitro are frequently multilayered , whether spontaneously assembled or nucleated by capsid templates ( Ganser-Pornillos et al . , 2011; Li et al . , 2016; and this study ) .",
"We speculate that the so-called cytoplasmic bodies that form upon overexpression of TRIM5α in cells ( Stremlau et al . , 2004; Campbell et al . , 2007 ) might also assemble in this manner .",
"Our finding that miniTRIMs can form quasi-equivalent dimers and trimers uncovers yet another mode of flexibility in TRIM5α self-assembly .",
"B-box dimers are clearly disfavored under our 'ideal' in vitro conditions wherein full-length TRIM5α assembles into hexagonal arrays .",
"In principle , however , B-box dimers provide a means for extending the TRIM array in regions where the local assembly environment disfavors trimers .",
"Indeed , apparent dimer linkages can be occasionally discerned in TRIM-coated capsids in vitro ( Li et al . , 2016 ) .",
"Interestingly , B-box dimerization is associated with local bending of the first two turns of the coiled-coil helix , as well as altered packing of the B-box against the coiled-coil domain .",
"This suggests a potential mechanism of allosteric communication that can link B-box oligomerization and coiled-coil dynamics .",
"Indeed , our observation that the L105A mutant could not assemble into flat arrays while remaining competent in forming curved arrays is another indication of a structural and functional link between B-box/B-box interactions , the coiled-coil dimer scaffold , and lattice curvature .",
"B-box mediated interactions also promote dimerization of the upstream RING domain and E3 ligase activity ( Ganser-Pornillos et al . , 2011; Pertel et al . , 2011; Yudina et al . , 2015 ) .",
"The catalytically active RING configuration is more structurally compatible with a B-box dimer , and it is therefore possible that the B-box switches its oligomeric configuration to facilitate enzymatic function .",
"In principle , it is possible for a RING-containing subunit to act simultaneously as ligase and ubiquitination substrate within the same E2-Ub/E3 complex , as biochemically demonstrated for the RING domain of RNF4 ( Plechanovová et al . , 2011 ) .",
"Nevertheless , our modeling studies also support a mechanism by which the B-box trimer could contribute to TRIM5α self-ubiquitination .",
"Specifically , a B-box trimer could bring together three RINGs , with the first two acting as the E3 ligase and the third acting as the substrate .",
"More important , our analysis further suggests that folding of the RING/B-box linker into a 4-helix bundle restricts the RING dimer to the outer surface of the TRIM hexagonal lattice , i . e . , on the opposite side of the capsid binding surface .",
"We suggest that this spatial compartmentalization facilitates formation of exposed polyubiquitin chains that can recruit downstream cytosolic factors to promote restriction and/or interferon signaling ."
],
[
"Synthetic DNA ( Genewiz , Inc . , South Plainfield , NJ ) encoding the RBcc miniTRIM sequence in Figure 1—figure supplement 1 and upstream His-tag and yeast Smt3p ( SUMO ) leader sequence was subcloned into pET30a ( Novagen/EMD Millipore , Germany ) .",
"To create the Bcc miniTRIM plasmid , the RING domain open reading frame ( residues 1–88 ) was excised using a PCR-based linearization and religation protocol .",
"Point mutations were introduced using the Quikchange method ( Agilent , Santa Clara , CA ) .",
"All plasmid constructs were confirmed by sequencing with T7 and/or T7 terminator primers .",
"Transformed E . coli BL21 ( DE3 ) cells were grown in LB broth supplemented with appropriate antibiotics and 50 μM zinc acetate .",
"Cultures were shaken at 250 rpm and 37°C until the OD600 reached 0 . 8–1 . 0 .",
"The shaker was then cooled to 18°C during induction with 1 mM isopropyl β-D-1-thiogalactopyranoside ( IPTG ) .",
"Cells were harvested by centrifugation 4 hr after induction then stored at −80°C .",
"Frozen E . coli weighing 25–30 g were resuspended in 120 mL of 2× lysis buffer ( 100 mM Tris , pH 8 , 100 mM LiCl , 10% ( v/v ) glycerol , 1% ( v/v ) Triton X-100 , 20 mM β-mercaptoethanol ( βME ) , 2 mM phenylmethanesulfonylfluoride ( PMSF ) ) then lysed using a microfluidizer ( Microfluidics , Westwood , MA ) .",
"The lysate was diluted to 1× with 120 mL cold water .",
"Cell debris was pelleted by centrifugation at 45 , 000 g and discarded .",
"The supernatant was then incubated with nickel agarose beads ( Qiagen , Germany ) .",
"The beads were washed with 10 column volumes ( CV ) of Wash 1 buffer ( 50 mM Tris , pH 8 , 50 mM LiCl , 10 mM βME , 5% ( v/v ) glycerol ) , 2 CV of Wash 2 buffer ( Wash 1 + 1 M LiCl ) , and again with 5 CV of Wash",
"1 . Proteins were eluted by addition of 5 mL fractions of elution buffer ( Wash 1 + 250 mM imidazole ) .",
"The His-tag and SUMO leader sequences were cleaved off with SUMO-specific Ulp1 protease ( 3 μg/mL ) , during overnight dialysis in Wash 1 buffer .",
"The His-SUMO protein was removed by a 15 min incubation with nickel agarose .",
"The sample was then diluted 1 . 5× with water , and then applied to a HyperD anion exchange column ( Pall Lifesciences , Port Washington , NY ) .",
"Bound fractions were eluted with a linear gradient from 100% Wash 1 buffer to 70% Wash 1/ 30% Wash",
"2 . Fractions were combined and concentrated to 0 . 5 mL then purified to homogeneity by gel filtration on a Superdex 75 column ( GE Healthcare , Little Chalfont , UK ) in 10 mM Tris , pH 8 , 100 mM LiCl , 1 mM TCEP .",
"Major peak fractions were pooled and concentrated to 3–15 mg/mL , flash-frozen in liquid nitrogen , then stored at −80°C .",
"Typical yields were around 0 . 3 mg per L of culture for RBcc and around 1 mg per L for Bcc .",
"Protein stock solutions for crystallization trials generally consisted of about 3 mg/mL Bcc miniTRIM in 10 mM Tris , pH 8 , 100 mM LiCl , 1 mM TCEP .",
"Crystallization was performed in hanging drop format .",
"Initial hits were identified with commercial sparse matrix screens .",
"Optimized conditions are summarized in Supplementary file 1A .",
"Diffraction data were collected at beamlines 22BM or 22ID at the Advanced Photon Source , and processed using HKL2000 ( Otwinowski and Minor , 1997 ) .",
"We initially determined the structure of a dimeric Bcc miniTRIM ( P6222 form ) to 2 Å resolution by molecular replacement with a computational model derived from the rhesus TRIM5α B-box/coiled-coil structure ( PDB 4TN3 ) ( Goldstone et al . , 2014 ) and residues 49–79 of Thermus thermophilus seryl-tRNA synthetase ( PDB 1SRY ) ( Fujinaga et al . , 1993 ) .",
"This model was partially refined and then used as a molecular replacement search model for all the other structures ( Table 1 ) .",
"Structure determination and refinement were performed using the Phaser/AutoMR and phenix . refine modules of the PHENIX suite of programs ( version 1 . 9–1692 ) ( Adams et al . , 2010 ) .",
"Secondary structure hydrogen bonding restraints and zinc coordination ( bond and angle ) restraints were used during refinement .",
"Torsion angle ( local ) non-crystallographic symmetry ( NCS ) restraints were also used when appropriate .",
"Manual model building was performed with the program Coot ( Emsley et al . , 2010 ) .",
"Structure validation tools , as implemented in both PHENIX and Coot were used throughout the structure refinement process .",
"Mass measurements on Bcc miniTRIM were performed on a Dionex UltiMate3000 HPLC system with a UV detection module ( ThermoFisher , Waltham , MA ) , connected to a miniDAWN TREOS static light scattering detector ( Wyatt Technology , Santa Barbara , CA ) and Optilab T-rEX differential refractometer ( Wyatt Technology ) .",
"A sample volume of 40 μL at 0 . 9 mM concentration was applied to a Superdex 200 HR 10/300 GL column ( GE Healthcare ) and developed in 30 mM Tris , pH 8 . 0 , 100 mM NaCl at a flow rate of 0 . 4 mL/min .",
"Data were recorded and processed using ASTRA software ( Wyatt Technology ) .",
"Equilibrium sedimentation AUC experiments on Bcc miniTRIM were performed at 4°C using either XL-A or XL-1 analytical ultracentrifuges with absorbance optics ( Beckman Coulter , Brea , CA ) .",
"Sample cells with a six-channel centerpiece were filled with 110 μL of the protein samples at concentrations of 60 , 30 , and 15 μM , while 120 μL of sample buffer was loaded into the reference sectors .",
"Absorbance scans at 280 nm were taken after equilibrium was reached ( ~12 h ) at 14 , 000 , 21 , 000 , and 26 , 000 rpm .",
"Protein partial specific volume and solvent density were calculated using SEDNTERP ( version 20120828 BETA ) available online at sedenterp . unh . edu .",
"These values were used during curve fitting and data analysis using Heteroanalysis Software ( version 1 . 1 . 58 ) ( Cole , 2004 ) .",
"Restriction assays were performed using HeLa cells grown in DMEM ( Gibco/Thermofisher , Waltham , MA ) supplemented with 10% fetal calf serum ( Gibco ) at 37°C in 5% CO2 .",
"The cells were first transduced with a VSV-G pseudotyped lentiviral vector encoding rhesus TRIM5α with a C-terminal Flag-One-Strep ( FOS ) tag followed by an IRES sequence and DsRed ( CSII-IDR2-TRIM5α-FOS ) .",
"Three days after transduction , cells expressing TRIM5α-FOS were re-seeded in 24-well plates and transduced with increasing MOI of VSV-G pseudotyped HIV-GFP .",
"A sample of these cells were pelleted and resuspended in SDS-PAGE sample buffer for western blot analysis of TRIM5α expression using anti-FLAG M2 antibody ( Sigma , St . Louis , MO ) .",
"72 hr after HIV-GFP transduction , cells were trypsinized and analyzed for GFP expression ( to determine the extent of HIV-GFP infection ) and DsRed ( as a marker for TRIM5α positive cells ) by flow cytometry .",
"Lentiviral vectors for expressing TRIM5α in the above experiments were produced in the following manner .",
"HEK293T cells were plated in 6-well plates and transfected with 1 μg pCMV-delR8 . 2 , 0 . 4 μg pCMV-VSV-G , and 1 . 0 μg of the CSII-IDR2-TRIM5α-FOS plasmid .",
"At 18 hr post-transfection , cells were placed in fresh media .",
"At 48 hr post-transfection , media ( containing the lentiviral particles ) was removed and placed directly on HeLa cells for transduction and expression of TRIM5α-FOS and DsRed .",
"VSV-G pseudotyped HIV-GFP virions were produced in the same way as the TRIM5α expressing virions , but media harvested at 48 hr post-transfection was filtered through a 0 . 45 μ filter , layered on a 20% sucrose cushion in HS buffer ( 10 mM HEPES , pH 7 . 4 , 140 mM NaCl ) and centrifuged for 2 hr at 28 , 000 rpm in a Beckman SW32 Ti rotor .",
"After centrifugation , the pellet containing viral particles was resuspended in HS buffer , aliquoted , and frozen for storage at −80°C .",
"The number of infectious units was determined by titrating an aliquot on HeLa cells , and determining the fraction of HIV-GFP infected cells by flow cytometry three days later .",
"The open reading frame from a previously described plasmid clone of Strep-FLAG-tagged TRIM5-21R ( Ganser-Pornillos et al . , 2011 ) was transferred into pFastBac1 ( Invitrogen , Carlsbad , CA ) .",
"Mutations were made in this vector using the Quikchange method ( Agilent ) .",
"Baculoviruses were made using a modification of the Invitrogen Bac-to-Bac system ( Hanson et al . , 2007 ) .",
"Proteins were expressed by infecting SF9 cells in shake culture format for 48 h , and purified as described previously ( Ganser-Pornillos et al . , 2011 ) .",
"Spontaneous TRIM5-21R assembly was achieved by incubation of purified protein samples at 4°C , as described ( Ganser-Pornillos et al . , 2011 ) .",
"The templated assembly assay for flat TRIM5-21R arrays was performed as described ( Ganser-Pornillos et al . , 2011 ) .",
"Co-assembly of TRIM5-21R and HIV-1 CA was performed as described ( Li et al . , 2016 ) .",
"Grid preparation and projection imaging of samples made by the spontaneous or template driven assembly methods were performed as described ( Ganser-Pornillos et al . , 2011 ) .",
"Electron tomography was performed as follows .",
"Samples were mixed with BSA Gold Tracer ( 10 nm , Electron Microscopy Sciences , Hatfield , PA ) and applied to glow-discharged continuous carbon grids for 1 min , washed with 0 . 1 M KCl , and stained with 2% ( w/v ) uranyl formate for 30 s .",
"Tilt series were collected manually from –60° to +60° with a Tecnai F20 transmission electron microscope ( Philips/FEI , Hillsboro , OR ) operating at 120 kV .",
"Step sizes were 5° at low tilts ( 0° to 30° ) , 3 . 5° at medium tilts ( 30° to 40° ) , and 1° at high tilts ( 30° to 60° ) .",
"Images were recorded on a Gatan Ultrascan 4k × 4k CCD camera at a magnification of 29 , 000× ( 3 . 7 Å/pixel ) and defocus of –2 . 5 μm .",
"Images were aligned and binned by 4 using the IMOD software package ( Kremer et al . , 1996 ) , and reconstructions were calculated with 16 iterations of the simultaneous iterative reconstruction technique using the TOMO3D software package ( Agulleiro and Fernandez , 2011 ) .",
"Individual z-sections were visualized using the slicer option in IMOD .",
"To boost the contrast and make features more visible , up to 5 sequential slices were combined .",
"The centrifugation assay was based on previously published methods ( Ganser-Pornillos et al . , 2011; Stremlau et al . , 2006 ) , with modification ( Fribourgh et al . , 2014 ) .",
"Disulfide-stabilized HIV-1 CA tubes ( 1 mg/mL , 20 μL ) were incubated with TRIM5-21R ( 1 mg/mL , 5 μL ) in ice for 1 hr .",
"A 5-μL aliquot was removed , then mixed with equal volume of 2x SDS-PAGE sample buffer ( load sample ) .",
"The remaining sample was centrifuged at 16 , 000 g for 30 min at 4°C .",
"The supernatant ( 20 μL ) was removed and mixed with equal volume of 2x SDS-PAGE sample buffer .",
"The pellet was resuspended in 40 μL of 1x SDS-PAGE sample buffer .",
"Samples were boiled and then analyzed by SDS-PAGE ( well volumes: 10 μL of load , 20 μL each of supernatant and pellet fractions ) with Coomassie staining .",
"Stained gels were scanned and quantified using ImageJ ( Schneider et al . , 2012 ) .",
"Binding activity was expressed as the fraction of TRIM5-21R in the pellet .",
"By this method , we found that about 50% of freshly purified TRIM5-21R was consistently found in the pellet ( Figure 5 ) .",
"Upon storage , the co-pelleted fraction dropped to about 30% ( not shown ) .",
"Mutants were therefore analyzed fresh after purification , two or three a time in parallel with a WT control .",
"Model ensembles were calculated with the program RANCH as described ( Bernadó et al . , 2007 ) , using the miniTRIM trimer structure from this study .",
"Additional input files for the RING monomer and dimer ensembles , respectively , were the NMR structure of the RING monomer ( PDB 2ECV ) ( Lienlaf et al . , 2011 ) and the crystal structure of the RING dimer subunit ( PDB 4TKP ) ( Yudina et al . , 2015 ) .",
"Residues linking the RING and B-box domains were modeled as impenetrable spheres , with a Cα angle distribution consistent with disordered proteins ( Bernadó et al . , 2007 ) .",
"Thus , possible interactions with the linker were disregarded .",
"Since RANCH cannot model symmetry mismatched PDB files , the RING dimer orientations were modeled in the following manner .",
"An ensemble of 30 , 000 models was calculated in the same manner as the RING monomer .",
"The models were then computationally filtered to identify ones that contained a pair of RING domains wherein the distance of separation between two of the zinc lobes and their corresponding C-termini were consistent with the crystal structure of the RING dimer ( Yudina et al . , 2015 ) .",
"The RING dimer structure was then re-aligned manually , and then rotated to avoid steric clashes .",
"We found that the RING 4-helix bundle was an important restraint that led to identification of only a handful of plausible RING dimer/B-box trimer configurations .",
"The model for the self-ubiquitination complex was created by superimposing one of the RING dimer/miniTRIM trimer models , the crystal structure of the TRIM5α RING dimer in complex with Ubc13 ( Yudina et al . , 2015 ) , and the structure of the RNF4 RING dimer in complex with ubiquitin-conjugated Ubc5a ( Plechanovová et al . , 2012 ) .",
"To model the third , substrate RING , the ensemble of 10 , 000 RING monomer/miniTRIM trimer models was then screened computationally to identify an orientation that placed the appropriate RING lysine ( Lys45 or Lys51 ) within 8 Å of the E2-ubiquitin thioester bond without steric clashes between any of the structural elements .",
"Coordinates and structure factors are available from http://www . rcsb . org: P212121 trimer , 5IEA; C2 dimer , 5EIU; P1 dimer , 5F7T ."
]
] | [
"Restriction factors and pattern recognition receptors are important components of intrinsic cellular defenses against viral infection .",
"Mammalian TRIM5α proteins are restriction factors and receptors that target the capsid cores of retroviruses and activate ubiquitin-dependent antiviral responses upon capsid recognition .",
"Here , we report crystallographic and functional studies of the TRIM5α B-box 2 domain , which mediates higher-order assembly of TRIM5 proteins .",
"The B-box can form both dimers and trimers , and the trimers can link multiple TRIM5α proteins into a hexagonal net that matches the lattice arrangement of capsid subunits and enables avid capsid binding .",
"Two modes of conformational flexibility allow TRIM5α to accommodate the variable curvature of retroviral capsids .",
"B-box mediated interactions also modulate TRIM5α’s E3 ubiquitin ligase activity , by stereochemically restricting how the N-terminal RING domain can dimerize .",
"Overall , these studies define important molecular details of cellular recognition of retroviruses , and how recognition links to downstream processes to disable the virus ."
] | [
"After infecting a cell , a virus reprograms the cell to produce new copies of the virus , which then spread to other cells .",
"However , cells have evolved ways to fight back against this infection .",
"For example , many mammalian cells contain proteins called restriction factors that prevent the virus from multiplying .",
"The TRIM5 proteins form one common set of restriction factors that act against a class of viruses called retroviruses .",
"HIV-1 and related retroviruses have a protein shell known as a capsid that surrounds the genetic material of the virus .",
"The capsid contains several hundred repeating units , each of which consists of a hexagonal ring of six capsid proteins .",
"Although this basic pattern is maintained across different retroviruses , the overall shape of the capsids can vary considerably .",
"For instance , HIV-1 capsids are shaped like a cone , but other retroviruses can form cylinders or spheres .",
"Soon after a retrovirus enters a mammalian cell , TRIM5 proteins bind to the capsid .",
"This causes the capsid to be destroyed , which prevents replication of the virus .",
"Previous research has shown that many TRIM5 proteins must link up with each other via a region of their structure called the 'B-box 2' domain in order to efficiently recognize capsids .",
"How this assembly process occurs , and why it enables the TRIM5 proteins to recognize different capsids was not fully understood .",
"Now , Wagner et al . ( and independently Li , Chandrasekaran et al . ) have investigated these questions .",
"Wagner et al . engineered short versions of a type of TRIM5 protein called TRIM5α and used a technique called X-ray crystallography to determine the structure of its B-box domain .",
"This revealed that the B-box present in one molecule of TRIM5α can associate with the B-boxes on two other TRIM5α molecules .",
"By working in groups of three ( or trimers ) , the B-box domains connect several TRIM5α proteins to form a hexagonal net .",
"The TRIM5α net matches the arrangement of the capsid proteins in the shell of the virus , which enables TRIM5α to bind strongly to HIV-1 capsids .",
"Wagner et al . also found that B-box trimers are flexible , which allows the TRIM5α net to adapt to the shape of the HIV-1 capsid and wrap around regions where it curves .",
"In addition , computer modelling suggested that the B-box trimer may also enable TRIM5α to carry out the next steps in the process of disabling the virus .",
"Further work is now needed to understand in more detail how the trimers have this effect ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"tools and resources",
"genetics and genomics"
] | Efficient single-copy HDR by 5’ modified long dsDNA donors | elife-39468-v2 | [
[
"The implementation of the bacterial CRISPR/Cas9 system in eukaryotes has triggered a quantum leap in targeted genome editing in literally any organism with a sequenced genome or targeting region ( Cong et al . , 2013; Jinek et al . , 2012; Mali et al . , 2013 ) .",
"Site-specific double-strand breaks ( DSBs ) are catalyzed by the Cas9 enzyme guided by a single RNA with a short complementary region to the target site .",
"In response , the endogenous non-homologous end joining ( NHEJ ) DNA repair machinery seals the DSB .",
"Since perfect repair will restore the CRISPR/Cas9 target site , mutations introduced by imprecise NHEJ are selected for .",
"To acquire precise genome editing the initial DSB should be fixed via the homology-directed repair ( HDR ) mechanism which is preferentially active during the late S/G2 phase of the cell cycle ( Hustedt and Durocher , 2017 ) .",
"Donor DNA templates with flanking regions homologous to the target locus are used to introduce specific mutations or particular DNA sequences .",
"Injected ( linear ) dsDNA rapidly multimerizes ( Winkler et al . , 1991 ) , which likely also happens in CRISPR/Cas9 based approaches .",
"Additionally , the high activity of NHEJ re-ligating CRISPR/Cas9 mediated DSBs can multimerize injected ( linear ) dsDNA donor templates .",
"This poses a problem since consequently , the precise HDR-mediated recombination of single-copy donor templates is rather rare .",
"Several strategies have been followed to avoid NHEJ and/or favor HDR .",
"NHEJ was interfered with by pharmacological inhibition of DNA ligase IV ( Maruyama et al . , 2015 ) .",
"Conversely , HDR was meant to be favored by fusing the HDR-mediating yeast protein Rad52 to Cas9 ( Wang et al . , 2017 ) .",
"Similarly , removal of Cas9 in the G1/S phase ( Gutschner et al . , 2016 ) by linking it to the N-terminal region of the DNA replication inhibitor Geminin , should restrict the introduction of double-strand cuts to the G2 phase , when HDR is most prominently occurring ( Hustedt and Durocher , 2017 ) .",
"Those approaches improved HDR-mediated integration of the homology flanks , while they did not tackle reported integration of multimers ( Auer et al . , 2014 ) arising after injection of dsDNA templates such as plasmids or PCR products ( Winkler et al . , 1991 ) ."
],
[
"To enhance HDR without interfering with the endogenous DNA repair machinery , we aimed at establishing DNA donor templates that escape multimerization or NHEJ events .",
"We thus blocked both 5´ends of PCR amplified long dsDNA donor cassettes using ‘bulky’ moieties like Biotin , Amino-dT ( A-dT ) and carbon spacers ( e . g . Spacer C3 , SpC3 ) .",
"This should shield the DNA donor from multimerization and integration via NHEJ , thus favoring precise and efficient single-copy integration via HDR ( Figure 1A ) .",
"We first addressed the impact of the donor 5’ modification on the formation of multimers in vivo .",
"We injected modified and unmodified dsDNA donors into one-cell stage medaka ( Oryzias latipes ) embryos and analyzed the conformational state of the injected material during zygotic development .",
"dsDNA donors were generated by PCR employing 5’ modified and non-modified primers respectively , and by additionally providing traces of DIG-dUTP for labeling of the resulting PCR product .",
"The conformation of the injected dsDNA donors was assayed in the extracted total DNA after 2 , 4 and 6 hr post-injection , respectively .",
"The DNA was size fractionated by gel electrophoresis and donor DNA conformation was detected after blotting the DNA to a nylon membrane by anti-DIG antibodies ( Figure 1B ) .",
"In unmodified DIG-labelled control donors , we uncovered multimerization already at 2 hr post-injection as evident by a ladder of labeled donor DNA representing different copy number multimers ( Figure 1B ) .",
"In contrast , Biotin and SpC3 modification of DIG-labelled donors prevented multimerization within six hours post-injection ( Figure 1B ) .",
"dsDNA donors established by A-dT modified primes , however , multimerized and produced similar results as unmodified DIG-labelled dsDNA donors ( Figure 1B ) .",
"Our results reveal that Biotin and SpC3 5’ modifications efficiently prevent donor multimerization in vivo .",
"While strongly blocking multimerization , the 5’ modification of dsDNA did not apparently enhance the stability of the resulting dsDNA ( compare modified and unmodified donors over time , Figure 1B ) .",
"To test whether 5’ modification not only reduces the degree of multimerization but also impacts on single-copy HDR-mediated integration of long dsDNA donors , we designed gfp containing donor cassettes for an immediate visual readout .",
"We generated gfp in-frame fusion donors for four different genes: the retinal homeobox transcription factors rx2 and rx1 ( Reinhardt et al . , 2015 ) , the non-muscle cytoskeletal beta-actin ( actb ) ( Stemmer et al . , 2015 ) and the DNA methyltransferase 1 ( dnmt1 ) .",
"Donor cassettes contained the respective 5’ homology flank ( HF ) ( 462 bp for rx2 , 430 bp for rx1 , 429 bp for actb , 402 bp for dnmt1 ) , followed by the in-frame gfp coding sequence , a flexible linker in case of rx2 , rx1 and dnmt1 , and the corresponding 3’ HF ( 414 bp for rx2 , 508 bp for rx1 , 368 bp for actb , 405 bp for dnmt1; schematic representation in Figure 1A , Figure 1—figure supplement 1 for detailed donor design ) .",
"To amplify the long dsDNA donors we employed a pair of universal primers ( 5’ modified or unmodified as control ) complementary to the backbone of the cloning vectors ( pDestSC-ATG [Kirchmaier et al . , 2013] or pCS2+ [Rupp et al . , 1994] ) encompassing the entire assembled donor cassette ( Figure 1—figure supplement 1 ) .",
"Modified or unmodified long dsDNA donors were subsequently co-injected together with Cas9 mRNA and the respective locus-specific sgRNA into medaka one-cell stage zygotes ( Loosli et al . , 1999; Rembold et al . , 2006 ) .",
"For all four loci ( and all 5’ modifications ) tested we observed efficient targeting as apparent by the GFP expression within the expected expression domain ( Figure 2A , Supplementary file 1 , Figure 2—figure supplement 1 ) .",
"The survival rates of embryos injected with Biotin and SpC3 5’ modified dsDNA donors did not differ significantly from embryos injected with the unmodified dsDNA control donors ( Supplementary file 1 , Figure 2—figure supplement 1 ) .",
"In contrast , the injection of the A-dT 5’ modified dsDNA donors resulted in high embryonic lethality ( Supplementary file 1 , Figure 2—figure supplement 1 ) .",
"We next analyzed the frequency of single-copy HDR events following a careful , limited cycle PCR approach on genomic DNA of GFP expressing embryos injected with unmodified , Biotin or SpC3 5’ modified dsDNA donors .",
"Our approach allowed distinguishing alleles without gfp integration ( i . e . size of wild-type locus ) from those generated by HDR and NHEJ respectively and addressed the size of the integration by a locus spanning PCR with a reduced number of PCR cycles ( <=30 ) to omit in vitro fusion-PCR artefacts ( own data and [Won and Dawid , 2017] ) .",
"To determine the predictive power of GFP expression for perfect integration , we genotyped randomly selected , GFP-expressing embryos using locus primers ( Lf/Lr ) located distal to the utilized HFs ( Figure 1—figure supplement",
"1 ) and addressed the fusion of the gfp donor sequence to the target genes ( Figure 2B , Figure 2—figure supplements 2 and 3 ) .",
"Employing unmodified donors , the rate of HDR was very low as evidenced by the predominant amplification of the alleles without gfp integration ( Figure 2B , Figure 2—figure supplement 2 ) .",
"In strong contrast , the 5’ modified long dsDNA donors resulted in efficient HDR already detectable in the injected generation for all targeted loci .",
"For the gfp tagging of rx2 , 6 out of 10 randomly selected , GFP-expressing embryos showed precise HDR-mediated single-copy integration in F0 ( Figure 2—figure supplement",
"2 ) as sequence confirmed by the analysis of the locus ( Figure 2—figure supplement 3 ) .",
"Thus , 9 . 5% of injected and surviving zygotes showed precise HDR-mediated single-copy integration ( 15 . 8% of the injected zygotes expressed GFP; 60% of those showed the precise single-copy integration; Supplementary file 1 ) .",
"For the gfp tagging of actb 46 . 5% of the injected zygotes expressed GFP , 35% of which ( 7 out of 20 randomly selected , GFP-expressing embryos ) showed precise HDR-mediated single-copy integration in F0 , accounting for 16 . 3% of the initially injected zygotes ( Supplementary file 1 ) .",
"In the case of dnmt1 , the rate of precise HDR-mediated single-copy integration was even higher , since full gene functionality is required for the progression of development and embryonic survival .",
"Here , strikingly , all GFP-expressing embryo showed the desired perfect integration .",
"We observed the highest efficiency of HDR targeting by 5’ modified dsDNA for all loci tested for 5’Biotin modified donors ( Figure 2—figure supplement 2 ) .",
"Already in the injected generation we prominently detected and validated the HDR-mediated fusion of the long dsDNA donors with the respective locus ( Figure 2—figure supplement 2 ) .",
"While still giving rise to a high percentage of HDR events , SpC3 5’ modified dsDNA donors also resulted in elevated levels of additional bands indicative for the integration of higher order multimers and NHEJ events ( Figure 2—figure supplement 2 ) .",
"Even though increasingly popular , in our hands the use of RNPs ( Cas9 protein and respective sgRNA ) did not even get close to the efficiency achieved by co-injection of Cas9 mRNA and the corresponding sgRNA assessed by gene targeting as described above .",
"The precise integration detected in the injected generation was successfully transmitted to the next generation ( Figure 3 , Figure 2—figure supplement 3 , Figure 3—figure supplement 1 ) .",
"For gfp-rx2 , 9% of fish originating from the initially injected embryos successfully transmitted the precisely modified locus to the next generation ( 15 . 8% of the injected embryos expressed GFP; 4 out of 7 GFP transmitting founder fish were also transmitting the precise single integration of the gfp donor cassette ) .",
"For gfp-rx1 , 3 . 9% of fish originating from the initially injected embryos were transmitting the precise single copy integrate to the next generation ( 13 . 5% of injected embryos expressed GFP; 2 out of 7 GFP transmitting founder fish were also transmitting the precise single integration of the gfp-rx1 donor cassette ) .",
"For dnmt1 , the high rate of precise HDR-mediated single-copy integration observed in the injected generation was fully maintained in the transmission to the next generation due to the absolute requirement of a functional/functionally tagged version of the locus .",
"As we found in the course of our study , the precise integration of the gfp donor cassette into the actb locus results in late embryonic lethality .",
"Consequently , stable transgenic lines could not be established .",
"To estimate the timepoint of HDR events in the injected embryos , we investigated the actual rate of mosaicism in the germline as reflected by the germline transmission rate .",
"For gfp-rx2 , this rate ranged from 0 . 8% up to 12 . 9% indicating an HDR event earliest at the 4-cell stage ( assuming that only a single event occurred per blastomere ) .",
"In the case of gfp-rx1 , HDR did not occur at the one-cell stage but rather later ( 2–8 cell stage as reflected by germline transmission rates of 23 . 9% and 5 . 8% respectively ) .",
"Thus , for both cases , the transmission rates of the perfectly tagged locus reflect a level of mosaicism indicative for an HDR event between the 4- and 32-cell stage .",
"We addressed the nature of the insertion predicted to be single-copy by a combination of PCR and expression studies .",
"We validated the genomic organization of the gfp-rx2 ( Figure 3A ) and gfp-rx1 knock-in in homozygous F2 animals by genomic sequencing ( Figure 2—figure supplement 3C ) and Southern Blot ( Southern , 2006 ) analysis ( Figure 3B and B’ , Figure 3—figure supplement 1 ) .",
"In both cases , we detected a single band indicative for a single-copy HDR-mediated integration , when probing digested genomic DNA of F2 gfp-rx2+/+ and gfp-rx1+/+ knock-in fish respectively .",
"We used enzyme combinations releasing the gfp-rx2 region including the HFs ( BglII/HindIII , Figure 3B and B’ ) or cutting in the 5’ HF ( ScaI/HindIII , Figure 3B , B’ ) .",
"For gfp-rx1 we released the 5’ flanking region including the donor cassette ( HindIII/XmaI ) or the respective 3’ flanking region and donor cassette ( NcoI/EcoRI ) ( Figure 3—figure supplement 1 ) .",
"This analyses crucially validated the PCR based predictions and sequencing results .",
"Furthermore , transcript analysis in gfp-rx2 homozygous F3 embryos exclusively uncovered a single fusion gfp-rx2 transcript ( Figure 3C and C’ ) .",
"This molecular analysis confirmed that the 5’Biotin modification of the long dsDNA donor promoted precise single-copy HDR-mediated integration with high efficiency .",
"Taken together the simple 5’Biotin modification at both ends of long dsDNA donors by conventional PCR amplification presented here provides the means to favor HDR without interfering with the cellular DNA repair machinery ."
],
[
"For efficient recombination the quality of the modified primers , that is the fraction of primers actually labeled with biotin and therefore the quality of the 5’ protected long ds DNA PCR product , is essential .",
"The integration of 5’ modified PCR fragments larger than 2 kb does in principle not pose a problem .",
"We already successfully integrated cassettes of up to 8 . 6 kb ( data not shown ) via HDR by in vivo linearization of the donor plasmid ( Stemmer et al . , 2015 ) .",
"In the approach presented here , the quality of the end-protected PCR product is likely to drop with higher length in part due to the ( UV– ) nicking in the extraction and purification process .",
"Also , the rapid validation by locus spanning PCR is size limited but eventually Southern blot analysis will resolve the question of a single copy , perfect integration .",
"Irrespective of the donor cassette size , the PCR cycles used to probe for the successful single integration via HDR must not exceed a total of 30 to avoid misleading in vitro fusion-PCR artifacts ( own data and [Won and Dawid , 2017] ) .",
"As already mentioned earlier , HDR preferentially occurs during the late S/G2 phase of the cell cycle ( Heyer et al . , 2010; Hustedt and Durocher , 2017 ) .",
"Varying efficiencies of genome editing via HDR could be due to species-specific differences , in particular of the S/G2 phase of the cell cycle that likely crucially impacts on the rate of HDR .",
"As reported extensively , there is no apparent G2 phase before mid-blastula-transition in amphibia ( Xenopus ) and fish ( Danio rerio ) with the marked exception of the first cleavage , where a G2 phase has been reported ( Kimelman , 2014 ) .",
"The germline transmission rates obtained for the tagging of Rx1 and Rx2 indicated HDR events after the two-cell stage , predominantly at the 4-cell and subsequent stages .",
"The slower cycling of medaka compared to zebrafish results in an extension of the G2 phase in the first cell cycle by more than 150% ( total cycle zebrafish: 45 min [Kimmel et al . , 1995] , medaka: 65 min [Iwamatsu , 2004] ) .",
"Assuming a comparable rate of DNA synthesis between the two species , there is more than double the time for S- and M-phase to replicate a genome encompassing roughly a third of the size of zebrafish .",
"This extended cell cycle length is in particular apparent also in the subsequent cycles in medaka ( 15 min zebrafish [Kimmel et al . , 1995] , 40 min medaka [Iwamatsu , 2004] ) .",
"Interestingly , phosphorylation of RNA polymerase , a prerequisite for the expression of the first zygotic transcripts has been reported to start at the 64cell stage in medaka ( Kraeussling et al . , 2011 ) and asynchronous divisions , a hallmark of MBT , are observed already at the transition of the 16- to 32-cell stage ( Kraeussling et al . , 2011 ) , suggesting species-specific differences in the onset of zygotic transcription and consequently the lengthening of the cell cycle .",
"Our approach facilitates the highly efficient detection of HDR events by GFP tagging even in non-essential genes .",
"Other than in non-modified dsDNA donors , the locus-specific expression of GFP is an excellent predictor for precise , single-copy HDR-mediated integration already in the injected generation .",
"This allows an easy selection and results in HDR rates in GFP positive embryos of up to 60% in the injected generation .",
"This rate can even be higher in essential loci , such as dnmt1 , where the full functionality of the ( modified ) gene under investigation is required for the survival of the affected cells , tissues , organs or organisms , thus providing the means for effective positive selection for single , fully functional integrates .",
"It is interesting to note that approximately 400 bp flanking the insertion site in 5’ and 3’ direction are sufficient for HDR .",
"The use of modified universal primers for the two donor template vectors employed allowed a flexible application of the procedure and ensured a high quality of the PCR generated 5’ modified long dsDNA donors .",
"Strikingly , the primer-introduced non-homology regions in the very periphery of the long dsDNA donors did not negatively impact on the HDR efficiency .",
"Taken together , the simplicity and high reproducibility of our proof-of-concept analysis highlight that the presented protection of both 5’ ends of long dsDNA donors prevent multimerization and promote precise insertion/replacement of DNA elements , thus facilitating functional studies in basic research as well as therapeutic interventions ."
],
[
"All fish are maintained in closed stocks at Heidelberg University .",
"Medaka ( Oryzias latipes ) husbandry ( permit number 35–9185 . 64/BH Wittbrodt ) and experiments ( permit number 35–9185 . 81/G-145/15 Wittbrodt ) were performed according to local animal welfare standards ( Tierschutzgesetz §11 , Abs . 1 , Nr . 1 ) and in accordance with European Union animal welfare guidelines ( Bert et al . , 2016 ) .",
"The fish facility is under the supervision of the local representative of the animal welfare agency .",
"Embryos of medaka of the wild-type Cab strain were used at stages prior to hatching .",
"Medaka was raised and maintained as described previously ( Koster et al . , 1997 ) .",
"Rx2 and actb template plasmids for gfp donor cassette amplification are described in Stemmer et al . ( 2015 ) and were generated by GoldenGATE assembly into the pGGDestSC-ATG destination vector ( addgene #49322 ) according to Kirchmaier et al . ( 2013 ) .",
"See Supplementary file 2 for primers used to amplify respective homology flanks .",
"The dnmt1 gfp plasmid was cloned with homology flanks ( 5’ HF 402 bp , primers dnmt1 5’HF f/dnmt1 5’HF r; 3’ HF 405 bp , dnmt1 3’HF f/dnmt1 3’HF r ) that were PCR amplified with Q5 polymerase ( New England Biolabs , 30 cycles ) from wild-type medaka genomic DNA .",
"mgfp-flexible linker was amplified with primers mgfpf/mgfpr .",
"The respective restriction enzyme was used to digest the amplicons ( 5’HF: SalI HF ( New England Biolabs ) , AgeI HF ( New England Biolabs ) ; mgfp-flexible linker: AgeI HF ( New England Biolabs ) , SpeI HF ( New England Biolabs ) ; 3’HF: SpeI HF ( New England Biolabs ) , NotI HF ( New England Biolabs ) ) followed by gel purification ( Analytik Jena ) and ligation into pCS2+ ( Rupp et al . , 1994 ) ( digested with SalI HF ( New England Biolabs ) , NotI HF ( New England Biolabs ) ) .",
"The rx1 gfp plasmid was cloned with homology flanks ( 5’HF 430 bp , primers rx1 5’HF f/rx1 5’HF r; 3’ HF 508 bp , rx1 3’HF f/rx1 3’HF r ) that were PCR amplified with Q5 polymerase ( New England Biolabs , 30 cycles ) from wild-type medaka genomic DNA .",
"All primers were obtained from Eurofins Genomics .",
"We designed universal primers that match the pGGDestSC-ATG ( Kirchmaier et al . , 2013 ) ( addgene #49322 ) or pCS2+ ( Rupp et al . , 1994 ) backbone encompassing the assembled inserts ( i . e . the gfp donor cassette ) .",
"Unmodified control primers ( pDest f , pDest r , pCS2 f , pCS2 r ) were ordered from Eurofins Genomics .",
"Modified primers obtained from Sigma-Aldrich ( pDest f mod , pDest r mod , pCS2 f mod , pCS2 r mod ) consist of the same sequences with phosphorothioate bonds in the first five nucleotides and 5’moiety extension: 5’Biotin , 5’Amino-dT or 5’Spacer C3 .",
"The dsDNA donor cassettes were amplified by PCR using 1x Q5 reaction buffer , 200 µM dNTPs , 200 µM primer forward and reverse and 0 . 6 U/µl Q5 polymerase ( New England Biolabs ) .",
"Conditions used: initial denaturation at 98°C 30 s , followed by 35 cycles of: denaturation at 98°C 10 s , annealing at 62°C 20 s and extension at 72°C 30 s per kb and a final extension step of 2 min at 72°C .",
"The PCR reaction was treated with 20 units of DpnI ( New England Biolabs ) to remove any plasmid template following gel purified using the QIAquick Gel Extraction Kit ( Qiagen , 28706 ) and elution with 20 µl nuclease-free water .",
"The LacZ cassette of the pGGDestSC-ATG ( Kirchmaier et al . , 2013 ) ( addgene #49322 ) which served as DIG labelled dsDNA fragment to test in vivo multimerization was amplified via Q5-PCR as above using a mixture of 200 µM dATP , dCTP , dGTP , 170 µM dTTP and 30 µM DIG-dUTP and purified as detailed .",
"Dnmt1 sgRNAs were designed with CCTop as described in Stemmer et al . ( 2015 ) .",
"sgRNAs for rx2 and actb were the same as in Stemmer et al . ( 2015 ) .",
"The following target sites close to the translational start codons were used ( PAM in brackets ) : rx2 ( GCATTTGTCAATGGATACCC[TGG] ) , actb ( GGATGATGACATTGCCGCAC[TGG] ) , dnmt1 ( TGACATCGTCTGGCAAAGAC[AGG] ) and rx1 ( AAATGCATGAGAGCGTCT[GGG] and CTCTCATGCATTTATCAC[TGG] ) .",
"Cloning of sgRNA templates and in vitro transcription was performed as detailed in Stemmer et al . ( 2015 ) .",
"The pCS2 +Cas9 plasmid was linearized using NotI and the mRNA was transcribed in vitro using the mMessage_mMachine SP6 kit ( ThermoFisher Scientific , AM1340 ) .",
"Medaka zygotes were injected with 10 ng of DIG-labelled donors and were allowed to develop until 2 , 4 and 6 hr post injection .",
"For the CRISPR/Cas9 experiments , medaka zygotes were injected with 5 ng/µl of either unmodified and modified long dsDNA donors together with 150 ng/µl of Cas9 mRNA and 15–30 ng/µl of the gene-specific sgRNAs .",
"Injected embryos were maintained at 28°C in embryo rearing medium ( ERM , 17 mM NaCl , 40 mM KCl , 0 . 27 mM CaCl2 , 0 . 66 mM MgSO4 , 17 mM Hepes ) .",
"One day post-injection ( dpi ) embryos were screened for survival , GFP expression was scored at two dpi .",
"In order to check for multimerization of unmodified and modified donors , we used a modified Southern Blot approach .",
"In brief , embryos were injected with DIG-labelled donors which were PCR-amplified from pGGDestSC-ATG ( addgene #49322 ) using primers pDest f/pDest r ( LacZ cassette ) harboring either no 5’ moiety or one of the following: 5’Biotin , Amino-dT or Spacer C3 .",
"2 , 4 and 6 hr post injection , 30 embryos were lysed in TEN buffer plus proteinase K ( 10 mM Tris pH 8 , 1 mM EDTA , 100 mM NaCl , 1 mg/ml proteinase K ) at 60°C overnight .",
"DNA was ethanol precipitated after removal of lipids and proteins by phenol-chloroform extraction .",
"Total DNA was resuspended in TE buffer ( 10 mM Tris HCl pH 8 . 0 , 1 mM EDTA pH 8 . 0 ) .",
"200 ng of each sample was run on a 0 . 8% agarose gel .",
"As a control , 100 pg of uninjected donor PCR product were loaded .",
"The agarose gel was transferred to a nylon membrane overnight using 10x SSC ( 1 . 5 M NaCl , 0 . 15 M C6H5Na3O7 ) as transfer solution .",
"The cross-linked membrane was directly blocked in 1% w/v blocking reagent ( Roche ) in 1x DIG1 solution ( 0 . 1 M maleic acid , 0 . 15 M NaCl , pH 7 . 5 ) and the labeled DNA was detected using CDP star ( Roche ) following the manufacturer’s instructions .",
"In order to check for copy number insertions in the gfp-rx2 and gfp-rx1 transgenic lines , genomic DNA was isolated as described above from F2 embryos expressing GFP .",
"10 µg digested genomic DNA were loaded per lane on a 0 . 8% agarose gel and size fractionated by electrophoresis .",
"The gel was depurinated in 0 . 25 N HCl for 30 min at room temperature , rinsed with H2O , denatured in 0 . 5 N NaOH , 1 . 5 M NaCl solution for 30 min at room temperature and neutralized in 0 . 5 M Tris HCl , 1 . 5 M NaCl , pH 7 . 2 before it was transferred overnight at room temperature onto a Hybond membrane ( Amersham ) .",
"The membrane was washed with 50 mM NaPi for 5 min at room temperature , then crosslinked and pre-hybridized in Church hybridization buffer ( 0 . 5 M NaPi , 7% SDS , 1 mM EDTA pH 8 . 0 ) at 65°C for at least 30 min .",
"The probe was synthesized from the donor plasmid with primers gfp probe f and gfp probe r using the PCR DIG Probe Synthesis Kit ( Roche , 11636090910 ) and the following PCR protocol: initial denaturation at 95°C for 2 min , 35 cycles of 95°C 30 s , 60°C 30 s , 72°C 40 s and final extension at 72°C 7 min .",
"The probe was boiled in hybridization buffer for 10 min at 95°C and the membrane was hybridized overnight at 65°C .",
"The membrane was washed with pre-heated ( 65°C ) Church washing buffer ( 40 mM NaPi , 1% SDS ) at 65°C for 10 min , then at room temperature for 10 min and with 1x DIG1% and 0 . 3% Tween for 5 min at room temperature .",
"The membrane was blocked in 1% w/v blocking reagent ( Roche ) in 1x DIG1 solution at room temperature for at least 30 min .",
"The membrane was incubated with 1:10 , 000 anti-digoxigenin-AP Fab fragments ( Roche ) for 30 min at room temperature in 1% w/v blocking reagent ( Roche ) in 1x DIG1 solution .",
"Two washing steps with 1x DIG1% and 0 . 3% Tween were performed for 20 min at room temperature , followed by a 5 min washing step in 1x DIG3 ( 0 . 1 M Tris pH 9 . 5 , 0 . 1 M NaCl ) at room temperature .",
"Detection was performed using 6 µl/ml CDP star ( Roche ) .",
"Single injected GFP positive embryos were lysed in DNA extraction buffer ( 0 . 4 M Tris/HCl pH 8 . 0 , 0 . 15 M NaCl , 0 . 1% SDS , 5 mM EDTA pH 8 . 0 , 1 mg/ml proteinase K ) at 60°C overnight .",
"Proteinase K was inactivated at 95°C for 10 min and the solution was diluted 1:2 with H2O .",
"Genotyping was performed in 1x Q5 reaction buffer , 200 µM dNTPs , 200 µM primer forward and reverse and 0 . 012 U/µl Q5 polymerase and 2 µl of diluted DNA sample and the respective locus primers .",
"The conditions were: 98°C 30 s , 30 cycles of 98°C 10 s , annealing for 20 s and 72°C 30 s per kb ( extension time used would allow for detecting potential NHEJ events on both ends of the donor ) ( rx2 Lf/rx2 Lr: 68°C annealing , 90 s extension time; rx1 Lf/rx1 Lr: 66°C annealing , 90 s extension time; actb Lf/actb Lr: 66°C annealing , 84 s extension time; dnmt1 Lf/dnmt1 Lr: 65°C annealing , 90 s extension time ) and a final extension of 2 min at 72°C .",
"PCR products were analyzed on a 1% agarose gel .",
"Diagnostic GFP PCR here: 63°C annealing , 500 bp , 15 s extension Total RNA was isolated from 60 homozygous embryos ( stage 32 ) by lysis in TRIzol ( Ambion ) and chloroform extraction according to the manufacturer’s protocol .",
"RNA was precipitated using isopropanol and resuspended in H2O .",
"cDNA was reverse transcribed with Revert Aid Kit ( Thermo Fisher Scientific ) after DNAse digestion and inactivation following the manufacturer’s instructions .",
"PCR was performed using 5’UTRf , 3’UTRr and Q5 polymerase ( New England Biolabs ) : 98°C 30 s , 35 cycles of 98°C 10 s , annealing 65°C for 20 s and 72°C 210 s and a final extension of 2 min at 72°C .",
"PCR products were analyzed on a 1 . 5% agarose gel .",
"Plasmids and PCR fragments were sequenced with the indicated primers by a commercial service ( Eurofins Genomics ) ."
]
] | [
"CRISPR/Cas9 efficiently induces targeted mutations via non-homologous-end-joining but for genome editing , precise , homology-directed repair ( HDR ) of endogenous DNA stretches is a prerequisite .",
"To favor HDR , many approaches interfere with the repair machinery or manipulate Cas9 itself .",
"Using Medaka we show that the modification of 5’ ends of long dsDNA donors strongly enhances HDR , favors efficient single-copy integration by retaining a monomeric donor conformation thus facilitating successful gene replacement or tagging ."
] | [
"CRISPR/Cas9 technology has revolutionized the ability of researchers to edit the DNA of any organism whose genome has already been sequenced .",
"In the editing process , a section of RNA acts as a guide to match up to the location of the target DNA .",
"The enzyme Cas9 then makes a cut in both strands of the DNA at this specific location .",
"New segments of DNA can be introduced to the cell , incorporated into DNA ‘templates’ .",
"The cell uses the template to help it to heal the double-strand break , and in doing so adds the new DNA segment into the organism’s genome .",
"A drawback of CRISPR/Cas9 is that it often introduces multiple copies of the new DNA segment into the genome because the templates can bind to each other before being pasted into place .",
"In addition , some parts of the new DNA segment can be missed off during the editing process .",
"However , most applications of CRISPR/Cas9 – for example , to replace a defective gene with a working version – require exactly one whole copy of the desired DNA to be inserted into the genome .",
"In order to achieve more accurate CRISPR/Cas9 genome editing , Gutierrez-Triana , Tavhelidse , Thumberger et al . attached additional molecules to the end of the DNA template to shield the DNA from mistakes during editing .",
"The modified template was used to couple a stem cell gene to a reporter that produces a green fluorescent protein into the genome of fish embryos .",
"The fluorescent proteins made it easy to identify when the coupling was successful .",
"Gutierrez-Triana et al . found that the additional molecules prevented multiple templates from joining together end to end , and ensured the full DNA segment was inserted into the genome .",
"Furthermore , the results of the experiments showed that only one copy of the template was inserted into the DNA of the fish .",
"In the future , the new template will allow DNA to be edited in a more controlled way both in basic research and in therapeutic applications ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"developmental biology"
] | Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa | elife-00824-v1 | [
[
"Usher syndrome is a genetically heterogeneous autosomal recessive disorder characterized by early onset sensorineural hearing loss and later onset retinitis pigmentosa ( RP ) .",
"Mutations in the USH2A gene are the most common cause of Usher syndrome type I ( Aller et al . , 2006; Baux et al . , 2007; DePristo et al . , 2011 ) and are also a common cause of non-syndromic RP ( McGee et al . , 2010; Vaché et al . , 2012 ) .",
"The combination of hearing loss and retinitis pigmentosa in Usher syndrome creates an unusual opportunity for the development of effective gene replacement therapy .",
"Unlike many other forms of retinitis pigmentosa in which a large fraction of the photoreceptors have already been lost by the time a diagnosis is made , newborn hearing tests coupled with increasingly sensitive molecular testing have the potential to identify patients affected with Usher syndrome early enough that the majority of their photoreceptors are still amenable to gene replacement therapy .",
"The obstacles to such treatment include the large size of the USH2A gene , which precludes the use of the types of viral vectors currently employed for retinal gene therapy .",
"Large genes also frequently harbor a number of rare variants of uncertain pathogenicity in the population , and these can make it difficult to establish a molecular diagnosis with sufficient certainty to undertake a therapy as invasive as subretinal injection of therapeutic viruses .",
"Another obstacle to treatment is the relative paucity of information about the normal function of the protein encoded by USH2A ( usherin ) and the degree to which it can be overexpressed in human cells without causing harm .",
"The advent of induced pluripotent stem cells ( iPSCs ) ( Takahashi and Yamanaka , 2006 ) and the ability to make tissue-specific progenitors from these cells have created a path for overcoming many of these obstacles .",
"It is now possible to investigate the function and dysfunction of a disease-associated gene in tissues such as retina that are inaccessible to molecular analysis in living patients ( Tucker et al . , 2011; Jin et al . , 2012; Singh et al . , 2013 ) .",
"For instance , in a recent study , we sequenced the exome of an individual with RP who had no family history of eye disease and only one living sibling and identified a likely disease-causing homozygous mutation in a gene ( MAK ) that had not been previously reported to be associated with disease ( Tucker et al . , 2011 ) .",
"We validated this finding in a large cohort of RP patients and then used fibroblast-derived iPSCs from the proband to investigate the mechanism through which the mutation causes disease ( Tucker et al . , 2011 ) .",
"The generation of iPSCs from older individuals is more difficult and less efficient than the generation of iPSCs from young individuals ( Mahmoudi and Brunet , 2012 ) .",
"To help overcome this limitation , a variety of different reprogramming factors , reprogramming enhancers , and cell types have been evaluated ( Liao et al . , 2008; Judson et al . , 2009; Mali et al . , 2010; Cheng et al . , 2011; Niibe et al . , 2011; Szablowska-Gadomska et al . , 2011; Zhang et al . , 2011; Li and Rana , 2012; Lin et al . , 2012; Liu et al . , 2012; Mahmoudi and Brunet , 2012; Okita et al . , 2013; Zhang and Wu , 2013 ) .",
"Of the accessible cell types used for iPSC generation , the reprogramming of keratinocytes has been shown to be as much as 100-fold more efficient and at least twofold faster than the reprogramming of dermal fibroblasts ( Aasen et al . , 2008 ) .",
"In addition , it has recently been shown that keratinocyte-derived iPSCs are more similar to embryonic stem cells than those generated from fibroblasts ( Barrero et al . , 2012 ) .",
"The ability to create otherwise inaccessible tissues like retina from patient-derived iPSCs provides a valuable tool for the study of disease pathophysiology and the development of treatment .",
"However , the utility of this approach is limited by one’s ability to generate relatively pure cultures of the cell type of interest .",
"As clearly demonstrated in a variety of recent publications , early passage iPSCs tend to retain an epigenetic profile that is characteristic of their somatic tissue of origin ( Marchetto et al . , 2009; Kim et al . , 2011 ) .",
"An excellent example of how epigenetic memory can influence retinal differentiation is the recent study by Clegg et al . ( Hu et al . , 2010 ) , who showed that iPSCs generated from retinal pigmented epithelium ( RPE ) would preferentially re-differentiate into mature RPE .",
"Although viable retinal tissue suitable for iPSC generation would be difficult to obtain for the purpose of patient-specific disease modeling and therapy , accessible somatic cells from the same germ lineage as the neural retina are readily available in the form of keratinocytes .",
"In addition to being a useful tool for investigation of disease pathophysiology , iPSC technology provides a means for future autologous photoreceptor transplantation for the treatment of retinal degeneration .",
"Work from Ali et al . clearly demonstrates that the post-mitotic photoreceptor precursor cell is the optimal cell type for efficient rod photoreceptor cell replacement ( Lakowski et al . , 2011; Pearson et al . , 2012 ) .",
"A variety of different protocols , utilizing both two- and three-dimensional culture systems , have succeeded in deriving photoreceptor precursor cells from less differentiated precursors ( Osakada et al . , 2008; Hirami et al . , 2009; Meyer et al . , 2009; Osakada et al . , 2009; Lamba et al . , 2010; Meyer et al . , 2011; Tucker et al . , 2011; Nakano et al . , 2012; Phillips et al . , 2012; Sasai et al . , 2012; Homma et al . , 2013; Mekala et al . , 2013; Tucker et al . , 2013 ) .",
"Although cultured three-dimensional eyecups will undoubtedly have many applications in developmental biology ( Nakano et al . , 2012; Sasai et al . , 2012 ) , two-dimensional systems have the advantage of easier identification and isolation of specific cell types for post-differentiation subculture .",
"We and others have shown that following transplantation , iPSC-derived photoreceptor precursor cells give rise to rod and cone photoreceptor precursors , which integrate within the dystrophic retina , form synapses with host bipolar cells , and induce a partial restoration of electrophysiological and anatomical correlates of retinal function ( Lamba et al . , 2010; Tucker et al . , 2011; Homma et al . , 2013 ) .",
"Isolation of specific cell types from two-dimensional iPSC-derived eyecups followed by transplantation into animal eyes provides a means for exploring human retinal pathophysiology in unprecedented detail , as well as a means for efficiently evaluating gene-based and cell-based therapies .",
"In this study , we combined Sanger sequencing , next-generation sequencing , and iPSC technologies to identify the disease-causing USH2A mutations in a 62-year-old patient with retinitis pigmentosa and to demonstrate that the mutations we identified are present in the patient’s retinal transcripts .",
"In addition , we were able to show that the patient’s iPSC-derived rod photoreceptor precursor cells could integrate within the immune suppressed developing mouse retina and give rise to morphologically and immunohistochemically recognizable photoreceptor cells .",
"These findings suggest that the USH2A mutations in this patient act via post-developmental photoreceptor degeneration rather than an early developmental abnormality ."
],
[
"Genomic DNA from a patient affected with autosomal recessive retinitis pigmentosa ( RP ) was fragmented and hybridized to an Agilent exome capture reagent ( v2 ) , and the eluted fragments were sequenced on an Illumina sequencing instrument .",
"This experiment yielded 71 million uniquely mapped paired-end sequences , each end 50 bp in length .",
"These sequences were aligned to the reference human genomic sequence ( hg19 ) using BWA ( Li and Durbin , 2009 ) and BFAST ( Homer et al . , 2009 ) .",
"Departures from the reference were identified with GATK ( DePristo et al . , 2011 ) .",
"More than 20 , 000 sequence variations were detected ( Supplementary file 1B ) .",
"These variants were prioritized using the following criteria: GATK variation quality score greater than 50 , presence in coding sequence or within 5 bp of a splice junction , annotated frequency of less than 2% in all available population databases , presence in two or fewer exome sequences from other individuals analyzed by our laboratory , predicted to alter protein structure , and lack of complete genotype sharing with the proband’s unaffected sibling ( as determined by genomewide SNP genotyping ) ( Supplementary file 1B ) .",
"After applying these filters to the data , more than 400 plausible disease-causing sequence variations , in more than 300 genes , remained for further consideration .",
"There were no instances of two plausible disease-causing variants in a gene previously associated with RP , but two human retinal degeneration genes ( ABCA4 and USH2A ) each harbored a single plausible disease-causing variant ( Thr1428Met ACG>ATG and Arg4192His CGC>CAC , respectively ) .",
"Sanger sequencing of the entire coding sequence of both of these genes confirmed the presence of the heterozygous variants identified by exome sequencing but failed to detect a second disease-causing variation in either gene .",
"We then began evaluating genes that had not been previously associated with human retinal disease that contained two protein-altering variants in the proband .",
"As we were screening additional RP patients and controls for these variants , two things occurred that re-directed our attention to USH2A .",
"First , we discovered a second RP patient with the Arg4192His variant in USH2A , and this individual had a second well-established disease-causing mutation on the opposite USH2A allele .",
"More importantly , Vaché et al . ( 2012 ) published their discovery of a disease-causing variant in intron 40 of USH2A .",
"Sanger sequencing of intron 40 in our patient revealed this intronic variant to be present in trans to his Arg4192His mutation .",
"To validate the pathogenicity of these mutations in retinal tissue , and to begin developing a cell replacement therapy , we developed an iPSC line from our patient using a primary culture of keratinocytes as the starting material .",
"Like retina , keratinocytes are of ectodermal origin and are significantly easier to reprogram than fibroblasts ( Aasen et al . , 2008 ) .",
"This is especially important when trying to generate iPSCs from older individuals .",
"The proband’s keratinocytes ( Figure 1A ) were expanded for three passages prior to being reprogrammed into iPSCs ( Tucker et al . , 2013 ) .",
"For iPSC generation , passage-4 cells were plated at a density of 30 , 000 cells/cm2 and transduced with the transcription factors OCT4 , SOX2 , KLF4 , and C-MYC .",
"Approximately , 7 days post-transduction , cells were passaged at a density of 10 , 000 cells/cm2 onto fresh Synthemax cell culture plates and fed every other day with fresh iPSC induction media .",
"2–3 weeks following the initial passage , small , morphologically distinct cell clusters were present ( Figure 1B ) .",
"2–3 weeks later , cell colonies large enough for mechanical isolation were dissected from the surrounding differentiated keratinocyte layer .",
"Each isolated colony was dissociated into 150–200 μm square cell clusters and cultured in individual wells of a 24-well Synthemax cell culture plate .",
"Each well was maintained as a separate clonally expanded line for four passages prior to analysis .",
"At passage 4 , cultures contained well-defined densely packed colonies consisting of cells with a high nucleus to cytoplasm ratio ( Figure 1C , RP-iPSC ) .",
"To test pluripotency , rt-PCR and teratoma assays were performed .",
"rt-PCR analysis confirmed expression of the pluripotency markers DNMT , LIN28 , OCT4 , KLF4 , SOX2 , Nanog , and C-MYC ( Figure 1D ) . 10 . 7554/eLife . 00824 . 003Figure 1 . Derivation of iPSCs from keratinocytes of a patient affected with USH2A-associated RP .",
"( A–H )",
"Microscopic analysis of human keratinocytes ( A ) , early keratinocyte-derived iPSC colonies ( B , arrows ) , and purified keratinocyte-derived iPSC cultures ( C ) .",
"At 2–3 weeks post-viral transduction , ES-cell-like iPSC colonies begin to emerge ( B , arrows ) .",
"iPSC colonies isolated , subcultured , and expanded on Synthemax cell culture surfaces maintain a pluripotent morphology ( C ) express the pluripotency markers DNMT , LIN28 , OCT4 , KLF4 , SOX2 , NANOG , and C-MYC ( D ) , and form teratomas consisting of tissues of ectoderm ( E , arrow and G , GFAP in green ) , mesoderm ( F , asterisk and H , SMA in red ) , and endoderm ( F , arrows ) each of the three embryonic germ layers ( E–H ) .",
"Scale bar = 400 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 00824 . 003 Histologic analysis of teratomas revealed tissues specific to each of the three embryonic germ layers ( Figure 1E , F ) .",
"Similarly , immunohistochemical staining revealed GFAP positive neural tissue ( Figure 1G ) and αSMA positive vascular structures ( Figure 1H ) .",
"To derive retinal neurons from these iPSCs for pathophysiologic studies and the development of patient-specific therapy , a slightly modified version of our previously published stepwise differentiation protocol was used ( Figure 2A ) ( Tucker et al . , 2011 , 2011 , 2013 ) .",
"This protocol is designed to maximize the percentage of retinal cells produced by taking into account: ( 1 ) the role of bone morphogenic protein ( BMP ) and Wnt signaling pathway inhibition in neuroectodermal development ( noggin and DKK1 , respectively ) ( Lamb et al . , 1993; Mukhopadhyay et al . , 2001; Anderson et al . , 2002 ) ; ( 2 ) the role of IGF-1 in anterior neural/eye field development ( Pera et al . , 2001 ) ; and ( 3 ) Notch pathway inhibition ( DAPT—gamma secretase inhibitor ) in photoreceptor cell development ( Jadhav et al . , 2006 ) .",
"Although retinal differentiation in mouse can be accomplished in 30 days ( Tucker et al . , 2011 , 2013 ) , retinal differentiation of human cells takes significantly longer .",
"During differentiation , these keratinocyte-derived iPSCs formed eyecup-like structures with clearly defined layers of pigmented RPE and non-pigmented neural retina ( Figure 2B–E ) .",
"This was a clear departure from our previous experience with fibroblast-derived iPSCs , which never develop eyecup-like structures under very similar culture conditions .",
"The eyecups began as small pigmented foci that could be detected as early as 45 days post-differentiation ( Figure 2B ) .",
"Following passage and continued differentiation , these pigmented clumps expanded and elongated over 150 days ( Figure 2C ) and were eventually joined by a clump of neural rosettes ( Figure 2C , arrow ) on one side of the pigmented structure .",
"In some cases , these RPE/neural units extended into C-shaped eyecup-like structures ( Figure 2D , E ) while in others , the central cavity did not form and the structure consisted of a circular sheet of neural cells surrounded by a pigmented epithelium ( Figure 2—figure supplement 1 ) .",
"In many cases , multiple eyecups at different developmental stages were present within the same well of a differentiating six-well plate ( Figure 2D , arrows ) .",
"The reason that these eyecup-like structures develop in a planar fashion instead of the spheres reported by both Meyer et al . and Eiraku et al . is that we grow these cells under adherent conditions in tissue culture dishes coated with collagen , laminin , and fibronectin , while spheres are grown in suspension ( Meyer et al . , 2011; Eiraku and Sasai , 2012 ) .",
"Although there will undoubtedly be advantages of both approaches in different situations , two advantages of this planar system is that the neural cells in the center of the eyecup do not necrose for lack of oxygen , and the firm attachment to the underlying substrate allows homogeneous biopsies to be taken from the eyecups for subculture and analysis . 10 . 7554/eLife . 00824 . 004Figure 2 . Differentiation of human USH2A-associated iPSCs into eyecup-like structures .",
"( A ) Schematic diagram illustrating the differentiation paradigm utilized to generate human eyecup-like structures .",
"( B–E )",
"Morphological analysis of USH2A-associated iPSC-derived eyecups .",
"iPSC-derived eyecups form pigmented cell clumps ( B ) that extend and wrap in a C shape around newly formed neural rossettes ( C ) .",
"Following this protocol , a typical six-well cell culture dish will have two to four eyecups/well , each at slightly different stages of development ( D , arrows , a low magnification image of a typical well of a six-well plate with developing eyecups ) .",
"At 150 days post-differentiation , complete eyecups with clearly defined neural retina and RPE layers can be identified ( D , top right arrow , and E ) .",
"Scale bar , B and C = 200 μm , D = 400 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 00824 . 00410 . 7554/eLife . 00824 . 005Figure 2—figure supplement 1 . Additional examples of human USH2A-associated iPSC-derived eyecup-like structures .",
"( A–C )",
"At 150 days post-differentiation , extensive neural rosette formation was present .",
"( D–F )",
"Immunocytochemical analysis targeted against recoverin and rhodopsin confirms that neural rosettes consisted predominantly of rod photoreceptor precursor cells . DOI: http://dx . doi . org/10 . 7554/eLife . 00824 . 005 For example , to explore the cellular makeup of the pigmented layer of the eyecups ( Figure 3A ) , biopsies were taken , gently dissociated , and subcultured for subsequent expansion and microscopic analysis .",
"24 hr after plating , pigment-containing cell clusters adhered to the culture surface and began to give rise to non-pigmented cells with a fibroblastic morphology ( Figure 3B ) .",
"By 72–96 hr post-plating , extensive cell spreading and a complete loss of pigmentation was noted ( Figure 3C ) .",
"By 2 weeks post-plating , confluent cultures of densely pigmented hexagonal epithelial cells were present ( Figure 3D ) .",
"Immunocytochemical analysis of these cultures revealed cells expressing the tight junction marker ZO1 ( Figure 3E ) , the transcription factor PAX6 ( Figure 3F ) , the RPE-specific channel bestrophin ( Figure 3—figure supplement 1A ) and the RPE visual cycle protein RPE65 ( Figure 3—figure supplement 1B ) .",
"To further investigate the identity of cells within the pigmented layer of patient-specific eyecups , TEM analysis was performed .",
"As shown in Figure 3 , cells within the pigmented layer contained pigment granules ( G , asterisks ) , tight junctions with neighboring cells ( G and H , arrows ) , and apical microvilli ( G and H , arrowheads ) , all of which are characteristic of native RPE .",
"Collectively , these results indicate that the pigmented layer of cells located at the perimeter of patient-specific eyecups is patient-specific RPE . 10 . 7554/eLife . 00824 . 006Figure 3 . Cells contained within the pigmented layer of USH2A-associated eyecups are of RPE origin .",
"( A ) A high magnification phase image of the RPE layer of an USH2A eyecup prior to biopsy and subculture .",
"( B–D )",
"Area of the RPE presented in panel A was picked and subcultured in fresh RPE culture media on collagen , laminin , and fibronectin coated six-well culture dishes .",
"24 hr after plating , RPE cells spread and take on a fibroblastic morphology ( B ) .",
"By 72 hr post-plating , RPE cells lose their pigmentation and begin to form cell–cell contacts ( C ) .",
"2 weeks post-plating , a confluent monolayer of RPE cells are present that have taken on the typical cuboidal RPE morphology and regained pigmentation ( D ) .",
"( E–F )",
"Immunocytochemical analysis of USH2A-associated RPE cells with antibodies targeted against the tight junction marker ZO1 ( E ) and the transcription factor PAX6 ( F ) .",
"( G–H )",
"TEM analysis of RPE cells within the intact RPE layer of USH2A eyecups ( H is a high magnification view of the upper right corner of panel G ) .",
"RPE cells are polarized , have apical microvilli , make tight junctions with neighboring RPE cells ( G and H , arrows ) and contain pigment granules within their cytoplasm ( G , asterisk ) .",
"Scale bar , B–D = 200 μm , G = 0 . 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 00824 . 00610 . 7554/eLife . 00824 . 007Figure 3—figure supplement 1 . Pigmented cells isolated from USH2A-associated eyecups express bestrophin 1 and RPE65 . ( A and B ) Immunocytochemical analysis of subcultured USH2A-associated RPE cells with antibodies targeted against bestrophin 1 ( A ) and RPE65 ( B ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00824 . 007 To determine whether cells contained within non-pigmented neural rosettes ( Figure 4A ) had adopted a photoreceptor cell fate , patient-specific eyecups were fixed and analyzed with both TEM ( Figure 4B ) and confocal microscopy using antibodies to the rod photoreceptor markers recoverin and rhodopsin ( Figure 4C–E , and Figure 2—figure supplement 1D–F ) , and recoverin the connecting cilium marker acetylated tubulin ( Figure 4F–H ) .",
"Densely packed neural rosettes contained polarized recoverin positive cells ( Figure 4C , E , F , and H , and Figure 2—figure supplement 1D–F , green ) with acetylated tubulin ( Figure 4D , E , red ) and rhodopsin ( Figure 4G , H , and Figure 2—figure supplement 1D–F , red ) positive structures concentrated at the luminal surface of the rosettes .",
"TEM analysis further confirmed the existence of cilia with clearly identifiable basal bodies ( Figure 4B , arrowhead ) . 10 . 7554/eLife . 00824 . 008Figure 4 . iPSC-derived USH2A-associated neural retinal rosettes consist predominantly of rod photoreceptor cells .",
"( A ) Morphological depiction of the neural retina at 120 days post-differentiation .",
"( B ) TEM analysis of neural rosettes demonstrates the existence of cilia with clearly identifiable basal bodies .",
"( C–H )",
"Immunocytochemical analysis targeted against the rod photoreceptor markers recoverin and rhodopsin ( C–E and Ei-high magnification inlay ) , and the rod photoreceptor marker recoverin and the connecting cilia marker acetylated tubulin ( F–H and Hi-high magnification inlay ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00824 . 008 In an attempt to determine the developmental timeline of retinal gene expression , a series of rt-PCR and western blot analyses were performed on retinal progenitor cultures isolated at 60 , 90 , and 120 days post-differentiation .",
"The retinal transcripts PAX6 , OTX2 , CRX , NRL , recoverin , and rhodopsin were detected in differentiating patient-specific iPSCs as early as 60 days post-differentiation ( Figure 5A ) .",
"However , the mature rod photoreceptor protein recoverin and the rod photopigment rhodopsin were not detected until 90 and 120 days post-differentiation , respectively ( Figure 5B ) .",
"Similarly , the cone photopigments blue opsin and red/green opsin were first detected at 90 and 120 days post-differentiation , respectively ( Figure 5B ) . 10 . 7554/eLife . 00824 . 009Figure 5 . Developmental timeline of neural retina marker expression .",
"( A ) RT-PCR analysis of USH2A and human control neural retina for expression of the retinal transcription factors/photoreceptor markers PAX6 , OTX2 , CRX , NRL , recoverin , and rhodopsin at 60 days post-differentiation .",
"( B ) Western blot analysis of USH2A neural retina for expression of the retinal photoreceptor markers recoverin , rhodopsin , blue cone opsin and red/green cone opsin at 60 , 90 , and 120 days post-differentiation .",
"Although retinal transcripts can be detected as early as 60 days post-differentiation , mature photoreceptor proteins such as recoverin , rhodopsin , and the cone opsins could not be detected until 90 to 120 days post-differentiation . DOI: http://dx . doi . org/10 . 7554/eLife . 00824 . 009 To determine whether USH2A was expressed in iPSC-derived neural retina and whether the mutations we identified in the genomic DNA affected the USH2A gene product , rt-PCR analysis of RNA isolated from human control retina , control iPSC-derived photoreceptor precursor cells , and patient-specific iPSC-derived photoreceptor precursor cells were performed .",
"As shown in Figure 6 , RNA isolated from patient-specific iPSC-derived photoreceptor precursor cells revealed that the suspected splice site mutation identified in intron 40 of the proband caused exonification of the intron ( Figure 6A ) .",
"Introduction of intronic sequence between exons 40 and 41 results in a frame shift and introduction of a premature stop codon .",
"Similarly , Sanger sequencing of rt-PCR products generated from the iPSC-derived photoreceptor precursor cells allowed us to confirm the single point mutation identified within exon 63 of the patients’ DNA ( Figure 6B ) .",
"In an attempt to determine the pathophysiological mechanism of these mutations , a series of western blot experiments were performed to look for evidence of mutation-induced apoptosis , ubiquitination , and ER stress .",
"As shown in Figure 6C , when compared to normal human retina , iPSC-derived photoreceptor precursor cells from a normal control and a separate RP patient with known disease-causing mutations and pathophysiology ( MAK associated RP caused by nonsense mediated decay of the transcript ) , the proband was found to have increased expression of the markers GRP78 and GRP94 indicative of protein misfolding and subsequent ER stress ( Obeng et al . , 2006; Lind et al . , 2013 ) . 10 . 7554/eLife . 00824 . 010Figure 6 . Confirmation of genomic USH2A variants in iPSC-derived neural retina .",
"( A ) RT-PCR analysis of USH2A exons 39 to 41 in human control retina ( DePristo et al . , 2011 ) , human control iPSC-derived neural retina ( Baux et al . , 2007 ) , and human RP iPSC-derived neural retina .",
"An intronic splice site mutation in intervening sequence 40 of the USH2A gene results in the introduction of a pseudoexon ( IVS40 Red ) causing a translation frameshift and a premature stop codon .",
"( B ) RT-PCR analysis of USH2A exon 62 to 63 in human control human retina ( DePristo et al . , 2011 ) , human control iPSC-derived neural retina ( Baux et al . , 2007 ) , and human RP iPSC-derived neural retina .",
"A single heterozygous point mutation identified by whole exome sequencing ( Arg4192His ) was confirmed in the proband’s retinal transcript .",
"( C ) Western blot analysis of protein isolated from human control retina and iPSC-derived photoreceptor precursor cells obtained from the proband , an unaffected control , and a separate RP patient with known disease pathophysiology for expression of the ER-stress markers GRP78 and GRP94 .",
"Elevated expression of both GRP78 and GRP94 suggests that the mutations identified within the proband result in protein misfolding and ER-stress .",
"**p<0 . 001DOI: http://dx . doi . org/10 . 7554/eLife . 00824 . 010 For patients who have lost the majority of their retinal photoreceptor cells and in turn the majority of their vision , our ultimate goal is to develop a patient-specific autologous cell replacement strategy that can repopulate the outer retina with functional photoreceptors .",
"The ability to transplant such cells into animals with retinal degeneration will also be valuable as part of the vision scientist’s armamentarium for exploring the pathophysiologic mechanism of specific mutations that are identified in patients .",
"To test whether patient-specific photoreceptor precursor cells isolated from the neural retina layer of human iPSC-derived eyecups could give rise to new photoreceptor cells in animals for the latter purpose , a series of transplantation experiments were performed .",
"It has been previously shown that the optimal cell type for retinal transplantation is the post-mitotic photoreceptor precursor cell ( MacLaren et al . , 2006; Pearson et al . , 2012 ) .",
"Prior to transplantation , 150-day neural rosettes were dissected free from their surrounding tissues and plated onto fresh tissue culture plates to determine whether these cells would maintain a photoreceptor cell identity following dissociation .",
"To test this , cell cultures were infected 3 days after plating with a lentiviral vector driving expression of GFP under control of the rhodopsin kinase promoter .",
"2 weeks after plating , post-mitotic rhodopsin kinase positive photoreceptor precursor cells were abundant ( Figure 7A , B ) .",
"In many instances , clusters of the rhodopsin kinase positive cells realigned in a polarized photoreceptor cell fashion and extended axon-like projections ( Figure 7B , arrowhead ) and outer-segment-like processes ( Figure 7B , arrow ) .",
"Following the confirmation of a stable photoreceptor cell fate , newly generated 150-day photoreceptor precursor cells were transplanted into the subretinal space of P4 immunodeficient Rag1−/− x Crb1−/− mice .",
"Crb1−/− mice were chosen as recipient animals because they exhibit a relatively slow retinal degeneration and also because they are known to be more conducive to cellular integration with proper rod photoreceptor morphology following transplantation than other retinal degenerative strains ( Barber et al . , 2013 ) . 10 . 7554/eLife . 00824 . 011Figure 7 . USH2A-associated photoreceptor precursor cells integrate into the dystrophic mouse retina and develop into mature photoreceptor cells .",
"( A and B )",
"Microscopic analysis of rhodopsin kinase GFP expression in 150-day photoreceptor precursor cells at 14 days post-plating .",
"( C–E )",
"Immunocytochemical analysis performed on the retinas of Rag−/− x Crb1−/− degenerative eyes 14 days after receiving subretinal injections of patient-specific photoreceptor precursor cells targeted against expression of the human cell antigen Tra-1-85 ( C–F ) and the photoreceptor marker recoverin ( D and E ) .",
"Scale bar = 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 00824 . 01110 . 7554/eLife . 00824 . 012Figure 7—figure supplement 1 . USH2A-associated photoreceptor precursor cells integrate into the dystrophic mouse retina . Immunocytochemical analysis performed on the Rag−/− x Crb1−/− degenerative eyes 14 days after receiving subretinal sham injections targeted against expression of the human cell antigen Tra-1-85 .",
"ONL , outer nuclear layer; OPL , outer plexiform layer; and INL , inner nuclear layer . DOI: http://dx . doi . org/10 . 7554/eLife . 00824 . 012 2 weeks after transplantation , extensive cellular integration was detected using the human Tra-1-85 blood antigen as a marker of human cells ( Figure 7C–F , Figure 7—figure supplement 1 , Sham injection control , red ) .",
"The integrated cells expressed the mature photoreceptor marker recoverin , extended axonal projections toward the inner plexiform layer ( Figure 7D–F arrowheads ) , and developed inner- and outer-segment-like projections that extended toward the underlying RPE layer ( Figure 7D–F arrows ) ."
],
[
"Patient-specific iPSC-derived retinal cells are a valuable new tool for investigators seeking to understand and treat degenerative retinal diseases .",
"These cells will allow scientists to explore the pathophysiology of human diseases in ways that were previously possible only in animal models .",
"They will also be useful for evaluating potential therapies ranging from high-throughput screens of small molecule drugs to the comparison of gene replacement constructs containing different promoters or packaged in different vectors .",
"For rare autosomal recessive disorders such as USH2A-associated RP , there is often little statistical evidence for the pathogenicity of one or both of a patient’s putative disease-causing mutations .",
"In such cases , iPSC-derived retinal cells may be useful for confirming the pathogenicity of an unusual genotype before embarking upon an invasive therapy like subretinal administration of viral-mediated gene therapy .",
"In this study , we combined sequencing and iPSC technologies to identify and confirm the pathogenicity of disease-causing mutations in an isolated patient with autosomal recessive RP .",
"Exome sequencing of this individual revealed more than 400 plausible disease-causing sequence variations in more than 300 genes .",
"Two of these were present in genes known to cause autosomal recessive RP ( an Arg4192His variant in USH2A and a Thr1428Met in ABCA4 ) , but a second disease-causing allele could not be found in either of these genes despite Sanger sequencing of the entire coding sequence .",
"We were fortunate that Vaché et al . identified and reported the USH2A variant in IVS 40 that proved to be our patient’s second disease-causing allele .",
"Non-exomic mutations are not an uncommon cause of recessive diseases .",
"In some instances ( e . g . , CEP290-associated LCA ) they account for the largest proportion of disease-causing alleles ( Stone , 2007 ) .",
"As the iPSC technology becomes more routine , the evaluation of RNA from iPSC-derived retina may become a common step in the analysis of exome sequencing data when two clearly disease-causing alleles cannot be identified in known disease genes .",
"To confirm the pathogenicity of the USH2A variants we observed in our patient , USH2A transcripts were analyzed using RNA isolated from iPSC-derived photoreceptor precursor cells obtained from the proband , an unaffected control and human donor retina .",
"The suspected splice site mutation within IVS40 was shown to cause exonification of the intron , a translation frameshift and a premature stop codon .",
"The transcription of the patient’s USH2A missense mutation was confirmed in a similar fashion .",
"In combination , these mutations resulted in an upregulation of the markers GRP78 and GRP94 indicative of protein misfolding and subsequent ER stress .",
"Following transplantation into neonatal retinal degenerative Crb1 mutant mice , our patient’s photoreceptor precursor cells integrated into the outer nuclear layer and differentiated into morphologically and immunohistochemically recognizable photoreceptors .",
"This finding is compatible with our patient’s history of normal vision until the third decade of life; that is , his USH2A mutations do not appear to cause a gross developmental abnormality of photoreceptor cells .",
"It is also noteworthy that transplantable photoreceptor precursor cells could be generated from the skin of a patient in the seventh decade of life .",
"The transplantation of photoreceptor precursor cells derived from early postnatal retina or embryonic stem cells has been accomplished before by other groups ( MacLaren et al . , 2006; La Torre et al . , 2012; Pearson et al . , 2012 ) , but to our knowledge , this is the first time that photoreceptor precursor cells have been derived from an adult human RP patient and successfully transplanted into mice .",
"However , much work remains to be done before such transplants could be considered evidence for the feasibility of sight-restoring iPSC-derived treatments in humans .",
"For example , synaptic connectivity will need to be demonstrated ultrastructurally and electrophysiologically , and useful vision in treated animals will need to be demonstrated with an array of psychophysical approaches .",
"Longevity of the transplanted cells will be important to demonstrate as will the cells’ lack of tumorigenicity .",
"For such transplants to have practical clinical utility it will also be important to show that the transplanted cells can integrate and function in the retinas of animals with advanced stages of many different molecular types of retinal degeneration .",
"Unlike many of the genes targeted in current clinical trials , USH2A is very large and will be impossible to package into the viral vectors in current clinical use .",
"The coding sequence of USH2A is >18 kb , and the packaging limits of AAV and EIAV vectors are about 3 kb and 10 kb , respectively .",
"For genes such as USH2A , vectors with large carrying capacities such as HSV1 , which can accommodate inserts of up to 40 kb in size ( Thomas et al . , 2003 ) , may be useful .",
"In addition to the vector limitations , the proper stoichiometry of the delivered message will also be critical for some genes .",
"It is possible that patients like the one reported here , with a truncated protein encoded by one allele and a misfolded protein encoded by the other , may require a different promoter and multiplicity of infection than a different patient who harbors two stop mutations in USH2A .",
"Patient-specific iPSC-derived retinal cells may be useful in choosing the optimal gene therapy strategy for each individual .",
"For late-stage USH2A-associated RP where significant photoreceptor cell loss has occurred , cell replacement strategies will be required to restore vision .",
"In some cases , correcting the genetic defect responsible for the photoreceptor degeneration will be necessary before transplanting photoreceptor cells into the patient .",
"The development of site specific TALEN- or CRISPR-based genome editing approaches , which allow for the targeted correction of patient-specific cells in vitro , is proving to have great utility for endogenous gene correction ( Hockemeyer et al . , 2011; Ding et al . , 2013; Wang et al . , 2013 ) .",
"For instance , Hockemeyer et al . recently demonstrated that for the five genomic sites targeted , pluripotent iPSC clones carrying transgenes solely at TALEN-specified loci could be obtained ( Hockemeyer et al . , 2011 ) .",
"Similarly using the CRISPR/CAS-9 system , Wang et al . recently demonstrated the ability to simultaneously manipulate five separate genetic loci in mouse ES cells ( Wang et al . , 2013 ) .",
"A major advantage of these approaches is that unlike exogenous gene addition in which promoter strength and multiplicity of infection may require patient-specific adjustment , TALEN- and CRISPR-mediated correction have the advantage that the gene remains under the control of the endogenous promoter .",
"For recessive diseases such as USH2A-associated RP , correction of a single disease-causing allele should be sufficient for a therapeutic effect .",
"One could test this directly by transplanting corrected cells into retinal degenerative mice and determining whether cells with both alleles corrected behave differently than those with one or neither allele corrected .",
"It is possible that for some individuals with late-onset disease , such as the patient described in this report , genetic correction prior to transplantation may not be required .",
"That is , if the patient’s native photoreceptor cells develop and function normally until the third decade of life , as suggested by our current data and the patient’s clinical history , it is possible that replacement of lost photoreceptors with iPSCs that have not been genetically modified could be a reasonably durable treatment .",
"An alternative approach to patient-specific autologous cell replacement that would also not require genetic manipulation of the donor cell population prior to delivery would be the use of either genetically unmatched ES or tissue-specific precursor cells .",
"This approach is currently in clinical trial for the treatment of Stargardt macular dystrophy and age-related macular degeneration ( AMD ) ( Schwartz et al . , 2012 ) .",
"In these studies , ES cell-derived retinal-pigmented epithelial cells are delivered to immune suppressed patients as subretinal bolus cell injections ( Schwartz et al . , 2012 ) .",
"However , there are considerable disadvantages to using allogeneic cells .",
"Retinas injured by inherited disease often have a loss of integrity of the blood retinal barrier , which would allow the patient’s peripheral immune system access to the transplanted cells .",
"This type of transplant approach would likely require life-long immunomodulation , ( Hambright et al . , 2012 ) putting patients at further risk .",
"In summary , by combining next-generation and Sanger sequencing with iPSC technologies we were able to demonstrate the pathogenicity of two disease-causing mutations in a patient with non-syndromic USH2A-associated RP .",
"We also demonstrated that keratinocytes cultured from a patient in the seventh decade of life can be reprogrammed into iPSCs and differentiated into a multi-layered eyecup-like structure with immunohistochemical and ultrastructural features of human retinal precursor cells .",
"Finally , we showed that these patient-derived retinal precursor cells have the ability to integrate into the developing mouse retina and to form morphologically and immunohistochemically recognizable photoreceptor cells .",
"These findings will enable patient-specific studies of disease mechanism , gene correction , and photoreceptor cell transplantation for many different types of human retinal degeneration ."
],
[
"All experiments were conducted with the approval of the University of Iowa Animal Care and Use Committee ( Animal welfare assurance #1009184 ) and the University of Iowa Internal Review Board ( IRB # 200202022 ) .",
"All experiments were consistent with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Treaty of Helsinki .",
"After informed consent , skin biopsies were collected from a 62-year-old patient with an unknown cause of RP , three patients with known causes of retinal disease and three individuals without eye disease , and these were used for fibroblast and keratinocyte isolation ( as described previously [Bickenbach , 2005; Tucker et al . , 2013] ) .",
"Cells were expanded and targeted for iPSC generation .",
"iPSCs were generated from human patient-specific keratinocytes via infection with four separate non-integrating Sendai viruses , each of which were designed to drive expression of one of four transcription factors: OCT4 , SOX2 , KLF4 , and c-MYC ( A1378001 , Invitrogen , Grand Island , NY ) .",
"Keratinocytes plated on six-well tissue culture plates were infected at an MOI of 5 .",
"At 12–16 hr post-infection , cells were washed and fed with fresh growth media ( Epilife media with keratinocyte supplement [Invitrogen] and 0 . 2% primocin [Invivogen] ) .",
"At 7 days post-infection , cells were passaged onto six-well Synthemax cell culture dishes at a density of 300 , 000 cells/well and fed every day with pluripotency media ( DMEM F-12 media [Gibco] , 20% knockout serum replacement [Gibco] , 0 . 0008% beta-mercaptoethanol [Sigma-Aldrich , St . Louis , MO] , 1% 100 × NEAA [Gibco] , 100 ng/ml bFGF [human] [R&D] , and 0 . 2% primocin [Invivogen] .",
"At 3 weeks post-viral transduction , iPSC colonies were picked , passaged , and clonally expanded on fresh Synthemax plates .",
"During reprogramming and maintenance of pluripotency , cells were cultured at 5% CO2 , 5% O2 , and 37°C .",
"To maintain pluripotency , adult-derived iPSCs were cultured in xeno/feeder free cell culture media .",
"To initiate differentiation , iPSCs were removed from the culture substrate via manual passage using Stem Passage manual passage rollers ( Invitrogen ) , resuspended in embryoid body ( EB ) media ( DMEM F-12 media [Gibco] containing 10% knockout serum replacement [Gibco] , 2% B27 supplement [Gibco] , 1% N2 supplement [Gibco] , 1% L-glutamine [Gibco] , 1% 100 × NEAA [Gibco] , 0 . 2% primocin [Invivogen] , 1 ng/ml noggin [R&D Systems , Minneapolis , MN] , 1 ng/ml Dkk-1 [R&D Systems] , 1 ng/ml IGF-1 [R&D Systems] , and 0 . 5 ng/ml bFGF [R&D Systems] ) , and plated at a density of ∼50 cell clusters/cm2 on ultra low adhesion culture plates ( Corning , Lowell , MA ) .",
"Cell clusters were cultured for 5 days as indicated above , after which the EBs were removed , washed , and plated at a density of 25–30 EBs/cm2 in fresh differentiation media 1 ( DMEM F-12 media [Gibco] , 2% B27 supplement [Gibco] 1% N2 supplement [Gibco] , 1% L-glutamine [Gibco] , 1% 100 × NEAA [Gibco] 10 ng/ml noggin [R&D Systems] , 10 ng/ml Dkk-1 [R&D Systems] , 10 ng/ml IGF-1 [R&D Systems] and 1 ng/ml bFGF [R&D Systems] ) in six-well Synthemax culture plates .",
"Cultures were fed every other day for 10 days with differentiation media 1 .",
"For the following 6 days , cultures were fed with differentiation media 2 ( differentiation media 1 + 10 μM of the Notch signaling inhibitor , DAPT [Calbiochem , Gibbstown , NJ] ) .",
"For the following 12 days , cultures were fed with differentiation media 3 ( differentiation media 2 + 2 ng/ml of aFGF [R&D Systems] ) .",
"To enhance pigmentation and eyecup-like structure formation , cells were passaged at day 50–70 in fresh cell culture plates coated as described above and cultured for up to 150 days in differentiation media 4 ( DMEM F-12 media [Gibco] , 2% B27 supplement [Gibco] 1% N2 supplement [Gibco] , 1% L-glutamine [Gibco] , 1% 100 × NEAA [Gibco] ) .",
"To validate that generated iPSCs were pluripotent , teratomas were generated by IM injection of 2 . 5 × 106 undifferentiated iPSCs into immunodeficient ( SCID ) mice .",
"After 90 days , tumors were excised , fixed , paraffin embedded , and sectioned .",
"Teratomas were fixed in 10% formalin for 24 hr prior to dehydration and mounting in paraffin wax ( VWR ) .",
"Samples were sectioned at 6 μm and H&E staining was performed using standard protocols .",
"Cells were fixed in a 4% paraformaldehyde solution and immunostained as described previously ( Tucker et al . , 2010 , 2011 , 2013 ) .",
"Briefly , cells/tissues were incubated overnight at 4°C with antibodies targeted against either GFAP ( MAB360; Millipore , Billerica , MA ) or αSMA ( ab5694; Abcam , Cambridge , MA ) for teratoma formation or ZO-1 ( MABT11; Millipore ) , PAX6 ( MAB5552; Millipore ) , bestrophin 1 ( AB14929; Abcam ) , RPE65 ( MAB5428; Millipore ) , acetylated tubulin ( t7451; Sigma Aldrich ) , recoverin ( AB5585; Millipore ) , and rhodopsin ( MAB5316; Millipore ) for retinal differentiation .",
"Subsequently , Cy2- or Cy3-conjugated secondary antibodies were used ( Jackson Immunochem , West Grove , PA ) , and the samples were analyzed using confocal microscopy .",
"Microscopic analysis was performed such that exposure time , gain , and depth of field remained constant between experimental conditions .",
"Eyecups at 150 days post-differentiation were fixed in one half strength Karnovsky fixative as described previously ( PMID 17591911 ) , followed by osmication , dehydration , and embedment in Epon resin .",
"All procedures took place in the tissue culture dish .",
"After polymerization , blocks were removed and trimmed , ultrathin sections were collected on formvar coated grids , and samples were imaged on a JEOL JEM1230 transmission electron microscope .",
"For Western blot analysis , undifferentiated and differentiated iPSCs were homogenized in lysis buffer ( 50 mM Tris-HCl , pH 7 . 6 , 150 mM NaCl , 10 mM CaCl2 , 1% triton X-100 , 0 . 02% NaN3 , [Sigma-Aldrich] ) and centrifuged .",
"Supernatants were isolated and protein concentrations determined using a BCA protein assay ( Pierce Chemicals , Rockford , IL ) .",
"Equivalent amounts of protein ( 50 μg ) were subjected to SDS-PAGE ( 8–10% acrylamide ) , transferred to PVDF , and probed with primary antibodies targeted against recoverin ( AB 5585; EMD Millipore ) , rhodopsin ( MAB5316; EMD Millipore ) , blue opsin ( AB5407; EMD Millipore ) , red/green opsin ( AB5405; EMD Millipore ) , GRP 78 ( SC-376768; Santa Cruz ) , GRP 94 ( SC-53929; Santa Cruz ) and Actin ( AB20272; Abcam; used as a loading control ) .",
"Blots were visualized with ECL reagents ( GE healthcare , Piscataway , NJ ) and exposed to X-ray film ( Fisher , Pittsburg , PA ) .",
"Total RNA was extracted using the RNeasy Mini-kit ( Qiagen , Valencia , CA ) following the provided instructions .",
"Briefly , cells were lysed , homogenized , and ethanol was added to adjust binding conditions .",
"Samples were spun using RNeasy spin columns , washed , and RNA was eluted using RNase-free water .",
"1 μg of RNA was reverse transcribed into cDNA using the random hexamer ( Invitrogen , Carlsbad , CA ) priming method and Omniscript reverse transcriptase ( Qiagen ) .",
"All PCR reactions were performed in a 40 μl reaction containing 1× PCR buffer , 1 . 5 mM MgCl2 , 0 . 2 mM dNTPs , 100 ng of DNA , 1 . 0 U of AmpliTaq Gold ( Applied Biosystems , Foster City , CA ) and 20 pmol of each gene-specific primer .",
"All cycling profiles incorporated an initial denaturation temperature of 94°C for 10 min followed by 35 amplification cycles with the following conditions , 30 s at 94°C , 30 s at annealing temperature of each primer , and 1 min at 72°C with a final extension at 72°C for 10 min .",
"PCR products were separated by electrophoresis on 2% agarose gels ( Invitrogen ) .",
"Gene-specific primers ( Invitrogen ) are given in Supplementary file 1A .",
"Blood samples were obtained from all subjects .",
"DNA was extracted by following the manufacturers specifications for whole blood DNA extraction using Gentra Systems’ Autopure LS instrument .",
"Targeted enrichment of exons was performed using the Agilent SureSelect All Exon Capture platform per manufacturer’s instructions .",
"This capture platform includes 38 Mb of targeted features .",
"Sequencing of the captured genomic DNA was performed following the manufacturer’s instructions on an Illumina HiSeq sequencer at the Hudson Alpha Institute in Huntsville , Alabama .",
"Variants detected by next-generation sequencing were confirmed using automated Sanger sequencing using dye termination chemistry on an ABI 3730 sequencer .",
"All sequencing was bi-directional .",
"Newly generated 150-day photoreceptor precursor cells were transplanted into the subretinal space of P4 immune compromised Rag1−/− x Crb1−/− mice .",
"2 weeks after transplantation , mice were sacrificed and eyes were removed for histological analysis , fixed in 4% paraformaldehyde for 12 hr , embedded and sectioned on a cryostat .",
"Sectioned mouse eyes were used for immunofluorescence analysis .",
"Sections were blocked in 10% goat serum 3% BSA and 0 . 2% triton X-100 for 1 hr , and then incubated with anti-Tra-1-85 ( MAB4385; Millipore ) , and recoverin antibodies ( AB5585; Millipore ) followed by visualization with Cy2- and Cy3-conjugated secondary antibodies ( Jackson Immunochem , West Grove , PA ) .",
"Sections were assessed by confocal microscopy ."
]
] | [
"Next-generation and Sanger sequencing were combined to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP .",
"Induced pluripotent stem cells ( iPSCs ) , generated from the patient’s keratinocytes , were differentiated into multi-layer eyecup-like structures with features of human retinal precursor cells .",
"The inner layer of the eyecups contained photoreceptor precursor cells that expressed photoreceptor markers and exhibited axonemes and basal bodies characteristic of outer segments .",
"Analysis of the USH2A transcripts of these cells revealed that one of the patient’s mutations causes exonification of intron 40 , a translation frameshift and a premature stop codon .",
"Western blotting revealed upregulation of GRP78 and GRP94 , suggesting that the patient’s other USH2A variant ( Arg4192His ) causes disease through protein misfolding and ER stress .",
"Transplantation into 4-day-old immunodeficient Crb1−/− mice resulted in the formation of morphologically and immunohistochemically recognizable photoreceptor cells , suggesting that the mutations in this patient act via post-developmental photoreceptor degeneration ."
] | [
"Retinitis pigmentosa is an inherited disorder in which the gradual degeneration of light-sensitive cells in the outer retina , known as photoreceptors , causes a progressive loss of sight .",
"Retinitis pigmentosa can also occur as part of a wider syndrome: patients with Usher syndrome , for example , suffer from early-onset deafness and then develop retinitis pigmentosa later in life .",
"Usher syndrome is caused by mutations in any of more than ten genes , but the most commonly affected is USH2A , which encodes a protein called usherin .",
"Mutations in USH2A can also cause retinitis pigmentosa on its own .",
"Clinical trials are underway to determine whether it is possible to treat various forms of inherited retinal degeneration using gene therapy .",
"This involves inserting a functional copy of the gene associated with the disease into an inactivated virus , which is then injected into the eye .",
"The virus carries the target gene to the light-sensitive photoreceptor cells where it can replace the faulty gene .",
"This could be particularly useful for conditions such as Usher syndrome , in which the early-onset deafness makes it possible to diagnose retinitis pigmentosa before substantial numbers of photoreceptor cells have been lost .",
"For gene therapy to become a widely used strategy for the treatment of retinal degenerative disease , identification and functional interrogation of the disease-causing gene/mutations will be critical .",
"This is especially true for large highly polymorphic genes such as USH2A that often have mutations that are difficult to identify by standard sequencing techniques .",
"Likewise , viruses that can carry large amounts of genetic material , or endogenous genome editing approaches , will need to be developed and validated in an efficient patient-specific model system .",
"Tucker et al . might have found a way to address these problems .",
"In their study , they used skin cells from a retinitis pigmentosa patient with mutations in USH2A to produce induced pluripotent stem cells .",
"These are cells that can be made to develop into a wide variety of mature cell types , depending on the exact conditions in which they are cultured .",
"Tucker et al . used these stem cells to generate photoreceptor precursor cells , which they transplanted into the retinas of immune-suppressed mice .",
"The cells developed into normal-looking photoreceptor cells that expressed photoreceptor-specific proteins .",
"These results have several implications .",
"First , they support the idea that stem cell-derived retinal photoreceptor cells , generated from patients with unknown mutations , can be used to identify disease-causing genes and to interrogate disease pathophysiology .",
"This will allow for a more rapid development of gene therapy strategies .",
"Second , they demonstrate that USH2A mutations cause retinitis pigmentosa by affecting photoreceptors later in life rather than by altering their development .",
"This suggests that it should , via early intervention , be possible to treat retinitis pigmentosa in adult patients with this form of the disease .",
"Third , the technique could be used to generate animal models in which to study the effects of specific disease-causing mutations on cellular development and function .",
"Finally , this study suggests that skin cells from adults with retinitis pigmentosa could be used to generate immunologically matched photoreceptor cells that can be transplanted back into the same patients to restore their sight .",
"Many questions remain to be answered before this technique can be moved into clinical trials but , in the meantime , it will provide a new tool for research into this major cause of blindness ."
] | 2013 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Rapid short-term reorganization in the language network | elife-25964-v2 | [
[
"The current knowledge of short- and long-term plasticity in the language network after stroke-induced aphasia is limited .",
"For instance , it is still a matter of debate if the temporary recruitment of neighbouring , ipsilateral networks and / or homologous right-hemispheric regions after a lesion of one critical node in the left hemisphere is adaptive or maladaptive for stroke recovery ( Chrysikou and Hamilton , 2011; Hamilton et al . , 2011 ) .",
"To investigate the potential for rapid short-term reorganization and flexible redistribution of the functional weight in the language network , we combined controlled , focal virtual lesions in the healthy brain with effective connectivity analyses of neuroimaging data .",
"We relied on an experimental task that required the rapid analysis of the meaning of sound patterns ( i . e . , semantic processing ) , which is crucial for efficient every-day communication in humans .",
"Previous studies associated semantic processing with increased neural activation of left-hemispheric temporal , inferior frontal and inferior parietal regions ( Binder et al . , 2009; Vigneau et al . , 2006 ) .",
"In particular , left angular gyrus ( AG ) and anterior inferior frontal gyrus ( aIFG ) were identified as semantic key nodes by means of focal perturbations induced with transcranial magnetic stimulation ( TMS ) ( Devlin et al . , 2003; Sliwinska et al . , 2015 ) .",
"Specifically , several TMS studies assigned the left aIFG a central role in semantic control processes ( Hoffman et al . , 2010; Whitney et al . , 2011 ) .",
"Others demonstrated that a number of temporal regions are also crucial for different semantic aspects .",
"These regions include the anterior temporal lobe ( ATL ) for core semantic representations and the posterior middle temporal gyrus ( pMTG ) as another key node for semantic control aspects ( Binney and Ralph , 2015; Davey et al . , 2015; Jung and Lambon Ralph , 2016; Whitney et al . , 2012 ) .",
"With respect to the interactions in the semantic network , it was shown that the contribution of the left aIFG to semantic decisions crucially depends on the functional integrity of the left AG ( Hartwigsen et al . , 2016 ) .",
"Hence , left AG was able to compensate for a focal perturbation of aIFG during semantic decisions unless it was additionally perturbed with TMS , indicating a strong interaction between both regions .",
"In contrast , TMS over neighbouring supramarginal gyrus ( SMG ) selectively delayed phonological decisions ( i . e . , decisions on the sound of words ) but not semantic decisions .",
"While the above-cited behavioural TMS studies clearly demonstrate , besides temporal regions , a critical contribution of AG and aIFG to semantic processing , they do not allow for any conclusions on the neural underpinnings of the observed flexible redistribution in the semantic network after a focal perturbation of the AG .",
"Moreover , the precise role of the AG in semantic processing remains elusive .",
"For instance , this region showed increased task-related activity for words as compared to non-words or concrete relative to abstract words ( Binder et al . , 2009; Seghier , 2013 ) , but deactivation for other semantic tasks , with the amount of deactivation being correlated with increased task-difficulty ( Humphreys et al . , 2015 ) .",
"Besides , the AG also contributes to other functions outside the core language domain like episodic memory and social cognitive tasks ( Bzdok et al . , 2016 ) .",
"Hence , it is unclear whether the task-related upregulation of left AG during word processing is necessary for semantic computations or rather reflects more general processes related to the level of task difficulty ( Graves et al . , 2017; Ralph et al . , 2017 ) .",
"To elucidate the brain’s ability for adaptive short-term plasticity in response to a controlled virtual lesion and the contribution of left AG to semantic processing , we applied focal continuous theta-burst stimulation ( cTBS ) over AG or neighbouring SMG prior to neuroimaging ( Figure 1 ) .",
"The inclusion of a phonological task allowed us to test for the task specificity of the perturbation effect .",
"cTBS was applied in healthy subjects to investigate immediate and transient perturbation effects .",
"The lasting cTBS-induced suppression of neuronal excitability should give rise to an acute adaptive reorganization within the non-affected functional nodes of the network to compensate for the cTBS-induced suppression of neuronal activity ( Siebner and Rothwell , 2003 ) . 10 . 7554/eLife . 25964 . 003Figure 1 . Experimental design and behavioural results .",
"( A ) Subjects received effective or sham cTBS either over supramarginal gyrus ( SMG ) or angular gyrus ( AG ) in different sessions .",
"Thereafter , they performed semantic and phonological tasks in two fMRI runs .",
"( B ) Tasks were divided into 10 miniblocks per task and run , each consisting of 6 stimuli ( e . g . ‘Katze’ ( ‘cat’ ) ) with varying stimulus onset asynchrony .",
"min=minutes; s=seconds .",
"( C ) Effects of cTBS over AG and SMG on reaction times ( RTs ) and accuracy .",
"*p<0 . 05; SEM= standard error of the mean . DOI: http://dx . doi . org/10 . 7554/eLife . 25964 . 003 We expected to find a functional double dissociation of cTBS over AG vs . SMG on semantic vs . phonological decisions .",
"Specifically , cTBS over AG should selectively inhibit task-related semantic activity at the targeted left AG , which might in turn lead to an upregulation of other important nodes in the semantic network ( e . g . left aIFG , pMTG or ATL ) .",
"Since our previous behavioural study ( Hartwigsen et al . , 2016 ) did not find a significant modulation of semantic response speed with a unifocal perturbation of AG , we did not expect any significant disruption of mean semantic response speed after cTBS of AG .",
"However , in accordance with the previous study , cTBS of SMG should delay phonological decisions , which might be reflected in the suppression of task-related activity in the phonological network .",
"As an alternative hypothesis , cTBS might also affect neural activity on a larger network level .",
"Indeed , combined TMS-fMRI studies often reveal widespread stimulation effects , involving neighbouring and distant interconnected brain regions ( Bestmann et al . , 2003 ) .",
"If this were the case , then we would expect that cTBS over AG should decrease neural activity not only at the stimulated site itself , but in a larger semantic network .",
"As cognitive processes are mediated by the dynamic interactions among relevant areas rather than by isolated regions , effective connectivity analyses that capture perturbation effects on the causal network level should be more closely related to the neurobiological mechanisms by which a ( virtual ) lesion changes a cognitive function ( Hartwigsen et al . , 2015 ) .",
"Such network effects might also mediate possible cTBS-induced changes on the behavioural level ."
],
[
"During fMRI , subjects performed semantic ( ‘natural or manmade ? ' ) and phonological tasks ( ‘two or three syllables ? ' ) on auditorily presented words .",
"Response speed for correct trials was investigated with a repeated measures ANOVA including the within-subject factors task ( semantic vs . phonological decisions ) and cTBS ( AG vs . SMG vs . sham ) .",
"Overall , phonological response speed was significantly longer than semantic response speed ( main effect of task: F1 , 14 = 13 . 69 , p=0 . 002; η2= 0 . 56 ) .",
"However , this effect significantly interacted with cTBS ( AG vs . SMG vs . sham ) , indicating that phonological and semantic decisions were differentially affected by cTBS ( F2 , 28 = 5 . 36 , p=0 . 011; η2= 0 . 28; Figure 1C ) .",
"Two-tailed post-hoc paired t-tests revealed that cTBS over SMG significantly delayed phonological decisions when compared with sham cTBS ( t14 = 2 . 76 , p=0 . 01; d = 0 . 73 ) or cTBS over AG ( t14 = 3 . 20 , p=0 . 006; d = 0 . 81 ) .",
"In contrast , for semantic decisions , there was a trend towards increased response latencies after cTBS of AG relative to sham ( t14 = 2 . 15; p=0 . 05; d = 0 . 34 ) but not SMG cTBS ( p=0 . 14; d = 0 . 13 ) .",
"Task accuracy was not affected by cTBS ( all p>0 . 05 ) .",
"We first investigated the effects of cTBS over AG on semantic decisions .",
"Relative to cTBS of neighbouring SMG , cTBS of AG significantly decreased task-related semantic activity not only at the stimulated area , but in a large network previously associated with semantic processing , including left AG ( x , y , z = −42 , –67 , 28; T = 5 . 21 ) ; aIFG ( x , y , z = −48 , 41 , –14; T = 6 . 64; x , y , z = −57 , 26 , 19; T = 6 . 48 ) and left posterior middle temporal gyrus ( pMTG: x , y , z = −60 , –43 , −2; T = 5 . 55 ) ( Figure 2A ) .",
"Similar results for left AG and aIFG were obtained when contrasting cTBS of AG with sham cTBS ( Figure 2—figure supplement 1A and Table 1 ) .",
"There was no significant difference in task-related activity between cTBS of SMG and sham cTBS during semantic decisions .",
"This demonstrates the anatomical specificity of the perturbation effect . 10 . 7554/eLife . 25964 . 004Figure 2 . Effects of cTBS on semantic decisions .",
"( A ) Relative to cTBS over SMG , cTBS of AG significantly decreased neural activity not only at the stimulated area , but in a larger network including AG and aIFG .",
"( B ) Relative to cTBS of SMG , cTBS of AG significantly increased neural activity in phonological regions , including the bilateral SMG and pIFG .",
"Lower panels display the respective parameter estimates ( arbitrary units ) for the different cTBS conditions that were extracted at the respective mean peak coordinates from the effect of interest for each task condition against rest .",
"p<0 . 001 for display reasons .",
"Sem=semantic , phon=phonological task . DOI: http://dx . doi . org/10 . 7554/eLife . 25964 . 00410 . 7554/eLife . 25964 . 005Figure 2—figure supplement 1 . Effects of cTBS on semantic decisions .",
"( A ) Inhibitory effects of cTBS on task-related neural activity during semantic decisions .",
"Relative to sham cTBS , cTBS of AG significantly decreased neural activity not only at the stimulated area , but in a larger semantic network .",
"( B ) Upregulation of the phonological network after AG cTBS during semantic decisions .",
"Relative to cTBS over SMG , cTBS of AG significantly increased neural activity in phonological regions , including the bilateral SMG and pIFG / ventral premotor cortex .",
"Right panels display the respective parameter estimates ( arbitrary units ) for the different cTBS conditions that were extracted at the respective mean peak coordinates from the effect of interest for each task condition against rest .",
"p<0 . 001 for display reasons . DOI: http://dx . doi . org/10 . 7554/eLife . 25964 . 00510 . 7554/eLife . 25964 . 006Figure 2—figure supplement 2 . Task-related activity changes after sham cTBS ( baseline ) .",
"( A ) , ( B ) Main effects of semantic or phonological decisions relative to rest trials .",
"( C ) , ( D ) Differential contrasts of semantic > phonological and phonological > semantic decisions .",
"Coordinates are given at respective peak activations .",
"p<0 . 05 , FWE corrected . DOI: http://dx . doi . org/10 . 7554/eLife . 25964 . 00610 . 7554/eLife . 25964 . 007Table 1 . Changes in task-specific neural activation patterns after cTBSDOI: http://dx . doi . org/10 . 7554/eLife . 25964 . 007RegionSideMNI coordinates X , Y , Z ( in mm ) TCluster sizeSemantic judgements: SMG > AG cTBS inferior frontal gyrus ( pars orbitalis ) L−4841−146 . 64115 inferior frontal gyrus ( pars triangularis ) L−5726106 . 4895 superior frontal gyrusL–9 44 435 . 7192 posterior middle temporal gyrusL−60−43–25 . 55255 angular gyrusL−42−67 285 . 21240Semantic judgements: sham > AG cTBS cerebellumR 24−85−385 . 21117 inferior frontal gyrus ( pars orbitalis ) L−45 38−145 . 1245 angular gyrusL−42−64 255 . 0555Semantic judgements: AG > SMG cTBS supramarginal gyrusL−45−40 465 . 30215 inferior frontal gyrus ( pars opercularis ) L−54 5 195 . 2899 supramarginal gyrusR 42−44 425 . 0078Semantic judgements: AG > sham cTBS supramarginal gyrusL−42−43 435 . 41225 supramarginal gyrusR 44−44 435 . 31118 inferior frontal gyrus ( pars opercularis ) L−57 8 165 . 28103 planum temporaleL−57−40 195 . 0135Phonological judgements: AG > SMG cTBS supramarginal gyrus / superior parietal lobeL−45−41 425 . 24222 supramarginal gyrus / superior parietal lobeR 36−40 405 . 23121 frontal operculum / posterior inferior frontal gyrusL−55 10 44 . 9787 frontal operculum / posterior inferior frontal gyrusR 57 11 44 . 9569 supplementary motor areaR 0 5 554 . 9192Phonological judgements: sham > SMG cTBS supramarginal gyrus / superior parietal lobeL−42−40 466 . 10334 frontal operculum / posterior inferior frontal gyrusL−51 8–25 . 9197 supramarginal gyrus / superior parietal lobeR 36−39 425 . 88169 frontal operculum / posterior inferior frontal gyrusR 57 8 75 . 4482 supplementary motor areaR 0 7 555 . 29101 middle frontal gyrusL−33 41 255 . 1257thresholded at p<0 . 05; FWE-corrected at the peak level , cluster extent >20 voxels .",
"A direct comparison of areas showing stronger activation increases after cTBS over left AG vs . SMG or sham cTBS during semantic decisions revealed an upregulation of several regions of the phonological network .",
"Specifically , after cTBS over left AG vs . SMG , increased task-related neural activity was found in bilateral supramarginal gyrus ( SMG , x , y , z = −45 , –40 , 46; T = 5 . 30; x , y , z = 42 , , –44 , 42; T = 5 . 00 ) and left posterior IFG/ventral premotor cortex ( pIFG: x , y , z= −54 , 5 , 19; T = 5 . 28; Figure 2B ) .",
"Similar results were found after cTBS over AG vs . sham cTBS ( Figure 2—figure supplement 1B and Table 1 ) .",
"This upregulation presumably helped to restore task processing .",
"The observed task-specific effects of cTBS over AG on semantic decisions are summarized in Figure 3A .",
"Notably , the individual suppression of task-related activity in AG after cTBS over AG relative to sham cTBS predicted the individual upregulation of semantic activity in SMG ( regression , normalized to sham cTBS , R2 = 0 . 37 , ß = −0 . 61 , t = 2 . 79 , p=0 . 016 , two-tailed; Figure 3B ) . 10 . 7554/eLife . 25964 . 008Figure 3 . Semantic network effects .",
"( A ) Illustration of the strong cTBS-induced suppression in the semantic network ( in blue ) and the upregulation of the phonological network ( in red ) .",
"( B ) The strength of the individual inhibition of left AG after cTBS ( effect sizes for AG/sham cTBS received from the effect of interest at x , y , z= −42 , –64 , 25 ) predicted the upregulation of left SMG ( effect sizes for AG/sham cTBS extracted from the effect of interest at x , y , z= −45 , –40 , 46 ) .",
"( C ) Three-dimensional tractography rendering illustrating the underlying anatomical fiber connections mediating the remote effects of cTBS .",
"AG and aIFG were most probably connected via the middle longitudinal fasciculus and extreme capsule . DOI: http://dx . doi . org/10 . 7554/eLife . 25964 . 008 Constrained probabilistic fiber tracking of diffusion tensor imaging data further revealed that the strong remote inhibitory effects of AG cTBS in the semantic network were most probably mediated by long-distance cortico-cortical association tracts .",
"Specifically , AG and aIFG were connected via a ventral pathway constituted by the middle longitudinal fasciculus and the extreme capsule ( Saur et al . , 2008 ) , also termed as inferior fronto-occipital fascicle ( Duffau et al . , 2005 ) ( Figure 3C ) .",
"For phonological decisions , we found a significant inhibition in the phonological network after cTBS over SMG ( Figure 4 and Figure 4—figure supplement 1 ) .",
"Specifically , when contrasted with cTBS over AG or sham cTBS , cTBS over SMG decreased task-specific activity in the bilateral SMG / superior parietal cortex , bilateral frontal operculum / pIFG and right supplementary motor area ( Table 1 ) .",
"We did not find any upregulation of task-related activity during phonological processing after cTBS of SMG vs . cTBS of AG or sham cTBS , even after reducing the threshold .",
"There were no significant differences in task-related activity between cTBS of AG and sham cTBS during phonological decisions . 10 . 7554/eLife . 25964 . 009Figure 4 . Effects of cTBS on phonological decisions .",
"Relative to cTBS over AG , cTBS over SMG significantly decreased neural activity in bilateral supramarginal gyrus , with the strongest effect at the stimulation site .",
"The right panel displays the parameter estimates ( arbitrary units ) for the different cTBS conditions that were extracted at the mean peak coordinates from the effect of interest for each task condition against rest .",
"p<0 . 001 for display reasons . DOI: http://dx . doi . org/10 . 7554/eLife . 25964 . 00910 . 7554/eLife . 25964 . 010Figure 4—figure supplement 1 . Inhibitory effects of cTBS on task-related neural activation during phonological decisions . Relative to sham cTBS , cTBS over SMG significantly decreased neural activity in bilateral supramarginal gyrus , with the strongest effect at the stimulation site .",
"The right panel displays the respective parameter estimates ( arbitrary units ) for the different cTBS conditions that were extracted at the respective mean peak coordinates from the effect of interest for each task condition against rest .",
"p<0 . 001 for display reasons . DOI: http://dx . doi . org/10 . 7554/eLife . 25964 . 010 We used dynamic causal modelling ( DCM ) to further explore how the inhibitory influence of cTBS over AG changed computations in the semantic network .",
"DCMs were based on the main peaks obtained from the second-level fMRI analyses ( i . e . , left AG , aIFG and SMG ) .",
"Among the 63 models tested ( Figure 5—figure supplement 1 ) , variational Bayesian model selection identified the model with driving input to SMG and modulation of the inhibitory connection from AG to aIFG as winning model .",
"Figure 5A shows the winning model with the mean parameter estimates that were significantly different from zero ( Table 2 ) .",
"These parameters included the intrinsic connection from AG to SMG ( regardless of cTBS site , mean: 0 . 03 , T = 3 . 27; p<0 . 006 ) and the modulation of the connection from AG to aIFG by cTBS of AG ( mean: −0 . 19 , T = 3 . 67 p<0 . 003 ) .",
"Further parameters that did not survive a Bonferroni-Holm correction were the driving input to SMG ( mean: 0 . 01 , T = 2 . 24 , p<0 . 046 ) and the intrinsic connection from AG to aIFG ( mean: 0 . 03 , T = 2 . 21 , p<0 . 049 ) .",
"Our winning model had an exceedance probability of 79% , while all other models had probabilities < 10% .",
"A family comparison pooled across different driving inputs identified the family with modulation of the inhibitory connection from AG to aIFG as winning family with an exceedance probability of 76% .",
"The winning model indicates a weak positive intrinsic connection from AG to aIFG during semantic decisions that was reversed to a strong inhibitory influence after cTBS of AG .",
"In contrast , cTBS of SMG did not significantly influence the connection from AG to aIFG ( p=0 . 23 ) .",
"A direct comparison of the parameter estimates for cTBS of AG vs . SMG confirmed that the connection between AG and aIFG was stronger modulated by cTBS of AG than SMG ( t14 = 2 . 92 , p<0 . 026 , paired t-test ) .",
"Importantly , the individual increase in the inhibitory drive from AG to aIFG after cTBS of AG predicted the individual delay in the mean semantic response speed after cTBS ( regression , R2 = 0 . 62 , ß = −0 . 79 , t = 4 . 39 , p=0 . 001 , two-tailed; Figure 5B ) .",
"These results show that the strong remote inhibition effects are behaviourally relevant . 10 . 7554/eLife . 25964 . 011Figure 5 . Effective connectivity in the semantic network .",
"( A ) The winning DCM model assumed modulation of the connection from left AG to aIFG by cTBS of left AG .",
"Mean parameter estimates are given for the significant driving input to SMG , the facilitatory intrinsic connections from AG to aIFG and SMG ( solid arrows ) and the inhibitory modulation of the connection from AG to aIFG by cTBS over AG ( red line ) , ( * ) survived a Bonferoni-Holm correction .",
"( B ) Regression analysis .",
"The increase in the inhibitory influence of AG on aIFG after AG cTBS predicted the individual semantic response delay .",
"( C ) The degree of the individual upregulation of left SMG after cTBS of AG ( effect sizes for AG/sham extracted from the effect of interest at x , y , z= −45 , –40 , 46 ) was significantly correlated with the delay in semantic response speed after AG/sham cTBS . DOI: http://dx . doi . org/10 . 7554/eLife . 25964 . 01110 . 7554/eLife . 25964 . 012Figure 5—figure supplement 1 . Illustration of the different DCM-models . Panel A displays seven models that differ with respect to the driving input regions ( indicated by fat solid arrows ) .",
"Panel B shows nine models with different external modulations by the cTBS conditions ( applied over AG or SMG , indicated by red lines ) .",
"The combination of both model types resulted in a total of 63 models .",
"All models had the same intrinsic connections ( shown as dotted arrows ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 25964 . 01210 . 7554/eLife . 25964 . 013Figure 5—figure supplement 2 . Regression analysis . The individual increase in the inhibitory influence of AG on aIFG after AG / sham cTBS predicted the individual semantic response delay . DOI: http://dx . doi . org/10 . 7554/eLife . 25964 . 01310 . 7554/eLife . 25964 . 014Table 2 . Mean parameter estimates of the winning model for semantic decisionsDOI: http://dx . doi . org/10 . 7554/eLife . 25964 . 014Connection / parameter RightMeanSD TPIntrinsic connectionsAG→aIFG 0 . 02950 . 0315 2 . 240 . 046 AG→SMG 0 . 03140 . 0140 3 . 270 . 006*aIFG→AG 0 . 11000 . 3965 1 . 070 . 30 aIFG→SMG 0 . 05290 . 1535 1 . 330 . 20 SMG→AG 0 . 24240 . 1642 2 . 100 . 056 SMG→aIFG 0 . 04130 . 1195 1 . 340 . 20 Modulation of connectivity from AG aIFG by cTBScTBS of AG−0 . 19000 . 1746−3 . 670 . 003*cTBS of SMG 0 . 01670 . 1471 1 . 270 . 23 Driving InputSMG 0 . 01360 . 0106 2 . 210 . 049 *significant at p<0 . 05; two-tailed; corrected with a Bonferroni-Holm correction for multiple comparisons .",
"To account for the variability in the individual semantic response speed at baseline ( i . e . , after sham cTBS ) , we calculated another regression analysis with the cTBS-induced changes in the individual connectivity strength from AG to aIFG as predictor and the ratio of semantic response speed after AG cTBS vs . sham cTBS ( i . e . , baseline corrected ) as dependent measure .",
"Results were still significant after baseline correction ( R2 = 0 . 48; ß = −0 . 69; t = 3 . 79 , p=0 . 006; two-tailed; see Figure 5—figure supplement 2 ) .",
"We found a significant correlation between the individual upregulation of SMG after cTBS over AG and the cTBS-induced delay in mean semantic response speed ( normalized to sham cTBS , r = 0 . 54 , p=0 . 036 , two-tailed; Figure 5C ) .",
"This result indicates that those subjects who showed a strong disruption of the semantic network displayed a stronger upregulation of the neighbouring SMG .",
"We speculate that this upregulation might have partially compensated for the cTBS-induced perturbation and enabled to maintain task processing .",
"To explore the role of the observed parieto-frontal networks in semantic and phonological processing at baseline , we contrasted each task with rest after sham cTBS .",
"Both tasks engaged widespread parieto-temporo-frontal networks of brain regions previously associated with both processes ( Devlin et al . , 2003; Price et al . , 1997; Vigneau et al . , 2006 ) ( Table 3 and Figure 2—figure supplement 2 ) .",
"Notably , we found increased task-related activity for ( bilateral ) AG and left aIFG during semantic decisions only , while phonological decisions increased task-specific activity in ( bilateral ) SMG and left pIFG ( Figure 2—figure supplement 2A , B ) .",
"A direct contrast of both tasks confirmed the role of AG and left aIFG ( among other regions ) as semantic areas ( Figure 2—figure supplement 2C ) and the contribution of SMG and left pIFG to phonological decisions ( Figure 2—figure supplement 2D ) .",
"This is well in line with previous fMRI studies that found similar regions for the respective contrasts of semantic and phonological decisions ( Devlin et al . , 2003; Price et al . , 1997; Vigneau et al . , 2006 ) . 10 . 7554/eLife . 25964 . 015Table 3 . Changes in task-specific neural activation patterns after sham cTBSDOI: http://dx . doi . org/10 . 7554/eLife . 25964 . 015RegionSideMNI coordinates X , Y , ZTCluster sizeSemantic judgements > restcerebellum ( lobule VIIa ) R 18−82−3510 . 762981angular gyrusL−44−66 2510 . 366144supplementary motor areaR 5 14 5210 . 21354superior frontal gyrusL–12 38 469 . 472900inferior frontal gyrus ( pars orbitalis ) L−48 38−149 . 423200inferior frontal gyrus ( pars triangularis ) L−57 26 104 . 85subclusterthalamusL−18−13 98 . 67135postcentral gyrusL−57−19 256 . 3368angular gyrusR 47−64 258 . 37150middle temporal gyrus ( posterior part ) L−63−43–25 . 29130precuneusL–3−52 164 . 871119Phonological judgements > restsupramarginal gyrus / superior parietal lobeL−48−38 4612 . 84462superior parietal lobeL−27−57 448 . 67subclustersupplementary motor areaR 3 14 4912 . 324936thalamusL−18−12 810 . 5495thalamusR 15−13 1310 . 9398precentral gyrus / posterior inferior frontal gyrus ( pars opercularis ) L−45 5 258 . 765222middle / inferior frontal gyrus ( pars triangularis ) L−48 33 268 . 23subclusteranterior insulaL−30 20 710 . 21326supramarginal gyrus / superior parietal lobeR 54−31 5210 . 22663precentral gyrus / primary motor cortexR 39−22 527 . 53subclusterprecentral gyrusR 36−19 677 . 37subclustercerebellum ( lobule VI ) R 30−64−2910 . 122582cerebellumR 39−52−329 . 92subclustercerebellumL−18−52−239 . 14subclusterinferior temporal gyrusL−51−52−146 . 53154middle frontal gyrusR 45 35 198 . 67345inferior frontal gyrus ( pars opercularis ) R 50 16 46 . 80subclusterpostcentral gyrusL−60−16 225 . 2429Semantic judgements > phonological judgementsinferior frontal gyurs ( pars triangularis ) L−53 26 106 . 68336angular gyrusL−45−67 286 . 21348superior frontal gyrusL–9 59 285 . 88292superior frontal gyrusR 9 41 494 . 95subclusterangular gyrusR 54−64 254 . 92123middle temporal gyrus ( posterior part ) L−63−40–24 . 90139middle temporal gyrus ( anterior part ) L−54–2−234 . 8828Phonological judgements > semantic judgementsinferior frontal gyrus ( pars opercularis ) / frontal operculumL−51 8 47 . 211235supramarginal gyrus / superior parietal lobeL−42−37 406 . 84158cerebellum ( lobule VIIb ) R 21−70−476 . 3497supplementary motor areaM 3 5 616 . 23383supramarginal gyrus / superior parietal lobeR 36−40 376 . 22548middle frontal gyrusL−33 41 255 . 83119inferior frontal gyrus ( pars opercularis ) R 54 11 75 . 67242cerebellum ( lobule VI ) R 18−70−175 . 64254middle frontal gyrusR 33 35 315 . 42166superior temporal gyrusL−60−15 104 . 8535thresholded at p<0 . 05; FWE-corrected at the peak level , cluster extent >20 voxels ."
],
[
"This study demonstrates functionally specific reorganization in the healthy semantic network after focal perturbation of a semantic key region .",
"Our main novel finding was that cTBS over AG significantly decreased task-specific neural activity in a larger network of semantic regions , including the stimulated AG and left aIFG .",
"Moreover , semantic suppression induced upregulation in neighbouring phonological regions that might have partially compensated for the disruptive effect on semantic decisions .",
"In contrast , cTBS over SMG selectively affected phonological decisions , leading to decreased neural activity at the stimulated site and other phonological regions without any upregulation of the semantic network , as well as a significant delay in response speed .",
"The observed functional-anatomical double dissociation during semantic vs . phonological decisions supports the notion that both processes are subserved by different networks ( Hartwigsen et al . , 2016 ) and suggests that these networks might differ in their potential to partially compensate for a focal disruption .",
"The distinct contribution of AG and SMG to semantic and phonological decisions was further supported by their differential engagement in these processes after sham cTBS and by the respective differential task comparisons .",
"Notably , the task-specific perturbation effect in the semantic network was underpinned by a strong increase in the inhibitory influence from AG to aIFG , which predicted an individual delay in semantic performance after cTBS .",
"In particular , individual response speed was prolonged as the inhibitory influence from left AG to aIFG increased .",
"These findings suggest that a focal perturbation over a semantic key region can induce large-scale inhibitory effects on the network level .",
"This provides new insight into the strong modulatory potential of cTBS .",
"Probabilistic fiber tracking revealed that this effect was most probably mediated via anatomical long-distance connections that are part of the ventral association fibre system that was previously associated with semantic processing ( Bajada et al . , 2015 ) .",
"We did not find a significant change in the mean semantic response speed after AG cTBS .",
"However , a trend towards delayed semantic response speed with cTBS over AG indicated that this region contributes to semantic processing .",
"We speculate that the relatively weak effect of AG cTBS on semantic decisions might be explained by the upregulation of the neighbouring phonological network that might have helped to maintain task processing .",
"Indeed , the subject-specific degree of AG suppression after cTBS predicted the individual upregulation of neighbouring SMG .",
"These results suggest that a topographically adjacent , yet functionally separate network may have the potential to partially support processing after disruption of a strategic key region in the language system .",
"This interpretation is in line with our finding that the individual delay in semantic performance was correlated with the upregulation of the SMG after cTBS over AG .",
"We conclude that a stronger individual disruption of the semantic network required a stronger contribution of the SMG to maintain task function .",
"Together , our findings may show the inherent potential of the language system to flexibly recruit neighbouring regions , thus providing new insight in the dynamic regulation of intrahemispheric interactions in two functionally segregated processing streams .",
"Specifically , the differential behavioural effects of cTBS over AG and SMG during semantic and phonological decisions suggest that lower-level resources like phonological working memory capacities ( Nixon et al . , 2004; Romero et al . , 2006 ) might be recruited to partially contribute to higher-level semantic tasks to preserve task processing , but not the other way round .",
"To the contrary , we observed a strong perturbation effect of SMG cTBS on task-related phonological activity and behaviour but no upregulation of semantic regions .",
"As an equally plausible alternative explanation , AG perturbation might have increased the overall task demands , thereby necessitating the contribution of more general executive control regions .",
"Indeed , the upregulation of left SMG after cTBS of AG extended into the intraparietal sulcus ( IPS ) and superior parietal lobe ( SPL ) that were previously associated with executively demanding tasks ( Humphreys et al . , 2015 ) and are part of a multi-domain control system ( Duncan , 2010; Whitney et al . , 2012 ) .",
"Hence , AG perturbation might have increased the overall task demands , thereby requiring the contribution of these regions .",
"In contrast , the strong perturbation effect on the phonological task after cTBS over SMG might be explained by the fact that ( higher-level ) semantic regions were not able to support task processing after disruption of the control network .",
"With respect to the role of the observed regions in semantic processing , both AG and aIFG were associated with executive control over semantic processing , although to a different degree ( Noonan et al . , 2013 ) .",
"Left AG corresponds to a high-level cross-modal convergence zone for concept retrieval and the integration of meaning and event representations and provides access to semantics ( Binder et al . , 2009 ) .",
"In particular , the ventral AG ( overlapping with our stimulation site ) was associated with automatic semantic retrieval , while a more dorsal subregion within the AG was assigned a role in controlled semantic retrieval ( Noonan et al . , 2013 ) .",
"aIFG , on the other hand , is an important node for top-down control during semantic processing and lexical retrieval ( Whitney et al . , 2012 ) .",
"Consequently , the observed network effect in our study might indicate that disruption of AG induced a functional disconnection by impairing the transfer of stimulus related activity from AG to aIFG , thereby necessitating the contribution of more domain-general resources under semantic conditions with increased task difficulty .",
"However , the precise role of the AG in semantic processing remains a conundrum .",
"A recent meta-analysis showed that most semantic tasks deactivate the core AG and rather activate neighbouring intraparietal sulcus ( among other regions ) ( Humphreys et al . , 2015 ) .",
"On the other hand , left AG is clearly engaged by non-semantic domains such as memory and social cognition ( Bzdok et al . , 2016; Humphreys et al . , 2015 ) .",
"Its precise contribution in semantic processing thus remains elusive .",
"In our study , the semantic task might have particularly engaged semantic working memory processes that contributed to the observed positive AG activation ( Vigneau et al . , 2006 ) .",
"We believe that our results are noteworthy for several reasons .",
"First , they implicate that the effects of a focal perturbation are not restricted to the stimulated area itself but might rather affect a larger network ( see Bestmann et al . , 2003 ) .",
"Specifically , our data indicate that remote effects may occur in distant cognitive network nodes .",
"Notably , the strong modulation of task-specific activity patterns in the absence of a significant disruption of the mean semantic response speed after AG cTBS indicates that behavioural measures alone might not be sufficient to map a cTBS-induced perturbation effect .",
"Rather , some modulatory effects induced by non-invasive brain stimulation might only be captured on the neural network level and most likely originate from a modulation of the effective connectivity in a network .",
"These results provide new insight into the neural mechanisms of the perturbation effect .",
"Indeed , our winning model revealed a positive intrinsic connection between AG and aIFG , indicating that without cTBS , there might be a ( weak ) facilitatory connectivity between both regions that turned into a strong inhibition after cTBS .",
"The individual magnitude of the induced inhibitory drive between AG and aIFG was able to predict the individual delay in semantic response speed after AG cTBS , demonstrating the functional relevance of this effect .",
"Secondly , the observed upregulation of a neighbouring parieto-frontal network for phonological processing might indicate some degree of degeneracy ( Price and Friston , 2002 ) in the language network that might have partly enabled cross-modal compensation even after inhibition of a large part of the semantic network .",
"This is compatible with the notion that cTBS may give rise to an acute adaptive reorganization within the non-targeted functional loops of another network to compensate for the cTBS-induced suppression of task-specific neuronal activity ( Hartwigsen and Siebner , 2012 ) .",
"The observed greater resilience of the semantic relative to the phonological network against the behavioural lesion effect was underpinned by a difference in the flexible recruitment of neighbouring networks that was selectively observed for the semantic task .",
"These findings are in line with previous behavioural results that a single perturbation of one semantic node did not affect semantic decisions , while phonological decisions were already impaired after perturbation of one key region ( Hartwigsen et al . , 2016 ) .",
"The difference in the flexible recruitment of neighbouring networks for semantic and phonological processes likely reflects the engagement of higher- vs . lower-level processes in both tasks .",
"Indeed , our optimal DCM identified the SMG as the most likely source of the driving input among the selected regions .",
"This indicates that left AG receives information from the neighbouring SMG , which is compatible with the above-discussed role of the AG as a heteromodal integration area ( Seghier , 2013 ) .",
"This might explain the absence of any upregulation of the semantic system during phonological processing after cTBS over SMG , as processing was already disrupted at a lower level .",
"Interestingly , disruption of this lower-level region was not sufficient to significantly affect semantic task processing .",
"We initially hypothesized that the virtual lesion of the AG might lead to an upregulation of other semantic key nodes .",
"However , this is not consistent with the observed strong remote effects of cTBS .",
"It is worth noting that few imaging studies have investigated short-term plasticity on the neural-network level in the healthy language network to date ( Binney and Ralph , 2015; Hallam et al . , 2016; Hartwigsen et al . , 2013; Jung and Lambon Ralph , 2016 ) .",
"Some of these studies ( Binney and Ralph , 2015; Hartwigsen et al . , 2013; Jung and Lambon Ralph , 2016 ) reported increased task-related activity of the right-hemispheric homologue after perturbation of a left-hemispheric key region for speech or language that was interpreted as adaptive short-term compensation .",
"Another study ( Hallam et al . , 2016 ) found that perturbation of left IFG increased the activation of left pMTG during the processing of weak semantic associations , demonstrating that compensation can also occur between different semantic nodes within the same left hemispheric network .",
"Moreover , Jung and Lambon Ralph , 2016 showed that after cTBS over left ATL , the right ATL increased its intrinsic facilitatory influence on left ATL , indicating a flexible , bilateral organization of the semantic system with a strong degree of adaptive plasticity .",
"In contrast to the above cited studies , we did not observe an upregulation of other left-hemispheric semantic areas or homologous right-hemispheric regions .",
"When lowering the threshold , neural activity in right AG was also decreased after cTBS of left AG , indicating a strong cTBS-induced inhibition in the semantic network .",
"This suggests that left AG is a strategic core region for semantic processes , a notion that is compatible with the key role of the AG in all aspects of semantic processing that require concept retrieval ( Binder et al . , 2009; Seghier , 2013 ) , including efficient retrieval of semantic information ( Davey et al . , 2015 ) .",
"Together , the previous findings and our results demonstrate the potential for a flexible recruitment after disruption of a semantic key node that may include regions within the same left-hemispheric network , as well as contralateral homologous regions or neighbouring regions from a different network .",
"The exact recruitment may depend on the degree of the disruption and the specific task demands .",
"Our findings have implications for the interpretation of reorganization effects in the lesioned language network .",
"The upregulation of neighbouring regions outside the core semantic network shows that aside from the specific contributions of a specialized network , neighbouring networks might also be beneficially recruited .",
"In this context , our finding of a positive intrinsic connection between AG and neighbouring SMG during semantic processing argues against the possible alternative explanation of the observed upregulation of SMG in terms of disinhibition .",
"Our findings are more compatible with the notion of an adaptive contribution of ipsilateral regions after a lesion of a strategic left-hemispheric area ( Heiss and Thiel , 2006 ) , rather than supporting the concept of maladaptive plasticity after a release from the inhibition of neighbouring regions .",
"Indeed , previous studies on post-stroke aphasia suggest that the upregulation of ipsi- vs . contralateral regions after stroke might depend on the site , size and extent of the lesion ( Chrysikou and Hamilton , 2011 ) .",
"Models of language recovery proposed that for smaller lesions of critical left-hemispheric language nodes , neighbouring perilesional regions might be adaptively recruited to subserve language function while more severe impairments might stronger draw on recruitment of homotopic regions ( Hamilton et al . , 2011 ) .",
"Our results show that even a relatively focal perturbation can already inhibit neural activity in a large network .",
"In our case , the recruitment of neighbouring areas seems to be related to a high level of adaptation ( i . e . , the absence of a strong behavioural effect ) , indicating a general flexibility of brain networks in terms of distributed processing .",
"In summary , our results shed important new light on the dynamic regulation of intrahemispheric interactions in the healthy human brain that are highly relevant for a variety of cognitive processes .",
"This is of potential relevance for understanding language recovery after left hemisphere stroke , indicating that neighbouring networks might bear the inherent potential to partially support task processing after a strategic lesion of one key region .",
"Our results help to unravel neural mechanisms of perturbation effects , indicating that cTBS not only influences neural activity in the stimulated region but also in remote areas .",
"Although the observed effects were transient , they were sufficient to influence task-related activity , connectivity , and behaviour , as evidenced by the significant correlation between individual connectivity strength and delay in response speed .",
"This demonstrates the value of combining TMS with effective connectivity analyses of fMRI data to map the neural consequences of a focal perturbation and might further inform models of language reorganization in post-stroke aphasia ."
],
[
"15 native , right-handed German speakers ( seven females , age range: 20–30 years ) with no history of neurological disorders or cTBS contraindications participated in our study .",
"All subjects were included in the final analyses .",
"Written informed consent was obtained before the experiment .",
"The study was performed according to the guidelines of the Declaration of Helsinki and approved by the local ethics committee ( Medical Faculty at the University of Leipzig ) .",
"We used a two ( task: semantic vs . phonological decisions ) by three ( cTBS: effective cTBS of AG or SMG and sham cTBS ) factorial within-subject design ( Figure 1 ) .",
"In three sessions ( inter-session interval >7 days ) , we applied effective or sham cTBS over left AG or SMG prior to fMRI ( Figure 1A ) .",
"Each fMRI session was divided into two runs with a short break .",
"During fMRI , subjects performed semantic ( ‘natural or manmade ? ' ) and phonological tasks ( ‘two or three syllables ? ' ) on auditorily presented words that were presented in short miniblocks of 6 items each ( Figure 1B ) .",
"Tasks were held constant within each miniblock and pseudorandomized across blocks such that no more than two blocks of the same task followed each other .",
"We used similar items for both tasks that were pseudorandomized across conditions and counterbalanced across subjects ( to the degree possible ) .",
"Each session consisted of 10 miniblocks of each task , leading to a total presentation of 120 stimuli per session ( 60 for each task ) .",
"Miniblocks were separated with 16 s rest periods .",
"At the end of each rest block , task instruction was auditorily presented .",
"Subjects were instructed to respond as quickly and accurately as possible by pressing a button on a response pad with their left middle or index finger .",
"The stimuli were taken from a previous study ( Hartwigsen et al . , 2010 ) .",
"Only highly frequent , unambiguous nouns from the CELEX lexical database for German ( Centre for Lexical Information , Max Planck Institute for Psycholinguistics , The Netherlands ) were selected .",
"In total , 60 two-syllable nouns and 60 three-syllable nouns were selected .",
"All words represented natural or manmade items ( i . e . , 50% of the two-syllable words and 50% of the three-syllable words , respectively ) and had a mean stimulus duration of 0 . 78 s ( range = 0 . 74–0 . 87s ) .",
"As a prerequisite for neuronavigated cTBS , all subjects first underwent structural MR imaging ( Siemens Verio 3-Tesla scanner; Siemens , Erlangen , Germany ) .",
"This included a high-resolution T1 weighted anatomical scan for each subject ( MPRAGE; 170 slices , voxel size = 1 × 1 × 1 . 5 mm , matrix = 240 × 240 pixel , TR = 1 . 3 s , TE = 3 . 46 ms ) .",
"Functional imaging was performed using a gradient EPI sequence ( repetition time [TR]/echo time [TE] = 2520/30 msec , flip angle = 90° , matrix = 64 × 64 pixel , voxel size = 3 × 3 x 3 mm; field of view = 192 mm ) with BOLD contrast for the acquisition of T2*-weighted images .",
"A total of 370 volumes consisting of 38 slices was acquired continuously during each run in descending order .",
"Finally , we acquired axial whole brain diffusion weighted images with a double spin echo sequence for probabilistic fiber tracking ( 60 directions; b-value = 1000 s/mm2; 88 slices; voxel size , 1 . 7 × 1 . 7 × 1 . 7 mm , no gap; TR = 12 . 9 s; TE = 100 ms; field of view = 220 × 220 mm2 ) plus seven volumes with no diffusion weighting ( b-value = 0 s/mm2 ) at the beginning of the sequence and interleaved after each block of 10 diffusion weighted images .",
"We used neuronavigated cTBS ( Brainsight; Rogue Research ) based on coregistered individual T1-weighted MRI images to navigate the TMS coil and maintain its exact location and orientation throughout all sessions .",
"Session order ( AG , SMG or sham cTBS ) was counterbalanced across subjects to the best possible degree .",
"cTBS was performed using the mean Montreal Neurological Institute ( MNI ) coordinates for the left AG ( x , y , z= −42 , –66 , 28 mm ) and SMG ( x , y , z= −45 , –39 , 45 mm ) described in our previous study ( Hartwigsen et al . , 2010 ) .",
"Using these stereotactic coordinates , the individual stimulation sites were determined by calculating the inverse of the normalization transformation and transforming the coordinates from standard to individual space for each subject .",
"The TMS coil was positioned with the handle pointing lateral and perpendicular to the midline over left SMG or AG , with the second phase of the biphasic pulse inducing a lateral to medial current flow ( Hartwigsen et al . , 2010 ) .",
"We applied cTBS ( 600 stimuli at 50 Hz in trains of three stimuli at an inter-burst interval of 200 ms for 40 s ) at a stimulation intensity of 80% of the individual active motor threshold ( AMT ) .",
"AMT was defined as the lowest stimulus intensity producing a motor evoked potential of 150–200 μV in the tonically active first dorsal interosseus muscle ( 20% of maximum contraction ) .",
"A figure-of-eight-shaped coil ( double 60 mm; coil type CB-60 ) connected to a MagPro X100 stimulator ( MagVenture , Farum , Denmark ) was used in all cTBS conditions .",
"For sham cTBS , we used a specially designed figure-of-eight shaped placebo coil ( MCF-P-B-70; outer diameter 7 . 5 cm ) that creates a sound level identical to the CB-60 coil but provides an effective field reduction of 80% .",
"Half of the subjects received sham cTBS over AG and SMG , respectively .",
"The overall application of TMS pulses per sessions was well within safety limits .",
"Task-related changes in the blood oxygenation level-dependent signal were analyzed with SPM 8 and 12 ( Wellcome Trust Centre for Neuroimaging; www . fil . ion . ucl . ac . uk/spm/ ) implemented in Matlab 8 . 1 ( The Mathworks , Inc . , Natick , MA ) .",
"Preprocessing of the fMRI data included slice timing , data realignment , coregistration of the individual T1-weighted and mean functional EPI images , segmentation , normalization into standard space ( Montreal Neurological Institute ( MNI ) template ) and smoothing with an isotropic 8 mm FWHM Gaussian kernel .",
"Statistical analyses of the functional images were performed in two steps .",
"The individual first level included regressors for each task condition , the instruction and the realignment parameters .",
"All onsets in each regressor were convolved with a canonical hemodynamic response function as implemented in SPM8 .",
"Voxel-wise regression coefficients for all conditions were estimated using the least squares method within SPM8 , and statistical parametric maps of the t statistic ( SPM{t} ) were generated from each condition ( i . e . , main effects of tasks under the different cTBS conditions ) .",
"The data for the second stage of analysis comprised pooled parameter estimates for each of these contrasts across all participants in a random-effects analysis using a flexible factorial within-subject ANOVA design including a correction for nonsphericity .",
"We used the Restricted Maximum Likelihood method in the SPM8 design specification for sphericity correction at the second-level inference .",
"All comparisons were thresholded at a significance level of p<0 . 05 , FWE-corrected at the peak level .",
"The SPM anatomy toolbox ( Version 1 . 7 ) and the WFU PickAtlas Tool ( Version 2 . 4 Wake Forest University of School of Medicine ) were used for anatomical localization of activation peaks .",
"To investigate the modulatory effects of cTBS in the semantic network , we employed effective connectivity analyses using dynamic causal modeling ( DCM ) .",
"DCM allows for investigation of the interaction of a predefined set of brain regions in different experimental contexts .",
"The strength and direction of regional interactions are computed by comparing the observed regional blood oxygenation level-dependent ( BOLD ) responses with the BOLD responses predicted by a neurobiologically plausible model .",
"The model describes how activity in , and interactions among , regional neuronal populations are modulated by external inputs ( i . e . , the experimental task conditions ) , as well as how the ensuing neuronal dynamics translate into the measured BOLD signal ( Stephan et al . , 2010 ) .",
"Three types of parameters are calculated: the direct influences of the external input or stimuli on regional activity ( called driving input ) , the strength of the intrinsic connections between two regions in the absence of modulating experimental effects , and the changes in the intrinsic connectivity between regions induced by the experimental design ( i . e . , the modulatory effects of our cTBS conditions , including AG and SMG stimulation ) .",
"The semantic network of interest included both cTBS sites ( AG , SMG ) and the aIFG .",
"The model space for each task thus included 63 different models with full intrinsic connectivity ( Figure 5—figure supplement 1 ) .",
"The driving input for our DCM analysis was set to either of the three regions of interest alone and to all possible combinations between these regions ( Figure 5—figure supplement 1A ) and the modulatory effects of cTBS over AG or SMG on the connections between these regions were specified for each subject ( Figure 5—figure supplement 1B ) .",
"The seed areas were derived from the comparison of SMG and AG cTBS for each task individually for each subject ( mean coordinates for AG: x , y , z = −45 , –67 , 28; SMG: x , y , z= −45 , –40 , 46; aIFG: x , y , z = − −48 , 41 , –14 ) .",
"We first specified a new design matrix at the individual level that modelled the conditions of interest for the DCM analysis ( i . e . , one regressor modeling semantic judgements after cTBS over both AG and SMG , one regressor including only semantic judgements after cTBS of AG , and one regressor including only semantic judgements after cTBS of SMG ) .",
"Subsequently , we extracted the first eigenvariate of the fMRI signal for our seed regions at the individual level at a liberal threshold of p<0 . 01 uncorrected within a sphere of 8 mm around the group coordinates derived from the effect of interest .",
"These data were included in the model specification for our DCMs .",
"For model selection across subjects , we used a random-effects Bayesian model selection procedure ( Stephan et al . , 2010 ) to identify the most likely model among the candidate models given the observed fMRI data .",
"In this procedure , all models are tested against each other .",
"The models’ probabilities are estimated , and inference is based on exceedance probabilities , reflecting the probability that this model is a better fit to the data than any other model of those tested .",
"Finally , subject-specific estimates for the parameters of interest in the winning model ( i . e . , the driving input , the strength of the intrinsic connections between the seed regions , and the impact of the modulation on the relevant connections by cTBS of AG or SMG ) were entered into Bonferroni-corrected two-sided one-sample t-tests to test differences from zero .",
"We additionally performed a family-level inference based on model space partitioning ( Stephan et al . , 2010 ) to identify the most likely winning family .",
"Accordingly , models were grouped based on the cTBS-induced modulation of the connection between regions .",
"The family with the highest exceedance probability value again represents the model group that is most plausible given the data ( Stephan et al . , 2010 ) .",
"Diffusion weighted data sets were analyzed with the Leipzig Image Processing and Statistical Inference Algorithms ( https://www . cbs . mpg . de/institute/software/lipsia ) and the FMRIB's diffusion toolbox ( FDT , Oxford Centre for Functional Magnetic Resonance Imaging of the Brain Diffusion Toolbox , Version 5 . 0 .",
"Preprocessing of the data included motion correction based on the 7 b0 images , global registration to the T1 anatomy and averaging of gradient directions resulting in an isotropic voxel resolution of 1 mm .",
"Subsequently , a diffusion model was fitted to the preprocessed data with Bayesian estimation of diffusion parameters obtained using sampling techniques ( Behrens et al . , 2007 ) .",
"8 mm spheres ( seed masks ) were created around the mean peak activation derived from the fMRI group contrasts of semantic decisions after AG cTBS vs . SMG cTBS ( AG: x , y , z= −45 , –67 , 28; aIFG: x , y , z= −48 , 41 , –14 ) and transformed to the individual diffusion space .",
"Crossing fibre probabilistic tractography was performed based on each participant’s probability distributions of voxel-wise principal diffusion directions applying region-to-region connectivity ( Behrens et al . , 2007 ) .",
"Region-to-region connectivity between the two seeds was assumed if repeatedly generated streamlines that started from one seed were passing through the other seed , that is , we picked those streamlines that passed through both ROIs in the sense of a constrained tractography .",
"Finally , the estimated connectivity distributions were thresholded based on the total number of samples sent out that reached the respective other seed mask without being rejected .",
"To remove spurious connections , individual tractography results were binarized at 1 × 10−2 threshold percentage .",
"These tracts were transformed into standard MNI space , and overlaid across subjects ( threshold: > 8 out of 15 subjects ) using FSLView .",
"Anatomical pathway definition was based on white matter atlases ( Juelich Histological atlas and JHU White-Matter Tractography atlas distributed with FSL ) and on the literature ( Saur et al . , 2008 ) ."
]
] | [
"The adaptive potential of the language network to compensate for lesions remains elusive .",
"We show that perturbation of a semantic region in the healthy brain induced suppression of activity in a large semantic network and upregulation of neighbouring phonological areas .",
"After perturbation , the disrupted area increased its inhibitory influence on another semantic key node .",
"The inhibitory influence predicted the individual delay in response speed , indicating that inhibition at remote nodes is functionally relevant .",
"Individual disruption predicted the upregulation of semantic activity in phonological regions .",
"In contrast , perturbation over a phonological region suppressed activity in the network and disrupted behaviour without inducing upregulation .",
"The beneficial contribution of a neighbouring network might thus depend on the level of functional disruption and may be interpreted to reflect a differential compensatory potential of distinct language networks .",
"These results might reveal generic mechanisms of plasticity in cognitive networks and inform models of language reorganization ."
] | [
"Taking part in a conversation requires us to extract meaning from a complex series of sounds by recognising words and phrases .",
"We then need to decide on a response , and plan and execute the lip and tongue movements necessary to generate that response .",
"Each of these processes – from analysing the meaning of words to producing speech – requires a distinct set of brain regions to work together .",
"However , we know relatively little about how these regions interact with one another during specific language processes , or about what happens when key regions are damaged .",
"Hartwigsen et al . have now used a technique called TMS in healthy volunteers to temporarily disrupt the activity of individual brain regions with a role in language .",
"TMS involves applying small magnetic fields to the scalp over a target brain area .",
"The magnetic fields induce electrical currents in the underlying brain tissue and temporarily scramble its activity .",
"Hartwigsen et al . examined how this affected the volunteers’ performance on a language task , as well as the activity of other language areas .",
"Temporarily disrupting a single brain region involved in analysing the meaning of words reduced the activity of multiple other areas in the brain’s language networks .",
"The greater this reduction in activity , the more poorly the volunteers performed on a language task .",
"However , not all brain regions showed reduced activity .",
"Areas adjacent to the disrupted region , which normally process the sounds of words , increased their activity .",
"This increase may have partially compensated for the effects of the TMS to help limit the language impairments .",
"These findings offer insights into how the brain reacts and adapts when key language areas are damaged , for example as a result of stroke .",
"The next step is to determine the extent to which healthy brain tissue can take over from damaged regions , and whether we can target this process to improve recovery after stroke ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Prenatal methadone exposure disrupts behavioral development and alters motor neuron intrinsic properties and local circuitry | elife-66230-v2 | [
[
"Pregnant women and their developing fetuses represent a vulnerable population severely impacted by the opioid crisis .",
"From 1999 to 2014 , the annual prevalence of opioid use disorder ( OUD ) in pregnant women at delivery increased 333% ( Haight et al . , 2018 ) .",
"Mirroring this rise , neonatal opioid withdrawal syndrome ( NOWS ) increased from 1 . 2 to 5 . 8 per 1000 hospital births from 2000 to 2012 with some geographical regions near 33 cases per 1000 ( Ko et al . , 2016; Patrick et al . , 2015 ) .",
"Opioid maintenance therapies , such as methadone and buprenorphine , continue to represent the first-line treatments for pregnant women with OUD because these therapies benefit overall maternal-fetal outcomes at parturition ( ACOG , 2017 ) .",
"However , it is unknown exactly how prenatal methadone exposure ( PME ) impacts the brain and behavioral development as these infants mature .",
"Prenatal opioid exposure is associated with smaller head circumferences ( Towers et al . , 2019 ) , lower brain volumes ( Hartwell et al . , 2020; Sirnes et al . , 2017 ) , and deficits in white matter microstructure ( Monnelly et al . , 2018 ) , although these studies are limited by small sample sizes making it difficult to control for confounding variables .",
"A recent , large study using data from the Adolescent Brain Cognitive Development study reported reduced motor cortical volumes and surface area in children with prenatal opioid exposure ( Hartwell et al . , 2020 ) .",
"These differences remained significant when controlling for multiple additional factors ( e . g . socioeconomic factors and prenatal alcohol/tobacco ) , indicating opioid exposure in utero may specifically disrupt motor cortex development and potentially impact motor behavior ( Hartwell et al . , 2020 ) .",
"Prior studies revealed poorer motor performance in children exposed to opioids prenatally suggesting that prenatal opioid exposure produces long-lasting changes in both motor behavior and in brain regions associated with motor functioning ( Lee et al . , 2020; Yeoh et al . , 2019 ) .",
"However , clinical studies are often complicated by significant environmental variations which are difficult to control such as the extent of prenatal care or additional prenatal substance exposures which also impact neurodevelopmental outcomes of children ( Larson et al . , 2019 ) .",
"Therefore , a translationally relevant animal model of prenatal opioid exposure is desperately needed to examine the neurological and behavioral consequences of opioid exposure in the absence of confounding variables .",
"Animal models of prenatal opioid exposure have demonstrated deficits in several sensorimotor milestones ( Kunko et al . , 1996; Robinson et al . , 2020; Wallin et al . , 2019 ) , but the mechanisms underlying disrupted sensorimotor development are unclear .",
"Preclinical studies suggest that prenatal opioid exposure may prevent normal neuronal development ( Lu et al . , 2012; Ricalde and Hammer , 1990 ) and disturb myelination ( Jantzie et al . , 2020 ) , which could underlie the aberrant neurobehavioral development .",
"Unfortunately , the translational value of these rodent studies is limited by models that do not adequately model the majority of human prenatal opioid exposure cases .",
"For instance , a large proportion of studies initiate opioid delivery around mid-gestation or later and often utilize morphine , even though methadone and buprenorphine account for a growing majority of prenatal opioid exposure cases ( Byrnes and Vassoler , 2018; Duffy et al . , 2018 ) .",
"Initiating opioid exposure at later stages of pregnancy overlooks the effect of opioids on earlier embryonic developmental processes .",
"These weaknesses underlie the call for improved animal models of prenatal opioid exposure , which encompass all stages of prenatal brain development ( Larson et al . , 2019 ) .",
"We developed a more translational mouse model that resembles the typical pattern of opioid use in a pregnant woman who is first dependent on oxycodone , then begins methadone maintenance treatment , and subsequently becomes pregnant while maintained on methadone .",
"We found that PME reduces physical growth in offspring which persists into adolescence and disrupts the development of locomotor activity and ultrasonic vocalization ( USV ) during the preweaning period .",
"Furthermore , PME delays the development of specific sensorimotor milestones .",
"These impairments were concurrently associated with alterations in layer 5 motor cortical neuron intrinsic properties and local connectivity ."
],
[
"An overview of model development and workflow representing the total animals used for these studies is found in Figure 1 .",
"Table 1 summarizes pregnancy and litter characteristics .",
"Opioid treatment did not reduce pregnancy rates ( chi square test: χ2 ( 1 , n=95 ) =0 . 904 , p=0 . 34 ) or instances of obstructed labor ( chi square test: χ2 ( 1 , n=70 ) =2 . 14 , p=0 . 14 ) .",
"Litter sizes at birth ( unpaired t test: t61 = 1 . 36 , p=0 . 18 ) and total neonatal deaths ( chi square test: χ2 ( 1 , n=406 ) =0 . 201 , p=0 . 65 ) were not affected by opioid treatment .",
"There was a trend for fewer PME males surviving to P7 ( chi square test: χ2 ( 1 , n=336 ) =2 . 71 , p=0 . 10 ) .",
"However , the proportion of sexes at birth between treatment groups was not significantly different ( chi square test: χ2 ( 1 , n=284 ) =0 . 010 , p=0 . 92 ) suggesting the reduction of male offspring at P7 may be attributed to the greater postnatal deaths in males relative to females in PME litters ( chi square test: χ2 ( 1 , n=37 ) =4 . 79 , p=0 . 03 ) .",
"Methadone and 2-ethylidene-1 , 5-dimethyl-3 , 3-diphenylpyrrolidine ( EDDP , main metabolite of methadone ) concentrations in the placenta , plasma , and brain for dams and offspring on G18 , P1 , and P7 are presented in Supplementary file 1 and Figure 2a .",
"Maternal brain and plasma levels were highest at G18 ( 248 . 9 ± 54 . 1 ng/g and 63 . 5 ± 11 . 3 ng/mL , respectively ) and lower slightly after giving birth , remaining relatively stable at P1 and P7 .",
"Methadone accumulated in the brain relative to the plasma for dams at all timepoints ( Figure 2a ) .",
"The placenta significantly retained methadone ( 3862 . 1 ± 258 . 4 ng/g ) and EDDP ( 1124 . 0 ± 84 . 4 ng/g; Figure 2a , b ) .",
"The fetal brain methadone concentration was 2100 . 8 ± 237 . 6 ng/g which was substantially higher than the maternal brain on G18 ( Figure 2a ) .",
"Offspring brain levels dropped precipitously to 7 . 9 ± 0 . 6 ng/g and 3 . 1 ± 0 . 3 ng/g on P1 and P7 , respectively .",
"Although we were not able to collect a sufficient plasma volume on G18 , minimal methadone plasma levels in offspring were quantified postnatally .",
"Placental levels were predictive of fetal brain methadone levels at G18 ( R2 = 0 . 39 , p<0 . 0078; Figure 2c ) .",
"Offspring plasma methadone was predictive of offspring brain methadone at P1 ( R2 = 0 . 45 , p<0 . 0084; Figure 2d ) , but not on P7 ( R2 = 0 . 008 , p=0 . 74; Figure 2e ) .",
"The opioid treatment strategy did not significantly impact maternal weights during the course of the study ( rmANOVA: Treatment , F ( 1 , 42 ) =0 . 00117 , p=0 . 97; Time , F ( 2 . 92 , 122 . 4 ) = 266 . 1 , p<0 . 0001; Interaction , F ( 22 , 924 ) =0 . 510 , p=0 . 97; Figure 3a ) .",
"Measures of food consumption revealed a significant effect of opioid treatment ( rmANOVA: Treatment , F ( 1 , 33 ) =5 . 09 , p=0 . 03; Time , F ( 7 , 231 ) =413 , p<0 . 0001; Interaction , F ( 7 , 231 ) =2 . 48 , p=0 . 02; Figure 3b ) with an increased food consumption during the postnatal period in opioid-treated dams reaching significance on postnatal week 2 ( Sidak’s post-hoc test , p=0 . 004 ) .",
"Maternal care-related behavior on P3 was assessed to examine the potential impact of methadone on offspring care .",
"There were no apparent differences in nest quality ( Mann-Whitney test: U = 53 , p=0 . 15; Figure 3c ) or average latency to retrieve pups removed from the nest ( Mann-Whitney test: U = 77 , p=0 . 97; Figure 3d ) .",
"No placentas remained in the cages 24 hr after birth in either group indicating normal placentophagy .",
"Oxycodone treatment prior to gestation ( PG14-PG6; see Figure 1a ) induced opioid dependency in dams as demonstrated by the significantly increased withdrawal behaviors following naloxone treatment ( Figure 3e ) .",
"Offspring from mothers that were assessed for oxycodone dependency were not used in subsequent analyses to avoid any impact that pregestational opioid withdrawal could have on offspring outcomes .",
"Methadone maintained opioid dependency as evidenced by the significantly increased naloxone-precipitated withdrawal behaviors observed in opioid-treated females at post-weaning ( ANOVA: Treatment , F ( 1 , 26 ) =38 . 1 , p<0 . 0001; Stage , F ( 1 , 26 ) =0 . 361 , p=0 . 55; Interaction , F ( 1 , 26 ) =0 . 337 , p=0 . 56; Figure 3e ) .",
"The clinical evidence for the effect of prenatal opioid exposure on fetal growth has revealed mixed findings ( Yazdy et al . , 2015 ) ; therefore , we first examined the impact of PME on offspring physical development .",
"As there were no significant effects of sex , the data on weight and lengths during the pre-weaning period were pooled .",
"PME significantly reduced weights and reduced weight gain during the pre-weaning period ( rmANOVA: Exposure , F ( 1 , 111 ) =3 . 96 , p=0 . 049; Time , F ( 1 . 344 , 149 . 2 ) = 3177 , p<0 . 0001; Interaction , F ( 6 , 666 ) =2 . 89 , p=0 . 0087; Figure 4a ) .",
"The reduced body weight in PME offspring persisted into adolescence as weights remained consistently lower across sexes at P35 and P49 ( rmANOVA: Exposure , F ( 1 , 50 ) =7 . 52 , p=0 . 0085; Sex , F ( 1 , 50 ) =131 , p<0 . 0001 , Time , F ( 1 , 50 ) =673 , p<0 . 0001; Time x Sex , F ( 1 , 50 ) =62 . 3 , p<0 . 0001; Time x Exposure , F ( 1 , 50 ) =1 . 60 , p=0 . 21; Exposure x Sex , F ( 1 , 50 ) =0 . 00956 , p=0 . 92; Time x Exposure x Sex , F ( 1 , 50 ) =0 . 20 , p=0 . 66; Figure 4a ) .",
"Similarly , body length was reduced by PME ( rmANOVA: Exposure , F ( 1 , 111 ) =12 . 24 , p=0 . 0007; Time , F ( 2 . 676 , 297 . 0 ) = 4719 , p<0 . 0001; Interaction , F ( 6 , 666 ) =0 . 987 , p=0 . 433; Figure 4b ) .",
"As chronic opioid use is associated with bone pathology ( Mattia et al . , 2012 ) , we collected femurs from P7 and P35 offspring to assess bone structure and density .",
"Although femur length was not impacted by PME ( unpaired t test: t23 = 1 . 126 , p=0 . 27; Figure 4—figure supplement 1a ) , whole bone volume tended to be lower in P7 PME offspring ( unpaired t test: t23 = 1 . 89 , p=0 . 072; Figure 4—figure supplement 1b ) .",
"By P35 , structural bone measures were similar between exposure groups including distal femur metaphysis bone volume ( ANOVA: Exposure , F ( 1 , 16 ) =1 . 94 , p=0 . 18; Sex , F ( 1 , 16 ) =13 . 4 , p=0 . 0021; Interaction , F ( 1 , 16 ) =2 . 17 , p=0 . 16; Figure 4—figure supplement 1c ) , trabecular bone volume ( ANOVA: Exposure , F ( 1 , 16 ) =1 . 30 , p=0 . 27; Sex , F ( 1 , 16 ) =25 . 7 , p=0 . 0001; Interaction , F ( 1 , 16 ) =0 . 126 , p=0 . 73; Figure 4—figure supplement 1d ) , cortical bone area ( ANOVA: Exposure , F ( 1 , 16 ) =0 . 000 , p=0 . 99; Sex , F ( 1 , 16 ) =13 . 4 , p=0 . 0021; Interaction , F ( 1 , 16 ) =2 . 04 , p=0 . 17; Figure 4—figure supplement 1e ) , and cortical thickness ( ANOVA: Exposure , F ( 1 , 16 ) =0 . 0435 , p=0 . 84; Sex , F ( 1 , 16 ) =19 . 0 , p=0 . 0005; Interaction , F ( 1 , 16 ) =0 . 790 , p=0 . 39; Figure 4—figure supplement 1f ) .",
"A recent report suggested opioid-exposed infants have a higher prevalence of orofacial clefting which may reflect an effect of prenatal drug exposure on craniofacial development ( Mullens et al . , 2019 ) .",
"However , we did not observe any effect on the day both eyes opened ( unpaired t test: t49 = 1 . 67 , p=0 . 10 ) , both ears moved to their final erect position and external auditory canals were patent ( unpaired t test: t49 = 0 . 910 , p=0 . 37 ) , or when bottom incisor teeth erupted ( unpaired t test: t49 = 1 . 05 , p=0 . 30; Figure 4—figure supplement 2 ) indicating measures of mouse postnatal craniofacial development progress normally in our PME model .",
"As methadone levels rapidly dropped from G18 to P1 in offspring , we sought to determine if PME offspring demonstrated behaviors consistent with NOWS such as hyperthermia or myoclonic jerks ( twitching or jerking of the limbs or whole-body ) which are indicators of NOWS in humans ( Kocherlakota , 2014 ) .",
"PME offspring displayed a relative hyperthermia at baseline that was maintained after two minutes of isolation ( rmANOVA: Exposure , F ( 1 , 48 ) =34 . 1 , p<0 . 0001; Time , F ( 1 , 48 ) =577 , p<0 . 0001; Interaction , F ( 1 , 48 ) =0 . 127 , p=0 . 723; Figure 5a ) .",
"A higher number of twitches/jerks were observed in P1 PME offspring ( unpaired t test: t49 = 2 . 04 , p=0 . 047; Figure 5b ) reminiscent of the myoclonic jerks which are common among human neonates experiencing NOWS from methadone ( Kocherlakota , 2014 ) .",
"In association with a rapid drop in brain methadone levels from G18 to P1 , the relative hyperthermia and increased twitches/jerks suggests PME offspring may experience opioid withdrawal at P1 .",
"Preclinical studies have indicated locomotor activity may be different in prenatal opioid-exposed offspring ( Andersen et al . , 2020 ) ; however , no studies to date have examined the trajectory of locomotor development during early life in opioid-exposed offspring .",
"Using an open field , we measured the development of motor activity by repeatedly testing animals on P1 , P7 , P14 , and P21 .",
"Locomotor activity was significantly altered during the pre-weaning period in PME offspring ( rmANOVA: Exposure , F ( 1 , 49 ) =5 . 18 , p=0 . 027; Time , F ( 1 . 38 , 67 . 84 ) = 299 , p<0 . 0001; Interaction , F ( 3 , 147 ) =10 . 5 , p<0 . 0001 ) with PME offspring demonstrating significantly reduced activity at P1 but greater activity at P7 and P21 ( Sidak’s post hoc test: p=0 . 048 , p=0 . 037 , p=0 . 009 , respectively; Figure 5c ) .",
"Inspection of the P1 videos indicated the reduction in motor activity was likely a result of poor coordination as PME displayed a greater propensity to fall over earlier in the testing session .",
"Although 88% of PME and 80% of PSE offspring fell over by the end of five-minute session on P1 ( chi square test: χ2 ( 1 , n=51 ) =0 . 690 , p=0 . 41; Figure 5—figure supplement 1a ) , 53 . 5% of PME compared to 28% of PSE animals had fallen over immediately upon placement into the arena ( chi square test: χ2 ( 1 , n=51 ) =0 . 30 . 52 , p=0 . 061; Figure 5—figure supplement 1b ) .",
"There was a nonsignificant reduction in the latency to fall over in PME offspring ( Mann-Whitney test: U = 248 . 5 , p=0 . 136; Figure 5—figure supplement 1c ) .",
"In addition to the hyperactivity on P21 , PME offspring exhibited significantly more vertical jumps at P21 ( Mann-Whitney test: U = 53 , p<0 . 0001; Figure 5d ) further indicating a hyperactive phenotype is present in juvenile PME offspring similar to what has been reported in opioid-exposed children ( Azuine et al . , 2019; Sundelin Wahlsten and Sarman , 2013 ) .",
"These jumps may represent escape attempts as some animals were able to jump and reach the top of the arena walls .",
"When this occurred , the animal was outside of the frame of video and our tracking software was not able to record activity .",
"Therefore , the total distance traveled at P21 in the PME animals may actually be greater than we are able to report .",
"As increased anxiety-like behavior has also been reported in some prenatal opioid exposure models ( Andersen et al . , 2020; Byrnes and Vassoler , 2018 ) , we further examined open field videos for thigmotaxis , grooming , and rearing behaviors .",
"Thigmotaxis was affected by PME ( rmANOVA: Exposure , F ( 1 , 49 ) =0 . 17 . 33 , p=0 . 0001; Time , F ( 1 . 41 , 68 . 9 ) = 236 . 3 , p<0 . 0001; Interaction , F ( 2 , 98 ) =12 . 01 , p<0 . 0001 ) , with PME animals spending significantly more time at P7 near the arena walls than PSE animals ( Sidak’s post hoc test: p=0 . 0004 Figure 5—figure supplement 2a ) However , this likely reflects the relative hypoactivity of PSE animals which demonstrated very little activity on P7 and mostly remained in the arena center where offspring were placed at the beginning of each trial .",
"Both grooming ( Mann-Whitney test: U = 173 . 5 , p=0 . 0037 ) and unsupported rearing ( unpaired t test: t49 = 2 . 48 , p=0 . 017 ) , but not supported rearing ( unpaired t test: t49 = 0 . 352 , p=0 . 73 ) were reduced in PME mice which likely reflects an increase in time spent in active locomotion and jumping instead of grooming and rearing in PME mice as opposed to anxiolysis ( Figure 5—figure supplement 2b–d ) .",
"When removed from the dam , pups emit separation-induced USVs which signal distress and encourage retrieving behavior from the mother .",
"USVs typically peak around P8 and extinguish by the third week of life ( Ehret , 2005 ) .",
"The production of USVs in the open field was also significantly altered by PME ( rmANOVA: Exposure , F ( 1 , 47 ) =0 . 380 , p=0 . 54; Time , F ( 3 , 141 ) =13 . 3 , p<0 . 0001; Sex , F ( 1 , 47 ) =4 . 24 , p=0 . 045; Time x Exposure , F ( 3 , 141 ) =3 . 01 , p=0 . 032; Time x Sex , F ( 3 , 141 ) =0 . 636 , p=0 . 59; Exposure x Sex , F ( 1 , 47 ) =1 . 57 , p=0 . 22; Time x Exposure x Sex , F ( 3 , 141 ) =0 . 947 , p=0 . 42; Figure 5—figure supplement 3 and collapsed on sex in Figure 5e ) .",
"Female PME offspring vocalized significantly more than both male and female PSE offspring at P7 ( Sidak’s post hoc test; p=0 . 0045 and p=0 . 026 , respectively ) but this difference was not observed in male PME offspring relative to either sex of PSE offspring ( Sidak’s post hoc test; both p>0 . 999 , Figure 5—figure supplement 3 ) .",
"When collapsed on sex , vocalizations are nonsignificantly reduced at P1 and significantly increased at P7 in PME offspring ( Sidak’s post hoc test: p=0 . 079 and p=0 . 015 , respectively; Figure 5e ) .",
"Although female PME offspring do not produce significantly more USVs than male PME offspring at P7 ( Sidak’s post hoc: p=0 . 60 ) , the sex-collapsed effect described on P7 is likely driven by the high rate of USV production in female PME offspring ( Figure 5—figure supplement 3 ) .",
"We next performed further analysis on P7 USVs as this day differed between exposure groups .",
"Total activity correlated significantly with total calls on P7 across all animals ( Pearson’s r = 0 . 41 , p=0 . 0028; Figure 5—figure supplement 4a ) suggesting that there may be a relationship between hyperactivity and hyper-vocalizations among PME offspring .",
"USV call types were classified into categories based on frequencies , to cluster the types of USVs emitted on P7 ( Coffey et al . , 2019 ) .",
"The call classification analysis revealed the increase in USVs on P7 may be a result of a higher number of complex , flat , step down , and upward ramp calls emitted by PME offspring ( unpaired t tests: t49 = 2 . 54 , p=0 . 014; t49 = 2 . 98 , p=0 . 0045; t49 = 2 . 74 , p=0 . 0086; t49 = 2 . 09 , p=0 . 041; Figure 5—figure supplement 4b ) .",
"Markov chains of call type transitions ( ‘syntax’ ) was further analyzed on P7 which also revealed differences in the transition between call types between exposure groups ( Figure 5—figure supplement 4c ) .",
"Beginning on P3 and through P14 , offspring began a battery of daily developmental tests assessing the development of sensorimotor milestones .",
"Offspring with PME demonstrated delayed acquisition of surface righting ( unpaired t test: t48 = 4 . 69 , p<0 . 0001 ) , forelimb grasp ( Mann-Whitney test: U = 166 , p=0 . 0029 ) , and cliff aversion ( Mann-Whitney test: U = 161 , p=0 . 0026; Figure 5f ) .",
"No differences in the acquisition of the extinguishment of pivoting ( Mann-Whitney test: U = 238 , p=0 . 14 ) or negative geotaxis behavior ( Mann-Whitney test: U = 270 . 5 , p=0 . 42 ) were observed .",
"As basic locomotor activity is not impaired in PME offspring , these data indicate that PME offspring lack the ability to produce more complex , coordinated motor behaviors in response to multimodal sensory input .",
"A recent investigation of prenatal THC exposure revealed deficits in sensorimotor gating ( Frau et al . , 2019 ) ; therefore , as sensorimotor performance was impaired in PME offspring , we subsequently tested prepulse inhibition ( PPI ) , a sensorimotor gating task .",
"In a cohort of P28-P29 offspring , we found that PME did not alter the acoustic startle response ( rmANOVA: Exposure , F ( 1 , 36 ) =0 . 129 , p=0 . 72; Intensity , F ( 1 . 830 , 65 . 87 ) = 185 . 5 , p<0 . 0001; Interaction , F ( 6 , 216 ) =0 . 147 , p=0 . 99; Figure 5—figure supplement 5a ) .",
"No exposure-related effects were found on the percent PPI suggesting sensorimotor gating is intact in this model ( rmANOVA: Exposure , F ( 1 , 36 ) =0 . 282 , p=0 . 60; Prepulse Intensity , F ( 1 . 860 , 66 . 97 ) = 30 . 8 , p<0 . 0001; Interaction , F ( 2 , 72 ) =0 . 770 , p=0 . 47; Figure 5—figure supplement 5b ) .",
"Numerous brain regions and neural mechanisms could contribute to the altered activity and sensorimotor development described in PME offspring; therefore , we began our investigation by probing structural differences in gray matter regions across the brain using in vivo , ultra-high-field volumetric MRI .",
"Volumes of interest ( VOIs; See Figure 6—figure supplement 1 for segmentations ) were used to determine if differences in various gray matter regions were present in offspring at P28-P30 .",
"No significant differences in VOIs across any of the brain regions examined were discovered suggesting gross gray matter structure is mostly unaffected by PME ( unpaired t tests: see Supplementary file 2 for full test statistics; Figure 6 ) .",
"To determine whether PME could alter cortical development , we examined cortical laminations and neuron densities in distinctive cortical layers of P22-P24 offspring with triple immunostaining of NeuN ( neuronal marker ) , Cux1 ( a cortical layer 2/3–4 marker ) , and Draq5 ( nuclei stain ) .",
"We focused on the anterior cingulate cortex ( ACC ) , primary somatosensory cortex ( S1 ) , and primary motor cortex ( M1 ) for their involvement in sensorimotor behavior ( Hatsopoulos and Suminski , 2011; Paus , 2001; Umeda et al . , 2019; Figure 7 , Figure 7—figure supplement 1 , Figure 7—figure supplement 2 ) .",
"In M1 , there was no difference in the distributions of Cux1+ cells ( ANOVA: Exposure , F ( 1 , 34 ) =0 . 0943 , p=0 . 76; Sex , F ( 1 , 34 ) =1 . 68 , p=0 . 20; Interaction , F ( 1 , 34 ) =0 . 334 , p=0 . 57; Figure 7a–c ) .",
"Interestingly , we found a significant reduction in NeuN densities in cortical layers 2/3–4 ( ANOVA: Exposure , F ( 1 , 34 ) =4 . 40 , p=0 . 044; Sex , F ( 1 , 34 ) =7 . 56 , p=0 . 0095; Interaction , F ( 1 , 34 ) =5 . 014 , p=0 . 032; Figure 7a , b , d ) that was specific to PME females ( Sidak’s post hoc: p=0 . 0098 ) .",
"However , NeuN densities were not reduced in layer 5 ( ANOVA: Exposure , F ( 1 , 34 ) =0 . 673 , p=0 . 42; Sex , F ( 1 , 34 ) =0 . 733 , p=0 . 40; Interaction , F ( 1 , 34 ) =1 . 31 , p=0 . 26; Figure 7a , b , e ) .",
"In the ACC , there was a minor but significant Exposure x Bin interaction for both Cux1+ ( rmANOVA: Exposure , F ( 1 , 32 ) =1 . 03 , p=0 . 32; Bin , F ( 2 . 13 , 68 . 3 ) = 394 . 6 , p<0 . 0001; Interaction , F ( 9 , 288 ) =2 . 12 , p=0 . 028; Figure 7—figure supplement 1a–c ) and NeuN+ cell densities ( rmANOVA: Exposure , F ( 1 , 32 ) =0 . 424 , p=0 . 52; Bin , F ( 4 . 88 , 156 . 0 ) = 142 . 2 , p<0 . 0001; Interaction , F ( 9 , 288 ) =2 . 59 , p=0 . 0070; Figure 7—figure supplement 1a , b , d ) .",
"Although no posthoc tests quite reached the level of significance , there appeared to be an increase in NeuN+ cell densities in Bin 9 ( corresponding to approximately to the layer 5/6 border; Sidak’s post hoc: p=0 . 050; Figure 7—figure supplement 1a , b , d ) .",
"In S1 , the distribution patterns of Cux1 did not differ ( rmANOVA: Exposure , F ( 1 , 310 ) =0 . 498 , p=0 . 48; Bin , F ( 9 , 310 ) =346 . 5 , p<0 . 0001; Sex , F ( 1 , 310 ) =1 . 24 , p=0 . 27; Bin x Exposure , F ( 9 , 310 ) =0 . 860 , p=0 . 56; Bin x Sex , F ( 9 , 310 ) =1 . 54 , p=0 . 13; Exposure x Sex , F ( 1 , 310 ) =1 . 21 , p=0 . 27; Bin x Exposure x Sex , F ( 9 , 310 ) =1 . 72 , p=0 . 08; Figure 7—figure supplement 2a–c ) ; however , there was a slight exposure related effect on NeuN+ cell densities as a there was a complex three way interaction between Exposure , Bin , and Sex ( rmANOVA: Exposure , F ( 1 , 310 ) =3 . 30 , p=0 . 07; Bin , F ( 9 , 310 ) =63 . 6 , p<0 . 0001; Sex , F ( 1 , 310 ) =2 . 72 , p=0 . 10; Bin x Exposure , F ( 9 , 310 ) =0 . 811 , p=0 . 61; Bin x Sex , F ( 9 , 310 ) =0 . 840 , p=0 . 58; Exposure x Sex , F ( 1 , 310 ) =0 . 0007 , p=0 . 98; Bin x Exposure x Sex , F ( 9 , 310 ) =2 . 68 , p=0 . 0051; Figure 7—figure supplement 2a , b , d ) .",
"Similar to the ACC , no posthoc tests reached the level of significance .",
"These data suggest that there are subtle disruptions in cortical lamination development in PME for ACC , S1 , and M1 regions with a notable reduction in neuronal density of the upper cortical layer of M1 in female PME .",
"Given the significant alterations in sensorimotor and locomotor behavior in offspring with PME , we next examined intrinsic properties , local circuitry , and morphology of layer 5B thick-tufted pyramidal neurons of the M1 in P21-P26 offspring using whole-cell patch clamp electrophysiology and laser scanning photostimulation for optical mapping of local circuitry ( see Figure 8a , b for schematic diagram ) .",
"Thick-tufted layer 5B neurons indicate neurons with specific subcortical projection targets ( Hattox and Nelson , 2007; Morishima et al . , 2011; Oswald et al . , 2013 ) , and most likely represented M1 pyramidal tract corticospinal neurons ( Suter et al . , 2013 ) .",
"Representative current-clamp traces from whole-cell recordings of action potential firing and sub-threshold voltage responses are shown in Figure 8—figure supplement 1a and Figure 8c , respectively .",
"Recording analyses showed that L5 M1 neurons from PME mice displayed significantly reduced firing rates ( number of APs ) in response to injected current compared to PSE offspring ( rmANOVA: Exposure , F ( 1 , 66 ) =0 . 707 , p=0 . 40; Current , F ( 2 . 198 , 145 . 1 ) =1109 p<0 . 0001; Interaction , F ( 12 , 792 ) =1 . 78 , p=0 . 047 , Figure 8—figure supplement 1b ) .",
"These findings suggest PME neurons are characterized by reduced intrinsic excitability at higher injected currents; however , no posthoc tests reached the level of significance .",
"PME neurons exhibited significantly reduced input resistance compared to PSE cells ( Figure 8d , see Supplementary file 3 for stats ) , indicating that current was translated into a smaller change in voltage across the membrane of PME neurons .",
"Additionally , subthreshold responses of L5 M1 neurons showed prominent ‘sag’ and ‘overshoot’ of the membrane potential ( Figure 8c ) , which is characteristic of hyperpolarization-activated cyclic nucleotide-gated ( HCN ) channel current ( Ih ) expression ( Sheets et al . , 2011 ) .",
"Both voltage sag and voltage overshoot percentage were larger in L5 M1 neurons from PME mice ( Figure 8e , f , see Supplementary file 3 for stats ) , although voltage overshoot did not quite reach the level of significance .",
"Additional intrinsic properties of L5 M1 neurons in offspring can be viewed in Supplementary file 3 .",
"In addition to electrophysiological recordings , we measured the strength and organization of local synaptic inputs onto a single post-synaptic layer 5B M1 neuron by directing a UV laser beam ( 355 nm ) to focally uncage glutamate across a 16 × 16 stimulation grid centered around the recorded neuron ( Figure 8b ) .",
"Compared to PSE ( Figure 8g , h ) , PME ( Figure 8k , l ) modified the amplitude of local inputs onto layer 5 M1 neurons ( see PME minus PSE amplitude difference images for females and males in Figure 8i & m , respectively ) .",
"Row averages , which approximately correspond to subdivisions of cortex layers , were calculated by using the average amplitude of local inputs from rows 1:16 , columns 6:13 of the stimulation grid .",
"A significant interaction between exposure and row average was observed ( rmANOVA: Exposure , F ( 1 , 38 ) =0 . 120 , p=0 . 73; Row , F ( 3 . 759 , 142 . 8 ) = 16 . 79 , p<0 . 0001; Sex , F ( 1 , 38 ) =5 . 168 , p=0 . 029; Row x Exposure , F ( 15 , 570 ) =2 . 411 , p=0 . 0021; Row x Sex , F ( 15 , 570 ) =0 . 561 , p=0 . 905; Exposure x Sex , F ( 1 , 38 ) =0 . 397 , p=0 . 532; Row x Exposure x Sex , F ( 15 , 570 ) =1 . 126 , p=0 . 329; Figure 8j , n ) .",
"In particular , increased amplitude of excitatory responses were observed following stimulation of local neurons in L2/3 ( Figure 8i , j , m , n ) .",
"These electro-anatomical results suggest that the L2/3 → L5 local pathway is slightly enhanced in the M1 of juvenile PME mice .",
"Representative morphological reconstruction of recorded neurons can be viewed in Figure 9a , b ( PSE and PME females , respectively ) and Figure 9d , e ( PSE and PME males , respectively ) with all reconstructed thick-tufted L5 found in Figure 9—figure supplement 1 .",
"Sholl analyses suggest that morphology of layer 5 M1 neurons in PME mice is unchanged from PSE littermates as neither intersections ( rmANOVA: Exposure , F ( 1 , 27 ) =0 . 602 , p=0 . 44; Radius , F ( 16 , 432 ) =148 , p<0 . 0001; Sex , F ( 1 , 27 ) =7 . 34 , p=0 . 012; Radius x Exposure , F ( 16 , 432 ) =0 . 632 , p=0 . 86; Radius x Sex , F ( 16 , 432 ) =3 . 87 , p<0 . 0001; Exposure x Sex , F ( 1 , 27 ) =0 . 0821 , p=0 . 78; Radius x Exposure x Sex , F ( 16 , 432 ) =0 . 666 , p=0 . 83; Figure 9c , f top , for males and females , respectively ) nor length ( rmANOVA: Exposure , F ( 1 , 27 ) =1 . 09 , p=0 . 30; Radius , F ( 5 . 43 , 146 . 6 ) = 119 . 6 , p<0 . 0001; Sex , F ( 1 , 27 ) =6 . 56 , p=0 . 016; Radius x Exposure , F ( 16 , 432 ) =0 . 659 , p=0 . 83; Radius x Sex , F ( 16 , 432 ) =3 . 10 , p<0 . 0001; Exposure x Sex , F ( 1 , 27 ) =0 . 305 , p=0 . 59; Radius x Exposure x Sex , F ( 16 , 432 ) =0 . 586 , p=0 . 89; Figure 9c , f bottom , for males and females , respectively ) were different between PME and PSE treatments ."
],
[
"Given the rapid rise in infants born passively exposed to opioids during prenatal development , there is an urgent need to develop translationally relevant animal models of prenatal opioid exposure to begin to elucidate the impact of opioid exposure on development .",
"The present study adds to the limited body of work examining preclinical models of prenatal opioid exposure .",
"We provide the first longitudinal assessment of behavioral and neuronal development in offspring with PME combined with data on maternal and fetal opioid levels in the brain , placenta , and blood .",
"Furthermore , the present study builds upon previous models by providing a more comprehensive , clinically relevant characterization of how methadone , a commonly used opioid during pregnancy , impacts development when offspring are opioid-exposed during the entire gestational period .",
"Our findings reveal a disruption in physical , behavioral , and neuronal development in PME male and female offspring which persists beyond the immediate neonatal period and supports clinical studies that suggest babies born with opioid exposure are at higher risk for adverse developmental outcomes ( Larson et al . , 2019; Lee et al . , 2020; Yeoh et al . , 2019 ) .",
"Data on opioid levels in offspring are lacking in the preclinical literature .",
"Bearing in mind differences in metabolism between rodents and humans , the methadone dose utilized here was likely in the clinical range ( Devidze et al . , 2008; Dryden et al . , 2009 ) .",
"Although dam plasma levels were lower that what is typically recommended for treating OUD in humans ( ~400 ng/mL ) ( Eap et al . , 2002 ) , our dosing protocol produced opioid dependency in dams .",
"Others also reported an accumulation of methadone in the rodent fetal brain ( Kongstorp et al . , 2019 ) .",
"We added to these findings by demonstrating methadone accumulated in the placenta and provided evidence that the placenta may have predictive power to infer the degree of prenatal brain opioid exposure during gestation .",
"Although both methadone and EDDP accumulated in the placenta , EDDP did not concentrate in the fetal brain as methadone did .",
"This observation may suggest the increase in methadone resulted from reduced metabolism in offspring .",
"Methadone undergoes N-demethylation to form EDDP primarily via the cytochrome P450 enzyme CYP3A4 in humans ( Eap et al . , 2002 ) , but the expression of most cytochrome P450 enzymes in both mice and humans are dramatically lower during the prenatal period ( Hart et al . , 2009 ) .",
"Nevertheless , we are confident that methadone was present in the fetal brain at pharmacologically relevant levels in our model and likely contributed to the aberrant neurobehavioral development .",
"The persistent decrease in the size of offspring was striking , and other models of prenatal opioid exposure , including methadone , similarly demonstrated reduced body weight ( Byrnes and Vassoler , 2018; Jantzie et al . , 2020; Kunko et al . , 1996 ) .",
"In humans , prenatal opioid exposure is similarly associated with low birth weight and being small for gestational age ( Nørgaard et al . , 2015 ) , but it remains unclear what underlies this reduced size .",
"Given the delayed acquisition of other sensorimotor milestones , it is possible PME also delayed acquisition of the suckling reflex which could contribute to reduced growth .",
"An alternative hypothesis is that methadone disrupted the hypothalamic–pituitary–somatotropic axis .",
"Central administration of exogenous opioids modulates plasma levels of growth hormone and insulin-like growth factor in adult humans and rodents ( Abs et al . , 2000; Hashiguchi et al . , 1996 ) .",
"Work done by Vathy and colleagues revealed that prenatal morphine exposure interacts with other hypothalamic-pituitary axes altering the homeostatic balance of stress-related hormones ( Rimanóczy et al . , 2003; Slamberová et al . , 2004; Vathy , 2002 ) , but the effect of prenatal opioid exposure on growth-related hormones has not been studied and may represent an important area of study for future work .",
"Separation-induced USVs emitted by mouse pups are frequently used as a measure of distress ( Ehret , 2005 ) .",
"Production of these USVs is dependent on the endogenous opioid system .",
"Mu opioid receptor ( MOR ) agonists reduce USV rates , and mice lacking MORs do not vocalize when separated from the dam ( Carden et al . , 1991; Moles et al . , 2004 ) .",
"Others showed that PME reduces MOR-binding affinity and alters downstream signaling ( Kongstorp et al . , 2020a ) suggesting the differences in USV development in our model resulted from changes in the endogenous opioid system .",
"While the exact biological relevance of the increase in USV call rates and the alterations in call types and syntax on P7 is unclear at this time , these complex differences in USVs certainly reflected differential pup-dam communication between PME and PSE offspring .",
"PME offspring were hyperactive in the open field on P7 and P21 including a high number of jumps on P21 .",
"Combined with the increased USV call rate on P7 , these activity findings are indicative of a heightened sensory arousal and hyperactive state in juvenile PME offspring .",
"This hyperarousal phenotype may represent an enduring consequence of PME during the development of neurocircuitry controlling stress and/or sensorimotor activity .",
"Our data support clinical findings that report higher risks of attention-deficit/hyperactivity disorder and increased hyperactivity and impulsivity in children with opioid exposure in utero ( Azuine et al . , 2019; Ornoy et al . , 2001; Sundelin Wahlsten and Sarman , 2013 ) .",
"In our model , PME offspring experienced delayed development of several sensorimotor milestones .",
"Again , these findings mirror recent meta-analyses which support the negative impact of prenatal opioid exposure on psychomotor performance and motor function ( Lee et al . , 2020; Yeoh et al . , 2019 ) .",
"In regards to animal models , others also reported delayed development of certain sensorimotor milestones ( Kunko et al . , 1996; Robinson et al . , 2020; Wallin et al . , 2019 ) , but these reports slightly differ in which specific milestones are delayed and which develop normally in offspring .",
"For instance , offspring from our PME model did not exhibit differences in negative geotaxis , although in other models of prenatal buprenorphine and methadone exposure , negative geotaxis was disrupted ( Kunko et al . , 1996; Wallin et al . , 2019 ) .",
"Due to the differences in opioid used , duration and timing of exposure , and differences between rat and mouse development , identifying why specific milestones are delayed and why others are not remains challenging .",
"The neural mechanisms that could mediate the sensorimotor deficits and hyperactivity observed in our model are diverse .",
"M1 has a central role in the preparation , execution , and adaptation of motor movements .",
"Although the intrinsic properties of neurons can be affected by numerous ion channels and ion pumps , slice recording data indicated that altered intrinsic properties of M1 L5 pyramidal neurons from PME mice may be mediated by enhanced HCN channel-driven Ih .",
"In cortical pyramidal neurons , there is a gradient of HCN channel expression with the greatest being in the apical dendrites ( Berger et al . , 2001; Magee , 1998; Stuart and Spruston , 1998 ) .",
"In mouse M1 , HCN channels filter synaptic inputs onto L5 corticospinal neurons ( Anderson et al . , 2010; Sheets et al . , 2011 ) .",
"As a result , increased Ih in M1 L5 pyramidal neurons in PME mice suggests a narrowing of both spatial and temporal integration windows for both local and long-range synaptic inputs .",
"Local circuit maps collected here showed that PME enhanced the L2/3→ L5 excitatory pathway in M1 , which is the major local excitatory pathway for M1 ( Anderson et al . , 2010; Weiler et al . , 2008 ) .",
"Therefore , it is possible that the narrowing of synaptic integration windows leads to compensatory mechanisms that enhanced the strength of synaptic inputs .",
"These findings may suggest HCN channels represent a therapeutic target to prevent the potential neurobehavioral consequences of PME .",
"In addition , methadone inhibits NMDA glutamate receptors ( Eap et al . , 2002 ) , which mediate experience-dependent synaptic pruning during maturation of the mouse cortex ( Yu et al . , 2013 ) .",
"Therefore , PME-induced changes to dendritic pruning may also contribute to the altered local circuits of layer 5 M1 neurons , which could be further mediated by changes to HCN expression or function .",
"While our morphological analysis revealed no effect of PME on L5 M1 neurons , measurements were highly variable indicating that neurons analyzed were a heterogeneous population of thick-tufted neurons .",
"Although methadone may be a necessary and effective treatment for maternal OUD , these findings suggest this treatment during pregnancy may come at the expense of persistent disruptions in offspring development .",
"As clinical evidence strongly indicates the use of opioid maintenance therapies for treating OUD improves pregnancy outcomes ( ACOG , 2017 ) , these findings which indicate PME impairs physical and neurobehavioral outcomes in offspring should not be interpreted as evidence against the use of opioid maintenance therapies in pregnancy .",
"Instead , early and preventative clinical intervention programs such as neurodevelopmental follow-up programs may be needed to support the healthy development of infants exposed to opioids in utero .",
"Further research is necessary to determine if additional prenatal pharmacotherapies may prevent pathology and behavioral impairments in offspring .",
"There are several limitations which must be considered when interpreting our findings .",
"Prior reports indicated opioid treatment alters maternal behavior which could impact offspring development ( Slamberová et al . , 2001; Wallin et al . , 2019 ) .",
"However , we and others ( Alipio et al . , 2021; Kongstorp et al . , 2020b; Tan et al . , 2015 ) , did not observe differences in maternal care suggesting the differences found in offspring are a result of passive methadone exposure during gestational development .",
"As maternal stress can have wide-ranging and persistent effects on physical , behavioral , and neurological development , additional longitudinal assessments of maternal care including maternal-offspring interactions throughout the preweaning period may provide additional information on maternal care .",
"Alternatively , future work is necessary to examine how repeated opioid treatment may disrupt neuroendocrine physiology ( e . g . HPA axis dysfunction or corticosterone production ) which may indirectly disrupt offspring development .",
"We cannot be confident that subtle differences in maternal behavior interacted with methadone exposure to alter offspring outcomes .",
"We discovered few sex-specific effects in our assessments of offspring .",
"This may be a result of the age at which the animals were tested or , if sex differences were very subtle , we many have been inadequately powered to detect such small differences .",
"Next , we modeled PME via noncontingent treatment using twice daily injections .",
"Although free-access or self-administration would be more translational , noncontingent exposure reduces variability in methadone exposure between pregnant females .",
"The interaction of PME with the stress of injections and repeated handling of dams could produce the differential offspring outcomes .",
"Given that medically supervised opioid withdrawal is not recommended for OUD , our choice of prenatal opioid exposure model was based on epidemiological trends which supports the high usage of methadone by pregnant women ( Duffy et al . , 2018 ) .",
"However , there exists numerous variations in human opioid use during pregnancy which our model does not reflect ( e . g . women may not initiate opioid maintenance therapy until partway through pregnancy; they may experience a relapse and/or use other recreational opioids at variable intervals during pregnancy; buprenorphine , which has a different pharmacological profile than methadone , is an increasingly frequent choice for opioid maintenance therapy ) .",
"Further work by our group and others will be required to examine these variants of prenatal opioid exposure .",
"The differences between rodent and human gestational development are an inherent limitation of our model that cannot be overcome .",
"The first week of rodent postnatal life is roughly equivalent to the third trimester ( Robinson et al . , 2020 ) .",
"In an attempt to maintain a sufficient level of methadone exposure to the newborn pups during P0-P7 , the dams were maintained at their highest gestational dose after giving birth and up to P7; however , it is unlikely this strategy completely replicates in utero third trimester opioid exposure in humans .",
"Lastly , our findings in offspring were collected in the preweaning and early adolescent periods .",
"Future studies will examine if alterations in physical , behavioral , and neuronal outcomes persist into adulthood or if PME offspring are able to eventually overcome these differences ."
],
[
"Animal care and research were conducted in accordance with guidelines established by the National Institutes of Health and protocols were approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee .",
"Eight-week-old female C57BL/6J mice were acquired from Jackson Laboratories ( Bar Harbor , Maine ) , single housed , and randomly assigned to either saline ( 10 mL/kg ) or oxycodone treatments .",
"Oxycodone dependence was induced by a dose-ramping procedure with a dose of 10 mg/kg administered on pregestational day ( PG ) 14 , 20 mg/kg on PG13 , and then maintained on 30 mg/kg for PG12-6 .",
"All saline or oxycodone doses were administered subcutaneously twice daily at least 7 hr apart .",
"On PG5 , oxycodone-treated mice were transitioned to 10 mg/kg methadone while saline-treated animals continued to receive saline injections ( s . c . b . i . d . ) .",
"All oxycodone and methadone solutions were prepared in saline .",
"Oxycodone and methadone were obtained from the National Institute on Drug Abuse Drug Supply Program .",
"Five days following initiation of methadone treatment , an 8-week-old C57BL/6J male mouse ( also acquired from Jackson Laboratories ) was placed into the cage of each female for four days .",
"Mucous plugs were assessed each morning to approximate gestational day ( G ) 0 .",
"Food consumption was examined weekly in a subset of these female mice .",
"Female mice were weighed every Monday , Wednesday , and Friday throughout the study with the exception of the mating period .",
"Cages were examined for the presence of pups at the time of each morning and afternoon injection , and the day of birth was designated postnatal day ( P ) 0 .",
"Data on litter size , sexes , and neonatal deaths were collected .",
"The presence of unconsumed placentas at 24 hr following birth was also examined .",
"Only litters between three and eight pups were used in subsequent studies of offspring .",
"In an attempt to maintain a sufficient level of methadone exposure to the newborn pups during P0-P7 , the dams were maintained at their highest gestational dose after giving birth and up to P7 .",
"After P7 , the dose of methadone administered to dams was adjusted to their body weight .",
"All treatments to dams were discontinued at weaning ( ~P28 ) .",
"All tissue and blood samples were collected 2 . 5 hr following morning administration of methadone .",
"On G18 , three dams were anesthetized with ketamine/xylazine and fetuses were rapidly removed from the uterine cavity to examine brain and placental drug levels .",
"On P1 and P7 , three dams and their litters were sacrificed for the collection of trunk blood and brains .",
"Blood samples were centrifuged for five minutes at 4500 g to collect 20 µL of plasma .",
"Plasma , placenta , and whole brains were frozen in isopentane on dry ice and stored in −80°C until processing .",
"The samples were examined for the presence of methadone and EDDP via high-performance liquid chromatography tandem mass spectroscopy ( HPLC–MS/MS; Sciex 5500 QTRAP , Applied Biosystems , Foster City , MA ) .",
"The analytical method for plasma samples has previously been described ( Metzger et al . , 2020 ) .",
"Tissue samples and standards were prepared by adding diphenhydramine ( internal standard , 0 . 1 ng/µL in methanol ) and 0 . 1 M phosphate buffer ( 200 µL , pH 7 . 4 ) to tissue samples .",
"Samples were then extracted using a liquid-liquid procedure with 3 mL ethyl acetate .",
"The supernatant was transferred to a clean tube and evaporated to dryness , then reconstituted in acetonitrile with 0 . 1% formic acid ( pH 6 . 5 ) .",
"Ten microliters of sample were then injected and the remaining protocol follows the previously published procedure ( Metzger et al . , 2020 ) .",
"The limit of quantification for methadone and EDDP detection was 0 . 1 ng/mL and 0 . 05 ng/mL in the plasma , respectively , and 0 . 08 ng/sample and 0 . 04 ng/sample of placenta and brain , respectively for methadone and EDDP .",
"Dams were given a new compressed paper nestlet , and twelve hours later , the ability of dams to build a new nest on P3 was assessed using a five-point nest rating system where one represents a nestlet untouched and a five requires >90% of nestlet to be torn creating a crater with walls higher than the height of the mouse for >50% of the circumference .",
"Further criterion are described elsewhere ( Deacon , 2006 ) .",
"A pup retrieval task was completed by first removing one pup and the dam and then placing the pup at one end of the cage ( ~30 cm away ) .",
"The dam was then released at the other end of the cage and the time required to retrieve the missing pup was recorded .",
"This was repeated with two other pups and an average pup retrieval latency score was calculated .",
"To demonstrate that the oxycodone dosing strategy induced dependency , a subset of mice underwent naloxone-precipitated withdrawal prior to methadone transition .",
"Sixty to 90 min following the final 30 mg/kg oxycodone dose on PG6 , all mice were administered naloxone ( 5 mg/kg , i . p . ) and somatic signs of withdrawal were assessed for 10 min using a previously described protocol ( Berrendero et al . , 2003 ) .",
"These behavioral signs included number of paw shakes , wet dog shakes , and jumps and the presence of ptosis , body tremor , teeth chattering , piloerection , and diarrhea .",
"The global withdrawal score was calculated using the following equation: ( jumps*0 . 8+wetdogs shakes*1+diarrhea*1 . 5+paw shakes*0 . 35+ptosis*1 . 5+teeth chattering*1 . 5+body tremor*1 . 5+piloerection*1 . 5 ) .",
"These mice which underwent precipitated withdrawal were not used in any subsequent studies .",
"Similarly , following the weaning of offspring , a subset of dams underwent the naloxone-precipitated withdrawal procedure to demonstrate that the transition to the 10 mg/kg methadone dosing regimen maintained opioid dependency .",
"Beginning at P0 and throughout the pre-weaning period , offspring were marked for identification on the paws with a red or black marker .",
"The progression of weight gain and body length on P0 , P3 , P7 , P10 , P14 , P18 , and P21 was examined .",
"A subset of the offspring was also weighed at P35 and P49 .",
"From P3 to P18 , offspring were examined for the first day both eyes were open , the first day both ear pinnae completely unfolded and the ear canal was patent , and the first day the lower incisors emerged .",
"The right femur of P7 and P35 offspring was scanned on a SkyScan 1172 ( Bruker , Billerica , MA , USA ) with a 0 . 5 aluminum filter and a 6 µm voxel size .",
"For P7 samples , mineralized femur length was obtained from a coronal view of the scan .",
"Whole bone volume was obtained in the transaxial plane incorporating the entire mineralized bone .",
"For P35 samples , a 0 . 5 mm region of interest at the distal femur was selected proximal to the growth plate .",
"Distal femur metaphysis bone volume was obtained in the transaxial plane from this whole region including both trabecular and cortical bone .",
"Trabecular bone was isolated in the same region for trabecular bone volume .",
"Cortical bone in P35 samples was measured from five slices 2 mm proximal the end of the metaphysis region of interest .",
"Cortical bone area and cortical thickness were measured at this site .",
"Volumetric MRI studies were performed using a 9 . 4 T Bruker system ( Bruker BioSpin MRI GmbH , Germany ) equipped with a BGA-12S gradient .",
"A Bruker two-channel surface mouse head coil was used for high resolution brain imaging .",
"Animals were anesthetized and maintained with 1 . 5–2% isoflurane during MRI sessions .",
"T2-weighted images with a three-dimensional Rapid Acquisition with Relaxation Enhancement ( RARE ) sequence were acquired for volumetric measurements ( TR/TE = 1500/11 ms , Rare Factor = 12 , FOV = 20×20 x 7 mm with an isotropic voxel of 120 µm3 , number of averages = 1 , scan time = 11 . 8 min ) .",
"A mouse brain atlas for 4-week-old mice was created by modifying PMOD ( PMOD Technologies LLC , Switzerland ) Ma-Benveniste-Mirrione mouse brain atlas .",
"Specifically , the T2-weighted MRI images of a 4-week-old control mouse were normalized to the PMOD built-in mouse brain template using a Deform function in the ‘Fuse It’ module .",
"VOIs were then manually adjusted to fit the 4-week-old mouse brain and to create a 4-week mouse atlas for this study .",
"To obtain the brain volume measurements , T2-weighted anatomical images from individual mouse in the native space were normalized to the 4-week mouse template and an inverse transformation matrix was saved in Fuse It module .",
"The native space image data were loaded again in the View module then 4-week-mouse atlas with VOIs was loaded and inverse transformed to obtain the volume measurements ( see Figure 6—figure supplement 1 for segmentation ) .",
"Raw volume measurements were then utilized for statistical comparisons .",
"On P22-24 , offspring were anesthetized with isoflurane and perfused with 4% paraformaldehyde prepared in PBS for 10 min at a pump rate of ~2 mL/min .",
"Fixed brains were sectioned into 100 μm sections in the coronal plane using a Leica VT-1000 vibrating microtome ( Leica Microsystems ) and stored in PBS until later analysis .",
"Sections were permeabilized with 0 . 3% Triton X100 , then incubated with a blocking solution ( 3% normal goat serum prepared in PBS with 0 . 3% Triton X-100 ) and then incubated overnight with primary antibody ( see Key Resources Table ) prepared in blocking solution .",
"An appropriate secondary antibody conjugated with an Alexa series fluorophore ( see Key Resources Table ) was used to detect the primary antibody .",
"Draq5 ( 1:10 , 000 dilution , Cell Signaling ) was included in the secondary antibody solution to stain nuclei .",
"Z-stack confocal images were acquired from both hemispheres with a Leica confocal microscope with a 10X/NA0 . 75 objective .",
"The Z-stacks were taken at 0 . 5 µm intervals , 5 µm-total thickness was imaged .",
"Projection images of 5 µm thickness were used for image quantification by using NIH ImageJ software .",
"The regions of interest were defined as bins 1–10 for the ACC and S1 and defined as layer 2/3–4 and layer 5 in M1 .",
"Experimenters were blinded to treatment/exposure group for data collection of all studies .",
"Statistical analyses were conducted using GraphPad Prism 8 ( San Diego , CA ) .",
"Data are graphically presented as the mean ± SEM or as box plots extending from the 25th to 75th percentiles with whisker characterizing minimum and maximum values .",
"The level of significance was set at p<0 . 05 .",
"All experiments were performed using both male and female offspring .",
"To minimize potential litter effects in all completed studies , offspring from four or more litters per exposure were utilized as previously done ( Ricalde and Hammer , 1990 ) .",
"For offspring weight and length , offspring from eight to nine litters per exposure were examined .",
"For all behavioral studies , offspring were taken from four to five different litters per exposure .",
"For neuroimaging and immunohistochemistry studies , offspring from four litters per exposure were utilized .",
"For electrophysiology , mapping , morphology , and imaging , neurons were recorded and imaged from offspring of nine different litters per exposure .",
"With the exception of MRI data , all studies were sufficiently powered to detect sex differences .",
"Therefore , all analyses ( with the exception of MRI data ) were first completed treating sex as a factor .",
"However , for clarity of focusing on the effect of prenatal exposure , when no main effects of sex or interactions with sex were found in offspring , the data was collapsed on sex and re-analyzed .",
"Chi-square tests for categorical data was used for litter characteristics .",
"A linear regression was performed to assess the relationship between placental methadone levels and offspring brain levels on G18 , and offspring plasma and brain levels on P1 and P7 .",
"Two-tailed unpaired t-tests were used to analyze normally distributed data and Mann-Whitney U tests or Wilcoxon rank sum test ( for electrophysiology data only ) were used to analyze non-normally distributed data .",
"For data with multiple groups and/or repeated measures , ANOVAs with Sidak’s post hoc tests were used ."
]
] | [
"Despite the rising prevalence of methadone treatment in pregnant women with opioid use disorder , the effects of methadone on neurobehavioral development remain unclear .",
"We developed a translational mouse model of prenatal methadone exposure ( PME ) that resembles the typical pattern of opioid use by pregnant women who first use oxycodone then switch to methadone maintenance pharmacotherapy , and subsequently become pregnant while maintained on methadone .",
"We investigated the effects of PME on physical development , sensorimotor behavior , and motor neuron properties using a multidisciplinary approach of physical , biochemical , and behavioral assessments along with brain slice electrophysiology and in vivo magnetic resonance imaging .",
"Methadone accumulated in the placenta and fetal brain , but methadone levels in offspring dropped rapidly at birth which was associated with symptoms and behaviors consistent with neonatal opioid withdrawal .",
"PME produced substantial impairments in offspring physical growth , activity in an open field , and sensorimotor milestone acquisition .",
"Furthermore , these behavioral alterations were associated with reduced neuronal density in the motor cortex and a disruption in motor neuron intrinsic properties and local circuit connectivity .",
"The present study adds to the limited body of work examining PME by providing a comprehensive , translationally relevant characterization of how PME disrupts offspring physical and neurobehavioral development ."
] | [
"The far-reaching opioid crisis extends to babies born to mothers who take prescription or illicit opioids during pregnancy .",
"Opioids such as oxycodone and methadone can freely cross the placenta from mother to baby .",
"With the rising misuse of and addiction to opioids , the number of babies born physically dependent on opioids has risen sharply over the last decade .",
"Although these infants are only passively exposed to opioids in the womb , they can still experience withdrawal symptoms at birth .",
"This withdrawal is characterized by irritability , excessive crying , body shakes , problems with feeding , fevers and diarrhea .",
"While considerable attention has been given to treating opioid withdrawal in newborn babies , little is known about how these children develop in their first years of life .",
"This is , in part , because it is difficult for researchers to separate drug-related effects from other factors in a child’s home environment that can also disrupt their development .",
"In addition , the biological mechanisms underpinning opioid-related impairments to infant development also remain unclear .",
"Animal models have been used to study the effects of opioid exposure during pregnancy ( termed prenatal exposure ) on infants .",
"These models , however , could be improved to better replicate the typical pattern of opioid use among pregnant women .",
"Recognizing this gap , Grecco et al . have developed a mouse model of prenatal methadone exposure where female mice that were previously dependent on oxycodone were treated with methadone throughout their pregnancy .",
"Methadone is an opioid drug commonly prescribed for treating opioid use disorder in pregnant women and was found to accumulate at high levels in the fetal brain of mice , which fell quickly after birth .",
"The offspring also experienced withdrawal symptoms .",
"Grecco et al . then examined the physical , behavioral and brain development of mice born to opioid-treated mothers .",
"These included assessments of the animals’ motor skills , sensory reflexes and behavior in their first four weeks of life .",
"Additional experiments tested the properties of nerve cells in the brain to examine cell-level changes .",
"The assessments showed that methadone exposure in the womb impaired the physical growth of offspring and this persisted into ‘adolescence’ .",
"Prenatal methadone exposure also delayed progress towards key developmental milestones and led to hyperactivity in three-week-old mice .",
"Moreover , Grecco et al . found that these mice had reduced neuron density and cell-to-cell connectivity in the part of the brain which controls movement .",
"These findings shed light on the potential consequences of prenatal methadone exposure on physical , behavioral and brain development in infants .",
"This model could also be used to study new potential treatments or intervention strategies for offspring exposed to opioids during pregnancy ."
] | 2021 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cancer biology"
] | KRAS-dependent sorting of miRNA to exosomes | elife-07197-v2 | [
[
"An emerging paradigm in the study of cell signaling is the potential role for post-transcriptional gene regulation by extracellular RNAs .",
"microRNAs ( miRNAs ) are perhaps the best characterized class of small noncoding RNAs ( ncRNAs ) that have been detected in extracellular fluids ( Valadi et al . , 2007 ) .",
"Mature miRNAs are 21–23 nucleotides in length and bind to target mRNAs to inhibit their expression ( Krol et al . , 2010 ) .",
"Because miRNAs imperfectly pair with their mRNA targets , they can potentially regulate hundreds of transcripts within a genome ( Bartel and Chen , 2004 ) .",
"However , individual miRNAs exhibit exquisite tissue-specific patterns of expression ( Wienholds et al . , 2005 ) , control cell fate decisions ( Alvarez-Garcia and Miska , 2005 ) , and are often aberrantly expressed in human cancers ( Thomson et al . , 2006 ) , affording possible disease-specific signatures with diagnostic , prognostic , and therapeutic potential ( Lu et al . , 2005; Volinia et al . , 2006 ) .",
"In addition to their intracellular roles , recent experiments have identified miRNAs outside the cell in extracellular vesicles ( EVs ) including exosomes or larger vesicles ( Valadi et al . , 2007; Crescitelli et al . , 2013 ) , in high-density lipoprotein particles ( Vickers et al . , 2011 ) , or in smaller complexes with Argonaute 2 protein ( Arroyo et al . , 2011 ) .",
"Exosomes are small 40–130 nm vesicles of endosomal origin that are secreted by all cells and can fuse and be internalized by recipient cells ( Valadi et al . , 2007; Kosaka et al . , 2010; Higginbotham et al . , 2011; Mittelbrunn et al . , 2011; Montecalvo et al . , 2012 ) .",
"It has been suggested that protein cargo transfer by exosomes between cells is associated with tumor aggressiveness and metastasis ( Skog et al . , 2008; Higginbotham et al . , 2011; Luga et al . , 2012; Hoshino et al . , 2013; Costa-Silva et al . , 2015 ) .",
"With the discovery that miRNAs and other RNAs can also be packaged into EVs , or exported by other extracellular mechanisms , it remains unclear the extent to which RNA cargo is sorted for export and how it is dysregulated in disease conditions , such as cancer .",
"Despite accumulating evidence that exosomes are biologically active , little is known regarding how oncogenic signaling affects the repertoire of miRNAs or proteins that are selected for secretion .",
"Given the potential of cancer-derived secreted RNAs to modulate the tumor microenvironment , elucidation of the potential mechanisms for selective sorting of cargo into exosomes is critical to understanding extracellular signaling by RNA .",
"KRAS mutations occur in approximately 34–45% of colon cancers ( Wong and Cunningham , 2008 ) .",
"We have previously shown that exosomes from mutant KRAS colorectal cancer ( CRC ) cells can be transferred to wild-type cells to induce cell growth and migration ( Higginbotham et al . , 2011; Demory Beckler et al . , 2013 ) .",
"Compared to exosomes derived from isogenically matched wild-type cells , exosomes derived from mutant KRAS cells contain dramatically different protein cargo ( Demory Beckler et al . , 2013 ) .",
"Here , we show that KRAS status also prominently affects the miRNA profile in cells and their corresponding exosomes .",
"Exosomal miRNA profiles are distinct from cellular miRNA patterns , and exosomal miRNA profiles are better predictors of KRAS status than cellular miRNA profiles .",
"Furthermore , we show that cellular trafficking of miRNAs is sensitive to neutral sphingomyelinase ( nSMase ) inhibition in mutant , but not wild type , KRAS cells and that transfer of miRNAs between cells can functionally alter gene expression in recipient cells ."
],
[
"Because small RNAs are thought to be sorted at endosomal membranes and since KRAS signaling can also occur on late endosomes ( Lu et al . , 2005 ) , we hypothesized that oncogenic KRAS signaling could alter RNA export into exosomes .",
"We prepared small RNA libraries from both exosomes and whole cells using isogenically matched CRC cell lines that differ only in KRAS status ( Figure 1—source data",
"1 ) ( Shirasawa et al . , 1993 ) .",
"Exosomes were purified using differential centrifugation and consisted of vesicles ranging in size from 40 to 130 nm ( Higginbotham et al . , 2011; Demory Beckler et al . , 2013 ) .",
"These preparations exclude larger microvesicles but contain smaller lipoproteins and probably other small RNA–protein complexes ( unpublished observation ) .",
"Comprehensive sequencing analyses of both cellular and exosomal small RNAs from all three cell lines revealed that more than 85% of the reads from the cellular RNA libraries mapped to the genome , compared to only 50–71% from the exosomal libraries ( Figure 1A ) .",
"The non-mappable reads consisted largely of sequences that contain mismatches to genomic sequences . 10 . 7554/eLife . 07197 . 003Figure 1 . Small RNA sequencing analysis of cellular and exosomal RNAs from CRC cell lines . Shown are ( A ) total read numbers ( y-axis ) and the total percentage of mappable reads ( red ) , percentage of unique mappable reads ( green ) , reads that map to multiple genomic locations ( dark blue ) , and those that could not be mapped ( cyan ) .",
"( B ) The majority of mappable small RNA reads were derived from noncoding RNAs in cells and exosomes .",
"In cells , the majority of small RNA reads mapped to microRNAs ( miRNAs ) ( miRbase 19 ) , whereas in exosomes , the majority of small RNA reads mapped to repetitive elements .",
"( C ) The origin of repetitive reads from exosomal small RNA sequencing is shown .",
"Repeat reads were annotated based on RepeatMasker and Rfam classified into tRNAs , rRNAs , snRNAs , and others .",
"( D ) The length distribution of reads mapping to miRNA hairpins was determined for small RNA reads from the three CRC cell lines and their purified exosomes .",
"Colors represent the nucleotides identified for the 5′ base , T ( cyan ) , A ( red ) , G ( green ) , and C ( blue ) .",
"Figure 1—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 00310 . 7554/eLife . 07197 . 004Figure 1—source data 1 . Colorectal cancer ( CRC ) cell lines . Small RNA sequencing libraries were prepared from three isogenic CRC cell lines with the indicated alleles of KRAS .",
"Table is based on work done in Demory Beckler et al . ( 2013 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 00410 . 7554/eLife . 07197 . 005Figure 1—figure supplement 1 . Length distribution of small RNA reads to genome . The small RNA read length distribution from serum-starved cells and from purified exosomes was determined , as well as the 5′ nucleotide from each read .",
"The pattern from total cellular small RNA is consistent with primarily miRNA reads , the distribution from exosomes is much broader , encompassing a number of small reads derived from many sources ( see Figure 1C , D ) .",
"Colors represent the nucleotides identified for the 5′ base , T ( cyan ) , A ( red ) , G ( green ) , and C ( blue ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 005 The global small RNA profiles identified reads from various classes of RNA , including miRNAs , with differential enrichment of specific RNAs in both the cellular and exosomal fractions .",
"Compared to cellular RNA samples , which displayed an enrichment of miRNA sequences ( ∼70% ) , miRNA reads in exosomal samples comprised a smaller percentage of the total small reads ( 5–18% ) compared to other ncRNA classes ( e . g . , tRNAs , rRNAs , snRNAs ) ( Figure 1B , C , Supplementary file 1 ) .",
"Most of these reads appear to be the fragments of larger RNAs , both cytoplasmic and nuclear .",
"It is not clear how these RNAs are associated with and/or deposited into exosomes .",
"The size distribution of cellular small RNA matched that expected from miRNA-derived reads ( 21–23 nucleotides ) .",
"However , the small RNA read distribution from exosomes was much broader with many reads smaller than 22 nucleotides in length ( Figure 1—figure supplement 1 ) .",
"Given that these reads map to RNAs other than known miRNAs , these data suggest that a large proportion of small exosomal RNA reads is derived from processing of other RNAs , in addition to post-transcriptionally modified miRNA reads that are apparently subject to editing , trimming , and/or tailing ( Koppers-Lalic et al . , 2014 ) .",
"Consistent with this , when read identity was restricted to miRNAs by mapping back to known miRNA hairpin sequences , the length distribution of mappable reads was nearly identical between cells and exosomes ( Figure 1D ) .",
"Focusing on mappable reads , we sought to ascertain whether miRNAs might be differentially represented when comparing cells to their secreted exosomes .",
"For this , we quantified the relative abundance of individual miRNAs and made pairwise comparisons between normalized miRNA reads .",
"Spearman correlation analyses demonstrated high correlation between replicates of individual cell lines ( r = 0 . 95–0 . 96 ) and between cellular data sets differing only in KRAS status ( r = 0 . 92–0 . 96 ) ( Figure 2—figure supplements 1–3 ) .",
"In contrast , the miRNA profiles from exosomes compared to their parent cells were less correlated ( DKO-1 r = 0 . 67–0 . 81 , DKs-8 r = 0 . 64–0 . 71 , DLD-1 r = 0 . 64–0 . 69 ) ( Figure 2—figure supplements 1 , 2 , 4 ) .",
"We next utilized principal component ( PC ) analysis to determine whether the overall miRNA profiles could distinguish between cells and exosomes and/or between wild-type and mutant KRAS status .",
"The miRNA profiles from the three cell lines all clustered close to one another indicating that overall miRNA expression profiles are fairly similar among the different cell types ( Figure 2 ) .",
"In marked contrast , PC analysis revealed that exosomal miRNA profiles clearly segregate according to KRAS status ( Figure 2 ) .",
"Relatively , minor differences between cellular miRNA expression profiles become much more prominent when comparing exosomal miRNA patterns ( Figure 2—figure supplement 3 ) .",
"This indicates that the presence of a mutant KRAS allele alters sorting of specific miRNAs to exosomes , a finding that has potentially important implications for biomarker development . 10 . 7554/eLife . 07197 . 006Figure 2 . Small RNA composition segregates with KRAS status . Principal component analysis was performed comparing small RNA sequencing data sets from CRC cells and exosomes .",
"The small RNA composition from cells differed significantly from exosomes .",
"Nevertheless , clustering showed that mutant KRAS status could be inferred from small RNA composition .",
"Also see Figure 2—figure supplements 1–4 . DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 00610 . 7554/eLife . 07197 . 007Figure 2—figure supplement 1 . Spearman correlations between miRNA expression profiles in cells and exosomes . Pairwise similarity between the RNA sequencing data sets derived from cells and exosomes .",
"Spearman correlations are shown between the cell samples ( R = 0 . 93–0 . 96 ) , between exosomes and cognate cells ( R = 0 . 64–0 . 83 ) and between exosome samples ( R = 0 . 74–0 . 86 ) .",
"Results were generated using the DESeq ‘pooled’ method . DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 00710 . 7554/eLife . 07197 . 008Figure 2—figure supplement 2 . Spearman correlations between miRNA expression profiles in cells and exosomes . Similar to supplemental Figure 2A .",
"Differential analysis using the DESeq ‘per condition’ method . DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 00810 . 7554/eLife . 07197 . 009Figure 2—figure supplement 3 . Spearman correlations between miRNA expression profiles in cells ( top ) and exosomes ( bottoms ) in reads per million ( RPM ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 00910 . 7554/eLife . 07197 . 010Figure 2—figure supplement 4 . Spearman correlations comparing miRNA expression profiles in exosomes to parent cell in RPM . DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 010 To gain more insight into the relative abundance of miRNAs in cells vs matched exosomes , we examined the most abundant miRNA species in the various sequencing libraries ( determined by mean reads of individual miRNAs ) .",
"For many miRNAs , exosomal abundance correlated with cellular abundance ( Supplementary file 2 ) .",
"However , calculation of fold changes among the three isogenic KRAS cell lines , and exosomes released from these cells , showed that distinct subsets of miRNAs are enriched in either exosomes or cells ( Tables 1 , 2 ) .",
"For all three cell lines , 25 miRNAs were consistently upregulated in cells and 29 miRNAs were consistently upregulated in exosomes ( Figure 3A , B ) .",
"Additionally , the diversity of miRNAs was substantially greater in mutant KRAS DKO-1 exosomes ( 94 unique miRNAs ) compared to parental DLD-1 or wild-type KRAS DKs-8 exosomes ( Figure 3B ) .",
"A select subset of cell and exosomally targeted miRNAs were validated separately by quantitative reverse-transcription PCR ( qRT/PCR ) ( Figure 3C ) .",
"Collectively , these data indicate that the miRNA profiles observed in exosomes are distinct from their parental cells with specific miRNAs preferentially overrepresented or underrepresented in exosomes .",
"We observed a mutant KRAS-specific pattern of secreted miRNAs , consistent with the hypothesis that dysregulation of miRNA metabolism is associated with tumorigenesis , a previously unrecognized feature of mutant KRAS . 10 . 7554/eLife . 07197 . 011Table 1 . Differential expression of miRNAs in colorectal cancer cells*DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 011DKO-1 hsa-miR-548uhsa-miR-16-1-3phsa-miR-33a-3phsa-miR-33a-5p hsa-miR-31-5phsa-miR-181b-3phsa-miR-450a-5phsa-miR-424-5p hsa-miR-9-5phsa-miR-219-5phsa-miR-190ahsa-miR-573 hsa-miR-30d-3phsa-miR-204-5phsa-miR-1226-3phsa-miR-499a-5p hsa-miR-450b-5phsa-miR-499b-3phsa-miR-3662hsa-miR-20a-3p hsa-miR-27b-5phsa-miR-5701hsa-miR-4677-3phsa-let-7i-5p hsa-miR-331-3phsa-miR-31-3phsa-miR-651hsa-miR-1306-5p hsa-miR-147bhsa-miR-3611hsa-miR-1305hsa-miR-148a-3p hsa-miR-27b-3phsa-miR-1306-3phsa-miR-374b-3phsa-miR-1260b hsa-miR-3940-3phsa-miR-200c-5phsa-miR-548ar-3pDKs-8 hsa-miR-132-5phsa-miR-484hsa-miR-374a-5phsa-miR-1180 hsa-miR-1307-3phsa-miR-200a-5phsa-miR-548o-3phsa-miR-149-5p hsa-miR-3615hsa-miR-100-5phsa-miR-197-3phsa-miR-378a-5p hsa-let-7a-3pDLD-1 hsa-miR-141-3phsa-miR-26b-5phsa-miR-24-3phsa-miR-3074-5p hsa-miR-15a-5phsa-miR-27a-3phsa-miR-3613-5phsa-miR-30b-5p hsa-miR-29a-3phsa-miR-301a-5phsa-let-7i-3phsa-miR-185-5p hsa-let-7g-5phsa-miR-23b-3phsa-miR-22-3pDKO-1 and DKs-8 hsa-miR-141-5phsa-miR-582-5pDKO-1 and DLD-1 hsa-miR-556-3phsa-miR-374a-3phsa-miR-106b-5phsa-miR-17-3p hsa-miR-24-1-5phsa-miR-340-3pDLD-1 and DKs-8 hsa-miR-24-2-5phsa-miR-106a-5phsa-miR-30e-5phsa-miR-107 hsa-miR-429hsa-miR-98-5phsa-miR-425-5phsa-miR-140-5p hsa-miR-93-5phsa-miR-210hsa-miR-126-3phsa-miR-194-5p hsa-miR-29b-3phsa-miR-15b-5phsa-miR-362-5phsa-miR-27a-5p hsa-miR-454-3phsa-miR-452-5phsa-miR-196b-5pDKO-1 , DLD-1 and DKs-8 hsa-miR-32-5phsa-miR-582-3phsa-miR-542-3phsa-miR-96-5p hsa-miR-101-3phsa-miR-18a-5phsa-miR-3529-3phsa-miR-7-5p hsa-miR-19a-3phsa-miR-142-3phsa-miR-20a-5phsa-miR-32-3p hsa-miR-130b-5phsa-miR-1278hsa-miR-7-1-3phsa-miR-590-3p hsa-miR-4473hsa-miR-17-5phsa-miR-103a-3phsa-miR-103b hsa-miR-19b-3phsa-miR-340-5phsa-miR-200a-3phsa-miR-34a-5p hsa-miR-372*miRNAs differentially enriched in cells when comparing mean reads in exosomes vs cell . miRNAs expression patterns were compared between DKs-8 , DKO-1 , and DLD-1 cells .",
"miRNAs were identified that were enriched in just one of the three cell types or that overlapped between combinations of cells .",
"For miRNAs , 25 were identified that are expressed in all three cell types , 13 were enriched in DKs-8 cells , 15 in DLD-1 cells , and 39 were unique to DKO-1 cells . 10 . 7554/eLife . 07197 . 012Table 2 . Differential distribution of miRNAs in exosomes*DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 012DKO-1 hsa-miR-139-5phsa-miR-3178hsa-miR-151bhsa-miR-125b-1-3p hsa-miR-193b-3phsa-miR-935hsa-miR-130b-3phsa-miR-628-3p hsa-miR-139-3phsa-let-7d-3phsa-miR-589-3phsa-miR-4532 hsa-miR-451ahsa-miR-6087hsa-miR-151a-5phsa-miR-940 hsa-miR-222-3phsa-miR-766-5phsa-miR-505-5phsa-miR-3187-3p hsa-miR-125a-3phsa-miR-3679-5phsa-miR-4436b-3phsa-miR-4787-3p hsa-miR-2277-3phsa-miR-361-5phsa-miR-1293hsa-miR-3183 hsa-miR-3162-5phsa-miR-642a-3phsa-miR-642b-5phsa-miR-197-5p hsa-miR-324-3phsa-miR-145-3phsa-miR-3182hsa-miR-3127-3p hsa-miR-3127-5phsa-miR-4728-3phsa-miR-3184-5phsa-miR-125b-5p hsa-miR-186-5phsa-miR-1hsa-miR-100-5phsa-miR-423-3p hsa-miR-766-3phsa-miR-4753-5phsa-miR-145-5phsa-miR-4724-5p hsa-miR-373-3phsa-miR-223-5phsa-miR-1307-5phsa-miR-1914-3p hsa-miR-3121-3phsa-miR-3613-3phsa-miR-205-5phsa-miR-98-3p hsa-miR-23a-3phsa-miR-3124-5phsa-miR-3656hsa-miR-3918 hsa-miR-4449hsa-miR-378chsa-miR-3138hsa-miR-1910 hsa-miR-3174hsa-miR-4466hsa-miR-3679-3phsa-miR-3200-5p hsa-miR-6511b-5phsa-miR-1247-5phsa-miR-22-3phsa-miR-877-5p hsa-miR-4687-3phsa-miR-1292-5phsa-miR-181c-5phsa-miR-6131 hsa-miR-6513-5phsa-miR-3661hsa-miR-132-3phsa-miR-214-3p hsa-miR-574-3phsa-miR-3190-3phsa-miR-326hsa-miR-3191-5p hsa-miR-3198hsa-miR-3928hsa-miR-629-3phsa-miR-4489 hsa-miR-4700-5phsa-miR-5006-5phsa-miR-5088hsa-miR-2110 hsa-miR-3911hsa-miR-3146DKs-8 hsa-miR-1224-5phsa-let-7b-5phsa-miR-155-5phsa-let-7c hsa-let-7a-5phsa-miR-146b-5phsa-miR-4647hsa-miR-4494 hsa-miR-711hsa-miR-1263DLD-1 hsa-miR-1226-5phsa-miR-4745-5phsa-miR-4435hsa-miR-939-5p hsa-miR-409-3phsa-miR-1304-3pDKO-1 and DKs-8 hsa-miR-146a-5phsa-miR-4508hsa-miR-224-5phsa-miR-4429 hsa-miR-222-5phsa-miR-629-5phsa-miR-4492hsa-miR-3653 hsa-miR-320ahsa-miR-1290hsa-miR-1262hsa-miR-5010-5p hsa-miR-204-3phsa-miR-4461hsa-miR-5187-5pDKO-1 and DLD-1 hsa-miR-483-5phsa-miR-4658hsa-miR-4758-5phsa-miR-492 hsa-miR-5001-5phsa-miR-371a-5phsa-miR-1323hsa-miR-371b-3p hsa-miR-501-3phsa-miR-4446-3phsa-miR-6511a-5phsa-miR-30a-3p hsa-miR-4727-3pDLD-1 and DKs-8 hsa-miR-28-3phsa-miR-3934-5pDKO-1 , DLD-1 and DKs-8 hsa-miR-658hsa-miR-320dhsa-miR-4792hsa-miR-1246 hsa-miR-320ehsa-miR-4516hsa-miR-320bhsa-miR-4488 hsa-miR-1291hsa-miR-320chsa-miR-4634hsa-miR-3605-5p hsa-miR-4741hsa-miR-3591-3phsa-miR-122-5phsa-miR-486-3p hsa-miR-184hsa-miR-223-3phsa-miR-3651hsa-miR-486-5p hsa-miR-3180hsa-miR-3180-3phsa-miR-3168hsa-miR-4497 hsa-miR-423-5phsa-miR-3184-3phsa-miR-150-5phsa-miR-664a-5p hsa-miR-182-5p*miRNAs differentially enriched in exosomes when comparing mean reads in exosomes vs cell . miRNAs expression patterns were compared between exosomes purified from DKs-8 , DKO-1 , and DLD-1 cells .",
"miRNAs were identified that were enriched in exosomes from just one of the three cell lines or that overlapped between combinations of cell lines .",
"29 miRNAs were common between exosomes from all three cell lines .",
"94 were enriched in exosomes from DKO-1 cells , 10 in exosomes from DKs-8 cells , and only 6 in exosomes from DLD-1 cells . 10 . 7554/eLife . 07197 . 013Figure 3 . KRAS-dependent regulation of miRNAs in exosomes and cells . Differentially distributed miRNAs in ( A ) cells and ( B ) exosomes from the three CRC cell lines differing in KRAS status .",
"( C ) qRT-PCR validation of selected miRs from DKs-8 and DKO-1 cellular and exosomal RNA samples normalized to U6 snRNA .",
"Fold changes were calculated using the ΔΔC ( t ) method comparing exosomes to cells .",
"Negative fold changes indicate greater enrichment in cells , and positive fold changes indicate greater enrichment in exosomes .",
"Also see Supplementary file 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 013 Because we observed differential export of specific miRNAs , we investigated whether there might be miRNA sequence-specific sorting signals .",
"Previous reports have shown differential accumulation of 5p or 3p strands in exosomes compared to parental cells ( Ji et al . , 2014 ) .",
"Thus , we analyzed our data sets to test whether exosomes might be preferentially enriched for one strand over the other .",
"We were able to identify individual miRNAs where the two strands differentially sorted between cells and exosomes .",
"For example , the -5p strands of miR-423 were overrepresented in DKO-1 exosomes but in exosomes from DKs-8 cells , both strands were overrepresented compared to cells ( data not shown ) .",
"This indicates that KRAS status may differentially affect selection of passenger or guide strands for sorting into exosomes for select individual miRNAs .",
"Individual miRNAs often exist as populations of variants ( isomiRs ) that differ in length and/or nucleotide composition generated by template- or non-template-directed variation ( Burroughs et al . , 2010; Newman et al . , 2011; Neilsen et al . , 2012 ) .",
"When we analyzed our sequencing data sets , we did not detect differential accumulation of isomers with variable 5′ termini ( data not shown ) .",
"For cellular miRNAs , most reads were full length with a slight enrichment in 3′ non-templated addition of A-tailed miRNAs , regardless of KRAS status ( Figure 4; Figure 4—figure supplement 1 ) .",
"For exosomes , we observed a slight enrichment for C residues added to the 3′ ends of miRNAs from wild-type KRAS cells ( Figure 4—figure supplement 1 ) .",
"We did not observe this in mutant KRAS exosomes , where instead , we noticed an increase in 3′ trimming of miRNAs ( Figure 4 , Figure 4—figure supplement 2 ) .",
"Overall , it remains to be determined whether such modifications constitute a global exosomal sorting signal in these cells . 10 . 7554/eLife . 07197 . 014Figure 4 . Comparison of miRNA 3′ trimming and tailing between cells and exosomes . Data from the heat maps shown in Figure 4—figure supplement 2 were pooled to illustrate overall changes in either 3′ nucleotide additions ( tailing ) or 3′ resection ( trimming ) compared to full length miRNA sequences ( intact ) .",
"Overall , the patterns between cells and between exosomes are very similar .",
"A comparison of cells to exosomes shows that exosomes display a slight increase in trimmed miRNAs .",
"Also see Figure 4—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 01410 . 7554/eLife . 07197 . 015Figure 4—figure supplement 1 . Non-templated addition ( NTA ) of nucleotides to 3′ ends of miRNAs . 3′ NTA of A-tailed miRNAs is enriched in cells independent of KRAS status , whereas NTA of C residues are more abundant in wild-type KRAS DKs-8 exosomes .",
"miRNAs with read counts ≥500 reads in both cells and exosomes were used in the analysis .",
"Reads mapping to hairpin sequences were considered as templated miRNAs ( untrimmed ) .",
"Reads ≥18 nucleotides that did not map to hairpin or genome sequences were trimmed one nucleotide at the 3′-termini and mapped again to hairpin sequences .",
"This was repeated three times to account for NTA's extending up to three nucleotides from the 3′-terminus .",
"miRNAs significantly enriched in exosomes or cells are represented by red and blue circles , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 01510 . 7554/eLife . 07197 . 016Figure 4—figure supplement 2 . Comparison of miRNA 3′ trimming and tailing between cells and exosomes . Heat maps show the extent of either 3′ nucleotide additions ( tailing ) or 3′ resection ( trimming ) compared to full-length miRNA sequences ( intact ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 01610 . 7554/eLife . 07197 . 017Figure 4—figure supplement 3 . MEME analysis of miRNA sequence in exosomes . MEME analysis was performed to attempt to identify sequence motifs that might target miRNAs for export into exosomes .",
"The top most abundant motifs found in miRNAs in Tables 1 , 2 are shown for both cells and exosomes from DKO-1 , DKs-8 , or DLD-1 cell lines .",
"( Upregulated in DKO-1 exo: 51 , 1 . 5e-009; Upregulated in DKO-1 cell: 28 , 1 . 5e-007; Upregulated in DKs-8 exo , 22 , 1 . 7e-010; Upregulated in DKs-8 cell , 19 , 7 . 6e-007; Upregulated in DLD-1 exo , 8 , 2 . 3e-003; Upregulated in DLD-1 cell 23 , 3 . 3e-012 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 017 Consistent with published data , we have shown that miRNA expression patterns vary between parental cells and their cognate exosomes ( Tables 1 , 2 , Figure 3A , B ) ( Valadi et al . , 2007; Mittelbrunn et al . , 2011; Ekstrom et al . , 2012; Montecalvo et al . , 2012; Squadrito et al . , 2014 ) .",
"Differential export suggests that specific signals must exist to sort distinct miRNAs ( Batagov et al . , 2011; Villarroya-Beltri et al . , 2013 ) .",
"We therefore conducted MEME analysis to attempt to identify sequence motifs that might serve as targeting signals .",
"When we examined all miRNA reads detected in exosomes , we did not find any global enrichment for specific sequences or motifs , including those reported to be bound by hnRNP A2B1 ( GGAG or U/CC ) ( Bolukbasi et al . , 2012; Villarroya-Beltri et al . , 2013 ) ( Figure 4—figure supplement 3 ) .",
"However , when we analyzed miR-320 because it is preferentially exported to exosomes independent of KRAS status , we were able to identify the GGAG sequence contained within the 3′ end of the mature sequence .",
"Additionally , upon restricting our analysis to reads from the most differentially expressed miRNAs when comparing exosomes to cells , we found a slight enrichment for C residues , possibly alternating C residues in exosomal miRNAs ( Figure 4—figure supplement 3 ) .",
"Although little is understood regarding the molecular mechanisms for packaging exosomal miRNAs , recent evidence suggests that the secretion of miRNAs in exosomes is dependent on ceramide via its production by neutral sphingomyelinase 2 ( nSMase",
"2 ) ( Kosaka et al . , 2010; Mittelbrunn et al . , 2011 ) .",
"Inhibition of de novo ceramide synthesis by treatment with a nSMase inhibitor impaired exosomal miRNA release , apparently due to decreased formation of miRNA-containing exosomes ( Kosaka et al . , 2010; Mittelbrunn et al . , 2011 ) .",
"To test the role of nSMase in miRNA secretion in our system , we treated CRC cells with the nSMase inhibitor , GW4869 .",
"We determined the effect of this inhibitor on miR-10b since it is preferentially found in wild-type KRAS DKs-8 exosomes , miR-100 since it is preferentially found in mutant KRAS DKO-1 and DLD-1 exosomes , and miR-320 which sorts into exosomes regardless of KRAS status .",
"For miR-10b , we did not observe significant changes in its cellular levels after treatment with GW4869 in either wild-type KRAS DKs-8 or mutant KRAS DKO-1 cells ( Figure 5C ) .",
"In contrast , inhibition of nSMase caused a ∼threefold increase in intracellular levels of miR-100 in mutant KRAS DKO-1 cells but remained unchanged in wild-type DKs-8 KRAS cells ( Figure 5A , B , C ) .",
"Similarly , miR-320 levels were found to increase ( ∼2 . 5 fold ) only in GW4869-treated mutant KRAS DKO-1 cells ( Figure 5C ) .",
"These data are most consistent with the hypothesis that impaired ceramide synthesis alters cellular accumulation of miRNAs dependent on mutant KRAS and suggest that multiple biogenic routes exist for miRNA secretion . 10 . 7554/eLife . 07197 . 018Figure 5 . Ceramide-dependent miRNA export into exosomes . DKO-1 or DKs-8 cells were treated with an inhibitor of neutral sphingomyelinase 2 ( nSMase 2 ) , GW4869 .",
"After treatment , in situ hybridization experiments were performed with probes against miR-100 ( A , B ) .",
"( C ) qRT-PCR for miR-10b , miR-100 , and miR-320a was performed on cells treated with GW4869 or DMSO , and fold change in expression was determined in treated vs untreated cells .",
"In wild-type KRAS cells ( DKs-8 ) , inhibition of nSMase 2 had little or no effect on the cellular levels of these miRNAs .",
"In contrast , mutant KRAS cells ( DKO-1 ) showed an increase in cellular miRNA levels after inhibition of nSMAse 2 .",
"Data were derived from three biological replicates and performed in technical triplicates for qRT-PCR .",
"Significance was determined by two-tailed , paired t-tests where * are p values ≤ 0 . 05 and ** ≤0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 018 Several reports have found that extracellular miRNAs can be taken up by recipient cells to mediate heterotypic cell–cell interactions and facilitate target repression in neighboring cells ( Mittelbrunn et al . , 2011; Boelens et al . , 2014; Squadrito et al . , 2014 ) .",
"To determine whether secreted miRNAs function in recipient cells , we designed luciferase ( Luc ) constructs containing either 3 perfect miR-100 recognition elements ( MREs ) in the 3′ untranslated region ( UTR ) ( Luc-100-PT ) or scrambled 3′UTR sequences that do not match any known miRNAs ( Luc-CTL ) .",
"These constructs were expressed in wild-type KRAS DKs-8 cells ( recipient cells ) in the presence or absence of donor cells .",
"Baseline repression of Luc in the absence of donor cells was first analyzed to determine the levels of repression from endogenous miR-100 in DKs-8 cells .",
"Compared to the scrambled control ( Luc-CTL ) , strong Luc repression in the absence of donor cells was observed with perfect MREs ( miR-100-PT ) ( Figure 6A ) .",
"This supports our finding that miR-100 is expressed and retained in DKs-8 cells . 10 . 7554/eLife . 07197 . 019Figure 6 . Transfer of extracellular miRNAs by mutant DKO-1 cells promotes target repression in wild-type DKs-8 cells . Transwell co-culture of DKs-8 recipient cells with or without DKs-8 or DKO-1 donor cells .",
"Luciferase ( Luc ) expression was measured in DKs-8 recipient cells transiently expressing ( A ) Luc fused to three perfectly complementary synthetic miR-100 target sites ( miR-100-PT ) or ( B ) Luc fused to the 3′UTR of mTOR , which harbors 3 endogenous target sites for miR-100 .",
"( C ) Luc expression increased upon mutation of two ( MS2 ) sites with full expression upon mutation of all three sites ( MS3 ) .",
"Luc-CTL contains three random scrambled target sites that do not match any known miRNA sequence .",
"( D ) Luc expression was restored in recipient cells expressing miR-100-PT upon pretreatment of donor DKO-1 cells with 100 nM miR-100 antagomirs ( AI-100 ) compared to pre-treatment of donor DKO-1 cells with 100 nm control antagomirs ( AI-CTL ) targeting cel-miR-67 .",
"( E ) Taqman qRT-PCR for miR-100 .",
"Compared to DKs-8 recipient cells grown without donor cells , mir-100 levels increased by approximately 34% in the presence of mutant DKO-1 donor cells pre-treated with AI-CTL compared to an 8% increase in AI-100 pre-treated donor cells .",
"Y axis is % increase in miR-100 = ( CPAI-CTL or CPAI-100 − CPno donor/CPno donor ) *100 , where CP = absolute copy number .",
"All Luc values were normalized to co-transfected vectors expressing β-galactosidase; n = 3 independent experiments in A–C and n = 4 in D , E . All Luc assays were performed in technical triplicate .",
"Significance was determined by two-tailed , paired t-tests where * are p values ≤ 0 . 05 and ** ≤0 . 01 .",
"Also see Figure 6—figure supplements 1–3 . DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 01910 . 7554/eLife . 07197 . 020Figure 6—figure supplement 1 . miR-100 binding sites in the mTOR 3′UTR . miR-100 binding sites within the mTOR 3′UTR .",
"Mutated nucleotides indicated in red ( oligonucleotide sequences in Supplementary file 3 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 02010 . 7554/eLife . 07197 . 021Figure 6—figure supplement 2 . Presence of mutant DKO-1 donor cells augments miR-100 levels in DKs-8 recipient cells . Taqman qRT-PCR for miR-100 .",
"miR-100 levels increased by approximately 114 copies in recipient cells cultured with DKO-1 donor cells pre-treated with AI-CTL compared to recipient cells cultured without donor cells .",
"Pre-treatment of mutant DKO-1 donor cells with miR-100 antagomir inhibitor ( AI-100 ) attenuated this effect .",
"Absolute levels of miR-100 determined by standard curve generation of synthetically derived miR-100 ( see ‘Materials and methods’ ) .",
"Approximately , 357 . 23 ± 16 . 65 , 442 . 58 ± 12 . 59 , and 329 . 48 ± 13 . 62 copies of miR-100 per input RNA were found in DKs-8 recipient cells cultured with DKO-1 donor AI-100 , DKO-1 donor AI-CTL and without donor cells , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 02110 . 7554/eLife . 07197 . 022Figure 6—figure supplement 3 . Transfer of extracellular miRNAs by mutant DKO-1 cells promotes target repression in wild-type DKs-8 cells . Transwell co-culture of DKs-8-recipient cells with or without DKs-8 or DKO-1 donor cells .",
"Luc expression was measured in DKs-8-recipient cells expressing Luc fused to three perfectly complementary synthetic miR-222 target sites ( miR-222-PT ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07197 . 022 To determine whether secretion of miR-100 by mutant KRAS DKO-1 donor cells could further augment miR-100 function in recipient wild-type cells , Transwell co-culture experiments were performed with DKs-8 recipient cells expressing the Luc reporters in the presence of DKO-1 donor cells ( Figure 6 ) .",
"Significantly increased repression of Luc was observed when the reporter construct containing three perfect miR-100 sites was used ( miR-100-PT ) ( Figure 6A ) .",
"Because exosomes released from DKO-1 cells contain abundant levels of miR-100 , increased Luc repression is consistent with transfer of additional copies of miR-100 .",
"Two control experiments were performed to test the hypothesis that additional copies of miR-100 are transferred between donor and recipient cells .",
"First , we treated donor cells with antagomirs that block production of miR-100 .",
"Luc repression was almost completely reversed upon pre-treatment of DKO-1 donor cells with a miR-100 hairpin antagomir inhibitor ( AI-100 ) ( Figure 6D ) .",
"Second , we performed qRT/PCR to calculate the increase in miR-100 levels in recipient cells .",
"Cells grown in the presence or absence of donor cells showed an approximate 34% increase in the levels of miR-100 ( Figure 6E and Figure 6—figure supplement 2 ) .",
"To further probe the repressive activity of miR-100 , we performed co-culture experiments in which the recipient Dks-8 cells express Luc fused to the 3′UTR of mTOR , an endogenous miR-100 target ( Nagaraja et al . , 2010; Grundmann et al . , 2011; Ge et al . , 2014 ) .",
"As observed with miR-100-PT repression , Luc-mTOR was significantly repressed in the presence of mutant KRAS DKO-1 but not in the presence of wild-type KRAS DKs-8 donor cells ( Figure 6B ) .",
"This suggests that miR-100-repressive activity is specific to the presence of mutant KRAS DKO-1 donor cells .",
"To confirm these results , we mutated the MREs within the mTOR 3′UTR and assayed for miR-100 activity ( Figure 6—figure supplement 1 ) .",
"Mutation of individual sites did not show significantly different Luc repression ( data not shown ) .",
"However , upon mutation of two MREs ( MS2 ) , we observed a partial rescue of Luc expression ( Figure 6C ) .",
"This was further augmented upon mutation of all three sites ( MS3 ) , with a complete rescue of miR-100-mediated repressive activity ( Figure 6C ) .",
"As a final test of miRNA transfer in the Transwell co-culture experiments , we created vectors expressing Luc fused to a 3′UTR containing perfect sites for miR-222 because miR-222 is not detectable in DKs-8-recipient cells , unlike miR-100 .",
"In this case , silencing of Luc should be due to transfer of miR-222 and not due to unforeseen changes in endogenous miRNA activity .",
"We observed a greater than twofold repression of the miR-222 Luc reporter in recipient cells ( Figure 6—figure supplement 3 ) .",
"These results support the hypothesis that miRNAs secreted by mutant KRAS cells can be transferred to recipient cells ."
],
[
"In this study , we comprehensively examined the composition of small ncRNAs from exosomes and cells of isogenic CRC cell lines that differ only in KRAS status .",
"By employing small RNA transcriptome analyses , we found that oncogenic KRAS selectively alters the miRNA profile in exosomes , and that ceramide depletion selectively promotes miRNA accumulation in mutant KRAS CRC cells .",
"Distinct miRNA profiles between cells and their exosomes may be functionally coupled to mitogenic signaling .",
"KRAS status-specific patterns of secreted miRNAs support the idea of using exosomes as potential biomarkers in CRC .",
"Our finding that miR-10b is preferentially enriched in wild-type KRAS-derived exosomes , while miR-100 is enriched in mutant KRAS-derived exosomes raises interesting questions regarding how they are selected for secretion .",
"miR-10b and miR-100 are both part of the miR-10/100 family and differ by only one base in the seed region , allowing regulation of distinct sets of target mRNAs ( Tehler et al . , 2011 ) .",
"Whether the accumulation or export of these miRNAs is a result or a consequence of oncogenic signaling remains unknown .",
"Preventing the export or retention of certain miRNAs , such as miR-100 and miR-10b , may serve a therapeutic role in reversing the tumorigenic effects seen with aberrant miRNA expression .",
"KRAS-dependent differential miRNA expression more prominently affected miRNA expression patterns observed in exosomes than in the parent cells .",
"This could reflect a mechanism by cells to selectively export miRNAs so as to maintain specific growth or gene expression states .",
"This is consistent with a recent report that found that the cellular levels of miR-218-5p could be maintained , despite changes in the abundance of its target , likely through a ‘miRNA relocation effect’ where unbound miRNAs that are in excess have the potential to be sorted to exosomes ( Squadrito et al . , 2014 ) .",
"Another mechanism may be through sequence-specific motifs that direct miRNA trafficking by interaction with specific chaperone proteins ( Bolukbasi et al . , 2012; Villarroya-Beltri et al . , 2013 ) .",
"Although we did not find any globally significant motif overrepresented in exosomal miRNAs , we cannot rule out that individual miRNAs might undergo sequence-specific export .",
"miR-320 family members all contain the GGAG motif that has been proposed to serve as an exosomal targeting signal ( Villarroya-Beltri et al . , 2013 ) .",
"We found that members of the miR-320 family are preferentially enriched in exosomes independent of KRAS status; however , the GGAG sequence was not found in other miRNAs that are targeted to exosomes .",
"It has been reported that the biogenesis of miR-320 family members occurs by a non-canonical pathway that requires neither Drosha ( Chong et al . , 2010 ) nor XPO5 ( Xie et al . , 2013 ) .",
"Instead , the 5′ ends contain a 7-methyl guanosine cap that facilitates nuclear–cytoplasmic transport through XPO1 ( Xie et al . , 2013 ) .",
"XPO1 is present in DKO-1 , DKs-8 , and DLD-1 exosomes as detected by mass spectrometry ( Demory Beckler et al . , 2013 ) .",
"It will be interesting to investigate whether alternate processing pathways and associated biogenic machinery contribute to the heterogeneity of EV cargo and affect miRNA secretion .",
"It was recently demonstrated that miRNAs in B-cell exosomes display enriched levels of non-template-directed 3′-uridylated miRNAs , while 3′-adenylated miRNA species are preferentially cell enriched ( Koppers-Lalic et al . , 2014 ) .",
"In certain contexts , the addition of non-templated uridine residues to cognate miRNAs accelerates miRNA turnover ( Baccarini et al . , 2011; Wei et al . , 2012 ) .",
"Thus , it is possible that the stability/half-life of a miRNA affects whether it is retained or secreted .",
"While the exact functional significance of 3′-end modifications of miRNAs detected in both cells and exosomes remains to be determined , it could be that differential export of ‘tagged’ miRNAs could allow cells to export specific miRNAs .",
"However , the lack of any apparent motif upon global analysis of miRNAs enriched in exosomes , coupled to the finding that even untagged miRNAs are differentially exported , suggests multiple strategies for loading of miRNAs into EVs , and that not all EVs and exosomes contain identical cargo .",
"This further implies that different cell types secrete a heterogeneous population of vesicles .",
"Although the biological relevance of these findings remains to be determined , the specific sorting of miRNAs into exosomes may enable cancer cells to discard tumor-suppressive miRNAs so as to increase their oncogenic potential or perhaps modulate gene expression in neighboring and distant cells to promote tumorigenesis .",
"In support of this hypothesis , miR-100 , which we found to be enriched in mutant KRAS exosomes , was found to down-regulate LGR5 in CRC cells and thereby inhibit migration and invasion of such cells ( Zhou et al . , 2015 ) .",
"In this context , removal of miR-100 from the cell would be a tumor-promoting event .",
"In other contexts , miR-100 can have contradictory activities , both inducing EMT by down-regulating E-cadherin through targeting SMARCA5 and inhibiting tumorigenicity by targeting HOXA1 ( Chen et al . , 2014 ) .",
"Thus , although miR-100 can function as a tumor suppressor under normal conditions , augmenting its levels , for example , by EV uptake , could potentially promote EMT .",
"In this regard , the role of miR-100 in tumorigenesis would be twofold , where its secretion in exosomes could function to maintain low-intracellular levels within mutant cells , while inducing EMT in wild-type-recipient cells .",
"Along these lines , miR-100 is part of the miR-125b/let-7a-2/miR-100 cluster that is transcribed and expressed coordinately ( Emmrich et al . , 2014 ) .",
"Interestingly , in malignant colonic tissues from individuals with CRC , miR-100 levels were significantly decreased while let-7a levels were strongly upregulated ( Tarasov et al . , 2014 ) .",
"Based on our finding that there is differential accumulation of individual miRNAs within this cluster between mutant KRAS cells and exosomes , it will be interesting to determine whether cancer cells down-regulate specific miRNAs by active secretion , while simultaneously maintaining the levels of other miRNAs transcribed within the same cluster .",
"miRNAs are secreted from malignant breast epithelial cells after packaging into vesicles larger than conventional exosomes that are enriched in CD44 , whose expression is linked to breast cancer metastasis ( Palma et al . , 2012 ) .",
"Normal cells tend to release miRNAs in more homogenous types of exosomes , suggesting that malignant transformation may alter the formation of secreted vesicles that could alter miRNA export and lead to differences in exosome content and morphology ( Palma et al . , 2012; Melo et al . , 2014 ) .",
"In support of this , it was recently shown that in exosomes from breast cancer cells , CD43 mediates the accumulation of Dicer ( Melo et al . , 2014 ) .",
"These exosomes also contain other RNA-induced silencing complex ( RISC ) proteins and pre-miRNAs , indicating that miRNA processing can occur in exosomes ( Melo et al . , 2014 ) .",
"These components were absent in exosomes derived from normal cells .",
"It remains to be determined whether components of the RISC-loading complex assemble within endosomes before their secretion as exosomes or by the fusion of exosomes containing heterogeneous cargo after they are secreted .",
"The observation that cells can selectively release miRNAs and also release a heterogeneous population of vesicles raises the possibility that differential release of miRNAs is associated with different classes of exosomes and microvesicles .",
"Recently , quantitative analysis of secreted miRNAs suggested that the levels of extracellular miRNAs are limited and raise the question as to how such levels can alter gene expression in recipient cells ( Chevillet et al . , 2014 ) .",
"The results of our Transwell co-culture experiments are most consistent with extracellular transfer of specific miRNAs to alter expression of reporter constructs .",
"Nevertheless , the level of exosomal transfer that is needed to alter recipient cell gene expression in vivo remains an open question .",
"Our finding that mutant KRAS protein can be functionally transferred in exosomes indicates that the full effect of exosomes on recipient cells can be due to a combination of both RNA delivery and protein-based signaling ( Higginbotham et al . , 2011 ) .",
"This could include activation of Toll-like receptors with possible downstream effects following nuclear factor kappa-light-chain-enhancer of activated B cells or mitogen-activated protein kinase cascades ( Fabbri et al . , 2012; Chen et al . , 2013 ) .",
"The complexity of miR-100 function in the tumor microenvironment underscores this argument by its potential for inhibiting mTOR expression which is required for proliferation of Apc-deficient tumors in mouse models ( Faller et al . , 2015 ) .",
"In tumors where some cells have incurred activating mutations in KRAS , while others have not , miR-100 could accumulate in wild-type KRAS tumor cells through exosomal transfer , inhibiting mTOR and cell growth .",
"Conversely , miR-100 could be secreted from mutant KRAS cells giving them a growth advantage .",
"In this way , exosomal transfer of miRNAs might act to select for cells carrying specific tumor driver mutations .",
"Our studies have direct implications for CRC and , together with other studies , indicate that delivery of exosomes to recipient cells can induce cell migration , inflammation , immune responses , angiogenesis , invasion , pre-metastatic niche formation , and metastasis ( Kahlert and Kalluri , 2013; Boelens et al . , 2014; Melo et al . , 2014; Costa-Silva et al . , 2015 ) ."
],
[
"Exosomes were isolated from conditioned medium of DKO-1 , Dks-8 , and DLD-1 cells as previously described , with slight modification ( Higginbotham et al . , 2011 ) .",
"Briefly , cells were cultured in Dulbecco's Modified Eagle's Medium ( DMEM ) supplemented with 10% bovine growth serum until 80% confluent .",
"The cells were then washed three times with Phosphate buffered saline ( PBS ) and cultured for 24 hr in serum-free medium .",
"The medium was collected and replaced with ionomycin-containing medium for 1 hr , after which ionomycin-containing medium was collected and pooled with the previously collected serum-free medium .",
"Pooled media was centrifuged for 10 min at 300×g to remove cellular debris , and the resulting supernatant was then filtered through a 0 . 22-mm polyethersulfone filter ( Nalgene , Rochester , NY , USA ) to reduce microparticle contamination .",
"The filtrate was concentrated ∼300-fold with a 100 , 000 molecular weight cut-off centrifugal concentrator ( Millipore , Darmstadt , Germany ) .",
"The concentrate was then subjected to high-speed centrifugation at 150 , 000×g for 2 hr .",
"The resulting exosome-enriched pellet was resuspended in PBS containing 25 mM hydroxyethyl-piperazineethanesulfonic acid ( HEPES ) ( pH 7 . 2 ) and washed by centrifuging again at 150 , 000×g for 3 hr .",
"The wash steps were repeated a minimum of three times until no trace of phenol red was detected .",
"The resulting pellet was resuspended in PBS containing 25 mM HEPES ( pH 7 . 2 ) , and protein concentrations were determined with a MicroBCA kit ( Pierce/Thermo , Rockford , IL , USA ) .",
"The number of exosomes per μg of protein was determined by means of nanoparticle tracking analysis ( NanoSight , Wiltshire , UK ) .",
"Analysis was performed on three independent preparations of exosomes .",
"Total RNA from exosomes and cells was isolated using TRIzol ( Life Technologies/Thermo , Grand Island , NY ) .",
"In the case of exosomal RNA isolation , TRIzol was incubated with 100 μl or less of concentrated exosomes for an extended 15 min incubation prior to chloroform extraction .",
"RNA pellets were resuspended in 60 μl of RNase-free water and were then re-purified using the miRNeasy kit ( Qiagen Inc . , Valencia , CA , USA ) .",
"Final RNAs were eluted with two rounds of 30 μl water extraction .",
"Total RNA from each sample was used for small RNA library preparation using NEBNext Small RNA Library Prep Set from Illumina ( New England BioLabs Inc . , Ipswich , MA , USA ) .",
"Briefly , 3′ adapters were ligated to total input RNA followed by hybridization of multiplex single read ( SR ) reverse transcription ( RT ) primers and ligation of multiplex 5′ SR adapters .",
"RT was performed using ProtoScript II RT for 1 hr at 50°C .",
"Immediately after RT reactions , PCR amplification was performed for 15 cycles using LongAmp Taq 2× master mix .",
"Illumina-indexed primers were added to uniquely barcode each sample .",
"Post-PCR material was purified using QIAquick PCR purification kits ( Qiagen Inc . ) .",
"Post-PCR yield and concentration of the prepared libraries were assessed using Qubit 2 . 0 Fluorometer ( Invitrogen , Carlsbad , California , CA , USA ) and DNA 1000 chip on Agilent 2100 Bioanalyzer ( Applied Biosystems , Carlsbad , CA , USA ) , respectively .",
"Size selection of small RNA with a target size range of approximately 146–148 bp was performed using 3% dye free agarose gel cassettes on a Pippin Prep instrument ( Sage Science Inc . , Beverly , MA , USA ) .",
"Post-size selection yield and concentration of libraries were assessed using Qubit 2 . 0 Fluorometer and DNA high-sensitivity chip on an Agilent 2100 Bioanalyzer , respectively .",
"Accurate quantification for sequencing applications was performed using qPCR-based KAPA Biosystems Library Quantification kits ( Kapa Biosystems , Inc . , Woburn , MA , USA ) .",
"Each library was diluted to a final concentration of 1 . 25 nM and pooled in equimolar ratios prior to clustering .",
"Cluster generation was carried out on a cBot v8 . 0 using Illumina's Truseq Single Read Cluster Kit v3 . 0 .",
"Single-end sequencing was performed to generate at least 15 million reads per sample on an Illumina HiSeq2000 using a 50-cycleTruSeq SBSHSv3 reagent kit .",
"Clustered flow cells were sequenced for 56 cycles , consisting of a 50-cycle read , followed by a 6-cycle index read .",
"Image analysis and base calling were performed using the standard Illumina pipeline consisting of Real Time Analysis version v1 . 17 and demultiplexed using bcl2fastq converter with default settings .",
"Read sequence quality checks were performed by FastQC ( Babraham Bioinformatics [http://www . bioinformatics . babraham . ac . uk/projects/fastqc/] ) .",
"Adapters from the 3′ ends of reads were trimmed using Cutadpt with a maximum allowed error rate of 0 . 1 ( Martin , 2011 ) .",
"Reads shorter than 15 nucleotides in length were excluded from further analysis .",
"Reads were mapped to the human genome hg19 using Bowtie version 1 . 1 . 1 ( Langmead and Salzberg , 2012 ) .",
"Mapped reads were annotated using ncPRO-seq ( Chen et al . , 2012 ) based on miRbase ( Griffiths-Jones et al . , 2008 ) , Rfam ( Gardner et al . , 2011; Burge et al . , 2013 ) , and RepeatMasker ( http://www . repeatmasker . org/ ) , and expression levels were quantified based on read counts .",
"Mature miRNA annotation was extended 2 bp in both upstream and downstream regions to accommodate inaccurate processing of precursor miRNAs .",
"Reads with multiple mapping locations were weighted by the number of mapping locations .",
"DESeq Version 1 . 16 . 0 was used to perform PC analyses ( Anders and Huber , 2010 ) .",
"Differential expression was analyzed using DESeq Version 1 . 16 . 0 ( Anders and Huber , 2010 ) .",
"Negative binomial distribution was used to compare miRNA abundance between cells vs exosomes and wild-type vs mutant KRAS status .",
"The trimmed mean of M values method was used for normalization ( Robinson and Oshlack , 2010 ) .",
"Differential expression was determined based on log2 fold change ( log2 fold change ) and false discovery rate ( FDR ) with |log2 fold change| ≥ 1 and FDR ≤ 0 . 001 .",
"Trimming and tailing analysis was based on miRBase annotation ( Griffiths-Jones et al . , 2006 , 2008; Griffiths-Jones , 2010 ) .",
"Only high-confidence miRNAs ( 544 ) and corresponding hairpin sequences were used .",
"Bowtie version 1 . 1 . 1 with 0 mismatch was used for mapping .",
"miRNA reads were first mapped to hairpin sequences with unmapped reads , then mapped to the human genome hg19 .",
"Remaining reads were trimmed 1 bp from the 3′ end and remapped to hairpin sequences .",
"The remapping process was repeated 10 times .",
"Finally , all mapped reads were collected for further analysis .",
"Taqman small RNA assays ( Life Technologies ) ( individual assay numbers are listed below ) were performed for indicated miRNAs on cellular and exosomal RNA samples .",
"Briefly , 10 ng of total RNA was used per individual RT reactions; 0 . 67 μl of the resultant cDNA was used in 10 μl qPCR reactions .",
"qPCR reactions were conducted in 96-well plates on a Bio-Rad CFX96 instrument .",
"All C ( t ) values were ≤30 .",
"Triplicate C ( t ) values were averaged and normalized to U6 snRNA .",
"Fold changes were calculated using the ∆∆C ( t ) method , where ∆ = C ( t ) miRNA − C ( t ) U6 snRNA , and ∆∆C ( t ) = ∆C ( t ) exo − ∆C ( t ) cell , and FC = 2−∆∆C ( t ) .",
"Analysis was performed on three independent cell and exosomal RNA samples .",
"Taqman probe #: U6 snRNA: 001973; hsa-let-7a-5p: 000377; hsa-miR-100-5p: 000437; hsa-miR-320b: 002844; hsa-miR-320a: 002277 .",
"RNase-free , HPLC-purified 5′-phosphorylated miRNA oligoribonucleotides were synthesized ( Integrated DNA Technologies ) for human miR-100-5p ( 5′-phospho-AACCCGUAGAUCCGAACUUGUG-OH-3′ ) and cel-miR-39-3p ( 5′-phospho-UCACCGGGUGUAAAUCAGCUUG-OH-3′ ) .",
"Stock solutions of 10 μM synthetic oligonucleotide in RNase-free and DNase-free water were prepared according to the concentrations and sample purity quoted by the manufacturer ( based on spectrophotometry analysis ) .",
"Nine twofold dilution series beginning with 50 pM synthetic oligonucleotide were used in 10 µl RT reactions ( Taqman small RNA assays ) , and qPCR was performed .",
"Each dilution was performed in triplicate from three independent experiments .",
"Linear regression was used to determine mean C ( t ) values plotted against log ( miRNA copies/µl ) .",
"Cells were plated in 6-well plates containing coverslips at a density of ∼2 . 5 × 105 cells and cultured in DMEM supplemented with 10% bovine growth serum for 24 hr .",
"The cells were then washed three times with PBS and cultured for 24 hr in serum-free medium containing either 5 μM GW4869 ( Cayman Chemicals # 13127 , Ann Arbor , MI , USA ) or DMSO .",
"Medium was removed and cells were washed three times with PBS and fixed with 4% Paraformaldehyde ( PFA ) for ∼15 min at room temperature .",
"After , cells were washed three times in DEPC-treated PBS and permeabilized in 70% ethanol for ∼4 hr at 4°C , and rehydrated in DEPC-treated PBS for 5 min .",
"Pre-hybridization was performed in hybridization buffer ( 25% formamide , 0 . 05 M EDTA , 4× saline-sodium citrate ( SSC ) , 10% dextran sulfate , 1X Denhardt’s solution 1 mg/ml Escherichia coli tRNA ) in a humidified chamber at 60°C for 60 min .",
"Hybridization buffer was removed and replaced with 10 nM of probe ( probe numbers are listed below ) diluted in hybridization buffer and incubated at either 55°C ( miR-100 and miR-10b ) or 57°C for scrambled and U6 probes for 2 hr .",
"Coverslips were then washed in series with pre-heated SSC at 37°C as follows: 4× SSC briefly , 2× SSC for 30 min , 1× SSC for 30 min , and 0 . 1× SSC for 20 min . miRNA detection was conducted using Tyramide Signal Amplification ( Perkin Elmer , # NEL741001KT , Waltham , MA , USA ) .",
"Briefly , coverslips were blocked in blocking buffer ( 0 . 1 M TRIS-HCl , pH 7 . 5 , 0 . 15 M NaCl , 0 . 5% Blocking Reagent [Roche , #11096176001 , Basel , Switzerland] ) at 4°C overnight .",
"Blocking buffer was replaced with anti-DIG-POD ( Roche , # 11207733910 ) diluted 1:100 in blocking buffer and incubated for 60 min .",
"Coverslips were washed three times , 5 min per wash , in wash buffer ( 0 . 1 M Tris-HCl , pH 7 . 5 , 0 . 15 M NaCl , 0 . 5% Saponin ) followed by incubation with 1× Fluorescein diluted in 1× amplification reagent for 5 min .",
"Fluorescent coverslips were then washed two times , 5 min per wash , in wash buffer .",
"To preserve fluorescent signals , coverslips were fixed with 2% PFA containing 2% Bovine serum albumin in 1× PBS for 15 min .",
"After fixation , coverslips were washed 2 times , 5 min per wash , in wash buffer , followed by a final wash in 1× PBS for 5 min .",
"Coverslips were then mounted in Prolong Gold ( Life Technologies ) and visualized on a Zeiss LSM510 at 63× objective .",
"3′-DIG labeled probes for in situ hybridizations-U6 snRNA: 99002-05; Scramble: 99004-05; miR-10b-5p: 38486-05; miR-100-5p: 18009-05 ( Exiqon , Woburn , MA , USA ) .",
"Recipient cells were plated in six-well plates at a density of ∼2 . 5 × 105 cells and cultured in DMEM supplemented with 10% bovine growth serum for 24 hr .",
"Media was replaced and cells were co-transfected ( Promega , E2311 , Madison , WI , USA ) with 1 . 5 μg of Luc-reporter plasmid and 1 . 5 μg β-gal plasmid DNA/well .",
"Donor cells were plated in 0 . 4-μm polyester membrane Transwell filters ( Corning , 3450 , Corning , NY , USA ) at ∼2 . 5 × 105 cells/well for 24 hr .",
"Media from donor Transwells and recipient 6-well plates were removed and replaced with DMEM without FBS .",
"Co-culture of donor and recipient cells was conducted for either 24 or 48 hr before recipient cells were harvested .",
"Lysates were prepared in 1× Reporter lysis buffer ( Promega , E2510 ) , and Luc assays were performed according to the manufacturer's protocol ( Promega , E2510 ) .",
"β-gal expression was simultaneously determined from the lysates according to the manufacturer's protocol ( Promega , E2000 ) .",
"Differences in transfection efficiency were accounted for by normalizing Luc expression to β-Gal expression ( Luc/β-Gal ) .",
"All assays were performed on three biological replicates , each with three technical replicates .",
"Donor cells were plated in 0 . 4-μm polyester membrane Transwell filters ( Corning , 3450 , Corning , NY , USA ) at ∼1 . 4 × 104 cells/well for 24 hr .",
"Medium was replaced and donor cells were transfected with either miR-100 hairpin antagomirs ( # IH-300517-05 , GE Life Sciences ) or negative control hairpin antagomirs corresponding to cel-miR-67 ( # IN-001005-01 , GE Life Sciences ) to produce a final concentration of 100 nM of antagomir for 24 hr .",
"Medium from donor Transwells and recipient 6-well plates was removed and replaced with DMEM without FBS .",
"Co-culture of donor and recipient cells was conducted for 24 hr before recipient cells were harvested for RNA isolation .",
"For the pLuc-mTOR construct , the 3′UTR of mTOR was PCR amplified ( primer sequences in Supplementary file 3 ) from genomic DNA isolated from DKs-8 cells .",
"The amplicon was cloned into pMiR-Report ( Life Technologies ) via SpeI/HinDIII restriction sites .",
"Mutation of miR-100 binding sites in mTOR 3′UTR ( MS ) was performed on pLuc-mTOR using forward or reverse primers targeting either all three MRE's , or MRE 2 and 3 with QuikChange Lightning Multi-Site Directed Mutagenesis ( Agilent , Santa Clara , CA , USA ) according to manufacturer's protocol .",
"To create the reporter construct containing three miR-100 perfect sites ( miR-100-PT ) , oligonucleotides ( Supplementary file 3 ) were annealed to produce a synthetic fragment containing the perfect sites with CTAGT and AGCTT overhangs .",
"The fragment was cloned into pMiR-report via SpeI/HinDIII restriction sites .",
"All plasmids were sequence verified ( GeneWiz , South Plainfield , NJ , USA ) ."
]
] | [
"Mutant KRAS colorectal cancer ( CRC ) cells release protein-laden exosomes that can alter the tumor microenvironment .",
"To test whether exosomal RNAs also contribute to changes in gene expression in recipient cells , and whether mutant KRAS might regulate the composition of secreted microRNAs ( miRNAs ) , we compared small RNAs of cells and matched exosomes from isogenic CRC cell lines differing only in KRAS status .",
"We show that exosomal profiles are distinct from cellular profiles , and mutant exosomes cluster separately from wild-type KRAS exosomes .",
"miR-10b was selectively increased in wild-type exosomes , while miR-100 was increased in mutant exosomes .",
"Neutral sphingomyelinase inhibition caused accumulation of miR-100 only in mutant cells , suggesting KRAS-dependent miRNA export .",
"In Transwell co-culture experiments , mutant donor cells conferred miR-100-mediated target repression in wild-type-recipient cells .",
"These findings suggest that extracellular miRNAs can function in target cells and uncover a potential new mode of action for mutant KRAS in CRC ."
] | [
"Cells use several different methods to control which genes are expressed to produce the proteins and RNA molecules that they need to work efficiently .",
"The first step of gene expression is to transcribe a gene to form an RNA molecule .",
"Protein-coding mRNA molecules can then be translated to make proteins .",
"However , many RNA transcripts do not encode proteins .",
"One example of these non-coding RNAs is a class of small RNAs called microRNAs ( miRNAs ) , which are predicted to target more than 60% of protein-coding genes and can control which proteins are made .",
"It was once thought that miRNAs exist only within the cell where they are synthesized .",
"Recently , however , miRNAs have been found outside the cell bound to lipids and proteins , or encased in extracellular vesicles , such as exosomes .",
"Exosomes are small bubble-like structures used by cells to export material into the space outside of cells .",
"Exosomes containing miRNAs can circulate throughout the body , potentially transferring information between cells to alter gene expression in recipient cells .",
"Many colorectal cancer cells have mutations in a gene that encodes a protein called KRAS .",
"In 2011 and 2013 , researchers found that the contents of the exosomes released from these mutant KRAS colorectal cancer cells can influence normal cells in ways that would help a cancer to spread .",
"Furthermore , the exosomes released from the KRAS mutant cells contain different proteins than non-mutant cells .",
"Now , Cha , Franklin et al . —including several researchers who worked on the 2011 and 2013 studies—show that exosomes released by mutant KRAS cells also contain miRNAs , and that these miRNAs are different from the ones exported in exosomes by cells with a normal copy of the KRAS gene .",
"In particular , several miRNAs that suppress cancer growth in a healthy cell are found at lower levels in mutant KRAS cells .",
"Instead , these miRNAs are highly represented in the exosomes that are released by the KRAS mutant cells .",
"When cells with a normal copy of the KRAS gene were exposed to the contents of the exosomes released from KRAS mutant cells , an important gene involved in cell growth was suppressed .",
"This indicates that the miRNAs exported from cancerous cells can influence gene expression in neighboring cells .",
"Getting rid of such cancer-suppressing miRNAs could give cancer cells a growth advantage over normal cells to promote tumor growth .",
"Cha , Franklin et al . also suggest that it might be possible to create a non-invasive test to detect colorectal cancer by monitoring the levels of circulating miRNAs in patients .",
"Potential treatments for the disease could also target these miRNAs ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"stem cells and regenerative medicine",
"developmental biology"
] | Single-cell analysis uncovers that metabolic reprogramming by ErbB2 signaling is essential for cardiomyocyte proliferation in the regenerating heart | elife-50163-v2 | [
[
"Within the animal kingdom , regenerative capacity of damaged organs and body parts differs widely .",
"While regenerative capacity is generally low in mammalian species , this can be very efficient in specific fish and amphibians ( Poss , 2010 ) .",
"Even amongst animals of the same species , such as Astyanax mexicanus a teleost fish like the zebrafish , regenerative capacity can vary significantly ( Stockdale et al . , 2018 ) .",
"A better understanding of these differences and the cellular and molecular processes during tissue regeneration might ultimately help to improve the regenerative capacity of organs and tissues with poor intrinsic regenerative capacity .",
"The mammalian adult heart has very little regenerative capacity .",
"The myocardium lost after an injury is replaced by scar tissue , which does not contribute to cardiac contraction resulting in reduced pumping efficiency and ultimately heart failure .",
"Although a low level of cardiomyocyte turnover has been observed , there is no evidence of a stem cell population in the mammalian heart ( Kretzschmar et al . , 2018; van Berlo et al . , 2014 ) .",
"Instead , a rare population of endogenous cardiomyocytes retains the capacity to proliferate to maintain cardiac homeostasis ( Bergmann et al . , 2009; Bergmann et al . , 2015; Kimura et al . , 2015; Senyo et al . , 2013 ) .",
"Contrary to the adult heart , the neonatal mouse and potentially human heart still have the capacity to regenerate after injury ( Haubner et al . , 2016; Porrello et al . , 2011 ) .",
"This regenerative capacity of the neonatal heart involves proliferation of pre-existing cardiomyocytes and is lost soon after birth most likely due to a sharp decrease in cardiomyocyte proliferation ( Porrello et al . , 2011; Soonpaa et al . , 1996 ) .",
"Recent efforts to enhance proliferation of cardiomyocytes by either inhibiting Hippo signaling or activating the Nrg1/ErbB2 signaling pathway in the adult mammalian heart have been successful and show the potential of existing cardiomyocytes to reenter the cell cycle .",
"( Bersell et al . , 2009; D'Uva et al . , 2015; Heallen et al . , 2011 ) .",
"The zebrafish heart regenerates very efficiently and will regrow cardiac muscle without scarring ( Poss et al . , 2002 ) .",
"Lineage tracing experiments revealed a model in which proliferation of preexisting cardiomyocytes replaces the myocardium that was lost during the injury ( Jopling et al . , 2010; Kikuchi et al . , 2010 ) .",
"While cardiomyocyte proliferation in the uninjured adult zebrafish heart is very low , 3 days after the injury ( dpi ) cardiomyocytes near the injury area ( so-called border zone ) start to reenter the cell cycle as observed by the induction PCNA expression , phosphorylated histone three and BrdU incorporation and proliferation peaks at 7 days post injury ( dpi ) ( Jopling et al . , 2010; Kikuchi et al . , 2010; Chablais et al . , 2011; González-Rosa et al . , 2011; Schnabel et al . , 2011; Wu et al . , 2016 ) .",
"At the time when proliferation is observed , cardiomyocytes located in the border zone start to express cardiac transcription factors known for their role in embryonic heart development such as nkx2 . 5 and tbx20 and activate gata4 enhancer elements ( Kikuchi et al . , 2010; Lepilina et al . , 2006 ) .",
"In addition , border zone cardiomyocytes show signs of dedifferentiation such as disorganization of sarcomere structures and the reexpression of embryonic myosins ( Jopling et al . , 2010; Wu et al . , 2016 ) .",
"There is increasing evidence that other ( non-muscle ) cells in the heart secrete growth factors that stimulate cardiomyocyte proliferation including retinoic acid , TGF-b ligands , insulin-like growth factor , Hedgehog , and Neuregulin ( Chablais and Jazwinska , 2012; Choi et al . , 2013; Dogra et al . , 2017; Gemberling et al . , 2015; Lepilina et al . , 2006; Wu et al . , 2016; Zhao et al . , 2019; Zhao et al . , 2014 ) .",
"In addition to these growth factors , prolonged hypoxia stimulates cardiomyocyte proliferation ( Jopling et al . , 2012; Marques et al . , 2008 ) .",
"The proliferating cardiomyocytes exist within a heterogeneous cell population including non-proliferating cardiomyocytes , endothelial cells and immune cells , hampering the discovery of genetic programs specific for these proliferating cardiomyocytes using whole tissue or spatially resolved RNA-sequencing ( RNA-seq ) approaches ( Kang et al . , 2016; Lien et al . , 2006; Sleep et al . , 2010 ) .",
"To identify molecular processes that differ between proliferating and non-proliferating cardiomyocytes , we explored a single-cell RNA-seq approach using the regenerating zebrafish heart .",
"We found that upon injury , adult border zone cardiomyocytes dedifferentiate and resemble embryonic cardiomyocytes on a transcriptomic level .",
"In addition , while adult cardiomyocytes mainly rely on fatty acid metabolism and mitochondrial oxidative phosphorylation ( OXPHOS ) , border zone cardiomyocytes have reduced mitochondrial OXPHOS activity while genes encoding enzymes for glycolysis are induced and glucose uptake is enhanced .",
"Importantly , Nrg1/ErbB2 signaling is sufficient to induce metabolic reprogramming in adult cardiomyocytes of both zebrafish as well as the murine hearts .",
"In addition , the metabolic reprogramming from mitochondrial OXPHOS to glycolysis is required for efficient cardiomyocyte proliferation .",
"Together , these data support a model in which cardiomyocytes located in the border zone of the regenerating zebrafish heart undergo metabolic reprogramming , which is essential for cardiomyocyte proliferation and that this mechanism is conserved in a murine model with Nrg1/ErbB2 induced regeneration ."
],
[
"The border zone comprises only a small fraction of the total number of cardiomyocytes in the injured ventricle ( Wu et al . , 2016 ) .",
"Several genes and regulatory sequencing have been identified that mark border zone cardiomyocytes , including nppa , which encodes for the cardiac stress hormone ANF ( Kikuchi et al . , 2010; Wu et al . , 2016 ) .",
"To mark these borderzone cardiomyocytes we generated a transgenic zebrafish nppa reporter line ( TgBAC ( nppa:mCitrine ) ) in which mCitrine expression recapitulates endogenous cardiac expression of nppa ( Figure 1—figure supplement 1a–e ) .",
"While low nppa:mCitrine expression was observed in trabecular cardiomyocytes of the remote area , higher expression was detected in the trabecular and cortical cardiomyocytes close to the injured area ( Figure 1a and Figure 1—figure supplement 1e ) .",
"Moreover , expression of nppa correlates with previously reported border zone activity of gata4 regulatory elements ( Figure 1—figure supplement 1f ) ( Kikuchi et al . , 2010 ) .",
"Histochemical analysis of cryo-injured adult hearts revealed that 75% ( ±7% , n = 3 ) of the cardiomyocytes expressing high levels of nppa:mCitrine reentered the cell cycle ( Figure 1a ) .",
"To obtain border zone ( proliferating ) and remote ( non-proliferating ) cardiomyocytes from the same tissue for further analysis , we cryo-injured nppa:mCitrine hearts followed by cell dissociation and FACS sorting for both mCitrinehigh and mCitrinelow cells ( Figure 1b ) .",
"Individual , living cells were sorted , followed by single-cell mRNA-sequencing using the SORT-seq ( SOrting and Robot-assisted Transcriptome SEQuencing ) platform ( Muraro et al . , 2016 ) ( Figure 1—source data 1 ) .",
"In total 768 cells where sequenced in which we detected 19257 genes .",
"We detected an average of 10 , 443 reads per cell and we introduced a cutoff at minimally 3500 reads per cell before further analysis , which resulted in the analysis of 352 cells .",
"To identify the cardiomyocytes amongst the other cell types , we first identified the different cell types based on their transcriptomes .",
"k-medoids clustering of the single cell transcriptomes by the RaceID clustering algorithm was used ( Grün et al . , 2015 ) ( Figure 1c and Figure 1—source data 2 ) , and visualized in two dimensions using t-distributed stochastic neighbor embedding ( t-SNE ) ( Figure 1d ) .",
"A total of 12 cell clusters were identified , including a large group of cardiomyocytes ( clusters 1 , 2 , 4 , 7 and 9 ) , a smaller group of endothelial cells ( clusters 5 , 6 , 8 , 10 and 12 ) , and some fibroblasts ( cluster 3 ) and immune cells ( cluster 11 ) using the expression of marker genes for specific cell types ( Figure 1—figure supplement 2 ) .",
"Based on the transcriptome clustering , the cardiomyocytes fell into four main transcriptionally-defined clusters ( 1 , 2 , 4 and 7 ) , indicating that the injured heart contained subgroups of cardiomyocytes .",
"To address whether the border zone cardiomyocytes were enriched in one of the four cardiomyocyte clusters we compared the mCitrine fluorescence intensity ( recorded during FACS sorting ) of the cardiomyocyte and found that the average intensity was highest in cluster 7 and lowest in cluster 2 ( Figure 1—figure supplement 3 ) .",
"In addition , we analysed the single-cell transcriptome data for the expression of nppa and compared this to the expression of vmhc and mustn1b , which mark border zone cardiomyocytes , and again found that cells expressing these genes were mostly in cluster 7 ( Figure 1e and Figure 1—figure supplement 4 ) .",
"Together , these results indicate two things: first , the border zone cardiomyocytes ( grouped in cluster 7 ) can be identified as a separate group in the single-cell RNA-seq data .",
"Secondly , these border zone cardiomyocytes are transcriptionally distinct from remote cardiomyocytes ( grouped in cluster 2 ) , while two intermediate cardiomyocyte clusters lie in between .",
"Cardiomyocytes in the border zone disassemble sarcomeric structures and re-express markers of embryonic cardiomyocytes suggesting their dedifferentiation .",
"We therefore wanted to address the level of dedifferentiation of cluster seven cardiomyocytes by comparing their transcriptome with embryonic cardiomyocytes .",
"To obtain embryonic cardiomyocytes we performed FACS sorting on embryos expressing the cardiomyocyte specific marker Tg ( myl7:GFP ) .",
"Single-cell mRNA-sequencing was performed and combined with the single-cell data from the injured adult hearts ( Figure 2a ) .",
"The RaceID algorithm identified several cell clusters with separate clusters for the embryonic and adult cardiomyocytes ( Figure 2b , c and",
"d ) .",
"Importantly , the cluster seven cardiomyocytes identified in the adult data analysis had a transcriptome that was highly similar to embryonic cardiomyocytes , as shown by pairwise correlation of the differentially expressed genes between the cardiomyocyte clusters: only 257 genes ( p-value<0 . 01 ) , out of 23 , 786 total detected genes , were differentially expressed between the embryonic and the cluster 7 ( border zone ) adult cardiomyocytes ( Figure 2d and Figure 2—source datas 1 , 2 , 3 and 4 ) suggesting a dedifferentiation of border zone cardiomyocytes to embryo--like cells .",
"In contrast , over 1000 genes ( p-value<0 . 01 ) were differentially expressed between embryonic and cluster 2 ( remote zone ) adult cardiomyocytes .",
"A heatmap with unbiased hierarchical clustering on the 500 most differentially expressed genes between the three clusters confirmed that cluster 7 cardiomyocytes were more closely related to embryonic than cluster 2 cardiomyocytes ( Figure 2e ) .",
"Corroborating the observation that border zone cardiomyocytes resemble embryonic cardiomyocytes we found that genes encoding sarcomere proteins and cardiac-specific factors highly expressed in the embryo were re-expressed in cluster 7 ( border zone ) cardiomyocytes ( Figure 2f , g and h ) .",
"To identify cellular events that occur during this dedifferentiation we used part of the RaceID algorithm ( StemID ) that uses the single cell transcriptome data and cell clustering to derive a branched lineage tree ( Grün et al . , 2016 ) .",
"The algorithm is based on the premise that stem cells and less differentiated cells tend to exhibit more uniform transcriptomes than differentiated cells , which express smaller numbers of genes at higher rates ( Banerji et al . , 2013 ) .",
"Using this approach , we found large differences in transcriptome entropy , resulting in low ( cluster 2 ) , intermediate ( clusters 1 and 4 ) and high ( cluster 7 ) StemID scores ( Figure 3a ) .",
"This gradual increase suggests a dedifferentiation axis from cells in cluster 2 ( remote myocardium ) to cells in cluster 7 ( border zone myocardium ) and is in good agreement with our finding that the transcriptome of cluster 7 cardiomyocytes resembles an embryonic cardiomyocyte transcriptome ( Figure 3b ) .",
"Together , these results indicate that clusters 4 and 7 are enriched for dedifferentiated and proliferative border zone cardiomyocytes while clusters 1 and 2 are enriched for differentiated remote cardiomyocytes .",
"While cardiomyocytes undergo a well-defined sequence of morphological and transcriptional changes during differentiation , very little is known about the reverse process .",
"Ordering whole-transcriptome profiles of single cells with an unsupervised algorithm can resolve the temporal resolution during differentiation by identifying intermediate stages of differentiation without a priori knowledge of marker genes ( Trapnell et al . , 2014 ) .",
"In this manner , the single-cell mRNA-seq experiment will constitute an in-silico time series , with each cell representing a distinct state of differentiation along a continuum .",
"To analyze the transcriptional changes occurring during this apparent dedifferentiation , the most likely dedifferentiation path , based on the StemID scores , was chosen starting at cluster two and progressing through clusters 1 , 4 and 7 .",
"Next , gene expression profiles along this pseudo-temporal order were computed for all detected genes using the single-cell transcriptomes .",
"These gene expression profiles were grouped into modules of co-expressed genes using self-organizing maps ( SOMs ) , resulting in 14 modules ( Figure 3c , Figure 3—source data 1 ) .",
"Corroborating our hypothesis of varying differentiation states , we observed that gene expression within these modules changed smoothly over pseudo time .",
"We next analyzed the temporally-ordered expression profiles and identified four groups of genes that shared the same dynamics of expression during this differentiation trajectory .",
"The first group ( modules 1 ,",
"2 ) contained genes that were most highly expressed only in cells at the very beginning of the pseudo time line and their expression rapidly declined in cells that were positioned later .",
"This group contained many genes transcribed from mitochondrial DNA and with a role in energy metabolism , which indicates that the cells at the start of the pseudo time line are mature cardiomyocytes .",
"The second group ( module 6 ) contained genes that are induced early and expression stayed constant in cells further along the pseudo time line .",
"Many genes involved in translation and cell cycle regulation follow this expression pattern .",
"The third group showed an exponential increase in expression with the highest expression at the end of the pseudo time line ( module 10 ) .",
"This group contained genes with a function in cardiac muscle fiber development and heart contraction .",
"The fourth group ( modules 11 and 14 ) contained genes with a rapid increase in expression that peaks before the end of the pseudo time , suggestive for an early role during the dedifferentiation process .",
"Interestingly this group contained many genes with a known role in glycolysis .",
"Together , these data suggest that border zone cardiomyocytes undergo profound metabolic changes .",
"This was confirmed by GO-term analysis between cluster 7 and cluster 2 cells ( Figure 4—figure supplement 1a and b , Figure 1—source data 3 ) .",
"To validate the functional consequences of the observed changes in mitochondrial gene expression , we measured succinate dehydrogenase ( SDH ) enzyme activity , located in the inner mitochondrial membrane that functions in both the citric acid cycle and electron transport chain .",
"We observed a 40% reduction in SDH activity specifically in the border zone cardiomyocytes as compared to the remote cardiomyocytes ( Figure 4a ) .",
"In agreement with the reduced mitochondrial OXPHOS activity , transmission electron microscopy ( TEM ) imaging revealed more immature mitochondria in border zone cardiomyocytes evidenced by their altered morphology and reduced cristae density ( Figure 4b ) , which is consistent with previous reports linking mitochondrial function with morphology ( Giraud et al . , 2002; Paumard et al . , 2002 ) .",
"Since the pseudotime analysis suggested an upregulation of glycolytic gene expression in the border zone cluster ( #7 ) , we performed gene set enrichment analysis ( GSEA ) for glycolysis genes .",
"The GSEA revealed a strong and significant enrichment in the expression of glycolytic genes in cluster 7 cells compared to cluster 2 cells ( Figure 4—figure supplement 1c ) .",
"By in situ hybridization we confirmed the induced expression in border zone cardiomyocytes of the rate-limiting enzymes hexokinase ( hk1 ) , pyruvate kinase M1/M2a ( pkma ) and pyruvate dehydrogenase kinase ( pdk2a ) , which diverts pyruvate away from the TCA cycle ( Figure 4c and Figure 1—figure supplement 4b ) .",
"Corroborating the suggested enhanced glycolysis , we observed induced expression of glucose importer genes ( glut1a/slc2a1a and glut1b/slc2a1b ) in cluster 7 cells ( Figure 4—figure supplement 1d ) and enhanced in vivo glucose uptake of border zone cardiomyocytes ( Figure 4d ) .",
"Furthermore , genes encoding lactate transporters and their proteins were upregulated in cluster seven and border zone cardiomyocytes ( Figure 4e and Figure 4—figure supplement 1d ) .",
"Together , these data indicate that during regeneration border zone cardiomyocytes switch energy metabolism from mitochondrial OXPHOS to glycolysis and lactate fermentation .",
"The pseudo time line analysis suggested that glycolysis genes induction precedes the induction of embryonic cardiac gene expression .",
"Indeed , ISH analysis showed that glycolysis gene expression is already induced at 3 dpi and thereby precedes expression of embryonic cardiac gene expression and cardiomyocyte proliferation , which peaks at 7 dpi ( Figure 3—figure supplement 1 ) .",
"To address the functional importance of glycolysis we inhibited glycolysis in injured fish with the glucose analogue 2-Deoxyglucose ( 2-DG ) , a general inhibitor of glycolysis , and analyzed its effect on cardiomyocyte proliferation ( Figure 5a ) .",
"We observed that repeated injections of 2-DG in the adult zebrafish with a cryoinjured heart significantly impaired cardiomyocyte proliferation in the border zone ( Figure 5b , c ) , suggesting that glycolysis is necessary for cell cycle reentry .",
"Next , we investigated the upstream signals that drive the observed metabolic reprogramming in border zone cardiomyocytes during cell cycle reentry .",
"Hypoxia is a well-known stimulus for metabolic reprogramming during cancer and promotes cardiomyocyte proliferation during cardiac regeneration ( Jopling et al . , 2012; Nakada et al . , 2017; Vander Heiden et al . , 2009 ) .",
"The responses to hypoxia are induced by the transcription factor , hypoxia inducible factor ( HIF ) , whose activity can be visualized using the Tg ( phd3:GFP ) reporter line ( Santhakumar et al . , 2012; Wang and Semenza , 1993 ) .",
"When analysing cryo-injured hearts of Tg ( phd3:GFP ) fish 7 days post injury we did not observe a good correlation between pdh3:GFP reporter activity and the induction of ldha expression in border zone cardiomyocytes suggesting that HIF signaling is not required for the observed induction of glycolysis gene expression in border zone cardiomyocytes ( Figure 6—figure supplement 1 ) .",
"Injury-induced Neuregulin 1 ( Nrg1 ) expression is another potent mitogen that induces cardiomyocyte dedifferentiation and cell cycle reentry by activating ErbB2 receptor signaling ( Gemberling et al . , 2015 ) .",
"Furthermore , in vitro experiments suggest that Nrg1 can induce glucose metabolism ( Cote et al . , 2005; Suárez et al . , 2001 ) .",
"To address whether Nrg1 can induce metabolic reprogramming in vivo , we used a previously described transgenic zebrafish model in which Nrg1 overexpression ( OE ) can be induced in a heart specific manner , tg ( cmlc2:CreER; β-act2:BSNrg1 ) ( Gemberling et al . , 2015 ) .",
"In this model especially cortical cardiomyocytes start to divide after tamoxifen injection leading to thickening of this layer .",
"We observed a profound and consistent upregulation of glycolysis genes in the cortical myocardium coinciding with the reported cardiomyocyte dedifferentiation and proliferation in this layer ( Figure 6a ) .",
"Expression of ldha and hk1 , encoding the rate limiting glycolytic enzyme hexokinase , was strongly induced in the ventricular wall of Nrg1 OE hearts , which correlates well with the observed induction of cardiomyocyte dedifferentiation and proliferation in this region ( Gemberling et al . , 2015 ) .",
"Next , we assessed whether blocking Nrg1/ErbB2 signaling impairs glycolytic upregulation in the zebrafish border zone after cryoinjury .",
"qPCR confirmed the profound upregulation of glycolytic genes in border zone cardiomyocytes compared to their expression in cardiomyocytes from uninjured hearts ( Figure 6b ) .",
"Importantly the ErbB2 inhibitor AG1478 inhibited the induction of glycolytic gene expression in border zone cardiomyocytes ( Figure 6b ) .",
"In contrast to the other glycolysis genes , hk1 expression was not reduced after AG1478 treatment likely as a result of redundant signaling pathways in the border zone .",
"From these results , we conclude that glycolysis gene expression can be induced by Nrg1/ErbB2 signaling even in the absence of cardiac injury and that endogenous Nrg1/ErbB2 signaling is an important mediator of metabolic rewiring during zebrafish heart regeneration .",
"The regenerative capacity of the adult murine heart is very low , but cardiomyocyte dedifferentiation and proliferation can be stimulated by cardiomyocyte specific overexpression of a constitutively active ErbB2 receptor ( caErbB2 OE ) ( D'Uva et al . , 2015 ) .",
"To address whether this is correlated with metabolic changes we performed qPCRs for metabolic genes on cardiac tissue from caErbB2 mice .",
"Indeed , we observed that critical glycolysis genes ( e . g . Pfkp , Pdk3 and Pkm2 ) including glucose and lactate transporters ( Slc16A3 and Slc2A1 ) were significantly upregulated in caErbB2 OE cardiomyocytes while genes transcribed from mitochondrial DNA were downregulated ( Figure 6c ) .",
"Pdk3 encodes a pyruvate dehydrogenase kinase , which phosphorylates pyruvate dehydrogenase ( PDH ) .",
"PDH is a mitochondrial multi-enzyme complex that converts pyruvate to Acetyl-CoA and provides a primary link between glycolysis and the TCA cycle .",
"Upon phosphorylation by PDK , p-PDH is inactivated and pyruvate is diverted away from the TCA cycle resulting in enhanced lactate production .",
"Consistent with the increase in pdk3 expression , an increased phosphorylation of PDH was observed in the inner myocardial layer of caErbB2 OE hearts ( Figure 6d ) .",
"Next , we addressed whether the observed switch in metabolic gene expression correlated with enhanced regenerative capacity after injury .",
"Therefore , we performed myocardial infarction ( MI ) in wild type and caErbB2 OE hearts .",
"Even though glucose uptake and glycolytic enzyme activity in the ischemic area are increased by MI ( Schelbert and Buxton , 1988 ) ( Owen et al . , 1969 ) , we observed a stronger upregulation of glycolytic gene expression and decreased mitochondrial gene expression in caErbB2 OE hearts with MI compared to wild type hearts with MI ( Figure 7—figure supplement 1 ) .",
"This stronger and consistent upregulation of glycolytic gene expression in caErbB2 OE hearts correlates with the reported enhanced cardiomyocyte proliferation and improved regeneration ( D'Uva et al . , 2015 ) .",
"These findings imply that the enhanced glucose uptake and anaerobic glycolysis observed after MI might be sufficient for cell survival , but that a stronger induction of glycolysis gene expression is required to fully stimulate cardiomyocyte proliferation and regeneration .",
"Finally , we addressed whether the observed metabolic switch to glycolysis in caErbB2 OE cardiomyocytes is required for their reentry into the cell cycle ( Figure 7a ) .",
"Corroborating our model , we indeed observed that treating caErbB2 OE cardiomyocytes in vitro with the glycolysis inhibitors 2-DG or lonidamine strongly and significantly impaired cell cycle reentry ( Figure 7b and c ) and cytokinesis ( Figure 7d and e ) .",
"Together these results indicate that in murine cardiomyocytes , ErbB2 signaling drives a metabolic switch towards glycolysis , which is required for their cell cycle reentry .",
"These results suggest that this metabolic switch in cardiomyocytes is beneficial for heart regeneration ."
],
[
"Heart regeneration in zebrafish is very efficient and relies on the proliferation of preexisting cardiomyocytes .",
"The number of proliferating cardiomyocytes that is induced by injury is very small and they comprise only a small portion of the total cardiomyocytes , which has hampered their characterization .",
"Thus far , RNA-seq data sets and differential gene analysis was based on whole tissue preparations or cryosections of uninjured and injured hearts ( Goldman et al . , 2017 Kang et al . , 2016; Lai et al . , 2017; Lien et al . , 2006; Sleep et al . , 2010; Wu et al . , 2016 ) .",
"Not only cardiomyocytes but other cell types in the heart such as epicardial and endocardial cells respond to the injury by the upregulation of injury-induced genes ( Lepilina et al . , 2006 ) .",
"Furthermore , the injured heart is infiltrated by immune cells and fibroblasts appear ( González-Rosa et al . , 2011; Lai et al . , 2017 ) .",
"Together , this complicates the detection of cardiomyocyte specific gene responses .",
"The use of single cell transcriptomics can overcome these limitations and has allowed us to identify and characterize the different cardiomyocyte populations in the regenerating zebrafish heart .",
"Here we have generated a unique RNA-seq dataset at single-cell resolution of cardiomyocytes during heart regeneration , which can be used as a resource to study and identify novel mechanisms .",
"Activation of Yap signaling in the murine heart promotes cardiomyocyte proliferation , which involves a partial reprogramming and dedifferentiation of adult cardiomyocytes towards a neonatal fate ( Monroe et al . , 2019 ) .",
"Interestingly , single-cell RNA-seq analysis of connective tissue cells during limb blastema formation revealed that their transcriptome is highly similar to embryonic limb precursor cells ( Gerber et al . , 2018 ) .",
"Together with our results that a similar reprogramming takes place during injury induced heart regeneration in zebrafish it suggests a common theme in which adult tissues that lack a clear stem cell use an alternative strategy to regenerate the missing tissue by reprogramming differentiated cells into embryo-like cells .",
"This might involve different mechanisms since Yap signaling is not required for cardiomyocyte proliferation during zebrafish heart regeneration ( Flinn et al . , 2019 ) .",
"The presence of embryo-like cardiomyocytes in the border zone after injury can explain the observed switch from trabecular cardiomyocytes into cortical cardiomyocytes during zebrafish heart regeneration , since embryonic cardiomyocytes give rise to both trabecular and cortical cardiomyocytes ( Sánchez-Iranzo et al . , 2018; Staudt et al . , 2014 ) .",
"Increased mitochondrial OXPHOS activity promotes cardiomyocyte maturation and reduces the proliferative capacity of cardiomyocytes ( Mills et al . , 2017 ) .",
"This correlates well with the loss of regenerative capacity of the murine heart in the first week after birth at which time the metabolism in cardiomyocytes changes from predominantly glycolysis to mitochondrial OXPHOS ( Lopaschuk et al . , 1992; Menendez-Montes et al . , 2016; Porrello et al . , 2011 ) .",
"Reactive oxygen species ( ROS ) generated by mitochondrial OXPHOS induce DNA damage causing cardiomyocyte cell-cycle arrest ( Nakada et al . , 2017; Puente et al . , 2014; Tao et al . , 2016 ) .",
"Our pseudo time line analysis shows a rapid downregulation of mitochondrial gene expression together with an increase in genes encoding glucose transporters , glycolytic enzymes and lactate transporters .",
"A direct role for glycolysis and lactate fermentation in cardiomyocyte proliferation had not been addressed and our results that pharmacological inhibition of glycolysis impairs cardiomyocyte proliferation suggest that activation of glycolysis can drive the reprogramming of cardiomyocytes .",
"It also raises the question why cardiomyocytes need to switch their metabolism to glycolysis to reenter the cell cycle ?",
"Interestingly , a similar metabolic shift from mitochondrial OXPHOS to glycolysis and lactate fermentation occurs in proliferating tumor cells , and was first described by Otto Warburg ( Warburg et al . , 1927 ) .",
"Since this glucose to lactate transformation occurs regardless of whether oxygen is present it is referred to as aerobic glycolysis .",
"While glycolysis generates much less ATP compared to fatty acid oxidation , it is thought that glycolysis and the connected pentose-phosphate pathway provides essential metabolites that are needed to create sufficient biomass to sustain proliferation of the tumor cells ( Vander Heiden et al . , 2009 ) .",
"Furthermore , progenitor cells in the developing embryo as well as induced pluripotent stem cells depend on glycolysis to maintain proliferation and their potency ( Folmes et al . , 2011; Gu et al . , 2016; Mathieu and Ruohola-Baker , 2017 ) .",
"In addition , glycolytic enzymes such as PKM2 and PFKFB4 can also directly interact with cell cycle regulators to promote proliferation ( Yang et al . , 2011; Dasgupta et al . , 2018 ) .",
"The precise role for glycolysis in driving the cellular reprogramming during heart regeneration needs to be further investigated using genetic loss- and gain-of-function experiments combined with metabolomics , which is challenging given the low number of proliferating cardiomyocytes in the regenerating heart .",
"Activation of Nrg1/ErbB2 signaling in either zebrafish or mouse hearts induces cardiomyocyte dedifferentiation and proliferation ( D'Uva et al . , 2015; Gemberling et al . , 2015 ) .",
"Our results build upon these observations and indicate that Nrg1/ErbB2 signaling induces profound metabolic reprogramming in cardiomyocytes and that this is required for efficient cardiomyocyte proliferation .",
"Future work should address how Nrg1/ErbB2 signaling induces metabolic reprogramming in cardiomyocytes , which could have important implications for stimulating mammalian heart regeneration ( Polizzotti et al . , 2015 ) ."
],
[
"The following fish lines were used: TL , Tg ( phd3:GFP ) , Tg ( gata4:EGFP ) ae1 and myl7:GFPtwu34Tg ( Huang et al . , 2003 ) ( Kikuchi et al . , 2010; Santhakumar et al . , 2012 ) .",
"The Tg ( cmlc2:CreER; β-act2:BSNrg1 ) line was used as described before ( Gemberling et al . , 2015 ) .",
"The TgBAC ( nppa:mCitrine ) line was generated essentially as described previously ( Bussmann and Schulte-Merker , 2011 ) .",
"In short , an iTOL2_amp cassette for pTarBAC was inserted in the vector sequence of bacterial artificial chromosome ( BAC ) CH211-70L17 , which contains the full nppa locus .",
"Subsequently , a mCitrine_kan cassette was inserted at the ATG start codon of the first exon of the nppa gene .",
"Amplification from a pCS2+mCitrine_kanR plasmid was achieved with primers: FWD_NPPA_HA1_GFP 5′-gagccaagccagttcagagggcaagaaaacgcattcagagacactcagagACCATGGTGAGCAAGGGCGAGG-3′ and REV_NPPA_HA2_NEO 5′-gtctgctgccaaaccaggagcagcagtcctgtcagaattagtcccccggcTCAGAAGAACTCGTCAAGAAGGCGATAGAA −3’ .",
"Sequences homologous to the BAC are shown in lower case .",
"Recombineering was performed following the manufacturer’s protocol ( Red/ET recombination; Gene Bridges GmbH , Heidelberg , Germany ) with minor modifications .",
"BAC DNA isolation was carried out using a Midiprep kit ( Life Technologies BV , Bleiswijk , The Netherlands ) .",
"BAC DNA was injected at a concentration of 300 ng/µl in the presence of 25 ng Tol2 mRNA .",
"At three dpf , healthy embryos displaying robust nppa-specific fluorescence in the heart were selected and grown to adulthood .",
"Subsequently , founder fish were identified by outcrossing and their progeny grown to adulthood to establish the transgenic line .",
"Zebrafish of ~4 to 18 months of age ( males and females , TL strain ) were used for regeneration experiments .",
"Cryoinjuries were performed as previously described ( Schnabel et al . , 2011 ) , except that a liquid nitrogen-cooled copper filament of 0 . 3 mm diameter was used instead of dry ice .",
"Sample sizes were chosen to accommodate the generally accepted standards in the field: five or more cryoinjured hearts per condition .",
"Animals were only excluded from experiments in case of severe sickness/infection/aberrant behavior ( according to animal experiment guidelines ) .",
"Doxycycline-inducible CM-restricted overexpression of a constitutively active Erbb2 ( caErbb2 ) was generated by crossing the TetRE–caErbb2 ( Xie et al . , 1999 ) mouse line with MYH6– tTA which expresses the tetracycline-responsive transcriptional activator ( tTA ) under the control of the human alpha myosin heavy chain promoter ( MYH6 ) ( Yu et al . , 1996 ) .",
"Doxycycline ( DOX , Harlan Laboratories , TD02503 ) was administered in the food to repress transgene expression .",
"For cultures derived of OE/WT hearts , we additionally intercrossed the MYH6-cre ROSA26-tdTomato transgenes in order to visualize CMs .",
"For myocardial infarction , mice were sedated with isoflurane ( Abbott Laboratories ) and were artificially ventilated following tracheal intubation .",
"Experimental myocardial infarction was induced by ligation of the left anterior descending coronary artery ( LAD ligation ) .",
"Following the closure of the thoracic wall mice were warmed for several minutes until recovery .",
"ADULT: For immunofluorescence , hearts were extracted , fixed in 4% PFA at room temperature for 1 , 5 hr and cryosectioned into 10 µm sections .",
"Heart sections were equally distributed onto seven serial slides so each slide contained sections representing all areas of the ventricle .",
"Primary antibodies used were anti-AuroraB kinase ( BD Transduction laboratories #611082 , 1:200 ) , anti-Ki67 ( Cell Marque #275R , 1:200 ) , anti-MCT4 ( Santa Cruz #SC50329 , 1:200 ) , anti-PCNA ( Dako #M0879 , 1:800 ) , anti-GFP ( aves #GFP-1010 , 1:1000 ) , and anti-Mef2c ( Santa Cruz #SC313 , Biorbyt #orb256682 both 1:1000 ) .",
"Antigen retrieval was performed by heating slides containing heart sections at 85°C in 10 mM sodium citrate buffer ( pH 6 ) for 10 min .",
"Secondary antibodies conjugated to Alexa 488 ( ThermoFisher Scientific ) , Cy3 or Cy5 ( Jackson Laboratories ) were used at a dilution of 1:1000 .",
"Nuclei were shown by DAPI ( 4’ , 6-diamidino-2-phenylindole ) staining .",
"Images of immunofluorescence stainings are single optical planes acquired with a Leica Sp8 or Sp5 confocal microscope .",
"Quantifications of PCNA , Mef2 , and mCitrine expression were performed in cardiomyocytes situated within 150 µm from the wound border on 3 sections of >4 hearts .",
"Sections were masked before quantification .",
"EMBRYONIC: Live embryos were immobilized using ms222 and embedded in nitrocellulose + E3 to be mounted on a Leica SPE confocal microscope , followed by a Z-stack maximum projection ( step size 2 µm ) .",
"MAMMALIAN P7 CARDIAC CULTURES: For immunofluorescence , cardiac cultures were fixed with 4% PFA for 10 min on room temperature on the shaker , followed by permeabilization with 0 . 5% Triton X-100 in PBS for 5 min , and blocking with 5% bovine serum albumin ( BSA ) in PBS containing 0 . 1% Triton for 1 hr at room temperature .",
"Masking was performed before quantification .",
"ADULT ZEBRAFISH: nppa:mCitrine zebrafish were cryoinjured and received two overnight pulses of DMSO ( 1:2000 ) or 5 µM AG1478 ( 10 mM stock in DMSO; Selleck Chemical , Houston , TX ) from 1dpi to 2dpi and from 2dpi to 3dpi .",
"Then , hearts were extracted for both conditions ( n = 9 ) as well as uninjured cmlc2:dsRED controls ( n = 4 ) , dissociated and single cells were FACS sorted for mCitrine and dsRED expression respectively .",
"RNA was isolated from sorted cells using Trizol ( Life Technologies BV , Bleiswijk , The Netherlands ) .",
"RNA was quantified using a NanoDrop spectrophotometer .",
"Superscript III First Strand Synthesis System ( ThermoFisher Scientific ) was used to reverse transcribe 200 ng of purified RNA per condition following manufacturer’s protocol .",
"qPCR reactions were performed using Fast SYBR Green PCR Master Mix ( Applied Biosystems ) .",
"Oligonucleotide sequences for real-time PCR analysis performed in this study are listed in Figure 6—source data",
"1 . MOUSE: RNA from whole hearts was isolated using the MiRNeasy kit ( Qiagen , 217004 ) , according to the manufacturer’s instructions .",
"RNA was quantified using a NanoDrop spectrophotometer .",
"A High Capacity cDNA Reverse transcription kit ( Applied Biosystems , 4374966 ) was used to reverse transcribe 1 µg of purified RNA according to the manufacturer’s instructions .",
"qPCR reactions were performed using Fast SYBR Green PCR Master Mix ( Applied Biosystems ) .",
"Oligonucleotide sequences for real-time PCR analysis performed in this study are listed in Figure 6—source data",
"1 . PARAFFIN: After o/n fixation in 4% PFA , hearts were washed in PBS twice , dehydrated in EtOH , and embedded in paraffin .",
"Serial sections were made at 10 µm .",
"In situ hybridization was performed on paraffin-sections as previously described ( Moorman et al . , 2001 ) except that the hybridization buffer used did not contain heparin and yeast total RNA .",
"CRYOSECTIONS: Sections were obtained as described earlier .",
"In situ hybridization was performed as for paraffin , however sections were pre-fixed for 10 min in 4% PFA + 0 . 25% glutaraldehyde before Proteinase K treatment .",
"Moreover , slides were fixed for 1 hr in 4% PFA directly after staining .",
"When in situ hybridization was combined with immunofluorescence , Fast Red staining solution was used instead of NBT-BCIP .",
"Cryoinjured hearts ( n = 13 ) were extracted at seven dpi .",
"Cells were dissociated according to Tessadori et al . ( 2012 ) .",
"For cell sorting , viable cells were gated by negative DAPI staining and positive YFP-fluorescence .",
"In brief , the FACS gating was adjusted to sort cells for nppa:mCitrinehigh ( to enrich for proliferating cardiomyocytes ) and nppa:mCitrinelow ( remote cardiomyocytes and other cell types ) cells .",
"In total n = 576 mCitrinehigh and n = 192 mCitrinelow cells were sorted into 384-well plates and processed for mRNA sequencing as described below .",
"Transgenic tg ( myl7:GFP ) 2-day-old embryos ( n = 200 ) were dechorionated and digested in HBSS Ca2+/Mg2+ free media containing 0 . 1% collagenase type II ( Gibco ) at 32°C for 30–40 min followed by 1X TrypLE Express ( Gibco ) for 15–30 min at 32°C with agitation .",
"Dissociated cells were then FACSorted and subjected to single-cell mRNA-seq .",
"Single-cell sequencing libraries were prepared using SORT-seq ( Muraro et al . , 2016 ) .",
"Live cells were sorted into 384-well plates with Vapor-Lock oil containing a droplet with barcoded primers , spike-in RNA and dNTPs , followed by heat-induced cell lysis and cDNA syntheses using a robotic liquid handler .",
"Primers consisted of a 24 bp polyT stretch , a 4 bp random molecular barcode ( UMI ) , a cell-specific 8 bp barcode , the 5’ Illumina TruSeq small RNA kit adapter and a T7 promoter .",
"After cell-lysis for 5 min at 65 °C , RT and second strand mixes were distributed with the Nanodrop II liquid handling platform ( Inovadyne ) .",
"After pooling all cells in one library , the aqueous phase was separated from the oil phase , followed by IVT transcription .",
"The CEL-Seq2 protocol was used for library prep ( Hashimshony et al . , 2016 ) .",
"Illumina sequencing libraries were prepared with the TruSeq small RNA primers ( Illumina ) and paired-end sequenced at 75 bp read length on the Illumina NextSeq platform .",
"Mapping was performed against the zebrafish reference assembly version 9 ( Zv9 ) .",
"To analyze the single-cell RNA-seq data , we used the previously published RaceID algorithm ( Grün et al . , 2015 ) .",
"For the adult hearts we had a dataset consisting of two different libraries of 384 cells each for a combined dataset of 768 cells , in which we detected 19257 genes .",
"We detected an average of 10 , 443 reads per cell .",
"Based on the distribution of the log10 total reads plotted against the frequency , we introduced a cutoff at minimally 3500 reads per cell before further analysis .",
"This reduced the number of cells used in the analysis to 352 .",
"Next , we downsampled reads to 3500 unique ( UMI corrected ) transcripts per cell , as means of normalization .",
"Moreover , we discarded genes that were not detected at >3 transcripts in >1 cell and These cutoffs is a stringent normalization method that allows us to directly compare detected transcripts between cells from different cell types and libraries .",
"Batch-effects were analyzed and showed no plate-specific clustering of certain clusters .",
"For embryonic cardiomyocytes , two libraries of 384 cells were combined to obtain a set of 768 cells , in which 22271 genes could be detected .",
"An average of 4412 reads per cell was detected .",
"After downsampling of this library to 3500 reads per cell 302 cells were included for further analysis .",
"Further analysis was performed by combining the embryonic heart data with the injured adult heart data .",
"The StemID algorithm were used as previously published ( Grün et al . , 2016 ) .",
"In short , StemID is an approach developed for inferring the existence of stem cell populations from single-cell transcriptomics data .",
"StemID calculates all pairwise cell-to-cell distances ( 1 – Pearson correlation ) and uses this to cluster similar cells into clusters that correspond to the cell types present in the tissue .",
"The StemID algorithm calculates the number of links between clusters .",
"This is based on the assumption that cell types with less links are more canalized while cell types with a higher number of links have a higher diversity of cell fates .",
"Besides the number of links , the StemID algorithm also calculates the change in transcriptome entropy .",
"Differentiated cells usually express a small number of genes at high levels in order to perform cell specific functions , which is reflected by a low entropy .",
"Stem cells and progenitor cells display a more diverse transcriptome reflected by high entropy ( Banerji et al . , 2013 ) .",
"By calculating the number of links of one cluster to other clusters and multiplying this with the change in entropy , it generates a StemID score , which is representative to ‘stemness’ of a cell population .",
"Differential gene expression analysis was performed using the ‘diffexpnb’ , which makes use of the DESeq algorithm .",
"P-values were Benjamini-Hochberg corrected for false discovery rate to make the cutoff .",
"For the comparison between the embryonic and adult cardiomyocyte clusters , the number of differentially expressed genes between two clusters was calculated as described above and this was used as a measure of similarity between clusters .",
"To identify modules of co-expressed genes along a specific differentiation trajectory ( defined as a succession of significant links between clusters as identified by StemID ) all cells assigned to these links were assembled in pseudo-temporal order based on their projection coordinate .",
"Next , all genes that are not present with at least two transcripts in at least a single cell are discarded from the sub-sequent analysis .",
"Subsequently , a local regression of the z-transformed expression profile for each gene is computed along the differentiation trajectory .",
"These pseudo-temporal gene expression profiles are topologically ordered by computing a one-dimensional self-organizing map ( SOM ) with 1000 nodes .",
"Due to the large number of nodes relative to the number of clustered profiles , similar profiles are assigned to the same node .",
"Only nodes with more than three assigned profiles are retained for visualization of co-expressed gene modules .",
"Neighboring nodes with average profiles exhibiting a Pearson’s correlation coefficient >0 . 9 are merged to common gene expression modules .",
"These modules are depicted in the final map .",
"Analyses were performed as previously published ( Grün et al . , 2016 ) .",
"Accession numbers mRNA-seq data are deposited on Gene Expression Omnibus , accession number GSE139218 .",
"Samples FK1 and FK2 represent adult cardiomyocytes .",
"Samples LG-A and LG-B represent embryonic cardiomyocytes .",
"Hearts were excised and immediately chemically fixated at room temperature with 2 , 5% glutaraldehyde and 2% formaldehyde ( EMS , Hainfield USA ) in 0 . 1M phosphate buffer pH 7 . 4 for 2 hr .",
"Next , hearts were post fixed with 1% OsO4 ( EMS , Hainfield USA ) /1 . 5% K3Fe ( CN ) 6 in 0 . 065 M phosphate buffer for 2 hr at 4°C and finally 1 hr with 0 , 5% uranyl acetate .",
"After fixation , hearts were dehydrated in a graded series of acetone and embedded in Epon epoxy resin ( Polysciences ) .",
"Ultrathin sections of 60 nm were cut on a Leica Ultracut T ( Leica , Vienna , Austria ) and contrasted with uranyl acetate ( 0 . 4% in AD , EMS , Hainfield USA ) and lead citrate ( Leica Vienna , Austria ) using the AC20 ( Leica Vienna , Austria ) and examined with a Jeol 1010 electron microscope ( Jeol Europe , Nieuw Vennep , The Netherlands ) .",
"In every heart , each in the borderzone and remote myocardial region , 100 well-deliniated mitochondria with clearly visible outer and inner membranes were selected .",
"Mitochondrial perimeter and surface were measured using the freehand tool of Image J . The perimeter to surface ratio was calculated and used as a factor that describes the pluriformity of mitochondria .",
"The amount of cristae was estimated by counting the number of cristae intersected by a line of 0 . 5 µm length in 40 mitochondria per region .",
"Serial cryosections of the heart were cut 7 µm thick and either fixed in formalin , stained with Meyer’s hematoxylin and eosin ( HE ) , dehydrated and mounted in Entellan , or incubated for enzyme histochemistry .",
"Chemicals for histochemistry of succinate dehydrogenase ( SDH ) activity were obtained from Sigma Aldrich .",
"Sections for SDH activity were incubated for 20 min at 28°C in 37 . 5 mM sodium phosphate buffer pH 7 . 60 , 70 mM sodium succinate , 5 mM sodium azide and 0 . 4 mM tetranitro blue tetrazolium ( TNBT ) .",
"The reaction was stopped in 10 mM HCl .",
"Controls without succinate did not stain .",
"The incubated sections were mounted in glycerine gelatin .",
"The absorbances of the SDH-reaction product in the sections were determined at 660 nm using a calibrated microdensitometer and ImageJ .",
"ZEBRAFISH: Zebrafish were injured and received intraperitoneal ( i . p . ) injections twice daily with either PBS or 2-Deoxy-D-Glucose ( Sigma-Aldrich , 1 mg/g ) from days 3 to 6 and one more injection on day seven after injury , two hours before fish were euthanized and hearts harvested .",
"I . p . injections were performed using a Hamilton Syringe ( gauge 30 ) as described in literature ( Kinkel et al . , 2010 ) .",
"Injection volumes were corrected to body weight ( 30 μl/g ) .",
"MAMMALIAN P7 CARDIAC CULTURES: Primary cardiac cultures were isolated from P7 mice using a neonatal dissociation kit ( Miltenyi Biotec , 130-098-373 ) using the gentleMACS homogenizer , according to the manufacturer’s instructions and cultured in Gelatin-coated ( 0 . 1% , G1393 , Sigma ) wells with DMEM/F12 medium supplemented with L-glutamine , Na-pyruvate , nonessential amino acids , penicillin , streptomycin , 5% horse serum and 10% FBS ( ‘complete-medium’ ) at 37◦C and 5% CO2 for 24 hr .",
"Afterwards , medium was replaced with FBS-depleted medium ( otherwise same composition ) for additional 48 hr of culture in either 3 mM 2DG ( Sigma-Aldrich ) or 80 μM lonidamine ( Sigma-Aldrich ) before further processing .",
"Fish were euthanized on ice water before hearts were extracted in PBS + heparin and were allowed to bleed out for 15 min .",
"Hearts were then transferred into fresh PBS + 10%KCl to stop the heart from beating and mounted directly on a glass bottom cell culture dish in 1% agarose .",
"Thereafter , 2NBDG ( Caymanchem #11046 , 400 μM ) was added to the dish and the hearts were taken directly for imaging .",
"Imaging was performed using a Leica SP5 multiphoton microscopy using 930 nm laser excitation wavelength .",
"150 μm z-stacks were made with z-step size 5 μm every 5 min for 2 hr .",
"GSEA ( Genepattern , Broad Institute ) was performed to assess enrichment for glycolytic genes upregulated between cluster 7 and",
"2 . A list of genes involved in zebrafish glycolysis was obtained from KEGG .",
"As number of permutations 1000 was used , which means p=0 indicates p<0 . 001 .",
"All statistical testing was performed by unpaired T-tests besides zebrafish qPCR data for which a one-way ANOVA was performed ."
]
] | [
"While the heart regenerates poorly in mammals , efficient heart regeneration occurs in zebrafish .",
"Studies in zebrafish have resulted in a model in which preexisting cardiomyocytes dedifferentiate and reinitiate proliferation to replace the lost myocardium .",
"To identify which processes occur in proliferating cardiomyocytes we have used a single-cell RNA-sequencing approach .",
"We uncovered that proliferating border zone cardiomyocytes have very distinct transcriptomes compared to the nonproliferating remote cardiomyocytes and that they resemble embryonic cardiomyocytes .",
"Moreover , these cells have reduced expression of mitochondrial genes and reduced mitochondrial activity , while glycolysis gene expression and glucose uptake are increased , indicative for metabolic reprogramming .",
"Furthermore , we find that the metabolic reprogramming of border zone cardiomyocytes is induced by Nrg1/ErbB2 signaling and is important for their proliferation .",
"This mechanism is conserved in murine hearts in which cardiomyocyte proliferation is induced by activating ErbB2 signaling .",
"Together these results demonstrate that glycolysis regulates cardiomyocyte proliferation during heart regeneration ."
] | [
"Heart attacks are a common cause of death in the Western world .",
"During a heart attack , oxygen levels in the affected part of the heart decrease , which causes heart muscle cells to die .",
"In humans the dead cells are replaced by a permanent scar that stabilizes the injury but does not completely heal it .",
"As a result , individuals have a lower quality of life after a heart attack and are more likely to die from a subsequent attack .",
"Unlike humans , zebrafish are able to regenerate their hearts after injury: heart muscle cells close to a wound divide to produce new cells that slowly replace the scar tissue and restore normal function to the area .",
"It remains unclear , however , what stimulates the heart muscle cells of zebrafish to start dividing .",
"To address this question , Honkoop , de Bakker et al . used a technique called single-cell sequencing to study heart muscle cells in wounded zebrafish hearts .",
"The experiments identified a group of heart muscle cells close to the site of the wound that multiplied to repair the damage .",
"This group of cells had altered their metabolism compared to other heart muscle cells so that they relied on a pathway called glycolysis to produce the energy and building blocks they needed to proliferate .",
"Blocking glycolysis impaired the ability of the heart muscle cells to divide , indicating that this switch is necessary for the heart to regenerate .",
"Further experiments showed that a signaling cascade , which includes the molecules Nrg1 and ErbB2 , induces heart muscle cells in both zebrafish and mouse hearts to switch to glycolysis and undergo division .",
"These findings indicate that activating glycolysis in heart muscle cells may help to stimulate the heart to regenerate after a heart attack or other injury .",
"The next step following on from this work is to develop methods to activate glycolysis and promote cell division in injured hearts ."
] | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology",
"microbiology and infectious disease"
] | Coupling chemosensory array formation and localization | elife-31058-v2 | [
[
"Chemotaxis is one of the primary means by which motile bacteria sense , respond , and adapt to changing environmental conditions .",
"This process enables motile bacteria to perceive changes in local concentrations of chemicals; as a result , they can bias their movement away from unfavorable chemical stimuli and towards more favorable compounds ( Wadhams and Armitage , 2004; Sourjik and Armitage , 2010 ) .",
"In the best studied model organism Escherichia coli , chemotaxis is mediated by an array of highly organized macromolecular complexes built from core chemotaxis units .",
"The core units are themselves composed of a highly organized set of chemotaxis signaling proteins ( Figure 1A–B ) .",
"In general , the chemotaxis signaling cascade is initiated upon the detection of chemotactic stimuli by methyl-accepting chemotaxis proteins ( MCPs ) .",
"These membrane-spanning receptors then interact with a cytoplasmic histidine kinase , CheA , while the adaptor protein CheW stabilizes this interaction and participates in regulating CheA kinase activity ( Ortega et al . , 2013; Parkinson et al . , 2015 ) .",
"A phosphosignaling cascade is initiated via CheA and its cognate response regulator CheY .",
"Phosphorylated CheY induces a change in flagellar rotation and consequently in the direction of bacterial swimming , which over time results in net movement towards a more favorable environment ( Wadhams and Armitage , 2004; Sourjik and Armitage , 2010 ) .",
"MCPs usually consist of a variable N-terminal extracellular ligand binding domain , a cytoplasmic HAMP domain , and a well conserved signaling domain ( or kinase control domain ) with a highly conserved protein interaction tip that directs the assembly and action of receptor signaling complexes ( Figure 1A ) ( Kim et al . , 1999; Falke and Hazelbauer , 2001; Alexander and Zhulin , 2007; Hazelbauer et al . , 2008 ) .",
"Importantly , the tip contains sites for forming trimers of receptor dimers ( Kim et al . , 1999; Parkinson et al . , 2015 ) , and for binding to CheA and CheW ( Miller et al . , 2006; Park et al . , 2006; Vu et al . , 2012; Wang et al . , 2012; Li et al . , 2013; Piasta et al . , 2013; Pedetta et al . , 2014; Cassidy et al . , 2015 ) ( Figure 1B ) .",
"The histidine kinase CheA is comprised of five separate domains ( P1 to P5 ) with specific functions ( Figure 1B , green ) .",
"P1 is the phosphotransfer domain and contains the substrate histidine for autophosphorylation; P2 binds CheY for phosphotransfer from P1 ( Swanson et al . , 1993; Morrison and Parkinson , 1994; Bilwes et al . , 1999 ) ; P3 is the dimerization domain ( Park et al . , 2006; Cassidy et al . , 2015 ) ; P4 is the kinase or ATP binding domain; and P5 is an SH3-like regulatory domain , which binds the signaling tip of MCPs ( Borkovich et al . , 1989; Gegner et al . , 1992; Bilwes et al . , 1999; Zhao and Parkinson , 2006 ) .",
"The adaptor protein CheW ( Figure 1B , red ) consists of a single SH3-like domain , and is structurally similar to P5 of CheA ( Griswold et al . , 2002; Li et al . , 2013; Cassidy et al . , 2015 ) .",
"Together , MCPs , CheA , and CheW form stable core signaling complexes .",
"As shown in Figure 1B , one CheA dimer joins two MCP trimer-of-dimers and two CheW proteins .",
"The helix formed by the dimerization of the P3 domains of CheA positions itself between the two MCP dimer-of-trimers ( Briegel et al . , 2011; Li and Hazelbauer , 2011; Briegel et al . , 2012; Liu et al . , 2012 , 2013; Briegel et al . , 2014a ) and each P5 domain of a CheA dimer binds to one CheW .",
"Therefore , a single core unit is arranged in a hexagonal structure held together by contacts between:",
"( i ) CheA-MCPs ,",
"( ii ) CheW-MCPs and",
"( iii ) CheA-CheW .",
"According to the current model , further hexagonal core units then join to form a super-lattice structure , commonly known as the chemosensory array ( Briegel et al . , 2009 , 2012; Liu et al . , 2012 , 2013; Briegel et al . , 2014a , 2014b; Piasta and Falke , 2014 ) ( Figure 1B ) .",
"In vivo and in vitro observations indicate that CheA-CheW interactions bridge the two receptor trimers of every core and give the array its characteristic stability and high sensitivity ( Zhao and Parkinson , 2006; Hazelbauer et al . , 2008; Erbse and Falke , 2009; Briegel et al . , 2009; Li and Hazelbauer , 2011; Briegel et al . , 2012; Slivka and Falke , 2012; Sourjik and Wingreen , 2012; Liu et al . , 2012; Briegel et al . , 2014a; Piasta and Falke , 2014 ) .",
"However , while there is much knowledge of array structure , the mechanisms that underlie the formation and localization of these elaborate structures are incompletely understood , especially in systems other than E . coli .",
"As mentioned above , chemotaxis has been extensively studied in E . coli , a peritrichously flagellated bacterium .",
"Here , array formation is thought to be a stochastic process in which individual receptors are inserted randomly in the membrane , and subsequently diffuse freely until they either join existing arrays or nucleate new ones ( Thiem and Sourjik , 2008 ) .",
"This process results in a non-uniform distribution of signaling arrays at cell poles and randomly along the cell length ( Sourjik and Berg , 2000 ) , and likely ensures that sensory arrays are in close proximity to the lateral flagella .",
"In organisms such as Caulobacter crescentus , Pseudomonas aeruginosa , and Rhodobacter sphaeroides , and several Vibrio species , chemosensory arrays are actively localized to the cell poles ( Alley et al . , 1992; Maddock and Shapiro , 1993; Wadhams et al . , 2003; Bardy and Maddock , 2005; Ringgaard et al . , 2011; Ringgaard et al . , 2014 ) .",
"In the polarly flagellated pathogens Vibrio cholerae and Vibrio parahaemolyticus , we recently reported that chemosensory arrays are exclusively localized at one or both cell poles by a mechanism that depends on the partner proteins ParC and ParP , both of which are encoded within the chemotaxis operon ( Ringgaard et al . , 2011; Yamaichi et al . , 2012; Ringgaard et al . , 2014 ) .",
"For V . cholerae , chemotaxis proteins encoded by chemotaxis operon II , e . g . CheA1 and CheW1 , are directed to the cell pole by ParC and ParP ( Ringgaard et al . , 2015; Briegel et al . , 2016 ) , and from here on , CheA and CheW will be used instead of CheA1 and CheW1 , respectively .",
"In newborn Vibrio cells , these signaling arrays are exclusively localized to the old flagellated cell pole , then recruited to the new cell pole as cells enlarge , resulting in a bi-polar localization pattern .",
"Thus , at cell division each daughter cell inherits a signaling array positioned at its old pole ( Ringgaard et al . , 2011 , 2014 ) .",
"In the absence of either ParC or ParP , the chemotaxis arrays are no longer properly recruited to the cell poles .",
"Instead , signaling arrays form and localize randomly along the cell length , and bi-polar localization is not established prior to cell division .",
"Therefore , daughter cells do not faithfully inherit a signaling array at their old poles , resulting in altered motility and decreased chemotaxis ( Ringgaard et al . , 2011 , 2014 ) .",
"ParC mediates polar localization of ParP , which in turn interacts with a specific domain of CheA that is only present in CheA proteins with an associated ParC/ParP-system ( CheA-LID ) ( Ringgaard et al . , 2014 ) .",
"ParP prevents dissociation of CheA from chemotaxis arrays and disruption of either ParP-ParC or ParP-CheA interactions results in defective recruitment of chemotaxis arrays to the cell poles , leading to their random instead of polar localization ( Ringgaard et al . , 2011 , 2014 ) .",
"However , the molecular mechanisms by which this protein interaction network governs the dynamic localization of chemotactic signaling arrays remain to be elucidated .",
"Notably , there is little knowledge of how factors promoting array positioning are able to access and guide localization of chemotaxis proteins .",
"In particular , it is not clear how such factors are integrated within the widely conserved structure of signaling arrays .",
"Here , using V . cholerae as a model organism , we analyze how ParP is able to gain access to and interact with chemotaxis proteins positioned within the highly ordered structure of signaling arrays and how it mediates their intracellular localization .",
"We identify MCP proteins as a new interaction partner for ParP .",
"Via interactions with MCPs and CheA , ParP is a part of the chemotaxis core unit and integrates into the chemotactic signaling arrays .",
"Importantly , ParP integrates into arrays and promotes their formation via a C-terminal Array Integration and Formation ( AIF ) domain , which is linked to ParP’s N-terminal ParC interaction domain .",
"Linkage of these domains within ParP couples array formation and localization and results in localized formation of arrays at the cell poles and thus promotes cell pole maturation ."
],
[
"To address how ParP is able to access chemotaxis proteins within signaling arrays in V . cholerae , we analyzed array localization in wild-type , cheA1 , parP and cheA1 parP deletion backgrounds using a functional ( Ringgaard et al . , 2011 ) YFP-CheW1 fusion as a marker for array localization and formation .",
"In wild-type cells YFP-CheW1 mainly localized in clusters at the cell poles ( Figure 1C–E ) .",
"In contrast to localization in wild-type cells , in the absence of ParP , YFP-CheW1 clusters were not recruited to the cell poles , but were instead mislocalized along the cell length or completely absent in 74% of cells ( Figure 1C–E ) .",
"In a strain lacking cheA1 , YFP-CheW1 still formed clusters at the cells poles in a manner indistinguishable to that observed in wild-type cells ( Figure 1C–E ) , suggesting that chemotaxis arrays still form in the absence of CheA .",
"To analyze if arrays are still properly formed in the absence of CheA , we performed cryo-electron microscopy ( cryo-EM ) on wild-type and ΔcheA cells ( Figure 1—figure supplement 1 ) .",
"For both strains , chemotaxis arrays were detectable and indistinguishable in structure , consisting of an inner membrane-anchored array of MCP proteins and an associated cytosolic baseplate .",
"Out of 61 cells imaged with cryo-EM for each strain , there was a 60% reduction in the number of cells with observable arrays in the ΔcheA background compared to wild-type – consistent with a role of CheA in stimulating array formation .",
"However , the cryo-EM experiments reveal that ordered signaling arrays can still form in the absence of CheA .",
"Furthermore , these cryo-EM images strongly suggest that the YFP-CheW1 clusters reflect the localization and formation of properly structured arrays in the absence of CheA , although we cannot formally exclude the possibility that YFP-CheW1 clusters may reflect misformed or variant states of supramolecular complexes in some cells .",
"Strikingly , in the double deletion strain ∆cheA1 ∆parP , YFP-CheW1 did not form clusters but was localized diffusely in the cytoplasm ( Figure 1CE , bottom ) .",
"Immunoblot analysis showed that the difference in localization of YFP-CheW1 was not due to differences in expression levels or cleavage of the YFP moiety from the YFP-CheW1 fusion construct ( Figure 1F ) .",
"These data indicate that formation of signaling arrays is severely compromised in the absence of both ParP and CheA , and that CheW1 alone only has a minor effect on array formation but requires the presence of either ParP or CheA , which individually are sufficient for promoting array formation .",
"These data are supported by cryo-EM analyses of the ∆cheA1 ∆parP strain , in which out of 61 imaged cells there was an 85% reduction in the number of cells with detectable signaling arrays compared to wild-type .",
"Together , these observations suggest that ParP participates in the process of array formation in addition to its previously known function in promoting polar localization of signaling arrays .",
"To further investigate how ParP contributes to array formation and localization , we performed a screen to identify additional ParP interaction partners .",
"We developed a bacterial-two-hybrid blue/white-colony screen in E . coli , using ParP as bait against a chromosomal library from V . cholerae ( Figure 2A and Figure 2—figure supplement 1 ) .",
"Bacteria harboring a plasmid expressing a ParP interaction partner give rise to blue colonies ( Figure 2A ) .",
"It is important to note that E . coli does not encode homologs of either ParP or ParC , thus reducing the possibility of indirect interactions mediated by an endogenous E . coli factor , and suggesting that interaction partners identified in this assay likely interact directly with ParP .",
"One hundred blue colonies were picked and the candidate ParP interaction partners identified by sequencing .",
"Of the 100 blue colonies sequenced , 95 contained plasmids with genes encoding MCP proteins , corresponding to 15 distinct MCPs ( Figure 2B ) .",
"While the fragments of all the mcp genes hit in the screen covered varying regions of the respective genes , all hits included the regions encoding the signaling domains of the MCP proteins .",
"Therefore , we assessed whether signaling domains ( including the conserved interaction tip ) from four MCPs were sufficient to mediate interactions with ParP ( Figure 2C ) .",
"All four MCP signaling domains interacted with ParP ( Figure 2C ) , confirming that MCPs are a newly identified ParP interaction partner and that interaction occurs via the MCP signaling domain .",
"No interaction between ParC and MCPs was observed , suggesting only ParP , but not ParC interacts with MCP proteins .",
"To test whether ParP and MCP could interact independently of other chemotaxis proteins , we co-expressed YFP-ParP and mCherry-MCP-VC1898 ( denoted mCherry-MCP ) and assayed for co-localization in an E . coli strain deleted for all native chemotaxis proteins ( strain VS296 ) .",
"When expressed alone , YFP-ParP was diffusely localized in the cytoplasm in 100% of cells , and mCherry-MCP localized as distinct clusters ( Figure 2—figure supplement 2 ) .",
"Strikingly , when YFP-ParP was co-expressed with mCherry-MCP , YFP-ParP also localized in clusters that always co-localized with mCherry-MCP clusters ( Figure 2D–E ) .",
"Therefore , in addition to interacting with CheA and ParC , ParP also interacts ( likely in a direct fashion ) with MCP proteins .",
"Since ParP interacts with both CheA and MCPs , we hypothesize that ParP forms part of the core chemotaxis unit .",
"Next , we investigated which MCP residues are required for MCP-ParP interaction .",
"Interestingly , the C-terminal part of ParP consists of a predicted SH3-like domain ( hereafter named array integration and formation domain – AIF domain ) similar to CheW and the P5 domain of CheA .",
"The highly conserved protein interaction tip within the MCP signaling domain is responsible for interactions with CheW and CheA-P5 proteins ( Kremer et al . , 1996; Kim et al . , 1999; Li et al . , 2007 , 2011; Li and Hazelbauer , 2011; Briegel et al . , 2012; Liu et al . , 2012 , 2013; Cassidy et al . , 2015 ) .",
"Furthermore , several residues in the MCP TM1143 from T . maritima have been shown to be important for these interactions: L362 , L365 , N366 , and A368 ( Figure 2F ) .",
"Multiple sequence alignment of all predicted V . cholerae MCPs with the sequence of MCP TM1143 from T . maritima , revealed that these four residues are conserved in all of the MCPs identified in the two-hybrid screen and all but two putative MCPs found in V . cholerae ( Figure 2—figure supplement 3 ) .",
"We chose MCP VC1898 , the MCP with the strongest signal for interaction with ParP , to create individual amino acid substitution variants ( VC1898-L518R , L521R , N522R , A524R; Figure 2F ) and tested their interaction capabilities .",
"Three of the four substitutions ( L518R , L521R , and N522R ) disrupted the capacity of the MCP to interact with CheW1 , but not with itself ( Figure 2G ) .",
"Notably , the same substitutions also abolished the interaction between the MCP and ParP ( Figure 2G ) .",
"Since the MCP variants retained the ability to self-interact , the effect on their interactions with CheW1 and ParP is likely not due to reduced expression levels of the MCP variants .",
"Moreover , we tested the L518R variant for interaction with ParP in the E . coli VS296 co-expression assay .",
"Notably , YFP-ParP no longer formed clusters co-localizing with mCherry-MCP-L518R clusters , but instead localized diffusely in the cytoplasm ( Figure 2D ) in 95% of cells ( Figure 2E ) , indicating that the L518R substitution abrogates the capacity of YFP-ParP and mCherry-MCP to interact .",
"Altogether , these observations suggest that ParP-AIF targets the same MCP residues that mediate MCPinteractions with CheW and CheA , and thus lends support to the idea that ParP is a component of the chemotaxis core unit of signaling arrays .",
"While ParP-AIF domains form their own distinct clade of SH3-domains , they are more similar to the P5 domain of CheAs than to CheWs ( Figure 3A ) .",
"CheW and CheA-P5 are each composed of two subdomains ( 1 and 2 ) and the junction between the two subdomains contains branched hydrophobic residues that form a groove mediating interaction with the MCP interaction tip ( in CheW from Thermotoga maritima MSB8: V27 , I30 , L14 , V33; Figure 3B , red residues ) ( Griswold et al . , 2002; Park et al . , 2006; Briegel et al . , 2012; Li et al . , 2013 ) .",
"The AIF domain of ParP is predicted to have a similar overall protein architecture as CheA-P5 and CheW , and we hypothesized that the corresponding hydrophobic amino acids ( L196 , L209 , L212A , and I215 ) in the junction between its putative subdomains function to promote ParP’s interactions with MCPs ( Figure 3—figure supplement 1 ) .",
"We replaced each of these amino acid residues with alanine , and evaluated each variant ParP’s capacity to interact with the MCP signaling domain ( MCP-SD; Figure 3C ) .",
"L196A , L209A , and to some extent L212A ( but not I215A ) , showed reduced interaction with the MCP-SDs , supporting that ParP interacts with the MCP protein interaction tip via residues in its putative interaction groove , in a manner similar to the way in which CheW and CheA-P5 interact with the MCPs .",
"Notably , this hybrid assay suggested that replacement of L209 with alanine ( ParPL209A ) completely disrupted interaction between ParP and MCP-SD ( Figure 3C ) .",
"Additionally , in the E . coli co-expression assay , YFP-ParPL209A did not co-localize with mCherry-MCP clusters , but were instead localized diffusely in the cytoplasm ( Figure 2D–E ) , further indicating that this residue is involved in mediating ParP-MCP interactions .",
"Thus , ParP appears to rely on analogous residues as CheW and CheA-P5 to interact with MCPs .",
"Interestingly , L196A , L209A , and L212A are almost 100% conserved amongst ParP proteins , suggesting it is a general property of ParP proteins to interact with the MCP-SD ( Figure 3—figure supplement 2 ) .",
"We next turned to analyzing the interaction between ParP and CheA .",
"Previous work had revealed that a single amino acid in V . parahaemolyticus ParP was critical for interaction with CheA ( Ringgaard et al . , 2014 ) , and we found that the corresponding amino acid in V . cholerae ParP ( W305 ) lies within the AIF domain .",
"V . cholerae ParPW305A did not interact with CheA; however , the single amino acid substitution in this variant had little influence on ParP’s capacity to interact with the MCP in the two-hybrid assay ( Figure 3D ) or in the E . coli co-expression assay ( Figure 2D–E ) .",
"Conversely , although ParPL209A did not interact with MCPs , it was still capable of interacting with CheA ( Figure 3D ) .",
"ParP carrying both substitutions ( ParPL209A-W305A , denoted ParP2PM ) did not interact with either CheA or the MCP ( Figure 3D ) .",
"Neither substitution – either singly or in combination – impeded ParP’s interactions with ParC ( Figure 3D ) , which is mediated by an N-terminal domain that is separated from AIF by a long proline-rich linker ( Ringgaard et al . , 2014 ) ( Figure 3E , Figure 3—figure supplement 2 ) .",
"Furthermore , since ParPL209A and ParPW305A were still observed to robustly interact with ParC , the effects observed on these variants’ capacities to interact with MCP and CheA proteins ( Figure 3D ) are not likely explained by their decreased expression .",
"Based on the similarity of ParP to CheW of Thermotoga maritima MSB8 , L209 and W305 are predicted to be positioned on opposite sides of the AIF domain ( Figure 3B , Figure 3—figure supplement 1 ) , supporting the idea that ParP-AIF contains distinct interfaces that direct its interactions with CheA-LID and MCPs , respectively ( Figure 3D ) .",
"Since ParP’s N-terminus mediates interaction with ParC ( Ringgaard et al . , 2014 ) , ParP has at least three distinct interaction interfaces .",
"These distinct interaction surfaces potentially allow ParP to simultaneously couple two critical signaling components ( MCP and CheA ) to the polar determinant ParC ( Figure 3E ) .",
"Thus , ParP is a protein of high connectivity upon which both the chemotactic signaling network , as well as the system responsible for cell pole development , depend ( Figure 3E ) .",
"We monitored localization of YFP-ParP and its variants co-expressed with CFP-CheW1 ( a marker of arrays ) , to address whether ParP interactions with MCPs and/or CheA are required for its capacity to associate with signaling arrays .",
"These experiments were done in a V . cholerae ΔparC background in order to investigate ParP’s association with arrays without interference from its interactions with ParC .",
"In ~75% of cells , YFP-ParP and CFP-CheW1 formed co-localized clusters ( Figure 4A , B ) .",
"Similarly , in ~50–55% of cells , CFP-CheW1 clusters were co-localized with those of YFP-ParPL209A or YFP-ParPW305A ( Figure 4A , B ) .",
"Thus , ParP’s association with signaling arrays can be mediated by its interaction with either MCPs or CheA , though its capacity to interact with both these array components likely enhances its association with arrays .",
"In striking contrast , when we expressed a ParP variant carrying both amino acid substitutions L209A and W305A ( ParP2PM ) , which is unable to interact with either CheA or MCPs , fused to YFP ( YFP-ParP2PM ) , almost no YFP-ParP2PM clusters were observed , despite the presence of CFP-CheW1 clusters in ~55% of cells .",
"( Figure 4A , B ) .",
"This result suggests that ParP’s association with chemotaxis signaling arrays is fully dependent upon its interactions with CheA and MCPs .",
"Furthermore , consistent with a function for ParP in stimulating array formation via its interactions with MCPs and CheA , there was a significant drop from ~75% of wild-type cells with YFP-CheW1 clusters , compared to ~50–55% only in cells expressing the ParPL209A , ParPW305A andParP2PM variants , respectively ( Figure 4A , B ) .",
"Our data indicate that either ParP or CheA are required for array formation .",
"Consistent with the idea that the AIF domain accounts for ParP’s activity in array formation , in the absence of CheA , chemotaxis clusters ( as visually detected by YFP-CheW1 ) did not form in strains deleted for either the whole entire parP gene ( ΔparP ΔcheA1 ) or only the ParP-AIF domain ( parP-ΔAIF ΔcheA1 ) ( Figure 5A–C ) .",
"Moreover , the ParP variant with an AIF-domain incapable of integrating into signaling arrays ( parP2PM ) was almost entirely incapable of stimulating formation of chemotaxis clusters in the absence of CheA1 ( strain parP2PM ΔcheA1 ) ( Figure 5A–C ) .",
"In similar analyses , we investigated which CheA domain promotes its recruitment into signaling arrays and found that the P5 domain is both required and sufficient for recruitment of CheA into signaling arrays ( Figure 5D ) .",
"Absence of the CheA-P5 domain alone ( cheA1-ΔP5 ) did not significantly influence array formation , however , combining deletion of CheA-P5 with deletion of ParP ( cheA1-ΔP5 ΔparP ) also led to diffuse localization of YFP-CheW1 and consequently no formation of chemotaxis clusters ( Figure 5A–C ) .",
"This indicates that CheA stimulates arrays formation via its P5 domain , and further supports that the presence of ParP alone is sufficient for stimulation of array formation .",
"Immunoblot analyses showed that the diffuse localization of YFP-CheW1 was not due to cleavage of the YFP moiety from the YFP-CheW1 fusion construct ( Figure 5—figure supplement 1 ) .",
"Taken together , these data indicate that the AIF domain of ParP promotes formation of signaling arrays via its interactions with MCPs and CheA as an integral part of the core unit .",
"If ParP enables polar localization of chemotaxis clusters by integrating into the core chemotaxis unit , we reasoned that fusion of ParP’s ParC-interaction domain to a different integral component of the core unit might also be capable of recruiting the chemotaxis clusters to the pole .",
"To test this hypothesis , we constructed a ParP variant in which the AIF-domain was swapped for the CheA P5-domain in a ΔcheA1 background ( Figure 5—figure supplement 2 , strain parP-P5/ΔcheA1 ) , and tested for array localization by imaging YFP-CheW1 ( Figure 5B–C ) .",
"Indeed , the presence of ParP-P5 restored localization of uni- and bipolar clusters in 65% of cells , compared to 0% in a ΔparP/ΔcheA1 background ( Figure 5B–C ) .",
"Thus , the ParC-interaction domain of ParP is capable of mediating polar localization of signaling arrays independent of the AIF-domain if fused to a protein that is part of the chemotaxis core unit and participates in array formation and structure ( the CheA P5-domain ) .",
"Collectively these observations suggest that ParP’s capacity to localize arrays at the cell pole ( mediated by its ParC-interaction domain ) can operate independently of its capacity to promote array formation ( mediated by AIF ) , and thus that ParP couples two distinct and separable functions .",
"To test if the incorporation of ParP into signaling arrays and its facilitation of array formation had functional consequences on the polar localization of arrays , the localization of signaling arrays was determined in a set of ParP interaction mutants .",
"Strain parP2PM , which produces the ParP2PM variant defective in interactions with both CheA and MCPs , exhibited a phenotype similar to that of ΔparP , with 65% of cells having mislocalized or absent arrays ( Figure 6A–B ) .",
"This deficiency in polar array localization , which is expected to preclude each daughter cell inheriting an array upon cell division , was largely dependent on ParP being unable to interact with both interaction partners; strains expressing a ParP variant defective in interaction solely with MCPs ( ParPL209A ) or CheA ( ParPW305A ) had a modest increase in mislocalized or absent arrays ( 9% and 20% of cells , respectively , compared to ~6% in wild-type cells ) ( Figure 6A–B ) .",
"These data suggest that integration of ParP into signaling arrays either via interaction with MCPs or CheA , though compromised , to some extent can suffice to enable ParP-mediated array formation and polar localization .",
"However , disruption of ParP’s interaction with both MCPs and CheA , and thus its integration into the arrays , results in defective recruitment of arrays to the cell pole .",
"Thus , ParP acts as an integral part of signaling arrays to couple the formation of signaling arrays and their polar localization , thereby ensuring their proper inheritance .",
"We next tested if ParP’s interactions with MCP and CheA influenced the intracellular localization of ParP itself .",
"Wild-type ParP and its variants ParPL209A , ParPW305A , and ParP2PM were fused to the C-terminus of YFP and expressed ectopically in a ΔparP strain background .",
"Wild-type YFP-ParP localized to the cell poles in a uni- or bi-polar manner in 97% of cells .",
"Consistent with their ability to still interact with ParC ( Figure 3D ) , ParPL209A , ParPW305A , and ParP2PM localized as clusters at the cell pole in about 60% of all cells .",
"However , in contrast to wild-type YFP-ParP , a significant proportion ( ~40% ) of cells only showed diffuse localization of the YFP-ParP variants whereas wild-type ParP was diffuse in only 3% of cells ( Figure 7A–B ) .",
"Furthermore , a larger proportion of ParPL209A , ParPW305A , and ParP2PM were diffusely localized in the cytoplasm and there was a significant reduction in the intensity of these YFP-ParP variants at the cell pole compared to wild-type YFP-ParP ( Figure 7C ) .",
"Thus , interactions of ParP with both CheA and MCPs promote proper polar localization of ParP , and disruption of either interaction results in a decreased proportion of ParP being tethered to the cell pole – even when interactions to recruit chemotaxis arrays to this site appear sufficient to some extent ( Figure 6 ) .",
"To determine the underlying reason for reduced polar localization of ParP variants incapable of interaction with MCPs and CheA , we analyzed the recruitment and release of ParP and ParP2PM to and from the cell pole respectively .",
"We performed FRAP ( fluorescence-recovery-after-photobleaching ) analysis on YFP-ParP and YFP-ParP2PM , to monitor the recruitment of new ParP molecules to the cell pole .",
"After photobleaching of polar YFP-ParP and YFP-ParP2PM polar foci , we monitored the recovery of polar YFP fluorescence ( Figure 7D–E ) .",
"These experiments showed that there was a continuous recruitment of new ParP and ParP2PM from the cytoplasm to the cell pole , however , no significant difference in recovery rate was observed between the two ParP variants ( Figure 7D–E ) .",
"Next we measured the release of YFP-ParP and YFP-ParP2PM from polar clusters by bleaching the cytoplasmic signal from YFP-ParP and YFP-ParP2PM in cells with uni-polarly localized foci .",
"The intensity of polar clusters was subsequently measured and plotted relative to the initial intensity as a function of time ( Figure 7F–G ) .",
"Post-bleach , the intensity of polar YFP-ParP and YFP-ParP2PM clusters decreased over time , demonstrating that both protein versions are continuously released from the polar clusters .",
"However , the decay curves for the two ParP variants differed significantly: ParP reached a steady state after about 5 min , while ParP2PM was released at a faster rate than wild-type ParP , and the YFP-ParP2PM intensity continued to drop for over 11 min .",
"This suggests ParP2PM is released from polar clusters to the cytoplasm to a much greater extent than wild-type ParP .",
"Together , these experiments show that there is a continuous release of ParP molecules from the pole to the cytoplasm and recruitment of new ParP from the cytoplasm to the cell pole .",
"Moreover , they reveal that ParP’s capacity to interact with MCPs and CheA ( and thereby integrate into signaling arrays ) prevents its release , and as such promotes its retention , at the cell pole and consequently stabilizes its localization at this site .",
"We also tested if ParP’s ability to interact with the chemotaxis proteins MCP and CheA influenced the intracellular localization of the polar localization determinant ParC by ectopically expressing a functional YFP-ParC fusion protein ( Ringgaard et al . , 2011 ) in wild-type and parP2PM background strains .",
"As previously reported , YFP-ParC localized in foci at the cell poles in wild-type V . cholerae ( Figure 8A–B ) ( Ringgaard et al . , 2011 ) .",
"Although polar foci were also often observed in a parP2PM background , a significantly higher proportion ( ~20% ) of cells exhibited diffuse localization of YFP-ParC compared to wild-type ( ~4% ) ( Figure 8A–B ) .",
"Furthermore , in the parP2PM strain , a larger proportion of YFP-ParC was diffusely localized in the cytoplasm and there was a significant reduction in the intensity of polar ParC foci ( Figure 8C ) .",
"Thus , integration of ParP within signaling arrays via its AIF-domain promotes the retention of ParC at the cell pole .",
"Altogether these data reveal that integration of ParP within signaling arrays not only couples chemotaxis array formation and localization but also modifies the dynamic localization of factors that govern cell pole development , such as ParC and ParP itself ."
],
[
"The highly ordered structure and distribution of signaling arrays within the cell is essential for proper chemotactic responses and bacterial competitiveness .",
"However , it is not well understood how factors responsible for array positioning are able to access chemotaxis proteins within arrays to mediate localization .",
"In addition to interacting with ParC and CheA , we found that ParP also interacts with MCP proteins .",
"ParP interacts with the MCP interaction tip domain in a manner analogous to CheA and CheW proteins and promotes array formation .",
"Thus , ParP , like CheA and CheW , appears to be a component of the chemotaxis core unit .",
"Mapping of ParP’s interaction interfaces revealed that its C-terminal SH3-like AIF domain includes distinct surfaces that enable interaction with MCPs and CheA .",
"Furthermore , its N-terminal ParC interaction domain is responsible for recruitment of ParP and signaling arrays to the cell pole .",
"The linkage of these domains within ParP couples array formation and localization and results in localized formation of arrays at the cell poles .",
"By stimulating polar array formation , ParP also promotes its own and ParC’s polar localization .",
"Collectively , by defining ParP’s interaction network and interfaces , we uncovered how this protein couples array formation and polar localization .",
"We identified MCP proteins as essential interaction partners for ParP .",
"The screen identified 15 distinct MCPs , and all but two MCPs of V . cholerae possess the motif within the conserved protein interaction tip that mediates ParP-MCP interaction .",
"The screen likely only identified 15 of the 45 predicted MCPs encoded by V . cholerae because only ~50 , 000 colonies were screened , and thus the screen was not comprehensive .",
"Furthermore , the screen only identifies ParP interaction partners that were fused in frame with the t18 gene during library generation .",
"These factors likely explain why a subset of MCPs , as well as known interaction partners ParC and CheA were not identified in the screen .",
"ParP integrates into signaling arrays through its interactions via its AIF domain with the conserved protein interaction tip of MCP proteins and with CheA .",
"Through these complex interactions , ParP promotes array formation rather than compromising array structure .",
"ParP-AIF’s similarity to CheW and CheA-P5 in regions that mediate interaction with MCP suggests that ParP might compete with CheW and CheA-P5 for MCP binding , and thereby to become part of the chemotactic core unit .",
"Previous studies indicate that other proteins with SH3-like structures compete to become part of the array in a comparable manner ( Levit et al . , 2002; Asinas and Weis , 2006; Erbse and Falke , 2009 ) ; e . g . , CheV can replace CheW ( Alexander et al . , 2010 ) and CheA-P5 ( Briegel et al . , 2012 ) within signaling arrays .",
"Although CheW , CheA-P5 and ParP-AIF all appear to have the capacity to recognize and bind the MCP interaction tip within the chemotactic core unit , all have distinct functions and interestingly form their own distinct clades of SH3-like domains .",
"Thus , evolution has exploited the ability of the SH3 domain to interact with MCPs within the core unit of arrays for diverse functions including signal transduction , array formation and the intracellular localization of signaling arrays .",
"Transactions between ParP and its array partners - MCPs and the LID domain of CheA - likely reflect the balancing of the requirement for an additional array component mediating array localization ( ParP ) with preservation of array structure and function .",
"Arrays can still form if one of ParP or CheA is absent , due to the presence of the other .",
"owever , in the absence of both proteins , the chemotaxis core unit is no longer assembled and signaling arrays are barely able to form .",
"Arrays form at almost wild-type levels in the absence of CheA , suggesting that ParP is able to fully replace CheA within the core unit .",
"However , we cannot rule out the possibility that ParP also is able to compete with CheW for integration into the core unit .",
"In the prevailing model of array structure , two CheA proteins are present within a core unit , dimerized through their P3 domain ( Figure 9 , type #1 ) – an interaction that contributes to array stability and signal transduction ( Briegel et al . , 2011; Li and Hazelbauer , 2011; Briegel et al . , 2012; Li et al . , 2013; Briegel et al . , 2014b ) .",
"If ParP-AIF replaces a CheA protein within the arrays , CheA dimerization , and its associated array stabilization , would be lost; however , it was shown that the ParP-CheA interaction ( via CheA-LID ) reduces dissociation of CheA from arrays ( Ringgaard et al . , 2014 ) and thereby provides an alternate means of stabilization .",
"Thus , our data are consistent with a model where ParP-AIF replaces some CheA-P5 in binding the MCP interaction tip within the chemotaxis core unit , and is tethered there through binding to the LID domain of the remaining CheA of the core unit .",
"Presumably this AIF-LID interaction is able to substitute for the absence of CheA-P3 dimerization within the core unit and thus maintains the stability of the array structure ( Figure 9 , type #2 ) .",
"Since ParP is able to dimerize via its N-terminus ( Ringgaard et al . , 2014 ) , it is also possible that a ParP dimer is able to replace the CheA dimer within the core unit of wild-type cells , resulting in a core unit comprised of two CheWs and a ParP dimer ( Figure 9 , type #3 ) .",
"Core units consisting only of CheW , ParP , and MCPs presumably constitute the arrays observed within a CheA-deficient strain , which form at close to wild-type levels .",
"Additional studies will be required to elucidate whether interactions between ParP/CheW and ParP/CheA-P5 occur and the factors that modulate the MCP interaction tip’s accessibility to different partners .",
"In a wild-type background arrays might consist of all three types of core units ( Figure 9—figure supplement 1 ) , whereas in the ΔparP and ΔcheA deletion backgrounds , arrays consist of type #1 and type #3 core units only , respectively ( Figure 9—figure supplement 1 ) .",
"Furthermore , it is also a possibility that the flexible dimerization domain of ParP could link ParP proteins from neighboring core units , and in this way augment retention of ParP itself and chemotaxis proteins within the array , thereby contributing to array stability and ultimately their sequestration at the cell pole via ParP’s ParC interaction domain .",
"Interestingly , the intracellular localization of cytosolic chemotaxis arrays in R . sphaeroides may have functional similarities to the Vibrio ParP/ParC system .",
"In R . sphaeroides , a ParA-like protein , PpfA , ( ParC is also a ParA-like protein ) is thought to ensure proper segregation and positioning of the cytosolic chemotaxis arrays over the bacterial nucleoid by means similar to that used by ParA proteins involved in plasmid segregation ( Thompson et al . , 2006; Ringgaard et al . , 2011; Roberts et al . , 2012 ) .",
"PpfA mediates array localization in concert with a predicted cytoplasmic chemoreceptor TlpT , which , in combination with a CheW , is required for array formation .",
"TlpT interacts with PpfA via its N-terminus , thereby likely stimulating PpfA ATPase activity – an action that is required for PpfA function ( Wadhams et al . , 2005; Thompson et al . , 2006; Roberts et al . , 2012 ) .",
"It is possible that TlpT integrates into the cytoplasmic arrays , bridging chemotaxis proteins and PpfA to promote array formation and localization in a manner similar to that of ParP .",
"Importantly , ParP’s interactions with the chemotaxis proteins in the core unit ( CheA and MCPs ) are also important for ParC-mediated sequestration of ParP at the poles and for the polar localization of ParC itself .",
"Disruption of ParP’s interactions with MCPs and CheA resulted in a much higher percentage of non-polar ( cytosolic ) ParC and ParP .",
"Thus , although ParC is still able to recruit ParP to the cell poles , sequestration of ParC and ParP at this site is diminished when ParP interactions with MCPs and CheA are disrupted .",
"As seen with ParC ( Ringgaard et al . , 2011 ) , here we show that there is a continuous exchange of ParP between the cell pole and the cytoplasm .",
"Our photobleaching-based comparisons of ParP and ParP2PM suggest that ParP’s capacity to integrate into signaling arrays does not influence its recruitment to the cell pole .",
"In contrast , the capacity of ParP to bind MCPs and CheA and integrate into signaling arrays had a significant impact on its release from the pole into the cytoplasm .",
"Particularly , that integration of ParP into signaling arrays prevents the release of ParP molecules from the cell pole and consequently promotes its retention at this site .",
"Thus , ParP’s integration into arrays modifies its own and likely in turn ParC’s subcellular localization dynamics , promoting their polar retention .",
"We have shown that ParP is a protein of high network connectivity and functions as an important nexus that facilitates chemotaxis array formation , array localization , and regulates the localization dynamics of its network elements .",
"ParP retains partial function as long as one of its network connections to the core unit exists ( i . e . to either CheA or MCPs ) .",
"Only loss of both ParP’s connections to the core unit results in a non-functional ParP variant .",
"In contrast , when either of its connections to the core unit are disrupted , the retention of ParP , and ParC , at the cell pole is compromised to the same extend as when both connections simultaneously are disrupted .",
"This suggests that when a ParP loses a network connection to the core unit , ParP is still able to function partially in mediating polar array localization due to the other connection , however , the ability of ParP to mediate retention of its network constituents at the cell pole is lost – ultimately resulting in its partial loss of function .",
"This further emphasizes the importance of ParP’s interconnectivity within the chemotaxis protein interaction network in regulating the polar retention of itself and its network constituents and in mediating the proper polar localization of chemotactic signaling arrays .",
"Taken together our findings show that ParP’s high connectivity allows it to serve as a critical nexus that regulates the temporal dynamics of its network constituents and stabilizes the polar localization of the cell-pole anchor ParC and itself .",
"Furthermore , it facilitates the localized assembly and inheritance of signaling arrays at the pole , hereby ensuring proper cell pole development ."
],
[
"If not otherwise stated , V . cholerae and E . coli were grown in LB media or on LB agar plates at 30°C or 37°C containing antibiotics in the following concentrations: streptomycin 200 μg/ml; kanamycin 50 μg/ml; ampicillin 100 μg/ml; chloramphenicol 20 μg/ml for E . coli and 5 μg/ml for V . cholerae .",
"When needed , L-arabinose was added to a final concentration of 0 . 2 % w/v .",
"E . coli strain DH5αλpir was used for cloning and E . coli strain SM10λpir was used to transfer plasmid DNA by conjugation from E . coli to V . cholerae ( Miller and Mekalanos , 1988 ) .",
"The wild-type strain of V . cholerae used was the El Tor clinical isolate N16961 and all mutants are derivatives of this strain .",
"Construction of V . cholerae deletion or point mutants was performed with standard allele exchange techniques using derivatives of plasmid pCVD442 ( Donnenberg and Kaper , 1991 ) .",
"All strains used are listed in Supplementary file",
"1 . All plasmids used in this study are listed in Supplementary file",
"1 . All primers used in construction of plasmids are listed in Supplementary file",
"2 . A schema explaining the bacterial-two-hybrid screen is presented in Figure 2A and Figure 2—figure supplement",
"1 . V . cholerae chromosomal DNA was digested with rare-cutter restriction enzymes and fragments in the size-range 1000–5000 bp purified and fused to the gene encoding the T25 fragment of adenylate-cyclase in vector pKT25 , thereby resulting in a library of chromosomal DNA fused to the gene encoding T25 .",
"The library was transformed into E . coli strain BTH101 ( Karimova et al . , 1998 ) expressing T18-ParP ( plasmid pAK2 ) and transformants were spread on indicator plates .",
"One hundred blue colonies were screened by sequencing for identification of the chromosomal DNA insert in pKT25 encoding a possible ParP interaction partner .",
"Fluorescence microscopy was carried out essentially as described in references ( Ringgaard et al . , 2015; Briegel et al . , 2016 ) .",
"For fluorescence microscopy in V . cholerae , fluorescent fusion proteins were ectopically expressed from plasmids .",
"Cells were grown for 12 hr in LB medium at 37° C with shaking .",
"Ten microliters were then used to inoculate 5 milliliter cultures .",
"When OD600 ≈ 1 . 0 , protein expression was induced by addition of 0 . 2% w/v final concentration of L-arabinose .",
"The cultures were incubated for one additional hour , at which point cells were ready for microcopy analysis .",
"For fluorescence microscopy of E . coli strain VS296 , a strain carrying the relevant plasmid for fluorescent protein expression was inoculated in 5 mL 10% LB in PBS buffer .",
"Expression of fluorescence proteins was induced by addition of 0 . 4% w/v final concentration of L-arabinose and 1 mM IPTG .",
"Cultures were incubated 8–10 hr , at which time-point cells were ready for microscopy analysis .",
"Cells ready for microscopy analysis were mounted onto 1% agarose ( in 20% PBS buffer with 10% LB ) on a microscopy slide before imagining .",
"Microscopy of YFP-CheW1 was performed using a Zeiss Axio Imager M1 fluorescence microscope .",
"Images were collected with a Cascade:1K CCD camera ( Photometrics ) , using a Zeiss αPlan-Fluar 100x/1 . 45 Oil DIC objective .",
"Images were analyzed using MetaMorph ( version 7 . 7 . 5 . 0; Molecular Devices ) .",
"Imaging of YFP-CheA1 variants was performed using a Zeiss Axioplan 2 microscope equipped with a 100 × a plan lens andHamamatsu cooled CCD camera .",
"All other microscopy was performed using a Nikon eclipse Ti inverted Andor spinning-disc confocal microscope equipped with a 100x lens and an Andor Zyla sCMOS cooled camera and an Andor FRAPPA system .",
"Microscopy images were analyzed using ImageJ imaging software ( http://rsbweb . nih . gov/ij ) and Metamorph Offline ( version 7 . 7 . 5 . 0 , Molecular Devices ) .",
"For comparison , all mutant strains were imaged with the same exposure time and light intensity as the wild-type background .",
"Generation of demographs was carried out precisely as described by ( Cameron et al . , 2014; Heering and Ringgaard , 2016; Heering et al . , 2017 ) .",
"For microscopy experiments counting the percentage of cells with distinct localization patterns a minimum of three biological experiments were performed and for each experiment >100 cells were counted in order to determine the percentages of cells with different localization patterns .",
"Cells and localization patterns were enumerated by hand .",
"The mean of the three experiments was then plotted with error bars indicating the standard-error-mean ( SEM ) .",
"A t-test was performed to calculate the p value .",
"The n-value indicates the total number of cells analyzed of the three independent experiments and is included for each sample in the respectively figures .",
"For microscopy experiments measuring the fluorescence intensity of polar foci relative to the cytosolic signal ( Figures 7C and 8C ) , relative intensity was measured in the total number of cells indicated ( n ) in the respective figures .",
"The mean was then plotted with error bars representing the SEM .",
"The p-value was calculated performing a Student’s t-test .",
"For demographic analysis the data from three biological experiments were pooled and for each experiment >100 cells were analyzed .",
"The total number of cells included ( n ) is mentioned for each demograph in the respective figures .",
"To test for stability and expression of YFP-CheW1 , bacterial samples were collected from cultures ready for fluorescence microscopy analysis .",
"Samples from different strains were normalized to optical density and subjected to western-blot analysis using JL8 anti-GFP antibodies ( also recognizing YFP ) .",
"As a positive control , a strain only expressing YFP was included .",
"Additionally , a strain not expressing any YFP variant was included as a negative control .",
"Photobleaching time-lapse experiments were performed using the Andor FRAPPA system .",
"Cells were treated and mounted on agarose pads as described for fluorescence microscopy of V . cholerae cells .",
"For FRAP experiments , a point-of-interest was bleached using a 515 nm laser at 7% intensity .",
"For bleaching of the cytoplasm , a region-of-interest corresponding to 2/3 of the cell length was chosen and bleached with 1 pulse using a 515 nm laser at 7% intensity .",
"Cells were then imaged over time .",
"For each time-point the fluorescence intensity at the cell pole was then calculated relative to the pre-bleach intensity and plotted as a function of time .",
"Graphs represent the average intensity of the indicated number of cells analyzed , with error-bars representing SEM .",
"The total number of cells analyzed ( n ) is mentioned in the figure .",
"In generation of multiple sequence alignments of V . cholerae MCPs and ParP orthologues , respectively , we used the MUSCLE tool at default settings ( Edgar , 2004 ) .",
"Phylogenetic trees were generated based on MUSCLE sequence alignments using Jalview Average Distance BLOSOM62 with default settings .",
"Phylogenetic trees generated in Jalview were displayed and colored using iTOL ( Letunic and Bork , 2011 ) .",
"ParP orthologs , in generation of the sequence alignment in Figure 3—figure supplement 2 and phylogenetic tree in Figure 3A , were chosen based on a STRING ( Jensen et al . , 2009 ) analysis of ParP and ParC from V . cholerae , using default settings .",
"Thus , all ParP orthologs included in the analysis are encoded by predicted parP genes , located within a chemotaxis operon , and with an associated parC gene immediately upstream , indicating that all ParPs included in the analysis are part of a ParC/ParP-system .",
"Furthermore , in generation of the phylogenetic tree in Figure 3A , we included CheWs and CheAs from chemotaxis operons that have an associated ParC/ParP-system , based on a STRING analysis of ParP and ParC from V . cholerae .",
"In Figure 3—figure supplement 1 , ParP from V . parahaemolyticus was aligned against CheW from T . maritima MSB8 .",
"T . maritima MSB8 CheW was chosen as reference for the alignment as amino acid residues from T . maritima MSB8 CheW important for mediating interactions to MCPs have been solved ( Griswold et al . , 2002; Park et al . , 2006; Briegel et al . , 2012; Li et al . , 2013 ) .",
"For imaging , V . cholerae strains were cultured overnight in 5 ml LB media at 37°C with 200 rpm shaking .",
"For each strain , 3 µl cell culture were applied to a freshly plasma-cleaned R2/2 copper Quantifoil grid ( Quantifoil Micro Tools , Jena , Germany ) .",
"Plunge freezing was carried out with a Leica EMGP ( Leica microsystems , Wetzlar , Germany ) .",
"Excessive liquid was wicked off from the grid by 1 s blotting inside the chamber set at room temperature and 95% humidity .",
"Grids were plunge frozen in liquid ethane at −183°C and then stored in liquid nitrogen until imaging .",
"Cryo EM images were collected on a Talos L120C transmission electron microscope ( Thermo Fisher Scientific ( formerly FEI ) , Hillsboro , OR , USA ) operating at 120 kV .",
"All targets were randomly picked , manually located and imaged in low dose mode ."
]
] | [
"Chemotaxis proteins organize into large , highly ordered , chemotactic signaling arrays , which in Vibrio species are found at the cell pole .",
"Proper localization of signaling arrays is mediated by ParP , which tethers arrays to a cell pole anchor , ParC .",
"Here we show that ParP’s C-terminus integrates into the core-unit of signaling arrays through interactions with MCP-proteins and CheA .",
"Its intercalation within core-units stimulates array formation , whereas its N-terminal interaction domain enables polar recruitment of arrays and facilitates its own polar localization .",
"Linkage of these domains within ParP couples array formation and localization and results in controlled array positioning at the cell pole .",
"Notably , ParP’s integration into arrays modifies its own and ParC’s subcellular localization dynamics , promoting their polar retention .",
"ParP serves as a critical nexus that regulates the localization dynamics of its network constituents and drives the localized assembly and stability of the chemotactic machinery , resulting in proper cell pole development ."
] | [
"Many bacteria live in a liquid environment and explore their surroundings by swimming .",
"When in search of food , bacteria are able to swim toward the highest concentration of food molecules in the environment by a process called chemotaxis .",
"Proteins important for chemotaxis group together in large networks called chemotaxis arrays .",
"In the bacterium Vibrio cholerae chemotaxis arrays are placed at opposite ends ( at the “cell poles” ) of the bacterium by a protein called ParP .",
"This makes sure that when the bacterium divides , each new cell receives a chemotaxis array and can immediately search for food .",
"In cells that lack ParP , the chemotaxis arrays are no longer placed correctly at the cell poles and the bacteria search for food much less effectively .",
"To understand how ParP is able to direct chemotaxis arrays to the cell poles in V . cholerae Alvarado et al . searched for partner proteins that could help ParP position the arrays .",
"The search revealed that ParP interacts with other proteins in the chemotaxis arrays .",
"This enables ParP to integrate into the arrays and stimulate new arrays to form .",
"Alvarado et al . also discovered that ParP consists of two separate parts that have different roles .",
"One part directs ParP to the cell pole while the other part integrates ParP into the arrays .",
"By performing both of these roles , ParP links the positioning of the arrays at the cell pole to their formation at this site .",
"The findings presented by Alvarado et al . open many further questions .",
"For instance , it is not understood how ParP affects how other chemotaxis proteins within the arrays interact with each other .",
"As well as enabling many species of bacteria to spread through their environment , chemotaxis is also important for the disease-causing properties of many human pathogens – like V . cholerae .",
"As a result , learning how chemotaxis is regulated could potentially identify new ways to stop the spread of infectious bacteria and prevent human infections ."
] | 2017 |
[
"Introduction",
"Results and discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology"
] | Biochemical basis for the regulation of biosynthesis of antiparasitics by bacterial hormones | elife-57824-v2 | [
[
"The rise of drug-resistant pathogens continues to compromise human health and is exacerbated by the decline in the rate of discovery of new anti-infectives ( Aminov , 2010; Tenover , 2006 ) .",
"A major limitation is the lack of tools that enable access to the vast library of bacterial natural product antibiotics .",
"Statistical surveys depict the number of antibiotics that are genetically encoded within the Streptomyces genus to be in excess of ~300 , 000 new molecules , but a large repertoire of these compounds cannot be produced when the strain is grown under standard laboratory conditions ( Govern , 2013; Watve et al . , 2001 ) .",
"The responsible biosynthetic genes are ‘silent’ under laboratory conditions and are regulated via unknown mechanisms ( Arakawa , 2018; Tyurin et al . , 2018 ) .",
"The diffusible small molecule γ-butyrolactone ( GBL ) A-factor plays an essential role in the biosynthesis of the antibiotic streptomycin in Streptomyces gresius .",
"The intracellular target of A-factor has been identified as a member of the TetR family , and these receptors have been shown to regulate antibiotic biosynthesis in several actinobacterial species ( Figure 1A; Tyurin et al . , 2018; Takano , 2006; Choi et al . , 2003; Onaka et al . , 1995 ) .",
"The use of exogenous GBLs has been shown to induce secondary metabolite production from otherwise silent clusters ( Thao et al . , 2017; Sidda and Corre , 2012 ) .",
"However , the alkali labile nature of γ-butyrolactones and the pleiotropic nature of GBL-mediated regulation limit the general use of these hormones .",
"Related classes of bacterial hormones include the 2-alkyl-3-methyl-4-hydroxybutenolides , the 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid , and the alkylbutenolides ( Figure 1B ) .",
"Butenolides , such as avenolide ( Figure 1B ) , trigger the production of secondary metabolites with a minimum effective concentration in the low nanomolar range in Streptomyces avermitilis ( Corre et al . , 2010; Arakawa et al . , 2012 ) .",
"Notably , butenolides show greater pH stability and generally regulate fewer processes than γ-butyrolactones .",
"The recent discovery of avenolide activity in about 24% of observed actinomycetes ( n = 51 ) , suggests that other active actinobacteria can also produce avenolide-like compounds to regulate secondary metabolism ( Thao et al . , 2017 ) .",
"For example , avenolide regulates the production of the anthelminitic compound avermectin ( Kitani et al . , 2011; Miller et al . , 1979 ) .",
"Ivermectin , a chemical derivative of avermectin , is on the World Health Organization’s list of essential medicines , and has lowered the incidence of otherwise untreatable parasitic infections , including river blindness , strongyloidiasis , and lymphatic filariasis ( elephantiasis ) ( Chen et al . , 2016; Callaway and Cyranoski , 2015 ) .",
"Enzymatic and synthetic routes towards the production of γ-butyrolactones have been described , but access to butenolides has been restrictive ( Kato et al . , 2007 ) .",
", ( Seitz and Reiser , 2005 ) Here , we describe a concise 22-step convergent route towards the total synthesis of avenolide , enabling biochemical and biophysical characterization of its interaction with the AvaR1 receptor .",
"We also present structures of AvaR1 , in isolation ( 2 . 4 Å resolution ) , in complex with avenolide ( 2 . 0 Å resolution ) , and bound to a synthetic DNA oligonucleotide ( 3 . 09 Å resolution ) derived from its natural binding site .",
"Using the primary sequence of AvaR1 and the synteny of genes that are likely to be involved in butenolide biosynthesis , we identify 89 additional putative butenolide receptors .",
"Mapping of residues at the ligand-binding site that form conserved sequences highlights their importance in ligand activation .",
"The identification of these putative avenolide-responsive strains may enable the production of novel metabolites in the presence of the hormone .",
"As proof of principle , we show that the supplementation of synthetic avenolide into growing cultures of two strains that contain homologous receptors results in visible changes to the production media ."
],
[
"We hypothesized that a detailed understanding of the mechanism of regulation of secondary metabolite production by avenolide would benefit from an understanding of the regulatory mechanism .",
"Moreover , as AvaR1 shares ~40% sequence identity with the γ-butyrolactone receptor ArpA , biochemical studies of AvaR1 may inform on other classes of bacterial hormone receptors while also providing the rationale for ligand specificity amongst these different classes .",
"The inherent temperature , acidic and alkali stability of butenolides over γ-butyrolactones prompted efforts towards the large-scale production of avenolide for biochemical studies .",
"However , little is known about the biosynthetic pathways that elaborate butenolides , as opposed to canonical GBLs that can be produced enzymatically from commercially available precursors .",
"To this end , we undertook a novel retro-synthetic strategy produce avenolide from the convergent synthesis of three key fragments: the iodide ( 11 ) , the aldehyde ( 12 ) and epoxy ( 10 ) ( Figure 1C ) .",
"We followed the protocol reported by Uchida et al . , 2011 , which begins with commercially available 1-methyl-2-butene .",
"A Sharpless asymmetric dihydroxylation ( Kolb et al . , 1994 ) produced the diol intermediate ( 1 ) in 82% yield in a single step ( Figure 1D ) .",
"The desired key intermediates aldehyde ( 12 ) , iodo alkene ( 11 ) and epoxy ( 10 ) were also made following the same reported protocol except that TBDPS rather than TBS was used for improved reaction monitoring using TLC .",
"The epoxy ( 10 ) was then stereospecifically converted into allyl alcohol ( 17 ) in a single step , by using titanocene dichloride and Zn powder ( Wang et al . , 2013; Katsuki and Sharpless , 1980 ) .",
"The final product , stereospecific ( 4S , 10R ) -avenolide ( 13 ) was then produced by ring-closing metathesis of molecule 19 , treating the dialkene with Grubb’s second-generation catalyst ( Sheddan and Mulzer , 2006 ) .",
"The synthetic strategy significantly reduced the number of steps from those reported in prior studies .",
"Specifically , the diol intermediate ( 1 ) was synthesized from the commercially available 1-methyl-2-butene in single step using Sharpless asymmetric dihydroxylation , avoiding multiple protection/deprotection steps .",
"Likewise , the aldehyde intermediate ( 12 ) was synthesized effectively in three steps ( as compared to ten steps in prior reports ) , avoiding the use of toxic CuCN .",
"Last , conversion of the epoxy intermediate ( 10 ) to the final product occurred through the intermediacy of an allyl alcohol ( 17 ) , reducing the strategy used in prior reports by four more steps .",
"The final yield of avenolide was 14 mg total from 15 g of starting material .",
"The identities of all intermediates , as well as that of the final product , were determined using 1H-NMR and 13C NMR that matched with the reported data .",
"Detailed experimental methods and NMR spectra can be found in Appendix 1 .",
"The structure of AvaR1 was determined to 2 . 4 Å resolution ( Figure 2A ) using crystallographic phases determined from anomalous diffraction data collected from SeMet-labeled protein crystals .",
"The overall structure is reminiscent of that of other TetR-family transcriptional repressors , and consists of an obligate homodimer ( Bhukya and Anand , 2017 ) .",
"Each monomer is entirely helical and consists of a DNA-binding domain ( DBD; composed of α helices 1–4 ) , and a ligand-binding domain ( LBD; consisting of α helices 5–13 ) .",
"The dimer interface is formed via interactions between the two LBDs and is formed mainly through hydrophobic packing interactions .",
"Co-crystallization efforts for AvaR1 bound to avenolide yielded crystals that diffracted to 2 . 0 Å resolution , and crystallographic phases were determined using molecular replacement ( Figure 2B; Thorn and Sheldrick , 2013 ) .",
"Clear density for the entire hormone can be visualized bound to the LBD of both monomers in the homodimer ( Figure 2C ) .",
"Notably , the structure of AvaR1 undergoes conformational shifting upon ligand binding , and a structure-based superposition against the ligand-free structure illustrates that binding of the hormone results in an ~10o shift in the DBD of each monomer ( Figure 2D ) .",
"This shift results in an increase in the distance between the two DBD in the dimer upon binding of the ligand , which would preclude DNA binding by the ligand-bound homodimer .",
"Additional local changes between the two structures included the movement of Gln165 , which swings into the binding pocket to make hydrogen-bonding interactions with the lactone ring , as well with Gln64 .",
"Last , Thr108 , which is harbored on helix α6 , also moves to accommodate interactions with the hormone .",
"The indole side chain of Trp127 likewise shifts to increase the volume of the binding cavity .",
"Other interactions include hydrogen bonds between Thr131 and the C10 hydroxy , and an interaction between Thr162 and the lactone ring of avenolide .",
"The superimposition suggests that the new contacts formed by these residues are likely to couple hormone binding to the conformational shift between the two monomers of AvaR1 ( Figure 2D ) .",
"We used isothermal titration calorimetry to characterize the binding interaction between the synthetic avenolide and AvaR1 ( measurements were conducted in triplicate ) .",
"The resultant binding isotherm shows the point of inflection at a molar ratio of N-1 , which suggests a 1:1 binding of ligand per monomer ( Figure 2D ) .",
"The strength of the binding is measured to be Kd = 42 . 5 nM ± 2 . 1 nM ( three independent trials ) , which correlates with the reported value of ~4 nM that was estimated from gel shift-based assays ( Kitani et al . , 2011 ) .",
"A structure-based sequence alignment of GBL-like receptors for which ligand specificity has been established reveals a strong conservation of residues that have been shown to be critical for ligand binding , suggesting a common mechanism for ligand recognition across disparate classes of receptors ( Figure 3A ) .",
"Residues that surround the alkyl chain of avenolide include Trp127 , Val158 , and Phe161 , which are almost universally conserved across all members of the receptor family , whereas Leu88 , which forms the opposite wall of the binding cavity , is always a hydrophobic residue but of variable size ( Figure 3B ) .",
"The chain length of the methylenomycin furans ( MMFs ) is shorter than those of the GBLs and butenolides; correspondingly , residues at the base of the ligand-binding cavity in the AvaR1 , such as Ala85 , His130 , and Thr131 , are replaced by bulkier the Glu107 , Leu150 , and Leu151 in the sequence of the MMF receptor MmrF .",
"Residue Thr161 is within hydrogen-bonding distance to the lactone ring of avenolide , and this residue is conserved in receptors that bind to γ-butyrolactones and butenolides , but absent from receptors for other classes of hormones such as MmrF .",
"Likewise , Gln64 in AvaR1 is positioned on the opposite site of the lactone and is conserved among receptors that bind to structurally related classes of hormones , but is absent from the structure of MmrF .",
"The latter sequence contains a Tyr85 at a near equivalent position , which may be necessary for interactions with the carboxyl group of MMF .",
"The lactone ring of avenolide is situated above helix α6 , which contains residues that are nearly universally conserved among GBL-like receptors , including Ser103 , Val104 , Arg105 , Leu106 , Val107 , and Asp108 .",
"Notably , this helix bridges the LBD and the DBD , suggesting that it plays a role in coupling ligand binding to DNA dissociation ( Figure 3C ) .",
"Specifically , movement of Gln64 and Thr108 into the ligand-binding cavity of AvaR1 upon engagement of the hormone results in the displacement of helix α6 away from the pocket .",
"The orientation of Arg105 , located on the opposite side of helix α6 , is established through multiple hydrogen-bonding interactions with the backbone carbonyls of conserved residues in helix α1 , including the universally conserved Phe22 , Gly26 , and Tyr27 .",
"Hence , the accommodation of hormone binding necessitates movement of the DBD , in order to preserve the suite of hydrogen-bonding interactions with helix α6 .",
"As noted , Gln64 and Thr107 are largely conserved among receptors that bind lactone-containing hormones , suggesting a common mechanism for coupling ligand binding to DNA dissociation .",
"In order to gain further insights into the mechanism of hormone-mediated de-repression , we also determined the structure of AvaR1 bound to a synthetic oligonucleotide derived from the autoregulator responsive element ( ARE ) sequence .",
"DNase foot-printing analysis had previously established the identity of the ARE located upstream of the aco gene , but this response element is pseudopalindromic ( Kitani et al . , 2011 ) .",
"Crystallization efforts with the symmetric AvaR1 homodimer yielded crystals that did not diffract beyond 8 Å , presumably as a result of the asymmetry of the ARE operator .",
"Efforts using an artificial palindromic sequence derived by inverting and repeating each half of the pseudo palindrome yielded crystals that diffracted to 3 . 09 Å resolution ( Figure 3D , Appendix 1—figure 2 , Appendix 1—table 1 ) , and the structure was determined by molecular replacement .",
"As a result of the use of this symmetric DNA , each homodimer in the crystallographic asymmetric unit is bound to a monomer from an adjacent ARE .",
"The structure shows that each DBD interacts with one half of the palindrome of the DNA duplex .",
"Numerous contacts are formed between helix α1 of the DBD and the duplex , including Arg5 , which inserts into the major groove and interacts with Thy7 , as well as between Lys43 and Ade15 of the ARE ( Figure 3D ) .",
"Additional non-specific interactions include those between Thr10 , Thr42 , and Tyr47 and the backbone phosphate of the duplex .",
"A comparison of the ligand-binding sites with that in the hormone-bound structure reveals that the binding pocket is further occluded through movements of Trp127 and the loop harboring Gln64 , consistent with the roles of these residues in effecting conformational movements of the DBD upon binding of the hormone .",
"Given the improved stability of butenolides over γ-butyrolactones , we speculate that these hormones may prove more amenable in attempts to activate antibiotic production .",
"In order to identify actinobacterial strains that are under butenolide regulatory control , we sought to use a bioinformatics approach based on the identification of the corresponding receptor .",
"However , as shown in Figure 3A , the sequence similarity between bona fide γ-butyrolactone receptors , such as ArpA , and butenolide receptors is high ( 40% sequence identity with AvaR1 ) precluding such analysis .",
"Orphan receptors called pseudo γ-butyrolactone receptors that are activated by multiple ligands likewise share 40% sequence identity with AvaR1 , confounding simple sequence-based analysis .",
"Prior efforts to discriminate between receptor classes have not proven fruitful , and phylogenetic analyses have failed to discriminate between positive and negative regulators .",
"In an effort to identify other actinobacteria that are under butenolide regulatory control , we utilized synteny of the putative butenolide biosynthetic genes to distinguish between receptor clades .",
"We first used the Enzyme Similarity Tool ( EST ) from the Enzyme Function Initiative to create a Sequence Similarity Network ( SSN ) of all members of the receptor class .",
"An E-value cutoff of 10−70 was used to produce an SNN in which characterized receptors of the various GBL families were segregated with mutually exclusive co-localization ( Figure 4A ) .",
"We used the resultant SSN as input for the EFI Genome Neighborhood Network ( GNN ) tool to identify nodes that are co-localized next to genes with PFams that are associated with putative avenolide biosynthetic genes .",
"The fact that the butenolide receptors often regulate the production of their own ligand further enabled this approach and allowed for inferences about the classes of ligands that are produced and recognized by receptors that have yet to be characterized .",
"Because the EFI tools are only integrated with the Uniprot database , we also manually searched sequences in Genbank for similar operonic architecture to identify putative butenolide receptors on the basis of genomic context .",
"Our analysis yielded a set of 90 putative actinobacterial genomes ( Appendix 1—table",
"2 ) that harbor a putative butenolide receptor that is likely to be under butenolide regulatory control ( Figure 4A ) .",
"We emphasize that , although this is orders of magnitude greater than the currently known number of butenolide receptors , this number represents a significant underestimate but the true number cannot be identified given the limitations of our approach .",
"Mapping of the sequence conservation amongst these 90 receptors onto the co-crystal structure of hormone bound to AvaR1 reveals that residues Gln64 , Thr108 , and Trp127 , which are proposed to couple ligand binding to domain shift , are conserved among all of these sequences ( Figure 4B ) .",
"The conservation score is highest for residues that are involved in interactions with the lactone , whereas those that interact with the alkyl tail are more divergent .",
"These data are consistent with the observations that the lactone ring and C10 hydroxyl are general features of butenolides , whereas the length and branching of the alkyl tail vary significantly .",
"The organization of the biosynthetic operon that harbors genes , which is hypothesized to be involved in butenolide biosynthesis , is largely conserved in these organisms ( Figure 4C and Appendix 1—table 2 ) .",
"Although the bacterial hormone A-factor was discovered nearly a half century ago , significant gaps remain in our understanding of how these signaling molecules regulate gene expression .",
"The discovery of the regulation of secondary metabolite biosynthesis by γ-butyrolactones inspired efforts to use these molecules as ex vivo effectors to induce otherwise silent biosynthetic gene clusters , but these efforts met with little success .",
"The labile nature of the lactone ring , as well as the often pleiotropic effects that γ-butyrolactones induce , have presumably subverted efforts to utilize these small molecules as chemical inducers .",
"By contrast , the structurally related butenolides show improved stability under strongly acidic and basic conditions .",
"However , the utility of butenolides in biotechnology efforts has been limited by an inability to access these molecules .",
"Here , we present here an efficient and convergent 22-step total synthetic route for the production of avenolide , which can be extrapolated for the total synthesis of other members of the butenolide class of small signaling molecules .",
"This effort allowed for detailed structure–function studies of the corresponding hormone receptor , including the first crystal structure of any GBL-type receptor bound to its cognate ligand .",
"The structural data informs on the mechanism by which hormone binding induces a conformational change in the AvaR1 receptor , resulting in the formation of a dimeric assembly that occludes efficient DNA binding .",
"We also elaborate a bioinformatics strategy using the genomic neighborhood context of butenolide biosynthetic genes as a marker to identify 90 actinobacterial strains that are likely to be under the regulatory control of butenolides .",
"The addition of avenolide to the growth media for two representative strains results in changes in the color of the culture supernatant .",
"These results support the validity of our bioinformatics approaches and set the framework for further efforts towards the use of butenolides to active antibiotic biosynthesis in otherwise silent gene clusters ."
],
[
"Wild-type protein AvaR1 was amplified from S . avermitilis genomic DNA by PCR using primers that were based on the published sequence of the polypeptide and inserted into a pET-28-MBP vector for expression in Escherichia coli as a maltose binding protein ( MBP ) -tagged fusion .",
"The resultant plasmid was transformed into E . coli containing the Rosetta plasmid for protein expression .",
"AvaR1 was produced by growing the cells in shaking flask of LB media at a temperature of 37°C .",
"When the cells reached an O . D . 600 of 0 . 6 , the cells were cooled on ice for 15 min .",
"Following the addition of 0 . 5 mM isopropyl β- d-1-thiogalactopyranoside ( IPTG ) , the cells were placed in an 18°C shaking incubator for 18 hr .",
"The cells were then harvested by centrifugation and re-suspended in a buffer composed of 500 mM NaCl , 20 mM Tris base ( pH 8 . 0 ) , and 10% glycerol .",
"Re-suspended cells were lysed by homogenization and the lysate was centrifuged at 14 , 000 rpm to remove cell debris .",
"The cleared cell lysate was loaded onto a HisTrap column , which was subsequently washed with 1 M NaCl , 30 mM imidazole , and 20 mM Tris base ( pH 8 . 0 ) .",
"MBP-tagged AvaR1 was eluted using a linear gradient beginning with 1 M NaCl , 20 mM Tris base ( pH 8 . 0 ) , and 30 mM imidazole and ending with 250 mM imidazole .",
"Pure fractions , as judged by SDS-PAGE , were combined and diluted two-fold before treatment with thrombin ( final ratio of 1:100 [w/w] ) for 18 hr at 4°C to cleave the N-terminal tag .",
"Tag-free AvaR1 was concentrated and loaded onto a size exclusion column ( Superdex S75 16/60 ) pre-equilibrated with 100 mM KCl and 20 mM HEPES free acid ( pH 7 . 5 ) .",
"Pure fractions were collected and concentrated to 25 mg/mL before storage in liquid nitrogen .",
"Production of SeMet-labeled AvaR1 was carried out by repression of methionine synthesis in defined media supplemented with selenomethionine ( Doublié , 2007 ) .",
"Preliminary crystals of AvaR1 were obtained using a sparse matrix screen .",
"Diffraction-quality crystals were grown using hanging drop vapor diffusion .",
"13 . 5 mg/mL AvaR1 was added at a 1:1 ratio to mother liquor containing 12% polyethylene glycol ( PEG ) 1000 , 0 . 1 M sodium citrate tribasic dehydrate ( pH 4 . 2 ) , 0 . 2 M LiSO4 and 4% ( v/v ) tert-butanol , and incubated against the same solution at 4°C .",
"Crystals were improved through multiple rounds of micro-seeding .",
"The SeMet-AvaR1 crystals were obtained using 12 mg/mL protein added to a 1:1 ratio of 30% PEG MME 2000 , and 0 . 15 M KBr .",
"Crystals were vitrified by direct immersion without the addition of any cryo-protectives .",
"All diffraction data were collected at Argonne National Laboratory ( IL ) .",
"The autoPROC ( Vonrhein et al . , 2011 ) software package was utilized for the indexing and scaling of the diffraction data .",
"Initial phases for AvaR1 were obtained using anomalous diffraction data collected on crystals of SeMet-labeled protein .",
"Initial models were built using Phenix and Parrot/Buccaneer .",
"Manual refinements were completed by the iterative use of COOT ( Emsley et al . , 2010 ) and Phenix . refine .",
"Cross-validation was utilized throughout the model-building process in order to monitor building bias .",
"The stereochemistry of all of the models was routinely monitored using PROCHECK .",
"Crystallographic statistics are provided in Appendix 1—table 3 .",
"For co-crystallization of the hormone-bound complex , purified AvaR1 ( 14 mg/ml ) was incubated with 3 mM avenolide for 30 min on ice .",
"Co-crystals were obtained by vapor diffusion methods and initial crystals were obtained in Index D5 ( 25% PEG3350 and 0 . 1M sodium acetate trihydrate [pH 4 . 5] ) .",
"Well-diffracting crystals were produced through optimization to a final solution of 23% PEG3350 and 0 . 1 M sodium acetate trihydrate ( pH 4 . 5 ) at 4°C using hanging drop crystallization .",
"Crystals were submerged briefly in the crystallization medium supplemented with 25% ethylene glycol prior to vitrification in liquid nitrogen .",
"The coordinates of apo AvaR1 were used to determine crystallographic phases .",
"AvaR1 was co-crystallized with different oligonucleotide sequences that were designed on the basis of the AvaR1 DBS upstream of aco gene .",
"Purified and concentrated dimeric protein ( 14 mg/mL ) was incubated with individual oligonucleotide duplexes ( Supporting Appendix 1—table 1 ) in 1:1 . 2 molar ratio , for 30 min on ice .",
"Palindromic DNA sequences were first self-annealed and then double stranded DNA was used from 1 mM stock prepared in 20 mM MgCl2 , 50 mM Tris ( pH 8 . 0 ) buffer .",
"The order in which reactants were added was: buffer , DNA and finally protein .",
"For some oligonucleotides , white turbid solution was obtained as soon as the protein was added , but the addition of a few microliters of ammonium acetate and incubating at either room temperature or on ice produced clear solution .",
"Crystallization trays were set up at 4°C and every oligonucleotide was crystallized in different conditions ( Appendix 1—figure 2B ) .",
"Ethylene glycol ( 25% v/v ) was used as cryoprotectant prior to the vitrification of crystals for all AvaR1–oligonucleotide co-crystals .",
"The oligonucleotide sequences used are listed in Appendix 1—table 1 and the sequences that produced diffraction-quality crystals are shown in Appendix 1—figure 2 .",
"Using the AvaR1 amino-acid sequence as a handle , tools from the Enzyme Function Initiative ( EFI ) were used to first create a Sequence Similarity Network ( SSN ) of 10 , 000 Uniprot sequences .",
"Once an SSN was created , an iterative process was undertaken to find an E-value that ensured that characterization of receptors of the various GBL families resulted in mutually exclusive co-localization .",
"The resulting E-value was 10−70 .",
"Using this SSN , the data was run through the EFI’s Genome Neighborhood Network ( GNN ) webtool .",
"Network visualization was performed in Cytoscape ( Shannon , 1971 ) .",
"Using Pfams associated with putative avenolide biosynthetic genes along with the knowledge that this family of receptors often regulates their own ligand production , inferences were made as to the class of ligand produced and recognized by uncharacterized receptors .",
"Because the EFI webtools are only integrated with the uniprot database , we also manually scoured through a number of Genbank sequence results derived from BLAST analysis for proper genomic context relative to butenolide production .",
"These BLAST searches used the sequences of any putative butenolide biosynthetic genes as handles .",
"The proper genomic context necessary for butenolide biosynthesis was defined as a TetR_N Pfam receptor surrounded by a gene in the p450 Pfam ( PF00067 ) , and either an Acyl-CoA_dh_1 ( PF00441 ) Pfam gene , an Acyl-CoA-dh_2 ( PF08028 ) Pfam gene , or an ACOX ( PF01756 ) Pfam gene .",
"A list of the 90 homologous strains that were identified is provided in Appendix 1—table 2 .",
"ITC measurements were performed at 25°C on a MicroCal VP-ITC calorimeter .",
"A typical experiment consisted of titrating 7 µL of a ligand solution ( 80 µM ) from a 250 µL syringe ( stirred at 300 rpm ) into a sample cell containing 1 . 8 mL of AvaR1 solution ( 8 µM ) with a total of 35 injections ( 2 µL for the first injection and 7 µL for the remaining injections ) .",
"The initial delay prior to the first injection was 60 s , with reference power 10 μCal/s .",
"The duration of each injection was 16 s and the delay between injections was 400 s .",
"All experiments were performed in triplicate .",
"Data analysis was carried out with Origin 5 . 0 software .",
"Binding parameters , such as the dissociation constant ( Kd ) , enthalpy change ( ∆H ) , and entropy change ( ∆S ) , were determined by fitting the experimental binding isotherms with appropriate models ( one-site binding model ) .",
"The ligand stock solution was prepared at 10 mM .",
"The buffer solutions for ITC experiments contained 300 mM KCl and 20 mM HEPES ( pH 7 . 5 ) .",
"The experimental procedures were adopted from Uchida et al . , 2011 with further optimizations and modifications as stated .",
"Detailed experimental procedures for relevant intermediates are specified in the Materials and methods section of Appendix 1 .",
"For synthesis of ( R ) −2-Methylbutane-1 , 2-diol ( 1 ) : To a stirred solution of the 2-methyl-1-butene ( 3 . 75 g , 53 . 47 mmol ) in t-BuOH: H2O ( 1:1 , 400 ml ) were added K3Fe ( CN ) 6 ( 52 . 81 g , 160 . 41 mmol ) , K2CO3 ( 22 . 17 g , 160 . 41 mmol ) , K2OsO4 ( OH ) 4 ( 197 mg , 0 . 54 mmol , 1 mol% ) and ( DHQD ) 2PHAL ( 416 . 5 mg , 0 . 54 mmol , 1 mol% ) were added to a stirred solution of 2-methyl-1-butene ( 3 . 75 g , 53 . 47 mmol ) in t-BuOH: H2O ( 1:1 , 400 ml ) at 0°C under Ar atmosphere .",
"The reaction mixture was stirred for 24 hr at 0°C using an Ar balloon .",
"The reaction was quenched with a saturated aqueous solution of Na2S2O3 and the aqueous phase was extracted with EtOAc ( 2 × 1 L ) .",
"The water layer was thoroughly washed with EtOAc , and combined organic extracts were washed with brine ( saturated NaCl ) and dried over anhydrous Na2SO4 and concentrated in vacuo .",
"The residue was purified by flash column chromatography with a gradient from 30% EtOAc/hexanes to 70% EtOAc/hexanes to 10% MeOH/DCM , to afford 1 ( 3 . 5 g , 82% ) as a colorless oil .",
"This molecule was obtained in single step from commercially available 2-methylbut-1-ene using Sharpless asymmetric dihydroxylation ( Kolb et al . , 1994 ) .",
"Spectroscopic characterization parameters agreed with the reported mass and chemical shift values .",
"[α] ( Bhukya and Anand , 2017 ) +4 . 96 ( c 1 . 0 , CHCl3 ) ; 1HNMR ( 500 MHz , CDCl3 ) δ 3 . 47 ( brs , 2H ) , 3 . 43 ( d , J = 11 . 1 Hz , 1H ) , 3 . 37 ( d , J = 11 . 1 Hz , 1H ) , 1 . 51 ( q , J = 7 . 3 Hz , 2H ) , 1 . 10 ( s , 3H ) , 0 . 89 ( t , J = 7 . 6 Hz , 3H ) ; 13C-NMR ( 125 MHz , CDCl3 ) δ 73 . 6 , 69 . 3 , 31 . 2 , 22 . 5 , 8 . 2 HRMS ( ESI+ , TFA-Na ) calcd for C5H12NaO2 127 . 0735 [M+Na]+ , found m/z 127 . 0740 .",
"For synthesis of aldehyde fragment 12 , the p-methoxybenzyl ( PMB ) -protected intermediates were synthesized according to the protocol reported by Uchida et al . , 2011 .",
"Iodo alkene fragment 11 was also synthesized following the reported protocol by substituting the use of the tert-butyl ( dimethyl ) silyl ( TBS ) protecting group with a tert-butyldiphenylsilyl group ( TBDPS ) to enable easy monitoring of the reaction by thin layer chromatography ( TLC ) visualization under UV light .",
"Epoxy fragment 10 was synthesized through intermediates 14 , 15 and 16 , as described in Appendix 1 .",
"( R ) −8- ( ( 2S , 3S ) −3- ( Hydroxymethyl ) oxiran-2-yl ) −3- ( ( 4-methoxybenzyl ) oxy ) −3-methyloctan-4-one ( 17 ) was synthesized following the protocol from the reported literature ( Wang et al . , 2013 ) .",
"Anhydrous ZnCl2 ( 2 mL , 1 M in Et2O , 2 mmol ) and zinc powder ( 350 mg , 6 . 72 mmol ) were added to a red solution of Cp2TiCl2 ( 1 . 26 g , 5 . 05 mmol ) in anhydrous tetrahydrofuran ( THF ) ( 15 mL ) .",
"The solution was stirred for 1 hr at room temperature until it turned green .",
"Epoxide 10 ( 590 mg , 1 . 68 mmol ) in anhydrous THF ( 5 mL ) was then added to the resultant mixture .",
"After stirring for 30 min at room temperature , the reaction was quenched with aqueous HCl ( 1 . 0 M , 3 mL ) and the mixture was extracted three times with Et2O ( 4 mL ) .",
"Collected Et2O fractions were combined and washed with water , 10% aqueous NaHCO3 , water and brine , dried over Na2SO4 and filtered and concentrated under reduced pressure .",
"The obtained residue was purified using flash column chromatography on silica gel ( 25% EtOAc/hexane to 40% EtOAc/hexane ) to obtain pure allyl alcohol compound 17 .",
"1H-NMR ( 500MHz , CDCl3 ) δ 7 . 27 ( d , J = 8 . 8 Hz , 2H ) , 6 . 89 ( d , J = 8 . 8 Hz , 2H ) , 5 . 87–5 . 81 ( m , 1H ) , 5 . 21 ( ddd , J = 17 . 2 , 1 . 4 , 1H ) , 5 . 10 ( ddd , J = 10 . 4 , 1 . 4 , 1H ) , 4 . 33 ( d , J = 10 . 8 Hz , 1H ) , 4 . 29 ( d , J = 10 . 8 Hz , 1H ) , 4 . 14–4 . 06 ( m , 1H ) , 3 . 81 ( s , 3H ) , 2 . 66 ( dt , J = 7 . 3 , 4 . 3 Hz , 2H ) , 1 . 84–1 . 70 ( m , 2H ) , 1 . 59–1 . 34 ( m , 6H ) , 1 . 33 ( s , 3H ) , 0 . 84 ( t , J = 7 . 5 Hz , 3H ) ; 13C-NMR ( 500MHz , CDCl3 ) δ 215 . 2 , 159 . 0 , 141 . 3 , 128 . 9 , 128 . 9 , 114 . 9 , 114 . 0 , 113 . 8 , 84 . 8 , 73 . 2 , 65 . 3 , 55 . 5 , 37 . 1 , 36 . 8 , 29 . 4 , 25 . 3 , 23 . 5 , 20 . 2 , 8 . 1; HRMS ( ESI+ , TFA-Na ) calcd for C20H30NaO4 357 . 2042 [M+Na]+ , found m/z 373 . 2032 .",
"This resulted in a simplified protocol for the synthesis of allyl alcohol 17 , which otherwise was reported to be made in two additional reaction steps starting from epoxy 10 .",
"An acrylic group was added to allyl alcohol 17 using DDQ by the reported procedure , which was followed by ring-closing metathesis reaction to yield stereospecific ( 4S , 10R ) -avenolide ( 13 ) .",
"Detailed synthetic schemes , experimental procedures and yields are reported in the Materials and methods section of Appendix 1 .",
"The obtained 1H and 13C NMR data support the reported values , and thus the spectra are provided only for key intermediates ."
]
] | [
"Diffusible small molecule microbial hormones drastically alter the expression profiles of antibiotics and other drugs in actinobacteria .",
"For example , avenolide ( a butenolide ) regulates the production of avermectin , derivatives of which are used in the treatment of river blindness and other parasitic diseases .",
"Butenolides and γ-butyrolactones control the production of pharmaceutically important secondary metabolites by binding to TetR family transcriptional repressors .",
"Here , we describe a concise , 22-step synthetic strategy for the production of avenolide .",
"We present crystal structures of the butenolide receptor AvaR1 in isolation and in complex with avenolide , as well as those of AvaR1 bound to an oligonucleotide derived from its operator .",
"Biochemical studies guided by the co-crystal structures enable the identification of 90 new actinobacteria that may be regulated by butenolides , two of which are experimentally verified .",
"These studies provide a foundation for understanding the regulation of microbial secondary metabolite production , which may be exploited for the discovery and production of novel medicines ."
] | [
"Bacteria that dwell in soil known as actinobacteria are the source of many drugs that are used to treat cancer and infectious diseases in humans .",
"In their natural environments actinobacteria produce these drugs , or at least similar compounds , to compete with neighboring microbes for food or to kill their enemies .",
"However , when researchers culture actinobacteria in the laboratory , the bacteria often produce little or none of these compounds .",
"Some actinobacteria produce a compound called avermectin .",
"This compound is closely related to a drug used to treat an infectious disease known as river blindness , which is a common cause of sight loss in people living in West Africa .",
"A bacterial hormone known as avenolide regulates when the bacteria produce avermectin by binding to a receptor known as AvaR1 .",
"But the precise details of how this process works remained unclear .",
"To investigate how avenolide binds to AvaR1 , Kapoor , Olivares and Nair developed a new strategy to produce large quantities of avenolide in the laboratory from commercially available molecules .",
"A three-dimensional structure of AvaR1 was then generated showing the receptor on its own , bound to avenolide or bound to a short DNA molecule .",
"In the absence of avenolide , AvaR1 sits on DNA .",
"However , binding to avenolide causes AvaR1 to move off the DNA .",
"This revealed how the binding of avenolide changes the receptor protein so that it can be released from DNA to allow the production and release of other small molecule compounds .",
"Further experiments used these structures as guides to identify 90 new species of actinobacteria that may respond to avenolide and other similar bacterial hormones .",
"Understanding how bacterial hormones stimulate actinobacteria to produce avermectin and other compounds will aid efforts to extract new compounds from soil bacteria that have the potential to treat cancer or infectious diseases ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"structural biology and molecular biophysics"
] | Recruitment of two dyneins to an mRNA-dependent Bicaudal D transport complex | elife-36306-v2 | [
[
"Mammalian cytoplasmic dynein-1 ( hereafter dynein ) is a 12 subunit , 1 . 4 MDa dimeric molecular motor complex of the AAA+ ATPase family that provides essential cellular functions including transport of vesicles , organelles , and mRNA ( reviewed in [Roberts et al . , 2013] ) .",
"Dynein is the predominant minus-end directed microtubule-based motor that traffics cellular cargoes for many microns at speeds of ~1 µm/s ( Allan , 2011 ) .",
"Molecular motors that transport cargo typically exhibit processive behavior when assayed in vitro , meaning that single motors remain bound to the polymer track for long distances without dissociating .",
"It was therefore surprising when in vitro studies showed that single molecules of mammalian dynein were at best weakly processive , even in the presence of the multi-subunit 1 . 2 MDa dynactin complex that is needed for most cellular functions of dynein ( King and Schroer , 2000; McKenney et al . , 2014; Miura et al . , 2010; Ross et al . , 2006; Schlager et al . , 2014; Trokter et al . , 2012; Wang et al . , 1995 ) .",
"It was not initially recognized that dynein , like other molecular motors , exists in an auto-inhibited state .",
"One of the first structural studies to investigate dynein auto-inhibition showed that the AAA+ motor domains are closely stacked in a structure called the phi particle ( Amos , 1989; Torisawa et al . , 2014 ) .",
"Several non-physiologic mechanisms of disrupting this interaction , such as coupling multiple dyneins to a DNA origami ( Amos , 1989; Torisawa et al . , 2014 ) or binding to a bead ( Belyy et al . , 2016; King and Schroer , 2000; Mallik et al . , 2004; Nicholas et al . , 2015 ) , converted dynein from a diffusive to a weakly processive motor by separating the stacked rings .",
"A major advance in understanding dynein function came from single molecule studies , which showed that an α-helical coiled-coil N-terminal fragment of the mammalian adaptor protein Bicaudal-D2 ( BicD2N ) couples dynein to dynactin .",
"This causes dynein to become highly processive , with 5–10 µm run lengths , and leads to motor accumulation at the microtubule minus-end ( McKenney et al . , 2014; Schlager et al . , 2014 ) .",
"This minimal tripartite complex is called DD ( BicD2N ) ( dynein-dynactin-BicD2N ) .",
"Dynein in the activated DD ( BicD2N ) complex produced 4 . 3 pN of force ( Belyy et al . , 2016 ) , considerably higher than the 0 . 5–1 . 5 pN forces reported earlier for dynein alone ( Mallik et al . , 2004; McKenney et al . , 2010; Ori-McKenney et al . , 2010; Rai et al . , 2013 ) , thus allowing dynein to successfully engage in a tug-of-war with a single kinesin .",
"Recent EM studies provided further insight into the structural basis for the inhibition and activation of dynein .",
"Cryo-EM studies showed that when the dynein motor domains are stacked in the phi particle they are locked in a conformation with low affinity for microtubules; disruption of the motor domain self-dimer creates an ‘open’ state that is still inhibited for motion ( Zhang et al . , 2017 ) .",
"Only when bound to dynactin and an ‘adaptor’ such as BicD2N are the dynein motor domains aligned in a parallel orientation on the microtubule that correlates with highly processive movement ( Chowdhury et al . , 2015 ) .",
"Cargo adaptor proteins such as BicD2 are thus central to control of dynein activity in the cell .",
"Although BicD2N artificially fused to mitochondria or peroxisome targeting sequences supports robust dynein-driven motility in HeLa cells ( Hoogenraad et al . , 2003 ) , full-length BicD has only a mild effect on organelle re-localization ( Hoogenraad et al . , 2003 ) .",
"It has been assumed that full length BicD2 assumes an auto-inhibited conformation that does not bind to or activate dynein-dynactin .",
"BicD2 is composed of three α-helical coiled-coil domains; the N-terminal domain ( CC1 ) is involved with dynein-dynactin binding and activation ( Urnavicius et al . , 2015 ) , whereas the C-terminal domain ( CC3 ) binds adaptor proteins that link dynein to cargo ( Liu et al . , 2013 ) .",
"Early yeast-2-hybrid studies showed that the CC1 domain interacts with the CC3 domain , leading to a model in which BicD forms N- to C-terminal auto-inhibitory interactions ( Hoogenraad et al . , 2001 ) .",
"Early metal-shadowed images of Drosophila BicD , in contrast , suggested interaction of CC3 with CC2 ( Stuurman et al . , 1999 ) .",
"The leading hypothesis for cellular activation of dynein-dynactin transport by BicD is that cargo-associated BicD2-binding proteins relieve auto-inhibition by freeing up CC1 .",
"Here , we test this cargo-activation model by reconstituting in vitro a biologically well-characterized system , localization of K10 mRNA in Drosophila .",
"During development , K10 mRNA is transported by dynein from nurse cells to the Drosophila oocyte , where it localizes to the anterior margin to establish the dorso-ventral axis of the Drosophila egg ( Cheung et al . , 1992 ) .",
"K10 contains a single 44 base-pair transport/localization element ( TLS ) in its 3’UTR that binds the mRNA-binding adaptor protein Egalitarian ( Egl ) , which in turn binds to CC3 of Drosophila BicD ( Dienstbier et al . , 2009; Serano and Cohen , 1995 ) .",
"To understand the molecular basis for dynein activation in this system , we reconstituted a motile mRNP ( messenger ribonucleotide protein ) complex in vitro from purified dynein , dynactin , full-length BicD , Egl , and synthesized K10 mRNA .",
"Single-molecule approaches coupled with electron microscopy suggest that the key to dynein activation is disruption of an auto-inhibited loop structure of BicD .",
"A single K10 mRNA can bind two Egl molecules to create a bivalent Egl that efficiently disrupts the BicD loop structure , freeing CC1 and favoring recruitment of two dynein motors to the complex .",
"The resulting mRNPs move at higher speeds and over longer distances than complexes with just one dynein .",
"K10 mRNA cargo thus orchestrates activation of the motile complex , an elegant mechanism ensuring that only motors properly complexed with cargo can undergo robust motility ."
],
[
"A single-molecule pulldown assay was used to determine if full-length Drosophila BicD ( hereafter called BicD ) binds to dynein-dynactin purified from bovine brain .",
"BicD is composed of predicted α-helical coiled-coil domains ( CC1 , CC2 , and CC3 , colored bars ) separated by non-coiled coil regions ( gray bars ) ( Figure 1A ) .",
"As positive controls , we tested the binding of a truncated version of Drosophila BicD ( hereafter called BicDCC1 ) , as well as a truncated version of mammalian BicD2 ( hereafter called BicD2N ) ( see Figure 1—figure supplement 1 for SDS-PAGE ) .",
"These same N-terminal fragments of Drosophila BicD ( Dienstbier et al . , 2009 ) and mammalian BicD2 ( Hoogenraad et al . , 2003 ) stimulate dynein-based transport when exogenously expressed in cultured cells , and BicD2N has been shown to couple dynein to dynactin and highly activate processive motility in vitro ( McKenney et al . , 2014; Schlager et al . , 2014 ) .",
"Full-length and truncated Qdot labeled-BicD constructs were incubated with dynein and dynactin , and applied to flow cells with surface-adhered microtubules in the presence of the ATP analog AMP-PNP , which causes dynein to bind strongly to microtubules .",
"Total internal reflection fluorescence ( TIRF ) microscopy was used to visualize and quantify associations with the microtubule ( Figure 1B , C ) .",
"Binding of BicD or BicDCC1 alone to microtubules was negligible ( Figure 1B , C ) , and thus BicD recruitment to microtubules is an accurate reporter of formation of a tripartite BicD-dynein-dynactin complex .",
"BicDCC1 showed an ~10 fold enhanced recruitment to microtubule-bound dynein-dynactin compared with full-length BicD , similar to the ~13 fold increase observed when mammalian BicD2N was compared with full-length BicD ( Figure 1D ) .",
"The truncated versions of Drosophila BicD and mammalian BicD2 thus behave similarly , suggesting that their mechanism of action is evolutionarily conserved .",
"Electron microscopy was used to determine the structural basis for BicD auto-inhibition .",
"Negatively stained EM images of YFP-BicD show two distinct globular densities at the N-terminus that correspond to YFP , confirming the formation of a parallel coiled-coil dimer .",
"The montage ( Figure 2A ) is arranged to show ( row",
"1 ) the most common ‘b’ orientation molecules , ( row",
"2 ) a less common ‘d’ orientation , ( row",
"3 ) the range over which the molecule can flex , ( row",
"4 ) some very compact molecules , and ( row",
"5 ) rare open molecules .",
"The most common feature of BicD is the loop which appears to be formed by parts of all three coiled coil segments , which is readily seen in averages of all molecules ( ‘global’ ) as well as averages of the most common ‘b’ orientation molecules ( Figure 2B , C , Figure 2—figure supplement 1 ) .",
"The YFP densities abut a fairly straight CC1 region followed by a flexible break in the coiled-coil from which CC2 and CC3 loop back to interact at a point approximately halfway along the CC1 coiled coil ( Figure 2D , E ) .",
"Our interpretation that the relatively straight portion of BicD is CC1 is consistent with contour length measurements .",
"The distance from YFP to the very bottom of the loop is 38 . 7 ± 3 . 8 nm ( ± SD , n = 150 ) , in good agreement with Paircoil2 analysis ( McDonnell et al . , 2006 ) that predicts the first 258 amino acids of BicD ( CC1 ) to be an α-helical coiled-coil 37 . 7 nm long ( based on 0 . 146 nm rise/residue ) .",
"The contour length from YFP to the point where the CC2-CC3 loop contacts CC1 is 16 . 5 ± 2 . 3 nm ( ± SD , n = 133 ) , and from here to the bottom of the loop is 19 . 1 ± 2 . 0 nm ( ± SD , n = 150 ) .",
"The sum of these lengths ( 16 . 5 + 19 . 1 nm ) is 35 . 6 nm , similar to the 38 . 7 nm measured from YFP to the base of the loop .",
"The contour length of the curved side of the loop is 24 . 5 ± 2 . 7 nm ( ± SD , n = 148 ) .",
"Together , these measurements predict that CC2-CC3 loop contacts CC1 near amino acid 113 ( 16 . 5 nm/0 . 146 nm rise per residue ) .",
"Pulldowns confirmed that the CC1 domain interacts with a construct containing the CC2-CC3 domains , but not with a construct composed only of CC2 ( Figure 2F ) , consistent with our interpretation of the EM images .",
"BicD and the mRNA-binding protein Egl can either be expressed separately and then reconstituted after purification , or co-expressed as a complex in Sf9 cells ( Figure 3—figure supplement 1 ) .",
"Electron microscopy of the co-expressed BicD-Egl complex revealed a remarkably similar overall conformation to that of BicD alone , with the loop remaining intact , and the paired N-terminal YFP domains confirming the BicD parallel coiled-coil ( Figure 3A , C–F ) .",
"An additional globular structure was often observed adjacent to the loop ( Figure 3A , F , Figure 3—figure supplement 4 ) .",
"The N-terminal region of Egl ( amino acids 1–79 ) binds BicD , and residues 557–726 of Egl contain a globular exonuclease homology region ( Dienstbier et al . , 2009 ) that may be the globular structure we observe .",
"Averages of molecules in the ‘b’ orientation ( Figure 3—figure supplement",
"4 ) are very similar to those seen in the absence of Egl ( Figure 2—figure supplement 1 ) .",
"Co-alignment and classification of BicD-Egl images with images of BicD alone revealed that the most consistent difference in density lies near the bottom center of the loop , suggesting that this region is where Egl is bound ( Figure 3G , Figure 3—figure supplement 5F ) .",
"Some raw images suggest attachment to BicD near to the top of the loop , but this was not evident in the difference map of the aligned images .",
"Classification using a mask adjacent to the loop intended to highlight the position of the Egl globular domain revealed it can occupy a range of positions , consistent with single-particle images ( Figure 3F , Figure 3—figure supplement 4 ) .",
"Positional variability can also be seen in a heatmap illustrating the location of the globular domain in aligned images ( Figure 3H ) .",
"Taken together , the EM data suggest the presence of a long flexible linker between the N-terminal BicD-binding region of Egl and the globular domain .",
"A potential complication is the presence of the YFP domains at the N-terminus of CC1 , which is itself flexible .",
"To rule out that the additional density corresponds to the YFPs , we aligned and classified images of Egl bound to BicD lacking YFP .",
"The appearance was strikingly similar to that of YFP-BicD-Egl but the two large YFP domains were no longer present .",
"By contrast , the globular domain adjacent to the loop was still observed ( Figure 3—figure supplement 2C ) .",
"This verified the assignment of the YFP domains and further demonstrated that the BicD structure is not significantly affected by the YFP tag .",
"Alignment and classification of this complex gave very similar results to those obtained for YFP-BicD-Egl , revealing once again the characteristic loop structure and adjacent globular domain ( Figure 3—figure supplement 5A–C ) .",
"Video 1 shows a low-resolution 3D map of negative stain EM data , which allows size comparison of the loop with existing structures for parts of the YFP-BicD-Egl complex .",
"The EM images imply that Egl alone is not sufficient to disrupt the auto-inhibited conformation of BicD .",
"This result predicts that the BicD-Egl complex will not bind to dynein-dynactin .",
"Single-molecule pulldowns confirmed that full-length YFP-BicD and Egl-Qdot were not recruited to microtubule-bound dynein-dynactin ( DDBE ) ( Figure 4A , B ) .",
"We next tested by single-molecule pulldowns whether mRNA cargo was required for BicD-Egl to bind to dynein-dynactin .",
"Dynein-dynactin , BicD , Egl ( DDBE ) plus K10 mRNA was applied to surface-adhered microtubules in the presence of AMP-PNP to visualize complexes associated with bound dynein-dynactin .",
"The presence of K10 mRNA enhanced Egl and BicD colocalization with microtubule-bound dynein-dynactin ( Figure 4A , B ) .",
"This observation implies that both mRNA and Egl are needed for BicD to adopt a conformation that can recruit dynein-dynactin .",
"Consistent with this , electron microscopy of the YFP-BicD-Egl-K10min mRNA complex showed a variety of flexible structures , with the auto-inhibited loop conformation seen for YFP-BicD alone or YFP-BicD-Egl largely absent ( Figure 3B vs . Figure 3A ) .",
"The same result was obtained with the construct lacking YFP ( Figure 3—figure supplement 2C vs . Figure 3—figure supplement 2D ) .",
"The highly variable appearance of the BicD-Egl-mRNA complexes hampered image alignment and resulted in heterogeneous class averages with insufficient features for detailed assignment of the individual components ( Figure 3—figure supplement 5D , E ) .",
"A small number of particles ( <10% ) of YFP-BicD-Egl-mRNA produced classes resembling the looped structure .",
"It is unclear if this represents incomplete occupancy of Egl with mRNA , or whether BicD retains a weak propensity for auto-inhibition even in the presence of Egl-mRNA .",
"We also examined YFP-BicD in the presence of mRNA and saw no disruption of the loop structure or binding of mRNA to the loop , indicating that only the combination of Egl and mRNA is sufficient to relieve BicD auto-inhibition ( Figure 3—figure supplement 2B ) .",
"Single-molecule pulldowns were further used to assess the requirement for a zip code in K10 mRNA for dynein-dynactin recruitment by BicD-Egl-mRNA complexes .",
"mRNPs reconstituted with a mutant K10 mRNA transcript lacking the transport/localization sequence ( TLS ) caused a ~2-fold reduction in the number of BicD-Egl complexes associated with microtubule-bound dynein-dynactin , compared with native K10 ( Figure 4A , B ) .",
"This suggests that Egl binds primarily to the TLS zip code but is also able to bind weakly to other regions of the mRNA .",
"The run frequencies of the reconstituted complexes were assayed by TIRF microscopy using either Qdot-labeled adaptor proteins ( BicD or Egl ) , or K10 mRNA synthesized with Alexa Fluor 488-UTP .",
"In the absence of either BicD or Egl , minimal movement of labeled K10 mRNA was observed ( Figure 4C ) .",
"In the presence of both BicD and Egl but absence of mRNA , a low-run frequency ( 20% of maximal ) was observed .",
"Inclusion of K10 mRNA enhanced run frequency fivefold .",
"The effect of addition of mRNA cargo is illustrated in kymographs ( Figure 4D ) .",
"As a final control , we confirmed that K10 mRNA and Egl colocalize in motile mRNP complexes ( Figure 4E ) .",
"Similar to the single-molecule pulldowns , K10 mRNA constructs lacking the TLS zip code showed a reduced run frequency ( 35% of maximal ) compared with wild-type K10 transcripts .",
"A similar low frequency was observed with two heterologous mRNAs , mammalian β-actin and S . cerevisiae ASH1 mRNA .",
"Robust cellular localization of K10 mRNA requires the TLS zip code ( Bullock and Ish-Horowicz , 2001; Serano and Cohen , 1995 ) , but even non-localizing mRNAs associate with dynein and move bidirectionally in Drosophila embryos ( Amrute-Nayak and Bullock , 2012 ) .",
"The motile properties of the fully reconstituted mRNPs ( dynein-dynactin-BicD-Egl-K10 mRNA ) were compared with the minimal complex containing dynein-dynactin-BicDCC1 , and in separate experiments with the previously characterized dynein-dynactin-BicD2N complex ( McKenney et al . , 2014; Schlager et al . , 2014 ) .",
"Kymographs demonstrating the observed motility are shown in Figure 5A and Figure 5—figure supplement 1 .",
"The speed of K10 mRNPs was not significantly different than the minimal DDBCC1 complex ( 0 . 42 ± 0 . 22 μm/s , n = 505 vs . 0 . 43 ± 0 . 26 μm/s , n = 516; p=0 . 25 , t-test , mean ± SD ) ( Figure 5B ) .",
"K10 mRNP run lengths were also similar to the minimal DDBCC1 complex ( 6 . 4 ± 0 . 25 μm , n = 71 vs . 5 . 3 ± 0 . 17 μm , n = 66; p=0 . 8 , Kolmogorov–Smirnov test , mean ± SE ) ( Figure 5C ) .",
"When the fully reconstituted mRNP was independently compared with dynein-dynactin-BicD2N , K10 mRNP motility speed was faster than the minimal DDBCC1 complex ( 0 . 45 ± 0 . 21 μm/s , n = 1126 vs . 0 . 35 ± 0 . 19 μm/s , n = 1147 , p<0 . 05 , t-test , mean ± SD ) ( Figure 5D ) .",
"Longer run lengths for K10 mRNP were also observed ( 7 . 2 ± 0 . 5 μm , n = 142 vs . 5 . 4 ± 0 . 7 μm , n = 137; p=0 . 053 , Kolmogorov–Smirnov test , mean ± SE ) ( Figure 5E ) .",
"Despite minor differences , all three complexes show long processive runs of ~5–7 µm at speeds of ~0 . 4 µm/s ( Figure 5B–E ) .",
"On this basis , we conclude that the fully reconstituted mRNP is as active as the minimal DDBCC1 or DD ( BicD2N ) complex .",
"BicD is a coiled-coil dimer , and thus has the potential to bind two molecules of Egl .",
"To determine the stoichiometry of Egl binding and test whether mRNP speed and run length is affected by Egl stoichiometry , we prepared Egl labeled with either a green ( 565 nm ) or a red ( 655 nm ) streptavidin-Qdot via a C-terminal biotin tag .",
"The samples were blocked with excess biotin to prevent additional Qdot binding .",
"K10 mRNPs were then reconstituted with equimolar red- and green-labeled Egl ( Figure 6A ) .",
"Motile complexes containing both green and red Qdots were observed , as were motile complexes with a single color ( Figure 6B ) .",
"Single-colored complexes can contain either one or two Egls of the same color .",
"After correction for complexes with two copies of the same color Egl ( see Materials and methods ) , 86 . 7% percent of complexes were determined to contain two molecules of Egl .",
"In rare cases , a moving complex started dual-colored , and then reduced to a single color in the same trajectory , indicating that Egl dissociation does not immediately terminate motion .",
"The speed and run length distributions of the single versus dual-colored complexes differed in an interesting way .",
"The speed histogram of the dual-colored complex was best fit with a double Gaussian distribution with a slow speed of 0 . 27 ± 0 . 10 μm/s , and a faster speed of 0 . 73 ± 0 . 16 μm/s ( n = 62 ) .",
"Speeds of the single-colored complexes were better fit to a broad single Gaussian distribution , with a speed of 0 . 38 ± 0 . 20 μm/s ( n = 81 ) ( Figure 6C ) .",
"Run length distributions of the single-colored runs were significantly shorter than the dual-colored runs ( 6 . 4 ± 0 . 7 μm , n = 81 vs . 12 . 1 ± 2 . 4 μm , n = 62; p=0 . 045 , Kolmogorov–Smirnov test , mean ± SE ) ( Figure 6D ) .",
"An explanation that would account for both the enhanced speed and run length is that binding of two Egl molecules favors recruitment of two dimeric dynein motors to the dynactin-BicD-mRNA complex ( Grotjahn et al . , 2018; Urnavicius et al . , 2018 ) .",
"This result is particularly striking in light of the fact that experiments performed up to this point used a molar ratio of 1 dynein per dynactin to assemble the mRNP , as this was the assumed stoichiometry of binding .",
"To further investigate whether K10 mRNA can recruit two molecules of Egl , we mixed the two components in solution , adhered them to a glass coverslip , and examined their color distribution via TIRF microscopy .",
"The numbers reported below reflect correction for complexes with two copies of the same color ( see Materials and methods ) .",
"Controls showed 9 . 9% colocalilzation of two Egl molecules ( Qdot-525 and Qdot-565 ) , and 6 . 8% colocalization of two K10 mRNAs ( Alexa 488 and Andy 647 ) ( Figure 6E ) .",
"In contrast , when two different colored Egl molecules ( Qdot-525 and Qdot-565 ) were incubated with unlabeled mRNA , 83% of complexes contained two Egl molecules , similar to the 87% of moving complexes described above .",
"Thus , both in the context of a fully reconstituted mRNP , and in a minimal complex containing only Egl and mRNA , K10 mRNA binds two Egl molecules .",
"Our results with dual-colored Egl molecules strongly suggest that BicD has the potential to recruit two dynein motors .",
"To test this directly , we labeled recombinant dynein containing an N-terminal biotin tag with either Alexa Fluor 647 ( red ) or Alexa Fluor 488 ( green ) ( Figure 7A ) .",
"K10 mRNPs were then reconstituted with equimolar red- and green-labeled dyneins , at a molar ratio of 2 dyneins per dynactin .",
"Single-molecule pulldowns in the presence of AMP-PNP revealed that after correction for complexes containing two copies of the same color dynein , 44% of mRNPs contained two dyneins ( n = 274 dual-colored ) ( Figure 7B ) .",
"Parallel analysis of dynein ( two colors ) , complexed with dynactin and truncated BicD2N showed that 46% of complexes contained two motors ( n = 114 dual-colored ) .",
"Finally , we examined the behavior of dynein-dynactin complexes containing Hook1; complexes with Hook3 have been shown to contain predominantly two dyneins ( Grotjahn et al . , 2018; Urnavicius et al . , 2018 ) .",
"We similarly observed that 50% of the complexes contained two motors ( n = 185 dual-colored ) .",
"The motile properties of mRNPs containing two colors of dynein were compared with single-colored complexes containing either one or two dyneins .",
"Figure 7C shows kymographs of moving mRNPs containing both single- and dual-colored dyneins .",
"The speed histogram of the dual-colored complexes was best fit with a single Gaussian distribution with a speed of 0 . 63 ± 0 . 26 μm/s ( n = 40 ) ( Figure 7D ) .",
"In contrast , the single-colored complexes were best fit to a double Gaussian distribution with speeds of 0 . 40 ± 0 . 14 and 0 . 74 ± 0 . 13 μm/s ( n = 44 ) , with the faster speed likely corresponding to a population containing two dyneins of the same color ( Figure 7D ) .",
"The speed of the dual-colored complexes was 58% faster than the single-colored complex slower speed .",
"Run lengths of dual-colored runs were 53% longer than the single-colored complexes ( 8 . 9 ± 0 . 5 μm , n = 40 , vs . 5 . 8 ± 1 . 1 μm , n = 44; p=0 . 036 , Kolmogorov–Smirnov test , mean ± SE ) ( Figure 7E ) .",
"Recruitment of two dyneins thus results in an mRNP that moves both faster and longer than an mRNP containing only one dynein .",
"Video 2 shows a clear example of a dual-colored complex that moves faster and longer than single-colored complexes .",
"Egl can bind the dynein light chain LC8 ( DDLC1 in Drosophila ) through a consensus light chain binding site on Egl at amino acids 963–969 ( 963AESQTLS969 ) ( Navarro et al . , 2004 ) .",
"To determine whether this interaction affects the motility of the mRNP we expressed recombinant dynein with or without LC8 ( Figure 8A ) , and reconstituted it into mRNPs at a molar ratio of 2 dyneins per dynactin .",
"As seen with the two-color dynein , the speed distributions were bimodal .",
"We detected no statistical difference in speed between mRNPs reconstituted with WT dynein versus dynein lacking LC8 ( 0 . 42 ± 0 . 13 μm/s and 0 . 71 ± 0 . 34 μm/s , n = 396 for WT dynein vs . 0 . 40 ± 0 . 13 μm/s and 0 . 79 ± 0 . 39 μm/s , n = 488 for dynein without LC8; p=0 . 57 , t-test , mean ±SD ) ( Figure 8B ) .",
"Likewise , the run lengths showed no statistical difference ( 6 . 2 ± 0 . 11 μm , n = 113 for WT dynein vs . 6 . 9 ± 0 . 14 μm , n = 102 for dynein without LC8; p=0 . 46 , Kolmogorov–Smirnov test , mean ± SE ) ( Figure 8C ) .",
"Thus , the putative interaction between Egl and the dynein LC8 light chain does not change the number of dyneins recruited nor does it change the speed or run length of the mRNP .",
"Using approaches similar to those described above , K10 mRNA was labeled with two different colors by incorporating either Andy 488-UTP or Andy 647-UTP ( Figure 9A ) .",
"The number of dual-labeled moving mRNPs was 5 . 5% of the total , the rest being single-colored ( Figure 9B ) .",
"After correction for single-colored complexes with two mRNAs , only 11% moving complexes have two mRNAs .",
"Kymographs illustrating this point are shown in Figure 9C .",
"Data for speed and run length were similar to experiments performed with unlabeled mRNA .",
"Because the complex was reconstituted with two dyneins per dynactin , the speed pattern was bimodal ( 0 . 36 ± 0 . 09 μm/s and 0 . 62 ± 0 . 38 μm/s , n = 187 ) ( Figure 9D ) .",
"Run lengths were 5 . 5 ± 0 . 09 μm ( n = 67 ) ( Figure 9E ) .",
"The observation that each mRNP contains only one mRNA , along with our previous data showing that 87% of motile mRNPS contained 2 Egls , suggests that a previously unidentified function of the mRNA cargo is to ensure recruitment of two Egl molecules to the complex , favoring disruption of the auto-inhibitory structure of the BicD coiled-coil .",
"To further explore the model that K10 mRNA ensures recruitment of two Egl molecules to the mRNP complex , we expressed an N-terminal fragment ( amino acids 1–121 ) of Egl containing the BicD binding site that was dimerized by addition of a leucine zipper ( Eglzip ) .",
"If this hypothesis is correct , then a motile complex should be formed by dynein-dynactin-BicD-Eglzip in the absence of mRNA .",
"Gratifyingly , dynein-dynactin-BicD-Eglzip complexes showed motile properties similar to a fully reconstituted mRNP , and were considerably more motile than control complexes composed of dynein-dynactin-BicD-Egl or dynein-dynactin-BicD , as illustrated by kymographs ( Figure 10A ) .",
"Although the frequency of movement was somewhat lower than for fully reconstituted mRNPs , it was higher than controls ( Figure 10B ) .",
"Speeds of dynein-dynactin-BicD-Eglzip were bimodal ( blue circles ) , with the slower population moving at 0 . 43 ± 0 . 21 μm/s and the faster population at 0 . 95 ± 0 . 15 μm/s ( n = 633 ) , whereas speeds of dynein-dynactin-BicD-Egl-K10 mRNA ( red circles ) were 0 . 44 ± 0 . 31 μm/s ( n = 560 ) ( p<0 . 001 , t-test , mean ± SD ) ( Figure 10C ) .",
"This result is notable for two reasons .",
"First , the fast speed observed with Eglzip was higher than previously seen in prior bimodal distributions with native mRNPs reconstituted with two dyneins .",
"Secondly , both complexes were reconstituted with one dynein per dynactin , yet a substantial portion of dynein-dynactin-BicD-Eglzip complexes moved at the fast speed indicative of two bound dyneins , solidifying our conclusion ( Figure",
"6 ) that the presence of two Egls favors recruitment of two dyneins .",
"Run lengths of dynein-dynactin-BicD-Eglzip ( blue circles ) were longer than those of dynein-dynactin-BicD-Egl-K10 mRNA ( red circles ) ( 7 . 6 ± 0 . 03 μm , n = 84 vs . 5 . 7 ± 0 . 09 μm , n = 106 , p=0 . 03 , Kolmogorov–Smirnov test , mean ± SE ) ( Figure 9E ) .",
"Because runs can terminate due to dissociation of the complex , the presence of the forced Egl dimer may enhance run length by stabilizing the assembled complex .",
"In summary , we conclude that the reason why mRNA is needed for robust mobility of the reconstituted mRNP is to ensure the presence of the two Egl molecules required to relieve BicD auto-inhibition ."
],
[
"EM showed that the auto-inhibited loop of BicD was disrupted only in the presence of Egl and mRNA , but not with mRNA alone .",
"This observation is consistent with the requirement of mRNA for high-frequency robust processive motion in single molecule experiments .",
"Experiments using two different colors of the same component , to allow copy number in motile mRNPs to be assessed , lent insight into why mRNA was required .",
"First , 87% of moving mRNPs contained two Egl molecules .",
"Second , two-color mRNA experiments showed that 89% of moving mRNPs contained only one mRNA .",
"Consistent with our in vitro studies , transported mRNAs in Drosophila extracts were shown to primarily contain a single mRNA ( Amrute-Nayak and Bullock , 2012; Soundararajan and Bullock , 2014 ) .",
"The observation that two Egls are incorporated into motile mRNPs at the nanomolar concentrations used in single-molecule assays suggested that interactions between components of the assembled complex may enhance affinity , with a likely candidate being the mRNA .",
"If one mRNA tethers two Egl molecules together as our data suggest , Egl essentially becomes bivalent in its interaction with the two-chained coiled-coil BicD , conferring the enhanced avidity associated with bivalent binding ( Siglin et al . , 2013 ) .",
"Thus , two Egl molecules may be required to overcome the auto-inhibition of BicD , and this is enabled by their simultaneous binding to one mRNA .",
"A proof-of-principle experiment that strongly supports this mechanism was the effect of a truncated Egl construct with a leucine zipper , which activates dynein-dynactin-BicD motility in the absence of mRNA to a similar extent as seen for the fully reconstituted mRNP .",
"Tantalizingly , the run lengths and speeds trended to higher values with the zippered Egl than observed with the fully reconstituted mRNP ( Figure 10 ) , suggesting that this artificial construct may stabilize the motile complex to a greater extent because one Egl can no longer dissociate , which would favor complex disassembly .",
"In vivo , a dynamic complex that needs to disassemble once cargo is transported to its destination may be advantageous and necessary .",
"In a concurrent elegant study , using different Drosophila mRNA transcripts and complementary techniques , Bullock and colleagues ( McClintock et al . , 2018 ) ( see accompanying paper ) also proposed that a single mRNA scaffolds the association of two Egls with BicD CC3 to facilitate activation of dynein .",
"The requirement for mRNA is biologically important because it ensures that motor activation is coupled to cargo binding , preventing futile dynein activity .",
"We previously showed that in an actomyosin-based mRNA transport system in budding yeast , mRNA was also required for processive motion , but for a different reason: mRNA stabilized the interaction of two single-headed class V myosins ( Myo4/She3 ) with the mRNA-binding adaptor protein She2 at physiological ionic strength ( Sladewski et al . , 2013 ) .",
"Although mechanistically the role of mRNA differs from what we show here with dynein-dynactin and BicD , in both cases the requirement for mRNA ensures that only cargo-bound motor complexes are motile .",
"Steric/competitive or allosteric mechanisms for disruption of the CC3-CC1 interaction by two Egl molecules can be considered .",
"A competitive mechanism suggests that two Egls compete effectively with CC1 for a common binding site on CC3 .",
"A competitive mechanism appears to be the case with Rab6GTP , an adaptor protein that links dynein-dynactin-BicD to vesicular cargo .",
"The Rab6GTP and CC1 binding sites on CC3 overlap ( Terawaki et al . , 2015 ) , implying that both cannot bind simultaneously .",
"Rab6GTP ( but not Rab6GDP ) in solution or bound to artificial liposomes released BicD2 from an auto-inhibited state to promote processive dynein-dynactin motion ( Huynh and Vale , 2017 ) .",
"The bound GTP in essence signals to BicD that the Rab is bound to cargo ( reviewed in [Grosshans et al . , 2006] ) .",
"Alternatively , an allosteric mechanism would postulate that binding of Egl-mRNA causes a propagated change in the BicD coiled-coil that weakens the CC3-CC1 interaction ( Liu et al . , 2013 ) .",
"This possibility is due to an unusual feature of part of the Drosophila BicD CC3 structure , in which the same residues in the two chains of the coiled-coil adopt different heptad registries , referred to as ‘heterotypic’ interactions .",
"It was proposed that this region may act as a molecular switch to promote weakening of the CC3-CC1 interaction following cargo binding to the adjacent ‘homotypic’ segment of coiled-coil ( Liu et al . , 2013 ) .",
"The highest frequency of moving complexes , and the highest number of complex associations detected in single-molecule pulldowns , were obtained with K10 mRNA containing the TLS zip code .",
"These values decreased ~two-fold when the zip code was removed , or with either of two heterologous mRNA transcripts , but remained higher than in the absence of mRNA , implying that motor complexes may also bind to sequences other than the TLS .",
"Studies using extracts from Drosophila embryos also concluded that the role of localization signals/zip codes is to increase the average copy number of dynein-dynactin recruited to an mRNP , but even non-localizing mRNAs associate with dynein and move bidirectionally ( Amrute-Nayak and Bullock , 2012 ) .",
"The function of zip codes in Drosophila appears to differ from that seen in budding yeast , where zip codes in the model mRNA ASH1 are essential for recruitment of myosin motors to the mRNA-binding adaptor protein She2 ( Sladewski et al . , 2013 ) .",
"When we began this study , we had assumed that a single dynein was recruited to the complex containing dynein and BicD , and thus used a 1:1 stoichiometry of dynein:dynactin for reconstitutions .",
"Surprisingly , when we reconstituted mRNPs with two different color Egls , we observed that dual-colored complexes had a distinct peak with faster speeds and longer run lengths than single-colored mRNPs containing a mixture of one or two Egls .",
"Separate experiments with dual-colored dyneins directly showed that the enhanced speed and run length can be attributed to the recruitment of two dyneins ( Figure 7 ) .",
"This result is consistent with recent studies showing that dynactin can recruit two dyneins to a dynein-dynactin-adaptor protein complex ( Grotjahn et al . , 2018; Urnavicius et al . , 2018 ) , and that complexes with two dyneins show faster movement and enhanced force ( Urnavicius et al . , 2018 ) .",
"In addition , our observation implies a link between a fully activated mRNP and enhanced dynein recruitment .",
"Experiments with a zippered Egl construct , which was also reconstituted with one dynein per dynactin , also showed a distinct faster peak indicative of recruitment of two dyneins , solidifying the link between full occupancy of BicD with two Egls and binding of two dyneins .",
"The putative interaction between Egl and LC8 of dynein does not , however , appear to contribute to dynein recruitment because mRNPs reconstituted with dynein lacking LC8 exhibited the same speed and run length as those containing wild-type dynein .",
"The cryo-EM studies of Grotjahn et al . ( 2018 ) visualized two dimeric dyneins bound to essentially all ( >97% ) of dynein-dynactin-BicD2N or dynein-dynactin-Hook3 complexes bound to a microtubule in the presence of AMP-PNP .",
"Interestingly , Carter and colleagues ( Urnavicius et al . , 2018 ) also showed that BicDR1 and Hook3 recruited two dimeric dynein motors , but found that only 18% of the BicD2N complexes bound two dyneins , which they related to the position of the N-terminus of the BicD2N fragment in the complex .",
"Our results showed the same number of two-dynein complexes with BicD versus Hook .",
"Factors that influence recruitment of the second dynein to Drosophila BicD and human BicD2 remain to be determined .",
"Our studies with full-length Drosophila BicD suggest that one factor influencing recruitment of two dimeric dynein motors in an mRNP is the presence of two bound Egls , thus establishing a link between cargo binding and full dynein occupancy on dynactin .",
"This in vitro reconstitution of an mRNP , from motor to bona fide biological cargo , provides an excellent model system to further test interactions between components of the complex , which likely are stabilized by multiple weak interactions that synergize to produce a robust transport complex ."
],
[
"Full-length Drosophila BicD ( NP_724056 . 1 or NM_165220 . 3 ) was cloned into pACSG2 for production of recombinant virus and expression in Sf9 cells .",
"Where indicated , full-length Drosophila BicD constructs contained an N-terminal monomeric YFP and a C-terminal FLAG tag , or an N-terminal FLAG followed by a biotin tag for conjugation to a streptavidin-Qdot ( Invitrogen ) .",
"The biotin tag is a 88 amino acid fragment of the biotin carboxyl carrier protein ( Cronan , 1990 ) .",
"The FLAG tag facilitated purification .",
"Truncated Drosophila BicDCC1 ( amino acids 21–380 ) , a derivative of the full-length clone described above , with N-terminal His and biotin tags , was cloned into pET19 for expression in bacteria .",
"Other truncations of the full-length Drosophila BicD were CC2 ( BicDCC2 , amino acids L318-Q557 ) , or CC2 and CC3 ( BicDCC2-CC3 , amino acids L318-F782 ) , which were cloned into pACSG2 for production of recombinant virus and expression in Sf9 cells .",
"Both BicDCC2 and BicDCC2-CC3 had an N-terminal Flag followed by a biotin tag .",
"Truncated human BicD2N ( NM_015250 and NP_056065 . 1 ) , amino acids 25–398 , was cloned into bacterial expression vector pET19 with either an N-terminal HIS and biotin tag , or an N-terminal HIS and monomeric YFP tag .",
"This human BicD2N construct aligns with Mus musculus BicD amino acids 25–400 .",
"Drosophila Egalitarian ( isoform B , NP_726360 . 3 ) was cloned into pACSG2 with either a C-terminal FLAG , or C-biotin followed by a HIS tag for production of recombinant virus and expression in Sf9 cells .",
"A truncated , dimerized variant of Egalitarian consisting of residues M1-S121 , followed by a linker and the GCN4 leucine zipper ( AAL09032 . 1 ) to ensure dimerization , and a C-terminal HIS tag was cloned into pET3 for bacterial expression .",
"The N-terminal 402 amino acids of mouse kinesin ( NP_032475 and NM_008449 . 2 ) with a G235A point mutation was cloned into pET21b for expression of a rigor kinesin used for attachment of microtubules to the flow cell surface .",
"Codon-optimized human dynein for expression in Sf9 cells ( DYNC1H1 ( DHC ) , DYNC1I2 ( DIC ) , DYNC1LI2 ( DLIC ) , DYNLT1 ( Tctex ) , DYNLRB1 ( Robl ) and DYNLL1 ( LC8 ) ) was a generous gift from Simon Bullock ( Schlager et al . , 2014 ) .",
"The heavy chain was modified to contain an N-terminal FLAG tag followed by either a biotin or SNAP tag to enable labeling of the heavy chain .",
"Separate recombinant viruses were produced to express each of the associated subunits ( except for Robl and Tctex which were present in the same virus ) .",
"All subunits were under the polyhedrin promoter except for Robl which was under the p10 promoter .",
"Reagents used for protein expression and purification include: 4- ( 2-aminoethyl ) benzenesulfonyl fluoride ( AEBSF , Fisher BioReagents 30827-99-7 ) , phenylmethylsulfonyl fluoride ( PMSF , Sigma-Aldrich P7626 ) , Tosyl-L-lysyl-chloromethane hydrochloride ( TLCK , Sigma-Aldrich T7254 ) , leupeptin ( Thermo Scientific 78435 ) , benzamidine ( Sigma-Aldrich B6506 ) , FLAG affinity resin ( Sigma-Aldrich A2220 ) , FLAG peptide ( Sigma-Aldrich , F3290 ) and HIS-Select resin ( Sigma-Aldrich P6611 ) , and biotin ( Sigma-Aldrich B4639 ) .",
"Cytoplasmic dynein and dynactin were purified from bovine brain as described previously ( Bingham et al . , 1998 ) , except that the preparation was scaled down to 1 . 5 brains ( ~300 g ) .",
"Alternatively , dynein was expressed in Sf9 cells as described below .",
"Bovine tubulin was purified from brain tissue as described previously ( Castoldi and Popov , 2003 ) .",
"Protein concentrations were determined using Bradford reagent with BSA as standard .",
"Dynein and accessory chains were co-expressed in Sf9 cells for ~72 hr at 27°C , harvested , and re-suspended in 10 mM imidazole , pH 7 . 0 , 0 . 3 M NaCl , 1 mM EGTA , 5 mM MgCl2 , 7% sucrose , 2 mM DTT , 0 . 5 mM AEBSF , 0 . 5 mM PMSF , 0 . 5 mM TLCK , 5 µg/ml leupeptin , and 1 . 3 mg/ml benzamidine .",
"Cells were lysed by sonication , and centrifuged at 257 , 000 x g for 40 min .",
"The clarified lysate was added to 4 ml FLAG affinity resin and incubated with mixing for 40 min .",
"Resin was transferred to a column and washed with 200 ml FLAG wash buffer ( 10 mM imidazole , pH 7 . 4 , 0 . 2 M NaCl , 1 mM EGTA ) and eluted with the same buffer containing 0 . 1 mg/ml FLAG peptide .",
"Peak fractions were concentrated using a Millipore Amicon Ultra-15 centrifugal filter and dialyzed against 5 mM NaPi , pH 7 . 2 , 0 . 2 M NaCl , 1 mM DTT , 0 . 1 µg/ml leupeptin , and 50% glycerol for storage at −20°C .",
"HIS-tagged Drosophila BicDCC1 and human BicD2N constructs were expressed in BL21 ( DE3 ) bacterial cells .",
"Cells were induced with 0 . 7 mM IPTG and grown overnight at room temperature in LB broth containing 0 . 024 mg/ml biotin .",
"Cells were harvested , pelleted , and re-suspended in HIS lysis buffer ( 10 mM NaPO4 , pH 7 . 4 , 0 . 3 M NaCl , 0 . 5% glycerol , 7% sucrose , 7 mM β-ME , 0 . 5 mM AEBSF , and 5 µg/ml leupeptin ) .",
"Cells were lysed by sonication , clarified at 33 , 000 x g for 30 min , and the supernatant bound to 3 . 5 ml of HIS-Select resin .",
"The resin was washed with wash buffer ( 10 mM NaPO4 , pH 7 . 4 , 0 . 3 M NaCl ) containing 10 mM imidazole , followed by four column volumes of wash buffer containing 30 mM imidazole .",
"Protein was eluted in wash buffer containing 200 mM imidazole , and concentrated using a Millipore Amicon Ultra-15 centrifugal filter .",
"Purified protein was clarified 487 , 000 x g for 20 min and dialyzed against 10 mM imidazole , pH 7 . 4 , 300 mM NaCl , 1 mM EGTA , 50% glycerol , 1 mM DTT , 0 . 1 µg/ml leupeptin for storage at −20°C .",
"Full-length Drosophila BicD , BicDCC2 , and BicDCC2-CC3 were expressed in Sf9 cells .",
"For constructs with a biotin tag , the media was supplemented with 0 . 2 mg/ml biotin .",
"Cells were grown for ~72 hr at 27°C , pelleted , and re-suspended in 10 mM imidazole , pH 7 . 4 , 0 . 3 M NaCl , 1 mM EGTA , 2 mM DTT , 0 . 5 mM AEBSF , 0 . 5 mM PMSF , 0 . 5 mM TLCK , 5 µg/ml leupeptin , 1 . 3 mg/ml benzamidine .",
"Cells were lysed by sonication and centrifuged at 257 , 000 x g for 40 min .",
"The clarified lysate was added to the FLAG affinity resin , and incubated with shaking at 4°C for 40 min .",
"The resin was transferred to a column and washed with 200 ml FLAG wash buffer ( 10 mM imidazole , pH 7 . 4 , 0 . 2 M NaCl , 1 mM EGTA ) and eluted with FLAG wash buffer containing 0 . 1 mg/ml of FLAG peptide .",
"Peak fractions were concentrated using a Millipore Amicon Ultra-15 centrifugal filter and dialyzed against storage buffer ( 10 mM Imidazole , pH 7 . 4 , 200 mM NaCl , 1 mM EGTA , 50% glycerol , 1 mM DTT , 0 . 1 µg/ml leupeptin ) for storage at −20°C .",
"Hook1 was expressed and purified as described for BicD .",
"Egl containing a C-terminal biotin and FLAG tag , or only a FLAG tag , was expressed in Sf9 cells and purified similarly except that cells were infected for only 48 hr .",
"Lysis and wash steps were done in buffers containing 0 . 3 M NaCl .",
"The protein was stored at −20°C .",
"in 25 mM imidazole , pH 7 . 4 , 300 mM NaCl , 1 mM EGTA , 1 mM DTT , 0 . 1 µg/ml leupeptin , 50% glycerol .",
"For Egl-BicD co-expression in Sf9 cells , Egl contained a C-terminal biotin and HIS tag , and BicD contained N-terminal YFP and FLAG tag , or only an N-terminal FLAG tag .",
"Following infection , cells were grown for ~72 hr at 27°C and then harvested .",
"The pellet was re-suspended in HIS lysis buffer ( 10 mM NaPO4 , pH 7 . 4 , 0 . 25 M NaCl , 0 . 5% glycerol , 7% sucrose , 0 . 1% NP40 , 0 . 5 mM DTT , 0 . 5 mM AEBSF , 0 . 5 mM PMSF , 0 . 5 mM TLCK , 5 µg/ml leupeptin , 1 . 3 mg/ml benzamidine ) .",
"The cells were lysed by sonication and the lysate was centrifuged 257 , 000 x g for 40 min .",
"A 5-ml HIS-Select column was prepared by washing the resin with five column volumes of water , three column volumes of 0 . 5 M imidazole pH 7 . 4 , 20 column volumes of water , and re-equilibrated in five column volumes of HIS lysis buffer without DTT and protease inhibitors .",
"The high-imidazole wash allows for subsequent use of DTT .",
"The clarified lysate was incubated with resin for 40 min and then washed in a column with ~100 ml of 10 mM imidazole wash buffer ( 10 mM NaPO4 , pH 7 . 4 , 0 . 25 M NaCl , 10 mM imidazole , 0 . 5 mM DTT , 0 . 5% glycerol , 0 . 1% NP40 ) .",
"Protein was eluted in 10 mM NaPO4 , pH 7 . 4 , 0 . 3 M NaCl , 200 mM Imidazole , 0 . 5 mM DTT , 0 . 5% glycerol , 0 . 1% NP40 and dialyzed overnight against 10 mM imidazole , pH 7 . 4 , 0 . 25 M NaCl , 0 . 5% glycerol , 0 . 1% NP40 , 1 mM DTT , 0 . 1 µg/ml leupeptin .",
"The dialyzed protein was then purified over a FLAG column to remove excess Egl by incubating with FLAG-affinity resin for 60 min , followed by washing and elution as described for BicD purification .",
"Peak elution fractions were combined , dialyzed against 30 mM HEPES , pH 7 . 4 , 0 . 25 M NaOAc , 2 mM MgOAc , 1 mM EGTA , 0 . 1 µg/ml leupeptin , and 2 mM DTT , snap frozen in liquid nitrogen , and stored at −80°C .",
"Some co-expressed preparations were purified only on HIS resin with extensive washing to remove extra BicD .",
"Elution fractions which showed approximately equal band intensities for BicD and for Egl were pooled and treated as described for the two column preparation .",
"For negative stain electron microscopy , protein was dialyzed against 30 mM HEPES pH 7 . 2 , 0 . 25 M KOAc , 2 mM MgOAc , 1 mM EGTA , 1 mM TCEP , 0 . 1 µg/ml leupeptin , centrifuged 487 , 000 x g for 20 min , and drop frozen into liquid nitrogen .",
"Rigor kinesin ( G235A ) , a mutant that binds to microtubules but does not dissociate in the presence of ATP or support microtubule motility , was cloned into pET21a .",
"Expression was induced with 0 . 4 mM IPTG overnight at room temperature in E . coli Rosetta ( DE3 ) ( Novagen ) in Terrific Broth Media ( Invitrogen 22711–022 ) containing kanamycin .",
"Cells were harvested , re-suspended in lysis buffer ( 10 mM Hepes pH 7 . 5 , 10 mM NaCl , 1 mM EGTA , 0 . 25 mM DTT with 0 . 5 mM AEBSF , 0 . 5 mM TLCK , and 5 µg/ml leupeptin ) , and lysed by sonication .",
"After clarification , the buffer was adjusted to a final concentration of 0 . 2 M NaCl and loaded onto a HIS-Select column equilibrated with lysis buffer containing 0 . 2 M NaCl .",
"The column was washed with the same buffer containing 10 mM imidazole , then eluted with lysis buffer containing 0 . 2 M imidazole .",
"Peak fractions were dialyzed against 50% glycerol , 10 mM imidazole , pH 7 . 4 , 0 . 2 M NaCl , 1 mM EGTA , 1 mM DTT , and 5 µg/ml leupeptin for storage at −20°C .",
"The 3’UTR of Drosophila K10 mRNA ( NM_058143 . 3 ) , 105–1165 nucleotides past the stop codon , was cloned after the SP6 promoter in the pSP72 vector ( Promega ) followed by a poly16A tail and an EcoRV site to allow the vector to be bluntly opened for use as a template for RNA transcription .",
"The 43 nucleotide transport/localization signal ( TLS ) zip code starts 679 base after the start of the 3’UTR ( Serano and Cohen , 1995 ) .",
"K10 mRNA constructs contain 574 bases before and 443 bases after the TLS .",
"For the K10 no zip construct , the TLS element ( CTTGATTGTATTTTTAAATTAATTCTTAAAAACTACAAATTAA ) was removed .",
"A minimal K10 mRNA construct ( K10min ) consists of 195 nucleotides that center the TLS element .",
"A minimal K10 mRNA construct lacking the zip code ( K10min no zip ) is the same sequence without the TLS and contains an additional 43 bases of 3’UTR sequence immediately following K10min so that K10min and K10min no zip are the same size .",
"The DNA template was bluntly linearized and transcribed using a phage SP6 RNA polymerase ( RiboMAX system , Promega ) .",
"Labeling of the K10 RNA was achieved by adding a mixture of Alexa Fluor 488 ( Molecular Probes , Invitrogen ) in a 1:10 molar ratio to unlabeled nucleotides .",
"For experiments with two different color mRNAs , K10 mRNA was labeled with Andy Fluor 488 UTP or Andy Fluor 647 UTP ( GenCopoeia ) .",
"For control mRNA experiments , two additional mRNAs were used .",
"The full rat β-actin ( Actb , NM_031144 . 3 ) gene , including 78 bases from the 3’UTR followed by the EcoRV site , was cloned into pSP72 .",
"The ASH1 gene of Saccharomyces cerevisiae ( NM_001179751 . 1 ) beginning with the start codon and ending at 1104 bp , minus the E1 zipcode ( 618–702 bp ) was cloned into pSP72 with a polyA11 tail followed by an EcoRV site .",
"PEGylated coverslips were made using methods adapted from ( Gestaut et al . , 2008 ) .",
"Glass cover slides ( Fisher Scientific 12–545 M ) were plasma cleaned for 5 min and transferred to glass Coplin jars containing 1 M KOH and then placed in a sonicating water bath for 20 min .",
"Slides were rinsed thoroughly with nanopure water , then 95% ethanol and dried using a nitrogen stream .",
"Slides were then placed in glass Coplin jars containing 1 . 73% 2-methoxy ( polyethyleneoxy ) propyltrimethoxysilane ( Gelest , Inc SIM6492 . 7–25 g ) and 0 . 62% n-butylamine ( Acros Organics 109-73-9 ) in anhydrous toluene ( Sigma-Aldrich 244511 ) , prepared with glass pipettes .",
"Coplin jars containing slides were then placed in plastic bags , purged with nitrogen and incubated for 1 . 5 hr at room temperature .",
"Following this incubation , the slides were dipped successively in two beakers containing anhydrous toluene and dried using a nitrogen stream .",
"The slides were immediately made into flow chambers , placed in 50 ml tubes and stored at −20°C .",
"This procedure produces a PEGylated slide surface that contains small gaps for the purpose of microtubule attachment .",
"Bovine tubulin was thawed and centrifuged at 400 , 000 x g for 5 min at 2°C .",
"Tubulin concentration was determined using Bradford reagent and diluted to 100 µM in ice cold BRB80 ( 80 mM PIPES , pH 6 . 9 , 1 mM EGTA , and 1 mM MgCl2 ) and supplemented with 1 mM GTP .",
"For generating labeled microtubules , unlabeled tubulin was mixed with 1 µM rhodamine-labeled tubulin ( Cytoskeleton , Denver , CO ) for a final labeled/unlabeled ratio of 1:100 .",
"The tubulin mixture was polymerized by transferring to 37°C water bath for 20 min and stabilized by adding 10 µM paclitaxel ( Cytoskeleton , Denver , CO ) .",
"Stabilized microtubules were kept at room temperature for experiments performed that day .",
"Microtubules could be stored at 4°C for use in experiments within 3 days .",
"Labeled or unlabeled microtubules were adhered to PEGylated flow chambers using rigor kinesin for attachment .",
"Rigor kinesin was diluted to 0 . 2 mg/ml in buffer B ( 30 mM HEPES , pH 7 . 4 , 25 mM KOAc , 2 mM MgOAc , 1 mM EGTA , 10% glycerol , 10 mM DTT ) and added to PEGylated flow chambers for 10 min at room temperature .",
"Flow chambers were then washed three times in buffer A containing 2 mg/ml BSA , 0 . 5 mg/ml κ-casein and 0 . 5% pluronic F68 .",
"Paclitaxel stabilized microtubules were diluted to a final concentration of 1 μM in buffer B containing 10 µM paclitaxel , and added to flow chambers and incubated for 10 min at room temperature .",
"Flow chambers were washed three times with buffer B containing 10 µM paclitaxel to remove unbound microtubules .",
"For DDBCC1 single-molecule motility , BicD2N containing an N-terminal biotin tag was diluted in buffer B300 ( 30 mM HEPES pH 7 . 4 , 300 mM KOAc , 2 mM MgOAc , 1 mM EGTA , 0 . 5% pluronic F68 , 20 mM DTT ) and centrifuged 400 , 000 x g for 20 min .",
"Protein concentration was determined using Bradford reagent and diluted to 1 µM in B300 .",
"Dynein , dynactin and BicD2N were mixed to a final concentration of 100 nM in buffer Go50 ( 30 mM HEPES pH 7 . 4 , 50 mM KOAc , 2 mM MgOAc , 1 mM EGTA , 2 mM MgATP , 20 mM DTT , 8 mg/ml BSA , 0 . 5 mg/ml κ-casein , 0 . 5% pluronic F68 , 10 µM paclitaxel and an oxygen scavenger system ( 5 . 8 mg/ml glucose , 0 . 045 mg/ml catalase , and 0 . 067 mg/ml glucose oxidase; Sigma-Aldrich-Aldrich ) ) .",
"Streptavidin-conjugated 655 quantum dots ( Invitrogen ) were added at 200 nM and incubated with proteins on ice for 30 min .",
"Samples were diluted in buffer Go50 to a final dynein concentration of 1 nM and added to microtubule adsorbed flow chambers for imaging .",
"For imaging complexes where K10 mRNA is labeled , BicD was diluted in B300 and centrifuged 400 , 000 x g for 20 min .",
"Egl was diluted in B300 supplemented with 40 mM DTT and incubated on ice for 1 hr before centrifuging 400 , 000 x g for 20 min .",
"Alternatively , co-expressed BicD-Egl complexes were used .",
"Protein was determined using Bradford reagent .",
"BicD and Egl were combined at 1 µM in B300 .",
"50 nM dynein and dynactin , 100 nM BicD-Egl and 50 nM K10 mRNA , synthesized with an Alexa Fluor 488 UTP for visualization , was mixed in buffer Go150 ( buffer Go50 adjusted to a final concentration of 150 mM KOAc ) containing 10 units of RNase Inhibitor ( Promega N261B ) and 0 . 25 mg/ml tRNA from E . coli ( Sigma-Aldrich R1753 ) .",
"The order of mixing is dynein-dynactin , BicD-Egl , RNase Inhibitor , tRNA , K10 mRNA .",
"The mixture was incubated on ice for 45 min and diluted to a final dynein concentration of 1 nM in Go80 ( buffer Go50 adjusted to a final concentration of 80 mM KOAc ) before imaging .",
"For imaging of complexes where the adaptors are labeled , either BicD or Egl containing a biotin tag were used for conjugation to quantum dots for visualization .",
"Mixtures contained 50 nM dynein and dynactin , 100 nM BicD and Egl , 50 nM unlabeled K10 mRNA and 200 nM Streptavidin-conjugated 655 quantum dots ( Invitrogen ) .",
"Complexes were incubated on ice for 45 min and diluted to a final dynein concentration of 1 nM in Go80 before imaging .",
"For single-molecule pulldowns on microtubules in the presence of AMP-PNP , BicD fused to an N-terminal YFP tag and Egl containing a C-terminal biotin tag were prepared as described above and pre-mixed at 1 µM in B300 .",
"Mixtures containing 50 nM dynein and dynactin , 100 nM BicD-Egl , 50 nM unlabeled K10 mRNA and 200 nM streptavidin-conjugated 655 quantum dots ( Invitrogen ) were diluted in Go150 supplemented with 10 units of RNase Inhibitor ( Promega N261B ) and 0 . 25 mg/ml tRNA ( Sigma-Aldrich R1753 ) .",
"Mixtures were diluted so that the final dynein concentration was 1 nM .",
"YFP was used to visualize YFP-BicD and 655 Qdots were used to visualize Egl on rhodamine-labeled microtubules .",
"For the dual-color Egl experiment , 1 µM of biotinylated Egl was mixed with either 1 µM streptavidin 565 or 655 nm Qdots for 10 min , blocked with a 10-fold molar excess of biotin , and then combined .",
"The combined labeled Egl was then added to a dynein-dynactin-BicD mixture followed by K10 mRNA .",
"The final molar ratio of components is dynein:dynactin:BicD:Egl:K10 = 1:1:1:2:1 .",
"Mixtures were diluted in buffer Go50 so that the final concentration of dynein in the assay was 1 nM .",
"The 565 and 655 nm channels were simultaneously recorded and combined later in ImageJ 1 . 47 v . For dual-color dynein motility experiments , expressed dynein with an N-terminal SNAP tag on the heavy chain was biotinylated with SNAP-biotin ( New England BioLabs , S9110S ) .",
"2 µM SNAP-dynein was incubated with 4 µM SNAP-biotin substrate ( 5 mM sodium phosphate , pH 7 . 5 , 140 mM NaCl , 1 mM DTT ) for 30 min at 37°C .",
"Excess reagent was removed by overnight dialysis at 4°C in 30 mM HEPES , pH7 . 4 , 300 mM KOAC , 20 mM DTT , followed by clarification at 350 , 000 x g for 20 min .",
"Dynein was incubated with either 525 nm or 655 nm streptavidin Qdots ( molar ratio of 1:2 ) for 20 min on ice , then blocked with a 20-fold molar excess of biotin for 10 min .",
"For single-molecule AMP-PNP pulldowns on microtubules to determine percent dual-labeled complexes , 1 µM dynein-biotin was incubated with 2 µM streptavidin Alexa Fluor 488 or Alexa Fluor 647 for 30 min on ice , followed by addition of a 20-fold molar excess of biotin to prevent further binding ( Invitrogen ) .",
"Equimolar amounts of the two different colored dyneins ( 200 nM total ) were incubated on ice with dynactin at a molar ratio of 2:1 .",
"Then BicD , Egl and K10 mRNA were added to the dynein-dynactin complex and incubated another 45 min .",
"The final molar ratio of dynein:dynactin:BicD:Egl:K10 mRNA in DDBE plus K10 mRNA complex was 2:1:1:2:2 .",
"RNase Inhibitor ( Promega N261B ) and tRNA from E . coli ( Sigma-Aldrich R1753 ) were added .",
"A minimal DDBCC1 complex was formed with similar stoichiometry ( 2:1:1 ) .",
"To observe movement , the complex was diluted in buffer Go50 to a final dynein concentration of 0 . 5–1 nM .",
"Motion was observed on rhodamine-labeled microtubules using TIRF microscopy and images of the Qdots ( 525 and 655 nm ) were recorded simultaneously .",
"For single-molecule pulldowns to show dynein stoichiometry , dynein was observed on unlabeled microtubules in the presence of 6 mM AMP-PNP .",
"For the two-color mRNA experiment , 1 µM of tissue-purified dynein was mixed with dynactin at a molar ratio of 2:1 , followed by BicD and Egl .",
"An equimolar mixture of Andy Fluor 488 and Andy Fluor 647 labeled K10 mRNA was then added to the complex .",
"Mixtures were incubated for 45 min .",
"The final molar ratio of components was dynein:dynactin:BicD:Egl:K10 =2:1:1:2:2 .",
"Mixtures were diluted in buffer Go50 so that the final concentration of dynein in the assay was 1 nM .",
"The Andy 488 nm and Andy 647 labeled K10 mRNA were recorded simultaneously using two separate channels ( 488 and 641 nm laser lines ) and combined later in Image J 1 . 47 v . For the zippered Egl experiment , 200 nM of tissue-purified dynein was mixed with dynactin at a molar ratio of 1:1 , followed by BicD-Bio and Eglzipper .",
"The complex was incubated for 45 min on ice .",
"Streptavidin-conjugated 655 quantum dots ( Invitrogen ) were added at 400 nM and incubated with proteins on ice for 30 min .",
"The same conditions and molar ratios were applied for the controls .",
"Imaging was carried out on a Nikon ECLIPSE Ti microscope , run by the Nikon NIS Elements software package , and equipped with through-objective type TIRF .",
"The samples were excited with the TIRF field of 405/488/561/640 nm laser lines , and emission was split by an image splitter ( 561 or 638 nm dichroic ) and recorded on two Andor EMCCD cameras ( Andor Technology USA , South Windsor , CT ) simultaneously at five frames/s with automatic focus correction .",
"The final resolution is 0 . 1066 µm/pixel .",
"Motile mRNPs were tracked with labeled mRNA or with adaptors labeled with a Qdot , and run lengths were measured with ImageJ and the particle-tracking plug-in MTrackJ ( Meijering et al . , 2012 ) .",
"For all processivity assays , frequencies were generated by counting the total number of runs in movies acquired no more than 15 min after dilution of the mRNP mixture .",
"The total number of runs was divided by the total microtubule length ( with a constant time and final dynein concentration for all samples being compared ) to generate a run frequency .",
"Speeds were measured by tracking run trajectories every 0 . 2 s with ImageJ using the particle-tracking plug-in SpotTracker ( Sage et al . , 2005 ) .",
"We performed experiments using two different colored Egl , two different colored dynein , or two different colored mRNA to determine what percent of complexes contain one versus two copies of these proteins or mRNA .",
"In each case , dual-colored complexes represent two copies of the protein ( or mRNA ) in the complex , while single-colored complexes represent a mixture of one and two-copies in the complex .",
"From quantifying the number of single- and dual-colored complexes , the following equations from Urnavicius et al . ( 2018 ) define the fraction of complexes with one or two copies .",
"Color 1obs= ( s × r ) + ( d × r2 ) Color 2obs= ( s × g ) + ( d × g2 ) Dual-coloredobs = d × 2 ( r × g ) r is the fraction of color one labeled subunit and g is the fraction of color two labeled subunit used to form the complex , with r + g = 1 .",
"Solving for d gives the corrected fraction of complexes that contain two copies , and s is the percentage of single-copy complexes .",
"YFP-BicD , YFP-BicD-Egl , BicD-Egl ( expressed and purified as intact complexes ) were imaged by diluting to 10–25 nM in buffer containing 30 mM HEPES , pH 7 . 2 , 250 mM KOAc , 2 mM MgOAc , 1 mM EGTA , 1 mM TCEP .",
"For experiments with mRNA , YFP-BicD-Egl complex was mixed with a twofold molar excess of mRNA prior to dilution .",
"A 3 µl volume of diluted samples were applied to UV-treated , carbon-coated copper grids and stained with 1% uranyl acetate .",
"Micrographs were recorded using an AMT XR-60 CCD camera at room temperature on a JEOL 1200EX II microscope at a nominal magnification of 40 , 000 .",
"Catalase crystals were used as a size calibration standard .",
"2D image processing was performed using SPIDER software as described previously ( Burgess et al . , 2004 ) .",
"Alignments are reference free and based on the SPIDER operations AP RA and AP SA .",
"Classifications are K-means and based on the SPIDER operation CL KM .",
"Data used in this study consisted of the following numbers of images: YFP-BicD = 4205 , YFP-BicD-Egl=4117 , YFP-BicD-Egl-mRNA=3345 , YFP-BicD-mRNA=1393 , BicD-Egl = 3697 , BicD-Egl-mRNA=3526 , mRNA = 2039 .",
"To compare YFP-BicD with YFP-BicD-Egl complex , 4205 images of YFP-BicD alone and 4117 images of YFP-BicD-Egl were combined into a single stack and subjected to reference-free alignment .",
"Aligned images were classified into 20 classes using K-means classification and classes in the most common orientation were selected .",
"A substack containing only these particles was generated .",
"This substack ( 4955 images ) was realigned and the BicD only images ( n = 2471 ) and BicD-Egl images ( n = 2484 ) were averaged ( Figure 3G – Global ) .",
"The aligned substack was classified into 10 classes using a mask around the loop .",
"Difference images shown in Figure 3G and Figure 3—figure supplement 4F are the result of subtracting the BicD averages from the BicD-Egl averages .",
"The heatmap shown in Figure 3H was created by marking the position of the Egl in 342 images of the aligned BicD-Egl stack described above .",
"To generate the low-resolution 3D map shown in Video 1 , 200 2D class averages of BicD were aligned to a starting model consisting of a second-order Gaussian ellipsoid .",
"The resulting model was then refined against 4205 raw images of BicD .",
"The median filtered volume and PDB files ( PDB ID: 2Y0G , 5AFU , 1YT3 ) were arranged manually using UCSF Chimera to create the final movie .",
"BicD , full-length Drosophila Bicaudal D; BicDCC1 coiled-coil 1 domain of Drosophila Bicaudal D; BicD2N , coiled-coil 1 domain of mammalian Bicaudal D2; DDBCC1 , dynein-dynactin-BicDCC1; DD ( BicdD2N ) , dynein-dynactin-BicD2N; DDB , dynein-dynactin-BicD complex; DDBE , dynein-dynactin-BicD-Egl complex; Egl , the mRNA-binding protein Egalitarian; Eglzip , a truncated Egl construct followed by a leucine zipper; K10min , 195 nucleotides of K10 that center the TLS element; Qdot , quantum dot; TLS , transport/localization sequence ."
]
] | [
"We investigated the role of full-length Drosophila Bicaudal D ( BicD ) binding partners in dynein-dynactin activation for mRNA transport on microtubules .",
"Full-length BicD robustly activated dynein-dynactin motility only when both the mRNA binding protein Egalitarian ( Egl ) and K10 mRNA cargo were present , and electron microscopy showed that both Egl and mRNA were needed to disrupt a looped , auto-inhibited BicD conformation .",
"BicD can recruit two dimeric dyneins , resulting in faster speeds and longer runs than with one dynein .",
"Moving complexes predominantly contained two Egl molecules and one K10 mRNA .",
"This mRNA-bound configuration makes Egl bivalent , likely enhancing its avidity for BicD and thus its ability to disrupt BicD auto-inhibition .",
"Consistent with this idea , artificially dimerized Egl activates dynein-dynactin-BicD in the absence of mRNA .",
"The ability of mRNA cargo to orchestrate the activation of the mRNP ( messenger ribonucleotide protein ) complex is an elegant way to ensure that only cargo-bound motors are motile ."
] | [
"Cytoplasmic dynein is a motor-like protein that uses energy to transport cargo where it is needed within cells .",
"It moves along protein filaments called microtubules , which act like miniature tracks .",
"Once dynein engages with microtubules , it then picks up cargo using adaptor proteins .",
"In fruit flies , this cargo includes a messenger RNA molecule known as K10 , which attaches to dynein via adaptors called Egalitarian and BicD ( short for “Bicaudal D” ) .",
"Egalitarian grabs hold of K10 , and BicD links Egalitarian to the dynein motor .",
"In the absence of a cargo , full-length BicD does not bind to dynein .",
"But , shortening BicD to remove its link to Egalitarian allows it to bind and activate the motor for transport .",
"Much of our current understanding of dynein comes from studies that use shortened adaptor proteins like these .",
"These proteins cannot bind cargo , so we know little about how the cargo and the adaptors control dynein activity .",
"To address this , Sladewski , Billington , Ali et al . purified the components of the K10 transport system and then recreated it in the laboratory .",
"This revealed that it is BicD that decides when dynein is ready to go .",
"First , imaging techniques showed that empty BicD forms a looped shape that hides the part of its structure that binds to dynein .",
"This essentially switches it “off” , preventing empty dynein motors from moving .",
"When the K10 cargo is ready for transport , it binds to two Egalitarian molecules , which work together to uncurl the BicD loop .",
"This frees up the end of the BicD molecule , allowing it to link up with the dynein motor .",
"The key to uncurling the BicD protein was the presence of two Egalitarian molecules .",
"And it was the cargo , K10 , that brought them together , ensuring that the motors only moved when the cargo was ready .",
"What is more , the uncurled BicD could bind not one but two dyneins .",
"This allowed the cargo to move faster , and over longer distances than cargo with one dynein motor .",
"Recreating molecular machines and imaging their molecules provides a way to understand how they work .",
"Studying how dynein moves cargo is key to understanding how molecules are transported within cells .",
"This , in turn , could reveal what happens when the system goes wrong .",
"Transport defects can cause diseases in humans , including neurodegenerative diseases .",
"As such , a better understanding of how the transport system works may one day open new avenues for health research ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion"
] | [
"cell biology"
] | The GARP complex is required for cellular sphingolipid homeostasis | elife-08712-v2 | [
[
"Eukaryotic membranes are composed of a complex mixture of lipids belonging to three major classes: sphingolipids , sterols , and glycerophospholipids .",
"The lipid composition of membranes is tightly regulated to achieve homeostasis .",
"Several mechanisms control intracellular levels of glycerophospholipids ( Loewen et al . , 2004; Martin et al . , 2007 ) and sterols ( Brown and Goldstein , 1999; Sever et al . , 2003; Hampton and Garza , 2009 ) .",
"How cells maintain appropriate sphingolipid levels , however , is much less understood .",
"Sphingolipids make up between 10 and 20% of the mammalian plasma membrane ( van Meer et al . , 2008 ) .",
"They are generated in the ER and Golgi and are subsequently delivered to the plasma membrane by vesicular transport ( Klemm et al . , 2009 ) .",
"Sphingolipids at the plasma membrane are internalized by endocytosis and delivered to the lysosome for catabolism .",
"Sphingolipids can also be recycled from endocytic vesicles back to the plasma membrane via the Golgi apparatus ( Choudhury et al . , 2002 ) .",
"Maintenance of membrane sphingolipid levels is important to ensure plasma membrane integrity and to facilitate normal membrane trafficking at the Golgi apparatus and throughout the endocytic pathway ( Trajkovic et al . , 2008; Klemm et al . , 2009; Shen et al . , 2014 ) .",
"In addition , intermediates of sphingolipid synthesis , including ceramide and sphingosine-1-phosphate , are important signaling molecules that mediate cell proliferation , differentiation , and death ( Hannun and Obeid , 2008; Maceyka and Spiegel , 2014; Montefusco et al . , 2014 ) .",
"How cells achieve sphingolipid homeostasis in membranes remains unclear .",
"However , recent studies have shed some light on this process .",
"In yeast , a reduction in plasma membrane sphingolipid levels results in activation of the Ypk1/2 kinases through the TORC2-signaling cascade ( Roelants et al . , 2011; Berchtold et al . , 2012 ) .",
"Substrates of these kinases , the ER-localized Orm1/2 proteins , are negative regulators of serine-palmitoyl transferase ( SPT ) , an enzyme that catalyzes the first and rate-limiting step of sphingolipid synthesis ( Breslow et al . , 2010 ) .",
"The phosphorylation of Orm1/2 releases these proteins from SPT , thereby initiating sphingolipid synthesis and the generation of the early synthesis intermediates , long-chain bases .",
"In addition , Ypk1/2 kinases phosphorylate key subunits of the sphingolipid metabolic enzyme ceramide synthase , increasing its activity and flux of more distal steps of sphingolipid biosynthesis ( Muir et al . , 2014 ) .",
"The physiological importance of sphingolipid homeostasis is underscored by the links of sphingolipid imbalances to disease .",
"For instance , a number of lysosomal storage diseases , such as Niemann–Pick type C or Gaucher's disease , result from lysosomal accumulation of different sphingolipid species ( Platt , 2014 ) .",
"These diseases preferentially affect the central nervous system ( Platt , 2014 ) suggesting that sphingolipid imbalance can lead to neurotoxicity .",
"There are few effective treatments .",
"Therefore , a more complete understanding of sphingolipids is needed and is of significant biomedical importance .",
"Here , we use a chemical genetics screen to uncover an important role of retrograde endosome-to-Golgi trafficking in maintaining cellular sphingolipid homeostasis .",
"We discover a critical role of the Golgi-associated retrograde protein ( GARP ) complex in sphingolipid homeostasis , and we provide mechanistic insight into a human mutation in this complex that causes the early-onset neurodegenerative disease progressive cerebello-cerebral atrophy type 2 ( PCCA2 ) ( Feinstein et al . , 2014 ) .",
"Remarkably , we find that inhibition of sphingolipid synthesis is sufficient to restore crucial aspects of membrane homeostasis when retrograde trafficking via the GARP complex is defective ."
],
[
"To identify genes involved in the regulation of sphingolipid homeostasis , we performed a quantitative , genome-wide screen in yeast for modulators of a growth defect caused by myriocin , an inhibitor of SPT , which catalyzes the first and rate-limiting step of sphingolipid synthesis ( Miyake et al . , 1995 ) .",
"Of 5500 yeast strains analyzed , 703 showed a significant interaction with myriocin ( p < 0 . 05 ) , including 326 that suppressed and 377 that exacerbated the growth defect ( Figure 1A and Supplementary file 3 ) . 10 . 7554/eLife . 08712 . 003Figure 1 . A chemical biology screen reveals the retrograde endosome to Golgi trafficking machinery as a key regulator of sphingolipid homeostasis .",
"( A ) A chemical genetic screen for interactions with myriocin .",
"Calculated T-scores are plotted against colony size on control plates .",
"Genes are color-coded according to their significance score .",
"Red p < 0 . 001 , orange p < 0 . 01 , light blue p < 0 . 05 , blue p > 0 . 05 .",
"( B ) A model for retromer- and Golgi-associated retrograde protein ( GARP ) -mediated retrograde endosome-to-Golgi trafficking .",
"Subunits of the GARP complex and retromer identified in our screen are denoted .",
"Genes are color coded according to their T-score .",
"( C ) Depletion of sphingolipids suppresses the growth defect of yeast strains harboring GARP mutations .",
"Wild-type cells and vps51Δ , vps52Δ , vps53Δ , and vps54Δ mutant cells were spotted on control plates and plates containing increasing concentrations of myriocin , as indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 08712 . 00310 . 7554/eLife . 08712 . 004Figure 1—figure supplement 1 . GO analysis of all suppressing mutants from the chemical genomic myriocin screen . Gene ontology ( GO ) analysis of the hits obtained in our genome-wide chemical genetic screen is shown .",
"Note , the GARP complex is strongly enriched among the suppressor mutants identified ( p < 10−5 ) , whereas the Golgi complex is not ( p > 10−3 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 08712 . 004 One of the strongest class of suppressors identified in the screen ( p < 10−7 ) contained factors mediating retrograde trafficking from endosomes to the Golgi ( Figure 1—figure supplement 1 ) .",
"This included mutants in each subunit of the GARP complex ( vps51Δ , vps52Δ , vps53Δ , and vps54Δ ) and in four of five subunits of retromer ( vps17Δ , pep8Δ , vps35Δ , vps29Δ; Figure 1B ) .",
"In addition , our screen identified two of the three SNARE proteins important for GARP-dependent trafficking ( tlg2Δ , vti1DAMP ) and VPS63 , a gene overlapping almost completely with the GTPase YPT6 that is involved in Golgi-endosomal trafficking .",
"Consistent with a function of Ypt6 maintaining sphingolipid homeostasis , deletion of one subunit of its guanine nucleotide exchange factor , RIC1 , suppressed the growth defect caused by sphingolipid biosynthesis inhibition .",
"However , YPT6 had no significant phenotype in our screen .",
"Similarly IMH1 , encoding a protein important for retrograde transport from endosomes to the Golgi had no phenotype ( Supplementary file 3 ) .",
"This could indicate that YPT6 and IMH1 are false negatives in our screen ( e . g . , due to problems of library yeast strains ) or indicate they are less critical when sphingolipid synthesis is inhibited .",
"In contrast to phenotypes for genes encoding GARP subunits , the disruption of genes involved in related vesicular trafficking machinery , such as the COG or TRAPP complexes ( Whyte and Munro , 2002; Sacher et al . , 2008 ) , resulted in little change in growth when sphingolipid synthesis was impaired by myriocin treatment ( Figure 1—figure supplement 1; Supplementary file 4 ) .",
"To validate these results , we spotted GARP complex mutants and control strains on plates containing myriocin .",
"The growth defects in yeast cells harboring GARP mutations were suppressed by myriocin , whereas wild-type cell growth remained impaired ( Figure 1C ) .",
"We hypothesized that the deficiency of the GARP complex may result in the accumulation of a toxic sphingolipid intermediate that is reduced by myriocin treatment .",
"To identify which lipids might contribute to this toxicity , we inhibited key steps of sphingolipid synthesis and examined their effect on cell growth ( for an overview see Figure 2—figure supplement 1 ) .",
"In contrast to myriocin treatment , the inhibition of downstream steps of sphingolipid synthesis , such as those catalyzed by Aur1 , an inositolphosphorylceramide synthase , or ceramide synthase , by using aureobasidin A ( Nagiec et al . , 1997 ) and fumonisin B1 ( Wu et al . , 1995 ) , respectively , strongly inhibited the growth of yeast harboring GARP mutations ( Figure 2A , B ) .",
"This suggests that vps53Δ cells accumulate a toxic intermediate upstream ceramide synthase and may not have adequate levels of the downstream products . 10 . 7554/eLife . 08712 . 006Figure 2 . The disruption of the GARP complex leads to the accumulation of early sphingolipid synthesis intermediates .",
"( A , B , C )",
"Blocking early steps of sphingolipid synthesis exacerbates GARP-associated growth defects .",
"( A ) GARP mutants are sensitive to IPC synthase inhibition .",
"Wild-type , vps51Δ , vps52Δ , vps53Δ , and vps54Δ cells were spotted on control plates and plates containing 0 . 05 μM aureobasidin A . ( B ) GARP mutants are sensitive to ceramide synthase inhibition .",
"Wild-type , vps51Δ , vps52Δ , vps53Δ , and vps54Δ cells were spotted on control plates and plates containing 100 μM fumonisin B1 .",
"( C ) VPS53 mutants are sensitive to overexpression of the alkaline ceramidase Ypc1 .",
"Wild-type or vps53Δ cells harboring an empty plasmid or a plasmid encoding YPC1 under control of the GAL10 promoter were spotted on glucose- or galactose-containing plates .",
"( D ) GARP mutants are sensitive to high levels of long-chain bases , early sphingolipid intermediates .",
"Wild-type , vps53Δ , and vps54Δ cells were spotted on control plates or plates containing 30 μM phytosphingosine ( PHS ) .",
"( E ) GARP complex deficiency results in an accumulation of long-chain bases .",
"The lipidomic analysis of sphingolipids from vps53Δ ( black ) compared with wild-type strains ( white ) is shown .",
"LCB = long chain base; CER = ceramide; IPC = inositolphosphorylceramide; MIPC = mannosylinositolphosphorylceramide; M ( IP ) 2C = mannose- ( inositol-P ) 2-ceramide .",
"*p < 0 . 05; n . s . not significant ( F , G ) Long-chain bases in GARP mutants are reduced upon myriocin treatment but remain elevated .",
"The levels of ( F ) dihydrosphingosine ( DHS ) and ( G ) PHS from wild-type ( white bars ) , vps52Δ ( light gray bars ) , vps53Δ ( dark gray bars ) , and vps54Δ cells ( black bars ) to myriocin treatment is plotted as fold change from wild-type .",
"*p < 0 . 05; n . s . not significant ( H ) Orm1/2 proteins are hyperphosphorylated in vps53Δ mutants .",
"Orm1-HA expressing wild-type or vps53Δ cells were analyzed by Western blotting against the HA tag or PGK1 as control .",
"Wild-type cells were treated with 5 μM myriocin as indicated .",
"Treatment of the cell lysates with λ-phosphatase as indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 08712 . 00610 . 7554/eLife . 08712 . 007Figure 2—figure supplement 1 . Model of Sphingolipid metabolism .",
"( A ) Schematic of sphingolipid synthesis is shown .",
"ER resident enzymes are indicated in blue .",
"Golgi resident enzymes are indicated in yellow .",
"Enzymes with multiple localizations are indicated in gray .",
"The steps of sphingolipid biosynthesis inhibited by myriocin , fumonisin B1 , and aureobasidin A are indicated in red . DOI: http://dx . doi . org/10 . 7554/eLife . 08712 . 00710 . 7554/eLife . 08712 . 008Figure 2—figure supplement 2 . The rate of serine palimtoyl-transferase inhibition is similar in WT and vps53∆ cells . Time-dependent response of DHS and PHS from wild-type ( white lines ) and vps53Δ cells ( black lines ) to myriocin treatment is plotted as fold change from time point 0 . DOI: http://dx . doi . org/10 . 7554/eLife . 08712 . 008 Consistent with this hypothesis , vps53Δ cells but not wild-type cells overexpressing the alkaline ceramidase Ypc1 , which is predicted to deplete ceramides and as a consequence downstream sphingolipids showed almost no detectable growth ( Figure 2C ) .",
"Also consistent with the hypothesis , vps53Δ and vps54Δ cells , but not wild type cells , were highly sensitive to addition of the upstream sphingolipid synthesis intermediate phytosphingosine ( PHS ) ( Figure 2D ) .",
"To directly assess whether upstream sphingolipid intermediates accumulate in GARP complex-deficient cells , we analyzed cellular lipids by mass spectrometry .",
"Strikingly , vps53Δ cells showed an eightfold increase in levels of total long-chain bases compared with wild-type controls ( Figure 2E ) .",
"Among the different long-chain base species , dihydrosphingosine ( DHS ) increased ∼tenfold and PHS increased ∼threefold ( Figure 2F , G ) .",
"In addition , vps53Δ cells had a ∼twofold reduction of the complex sphingolipid M ( IP ) 2C ( Figure 2E ) ; the levels of IPC , MIPC , and ceramides were unchanged .",
"Based on the current model of sphingolipid synthesis regulation , we expect that reduction of the complex sphingolipid M ( IP ) 2C in vps53Δ cells may activate the upstream sphingolipid synthesis enzyme SPT by releasing Orm1/2 inhibition , thereby exacerbating the GARP mutant phenotype .",
"Consistent with this model , we found that Orm1 was hyperphosphorylated in vps53Δ cells compared with controls ( Figure 2H ) .",
"Together , these results suggest that blocking the GARP-mediated recycling of lipids to the plasma membrane leads to a toxic build-up of long-chain bases , possibly due to a higher rate of degradation of complex sphingolipids in the vacuole and increased biosynthesis .",
"We therefore reasoned that myriocin treatment reduces this toxicity by lowering the levels of long-chain bases .",
"To evaluate this possibility , we determined the levels of long-chain bases before and after myriocin treatment in vps52Δ , vps53Δ , vps54Δ , and control cells .",
"Importantly , myriocin treatment greatly reduced the accumulation of long-chain bases in each of the GARP mutants ( Figure 2F , G ) .",
"The rate of long-chain base decrease during the myriocin treatment time course was similar in wild-type and vps53Δ cells ( Figure 2—figure supplement 2 ) , arguing that myriocin is equally effective in reducing sphingolipid synthesis in either strain .",
"However , even after prolonged , DHS levels remained elevated compared with untreated control cells , suggesting the pool of long-chain bases turns over more slowly ( Figure 2F ) .",
"We reasoned that complex sphingolipids fail to be recycled to the plasma membrane in GARP mutants and are instead rerouted for degradation in vacuoles causing accumulation of long-chain bases and triggering lipotoxicity .",
"A prediction from this hypothesis is that vps53Δ and wild-type cells would distribute exogenously added , fluorescently labeled sphingosine ( NBD-sphingosine ) differently .",
"Testing this possibility , we found that added NBD-sphingosine and FM4-64 , a marker of endocytic membranes both initially label the plasma membrane , but then segregate into different compartments in wild-type cells: as expected , after 60 min , FM4-64 stained the yeast vacuole , whereas the NBD-sphingosine signal localized in one or a few foci likely representing compartments of the endosomal/secretory pathway ( Figure 3A , top control panels ) .",
"In vps53Δ cells , however , both lipids co-localized after 60 min in what appeared to be highly fragmented vacuoles ( Conboy and Cyert , 2000; Conibear and Stevens , 2000 ) ( Figure 3A middle control panels ) .",
"Intriguingly , the abnormal vacuolar morphology in vps53Δ cells was partially rescued by 12-hr myriocin treatment , resulting in a few small vacuoles ( Figure 3A bottom-myrocin , 3B , 3C , and Figure 3—figure supplement 1 for characterization of vacuolar classes ) .",
"However , NBD-sphingosine still localized to the vacuoles in myriocin-treated vps53Δ cells , not to the plasma membrane as in wild-type cells ( Figure 3A ) .",
"Together , these results suggest that exogenously added long-chain bases are maintained to a large degree in the endosomal/secretory pathway of wild-type cells , but accumulate in the vacuole of GARP mutants .",
"The data also suggest that myriocin treatment of these cells does not restore proper endosome to Golgi recycling of sphingolipids , but partially rescues vacuolar dysfunction , as indicated by restoration of vacuole morphology . 10 . 7554/eLife . 08712 . 009Figure 3 . GARP mutants show altered vacuolar morphology and function .",
"( A ) Sphingolipid recycling remains blocked upon myriocin treatment and sphingolipids accumulate in vacuoles .",
"Wild-type cells ( top panels ) , wild-type cells treated with myriocin ( 1 μM , 12hr ) , vps53Δ cells , or vps53Δ cells treated with myriocin ( 1 μM , 12hr ) .",
"NBD-sphingosine , green .",
"Vacuoles ( FM4-64 ) , red .",
"Representative images are shown .",
"Scale bar = 2 . 5 μm .",
"( B , C )",
"Inhibition of sphingolipid biosynthesis partially restores vacuolar morphology in GARP mutants .",
"( B ) Quantification of the vacuolar phenotypes in vps53Δ mutants , mock treated or treated with myriocin is shown ( Classification , see Supplementary figure 2 ) .",
"( C ) Thin-section EM analysis of high-pressure frozen wild-type or vps53Δ cells mock-treated ( left panels ) or treated with myriocin ( 12hr , 1 μM; right panels ) .",
"Representative images are shown .",
"N = nucleus; V = vacuole; LD = lipid droplet .",
"White arrows indicate fragmented vacuoles .",
"Scale bars = 1 μM ( D ) .",
"Three targets of zinc transcription factor Zap1 are down regulated in the GARP mutant vps53Δ .",
"A proteomic analysis of vps53Δ and wild-type cells is shown .",
"Protein intensities are plotted against light/heavy SILAC ratios .",
"Significant outliers are colored in red ( P < 1−11 ) , orange ( P < 1−4 ) , or light blue ( p < 0 . 05 ) ; other proteins are shown in dark blue .",
"E ) GARP mutant zinc sensitivity is rescued by myriocin treatment .",
"Wild-type and vps53Δ cells were spotted on control plates or plates containing 1 μM myriocin , 4 mM zinc or 1 μM myriocin and 4 mM zinc . DOI: http://dx . doi . org/10 . 7554/eLife . 08712 . 00910 . 7554/eLife . 08712 . 010Figure 3—figure supplement 1 . Classification of vacuolar phenotypes used for quantification . Vacuoles labeled with Vma1-mars ( left panels ) , bright field images ( middle panels ) , and merged images ( right panels ) are shown .",
"Phenotypes were classified based on severity .",
"Cells with 1–3 round vacuoles were assigned as class I ( top panels ) , cells with multiple small round vacuoles were assigned as class II ( middle panels ) , and cells with a high degree of vacuolar fragmentation and no round vacuolar structures visible were assigned as class 3 ( lower panels ) .",
"Scale bar = 2 . 5 μM . DOI: http://dx . doi . org/10 . 7554/eLife . 08712 . 010 To further understand the physiological impact of trafficking defects caused by GARP complex deficiency , we analyzed the protein composition of vps53Δ mutant cells by mass spectrometry-based proteomics .",
"Among the 3347 proteins analyzed , we found that the levels of three proteins under control of the zinc-dependent transcription factor Zap1 ( Lyons et al . , 2000 ) were strongly decreased ( Adh4 , Zrt1 , Zps1; P < 1e−11; Figure 3D ) .",
"Since the yeast vacuole is the main storage organelle for intracellular zinc ( Simm et al . , 2007 ) , we hypothesized that the altered vacuolar morphology of vps53Δ mutants results in increased zinc release from the vacuole contributing to toxicity .",
"Consistent with previous studies ( Banuelos et al . , 2010 ) , we found that increased levels of zinc exacerbated the growth defect of vps53Δ mutants .",
"Importantly , this sensitivity is completely suppressed by myriocin treatment ( Figure 3E ) .",
"Our proteomic studies also detected significantly ( p < 0 . 05 ) decreased levels of several ER-resident , early sphingolipid metabolic enzymes , such as Tsc10 , Sur2 , Sur4 , or Phs1 , potentially indicating regulatory adaptations to high levels of intracellular long-chain bases ( Figure 3D ) .",
"Sphingolipids and sterols interact in the plasma membrane and are both internalized by endocytosis , suggesting that their levels might be coordinately regulated ( Simons and Vaz , 2004; Jacquier and Schneiter , 2012 ) .",
"We therefore reasoned that sphingolipid imbalance due to GARP deficiency could also lead to sterol accumulation or altered cellular distribution .",
"Indeed , filipin , a dye that binds sterols , accumulated in internal structures of vps53Δ and vps54Δ cells ( Figure 4A ) .",
"Inhibition of sphingolipid synthesis by myriocin in GARP mutants was sufficient to reverse sterol accumulation , as detected by filipin staining .",
"In contrast , wild-type cells accumulated filipin-positive structures during myriocin treatment .",
"This finding is consistent with the sterol accumulation phenotype of the temperature sensitive SPT mutant lcb1-100 ( Baumann et al . , 2005 ) . 10 . 7554/eLife . 08712 . 011Figure 4 . The disruption of the GARP complex alters sterol distribution in yeast .",
"( A ) Intracellular sterols accumulate in the GARP mutants vps53Δ and vps54Δ .",
"Wild-type ( left panels ) , vps53Δ cells ( middle panels ) , and vps54Δ cell ( right panels ) treated with methanol ( top panels ) or myriocin ( lower panels ) were stained with filipin and analyzed by epifluorescence microscopy .",
"Representative images are shown .",
"Scale bar = 2 . 5 μm .",
"( B ) Neutral lipids accumulate in the GARP mutant vps53Δ .",
"Lipidomic analysis of neutral lipids isolated from vps53Δ ( black ) expressed in fold change from the wild-type ( white ) .",
"ERG = ergosterol; EE = ergosterol ester; DAG = diacylglycerol , TAG = triacylglycerol .",
"*p < 0 . 005; n . s . not significant .",
"( C , D )",
"Lipid droplets accumulate in the GARP mutant vps53Δ .",
"Lipid droplets marked by Faa4-GFP .",
"( C ) Representative confocal midsections of wild-type ( left ) or vps53Δ cells are shown .",
"Scale bar = 2 . 5 μm .",
"( D ) Quantification of ( C ) .",
"The number of lipid droplets per cell compared to wild-type cells ( white bar ) is shown .",
"n = 100 cells .",
"*p < 0 . 001; n . s . not significant ( E ) Components of the GARP complex genetically interact with sterol synthesis genes .",
"Histogram of S-scores for VPS52 extracted from an EMAP ( Hoppins et al . , 2011 ) is shown .",
"( F ) Model of ergosterol metabolism .",
"Genes are color-coded according to correlation coefficient with the GARP subunit VPS52 .",
"Positive-correlated genes are indicated in yellow; anti-correlated genes are indicated in blue . DOI: http://dx . doi . org/10 . 7554/eLife . 08712 . 01110 . 7554/eLife . 08712 . 012Figure 4—figure supplement 1 . Components of the GARP complex genetically interact with ergosterol metabolism genes in yeast . Tetrad analysis of vps52Δ mutants crossed with erg3Δ mutants is shown . DOI: http://dx . doi . org/10 . 7554/eLife . 08712 . 012 Lipidomic analyses showed no significant change in cellular ergosterol levels in vps53Δ cells .",
"However , we detected a two to threefold increase in ergosterol esters in these cells , suggesting that neutral lipids accumulate due to GARP complex deficiency ( Figure 4B ) .",
"Consistent with this , vps53Δ mutants showed a threefold increase in lipid droplets per cell , as identified by the LD marker protein Faa4 , compared with wild-type cells ( Figure 4C , D ) .",
"To further explore the role of GARP in maintaining sterol homeostasis , we analyzed genetic interaction data ( Hoppins et al . , 2011 ) of vps52Δ and found synthetic growth defects with mutations in several genes encoding enzymes of ergosterol biosynthesis ( ERG2 , ERG20 , ERG25 , ERG26 ) ( Figure 4E ) .",
"Tetrad analyses of vps53Δ erg3Δ double mutants confirmed these synthetic growth defects ( Figure 4—figure supplement 1 ) .",
"In addition , VPS52 showed positive correlations with many genes in ergosterol metabolism ( ERG3 , ERG25 , ERG26 , ERG6 , ERG2 , and ERG4; Figure 4F ) , further suggesting a role in maintaining intracellular sterol levels .",
"Together , these results suggest that the GARP complex may help manage sterol levels in cells by recycling them from endosomes to the plasma membrane via the Golgi apparatus .",
"Compound heterozygous mutations of human VPS53 were reported recently to cause the neurodegenerative disease PCCA2 ( Feinstein et al . , 2014 ) .",
"In these patients , one Vps53 allele abolishes protein expression , and the second is a missense mutation ( Q695R ) in the highly conserved C-terminus of VPS53 .",
"To understand the molecular consequences of the latter mutation , we generated a yeast strain harboring an analogous mutation , Q624R , and investigated its effects on lipid homeostasis , retrograde trafficking of proteins between endosomes and the Golgi , and vacuolar morphology .",
"Similar to vps53Δ cells , cells harboring the Q624R mutation were resistant to myriocin treatment ( Figure 5A ) .",
"However , growth in the mutant was more impaired than in vps53Δ cells , suggesting a partial loss of GARP function in vps53 Q624R cells .",
"Subsequent proteomic analyses revealed that the vps53 Q624R mutant protein is expressed at similar levels as the wild-type protein , indicating that this effect is due to its impaired activity , not its instability ( Figure 5—figure supplement 1 ) . 10 . 7554/eLife . 08712 . 013Figure 5 . A PCCA2-causing GARP complex mutation is a partial loss of function allele .",
"( A ) A mutation analogous to the VPS53 allele causing progressive cerebello-cerebral atrophy type 2 ( PCCA2 ) in humans is partially resistant to sphingolipid biosynthesis inhibition induced by myriocin .",
"Wild-type cells harboring an empty plasmid and vps53Δ cells harboring an empty plasmid , a plasmid expressing Vps53 , or a plasmid expressing the mutant vps53 Q624R were spotted on myriocin-containing ( right ) plates or control plates containing methanol ( left ) ( B ) Endosome-Golgi trafficking is partially impaired in yeast cells expressing the analogous PCCA2-causing mutation vps53 Q624R .",
"The GFP-tagged CPY receptor Vps10-GFP ( top panels left and right ) was co-expressed with either Sec7-tomato ( Sec7-tom; middle left panels ) or Vps17-tomato ( Vps17-tom; middle right panels ) in wild-type and vps53 Q624R mutant cells .",
"Representative confocal midsections are shown; scale bar , 2 . 5 µm .",
"( C ) The CPY receptor Vps10 accumulates in vps53 Q624R endosomes .",
"Quantification of the distribution of Vps10-GFP between the Sec7-decorated Golgi ( white bars ) and retromer-decorated endosomes ( black bars ) in wild-type and vps53 Q624R cells .",
"*p < 0 . 005; n . s . not significant .",
"( D ) Mutations in the GARP complex cause vacuolar fragmentation .",
"Maximum projections of vacuoles marked with RFPmars-tagged V-ATPase subunit Vma1 ( left panels ) and the GFP-tagged CPY sorting receptor Vps10 ( middle panels ) in vps53Δ cells harboring a plasmid expressing Vps53 ( top panels ) , an empty plasmid ( middle panels ) or a plasmid expressing a vps53 Q624R mutant ( lower panels ) are shown; scale bar = 2 . 5 µm .",
"( E ) Quantification of ( D ) .",
"Cells with class I ( white bars ) , class II ( gray bars ) , and class III ( black bars ) vacuoles were counted and plotted as percentage of the total number .",
"n = 50 .",
"*p < 0 . 005; n . s . not significant .",
"For phenotypic classification , see Figure 3—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 08712 . 01310 . 7554/eLife . 08712 . 014Figure 5—figure supplement 1 . A GARP complex mutation analogous to the VPS53 allele causing PCCA2 vps53 Q624R does not impact protein stability . vps53Δ cells harboring a plasmid encoding vps53 Q624R or wild-type Vps53 were labeled with either light or heavy L-lysine .",
"Protein intensities are plotted against light/heavy SILAC ratios .",
"Significant outliers are colored in red ( P < 1−11 ) , orange ( P < 1−4 ) , or light blue ( p < 0 . 05 ) ; other proteins are shown in dark blue .",
"Note , peptides from wild-type Vps53 or vps53 Q624R were present in similar abundance . DOI: http://dx . doi . org/10 . 7554/eLife . 08712 . 014 To evaluate whether endosome-to-Golgi trafficking is also compromised in vps53 Q624R cells , we evaluated this process by assaying the localization of Vps10 , the sorting receptor for the vacuolar protease carboxypeptidase Y ( Chi et al . , 2014 ) .",
"In this assay , a green fluorescent protein ( GFP ) -tagged form of Vps10 is retrieved from endosomes to the Golgi apparatus by retrograde trafficking ( Conibear and Stevens , 2000 ) .",
"We quantified co-localization of Vps10-GFP with the Golgi apparatus ( marked with Sec7-tomato ) or the endosome ( marked by Vps17-tomato ) ( Figure 5B ) .",
"Using this assay , we found that cells harboring a vps53 Q624R mutant allele had significantly reduced Vps10 levels in the Golgi apparatus ( 28% , compared with 43% in wild-type cells ) and increased signal in the endosome ( 49% , compared with 24% in wild-type cells; Figure 5C ) , suggesting that retrograde trafficking is indeed impaired .",
"Also consistent with a partial loss of GARP function , vps53 Q624R mutant cells showed vacuolar fragmentation , highlighted by multiple small vacuoles in cells , which we found three times more frequently than in control cells ( 33% vs 11% , Figure 5D , E , Figure 3—figure supplement 1 for characterization of vacuolar classes ) .",
"This intermediate phenotype appears to be similar to vacuoles in vps53Δ cells depleted for sphingolipids ( Figure 3A ) and is much weaker than in vps53Δ mutants in which all cells have highly fragmented vacuoles with no typical round structures apparent ( Figure 5D , E ) .",
"To determine whether GARP's function in sphingolipid homeostasis is conserved in mammals , we assessed the phenotype of HeLa cells depleted of the GARP complex .",
"Similar to our findings in yeast , knock-down of the VPS53 GARP subunit caused accumulation of cholesterol in internal structures of HeLa cells , based on filipin staining ( Figure 6A and ( Perez-Victoria et al . , 2010 ) ) .",
"Notably , this accumulation was reduced dramatically by myriocin treatment , restoring filipin staining to levels similar to control cells treated with myriocin ( Figure 6B ) . 10 . 7554/eLife . 08712 . 015Figure 6 . The depletion of sphingolipid levels reduced lysosome clustering and sterol accumulation due to GARP complex deficiency in HeLa cells .",
"( A , B )",
"Myriocin treatment reduced build-up of free cholesterol due to GARP complex deficiency .",
"( A ) Filipin staining of unesterified cholesterol in control ( top panels ) or Vps53 knock-down ( lower panels ) in HeLa cells treated with 1 μM myriocin for 12 hr ( right panels ) or DMSO as a control ( left panels ) .",
"Representative images are shown .",
"Scale bar = 5 μm .",
"( B ) Quantification of the average free cholesterol filipin intensity/cell normalized to control cells .",
"n = 32 .",
"*p < 0 . 0005; n . s . not significant .",
"( C ) Myriocin treatment partially restored intracellular distribution of lysosomes in the GARP KD .",
"Control cells ( top panels ) or Vps53-KD cells ( lower panels ) were treated with 1 μM myriocin for 12 hr ( right panels ) or DMSO as a control ( left panels ) and stained with an antibody against the lysosomal protein LAMP-1 .",
"Representative confocal midsections are shown; scale bar = 2 . 5 μm .",
"( D ) Myriocin treatment reduced accumulations of early sphingolipid intermediates due to GARP complex deficiency .",
"Lipidomic analysis of mock-treated HeLa control cells ( white bars ) , myriocin-treated control cells ( light gray bars ) , mock-treated VPS53 KD cells ( dark gray bars ) , or myriocin-treated VPS53 KD cells ( black bars ) is shown .",
"Levels are plotted as fold change from that of mock-treated control cells .",
"Error bars represent the average of three independent experiments .",
"*p < 0 . 05; n . s . not significant .",
"( E ) Lipidomic analysis of mock-treated control fibroblasts ( white bars ) , myriocin-treated control fibroblasts ( light gray bars ) , mock-treated PCCA2 patient fibroblasts ( dark gray bars ) , or myriocin-treated PCCA2 patient fibroblasts ( black bars ) is shown .",
"Levels are plotted as fold change from mock-treated control fibroblasts .",
"Error bars represent the average of six independent experiments .",
"*p < 0 . 05; n . s . not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 08712 . 01510 . 7554/eLife . 08712 . 016Figure 6—figure supplement 1 . Myriocin treatment reduced accumulations of early sphingolipid intermediates due to GARP complex deficiency . Lipidomic analysis of mock-treated HeLa control cells ( white bars ) , myriocin-treated control cells ( light gray bars ) , mock-treated VPS53 KD cells ( dark gray bars ) , and myriocin-treated VPS53 KD cells ( black bars ) is shown .",
"Levels are plotted as mole percent .",
"Error bars represent the average of three independent experiments .",
"*p < 0 . 05; n . s . not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 08712 . 01610 . 7554/eLife . 08712 . 017Figure 6—figure supplement 2 . LAMP1 expression in VPS53 knock-down HeLa cells is not altered . HeLa control cells and HeLa VPS53 KD cells were analyzed by Western blotting against VPS53 , LAMP1 , or tubulin as control . DOI: http://dx . doi . org/10 . 7554/eLife . 08712 . 017 GARP complex deficiency results in a defect in retrograde transport , leading to defects in lysosome morphology in HeLa cells ( Perez-Victoria et al . , 2008 ) .",
"We therefore reasoned that lysosome dysfunction could contribute to the toxicity caused by GARP deficiency .",
"We confirmed that lysosomes appeared swollen and clustered in the juxtanuclear area of Vps53 knock-down cells , although LAMP1 protein levels were normal ( Figure 6C , Figure 6—figure supplement 2 ) .",
"Importantly , myriocin treatment resulted in a more uniform lysosome distribution and partially reduced the size of LAMP-1 positive lysosomes in Vps53 knock-down cells ( Figure 6C ) .",
"In contrast , lysosome morphology in control cells was not appreciably affected .",
"To test whether these defects could be caused by accumulation of sphingolipid metabolism intermediates , we isolated sphingolipids from myriocin- or mock-treated VPS53 knock-down and control cells and analyzed them by lipidomics .",
"We found a ∼2 . 5 fold increase in hexosylceramide and a significant increase in the levels of sphingosine and sphinganine , as well as a decrease in sphingomyelin ( Figure 6D and Figure 6—figure supplement 1 ) .",
"Importantly , myriocin treatment restored the levels of these sphingolipid intermediates in VPS53 knock-down cells ( Figure 6D ) .",
"To further evaluate the possibility that sphingolipid imbalances could , at least partially , contribute to development of PCCA2 , we analyzed sphingolipid levels in fibroblasts from PCCA2 patients and controls .",
"Similar to VPS53 knock-down in HeLa cells , PCCA2 fibroblasts exhibited increases in sphingosine , sphinganine , and ceramides compared with control fibroblasts .",
"Similar to what was observed for VPS53 knock-down in HeLa cells , myriocin treatment reduced the accumulation of sphingolipid intermediates in PCCA2 fibroblasts to levels similar to those found in untreated control fibroblasts ( Figure 6E ) ."
],
[
"Here , we show that GARP complex-mediated retrograde trafficking from endosomes to the Golgi is important for cellular sphingolipid homeostasis .",
"In yeast , loss of the GARP complex results in reduced levels of complex sphingolipids and the accumulation of sphingolipid synthesis intermediates , most notably long-chain bases .",
"Importantly , this accumulation is correlated with abnormal vacuolar morphology and function , suggesting this build-up may be toxic .",
"The depletion of a GARP complex subunit in mammalian cells results in similar phenotypes .",
"Retrograde trafficking likely influences the subcellular localization of a large number of proteins .",
"It is thus remarkable that many of phenotypes associated with GARP deficiency are greatly attenuated by pharmacological inhibition of SPT , the first and rate-limiting step in sphingolipid synthesis .",
"Together , these results suggest that accumulation of toxic lipids may underlie a substantial degree of cellular dysfunction due to genetic defects in retrograde trafficking and that restoration of sphingolipid balance may be a strategy to treat diseases due to these defects .",
"Our data suggest that the GARP complex is critical for recycling lipids between the plasma membrane , endosomes , and the Golgi apparatus .",
"In both yeast and mammalian cells , GARP complex deficiency disrupted sphingolipid and sterol homeostasis and resulted in the accumulation of sterols in the endo-lysosomal system .",
"Normally , lipids retrieved from the plasma membrane by endocytosis can be recycled via the retrograde endosome-to-Golgi pathway to supply lipids necessary for rapid membrane expansion during growth .",
"In GARP mutants , however , some membrane-derived lipids appear to be rerouted to vacuoles ( or lysosomes ) for degradation .",
"This rerouting of lipids likely reduces their levels in the plasma membrane and increases the levels of sphingolipids , and their break-down products , in the vacuole/lysosome .",
"This reduction in membrane sphingolipids may explain GARP's apparent role in plasma membrane organization , reported previously in genome-wide visual screens ( Grossmann et al . , 2008; Frohlich et al . , 2009 ) .",
"It is yet unclear whether sphingolipids and sterols follow bulk flow of membrane materials in the endocytic pathway or are preferably recycled by retromer and GARP complexes .",
"Regardless , our data are most consistent with the hypothesis that in the absence of GARP , sphingolipids and sterols are rerouted to vacuoles/lysosomes .",
"In lysosomes , sphingolipids are broken down by acid sphingomyelinase and ceramidase , likely contributing to the elevation in sphingoid bases .",
"However , it is also possible that missorting of sphingolipid synthesis enzymes or trafficking proteins in GARP mutants contributes to the observed phenotypes .",
"For instance , a protein important for the export of sphingolipid intermediates from lysosomes/vacuoles could be missorted in GARP mutants , thus increasing vacuolar sphingolipid accumulation .",
"Several of our findings suggest that the accumulation of early sphingolipid synthesis intermediates due to GARP deficiency may result in cellular toxicity .",
"We show that inhibition of SPT , which lowers long-chain bases in GARP mutants , suppresses the growth defect of these strains .",
"In contrast , the inhibition of later steps of sphingolipid synthesis , for example , by blocking ceramide synthase or inositolphosphoryl ceramide synthase , does not lower long-chain base levels and increases toxicity in GARP mutants .",
"In addition , treatment with the long-chain base PHS exacerbated this toxicity .",
"In yeast , low levels of plasma membrane sphingolipids activate TORC2/Slm/Ypk signaling to phosphorylate the Orm-proteins , relieving their inhibition of SPT , increasing sphingolipid synthesis ( Breslow et al . , 2010; Han et al . , 2010; Roelants et al . , 2011; Berchtold et al . , 2012 ) .",
"Consistent with a decrease of plasma membrane sphingolipids , we found that the Orm1/2 proteins are hyperphosphorylated in GARP mutants suggesting that de novo sphingolipid synthesis is also upregulated .",
"This increase may exacerbate GARP deficiency-induced lipotoxicity due to increased production of these early synthesis intermediates .",
"Why the accumulation of long-chain bases is toxic is currently unclear .",
"One possibility is that long-chain bases possess detergent-like properties , especially in an acidic environment ( Jimenez-Rojo et al . , 2014 ) , such as in the yeast vacuole .",
"In model membranes , such as giant unilamellar vesicles , this detergent-like action leads to vesicular leakage ( Contreras et al . , 2006 ) .",
"This could explain the findings of marked vacuolar fragmentation and potential leakage of zinc from GARP-deficient yeast vacuoles .",
"In support of this hypothesis , we found that inhibition of SPT and reduction of long-chain bases suppressed the zinc sensitivity in GARP mutants in yeast .",
"Also consistent with this hypothesis , the accumulation of sphingolipid intermediates impairs ion homeostasis in mammalian lysosomes of a cellular Niemann–Pick type-C disease model ( Lloyd-Evans et al . , 2008 ) .",
"Alternatively , long-chain bases are known to inhibit glycerolipid synthesis ( Wu et al . , 1993 ) , which might contribute to their cytotoxicity .",
"Since sphingolipid and sterol levels are coordinated in membranes , it is not surprising that we also found that GARP deficiency alters sterol metabolism .",
"Sphingolipids and sterols are thought to interact with each other in membranes .",
"Thus , GARP deficiency likely results in missorting of both to the vacuole/lysosome .",
"Our data are most consistent with a model where sphingolipids are degraded in the lysosome/vacuole leading to a toxic build-up of sphingolipid metabolites , whereas sterols are exported and stored in cytoplasmic lipid droplets as more inert fatty acid esters .",
"Consistent with this , lowering sphingolipid levels by myriocin treatment of wild-type cells increases sterols stained by filipin .",
"Possibly , this indicates that in the absence of sufficient sphingolipids , sterols that are not complexed with sphingolipids build-up and are stored in ester form .",
"A surprising finding is that the accumulation of sterols in GARP mutants is suppressed by inhibiting sphingolipid synthesis .",
"These data argue that in addition to sterol missorting in GARP mutants , accumulation of sphingolipid intermediates impairs normal sterol homeostasis , for example , by interfering with sterol export from the vacuole/lysosome or synthesis regulation .",
"This further highlights that a primary cause for defects due to GARP deficiency may be lysosomal/vacuolar dysfunction due to sphingolipid accumulation .",
"Consistent with our findings on sterol accumulation in GARP mutants , previous screens for lipid droplet phenotypes have identified mutations of several GARP subunit genes ( VPS51 , VPS53 , and VPS54 ) to be associated with neutral lipid accumulation in LDs ( Szymanski et al . , 2007; Fei et al . , 2008 ) , suggesting that GARP deficiency could modulate sterol metabolism .",
"In addition , mutations in the zebrafish homolog of Vps51 , fat-free , result in an increased number of lipid droplets in the liver and intestine ( Liu et al . , 2010 ) .",
"Mutations in one component of the GARP complex , VPS53 , cause PCCA2 , a severe neurodegenerative disease characterized by profound mental retardation , progressive microcephaly , spasticity , and early-onset epilepsy ( Feinstein et al . , 2014 ) .",
"The autosomal disease , linked thus far to two PCCA2 alleles , the missense Q695R mutation , and a splice donor mutation , occurs predominantly in Jewish people of Moroccan ancestry .",
"Our data show that one of these mutations , VPS53 Q695R , leads to defects in yeast that are similar to , albeit not as severe as , a Vps53 deletion .",
"In addition , we show that sphingolipid intermediate levels are elevated in PCCA2 patient fibroblasts , which can be remedied by sphingolipid biosynthesis inhibition with myriocin .",
"Thus , the pathogenesis of PCCA2 could be , at least partially , due to defects in lipid balance and storage .",
"Interestingly , the disruption of the GARP subunit Vps54 in mice leads to a ‘wobbler’ phenotype , indicative of neurodegenerative disease sharing characteristics of ALS , including progressive motor degeneration and motor neuron loss ( Perez-Victoria et al . , 2010; Moser et al . , 2013 ) .",
"Analyses of brain lipid profiles suggest sphingolipid intermediates and sterol esters accumulate in ALS patients ( Cutler et al . , 2002 ) .",
"In addition , mutations in the endo-lysosomal trafficking machinery have been implicated in neurodegenerative disorders including ALS and FTD ( CHMP2B , FIG4 , VAPB , and VCP ) ( Yang et al . , 2001; Parkinson et al . , 2006; Johnson et al . , 2010; Maruyama et al . , 2010 ) and defects in retrograde trafficking increase the risk for Parkinson's disease ( VPS35 ) ( Zimprich et al . , 2011 ) .",
"Our data therefore suggest that lipid imbalance and toxicity due to impaired endo-lysosomal trafficking could be a common feature of this group of neurodegenerative diseases .",
"Since inhibition of SPT reversed some aspects of cellular dysfunction in our studies , our findings suggest that inhibition of the initial step of sphingolipid synthesis may provide a useful therapeutic strategy to pursue in these diseases ."
]
] | [
"Sphingolipids are abundant membrane components and important signaling molecules in eukaryotic cells .",
"Their levels and localization are tightly regulated .",
"However , the mechanisms underlying this regulation remain largely unknown .",
"In this study , we identify the Golgi-associated retrograde protein ( GARP ) complex , which functions in endosome-to-Golgi retrograde vesicular transport , as a critical player in sphingolipid homeostasis .",
"GARP deficiency leads to accumulation of sphingolipid synthesis intermediates , changes in sterol distribution , and lysosomal dysfunction .",
"A GARP complex mutation analogous to a VPS53 allele causing progressive cerebello-cerebral atrophy type 2 ( PCCA2 ) in humans exhibits similar , albeit weaker , phenotypes in yeast , providing mechanistic insights into disease pathogenesis .",
"Inhibition of the first step of de novo sphingolipid synthesis is sufficient to mitigate many of the phenotypes of GARP-deficient yeast or mammalian cells .",
"Together , these data show that GARP is essential for cellular sphingolipid homeostasis and suggest a therapeutic strategy for the treatment of PCCA2 ."
] | [
"Every cell is enveloped by a membrane that forms a barrier between the cell and its environment .",
"This membrane contains fat molecules called ‘sphingolipids’ , which help to maintain the structure of the membrane and enable it to work correctly .",
"These molecules are also used as signals to send information around the interior of the cell and are required for the cell to grow and divide normally .",
"The levels of sphingolipids in the membrane have to be tightly controlled because any imbalance can cause stress to the cell and can lead to serious diseases .",
"Sphingolipids are made inside the cell and are then sent to a compartment called the Golgi before being delivered to the membrane .",
"To regulate the amount of sphingolipids in the membrane , these molecules are routinely returned to the interior of the cell in small structures called endosomes .",
"From here , they can either be broken down or recycled back to the membrane via the Golgi .",
"A group of proteins known as the Golgi-associated retrograde protein complex ( or GARP ) is involved in the movement of endosomes from the membrane to the Golgi .",
"People that have a mutation in the gene that encodes GARP suffer from a severe neurodegenerative disease known as ‘progressive cerebello-cerebral atrophy type 2’ ( PCCA2 ) in which brain cells die prematurely .",
"Researchers have assumed that the most important role of GARP is to sort proteins , and that the missorting of proteins leads to PCCA2 .",
"Here , Frohlich et al . used a combination of genetic analysis and biochemical techniques to study GARP in yeast cells .",
"The experiments show that GARP is critical for sphingolipid recycling , and that a lack of GARP leads to more sphingolipids being degraded , which results in a build-up of toxic molecules .",
"Frohlich et al . generated yeast cells that have the same mutations in the gene that encodes GARP as those in human patients with PCCA2 .",
"These cells grew much slower than normal yeast and were less able to transport sphingolipids from the endosome to the Golgi .",
"Like the yeast cells , human cells in which the gene that encodes GARP was less active also accumulated toxic molecules .",
"Together , these findings suggest that a build-up of toxic fat molecules may be responsible for the symptoms observed in PCCA2 patients .",
"A future challenge is to find out whether this also applies to patients with Alzheimer's disease and other conditions that also affect endosomes ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"cell biology"
] | Wnt/PCP controls spreading of Wnt/β-catenin signals by cytonemes in vertebrates | elife-36953-v2 | [
[
"Wnt signaling regulates development and tissue homeostasis in multicellular organisms ( Nusse and Clevers , 2017 ) , including processes such as cell fate specification , cell proliferation , morphogenesis , and maintenance of tissue integrity .",
"Dysregulation of Wnt/β-catenin signaling has been causally linked to multiple diseases , with Wnt signaling being one of the most frequently dysregulated pathways in several cancer types ( Anastas and Moon , 2013 ) , including colorectal cancers , pancreatic cancer , and gastric cancers ( Chiurillo , 2015; Madan and Virshup , 2015 ) .",
"The Wnt signaling network consists of several branches that can be classified according to the receptors involved and the specific signaling cascades that they activate .",
"Two major branches of this network are the β-catenin-dependent pathway and the β-catenin-independent Wnt/planar cell polarity ( PCP ) pathway ( Niehrs , 2012 ) .",
"The β-catenin dependent pathway is triggered by the interaction of Wnt with Frizzled ( Fzd ) receptors and the co-receptor Lrp6 ( Logan and Nusse , 2004 ) .",
"Wnt/β-catenin signaling regulates the expression of target genes such as axin2 and lef1 , as well as the expression of tissue-specific genes , and subsequently controls both cell proliferation and tissue patterning .",
"In the β-catenin-independent Wnt/PCP pathway ( Yang and Mlodzik , 2015 ) , Wnt proteins bind to Frizzled and to co-receptors such as the receptor-tyrosine kinase-like orphan receptor 2 ( Ror2 ) to regulate cytoskeleton organization by actin polymerization and cell polarity ( Grumolato et al . , 2010; Ho et al . , 2012; Oishi et al . , 2003 ) .",
"To this end , the small GTPases Rho , Rac1 , and Cdc42 are regulated to control the formation of filopodia and lamellipodia , as well as cell motility and morphogenetic movements of cells in vertebrates .",
"Although the PCP pathway and β-catenin signaling generally act in a mutually repressive fashion , by competing for similar hub proteins such as the effector protein Dishevelled ( Dvl ) ( van Amerongen and Nusse , 2009 ) , recent evidence suggests that PCP signaling can act — dependent on the context — either in opposition to , in concert with , or independently of β-catenin signaling .",
"The production and secretion of Wnt ligands requires lipid modification by the acyltransferase Porcupine ( Porcn ) followed by binding to Evi/Wls , which serves as a Wnt chaperone and facilitates its transport from the endoplasmic reticulum to the plasma membrane ( Bartscherer and Boutros , 2008; Bänziger et al . , 2006; Yu et al . , 2014 ) .",
"From there , lipophilic Wnt is transported through the neighboring tissue to exert its long-range signaling activity .",
"Extracellular binding proteins have been suggested to increase the solubility of Wg/Wnt in the aqueous extracellular space and facilitate this activity ( Mii et al . , 2009; Mulligan et al . , 2012 ) .",
"Other studies , however , point to membrane-associated mechanisms of Wg/Wnt delivery , which do not compromise the signaling capability of Wg/Wnt ( McGough and Vincent , 2016; Port and Basler , 2010; Stanganello and Scholpp , 2016 ) .",
"These trafficking routes include Wg/Wnt protein distribution on the plasma membrane of dividing source cells ( Alexandre et al . , 2014; Farin et al . , 2016 ) and actively migrating cells ( Serralbo and Marcelle , 2014 ) , or the dissemination of Wg/Wnt proteins on exovesicles ( Panáková et al . , 2005 ) ( more specifically on exosomes [Beckett et al . , 2013; Gross et al . , 2012; Korkut et al . , 2009] ) .",
"Wg/Wnt proteins and their receptors are also transported on cell protrusions in various tissues .",
"Lipid-modified Wnt proteins were found at the cell membrane of signaling filopodia — so-called cytonemes — in Xenopus and zebrafish ( Holzer et al . , 2012; Luz et al . , 2014; Stanganello et al . , 2015 ) , whereas Fzd receptor proteins can be localized to filopodia in Drosophila and chicken ( Huang and Kornberg , 2016; Sagar et al . , 2015 ) .",
"In zebrafish , an analysis of cytonemes demonstrates that these are specialized filopodia , with stabilizing actin bundles at their cores , which serve as a main transport device for the β-catenin ligand Wnt8a during neural plate patterning ( Stanganello et al . , 2015 ) .",
"Wnt8a is loaded on cytoneme tips and transferred to the neighboring cells by direct cell–cell contact .",
"At the contact sites , Wnt8a cytonemes induce Lrp6/Fzd receptor clustering into the Lrp6 signalosome to activate β-catenin signaling .",
"Although the lengths and numbers of cytonemes are crucial in determining the β-catenin signaling range during embryogenesis ( Stanganello et al . , 2015 ) , it is not yet clear what mechanism controls the formation of Wnt cytonemes in a tissue .",
"Here , we show that Wnt8a can activate both the PCP pathway by interaction with Ror2 and the β-catenin pathway by interaction with Lrp6 .",
"This dual function allows Wnt8a to control its own route of dissemination .",
"In the source cells , Wnt8a binds and activates the Ror2 co-receptor followed by activation of the PCP pathway .",
"Wnt8a-PCP influences convergent extension ( CE ) movement and activates the small GTPase Cdc42 , which leads to the outgrowth of signaling filopodia .",
"Wnt8a is loaded onto these cytonemes , and is transported through the tissue to bind to the β-catenin-specific co-receptor Lrp6 in the responding cells , examples being PAC2 fish fibroblasts and HEK293T human embryonic kidney cells , where it activates the β-catenin pathway in a paracrine fashion .",
"Activation of the β-catenin pathway by Wnt cytonemes leads to target-gene induction , which in zebrafish , regulates neural plate patterning .",
"Ror2-mediated Wnt cytonemes also regulate the proliferation of human gastric cancer cells .",
"We also show that Wnt cytonemes induced by Ror2/PCP signaling are required for the maintenance of murine intestinal crypt organoids .",
"We conclude that Ror2-regulated cytonemes are a critical transport route for Wnt proteins in vertebrates .",
"The molecular mechanism for Wnt cytoneme formation illustrates the co-dependent interactions of the different branches of the Wnt signaling pathways ."
],
[
"Cumulative evidence indicates that Wnt signal molecules are lipidated and remain associated with membranes during secretion , action and degradation ( Nusse and Clevers , 2017 ) .",
"Our previous work demonstrated that Wnt molecules can be distributed over distances of 100 µm through a tissue via cytonemes ( Stanganello et al . , 2015 ) .",
"Manipulation of the length or number of Wnt cytonemes led to alterations in Wnt-mediated tissue patterning and malformations of the zebrafish embryo .",
"Therefore , we hypothesized that the formation , emergence , and maintenance of cytonemes are tightly controlled .",
"To identify potential cytoneme regulators , we performed a cell-culture-based genetic screen ( Figure 1A , Figure 1—figure supplement 1A–J ) by co-expressing Wnt8a-GFP and glycophosphatidylinositol ( GPI ) -anchored , membrane-bound mCherry ( memCherry ) in PAC2 cells together with arrayed cDNA clones from a Medaka cDNA library consisting of 229 kinases ( Chen et al . , 2014; Souren et al . , 2009 ) .",
"We quantified the length and number of signaling filopodia of ten fibroblasts per cDNA 24 hr post transfection using automated filopodia detection software ( Figure 1B ) .",
"The tyrosine-protein kinase transmembrane receptor Ror2 was found to stimulate both filopodia number per cell as well as average filopodia length above the 85th percentile ( Figure 1C , D ) .",
"To validate the screening results , we co-transfected PAC2 fibroblasts with a zebrafish full-length Ror2 expression construct and GPI-memCherry as a membrane marker .",
"The number and length of filopodia were measured in living cells ( Figure 1E ) .",
"Ror2 expression significantly increased the average number and length of filopodia per cell ( Figure 1F , Figure 1—figure supplement 1A , B ) .",
"Ror2 requires homodimerization for transautophosphorylation and subsequent downstream signaling ( Liu et al . , 2007 ) , which can be inhibited by overexpressing a kinase-dead construct .",
"Transfection of the dominant-negative mutant Ror23I ( Hikasa et al . , 2002 ) caused a reduction in both the number of protrusions and their average length , consistent with an essential role of the Ror2 kinase activity in filopodia induction in PAC2 fibroblasts .",
"The Rho GTPase Cdc42 is crucial for organizing the actin cytoskeleton to stabilize Wnt cytonemes ( Stanganello et al . , 2015 ) and is thought to be a downstream target of the Wnt/Ror2 pathway regulating filopodia ( Schambony and Wedlich , 2007 ) .",
"To determine whether Ror2-induced filopodia require Cdc42 function in order to assemble an actin scaffold we co-transfected Ror2-stimulated fibroblasts with dominant-negative Cdc42T17N ( Nalbant et al . , 2004 ) .",
"Blockage of Cdc42 function reduced filopodia formation significantly ( Figure 1E , F ) .",
"BAR-domain containing proteins mold membranes into tube-like filopodia .",
"The insulin receptor tyrosine kinase substrate p53 ( IRSp53 ) is a BAR protein , as well as a Cdc42 effector , which connects filopodia initiation and maintenance by assembling the actin scaffold ( Yeh et al . , 1996 ) .",
"IRSp534Khas four former lysine residues that have been mutated to glutamic acid in the actin-binding sites , inhibiting Cdc42-mediated filopodia formation ( Disanza et al . , 2013; Kast et al . , 2014 ) .",
"IRSp534k expression , like Cdc42T17N transfection , blocks Ror2-induced filopodia formation ( Figure 1E , F ) .",
"Treatment of Ror2-expressing cells with ML141 , a GTPase inhibitor for Cdc42/Rac1 ( Surviladze et al . , 2010 ) , also caused a substantial reduction in both the number and the length of filopodia .",
"Thus , Ror2 is a crucial regulator of filopodia in PAC2 fibroblasts , and filopodia depend on a Cdc42-mediated actin scaffold for their outgrowth and maintenance .",
"Ror2 is a tyrosine kinase receptor that binds Wnt5a via its extracellular cysteine-rich domain ( CRD ) ( Hikasa et al . , 2002 ) and serves as a β-catenin independent Wnt co-receptor activating the PCP signaling pathway ( Schambony and Wedlich , 2007 ) .",
"To investigate the interaction between Ror2 and the β-catenin ligand Wnt8a , we expressed fluorescently tagged Wnt8a and Ror2 proteins in the zebrafish embryo during gastrulation .",
"The mRNA concentration of the injected fluorescent constructs was chosen in such a way that it did not induce phenotypic alteration after 24 hr ( Figure 3—figure supplement 1A ) .",
"Zebrafish ror2 is expressed ubiquitously in early development , with its expression peaking during gastrulation between 2 and 9 hours post fertilization ( hpf ) ( Bai et al . , 2014 ) .",
"Expression of the β-catenin Wnt ligand wnt8a is confined to the embryonic margin during zebrafish gastrulation , orchestrating patterning of the prospective neural plate ( Kelly et al . , 1995; Rhinn et al . , 2005 ) .",
"Confocal microscopy on live specimens revealed that Wnt8a-GFP displays a punctate pattern in the cytoplasm and at the membrane , including cytoneme tips , but when Wnt is absent , Ror2-mCherry is uniformly distributed in the cell membrane ( Figure 2A , Figure 2—figure supplement 1A ) .",
"When Wnt8a-GFP and Ror2-mCherry are co-expressed in the same cell , Ror2-mCherry accumulates in punctae along the membrane ( Figure 2A , Figure 2—video 1 ) .",
"Correlated fluorescence intensity analysis of Wnt8a-GFP and Ror2-mCherry suggests that both proteins predominantly co-localize in membrane-associated clusters ( Figure 2B ) .",
"This is supported by a Pearson-based correlation analysis of Wnt8a-GFP and Ror2-mCherry in embryonic tissue of a volume of 40 × 40 × 60 µm3 ( N=8 , Supplementary Figure 2G ) .",
"We found a similar intensity correlation between Wnt5b and Ror2 ( Figure 2—figure supplement 1B , C ) .",
"The CRD of Ror2 is crucial for interaction with Wnt ligands ( Hikasa et al . , 2002; Mikels et al . , 2009 ) .",
"To exclude non-specific clustering of fluorescent fusion proteins , we used a Ror2 construct with a deletion in the Fzd-like CRD .",
"We observed that Ror2-ΔCRD-GFP still localizes to the cell membrane and with Wnt8a-mCherry forming clusters therein .",
"However , image profile analysis showed a marked reduction of the intensity peaks at the cluster site , indicating that Ror2-CRD is required for the interaction with Wnt8a ( Figure 2B ) .",
"We further characterized Wnt8a/Ror2 protein–protein interactions in vivo using line-scanning fluorescence correlation spectroscopy ( lsFCS; Figure 2—figure supplement 1D ) , which measures the concentrations and diffusion coefficients of ligands and receptors in the presence of a membrane ( Dörlich et al . , 2015 ) .",
"We performed lsFCS analysis in two different spots , at a Ror2-positive membrane domain ( spot 1 ) and at a Wnt8a/Ror2 membrane cluster ( spot 2; Figure 2C ) .",
"A focused laser spot was scanned across the membrane for 390 s while the intensity was measured as a function of time .",
"After compensation for membrane fluctuations , the intensity time traces were time-correlated .",
"In spot 1 , we found intensity fluctuation from Ror2-mCherry emission ( Figure 2D ) .",
"A fit of the autocorrelation function revealed a receptor concentration ( area density ) Cr= ( 37 ± 3 ) µm−2 .",
"The diffusion coefficient , D= ( 0 . 28 ± 0 . 03 ) µm2 s−1 , is similar to values found for LRP6 receptors in the plasma membrane ( Dörlich et al . , 2015 ) .",
"There was no clear emission from Wnt8a-GFP molecules in spot 1 .",
"By contrast , lsFCS on spot 2 revealed clear autocorrelations in both color channels ( Figure 2E ) , indicating the presence of both Wnt8a-GFP and Ror2-mCherry at this site .",
"We found a dual-color cross-correlation between Wnt8a-GFP and Ror2-mCherry ( Figure 2E ) , indicating concerted intensity fluctuations in both color channels , which arise from their co-diffusion in the plasma membrane due to binding .",
"Therefore , the cross-correlation lsFCS data provide clear evidence of complex formation between Wnt8a and Ror2 .",
"Furthermore , the low diffusion coefficient of the bound species , D= ( 0 . 02 ± 0 . 01 ) µm2 s−1 , indicates that the complexes diffuse as large clusters .",
"We have previously shown that Fzd7 also interacts with Ror2 and enhances Ror2-mediated signaling during Xenopus gastrulation ( Brinkmann et al . , 2016 ) , suggesting that Fzd7a could be a part of the Wnt8a/Ror2 cluster in zebrafish .",
"To test this , we overexpressed Ror2-mCherry , Fzd7a-CFP and Wnt8a-GFP and found co-localization of all three proteins in the cell membrane ( Figure 2—figure supplement 1E , F ) .",
"From our data , we conclude that Wnt8a interacts with Ror2 by binding to its CRD .",
"Wnt8a and Ror2 co-migrate and form dense protein clusters .",
"The data suggest that the Wnt8a/Ror2 clusters are in close steric contact with other components of the Wnt signaling complex including Fzd7a .",
"The interaction of Wnt8a with Ror2 could trigger non-canonical PCP signaling via the Ror2 pathway .",
"PCP signaling plays a role in regulating tissue migration during gastrulation ( Tada and Heisenberg , 2012 ) .",
"PCP signaling via Ror2 activation regulates collective cell migration towards the embryonic midline , which is most pronounced in the mesodermal germ layer in zebrafish ( Bai et al . , 2014 ) .",
"We utilized this classical PCP-controlled process to observe the involvement of Wnt8/Ror2 in non-canonical signaling .",
"CE can be visualized by condensation of the no tail ( ntl ) -positive notochordal plate at the embryonic midline at 11 hpf ( Figure 3A ) .",
"To this end , we overexpressed the Ror2 receptor , which alone had a very small effect on the establishment of the ntl expression domain ( for classification see Figure 3—figure supplement 1B ) .",
"However , overexpression of Wnt8a leads to a broadening and shortening of the ntl expression domain .",
"This phenotype is reminiscent of Wnt5b activation .",
"A similar phenotype was observed when Wnt8a and Ror2 were co-expressed .",
"Categorization of the phenotypes suggests that the co-activation of Ror2/Wnt8a and Ror2/Wnt5b have similar effects ( Figure 3B ) .",
"Inhibition of Ror2 function by either Ror23I expression or a Morpholino-based Ror2 knock-down also led to CE defects .",
"Our data suggest that Fzd7a may be a member of the Ror2-Wnt8a signaling complex ( Figure 2—figure supplement 1E , F ) .",
"We found an enhanced broadening of the embryonic midline if Wnt8/Fzd7a and Wnt8a/Fzd7a/Ror2 were overexpressed ( Figure 3—figure supplement 1C ) .",
"This suggests that endogenous Ror2 is already expressed at high levels , and that the available Wnt ligand concentration is the key quantity-controlling step for PCP signaling during zebrafish CE .",
"During CE , cells intercalate in the notochordal plate ( convergence ) , push previously adjacent cells apart , and lengthen the field along the AP axis ( extension ) ( Glickman et al . , 2003 ) .",
"We investigated the shape of the notochord cells in embryos with ectopically expressing Wnt8/Ror2 signaling .",
"We found that the cells had a less bipolar shape and displayed a more circular form in embryos with Wnt8/Ror2 signaling , reminiscent of Ror2 activation by Wnt5a ( Figure 3C , D ) , suggesting that mediolateral narrowing of axial mesoderm is reduced .",
"Activation and inhibition of the PCP signaling pathway leads to a similar phenotype ( Tada and Heisenberg , 2012 ) .",
"Therefore , we were unable to distinguish how Wnt8a/Ror2 alters PCP signaling .",
"During Xenopus gastrulation , Wnt5A activates Ror2 downstream signaling , leading to Cdc42 activation , JNK phosphorylation , and ultimately , the enhancement of ATF2 transcription ( Hikasa et al . , 2002; Schambony and Wedlich , 2007 ) .",
"To test whether zebrafish Wnt8a is able to activate Ror2 signaling , we used a reporter assay with an ATF2-responsive element driving luciferase expression in Xenopus embryos ( Brinkmann et al . , 2016; Ohkawara and Niehrs , 2011 ) .",
"Wnt8a co-expressed with Ror2 produced a greater than five-fold induction of the ATF2 reporter in Xenopus ( Figure 3E ) .",
"Co-expression of Wnt5A/Ror2 leads to a similar activation of reporter expression , whereas expressed Ror2 without a co-expressed ligand did not alter expression of the ATF2 reporter .",
"We determined whether the kinase domain of Ror2 is required for Wnt8a-dependent activation of the PCP pathway by overexpressing Wnt8a together with dominant-negative Ror23I and observed a reduction of ATF2 reporter activation but an activation of Wnt8a/Ror2 .",
"Taken together , our data indicate that Wnt8a serves as a ligand for the receptor Ror2 and induces PCP signaling upon binding .",
"Thus , ectopic overexpression of Wnt8a modulates cell movements and cell morphology in zebrafish and gene transcription in Xenopus .",
"To study the dynamics of filopodia formation in the Wnt8a-positive germ ring during normal development in zebrafish ( Figure 4—video 1 ) , we quantified signaling filopodia during gastrulation in live embryos .",
"In confocal image stacks , filopodia were traced using a semi-automatic live wire approach ( Barrett and Mortensen , 1997 ) .",
"A precise measurement of filopodia protrusion lengths in 3D was obtained using manually selected nucleation start points and filopodia end points .",
"We found that the number of filopodia significantly increase from in the period from 4 hpf to 6 hpf , which comprises the neural-plate-patterning phase ( Figure 4A , B ) .",
"This coincides with increasing Ror2 expression levels during zebrafish development ( Bai et al . , 2014 ) .",
"We determined whether the formation of these filopodia was dependent on Ror2 function by manipulating Ror2 signaling and measured germ ring cell filopodia number at 6 hpf .",
"We found only a modest increase in filopodia number when Ror2 was activated , suggesting that Ror2 itself is not rate-limiting ( Figure 4C , D ) .",
"However , when Ror2 function was reduced by overexpression of Ror23I , we observed a significant reduction in filopodia number .",
"We conclude that Ror2 signaling is required for filopodia induction of embryonic marginal cells during zebrafish development .",
"We speculated that Ror2 might influence the formation of filopodia carrying Wnt8a protein ( Wnt8a-cytonemes ) during zebrafish gastrulation .",
"To visualize these cytonemes , we generated cell clones at the embryonic margin expressing Wnt8a-GFP and memCherry .",
"Wnt8a-GFP clusters were seen in the cell membrane and the cytoneme tips of germ ring cells ( Figure 4E ) .",
"Statistically , more filopodia carrying Wnt8a-GFP clusters on their tips were detected upon Ror2 overexpression within cells ( Figure 4E , F ) .",
"Conversely , we found a significant reduction in the number of cytonemes in Ror2-deficient marginal cells .",
"Wnt8a-GFP is still present at the plasma membranes of cells that have compromised Ror2 function , suggesting that intracellular routing of Wnt8a from the producing organelles to the cell membrane is independent of Ror2-dependent cytonemal transport .",
"Filopodia without detectable Wnt8a seemed to be unaffected by Ror2 signaling in zebrafish ( Figure 4F ) .",
"This suggests a function of Ror2 in regulating a specific subset of filopodia , those carrying Wnt8a ( the Wnt cytonemes ) , in the zebrafish embryo in vivo .",
"On the basis of these findings , we hypothesized that Ror2 function in the Wnt source cells is crucial for Wnt dissemination via cytonemes .",
"To test the consequences of altered Ror2 signaling quantitatively , we used a simulation of morphogen distribution via cytonemes ( Stanganello et al . , 2015 ) .",
"The simulation takes into account ligand transport by cytonemes , ligand decay and the migration of epiblast cells using a Monte-Carlo-based direct event simulation approach .",
"Employing cytonemes as the exclusive transport mechanism from the producing cell group to the target cell group , and assuming an unlimited source of the ligand , we found that cytonemes can distribute Wnt8a in a graded manner in the dynamically evolving target tissue ( Figure 4G ) .",
"We tested two scenarios with varying cytoneme number , based on our in vivo measurements after alteration of Ror2 function ( Figure 4F ) .",
"We found that ligand concentration within the morphogenetic field depends on the appearance of the cytonemes ( Stanganello et al . , 2015 ) .",
"We also found that increasing the number of cytonemes per cell ( by the experimentally determined factor of 1 . 61 ) after expression of Ror2 and Wnt8a in the source cells leads to an enhanced ejection ( 187% ) of the ligand into the target tissue when compared to that in the Wnt8a-producing cells ( Figure 4G ) .",
"Consistently , by using the measured cytoneme parameters after blockage of Ror2 function ( number scaled by 0 . 388 ) , we found a decrease to 36% of the Wnt8a concentration compared to the WT situation and a smaller range of the morphogen gradient within the tissue .",
"These data suggest that Ror2 signaling can specifically regulate the number of Wnt-positive cytonemes in vivo and thus represents a cytoneme-specific regulator .",
"Furthermore , on the basis of the simulations , we predicted a strong decrease in the range of Wnt signal activation in the neighboring tissue when Ror2 function is compromised in the Wnt8a source cells .",
"Next , we asked how Wnt8a and Ror2 interact to facilitate cytoneme-mediated transport .",
"We used a high-resolution imaging approach in the developing zebrafish embryo that involved the overexpression of tagged constructs ( Figure 5 ) .",
"The high-sensitivity of this image-based approach allowed us to reduce the expression levels of the tagged construct significantly , and morphological alterations of the embryonic phenotype were not observed at 24 hpf ( Figure 3—figure supplement 1A ) .",
"Using a time-lapse analysis , we observed the formation of Wnt8a-GFP-positive clusters , which transit in the plasma membrane of the secreting cell ( Figure 5B ) .",
"Then , Wnt8a co-localizes with Ror2 at the plasma membrane , suggesting Wnt8a-Ror2 cluster induction ( Figure 5C ) as previously described ( Figure 2A , B ) .",
"These clusters initiate cytoneme formation and , consequently , they decorate the tip of the outgrowing cytoneme .",
"The cytoneme contacts the target cell and Wnt8a-Ror2 forms a cluster at the receiving cell ( Figure 5C , Figure 5—video 1 ) .",
"Within minutes , this cluster is endocytosed into the target cell .",
"We wondered whether Wnt8a-Ror2 induces the Wnt signaling cascade in the target cell .",
"Therefore , we analyzed Lrp6-signalosome formation at the plasma membrane of the target cell by induction of a Wnt8a-secreting cell clone and a Lrp6-expressing receiving cell clone ( Figure 5D ) .",
"We find that cytonemal Wnt8a-mCherry induces Lrp6-GFP cluster formation at the membrane of the target ( Figure 5E , F ) .",
"We hypothesized that the source cell presents Wnt8a by clustering the ligand on Ror2-positive cytonemes .",
"Indeed , we observe a Lrp6-GFP cluster at the contact points of Ror2-positive cytonemes ( Figure 5E ) .",
"The following dynamics of the Lrp6-signalosome have been described recently in zebrafish development ( Hagemann et al . , 2014 ) .",
"Therefore , we conclude that Ror2 clusters on cytoneme tips to act as a platform to present Wnt8a to the target cell and to induce the Wnt signaling cascade therein .",
"On the basis of our simulations , we speculated that Ror2 signaling may have a function in Wnt ligand trafficking and , consequently , in paracrine β-catenin signaling during zebrafish gastrulation .",
"To test this , we overexpressed Ror2 and analyzed the effect of this overexpression on CE processes ( via PCP signaling ) and , simultaneously , on neural plate patterning ( via β-catenin signaling; Figure 6—figure supplement 1A ) during embryogenesis .",
"Overexpression of Ror2 by injection of low levels of mRNA did not induce gross morphological changes in zebrafish embryos , consistent with our findings that Ror2 without a suitable ligand only mildly impacts PCP-mediated processes ( Figure 3A , C , E ) .",
"Overexpression of Wnt8a resulted in a substantial alteration in neural plate patterning as described above , with β-catenin signaling being activated in the entire neural plate , marked by ubiquitous axin2 expression at 6 hpf ( Figure 6A ) .",
"As a consequence , we observed posteriorization of the developing nervous system , observed as an anterior shift of the fgf8a-positive midbrain-hindbrain boundary ( MHB ) at 9 hpf and a loss of the anterior pax6a-positive forebrain at 26 hpf .",
"In embryos co-expressing Wnt8a together with Ror2 , we still observed the posteriorization phenotype in the neural plate and , in addition , we found that CE is compromised , as the neural plate does not converge to the midline and , consequently , the expression domains of fgf8a at the MHB showed a pronounced gap .",
"We compared these observations to embryos expressing the β-catenin-independent ligand Wnt5a , and Wnt5a together with Ror2 .",
"Ror2-mediated Wnt5a signaling induces CE in Xenopus ( Hikasa et al . , 2002 ) and represses β-catenin signaling in mouse embryos ( Mikels et al . , 2009 ) .",
"In both settings , we observed a strong effect on CE movement in the zebrafish embryo .",
"In addition , Wnt5a/Ror2 overexpression led to reduced β-catenin signaling , causing a reduction in target gene expression ( axin2 ) and anteriorization of the neural plate , leading to a posterior shift of pax6a expression in the forebrain .",
"We conclude that Wnt8a can activate β-catenin signaling and PCP signaling via the Ror2 receptor during zebrafish development .",
"We showed that Wnt5b/Ror2-mediated PCP signaling represses Wnt/β-catenin signaling .",
"We hypothesized that Wnt8a function depends on the route of secretion .",
"However , global overexpression did not differentiate between autocrine and paracrine Wnt8a signaling mechanisms and on subsequent downstream activation .",
"To separate Wnt-producing from Wnt-receiving cells , we performed a co-cultivation assay using HEK293T cells , which are typically Wnt-Off due to low endogenous expression of Wnt ligands ( Voloshanenko et al . , 2017 ) .",
"Cytoneme regulators were transfected into HEK293T source cells ( Wnt-producing cells ) and co-cultivated with HEK293T cells expressing the SuperTOPFlash TCF/Wnt reporter , with seven TCF-responsive elements hooked up to nuclear mCherry ( 7xTRE-NLS-mCherry [Moro et al . , 2012] , Figure 6—figure supplement 1B ) .",
"Ror2 transfection into source cells did not alter the induction of 7xTRE-nucRFP in the receiving cells ( Figure 6B , C ) .",
"However , Wnt8a-producing cells led to the activation of signaling activity in the HEK293T reporter cells .",
"Reporter expression was further enhanced ( 147 . 3% compared to Wnt8a transfected source cells ) when Wnt-producing cells co-expressed Wnt8a and Ror2 , indicating a synergistic interaction between Wnt8a and Ror2 ( Figure 6D ) .",
"Co-transfection of Wnt8a with dominant-negative Ror23I resulted in a 34 . 6% decrease in reporter activation below the level seen in Wnt8a-transfected source cells .",
"These findings support our simulations regarding the available Wnt8a concentration in the target tissue after alteration of Ror2-dependent cytoneme appearance ( Figure 4G ) .",
"We conclude that Wnt8a can be transmitted via Ror2-dependent cytonemes in HEK293T cells , whereas Wnt5b and Wnt5b/Ror2 transfections were unable to activate the β-catenin signaling reporter in neighboring cells ( Figure 6D ) .",
"To test whether Ror2-mediated Wnt cytonemes affect β-catenin-dependent target gene activation in neighboring cells in vivo , we generated small-source clones by microinjecting cytoneme regulator mRNAs at the 16-cell stage ( Figure 6—figure supplement 1C ) .",
"By mid-gastrulation , the source cells were distributed over an area of the embryo and intermingled with WT host cells , generating many responding cells around a few source cells .",
"At 6 hpf , we analyzed the transcriptional profile of the embryos for the β-catenin target genes axin2 and lef1 .",
"Embryos with cells overexpressing Ror2 or Ror23I showed no alteration in axin2 or lef1 expression ( Figure 6D ) .",
"However , source cells overexpressing Wnt8a resulted in a significant increase in β-catenin-dependent target gene expression , which was not further enhanced by Ror2 addition .",
"However , blockage of cytoneme formation in the Wnt8a source cells by co-expression of Ror23I led to a significant reduction of β-catenin target gene induction in neighboring cells .",
"Blockage of filopodia per se by overexpression of the dominant-negative form of IRSp534K caused a similar reduction of activation of axin2 and lef1 expression in embryos .",
"This suggests that , during zebrafish gastrulation , the majority of Wnt8a protein is transmitted via cytonemes and that the formation of these Wnt cytonemes is Ror2 dependent .",
"Over-activation of canonical β-catenin signaling can be identified in one-third of gastric cancers ( Chiurillo , 2015 ) .",
"β-catenin signaling is essential for self-renewal of gastric cancer stem cells , leading to Wnt-mediated resistance to apoptosis , which may be responsible for recurrences of these tumors .",
"The β-catenin-independent branch plays a similarly important role in cancer progression: the key ligands Wnt5a and Ror2 are upregulated in various gastric cancers , regardless of the histological phenotype .",
"To determine whether Wnt ligands are transported on cytonemes between gastric cancer cells , we used the gastric cancer ( GC ) cells lines MKN7 , MKN28 and AGS .",
"Transfected Wnt8a-mCherry localized on filopodia in GC cell lines ( Figure 7A , Figure 7—figure supplement 1 ) .",
"Forced expression of Ror2 led to a strong increase in the number of filopodia on GC cells ( Figure 7B , C ) , whereas there was a significant reduction in cumulative filopodia length in cells expressing dominant-negative Ror23I ( Figure 7C ) , indicating that Ror2 also control filopodia formation in GC cells .",
"We focused on AGS cells because they show highly dynamic formation and retraction of filopodia , are particularly receptive to Ror2 manipulation , express Wnt1 at constant high levels , and , thus , have high endogenous β-catenin activity , which has been linked to the increased proliferation rate of this cell line ( Mao et al . , 2014 ) .",
"To assess whether cytoneme-mediated Wnt transport influences AGS cell behavior , and specifically proliferation , we co-cultivated Ror2-transfected cells with cells carrying the nuclear marker nucRFP .",
"Cells overexpressing Ror2 significantly increase cell proliferation in neighboring AGS cells ( Figure 7D , E ) .",
"Coexpression of the specific filopodia inhibitor IRSp534K dampened the increased proliferation rate induced by Ror2 expression .",
"Inhibition of Wnt signaling by the tankyrase inhibitor IWR1 abrogated the stimulatory effect of Ror2 expression , confirming that the Ror2 effect is due to increased Wnt signaling .",
"We conclude that Wnt is moved on cytonemes between GC cells to stimulate Wnt/β-catenin signaling and proliferation of neighboring cells .",
"Abrogation of this transport route has a consequence similar to that resulting from inhibition of Wnt signaling per se – it leads to reduced proliferation .",
"As the intestinal crypt requires a constant supply of Wnt signaling for tissue homeostasis ( Beumer and Clevers , 2016; Kuhnert et al . , 2004; Pinto et al . , 2003; Sailaja et al . , 2016 ) , we asked whether Wnt cytonemes operate in the mouse intestinal crypt .",
"In vivo , subepithelial myofibroblasts provide the major source of physiologically relevant Wnts , which maintain the crypt in vivo ( Kabiri et al . , 2014; Valenta et al . , 2016 ) .",
"It has been further demonstrated that these PdgfRα-positive myofibroblasts regulate the intestinal stem-cell niche by Wnts and RSPO3 ( Greicius et al . , 2018 ) .",
"The intestinal myofibroblasts form a large number of filopodia ( Figure 7F ) .",
"The formation of filopodia is inhibited by siRNA-mediated knock-down of Ror2 .",
"We used an organoid formation assay to analyze the requirement of Wnt-signaling filopodia in the intestinal crypt .",
"We used Wnt-deficient Porcn–/– crypt cells , co-cultivating them with Wnt3a-secreting L cells , to grow Wnt-deficient crypt organoids .",
"Co-culture of WT myofibroblasts with Wnt-deficient crypt cells leads to induction and maintenance of crypt organoids ( Kabiri et al . , 2014 ) .",
"These myofibroblasts extend filopodia to engulf crypt organoids ( Figure 7—figure supplement 1C ) .",
"When Ror2 was knocked down in the Wnt-producing myofibroblasts , we observed a substantial decrease in the number of organoids ( Figure 7G ) .",
"This suggests that the transport of Wnt signals on Ror2-dependent cytonemes from the myofibroblasts is crucial for the induction and maintenance of the intestinal crypt .",
"We conclude that cytonemes are vital for Wnt protein dissemination in vertebrates and that their appearance is regulated by Ror2-mediated PCP signaling ."
],
[
"Cytonemes are actin-based filopodia that transport an array of signaling molecules and their receptors , facilitating juxtacrine signaling .",
"Their appearance is highly dynamic during development ( Gradilla and Guerrero , 2013; Kornberg and Roy , 2014; Stanganello and Scholpp , 2016 ) .",
"A continuous adjustment of the number , length , rate of formation , direction , and retraction of cytonemes is crucial to regulate the exchange of signaling proteins within a tissue .",
"However , how cytoneme formation is controlled remains unclear .",
"Cytonemes change in response to signaling protein levels in the source cell ( Sato and Kornberg , 2002; Stanganello et al . , 2015 ) , suggesting that cytoneme formation is linked to signal production .",
"A signaling pathway that influences cytoneme emergence is the Wnt signaling network .",
"We have found that cytoneme initiation is linked to Wnt8a production .",
"Wnt8a activates the PCP signaling pathway in ligand-producing cells and , in turn , PCP induces cytonemes , which can be loaded with Wnt protein .",
"Data from Drosophila show that cytonemes require the PCP components Prickle and Vangl , which are essential for Fgf- and Dpp-positive cytonemes in the air sac primordium ( Huang and Kornberg , 2016 ) .",
"Prickle/Vangl modulate heparin proteoglycan content in the extracellular matrix to allow cytoneme outgrowth .",
"By contrast , we found that Wnt8a activates the PCP pathway via interaction with the Ror2 receptor to initiate cytoneme formation .",
"Our in vivo analysis suggests that activation of Ror2 in the zebrafish embryonic margin is necessary and sufficient for the formation of Wnt cytonemes , as other filopodia remain unperturbed .",
"We conclude that Ror2 is a specific cytoneme regulator .",
"Ror2 is a member of the family of orphan receptor tyrosine kinases , possessing an extracellular Fzd-like CRD , a cytoplasmic tyrosine kinase domain , and a proline-rich domain ( PRD ) ( Yoda et al . , 2003 ) .",
"Ror2 plays crucial roles in developmental morphogenesis in vertebrates .",
"Ror2-deficient mice exhibit skeletal , genital , and cardiovascular abnormalities caused by disrupted CE movements during gastrulation ( Oishi et al . , 2003; Takeuchi et al . , 2000 ) .",
"Ror2 is required for filopodia formation and XWnt5a-induced cell migration in Xenopus ( Nishita et al . , 2006 ) .",
"Irrespective of stimulation by Wnt proteins , ectopic expression of Ror2 can induce filopodia formation by actin polymerization via coupling of the Ror2-PRD to the actin-binding protein Filamin A . Our in vitro data show that Ror2 induces filopodia formation in PAC2 fibroblasts , myofibroblasts and gastric cancer cells .",
"Moreover , we show that Ror2 is crucial for Wnt cytoneme formation in vivo , and it is known to contribute to cytoskeleton remodeling and JNK activation in migrating cells ( Oishi et al . , 2003 ) .",
"We have previously shown that Wnt cytonemes require Cdc42 function to stabilize the intracytonemal actin skeleton ( Stanganello et al . , 2015 ) .",
"Here , we demonstrate that Ror2-dependent cytonemes also require Cdc42 function for the formation and maintenance of the actin scaffold .",
"Our data are supported by an analysis of CE movement in Xenopus: co-expression of Cdc42T17N rescues XRor2-induced CE alterations ( Hikasa et al . , 2002 ) , suggesting that Ror2 regulates Cdc42-dependent processes .",
"Ror2 acts as a receptor for Wnt5A in mice ( Mikels et al . , 2006 ) .",
"In Xenopus , Wnt5A/Ror2 activates the PCP signaling pathway , including transcription of ATF2 and PAPC ( Schambony and Wedlich , 2007 ) .",
"Although there is in vitro evidence that multiple Wnts can associate with the CRD domain of Ror2 , only a few Wnts have been demonstrated to elicit Ror2 activation in vivo .",
"For example , Wnt1 and Wnt3a bind to the CRD domain of Ror2 ( Billiard et al . , 2005 ) but neither altered receptor activity as assessed by levels of Ror2 autophosphorylation .",
"Xenopus Wnt8 binds to the ectodomain of Ror2 ( Hikasa et al . , 2002 ) but it is not known whether XWnt8 induces Ror2 signaling .",
"In zebrafish , evidence suggests that Wnt11 is a potential binding and signaling partner for Ror2 ( Bai et al . , 2014 ) .",
"Wnt11 activates Ror2 to modulate CE during zebrafish gastrulation .",
"We have observed that Wnt8a co-localizes with Ror2 in cytonemes .",
"Although tagged constructs have been examined at very low concentration , this does not necessarily imply correct subcellular localization .",
"However , our results are in line with studies on endogenous Wnt8a localization on cytonemes ( Stanganello et al . , 2015 ) and with studies on Ror2 localization on filopodia in mouse ( Paganoni and Ferreira , 2003; Laird et al . , 2011 ) and in Xenopus ( Nishita et al . , 2006; Brinkmann et al . , 2016 ) .",
"We have further observed that Wnt8a binds and activates Ror2 signaling in vitro and in vivo .",
"It is possible that other β-catenin-independent Wnts , such as Wnt5a and Wnt11 , might induce Ror2 cytonemes to regulate β-catenin Wnt ligand trafficking and thus β-catenin signaling .",
"Wnt5A and Wnt11 are known to regulate the dorso-ventral patterning of the neural tube and somites in mice ( Andre et al . , 2015 ) .",
"However , the observed patterning defect and AP axis shortening in Wnt5A/Wnt11 double-knockout mice was explained by migration alteration of the axial mesoderm .",
"Binding of Wnt proteins to their cognate Frizzled receptors activates several distinct signaling pathways , including the canonical Lrp6/β-catenin-dependent and the non-canonical Ror2/β-catenin-independent PCP pathways ( Niehrs , 2012 ) .",
"According to the conventional classification , Wnt1 , Wnt3a , and Wnt8a belong to the β-catenin-dependent Wnt signaling proteins , whereas Wnt5a and Wnt11 are representatives of the β-catenin-independent Wnt signaling proteins ( Kikuchi et al . , 2011 ) .",
"Activation of the Ror2/PCP signaling branch is assumed to repress the β-catenin signaling branch ( Niehrs , 2012 ) .",
"Both Wnt signaling branches use the same hub proteins as intracellular effectors , which may lead to competition and mutual repression .",
"For example , Wnt5a , which preferentially activates PCP signaling , competes with Wnt3a for Fzd2 and thus inhibits the β-catenin-dependent pathway ( Sato et al . , 2010 ) .",
"Furthermore , in Caenorhabditis elegans , Ror2 is thought to act by sequestering β-catenin Wnt ligands , a function that is independent of its intracellular domain ( Green et al . , 2007 ) .",
"In tissue culture , intracellular Ror2 signaling represses β-catenin signaling via its tyrosine kinase activity ( Mikels et al . , 2006 ) and its interaction with Dvl ( Witte et al . , 2010 ) .",
"Furthermore , intracellular binding partners such as Tak1 interact with the C-terminal region of Ror2 to further inhibit Wnt/β-Catenin signaling ( Winkel et al . , 2008 ) .",
"However , recent work reported that Ror2 signaling is able to enhance β-catenin-mediated signaling and proliferation in several tumor types ( Rasmussen et al . , 2013; Roarty et al . , 2017; Yan and Lin , 2008 ) .",
"These apparently contradictory results could be explained by cross-regulation of Ror2 and the β-catenin signaling being influenced by tissue heterogeneity .",
"This might occur , for example , during tumor progression ( Roarty et al . , 2017 ) when depending on the repertoire of Wnt signaling components present , Ror2-expressing tumor cells regulate the spatial distribution , duration , and amplitude of Wnt/β-catenin signaling within a tumor .",
"Our data provide a mechanistic explanation for this effect .",
"Although we cannot rule out the possibility of Ror2 activation within the Wnt source cells , we hypothesize that the Ror2/PCP signaling pathway is activated in an autocrine fashion on the basis of our observations of the behavior of individual cells in cell culture and in the embryo .",
"We further provide evidence that Ror2/PCP-dependent signaling is crucial for cytoneme emergence in Wnt source cells .",
"Then , cytonemes transmit Wnt ligands to neighboring cells to activate juxtacrine β-catenin signaling .",
"We demonstrate the importance of separating Wnt-producing from Wnt-receiving cells by showing that Ror2-mediated Wnt transport is required for controlling cell proliferation in AGS gastric cells .",
"AGS cells express Wnt ligands such as Wnt1 , display a high endogenous β-catenin level , and show enhanced proliferation ( Mao et al . , 2014 ) .",
"The anti-cancer drug salinomycin inhibits β-catenin signaling by inducing degradation of the Wnt co-receptor Lrp6 , resulting in reduced proliferation .",
"Autonomous activation of PCP signaling via Ror2 represses β-catenin signaling and reduces proliferation in AGS cells ( Yan et al . , 2016 ) , suggesting that activation of PCP signaling could be used as a further strategy to inhibit uncontrolled proliferation of gastric tumors .",
"However , gastric tumors display a high degree of tissue heterogeneity and we find that Ror2 can also enhance β-catenin signaling and consequently cell proliferation .",
"Our experimental approach is fundamentally different to that of the former analysis: Wnt-producing cells and Wnt-receiving cells are treated separately .",
"We find that Ror2 activation in the Wnt-producing cells increases proliferation in the Wnt-receiving cells .",
"This hypothesis is supported by recent findings showing that Wnt-Ror2-positive mesenchymal cells promote gastric cancer cell proliferation if co-cultivated ( Takiguchi et al . , 2016 ) .",
"Here , we describe Ror2/PCP-induced cytonemes as transport carrier for Wnt8a in zebrafish .",
"In cell culture experiments , we use PAC2 fibroblasts and HEK293T cells to provide further evidence for the importance of Ror2-dependent cytonemes in Wnt trafficking .",
"In addition , we show that human gastric cancer cells AGS , which primarily express the Wnt ligand Wnt1 , process paracrine Wnt signaling via cytonemes , which are influenced by Ror2 signaling .",
"Finally , we use murine intestinal stroma cells , which express Wnt2b to maintain the Wnt gradient operating in the intestinal crypt ( Aoki et al . , 2016; Kabiri et al . , 2014 ) .",
"We provide further evidence that Wnts from intestinal stroma utilize Ror2-dependent cytonemes for their transport .",
"In summary , we show that autocrine PCP pathway activation via Ror2 induces Wnt cytonemes in the Wnt source cell to transmit Wnt to the neighboring cell to activate paracrine Wnt/β-catenin signaling .",
"We propose cytonemes as an general mechanism for the mobilization of Wnt ligands in tissue homeostasis as well as in development in vertebrates ."
],
[
"The following plasmids were used: pCS2 + zfWnt8aORF1 ( Addgene 17048 ) , pCS2 + zfWnt8aORF1-GFP , pCS2 + zfWnt8aORF1-mCherry ( Stanganello et al . , 2015 ) , pCS2 + xWnt5 a-GFP , pCS2 + zfWnt5 b ( Wallkamm et al . , 2014 ) , xRor2-mCherry ( Feike et al . , 2010 ) , xRor23I ( Casella et al . , 1981 ) , mRor2-dCRD-GFP , pCS2 +Fz7 a-CFP , pcDNA3-EGFP-Cdc42T17N ( Addgene 12976 ) , 7xTRE Super TOPFlash-NLS-mCherry ( Moro et al . , 2012 ) , pmKate2-f-mem ( Evrogen ) , GPI-anchored mCherry in pCS2+ ( Scholpp et al . , 2009 ) , IRSp534K ( Casella et al . , 1981 ) .",
"To generate the pCS2 + xRor2 construct , the open reading frame of xRor2-mCherry was inserted into the ClaI/XhoI site of pCS2+ .",
"Breeding zebrafish ( Danio rerio ) were maintained at 28°C on a 14 hr light/10 hr dark cycle ( Brand et al . , 2002 ) .",
"To prevent pigment formation , embryos were raised in 0 . 2 mM 1-phenyl-2-thiourea ( PTU , Sigma , St Louis , MO 63103 , USA ) after 24 hpf .",
"The data we present in this study were acquired with wild-type zebrafish ( AB2O2 ) as well as with the transgenic zebrafish line tg ( −6gsc:EGFP-CAAX ) ( Smutny et al . , 2017 ) .",
"All animal work ( zebrafish husbandry and experimental procedures ) were undertaken under project and personnel licences granted by the UK Home Office under the United Kingdom Animals ( Scientific Procedures ) Act , in accordance with The University of Exeter’s ethical policies and were approved by the University of Exeter’s Animal Welfare and Ethical Review Body , and in accordance with the German law on Animal Protection approved by the Local Animal-Protection Committee ( Regierungspräsidium Karlsruhe , Az . 35–9185 . 64 ) and the Karlsruhe Institute of Technology ( KIT ) .",
"Experiments were performed in zebrafish PAC2 fibroblasts derived from 24-hr-old zebrafish embryos cultivated at 28°C without additional CO2 supply , primary gastric adenocarcinoma cells ( AGS ) , gastric tubular adenocarcinoma liver metastasis cells ( MKN28 and MKN7 [Motoyama et al . , 1986] ) and human embryonic kidney cells ( HEK293T; CRL-1573 ) cultivated at 37°C with 5% additional CO2 supply .",
"PAC2 were maintained in Leibowitz-15 media , HEK in DMEM , AGS and MKN28 in RPMI 1640 , all supplemented with 10% fetal bovine serum , 1% L-glutamine ( 2 mM ) and 1% penicillin/streptomycin .",
"All of the materials used for cell culture were purchased from Life Technologies , Gibco .",
"For transfection experiments , FuGENE HD Transfection Reagent ( Promega ) was used and cells were imaged after 48 hr .",
"For co-culture experiments , transfected cells were incubated for 24 hr , detached by Trypsin-EDTA ( 0 . 05% ) and incubated in a mixed population for another 48 hr before image acquisition .",
"Assays for SuperTOPFlash ( STF ) TCF/Wnt reporter expression and proliferation required initial co-cultivation of two distinct cell populations .",
"Cells were transfected with pDest7xTCF-NLS-mCherry or pCS2 +nucRFP plasmids , respectively , and incubated for 24 hr , detached by trypsin-EDTA ( 0 . 05% ) and further incubated in a mixed population for another 72 hr before image acquisition .",
"For one replication , seven 10x magnification images were taken per sample with identical laser settings .",
"Image locations were saved by the Mark and Find microscope feature to reproduce similar scanning setups .",
"For measuring TCF/Wnt reporter activation , fluorescent nuclei were processed using the Dot-Plugin in Imaris and the average grey value of the nuclei was determined or fluorescent nuclei were counted to measure cell proliferation .",
"For the chemical treatment , cell cultures were treated with GTPase inhibitor ML141 10 mM ( Merck Millipore ) or 50 µM IWR-1 ( Sigma ) to antagonise the Wnt signaling pathway .",
"For staining , cells were fixed with 4% PFA at RT , washed with PBS .",
"Cells were incubated with 50 µg/ml phalloidin ( P1951 , Sigma ) and 10 µg/ml DAPI ( D9542 , Sigma ) .",
"Myofibroblasts were prepared from C57BL/6-Tg ( Pdgfra-cre ) 1Clc/J/RosamTmG mice and cultured as previously described ( Greicius et al . , 2018 ) .",
"As confluence of cultured cells was reaching 80% , they were transfected with respective the siRNA ( Dharmacon mouse ROR2 siRNA pool Cat#LQ-041074-00-0002 , four siRNAs combined in equal parts at 10 nM ) using siRNAmax reagent ( Invitrogen Cat#13778–030 ) .",
"On day 2 post-transfection , myofibroblasts were mixed with Porcn-deficient intestinal epithelial cells and cultured using RSPO1-supplemented medium .",
"Organoid counting was performed at the time point when the group containing no stromal cells had no surviving organoids left ( the end of day 3/beginning of day 4 of co-culture ) .",
"siRNA-transfected cells were imaged using the OlympusLive Imaging system IX83 .",
"Acquired 3D image stacks were de-convoluted using cellSens Dimension ( Olympus ) and are presented as maximum intensity projections .",
"Cells and their attached filopodia were automatically detected in the RFP channel ( mem-mCherry ) of the acquired images ( Figure 4—figure supplement 1A ) .",
"The images were initially filtered using a Gaussian low-pass filter ( σ2=1 ) and subsequently used detect the cell body as well as the cell’s filopodia ( Figure 4—figure supplement 1B ) .",
"For the filopodia detection , we used an objectness filter ( σ=1 , α=1 , β=1 , γ=0 . 003 , N=2 ) that emphasized line-like structures based on the eigenvalues of the Hessian matrix at each pixel location ( Figure 4—figure supplement 1C , [Antiga , 2007] ) .",
"The obtained edge-enhanced image was then binarized ( Figure 4—figure supplement 1D ) using a local adaptive threshold filter that set pixels to foreground if their intensity value was larger than a regional mean intensity minus a multiple of the regional intensity standard deviation and otherwise to background ( window radius = 200 , std . dev . multiplier = 1 ) .",
"To segment the cell body , we applied the local adaptive threshold ( window radius = 200 , std . dev . multiplier = 1 ) on the smoothed input image ( Figure 4—figure supplement 1E ) and subsequently used a morphological opening operation ( kernel radius = 2 ) to get rid of noise and remaining filopodia parts ( Figure 4—figure supplement 1F , [Soille et al . , 2011] ) .",
"The cell body was given by the largest connected component in the opened binary image .",
"The segmentation mask of the cell body including filopodia was then constructed by combining the binarized edge-enhanced image with the binary cell body image ( Figure 4—figure supplement 1G ) .",
"The combined cell image was subsequently skeletonized to identify potential filopodia tips at the end points of the skeleton ( Figure 4—figure supplement 1H ) .",
"All of the above-mentioned preprocessing steps were implemented in the open-source software tool XPIWIT ( Bartschat et al . , 2016 ) .",
"The preprocessing results were then imported into a dedicated MATLAB tool that was developed to validate , correct and analyze this kind of images ( Figure 4—figure supplement 1I , J ) .",
"In order to trace filopodia from the identified tips to the cell body automatically , we used an adapted livewire algorithm ( Barrett and Mortensen , 1997 ) .",
"The output of the objectness filter was used as an edge map ( Figure 4—figure supplement 1C ) on which the livewire algorithm tried to find a maximally scoring path from the tip of the filopodium to the center of the cell body .",
"Based on the segmentation of the cell body ( Figure 4—figure supplement 1F ) , the automatic tracing was stopped as soon as the cell body was reached .",
"The interactive user interface was then used to add , remove and correct both segmentation masks and detected filopodia on a per-cell basis .",
"For each cell’s filopodia , we calculated the Euclidean distance along the the path from the tip to the cell body .",
"The same image preprocessing and tracing was applied to semi-automatically extract filopodia in 3D confocal images ( Figure 4—figure supplement 1K ) .",
"For 3D images , however , the start and end points of the filopodia were provided by the user via a graphical user interface and the livewire approach was applied twice: first on an axial maximum intensity projection ( z ) to obtain the lateral path ( xy ) , then the axial positioning of the filopodium was obtained by searching for the highest scoring path between the provided start and end points solely in the z-direction ( Figure 4—figure supplement 1K ) .",
"Multiple automatically traced filopodia can then be exported and used to obtain average statistics for all filopodia of interest .",
"For the FCS measurements , a home-built confocal microscope was used as previously described ( Dörlich et al . , 2015 ) , with slight modifications .",
"We used a water immersion objective ( HCX PL APO CS 63x/1 . 2 , Leica , Wetzlar , Germany ) instead of an oil immersion objective; the multimode fiber , which acts as a confocal pinhole , was modified accordingly to ensure a pinhole size of 1 AU .",
"Data were collected for 390 s by continuously scanning the focus perpendicularly through the membrane .",
"Each scan line consisted of 100 pixels , with a step size of 100 nm .",
"eGFP was excited with a 488 nm continuous wave ( cw ) laser and mCherry with a 561 nm cw laser .",
"After splitting the fluorescence signal into two color channels by using a 555 nm dichroic filter , 525/50 ( eGFP ) and 600/37 ( mCherry ) band pass filters were used for detection .",
"To avoid artefacts in the correlation curves caused by scanner flyback and wavelength switching , the membrane was always kept in the center of the field of view .",
"The intensity data were arranged as an x-t pseudo image , and the intensities of those pixels containing membrane fluorescence were integrated to obtain an intensity time trace for correlation analysis , as described earlier ( Hunter , 2007 ) .",
"The injection of mRNAs and Morpholino oligomers was performed according to the description in the text and in Mattes et al . ( 2012 ) .",
"Ror2 MO was used in a 0 . 5 mM concentration ( 5’-CAGTGTAACAACTTCCAAACTCTCC −3’ ) ( Gene Tools , Philomath , OR 97370 , USA ) .",
"Capped and in vitro transcribed mRNA ( mMessage Machine Kit , Ambion ) was microinjected into one cell or into the yolk for ubiquitous expression or into one of 16 blastomeres to generate cell clones .",
"Embryos were incubated at 28°C until used for image acquisition or fixed for whole-mount mRNA in situ hybridization ( ISH ) .",
"For ISH , hgg , ntl , axin2 , fgf8a and pax6a digoxigenin- and fluorescein-labeled probes were prepared from linearized templates using an RNA labelling and detection kit ( Roche ) as described by Scholpp and Brand ( 2003 ) .",
"Images were taken on an Olympus SZX16 microscope equipped with a DP71 digital camera by using Cell D imaging software .",
"For real-time quantitative PCR ( RT-qPCR ) , 50 embryos each were lysed in 1 ml TriZol ( Sigma ) , and total RNA was prepared using the Direct-zol RNA Mini Prep Kit from Zymo Research .",
"cDNA was prepared using MMLV reverse transcriptase from Promega and analysed in a Real-Time PCR system from LifeTechnologies ( ABI StepOnePlus ) .",
"Primers with the following sequence were used: beta-actin ( 5′-CCTTCCTTCCTGGGTATGG-3′; 5′-GGTCCTTACGGATGTCCAC-3′ ) , axin2 ( 5′-CAATGGACGAAAGGAAAGATCC-3′; 5′-AGAAGTACGTGACTACCGTC-3′ ) , lef1 ( 5′-CAGACATTCCCAATTTCTATCC-3′; 5′-TGTGATGTGAGAACCA ACC-3′ ) .",
"Results were analysed using the ΔΔCT method .",
"Computer simulations have been implemented in Python using the libraries numpy , scipy and matplotlib ( Hunter , 2007 ) and are based on a simulation of cytoneme-based ligand distribution described in detail in Stanganello et al . ( 2015 ) .",
"The modelling of the tissue is split up into two subproblems ( 1 ) the correct modelling of the dynamically forming tissue and ( 2 ) the morphogen transport through that tissue .",
"The tissue dynamics are modelled by a two-dimensional non-periodic plane of discrete cells .",
"The simulated area is 1 , 000 µm by 1 , 000 µm in size and each cell occupies a circle of 8 µm radius .",
"Possible cell positions are precomputed and kept fixed during the course of the simulation .",
"Cells can change positions by moving along those precomputed positions .",
"In every simulation step ( Δt = 1 s ) , it is possible for each individual cell to perform actions with predetermined probabilities , which are directly derived from experimental measurements .",
"Possible actions are:",
"( i ) signaling from the producing tissue ,",
"( ii ) cell insertion by division or intercalation of the receiving tissue ,",
"( iii ) cell migration — directed or non-directed nearest neighbor swapping of the receiving tissue , and",
"( iv ) morphogen decay in the receiving tissue .",
"Two different cell types are distinguished .",
"The marginal , Wnt active cells produce morphogen and are able to deposit morphogen via cytonemes into cells of the receiving tissue .",
"Once a signaling event is accepted for a marginal cell , an angle is randomly chosen from the respective distribution determined in Stanganello et al . ( 2015 ) .",
"The length is randomly chosen from a Gaussian distribution with the peak value lfilo .",
"A virtual filopodium is formed with those values originating from the marginal cell .",
"If the tip of the filopodium ends up in the vicinity of the surface of a receiving cell ( ±2 µm ) the Wnt content of that cell is increased , otherwise the filopodium is deleted .",
"For the ATF2 luciferase reporter assay , four-cellstage Xenopus embryos were injected into animal ventral blastomeres along with the 100 pg ATF2-luciferase reporter plasmid and 10 pg TK-Renilla-luciferase reporter plasmid .",
"The reporter plasmids were injected alone or together with 500 pg of the respective synthetic mRNAs .",
"Luciferase reporter assays were carried out using triplicates of five-gastrula-stage ( st . 12 ) embryos lysed to measure luciferase activity using the dual luciferase system ( Promega ) .",
"For confocal analysis , live embryos were embedded in 0 . 7% low melting agarose ( Sigma-Aldrich ) dissolved in 1x Ringer’s solution .",
"Images of cells and embryos were obtained with a Leica TCS SP5 X or SP8X confocal laser-scanning microscope using 20x or 63x dip-in objective or , for the kinase library screen , a Leica DMI600SD with 20x objective .",
"Image processing was performed with Imaris 9 . 1 software ( Bitplane AG , Switzerland ) .",
"Filopodia and cytoneme measurements from confocal z-stacks of living embryos was performed via the semi-automated filopodia analysis software described before .",
"Cell culture quantifications were carried out using Fiji software .",
"Roundness of notochordal embryo cells was determined by calculating the width to length ratio of each cell in Fiji .",
"All experiments were carried out at least in biological triplicates if not indicated otherwise .",
"Significance was calculated by Student’s t-test and asterisks are used to indicate p values .",
"One-way ANOVA was used to analyse groups of experimental data .",
"All groups are random samples from the same population .",
"Variances are similar across treatments and residuals are normally distributed .",
"P values , F values and degrees of freedom ( df ) are indicated .",
"Box plots: centre lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles , outliers are represented by dots ."
]
] | [
"Signaling filopodia , termed cytonemes , are dynamic actin-based membrane structures that regulate the exchange of signaling molecules and their receptors within tissues .",
"However , how cytoneme formation is regulated remains unclear .",
"Here , we show that Wnt/planar cell polarity ( PCP ) autocrine signaling controls the emergence of cytonemes , and that cytonemes subsequently control paracrine Wnt/β-catenin signal activation .",
"Upon binding of the Wnt family member Wnt8a , the receptor tyrosine kinase Ror2 becomes activated .",
"Ror2/PCP signaling leads to the induction of cytonemes , which mediate the transport of Wnt8a to neighboring cells .",
"In the Wnt-receiving cells , Wnt8a on cytonemes triggers Wnt/β-catenin-dependent gene transcription and proliferation .",
"We show that cytoneme-based Wnt transport operates in diverse processes , including zebrafish development , murine intestinal crypt and human cancer organoids , demonstrating that Wnt transport by cytonemes and its control via the Ror2 pathway is highly conserved in vertebrates ."
] | [
"Communication helps the cells that make up tissues and organs to work together as a team .",
"One way that cells share information with each other as tissues grow and develop is by exchanging signaling proteins .",
"These interact with receptors on the surface of other cells; this causes the cell to change how it behaves .",
"The Wnt family of signaling proteins orchestrate organ development .",
"Wnt proteins influence which types of cells develop , how fast they divide , and how and when they move .",
"Relatively few cells , or small groups of cells , in developing tissues produce Wnt proteins , while larger groups nearby respond to the signals .",
"We do not fully understand how Wnt proteins travel between cells , but recent work revealed an unexpected mechanism – cells seem to hand-deliver their messages .",
"Finger-like structures called cytonemes grow out of the cell membrane and carry Wnt proteins to their destination .",
"If the cytonemes do not form properly the target cells do not behave correctly , which can lead to severe tissue malformation .",
"Mattes et al . have now investigated how cytonemes form using a combination of state-of-the-art genetic and high-resolution imaging techniques .",
"In initial experiments involving zebrafish cells that were grown in the laboratory , Mattes et al . found that the Wnt proteins kick start their own transport; before they travel to their destination , they act on the cells that made them .",
"A Wnt protein called Wnt8a activates the receptor Ror2 on the surface of the signal-producing cell .",
"Ror2 then triggers signals inside the cell that begin the assembly of the cytonemes .",
"The more Ror2 is activated , the more cytonemes the cell makes , and the more Wnt signals it can send out .",
"This mechanism operates in various tissues: Ror2 also controls the cytoneme transport process in living zebrafish embryos , the mouse intestine and human stomach tumors .",
"This knowledge will help researchers to develop new ways to control Wnt signaling , which could help to produce new treatments for diseases ranging from cancers ( for example in the stomach and bowel ) to degenerative diseases such as Alzheimer’s disease ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology"
] | Molecular conservation of marsupial and eutherian placentation and lactation | elife-27450-v1 | [
[
"While placental morphology varies widely between different mammalian species , its functions as a center for embryonic respiration , nutrient uptake , waste removal and embryonic signaling are highly conserved ( Cross , 2006 ) .",
"Given this diverse set of tasks , it is intriguing how an organ with such great morphological diversity performs the same function across widely divergent taxa .",
"For example , one critical difference in placentation across species is invasive ability .",
"Human placental tissues invade deeply into maternal tissues where they remodel arteries , whereas other eutherian placentas , like that of the pig , do not invade ( Leiser and Kaufmann , 1994 ) .",
"In marsupials , most placentas , including that of the tammar , are non-invasive ( Freyer et al . , 2002; Freyer and Renfree , 2009 ) , but some , like the South American grey short-tailed opossum , do invade , albeit only for the last few days of pregnancy ( Tyndale-Biscoe and Renfree , 1987; Freyer et al . , 2002 , 2007 ) .",
"While the diversity of invasion is extreme between therian species , it is only one of the many functional , cellular , and morphological differences in placentation .",
"This extreme diversity raises interesting questions about how a wide array of structures and cell types can mediate something as complex as fetal development .",
"Reproductive diversity is even more pronounced when comparing morphology and function between eutherians and marsupials , not only because the placentas are different , but also because marsupials , generally , rely more heavily on lactation to support fetal development ( Renfree , 1983 ) .",
"The tammar wallaby ( Macropus eugenii ) , a key model for investigating marsupial reproduction ( Renfree et al . , 2011 ) , has a 26 . 5 day pregnancy followed by a 300–350 day period of lactation ( Tyndale-Biscoe and Renfree , 1987 ) .",
"By comparison , the mouse has a 20 day pregnancy followed by a 20–24 day period of lactation ( Crew and Mirskaia , 1930 ) , relying much less on lactation for offspring success .",
"The tammar placenta is derived from a fusion of the yolk sac and chorion , producing a structure that contains two different tissue types: the avascular bilaminar omphalopleure ( BOM ) and the vascular trilaminar omphalopleure ( TOM ) .",
"These two tissues each contain a single trophoblast cell layer ( TL ) and a single endodermal cell layer ( EL ) with TOM containing an additional mesodermal layer ( ML ) which gives rise to the vasculature ( Freyer et al . , 2002; Renfree , 2010 ) ( Figure 1 ) .",
"The cells of the trophoblast layer are characterized by large nuclei , consistent with eutherian trophoblast morphology which are typically large , polyploid or multinucleated ( Zybina and Zybina , 1996 ) ( Hannibal et al . , 2014 ) , while the cells of endodermal layer contain small nuclei ( Figure 1 ) ( Freyer et al . , 2002; Jones et al . , 2014 ) .",
"During early pregnancy nutrient transfer to the embryo occurs across a keratinous shell coat ( Renfree , 1980; Menzies et al . , 2011 ) .",
"At day 18 the embryo loses this shell coat , and the yolk sac becomes closely interlaced with the uterine epithelium and maternal blood supply , forming the yolk sac placenta .",
"The BOM then expands to increase the nutrient exchange surface area , while the TOM expands to further support the increased respiratory needs to the end of gestation over the next 7 days ( Renfree , 1973b ) . 10 . 7554/eLife . 27450 . 003Figure 1 . Tammar placental structure . Schematic of a day 24 tammar conceptus ( right ) highlighting placental cell types ( middle ) .",
"The tammar yolk sac placenta contains two cellular layers: the trophoblast ( TL; blue ) and the endoderm ( EL; yellow ) .",
"Further , the placental structure is divided into two halves .",
"The ‘top’ non-vascularized tissue is termed the bilaminar omphalopleure ( BOM ) and the ‘bottom’ vascularized tissue is termed the trilaminar omphalopleure ( TOM ) which also contains a mesodermal layer ( ML ) .",
"H&E sections ( left ) demonstrate the cell types within the BOM and TOM components ( Scale bars are 50 μm ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 27450 . 003 As the tammar fetus is underdeveloped at birth , it relies on lactation to complete its growth and development ( Trott et al . , 2003; Green and Merchant , 1988; Nicholas et al . , 1997 ) and to provide immune protection while the adaptive immune system matures ( Joss et al . , 2009 ) .",
"While lactation in tammar shares some characteristics of eutherian lactation , such as initial high immunoglobulin secretion ( Old and Deane , 2000 ) and nutrient density to promote growth ( Daly et al . , 2007 ) , tammar milk undergoes dramatic and dynamic changes over the course of lactation in both type and amount of carbohydrates ( Messer and Green , 1979 ) , proteins , and lipids ( Green et al . , 1980; Green and Renfree , 1982 ) .",
"The composition of tammar milk is exquisitely programmed to support the remainder of fetal development , with individual proteins and amino acids secreted for differing windows of time ( Nicholas , 1988; Renfree et al . , 1981b; Vander Jagt et al . , 2015 ) and is so potent that early pouch young fostered by a mother lactating for an older pouch young results in accelerated growth and development , including: increases in body size , head length , thicker fur , and accelerated maturation of the fore-stomach ( Nicholas et al . , 1997 ) ; Menzies et al . , 2007; Kwek et al . , 2009 ) .",
"To date , the signals within the milk that drive this acceleration are unknown , but the milk appears to be crafted to promote fetal morphogenesis in specific windows of time ( Green et al . , 1980 ) , which is related to variation in both resource availability in the environment ( Dennis and Marsh , 1997 ) and neural cues from the mother ( Renfree MB et al . , 1981a ) .",
"As marsupial milk is clearly supplied to support fetal development , a role fulfilled primarily by the placenta in eutherians , it has been proposed that dynamic and extended lactation periods evolved in marsupials as an alternative to the complex placentation seen in eutherians ( Renfree , 1983; Tyndale-Biscoe and Renfree , 1987; Renfree , 2010 ) .",
"To better understand the evolution of pregnancy and lactation in marsupials , we generated and analyzed transcriptome data from placental and mammary gland tissues of the tammar wallaby .",
"We find that the tammar yolk sac placenta shares striking molecular similarity to the chorioallantoic placenta of eutherians , despite major morphological and developmental differences .",
"Moreover , we find that established protein markers that distinguish decidua ( maternal ) and trophoblast ( fetal ) tissue in the eutherian placenta are instead co-localized in the yolk sac placenta , suggestive of an ancestral molecular program underlying placentation that has undergone further compartmentalization during eutherian evolution .",
"Furthermore , we provide additional molecular confirmation that marsupials do have fully functional placentas , which supports the growing body of evidence that eutherians and marsupials can both be classified as ‘placental mammals’ ( Padykula and Taylor , 1982; Freyer et al . , 2003; Renfree , 2010 ) .",
"Finally , we discover that genes crucial for eutherian placental function are expressed in the tammar mammary gland .",
"This provides the first comprehensive molecular evidence that marsupials favored a physiologically complex lactation to achieve the same developmental milestones that placentation has facilitated in eutherians ."
],
[
"To investigate how the marsupial placenta functions we used 3SEQ ( Beck et al . , 2010 ) to sequence the BOM and TOM transcriptomes at day 21–23 and day 25 .",
"To examine the specific molecular characteristics of the BOM and TOM components of the placenta , we pooled BOM ( 4 replicates ) and TOM ( 6 replicates ) sequences from all gestational time points .",
"Using DESeq2 , we find 445 differentially expressed transcripts; of which 83 were up-regulated in BOM tissue and 362 were up-regulated in TOM tissue ( Figure 2A ) .",
"The BOM up-regulated genes are associated with ontology related to endocytosis and metabolism ( p-adj = 0 . 0764 , 0 . 0390 , see Supplementary file 1 ) consistent with the hypothesis that BOM acts as the center for embryonic nutrient uptake ( Renfree , 1973b ) .",
"Conversely , the genes up-regulated in TOM are associated with ontology related to extracellular matrix organization , hematopoiesis , and response to oxygen levels ( p-adj = 1 . 49×10−7 , 0 . 00193 , 0 . 0688 , see Supplementary file 1 ) .",
"This is consistent with the idea that TOM , being the vascularized component of the tammar placenta , acts as the center of embryonic respiration ( Renfree , 1973b ) .",
"Overall , this supports the suggestion that BOM and TOM have specific and distinct functions throughout gestation ( Renfree , 1973b ) ( Figure 2—figure supplement 1A ) , consistent with previously observed morphology differences ( Freyer et al . , 2002 ) . 10 . 7554/eLife . 27450 . 004Figure 2 . Tammar placental gene expression differs between tissues and over time .",
"( A ) Transcripts differentially expressed between BOM and TOM ( n = 445 ) with blue indicating high expression and red low expression ( see Z-score insert upper right ) .",
"( B ) Transcripts differentially expressed between early ( day 21–23 ) and late ( day 25 ) placental time points ( n = 1705 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 27450 . 00410 . 7554/eLife . 27450 . 005Figure 2—figure supplement 1 . Tammar placenta transcriptome exhibits distinct signatures in both tissue type and time .",
"( A ) PCA plot depicting the 10 RNA-seq replicates colored by placental tissue type: BOM in red and TOM in blue , demonstrating tissue specific separation on principal component 1 ( B ) PCA re-plotted and colored by time point: early ( day 21–23 ) in red , and late ( day 25 ) in blue , demonstrating temporal separation on principal component 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 27450 . 005 After the shell coat is lost by gestational day 18 , TOM begins to expand through day 23 of gestation until it has covered 50% of the yolk sac ( Freyer et al . , 2002 ) .",
"We examined if the function of the placenta changed from day 21–23 , when TOM is still expanding , to day 25 , when TOM has finished expanding .",
"To this end , we pooled the 21–23 day sequences ( 4 replicates ) together and the 25 day sequences ( 6 replicates ) together , including both BOM and TOM .",
"Using DESeq2 , we find 1705 differentially expressed transcripts; of which 592 were up-regulated at day 21–23 ( early ) and 1113 were up-regulated at day 25 ( late ) ( Figure 2B ) .",
"Genes up-regulated at earlier time points exhibited no significant enrichment for any gene ontology , but trended toward intracellular and ion transport ( p-adj = 0 . 188 , 0 . 109 , see Supplementary file 1 ) .",
"In contrast , those up-regulated at later time points were enriched for cellular growth , differentiation , and junction organization ( p-adj = 0 . 0331 , 0 . 0162 , 5 . 79 × 10−4 , see Supplementary file 1 ) .",
"Overall , these findings reveal that the tammar placenta undergoes dynamic changes in gene expression throughout its development despite the short duration of its physiological activity ( Figure 2—figure supplement 1B ) .",
"Although the overall morphology and cellular structure of the chorioallantoic placenta in mouse and humans is vastly different from the yolk sac placenta in marsupials , we next tested whether they shared similarity at the molecular level .",
"To this end , we compared the tammar placenta transcriptome , pooling samples of all tissue types and time points , to publically available traditional RNA-Seq datasets for the term mouse ( ENCSR000BZP ) and human ( GSE56524 ) placenta transcriptomes .",
"We found 3894 transcripts shared by all three species ( Supplementary file 2 ) which included many factors that have documented roles in placental function ( Rawn and Cross , 2008 ) .",
"We uncovered the expression of trophoblast markers including: Gcm1 ( Anson-Cartwright et al . , 2000 ) , members of the Igf signaling pathway ( Reik et al . , 2003 ) , many placental cytokeratins ( Gauster et al . , 2013 ) , Wnt7b ( Parr et al . , 2001 ) , Stra13 ( Hughes et al . , 2004 ) , PTN ( Schulte et al . , 1996 ) , and Gjb3 ( Koch et al . , 2012 ) .",
"Cathepsins ( Mason , 2008 ) and Serpins ( Kaiserman et al . , 2002 ) a class of proteases and protease inhibitors respectively , which are important for mediating maternal fetal interactions were also present ( Hannibal and Baker , 2016 ) .",
"We also found markers of the maternal decidua such as Cebpb ( Lynch et al . , 2011 ) and Vim ( Can et al . , 1995 ) .",
"Additionally , the tammar and mouse placentas share the expression of Cdx2 , a transcription factor that is essential for early trophoblast development ( Strumpf et al . , 2005; Frankenberg et al . , 2013 ) , while the tammar and human placentas share expression of Nodal , a secreted factor important for trophoblast differentiation ( Ma et al . , 2001 ) .",
"These findings are consistent with the idea that a core set of genes is essential for placental function and development across all therian mammals and that , regardless of morphological differences , both marsupial and eutherian placentas share expression of key placental transcripts .",
"Eutherian and marsupial placentas have key differences in their cellular composition: the eutherian placenta is derived from both fetal trophoblast and maternal decidua cells , and the marsupial placenta is derived solely from fetal trophoblast and yolk sac endoderm cells .",
"Given our observation that the tammar placenta expresses genes that function in eutherian placentation , we next examined where these proteins are expressed at the cellular level .",
"To this end , we performed immunofluorescence using antibodies against the trophoblast markers - CDX2 , GCM1 , and Pan-Cytokeratins and the maternal decidual markers - CEBPB and Vimentin - on day 24 tammar BOM ( Figure 3 ) and TOM ( Figure 3—figure supplement 1 ) tissues .",
"We find that CDX2 , CEBPB and Vimentin are expressed in both the endodermal and trophoblast layers , with CDX2 and CEPBP being nuclear and Vimentin cytoplasmic .",
"This is particularly intriguing as CEBPB ( Lynch et al . , 2011 ) and Vimentin ( Can et al . , 1995 ) are known markers of maternal decidua in eutherians and have limited expression in the eutherian trophoblast .",
"Additionally , we find that the known eutherian markers for trophoblasts , Pan-Cytokeratins ( Gauster et al . , 2013 ) and GCM1 ( Anson-Cartwright et al . , 2000 ) are strongly expressed only in the endodermal layer , and are not expressed in the trophoblast layer .",
"Overall , protein localization in tammar reveals substantial findings including the expression of maternal decidual markers throughout the yolk sac placenta and fetal trophoblast markers expressed exclusively within the endoderm .",
"Taken together , these data suggest that the choriovitelline placenta in tammar has accomplished coordination of critical placental functions and may even include functions that are supplied maternally in eutherians . 10 . 7554/eLife . 27450 . 006Figure 3 . Tammar placenta expresses eutherian placental markers . Immunofluorescence of the eutherian placental markers: CDX2 , CEBPB , GCM1 , Pan- cytokeratins , and Vimentin , in day 24 tammar placenta .",
"The left column shows protein expression .",
"The middle is DAPI stain depicting the nucleus for the same section .",
"The right column is the merge .",
"No 1° Antibody is a negative control ( bottom row ) .",
"Scale bars are 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 27450 . 00610 . 7554/eLife . 27450 . 007Figure 3—figure supplement 1 . Tammar placenta expresses eutherian placental markers .",
"( TOM )",
"Immunofluorescence of key placenta development genes in day 24 wallaby TOM placenta tissue .",
"The first column of panels depicts the expression of each gene , the second column of panels depicts a DAPI nuclear stain , and the third column of panels depicts a merge of both images .",
"Genes depicted from top to bottom include: CDX2 , CEBPB , GCM1 , Pan-cytokeratins , Vimentin , and a negative control where no primary antibody was applied ( Scale bars are 50 μm ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 27450 . 007 We next examined whether the similarity between tammar and eutherian placentation exist during both the early and late stages of placentation .",
"While we demonstrate that the tammar placental transcriptome shares molecular similarities to the term eutherian placenta , the mouse and human placenta are molecularly distinct between the first half and second half of pregnancy , with the early stages primarily involved in cell cycle and metabolism and the later stages focused on reproduction ( Knox and Baker , 2008; Winn et al . , 2007 ) .",
"Therefore we tested whether the tammar placenta might share greater similarity with a specific stage of eutherian placentation .",
"To this end , we compared the transcriptome of all sequenced tammar time points with gene expression in the mouse placenta throughout gestation ( e8 . 0-term ) ( GSE11220 ) , and to the transcriptome of the adult mouse heart ( ENCFF204IFN ) .",
"We identified the transcripts expressed in both the tammar and mouse , ranked the expression of each transcript at each time point from highest to lowest , and compared the time points using a series of pairwise Spearman correlation tests .",
"The highest coefficients reveal that the transcriptome of the tammar placenta , regardless of gestational age , is more similar to that of an e10 . 5 mouse placenta than any other time point ( Figure 4A ) .",
"Additionally , we find that the lowest coefficients exist between the day 21 and 23 tammar placenta and the later stages of mouse development ( e17 . 0 , e19 . 0 , and birth ) ( Figure 4A ) .",
"Finally , we find that the mouse placenta has higher correlations at all time points with the tammar placenta than it does with the mouse heart ( Figure 4—figure supplement 1 ) .",
"This suggests we are detecting a true conservation of placenta-specific transcription and not just species-specific transcription .",
"While the Spearman coefficients are higher between the mouse e10 . 5 and tammar transcriptomes than in any other pairwise comparison , this difference is minimal and therefore we next sought to refine the analysis by comparing only placenta-specific transcripts , removing housekeeping and other transcripts that may contribute to noise .",
"In a previous study , we identified 410 transcripts are unique to the mouse placenta ( Knox and Baker , 2008 ) .",
"During gestation 340 of these transcripts are expressed prior to e12 . 5 and 70 are expressed after e12 . 5 of mouse gestation , demarcating an early and late placental signature ( Knox and Baker , 2008 ) .",
"We found 100 tammar orthologs of these 410 mouse placental transcripts expressed in our datasets at 21 , 23 and 25 days gestation .",
"Of these 100 , 90 are expressed only in the early mouse placenta ( prior to 12 . 5 ) and 10 are expressed only in the late mouse placenta ( after 12 . 5 ) ( Figure 4B ) .",
"When comparing the early transcripts ( 90/340 ) to the late placental transcripts ( 10/70 ) , we find a significant enrichment of early transcripts ( p<0 . 05 by chi-square test ) .",
"Overall , this more targeted analysis supports the idea that tammar placentation functionally resembles the earlier phases of mouse placentation . 10 . 7554/eLife . 27450 . 008Figure 4 . The tammar placenta is more similar to the midgestation mouse placenta .",
"( A ) Comparison of mouse and tammar placental transcriptomes at different gestational ages using Spearman correlations ( see values throughout graph ) .",
"Red indicates the greatest level of transcriptional similarity while yellow indicates the weakest transcriptional similarity .",
"The mouse heart transcriptome used as a control ( top ) .",
"( B ) Heat map depicting the expression of 410 transcripts that are specific for the mouse placenta of which 340 are expressed before and 70 are expressed after midgestation ( e12 . 5 ) .",
"The tammar placenta expresses orthologs of 100 of these transcripts , which are highlighted in the yellow boxes on the heat map .",
"90 are classified as developmental , or expressed early in mouse , and 10 are classified as mature , or expressed late in mouse .",
"Blue indicates high expression and red low expression ( see Z-score on right ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 27450 . 00810 . 7554/eLife . 27450 . 009Figure 4—figure supplement 1 . Tammar placenta exhbits conservation of placenta-specific gene expression . Heat map depicting a comparison of the mouse placental transcriptomes against tammar and adult mouse heart transcriptome to detect conservation organ-specific transcription .",
"Red indicates the greatest level of transcriptional similarity while yellow indicates the weakest transcriptional similarity . DOI: http://dx . doi . org/10 . 7554/eLife . 27450 . 009 The evolution of complex lactation in marsupials represents an independent strategy that has been proposed to mirror aspects of eutherian placentation ( Renfree , 1983 , 2010 ) .",
"Previously , it was demonstrated that Igf2 was imprinted in the tammar mammary gland ( Stringer et al . , 2012 , 2014 ) , a phenomenon previously determined to be essential for proper growth and development of the eutherian placenta ( Reik et al . , 2003 ) .",
"This suggests that the tammar mammary gland may indeed express similar transcripts as the placenta .",
"To identify any conservation of function between organ systems , we first performed transcriptome sequencing using 3SEQ on tammar mammary glands during early lactation periods ( days 36 , 60 , and 95 ) .",
"We next compared the mammary gland transcriptomes with the transcriptomes of tammar placenta , tammar liver , tammar testis , mouse placenta , mouse liver , mouse testis , and mouse mammary gland .",
"After ranking the expression of shared transcripts and calculating pairwise Spearman correlations , several findings stand out .",
"First , we find that the tammar and mouse placenta share the highest correlation of any cross-species organ pair ( Figure 5—figure supplement 1 ) , confirming the high degree of molecular similarity between the eutherian and tammar placenta that we found using immunofluorescence ( Figure 3 ) .",
"Second , although less so than the placenta , the tammar mammary gland shows the highest correlation with the mouse mammary gland when compared to any other mouse tissue , highlighting some functional conservation in this organ .",
"Among those genes with conserved expression in mammary glands , we found Gata3 ( Chiu and Chen , 2016 ) and Msh2 ( Tanaka et al . , 1997 ) , transcription factors known to be essential for placentation in mice .",
"Immunofluorescence in tammar mammary gland at day 60 of lactation finds GATA3 in the alveoli ( Figure 5B ) .",
"This pattern of expression is reminiscent of GATA3 expression in the mouse mammary gland ( Tlsty , 2007 ) , suggesting an ancestral role of GATA3 in mediating lactation .",
"Finally , the placentas of both tammar and mouse show the lowest within species correlation with mammary gland which suggests widely divergent roles for each organ in supporting reproductive physiology . 10 . 7554/eLife . 27450 . 010Figure 5 . Shuffling of reproduction genes between lactation and placentation .",
"( A ) Venn-diagram ( Left ) comparing the genes expressed in the lactating tammar and mouse mammary glands with the tammar and eutherian placenta .",
"Key ontology of genes in overlapping categories is highlighted .",
"Genes listed for each category in Supplementary file 3 .",
"The table ( Right ) depicts key categories of genes from the venn-diagram and whether or not they are expressed in the placenta and mammary gland tissues of the tammar and eutherians .",
"If the gene class is present in the tissue of a given lineage it is given a green ‘+” , if it is absent it is given a red “-“ .",
"( B ) Immunofluorescence of GCM1 and GATA3 .",
"The first column depicts the expression of each protein , the second column is DAPI stain for nuclear DNA , and the third column is a merge of both images .",
"The final row contains a negative control where no primary antibody was applied ( Scale bars are 50 μm ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 27450 . 01010 . 7554/eLife . 27450 . 011Figure 5—figure supplement 1 . Tammar placenta and mammary gland have exceptionally distinct molecular functions . Comparison of tammar and mouse organ transcriptomes including: placenta , mammary gland , liver and testis using Spearman correlations ( see values throughout graph ) .",
"Red indicates the greatest level of transcriptional similarity while yellow indicates the weakest transcriptional similarity . DOI: http://dx . doi . org/10 . 7554/eLife . 27450 . 011 Although the transcriptional repertoire is globally distinct between placenta and mammary gland in both tammar and mouse we next examined whether there were genes expressed in both organs that might reflect any shared functions .",
"First , we tested whether placental transcripts could be found specifically in the tammar mammary gland , suggesting a role in supporting tammar fetal morphogenesis .",
"To this end , we identified transcripts present in eutherian placenta ( Supplementary file 1 ) , tammar placenta , tammar mammary gland , but not in the mouse mammary gland ( GSE60450 ) , finding 77 genes expressed .",
"As we could find genes simply by chance that meet these criteria , we also compared placental transcripts to other organ systems in a similar fashion , including tammar liver , mouse liver , tammar testis , and mouse testis .",
"While we find gene expression shared in all categories , including 76 genes shared between tammar placenta , eutherian placenta , tammar liver , but not mouse liver , and 589 genes shared between tammar placenta , eutherian placenta , tammar testis , but not mouse testis , we only find significant functional associations when comparing placenta to mammary gland .",
"The 77 genes shared between the tammar placenta , eutherian placenta , and tammar mammary gland ( Figure 5A ) , are associated with nutrient transport ( GO analysis; p-adj = 0 . 0163 , see Supplementary file 3 ) and include the placental transcription factor Gcm1 , which is necessary for eutherian placentation ( Anson-Cartwright et al . , 2000 ) .",
"Localization of GCM1 by immunofluorescence demonstrates strong nuclear staining throughout the tammar mammary gland at day 60 of lactation .",
"Additionally , we find high expression of GCM1 in the alveoli ( Figure 5B ) , the site of milk ejection in the tammar mammary gland ( Findlay , 1982 ) .",
"This expression suggests a novel function of GCM1 in supporting multi-stage lactation in marsupials .",
"Therefore , we suggest that these 77 transcripts , including Gcm1 , represent genes that have been convergently co-opted by the marsupial mammary gland to expand its potential for fetal support or lost in the eutherian mammary gland where the placenta provides all the required support for fetal development .",
"We next tested whether there were transcripts shared only between eutherian placentas and the tammar mammary gland suggesting conservation of genes used for late fetal development in two different reproductive organs .",
"To this end , we identified 108 transcripts that were expressed in the eutherian placenta and the tammar mammary gland , but not in the tammar placenta and mouse mammary gland ( Figure 5A ) .",
"As we could find genes simply by chance that meet these criteria , we again compared placental transcripts to the other organ systems used in our previous analysis .",
"While we find gene expression shared in all categories , including 168 genes shared between eutherian placenta and tammar liver , but not tammar placenta and mouse liver , and 589 genes shared between eutherian placenta and tammar testis , but not tammar placenta and mouse testis .",
"GO analysis of these 108 genes did not unveil significant ontology , but trended toward processes critical for reproductive success including: embryonic morphogenesis and immune function ( p-adj = 0 . 163 , 0 . 146 , see Supplementary file 3 ) .",
"Among the genes associated with these two GO terms we find Dkk1 and Csf1r , transcripts with documented roles in placentation ( Pollheimer et al . , 2006; Sasmono et al . , 2003 ) .",
"Here we also find the expression of Igfbp1 , a molecule important for regulating insulin growth factor function , a process previously demonstrated to be important in both therian placentation ( Reik et al . , 2003 ) and marsupial lactation ( Stringer et al . , 2012 ) .",
"Conversely , the liver and testis analysis does not reveal any significant GO enrichment associated with placentation .",
"Together , these data provide molecular evidence that the tammar mammary gland performs some similar functions to the late eutherian placenta to support continued fetal development in the pouch ."
],
[
"The yolk sac structure of the tammar placenta has led to the idea that it does not function like the eutherian placenta and marsupials are therefore often misleadingly termed ‘non-placental’ .",
"However , we find that the tammar yolk sac placenta expresses many key markers of eutherian placental cell types , including those of maternal decidua .",
"This suggests that the tammar yolk sac placenta perform functions that the eutherian placenta has compartmentalized into more complex cellular domains .",
"Additionally we find that global gene expression undergoes dynamic shifts from day 21 and 23 to day 25 , during tammar gestation , a window critical for placental growth and proliferation .",
"This same type of dynamic change in placental gene expression has also been documented in the mouse ( Knox and Baker , 2008 ) and human ( Winn et al . , 2007 ) suggesting rapidly changing gene expression patterns may be conserved between therian placentas .",
"The presence and expression of core placental genes and the dynamic gene expression in the tammar placenta suggests that many molecular functions of the placenta may be conserved in the therian mammal stem species , as suggested previously ( Freyer et al . , 2002 , 2007 ) .",
"Furthermore , this strongly suggests that the use of the placenta to distinguish eutherians from marsupials is incorrect .",
"We suggest that reproductive genes have been shared between the placenta and the mammary gland throughout evolution to establish diverse lactation and placentation strategies to nourish mammalian young .",
"We show that these shifting events have occurred both to facilitate the multi-stage lactation of marsupials and to allow for the formation of placentas of diverse cell and tissue types that allow for longer pregnancy in eutherians .",
"The genes shared between these tissues are enriched for processes involved in nutrition , immune function and embryonic morphogenesis suggesting a functional convergence between placentation and lactation .",
"Our finding that the mammary gland of the tammar shares the expression of transcripts , including Gcm1 ( Anson-Cartwright et al . , 2000 ) and Gata3 ( Chiu and Chen , 2016 ) , that promote placental functions in eutherians suggests marsupial lactation is an alternate strategy that parallels eutherian placentation .",
"Taken together these data support the idea that reproductive genes that allow efficient exchange between mother and offspring have been respectively co-opted in placentation and lactation to facilitate proper offspring growth and development .",
"While anatomical work has described the tammar placenta , our data provides a molecular complement .",
"We first confirm ( Renfree , 1973b ) that BOM and TOM are discrete placental structures with independent functions .",
"Many of the genes up-regulated in BOM fall into ontologies that are associated with nutrient uptake and metabolism .",
"This is consistent with the hypothesis that BOM , with its large trophoblast layer , has a high cytoplasm-to-nucleus ratio allowing for greater uptake and subsequent metabolism of nutrients from uterine secretions ( Renfree , 1973b; Renfree and Renfree , 1973a; Renfree and Tyndale-Biscoe , 1973; Freyer et al . , 2002 ) .",
"Many of the genes up-regulated in TOM fall into ontologies that are associated with respiration .",
"This is consistent with the idea that TOM , having vasculature and a thin trophoblast layer , allows for rapid transport of oxygen thus acting as the primary site of embryonic respiration ( Renfree , 1973b; Renfree and Renfree , 1973a; Renfree and Tyndale-Biscoe , 1973; Freyer et al . , 2002 ) .",
"This is especially intriguing because the thinning of the trophoblast layer in TOM is reminiscent of the convergent thinning of the maternal-fetal interhemal distance in eutherian placentation ( Enders and Carter , 2004 ) , suggesting an additional layer of morphological convergence .",
"In addition , our work provides strong molecular support for the earlier suggestions that the endoderm and trophoblast layers of the tammar placenta have each adopted separate and critical placental functions .",
"Based on expression of both GCM1 and Pan-cytokeratins , we suggest that the endodermal layer plays a key role in tammar placenta function .",
"These genes mark critical components of the trophoblast ( or fetal ) component of a wide variety of eutherian placentas ( Enders et al . , 2006 ) .",
"GCM1 specifically marks the placental labyrinth ( Anson-Cartwright et al . , 2000 ) , the site of nutrient uptake from maternal blood ( Cross , 2006 ) , suggesting that the endodermal layer is serving as a center of nutrient trafficking in the tammar placenta , a function thought to be held by the trophoblast layer .",
"Because there is no decidualization in the tammar , it also is surprising to find CEBPB and Vimentin in the tammar trophoblast layer , as these are known decidua ( or maternal ) markers in eutherians .",
"Additionally , CEBPB is a direct regulator of decidualization in eutherians ( Wagner et al . , 2014 ) .",
"Further comparative studies will need to be performed to determine whether the tammar trophoblast layer gained maternal placenta function or whether eutherians expanded fetal placenta function to include the decidua .",
"Overall , the layer-specific expression of these proteins suggests that the endodermal and trophoblast layers of the tammar placenta play similar roles to the eutherian trophoblast and decidua tissues , respectively .",
"Our findings indicate that , despite its anatomical simplicity , the tammar placenta expresses a dynamic molecular program that is highly reminiscent of the eutherian placenta .",
"Additionally we provide evidence that genes underlying important organismal functions may move freely between different cell and tissue types throughout the course of morphological evolution .",
"This is highlighted in both the sub-functionalization of the tammar placenta to express the genes of both the maternal and fetal placenta tissues of eutherians in distinct cell layers and by the convergent co-option of key reproductive transcripts for use in placentation and lactation throughout mammalian evolution .",
"Overall , our study highlights the molecular conservation in mammalian placentation despite the enormous morphological variation and provides some of the first molecular data on how the marsupial lineage fits into this framework ."
],
[
"Pouch young were removed ( RPY ) from adult female tammars to reactivate their diapausing blastocysts .",
"Pregnant females were euthanized during the last third of gestation to collect placental tissues from days 21 , 23 , 24 , and 25 RPY .",
"Mammary gland tissue was collected from females carrying pouch young at days 36 , 60 , and 95 post partum , during phase 2A of lactation ( Green and Merchant , 1988 ) .",
"3SEQ captures only the sequence of the 3’ end of transcripts , yielding a single read per transcript , allowing for quantification of expression while using fewer total reads ( Finn et al . , 2014 ) .",
"For placenta tissue , one sample of day 21 bilaminar omphalopleure ( avascular yolk sac ) ( BOM ) , day 21 trilaminar omphalopleure ( vascular yolk sac ) ( TOM ) , day 23 BOM , day 23 TOM , two samples of day 25 BOM , and four samples of day 25 TOM liquid nitrogen frozen tissues were prepared for sequencing .",
"mRNA was isolated using Dynabeads Oligo ( dT ) 25 .",
"3SEQ libraries were prepared from this mRNA as previously described ( Chuong et al . , 2013 ) .",
"In brief , the samples were given a short heat-shearing treatment immediately followed by cDNA synthesis .",
"The resulting cDNA was repaired using 3’A-tailing and ligated to linkers containing unique Illumina barcodes for sequencing .",
"An E-gel SizeSelect agarose gel was used for size selection and the samples were then PCR amplified for 15 cycles and purified using AMPure XP beads .",
"Quality of the libraries was assed using both Qubit and Bioanlyzer technology , which were then sequenced on the Genome Analyzer IIx .",
"For mammary glands , a single sample from each of day 36 , 60 , and 95 paraformaldehyde fixed tissues were prepared for sequencing .",
"3SEQ proved especially useful in this experiment because it is known to be more effective than traditional RNA-seq when used on fixed or archived tissue samples ( Beck et al . , 2010 ) .",
"Fixed samples were initially subjected to a brief protease digestion to remove protein to nucleic acid cross-linking .",
"Total RNA was then extracted using the ambion RecoverAll Total Nucleic Acid Isolation kit and mRNA was subsequently isolated using Dynabeads Oligo ( dT ) 25 .",
"3SEQ libraries were prepared from this mRNA as previously described ( Chuong et al . , 2013 ) .",
"In brief , the samples were given a short heat-shearing treatment immediately followed by cDNA synthesis .",
"The resulting cDNA was repaired using 3’A-tailing and ligated to linkers containing unique Illumina barcodes for sequencing .",
"A 3% NuSieve GTG agarose gel was used for size selection and the samples were then PCR amplified for 17 cycles and purified using AMPure XP beads .",
"Quality of the libraries was assed using both Qubit and Bioanlyzer technology , which were then sequenced on the Illumina NextSeq .",
"The resulting sequences were aligned to the tammar wallaby genome ( m . eug_v1 . 0 ) ( Renfree et al . , 2011 ) using STAR ( Dobin et al . , 2013 ) ( version 2 . 5 . 1b , RRID:SCR_005622 ) .",
"Raw counts were assigned to significantly transcribed regions using UniPeak ( Foley and Sidow , 2013 ) v1 . 0 .",
"Regions were then associated with the nearest gene using HOMER ( Heinz et al . , 2010 ) ( version 4 . 7 , RRID:SCR_010881 ) .",
"Multiple regions mapping to the same gene were combined to give a final read count for each gene .",
"The resulting data was then normalized across samples and tissue types using the bioconductor package DESeq2 ( Love et al . , 2014 ) in R ( RRID:SCR_000154 ) .",
"The data files processed in this study were deposited at the Gene Expression Omnibus ( GEO ) accession number GSE90838 .",
"Differentially expressed transcripts were identified using two pairwise comparisons .",
"First we compared placental tissue types ( BOM vs . TOM ) and then we compared gestational time points: time during TOM expansion ( day 21 and 23 , termed ‘early’ ) vs . time after TOM expansion ( day 25 , termed ‘late’ ) .",
"While some samples had a single replicate sequenced our strategy of grouping the resulting data by both time and tissue type allowed us to achieve significant statistical power .",
"Differential expression was determined using the DEseq2 bioconductor package in R with any transcripts significantly up or downregulated ( Benjamini-adjusted p<0 . 05 ) being included in subsequent analyses .",
"The Ensembl transcript names were used to obtain associated gene names from Ensembl biomart .",
"The resulting gene lists for each comparison were entered into Enrichr ( Kuleshov et al . , 2016 ) for gene ontology ( GO ) analysis to assess any relevant differences in biological function picked up by our comparisons .",
"To compare our newly assembled tammar placenta and mammary gland transcriptomes to those of eutherian mammals we used several publically available gene expression datasets .",
"RNA-seq based transcriptome data for the term mouse placenta was obtained from ENCODE ( ENCSR000BZP ) .",
"RNA-seq based transcriptome data for the human term placenta was obtained from the gene expression omnibus ( GEO ) ( GSE56524 ) ( Metsalu et al . , 2014 ) .",
"RNA-seq based transcriptome data for mouse lactating luminal and basal cells of the mammary gland was obtained from GEO ( GSE60450 ) ( Zhou et al . , 2014 ) .",
"Read count tables were obtained for all of these data sets and the average number of reads per transcript was taken , after this those transcripts that were below 1% of the mean were excluded and dubbed not expressed .",
"For the mouse lactating mammary gland transcriptomes the basal and luminal cells were combined to get the best representation of total mammary gland function .",
"The remaining genes were dubbed expressed and used to compare against the tammar transcriptomes .",
"Resulting gene lists for meaningful comparisons were entered into Enrichr ( Kuleshov et al . , 2016 ) for gene ontology ( GO ) analysis to assess any relevant differences in biological function picked up by our comparative transcriptomics .",
"To compare gene expression throughout placental development we compared our tammar placenta transcriptome time course to microarray based transcriptome data documenting gene expression in the developing mouse placenta from GEO ( GSE11220 ) ( Knox and Baker , 2008 ) .",
"Additionally , we compared our data to ENCODE adult mouse heart transcriptome data ( ENCFF204IFN ) to be sure we were detecting conservation of organ-specific expression and not just species-specific expression .",
"To make these data sets comparable we reduced the data to include only those genes expressed in the developing tammar and mouse placentas and the adult mouse heart .",
"From here we ranked the expression of these genes in each time point of each species from highest to lowest and performed a series of pairwise Spearman correlations to assess the degree of similarity between each .",
"The resulting Spearman correlation coefficients comparing tammar and mouse were then used to assess where the greatest transcriptional similarities exist .",
"To compare the transcriptional similarity of the mammary glands and placenta we used the same approach .",
"We compared our tammar placenta and mammary gland transcriptomes to RNA-seq based transcriptomes from GEO for the tammar liver , testis ( GSE50747 ) , and mouse mammary gland ( GSE60450 ) and Encode data for the mouse placenta ( ENCSR000BZP ) , liver ( ENCSR216KLZ , ENCSR000BYS ) , and testis ( ENCSR266ESZ , ENCSR000BYW ) .",
"Liver and testis tissues were included to ensure that any relationship detected between the placenta and mammary gland are unique and not a relationship that occurs when comparing any two organs .",
"Samples for immunofluorescence ( IF ) were fixed overnight at 4°C in a 4% paraformaldehyde solution .",
"They were then processed while rocking at room temperature through five 10 min washes in PBS , one 1 hr wash in 70% ethanol , one 1 hr wash in 85% ethanol , one 1 hr wash in 90% ethanol , one 1 hr wash in 95% ethanol , three 20 min washes in 100% ethanol , and three 10 min washes in xylenes .",
"Samples were then placed at 65°C in paraffin for 1 hr , the paraffin was then replaced and left overnight , and the tissues were embedded in paraffin and put into blocks the following day .",
"Sections were then taken from these blocks on a Leica RM 2155 at a thickness of 5 μm and mounted on glass slides and left to dry overnight at 37°C .",
"Slides for immunofluorescence were processed as follows: two 10 min washes in xylenes , two 10 min washes in 100% ethanol , two 10 min washes in 95% ethanol , two 10 min washes in 80% ethanol , two 10 min washes in 70% ethanol , one 10 min wash in 50% ethanol , and two 10 min washes in deionized water .",
"The protocol for GCM1 on mammary gland tissue included antigen retrieval in sodium citrate buffer ( pH 6 . 0 ) for 13 min in a pressure cooker .",
"All slides were then blocked in PBS with either 5% normal goat serum , 1% bovine serum albumin , and 0 . 01% triton-X or 5% normal rabbit serum and 0 . 01% triton-X at room temperature for 1 hr .",
"Primary antibodies were then added to the same blocking serums and applied to the slides overnight at 4°C .",
"Slides were then washed three times in PBS containing 0 . 1% Tween 20 for 10 min .",
"After this , biotin conjugated secondary antibodies were added to the previous blocking solutions and applied to the slides for 1 hr at room temperature .",
"Slides were washed for 10 min three times in PBS-Tween after which , Vectastain ABC reagent was applied to the slides for 30 min at room temperature .",
"Slides were then washed for 10 min three times in PBS-Tween after which , Perkin Elmer Tyramide signal amplification , Cyanine-3 reagent was applied to the slides for 6 min at room temperature .",
"Slides were then washed for 10 min three times in PBS-Tween and are then mounted using ProLong gold antifade mountant with Dapi .",
"Slides were visualized using a Leica DMRX A2 upright light microscope and photographs were captured using LAS ( Leica Application Suite ) software package v4 . 2 . 0 .",
"Primary antibodies used for IF were: rabbit polyclonal anti-CDX2 ( Cell Signaling 3977 , RRID:AB_2077043 ) at a 1:50 dilution , rabbit polyclonal anti-CEBPB ( Santa Cruz Antibodies sc-150 , RRID:AB_2260363 ) at a 1:2500 dilution , goat polyclonal anti-GATA3 ( Santa Cruz Antibodies sc-22206 , RRID:AB_2108588 ) at a 1:950 dilution , mouse monoclonal anti-GCM1 ( abcam ab88748 , RRID:AB_2041368 ) at a 1:200 dilution for placental tissue and 1:100 for mammary gland tissue , mouse monoclonal anti-Pan-Cytokeratins ( AE1/AE3 ) ( BioLegend , 914201 , RRID:AB_2565152 ) at a 1:5000 dilution , and mouse monoclonal anti-Vimentin ( Sigma v5255 , RRID:AB_477625 ) at a 1:1000 dilution .",
"Secondary antibodies used for IF were: biotinylated goat anti-mouse igG ( Jackson Immunoresearch , 111-065-166 , RRID:AB_2338569 ) at a 1:1000 dilution , biotinylated goat anti-rabbit igG ( Jackson Immunoresearch , 111-065-144 , RRID:AB_2337965 ) at a 1:5000 dilution , and biotinylated rabbit anti-goat igG ( Vector , BA-5000 , RRID:AB_2336126 ) at a 1:2500 dilution ."
]
] | [
"Eutherians are often mistakenly termed ‘placental mammals’ , but marsupials also have a placenta to mediate early embryonic development .",
"Lactation is necessary for both infant and fetal development in eutherians and marsupials , although marsupials have a far more complex milk repertoire that facilitates morphogenesis of developmentally immature young .",
"In this study , we demonstrate that the anatomically simple tammar placenta expresses a dynamic molecular program that is reminiscent of eutherian placentation , including both fetal and maternal signals .",
"Further , we provide evidence that genes facilitating fetal development and nutrient transport display convergent co-option by placental and mammary gland cell types to optimize offspring success ."
] | [
"Before birth , mammals in their mother’s womb are provided with nutrients and oxygen via an organ called the placenta .",
"After birth , the mother produces milk to feed her young , which supports their continuing development .",
"The majority of living mammals , from mice to humans , belong to a group known as “eutheria” and have placentas made up of many different types of cells and tissues .",
"This complex placenta has caused many people to wrongly refer to this group as “the placental mammals” .",
"However , marsupials like kangaroos and koalas represent a second group of mammals that also have a placenta , albeit a much simpler one that consists of only a few layers of cells .",
"The simpler placenta means that marsupials give birth to young that are underdeveloped compared to eutherians .",
"These young must develop further inside the mother’s pouch , where they are fed with milk that changes over time to support the different stages of their development .",
"As a result , marsupials produce a more complicated range of milks than eutherians .",
"To date , scientists do not fully understand how these two groups of mammals evolved to nurture their offspring in two different ways .",
"Guernsey et al . have now studied which genes are active in the placenta and milk-producing “mammary” glands of the tammar wallaby , a small member of the kangaroo family .",
"Comparing the data with similar data from mice and humans revealed that marsupial and eutherian placentas are alike in many ways .",
"For example , both rapidly change which genes are active as the placenta matures , and both contain tissues that perform specific tasks such as providing offspring with nourishment for growth and oxygen for respiration .",
"The analysis also shows that mammary glands in marsupials use similar genes to those used by the eutherian placenta to support the development of offspring .",
"This suggests the placenta and the mammary gland have converged on a similar set of genes to allow female mammals to nourish their young .",
"Overall , the findings further scientific knowledge about the evolution of pregnancy and milk production in marsupials , paving the way for further research into the diversity of life on Earth .",
"The findings also confirm that marsupials have working placentas , stressing that eutherians and marsupials should both be referred to as “placental mammals” ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"plant biology"
] | Identification and characterisation of hypomethylated DNA loci controlling quantitative resistance in Arabidopsis | elife-40655-v2 | [
[
"Eukaryotic cytosine methylation plays an important role in the regulation of gene expression and genome stability .",
"In plants , this form of DNA methylation occurs at three sequence contexts: CG , CHG and CHH , where H indicates any base except guanine ( G ) ( Vanyushin , 2006; Law and Jacobsen , 2010 ) .",
"Patterns of plant DNA methylation in the plant genome can remain stable over multiple generations and influence heritable phenotypes ( Quadrana and Colot , 2016 ) .",
"Recent evidence has suggested that reduced DNA methylation increases the responsiveness of the plant immune system ( Espinas et al . , 2016 ) This ‘priming’ of plant defence enables an augmented induction of defence-related genes after pathogen attack , causing increased levels of quantitative resistance ( Prime-A-Plant Group et al . , 2006; Conrath et al . , 2015; Martinez-Medina et al . , 2016; Liégard et al . , 2018 ) .",
"In some cases , priming of defence-related genes is associated with post-translational histone modifications that mark a more open chromatin structure ( Jaskiewicz et al . , 2011; Luna et al . , 2012 ) .",
"Additional evidence for epigenetic regulation of plant immunity has come from independent studies reporting that disease-exposed Arabidopsis produces progeny that expresses transgenerational acquired resistance ( TAR ) , which is associated with priming of defence-related genes ( Luna et al . , 2012; Slaughter et al . , 2012 ) .",
"Furthermore , Arabidopsis mutants that are impaired in the establishment or maintenance of DNA methylation mimic TAR-related priming without prior priming stimulus ( Lopez et al . , 2011; Luna and Ton , 2012; López Sánchez et al . , 2016 ) .",
"By contrast , the hyper-methylated ros1-4 mutant , which is impaired in active DNA de-methytation , is more susceptible to biotrophic pathogens , affected in defence gene responsiveness , and impaired in TAR ( López Sánchez et al . , 2016; Yu et al . , 2013 ) .",
"Thus , DNA ( de ) methylation determines quantitative disease resistance by influencing the responsiveness of defence-related genes .",
"However , causal evidence that selected hypomethylated DNA loci are responsible for the meiotic transmission of this form of quantitative disease resistance is lacking .",
"Epigenetic Recombinant Inbred Lines ( epiRILs ) have been developed with the aim to study the epigenetic basis of heritable plant traits ( Reinders et al . , 2009; Johannes et al . , 2009 ) .",
"EpiRILs show little differences in DNA sequence , but vary substantially in DNA methylation .",
"A commonly used population of epiRILs is derived from a cross between the Arabidopsis wild-type ( Wt ) accession Col-0 and the decreased DNA methylation1-2 ( ddm1-2 ) mutant ( Johannes et al . , 2009 ) .",
"The DDM1 protein is a chromatin re-modelling enzyme that provides DNA methyltransferase enzymes access to heterochromatic transposable elements ( TEs ) ( Jeddeloh et al . , 1998; Brzeski and Jerzmanowski , 2003; Zemach et al . , 2013 ) .",
"Accordingly , the ddm1-2 mutation causes loss of pericentromeric heterochromatin and reduced DNA methylation in all sequence contexts ( Kakutani et al . , 1996; Ito et al . , 2015 ) .",
"Although the epiRILs from the ddm1-2 x Col-0 cross do not carry the ddm1-2 mutation , they contain stably inherited hypomethylated DNA regions from the ddm1-2 parent , which are maintained up to 16 generations of self-pollination ( Johannes et al . , 2009; Colomé-Tatché et al . , 2012; Latzel et al . , 2013 ) .",
"A core set of 123 epiRILs from this population at the eight generation of self-pollination in the wild-type ( Wt ) background has been characterized for differentially methylated region ( DMR ) markers , enabling linkage mapping of heritable hypomethylated loci controlling root growth , flowering and abiotic stress tolerance ( Liégard et al . , 2018; Cortijo et al . , 2014; Kooke et al . , 2015 ) .",
"In this study , we have characterised the core set of 123 lines from the ddm1-2 x Col-0 epiRIL population for resistance against the biotrophic downy mildew pathogen Hyaloperonospora arabidopsidis ( Hpa ) to search for heritable hypomethylated loci controlling disease resistance .",
"We identified four of these epigenetic quantitative trait loci ( epiQTLs ) , accounting for 60% of the variation in disease resistance .",
"None of these epiQTLs were associated with growth impairment , indicating that the resistance does not incur major physiological costs on plant development .",
"Further phenotypic characterisation and transcriptome analysis of selected Hpa-resistant epiRILs revealed that their resistance is associated with genome-wide priming of defence-related genes .",
"Interestingly , bisulfite sequencing did not reveal defence regulatory genes inside the epiQTL regions that were simultaneously primed and hypomethylated , suggesting that DDM1-dependent DNA methylation at the epiQTLs trans-regulates the responsiveness of distant defence genes ."
],
[
"To examine the role of DDM1-dependent DNA methylation in heritable disease resistance , 123 epiRILs from the ddm1-2 x Col-0 cross were analysed for Hpa resistance and compared to siblings of the ddm1-2 parent ( Figure 1a , red ) , the Wt parent ( Col-0 ) , and five progenies thereof ( Figure 1a , green ) .",
"Leaves of 3-week-old plants were inoculated with Hpa conidiospores and then collected for trypan-blue staining at 6 days post-inoculation ( dpi ) .",
"Microscopic classification of leaves into four classes of Hpa colonisation ( Figure 1—figure supplement 1 ) revealed 51 epiRILs with statistically enhanced levels of resistance compared to each susceptible Wt line ( Pearson’s Chi-squared tests , p<0 . 05 ) .",
"Of these , eight epiRILs showed similar levels of Hpa resistance as the ddm1-2 line ( Figure 1a , dark blue triangles; Pearson’s Chi-squared test , p>0 . 05 ) , whereas 43 epiRILs showed intermediate levels of resistance .",
"To identify the epiQTL ( s ) responsible for the observed variation in Hpa resistance , the categorical classification of Hpa infection was converted into a single value numerical resistance index ( RI; Figure 1a , bottom graph ) .",
"Using a linkage map of stably inherited DMR markers ( Colomé-Tatché et al . , 2012 ) ( Supplementary file 1 dataset S1 ) , interval mapping revealed four statistically significant epiQTLs on chromosomes I , II , IV and V ( Figure 1b ) .",
"The epiQTL on chromosome II had the highest logarithm of odds ( LOD ) value .",
"For all epiQTLs , the DMR markers with the highest LOD scores ( ‘peak markers’ ) showed a positive correlation between ddm1-2 haplotype and RI ( Figure 1c ) , indicating that the hypomethylated haplotype from ddm1-2 increases resistance against Hpa .",
"A linear regression model to calculate the percentage of RI variance explained by each peak marker ( R ( 2 ) ( g ) ) ( Cortijo et al . , 2014 ) confirmed that the DMR peak marker of the epiQTL on chromosome II had the strongest contribution to RI variation .",
"Using an additive model , the combined contribution of all epiQTL peak markers to RI variation ( R2 ( G ) ) ( Cortijo et al . , 2014 ) was estimated at 60 . 0% ( Figure 1d ) .",
"DNA methylation maintains genome stability by preventing transposition of TEs .",
"In the Col-0 x ddm1-2 epiRIL population , reduced methylation at the ddm1-2 haplotype occurs predominantly at long transposons in heterochromatic pericentromeric regions ( Zemach et al . , 2013; Colomé-Tatché et al . , 2012 ) .",
"Frequent transposition events in the epiRILs are nevertheless rare as most DNA hypomethylation occurs at relic transposons that have lost the ability to transpose , and the occurrence of independent transposition events at similar loci is extremely unlikely ( Colomé-Tatché et al . , 2012; Matzke and Mosher , 2014 ) .",
"However , it is possible that transposition events originating from the heavily hypomethylated ddm1-2 parent were crossed into the population , resulting into shared transposition events ( STEs ) between multiple epiRILs , which could have contributed to variation in resistance .",
"To account for this possibility , we compared the genomic DNA sequences of the four epiQTL intervals from 122 epiRILs ( LOD drop-off = 2 ) for the presence of STEs in more than two epiRILs , using TE-tracker software ( Gilly et al . , 2014 ) .",
"This analysis revealed three STEs in the epiQTL interval on chromosome I ( Supplementary file 1 dataset S2 ) , while no STEs could be detected in the other epiQTL intervals .",
"None of the STEs in the epiQTL on chromosome I showed statistically significant linkage with RI ( Supplementary file 1 dataset S2 ) .",
"Accordingly , we conclude that the segregating Hpa resistance in the epiRIL population is caused by epigenetic variation in DNA methylation , rather than genetic variation by STEs .",
"Expression of inducible defence mechanisms is often associated with physiological costs , resulting in reduced plant growth ( Huot et al . , 2014 ) .",
"To determine whether the resistance that is controlled by the four epiQTLs is associated with costs to plant growth , we quantified the green leaf area ( GLA ) of 12–15 individual plants per line at the stage of Hpa inoculation ( Figure 1—figure supplement 2 ) .",
"Subsequent interval mapping revealed one statistically significant epiQTL on chromosome I ( Figure 1b ) .",
"The corresponding peak marker ( MM150 ) showed a negative correlation between GLA and ddm1-2 haplotype ( Figure 1c ) , indicating that the hypomethylated ddm1-2 allele at this locus represses plant growth .",
"The growth epiQTL mapped to a different region than the resistance epiQTL on chromosome I ( Figure 1b , inset ) .",
"Furthermore , none of the eight most resistant epiRILs showed significant growth reduction compared to all Wt lines in the screen ( Figure 1—figure supplement 2 ) .",
"Hence , the resistance provided by the four hypomethylated epiQTLs is not associated with major physiological costs to plant growth .",
"Enhanced defence to one stress can lead to enhanced susceptibility to another stress , which is caused by antagonistic cross-talk between defence signalling pathways ( Koornneef and Pieterse , 2008 ) .",
"To examine whether Hpa resistance in the epiRIL population is associated with increased susceptibility to other stresses , we compared the eight most Hpa-resistant epiRILs ( Figure 1a; Figure 1—figure supplement 3a ) for resistance against the necrotrophic fungus Plectosphaerella cucumerina ( Pc ) and tolerance to salt ( NaCl ) .",
"At 9 dpi with Pc spores , epiRIL#193 showed a statistically significant reduction in necrotic lesion size compared to the Wt ( line #602 ) , indicating enhanced resistance ( Figure 1—figure supplement 3b ) .",
"The seven other epiRILs showed unaffected levels of Pc resistance that were similar to the Wt .",
"Salt tolerance was quantified by the percentage of seedlings with fully developed cotyledons at 6 days after germination on agar medium with increasing NaCl concentrations .",
"Remarkably , all Hpa-resistant epiRILs showed varying degrees of tolerance to the highest NaCl concentration compared to Wt plants ( Figure 1—figure supplement 3c ) .",
"Thus , the quantitative resistance to Hpa in the epiRIL population does not compromise resistance against necrotrophic pathogens or abiotic stress .",
"Basal resistance against Hpa involves a combination of salicylic acid ( SA ) -dependent and SA-independent defence mechanisms ( Knoth et al . , 2007; Coates and Beynon , 2010 ) .",
"To examine the role of SA-dependent defences , we profiled the expression of the SA-inducible marker gene PR1 at 48 and 72 hr post-inoculation ( hpi ) , which represents a critical time-window for host defence against Hpa ( Koch and Slusarenko , 1990; Soylu and Soylu , 2003 ) .",
"None of the epiRILs showed a statistically significant increase in basal PR1 expression after mock inoculation ( Figure 2a; Figure 1—figure supplement 4a ) , indicating that the resistance is not based on constitutive up-regulation of SA-dependent defence signalling .",
"However , in comparison to the Wt line , epiRILs #71 , #148 , #193 , #229 and #508 showed augmented induction of PR1 at 48 and/or 72 hpi with Hpa ( Figure 2a; Figure 1—figure supplement 4a ) , indicating priming of SA-inducible defences ( Martinez-Medina et al . , 2016 ) .",
"To assess the role of cell wall defence , all lines were analysed for effectiveness of callose deposition , which is a pathogen-inducible defence mechanism that is largely controlled by SA-independent signalling ( Luna et al . , 2011 ) .",
"Compared to the Wt line , all but one epiRIL ( #193 ) showed a statistically significant increase in the proportion of callose-arrested germ tubes ( Figure 2a; Figure 1—figure supplement 4b ) .",
"Hence , the eight most Hpa-resistant epiRILs are primed to activate differentially regulated defence responses , which explains the lack of major costs on growth and compatibility with other types of ( a ) biotic stress resistance in the epiRILs ( Figures 1b and 2a; Figure 1—figure supplements 2–4 ) .",
"The 123 epiRILs analysed for Hpa resistance had been self-pollinated for eight generations in a Wt ( Col-0 ) genetic background since the F1 x Col-0 backcross ( F9 ) ( Johannes et al . , 2009 ) .",
"To examine the transgenerational stability of the resistance phenotype over one more generation , five individuals from the eight most resistant epiRILs and the Wt line ( Figure 1a , Figure 1—figure supplement 3a ) were selected to generate F10 families , which were then tested for Hpa resistance .",
"Comparing distributions of pooled leaves from all five families per line confirmed that each epiRIL maintained a statistically enhanced level of resistance ( Figure 1—figure supplement 5; Pearson’s Chi-squared test , p<0 . 05; top asterisks ) .",
"However , when comparing individual F10 families to the Wt , 2 of the 40 F10 families ( line #71–2 and line #148–2 ) exhibited Wt levels of susceptibility , indicating that they had lost Hpa resistance from the F9 to the F10 generation .",
"Furthermore , four of the eight epiRILs tested ( #71 , #148 , #545 , and #508 ) displayed statistically significant variation in Hpa resistance between the 5 F10 families within the epiRIL ( Figure 1—figure supplement 5; Pearson’s Chi-squared test , p<0 . 05; † symbols ) , suggesting instability of the Hpa resistance .",
"To study the transcriptomic basis of the transgenerational resistance , Wt plants ( line #602 ) and 4 Hpa-resistant epiRILs ( #148 , #193 , #454 and #508 ) , each carrying different combinations of the four epiQTLs , were analysed by RNA sequencing at 48 and 72 hpi ( Figure 2a , bottom panel ) .",
"Principal component analysis ( PCA ) of biologically replicated samples ( n = 3 ) revealed clear separation between all treatment/time-point/epi-genotype combinations ( Figure 2b ) .",
"The first PCA axis explained 31% of the variation in transcript abundance , separating samples from mock- and Hpa-treated plants , whereas the second PCA axis explained 20% of the variation , mostly separating samples from the different lines ( Figure 2b ) .",
"This PCA pattern indicates that the response to Hpa infection had a bigger effect on global gene expression than epi-genotype .",
"Moreover , samples from Hpa-inoculated epiRILs showed relatively little difference between both time-points ( Figure 2b ) , whereas samples from Hpa-inoculated Wt plants at 48 hpi clustered between samples from mock-inoculated Wt plants and samples from Hpa-inoculated Wt plants at 72 hpi .",
"This pattern suggests a difference in the speed and/or intensity of the transcriptional response to Hpa .",
"To explore this possibility further , we performed three-factorial likelihood ratio tests ( q < 0 . 05 ) to select differentially expressed genes between all epigenotype/treatment/time-point combinations .",
"This analysis identified 20 , 569 genes , representing 61% of all annotated RNA-producing genes in the Arabidopsis genome , including transposable elements , non-coding RNA genes and pseudogenes ( Supplementary file 1 dataset S3 ) .",
"Of these , 9364 genes were induced by Hpa at 48 and/or 72 hpi in one or more lines ( Supplementary file 1 dataset S4 ) .",
"Subsequent hierarchical clustering of this gene selection revealed a large cluster of Hpa-inducible transcripts displaying augmented induction in the epiRILs at the relatively early time-point of 48 hr after Hpa inoculation ( Figure 2—figure supplement 1 ) .",
"To characterize further the pathogen-inducible transcriptome of the resistant epiRILs , we selected Hpa-inducible genes showing elevated levels of expression in the epiRILs during Hpa infection .",
"Within this gene selection , we distinguished two expression profiles .",
"The first group of genes had been selected for constitutively enhanced expression in the resistant epiRILs , using the following criteria ( Wald tests , q < 0 . 05 ) :",
"( i ) Hpa-inducible in the Wt ,",
"( ii ) not inducible by Hpa in the epiRIL and",
"( iii ) displaying enhanced accumulation in mock-treated epiRIL that is equal or higher than accumulation in the Hpa-inoculated Wt ( ‘Group 1’; Figure 2—figure supplement 2a ) .",
"The second group of genes had been selected for enhanced Hpa-induced expression in the epiRILs , using the following criteria ( Wald tests , q < 0 . 05 ) :",
"( i ) Hpa-inducible in the Wt ( #602 ) ,",
"( ii ) Hpa-inducible in the epiRIL ( s ) and",
"( iii ) displaying statistically increased accumulation in Hpa-inoculated epiRILs compared to Hpa-inoculated Wt plants ( ‘Group 2’; Figure 2—figure supplement 2a ) .",
"For each epiRIL , we identified more genes in Group 2 than in Group 1 ( Figure 2c; Figure 2—figure supplements 2b , 3 and 4; Supplementary file 1 datasets S5 and S6 ) .",
"This difference was most pronounced at 48 hpi , which represents a critical time-point for host defence against Hpa ( Koch and Slusarenko , 1990; Soylu and Soylu , 2003 ) .",
"Analysis of a statistical interaction between epi-genotype x Hpa treatment revealed that >92% of all genes in Group 2 are significant for this interaction term ( Supplementary file 1 dataset S7 ) , indicating a constitutively primed expression pattern .",
"Visualisation of the expression profiles in heatmaps confirmed this notion , showing that the induction of Group 2 genes by Hpa is strongly augmented in the resistant epiRILs compared to the Wt line ( Figure 2c; Figure 2—figure supplement 4 ) , which is consistent with the definition of plant defence priming ( Martinez-Medina et al . , 2016 ) .",
"To examine the functional contributions of the Hpa-inducible genes in Groups 1 and 2 , we employed gene ontology ( GO ) term enrichment analysis .",
"After exclusion of redundant GO terms ( Jantzen et al . , 2011 ) , we identified 469 GO terms , for which one or more of the sets showed statistically significant enrichment .",
"Group 2 genes at 48 hpi displayed dramatically enhanced GO term enrichment compared to all other sets , which was obvious for all epiRILs ( Figure 2d ) .",
"This enrichment was particularly pronounced for 111 GO terms relating to SA-dependent and SA-independent defence mechanisms ( Supplementary file 1 dataset S8 ) , which supports our phenotypic characterisation of SA-dependent and SA-independent defence markers ( Figure 1—figure supplement 4 ) .",
"Collectively , these results suggest that the quantitative resistance of the epiRILs is based on priming of Hpa-inducible defence genes .",
"Interestingly , compared to the other gene selections , a relatively large proportion of defence-related genes in Group 2 at 48 hpi was shared between all four epiRILs ( Figure 2—figure supplement 2b ) , pointing to relatively high similarity in the augmented immune response of the epiRILs .",
"Furthermore , only 5% of the genes in the Group 1% and 6 . 5% of the genes in Group 2 are physically located within the borders of the epiQTL intervals ( LOD drop-off = 2 ) .",
"The frequency of Group 1 and 2 genes relative to all other genes was significantly lower for the epiQTL regions compared to the entire Arabidopsis genome ( 14 . 6%; Pearson’s Chi-squared test , p<0 . 05 ) .",
"Thus , the majority of Hpa-inducible Group 1 and 2 genes showing enhanced expression in the more resistant epiRILs are ( trans- ) regulated by DNA methylation at the four epiQTLs .",
"Although 92% of all genes in Group 2 were located outside the physical borders of the four epiQTL intervals ( LOD-drop-off=2 ) , we hypothesized that a small set of defence regulatory genes inside the epiQTL regions are directly ( cis- ) regulated by DNA methylation to mediate augmented levels of defence in response to Hpa infection .",
"Since the Group 2 genes were strongly enriched with defence-related GO terms ( Figure 2d ) , we examined whether their augmented expression during Hpa infection is associated with the hypomethylated ddm1-2 haplotype .",
"To this end , we calculated for each gene in Group 2 the ratio of normalized transcript abundance between Hpa-inoculated epiRIL and the Hpa-inoculated Wt line , which is proportional to their level of augmented expression during Hpa infection .",
"Hierarchical clustering of these ratios enabled us to select for genes that exclusively show augmented expression when associated with the hypomethylated ddm1-2 haplotype of the corresponding epiQTL ( Figure 3a; Figure 3—figure supplement 1a ) .",
"The expression ratios of 279 epiQTL-localised genes did not correlate with the ddm1-2 haplotype ( Figure 3a , cluster II; Figure 3—figure supplement 1a; Supplementary file 1 dataset S9 ) , indicating that DNA methylation does not cis-regulate their augmented Hpa-inducible expression .",
"By contrast , 73 epiQTL-localised genes only showed augmented expression when associated with the hypomethylated ddm1-2 haplotype ( Figure 3a , cluster I; Figure 3—figure supplement 1a; Supplementary file 1 dataset S10 ) .",
"To confirm the hypomethylated status of these genes , we performed comprehensive bisulfite sequencing analysis of DNA methylation for the four epiRILs and the Wt line .",
"DMR analysis of the gene body ( GB ) , 2 kb promoter region ( P ) and 1 kb downstream ( D ) regions confirmed that the levels of augmented gene expression of the 279 genes in cluster II do not correlate positively with the extent of DNA hypomethylation ( Figure 3b , Figure 3—figure supplement 1b ) .",
"This notion was confirmed by linear regression analysis between the augmented expression ratio ( 48 hpi ) and the average level of DNA hypomethylation ( Figure 3—figure supplement 2 ) , indicating that the 279 genes in cluster II are regulated indirectly ( in trans ) by DNA methylation .",
"By contrast , the 73 epiQTL-based genes in cluster I showed a positive correlation between augmented expression ratio ( 48 hpi ) and DNA hypomethylation , which was statistically significant for each epiQTL ( p<0 . 05; Figure 3—figure supplement 2 ) .",
"These results indicate that the 73 genes in cluster I are regulated locally ( in cis ) by DNA methylation .",
"Nearly all cis-regulated genes in cluster I showed a TE-like pattern of DNA methylation in the Wt ( teM; methylation at CG , CHG and CHH contexts ) , whereas most cluster II genes showed either no methylation or a pattern of gene-body methylation in the Wt ( gbM; methylation at CG only; Figure 3b and Figure 3—figure supplement 1b ) .",
"Furthermore , dividing hypomethylation at gene bodies of Group 2 genes by type of DNA methylation ( i . e . either teM or gbM ) and plotting these values against augmented expression ratio revealed a statistically significant correlation between expression ratio and reduced teM ( p=1 , 06e−8; Figure 3—figure supplement 3 ) , whereas no such correlation was found for reduced gbM ( p=0 . 66; Figure 3—figure supplement 3 ) .",
"These results support the growing notion that reduced teM increases gene expression , whereas changes in gbM have no direct influence on gene expression ( Bewick et al . , 2017 ) .",
"The majority of in cis-regulated genes in cluster I genes were annotated as TEs , such as DNA transposons of the CACTA family , retrotransposons of the GYPSY or COPIA families , or TE-related genes , encoding transposases or enzymes necessary for TE function ( Supplementary file 1 dataset S10 ) .",
"Only six genes were annotated as protein-coding genes , of which two shared homology to known protein-encoding genes ( At2G07240 , cysteine-type peptidase; At2G07750 , RNA helicase ) .",
"However , none of these two genes has previously been associated with plant defence .",
"Furthermore , analysis of the genomic context of the six protein-coding genes revealed the presence of overlapping and/or nearby TEs ( Figure 3—figure supplement 4 ) , suggesting that their correlation between augmented expression and DNA hypomethylation is determined by association with TEs .",
"Since TE-encoded proteins have no antimicrobial activity or direct defence regulatory function , our results suggest that global defence gene priming by hypomethylated epiQTLs is not based on cis-regulation of defence regulatory genes , but rather on alternative trans-acting mechanisms by DNA methylation of the TE-rich epiQTL ."
],
[
"By screening the Col-0 x ddm1-2 epiRIL population for leaf colonisation by the downy mildew pathogen Hpa , we have identified four epiQTLs that provide quantitative disease resistance ( Figure 1b ) .",
"The combined contribution of all 4 DMR peak markers was estimated at 60% of the total variation ( Figure 1d ) , which is higher than previously reported variation in developmental plant traits for this population ( Latzel et al . , 2013; Cortijo et al . , 2014; Kooke et al . , 2015 ) .",
"It was previously shown that half of all stably inherited DMRs in the Col-0 x ddm1-2 epiRILs also occur in natural Arabidopsis accessions ( Latzel et al . , 2013; Schmitz et al . , 2013 ) .",
"Considering that the epiRIL population includes heritable variation in a range of ecologically important plant traits , including flowering , root growth , nutrient plasticity and ( a ) biotic stress resistance ( Latzel et al . , 2013; Cortijo et al . , 2014; Kooke et al . , 2015 ) , it is tempting to speculate that variation in DDM1-dependent DNA methylation contributes to natural variation and environmental adaptation of Arabidopsis .",
"Indeed , the phenotypic diversity in the Col-0 x ddm1-2 epiRIL population closely resembles that of natural Arabidopsis accessions ( Roux et al . , 2011; Mauch-Mani et al . , 2017 ) .",
"Furthermore , independent studies have shown that high levels of enduring ( a ) biotic stress can trigger transgenerational acquired resistance ( TAR ) in Arabidopsis ( Luna et al . , 2012; Wibowo et al . , 2016; Rasmann et al . , 2012 ) .",
"Interestingly , repeated inoculation of 2- to 5 weeks old Arabidopsis seedlings with the hemi-biotrophic leaf pathogen Pseudomonas syringae pv .",
"tomato causes TAR , which is associated with reduced transcription of DDM1 gene in local leaves that is maintained in the apical meristem of paternal plants ( Furci and Ton , unpublished results ) .",
"To what extent this this prolonged repression in DDM1 gene transcription causes heritable reduction in DNA methylation at the epiQTLs requires further study .",
"Aller et al . ( 2018 ) have recently used the same Col-0 x ddm1-2 epiRIL population to map the contribution of heritable variation in DNA methylation to the production of defence-related glucosinolate metabolites ( Aller et al . , 2018 ) .",
"Interestingly , the resistance epiQTL on chromosome I from our study partially overlaps with an epiQTL that influences basal production of the aliphatic glucosinolate 3-methylthiopropyl ( 3MTP ) ( Aller et al . , 2018 ) .",
"Glucosinolates contribute to defence against both herbivores and microbes ( Ishida et al . , 2014 ) .",
"Moreover , myrosinase-dependent breakdown products of indole-derived 4-methoxy-indol-3-ylmethylglucosinolate have been linked to the regulation of callose-mediated cell wall defence in Arabidopsis ( Clay et al . , 2009; Bednarek et al . , 2009 ) .",
"However , the 3MTP-controlling epiQTL identified by Aller et al . ( 2018 ) was relatively weak compared to the epiQTL controlling Hpa resistance ( Figure 1b ) , indicating that its contribution to Hpa resistance would at most be marginal .",
"Furthermore , our transcriptome analysis revealed that the largest variation in gene expression between epiRILs and the Wt line comes from the transcriptional response to Hpa , rather than differences in basal gene expression ( Figure 2b–c ) .",
"Moreover , the genes in Group 2 , which displayed enhanced Hpa-induced expression in the resistant epiRILs at the critical early time-point of 48 hpi , were strongly enriched with defence-related GO terms ( Figure 2d ) .",
"The majority of these Group 2 genes showed a statistically significant interaction between epi-genotype and Hpa treatment ( Supplementary file 1 dataset S7 ) , indicating that these epiRILs were primed to activate defence-related genes .",
"This notion was supported by the actual expression profiles of Group 2 genes ( Figure 2c; Figure 2—figure supplement 4 ) , as well as the defence phenotypes of the eight most resistant epiRILs in the population ( Figure 2a; Figure 1—figure supplement 4 ) .",
"Furthermore , our epiRIL screen for growth phenotypes demonstrated that the resistance-controlling epiQTLs do not have a major impacts on plant growth ( Figure 1b ) , which is consistent with previous findings that defence priming is a low-cost defence strategy ( van Hulten et al . , 2006 ) .",
"While we cannot exclude other mechanisms , these independent lines of evidence collectively indicate that genome-wide priming of defence genes is the most plausible mechanism by which the epiQTLs mediate quantitative disease resistance in the population .",
"Over recent years , various studies have established a link between DNA hypomethylation and plant immune priming ( Espinas et al . , 2016; Conrath et al . , 2015; López Sánchez et al . , 2016 ) .",
"However , causal evidence that heritable regions of reduced DNA methylation mediate transgenerational disease resistance is lacking .",
"Our study has shown that heritable regions of hypomethylated DNA are sufficient to mediate resistance in a genetic Wt background .",
"Furthermore , our study is the first to link phenotypic and epigenetic variation of selected epiRILs to profiles of global gene expression , revealing that epigenetically controlled resistance is associated with genome-wide priming of defence-related genes ( Figure 2b–d; Figure 2—figure supplement 1; Figure 2—figure supplement 4 ) .",
"The majority of these pathogenesis-related genes showed augmented induction at 48 hpi ( Figure 2c ) , which represents a critical early time-point in the interaction between Arabidopsis and Hpa , during which hyphae from germinating spores start to penetrate the epidermal cell layer and invade the mesophyll ( Koch and Slusarenko , 1990; Soylu and Soylu , 2003 ) .",
"Notably , this set of primed genes was substantially more enriched in SA-dependent and SA-independent defence GO terms than the set of Hpa-inducible genes that were constitutively up-regulated in Hpa-resistant epiRILs ( Figure 2d ) , corroborating the analysis of phenotypical defence markers ( Figure 2a; Figure 1—figure supplement 4 ) .",
"DNA methylation of TEs has been reported to cis-regulate expression of nearby genes in Arabidopsis ( Soppe et al . , 2000; Saze and Kakutani , 2007; Kinoshita et al . , 2007; Lei et al . , 2015; Williams et al . , 2015 ) .",
"By contrast , our study did not find evidence that DNA methylation in the epiQTLs cis-regulates the responsiveness of nearby of defence genes .",
"Firstly , the majority of primed defence genes in the Hpa-resistant epiRILs were located outside the epiQTL intervals ( 92% ) .",
"Secondly , of all primed genes within the epiQTLs , only 73 showed augmented induction that coincided with DNA hypomethylation ( Figure 3a; Figure 3—figure supplement 1; Figure 3—figure supplement 2; Supplementary file 1 dataset S10 ) .",
"Of these , 67 encoded TEs or TE-related genes , while the six protein-encoding genes were closely associated with one or more TEs and did not have functions related plant defence ( Figure 3a; Figure 3—figure supplement 1; Supplementary file 1 dataset S10 ) .",
"Since TEs do not encode defence signalling proteins , we propose that DNA hypomethylation at the TE-rich epiQTLs mediates augmented induction of defence genes across the genome via trans-acting mechanisms .",
"A recent transcriptome study of Hpa-infected Arabidopsis identified 166 defence-related genes that were primed in the hypomethylated nrpe1-11 mutant and/or repressed in hyper-methylated ros1-4 mutant ( López Sánchez et al . , 2016 ) .",
"The majority of these defence genes were not targeted by NRPE1- and/or ROS1-dependent DNA ( de ) methylation , indicating that their responsiveness is trans-regulated by DNA methylation .",
"Although NRPE1 and ROS1 target partially different genomic loci than DDM120 , this study supports our hypothesis that DNA methylation controls global defence gene responsiveness via trans-acting mechanisms .",
"There are various mechanisms by which DNA methylation could trans-regulate defence gene expression .",
"It is possible that transcribed TEs in the hypomethylated epiQTLs generate 21-22nt or 24nt small RNAs ( sRNAs ) that influence distant heterochromatin formation through via RDR6- and DCL3-dependent RdDM pathways ( Panda et al . , 2016 ) .",
"Support for trans-regulation by sRNAs came from a recent study , which reported that induction and subsequent re-silencing of pericentromeric TEs in Arabidopsis upon Pseudomonas syringae infection is accompanied with accumulation of RdDM-related sRNAs that are complementary to TEs and distal defence genes .",
"Interestingly , while the accumulation of these sRNAs coincided with re-silencing of the complementary TEs , the complementary defence genes remained expressed in the infected tissues ( Cambiagno et al . , 2018 ) .",
"These findings are supported by another recent study , which demonstrated that AGO1-associated small RNAs can trans-activate distant defence gene expression through interaction with the SWI/SNF chromatin remodelling complex ( Liu et al . , 2018 ) .",
"Apart from sRNAs , it is also possible that long intergenic noncoding RNAs ( lincRNAs ) from the hypomethylated epiQTLs regulate pathogen-induced expression of distant defence genes .",
"A recent study revealed that pericentromeric TEs of Arabidopsis can produce DDM1-dependent lincRNAs that are increased by abiotic stress exposure ( Wang et al . , 2017a ) .",
"Since lincRNAs can promote euchromatin and heterochromatin formation at distant genomic loci ( Heo et al . , 2013; Quinodoz and Guttman , 2014 ) , hypomethylated TEs within the epiQTLs could generate priming-inducing lincRNAs .",
"While knowledge about lincRNAs in plants remains limited , like sRNAs , their activity depends on sequence complementary with target loci ( Vance and Ponting , 2014 ) .",
"Unlike non-coding RNAs , long-range chromatin interactions can trans-regulate gene expression independently of sequence complementarity ( Harmston and Lenhard , 2013; Liu , 2016; Weber et al . , 2016; Wang et al . , 2017b ) .",
"Previous high-throughput chromosome conformation capture ( Hi-C ) analysis revealed that the ddm1-2 mutation has a profound impact on long-range chromatin interactions within and beyond the pericentromeric regions ( Feng et al . , 2014 ) .",
"Projection of these DDM1-dependent interactions onto the Arabidopsis genome shows extensive coverage of the resistance epiQTLs identified in this study ( Figure 3—figure supplement 5 ) .",
"Whether these long-range interactions contribute to trans-regulation of defence gene priming would require further study , including a fully replicated Hi-C analysis of the resistant epiRILs characterised in this study .",
"In conclusion , our study has shown that heritable DNA hypomethylation at selected pericentromeric regions controls quantitative disease resistance in Arabidopsis , which is associated with genome-wide priming of defence-related genes .",
"This transgenerational resistance is not associated with reductions in plant growth ( Figure 1b ) , nor does it negatively affect resistance to other types of ( a ) biotic stresses tested in this study ( Figure 1—figure supplement 3 ) .",
"However , whether this form of epigenetically controlled resistance can be exploited in crops depends on a variety of factors , including the stability of the disease resistance and potential non-target effects .",
"For instance , our experiments with Arabidopsis revealed that the resistance has limited stability and can erode over one more generation in some epiRILs ( Figure 1—figure supplement 5 ) .",
"Furthermore , the genomes of most crop species contain substantially higher numbers of TEs , rendering predictions about the applicability and potentially undesirable side effects on growth and seed production uncertain .",
"Future research will have to point out whether introgression of hypomethylated pericentromeric loci into the background of elite crop varieties allows for selection of meta-stable quantitative disease resistance without side-effects on agronomically important traits ."
],
[
"Epigenetic recombinant inbred lines ( epiRILs ) seeds of Arabidopsis ( Arabidopsis thaliana , accession Col-0 ) were purchased from Versailles Arabidopsis Stock Centre , INRA , France ( http://publiclines . versailles . inra . fr/epirils/index ) .",
"The epiRIL screen included siblings of the F4 ddm1-2 parental plant of the epiRIL population ( IBENS , France ) .",
"Arabidopsis seeds were stratified in water at 4°C in the dark for 3-5 days .",
"For pathogen bioassays , seeds were sown in a sand:compost mixture ( 1:3 ) and grown at short-day conditions for 3 weeks ( 8 . 5 hr light/15 . 5 hr dark , 21°C , 80% relative humidity , ~125 µmol s−1 m−1 light intensity ) .",
"To test transgenerational inheritance and stability of Hpa resistance in the eight most resistant epiRILs ( Figure 1—figure supplement 5 ) , five individual F9 plants were cultivated for 4 weeks at short-day conditions and then moved to long-day conditions to initiate flowering ( 16 hr light/8 hr dark , 21°C , 80% relative humidity , ~125 µmol s−1 m−1 light intensity ) .",
"Seeds of the 40 F10 families were collected for analysis of Hpa resistance ( see below ) .",
"Three-week-old seedlings were spray-inoculated with a suspension of asexual conidia from Hyaloperonospora arabidopsidis strain WACO9 ( Hpa ) at a density of 105 spores/ml .",
"Hpa colonizsation was quantified at 6 days post-inoculation ( dpi ) by microscopic scoring of leaves , as described previously ( López Sánchez et al . , 2016 ) .",
"Briefly , trypan blue-stained leaves were analysed with a stereomicroscope ( LAB-30 , Optika Microscopes ) and assigned to 4 Hpa colonisation classes: class I , no hyphal colonisation; class II , ≤50% leaf area colonized by pathogen hyphae without formation of conidiophores; class III , ≤75% leaf area colonized by hyphae , presence of conidiophores; class IV , >75% leaf area colonized by the pathogen , abundant conidiophores and sexual oospores ( Figure 1—figure supplement 1 ) .",
"At least 100 leaves per ( epi ) genotype were analysed , not including the cotyledons .",
"Statistically significant differences in frequency distribution of Hpa colonisation classes between lines were determined by Pearson’s Chi-squared tests , using R ( v . 3 . 5 . 1 ) .",
"Growth analysis of the epiRIL population was based on digital photos ( Canon 500D , 15MP ) of 3-week-old plants , which were taken on the day of Hpa inoculation .",
"Digital image analysis of total green leaf area ( GLA ) was performed using Adobe Photoshop 6 . 0 .",
"Green pixels corresponding to GLA were selected and converted into mm2 after colour range adjustment , using the magic wand tool .",
"Mapping of epiQTLs was performed using the ‘scanone’ function of the R/qtl package for R ( Broman et al . , 2003 ) ( Haley-Knott regression , step size: 2 cM ) , combining experimental phenotypical data with the recombination map of differentially methylated regions ( DMR ) generated previously ( Colomé-Tatché et al . , 2012 ) .",
"For analysis of Hpa resistance , the categorical scoring of Hpa resistance was first converted into a numeric resistance index ( RI ) , using the following formula:RI= ( fclassI∗4 ) + ( fclassII∗3 ) + ( fclassIII∗2 ) + ( fclassIV∗1 ) where f = relative frequency of Hpa colonisation class of each line , multiplied by an arbitrary weight value ranging from four for the most resistant category ( class I ) to one for most susceptible category ( class IV ) .",
"Mapping of epiQTLs controlling plant growth was based on average GLA values of each line before Hpa infection .",
"A logarithm of odds ( LOD ) threshold of significance for each trait was determined on the basis of 1000 permutations for each dataset ( α = 0 . 05 ) .",
"The proportion of phenotypic variance R2 ( G ) explained by the DMR markers with the highest LOD score ( peak markers ) of all four epiQTLs was calculated with the following formula ( Cortijo et al . , 2014 ) :R2G=1-n-1n-k+1∑in ( yi-[β^0+∑jkβjgij] ) 2∑inyi-y-2 where n = number of lines analysed , k = number of DMR markers tested; β0 = intercept of the multiple regression model; βj= QTL effect for each QTL j ( slopes for each marker in the multiple regression model ) ; gij = ( epi ) genotype of the jth marker for each individual i ( coded as ‘1’ for ddm1-2 epialleles and ‘-1’ for WT epialleles ) ; yi = phenotypic value of individual i; y- = mean of phenotypic values .",
"The contribution of each individual QTL j ( R2",
"( g ) ) was calculated , using the following formula:R2g=1-n-1n-k+1β^j2∑ingij-g-j2∑inyi-y-2 , as described by ( Cortijo et al . , 2014 ) , where n= number of lines analysed , k = number of markers tested; βj = QTL effect for each QTLj ( slopes for each peak marker in the multiple regression model ) ; gij = ( epi ) genotype of the jth marker for each individual i ( coded as ‘1’ for ddm1-2 epialleles and ‘-1’ for WT epialleles ) ; g-j = average of the ( epi ) genotypes values for the jth marker .",
"Covariance was calculated by subtracting the sum of the individual contributions of each QTL j on phenotypical variance ( i . e . R2 ( gQTL1 ) + R2 ( gQTL2 ) + R2 ( gQTL4 ) + R2 ( gQTL5 ) ) from the phenotypical variance explained by the full model ( i . e . R2 ( G ) ) .",
"TE-tracker software was used to interrogate available Illumina whole-genome sequencing data from 122 epiRILs for the presence of >2 shared transposition evens ( STEs ) within the epiQTLs intervals ( Gilly et al . , 2014 ) .",
"STEs were analysed for statistically significant linkage with resistance phenotypes ( RIs ) , using the same linear regression model as described above for DMR linkage analysis .",
"Plectosphaerella cucumerina ( Pc , strain BMM ( Ton and Mauch-Mani , 2004 ) ) was grown from frozen agar plugs ( −80° C ) on potato dextrose agar ( PDA; Difco , UK ) .",
"Inoculated plates were maintained at room temperature in the dark for at least 2 weeks .",
"Spores were gently scraped from water-inundated plates , after which spore densities were adjusted to 106 spores/ml using a hemocytometer ( Improved Neubauer , Hawksley , UK ) .",
"Four fully expanded leaves of similar age from 5-week-old plants were inoculated by applying 5 µl droplets , minimising variability due to age-related resistance .",
"After inoculation , plants were kept at 100% RH until scoring of lesion diameters .",
"Average lesion diameters at nine dpi were based on four leaves per plant from 12 plants per ( epi ) genotype ( n = 40–48 ) , using a precision caliper ( Traceable , Fischer Scientific ) .",
"Statistically significant differences in necrotic lesions diameter ( asterisks ) were quantified by two-tailed Student’s t-test ( p<0 . 05 ) in pairwise comparisons with Wt line ( #602 ) , using R ( v3 . 5 . 1 ) .",
"Seeds were sterilised by exposure for 4 hr",
"( h ) to chlorine vapours from a 200 ml bleach solution containing 10% v/v hydrochloric acid ( 37% v/v HCl , Fischer Scientific , 7732-18-5 ) .",
"Seeds were air-dried for 1 hr in a sterile laminar flow cabinet and plated on half strength MS plates ( Duchefa , M0221;+0 . 05% w/v MES , +1% w/v sucrose , pH 5 . 7 ) , containing increasing concentrations of NaCl ( 0 mM , 50 mM , 75 mM and 100 mM; Fischer Scientific , 7647-14-5 ) .",
"Plates were stratified for 4 days in the dark at 4°C and transferred to short-day growth conditions ( 8 . 5 hr light/15 . 5 hr dark , 21°C , 80% RH , light intensity 100–140 µmol s−1 m−1 ) .",
"Salt tolerance was expressed as percentage of seeds producing fully expanded cotyledons by 6 days after stratification .",
"Germination percentages of epi-genotypes were calculated from >50 seeds per treatment .",
"Statistically significant differences in germination rates ( asterisks ) were quantified by Fisher’s exact test ( p<0 . 05 ) in pairwise comparisons with Wt line ( #602 ) at each salt concentration , using R ( v3 . 5 . 1 ) .",
"Seedlings were collected at three dpi and cleared for >24 hr in 100% ethanol .",
"One day prior to analysis , samples were incubated for 30 min in 0 . 07 M phosphate buffer ( pH 9 ) , followed by 15 min incubation in a 4:1 mixture ( v/v ) of 0 . 05% w/v aniline blue ( Sigma-Aldrich , 415049 ) in 0 . 07M phosphate buffer ( pH 9 ) and 0 . 025% w/v calcofluor white ( Fluorescent brightener 28 , Sigma-Aldrich , F3543 ) in 0 . 1M Tris-HCL ( pH 7 . 5 ) .",
"After initial staining , samples were incubated overnight in 0 . 5% w/v aniline blue ( Sigma-Aldrich , 415049 ) in 0 . 07M phosphate buffer ( pH 9 ) and scored with an epifluorescence microscope ( Olympus BX 51 ) fitted with blue filter ( XF02-2; excitation 330 nm , emission 400 nm ) .",
"Germinated conidia ( germ tubes ) were divided between in two classes: non-arrested and arrested by callose .",
"In each assay , 10 leaves from different plants for each ( epi ) genotype were analysed , amounting to >150 conidia-callose interactions .",
"Statistically significant differences in resistance efficiency of callose ( asterisks ) were analysed using Pearson’s Chi-squared tests ( p<0 . 05 ) in pairwise comparisons with Wt line ( #602 ) , using R ( v3 . 5 . 1 ) .",
"Three biologically replicated samples for each genotype/treatment/time-point combination were collected at 48 and 72 hpi , each consisting of six to 12 leaves collected from different plants per pot .",
"Samples were snap-frozen in liquid nitrogen and ground to a fine powder , using a tissue lyser ( QIAGEN TissueLyser ) .",
"Total RNA was extracted using a guanidinium thiocyanate-phenol-chloroform extraction isolation protocol .",
"Frozen powder was vortexed for 30 s in 1 ml Extraction buffer: 1M guanidine thiocyanate ( Amresco , 0380 ) , 1M ammonium thiocyanate ( Sigma-Aldrich , 1762-95-4 ) , 0 . 1M sodium acetate ( Fisher Scientific , 127-09-3 ) , 38% v/v AquaPhenol ( MP Biomedicals , 108-95-2 ) and 5% v/v glycerol ( Fisher Scientific , 56-81-5 ) .",
"Samples were incubated at room temperature ( RT ) for 1 min and then centrifuged for 5 min at 16 , 500 g .",
"The supernatant was then transferred to a new tube , mixed with 200 μl chloroform and vortexed for 10–15 s .",
"After centrifuging for 5 min ( 16 , 500",
"g ) , the aqueous phase was transferred to new tubes , gently mixed by inversion with 350 μl 0 . 8M sodium citrate ( Sigma-Aldrich , 6132-04-3 ) and 350 μl isopropanol ( Fischer Chemicals , 67-63-0 ) and left at RT for 10 min for RNA precipitation .",
"Samples were centrifuged for 15 min at 16 , 500 g ( 4°C ) , after which pellets were washed twice in 1 ml 70% ethanol , centrifuged at 16 , 500 g for 1 min , and air-dried before dissolving in 50 μl nuclease-free water .",
"Total RNA was quantified , using a Nanodrop 8000 Spectrophotometer ( Thermo Scientific ) .",
"RNA extracts were treated with DNaseI , using the RQ1 RNase-Free DNase kit ( Promega , M6101 ) .",
"First-strand cDNA synthesis was performed from 1 μg RNA , using SuperScript III Reverse Transcriptase ( Invitrogen , 18080093 ) according to the supplier’s recommendations .",
"The qPCR reactions were carried out with a Rotor-Gene Q real-time PCR cycler ( Qiagen ) and the Rotor-Gene SYBR Green PCR Kit ( Qiagen , 204074 ) .",
"Relative PR1 gene expression was calculated , using Livak’s ΔΔCT method ( Livak and Schmittgen , 2001 ) with correction for average PCR efficiencies for each primer pair across experiment samples .",
"Gene expression was normalised against average expression values of At1G13440 ( GAPDH ) , At5G25760 ( UBC ) and At2G28390 ( SAND family protein ) ( Czechowski et al . , 2005 ) .",
"Reactions were performed using previously described primer sequences ( López Sánchez et al . , 2016 ) .",
"Statistically significant differences in relative expression ( asterisks ) were quantified by two-tailed Student’s t-test ( p<0 . 05 ) in pairwise comparisons with Hpa-treated Wt line ( #602 ) .",
"Samples for RNA sequencing were collected at 48 and 72 hpi of 3-week-old plants .",
"Every epi-genotype/treatment/time-point combination was based on three biologically replicated samples , each consisting of 6–12 shoots from different plants .",
"Initial RNA extraction was performed as described for RT-qPCR reactions .",
"Prior to library preparation , RNA concentration and integrity were measured , using 2100 Bioanalyzer ( Agilent ) with provided reagents kits and according to manufacturer’s instructions .",
"All RNA samples had RNA integrity numbers ( RIN ) >7 . 5 .",
"Sequencing libraries were prepared from total RNA , using the TruSeq Stranded Total RNA kit and Ribo-Zero Plant leaf kit ( Illumina , RS-122–2401 ) , according to the manufacturer’s instructions .",
"Sequencing runs were performed on a HiSeq1500 platform ( Illumina ) , generating paired-end reads of 125 bp and an average quality score ( Q30 ) >93% .",
"Each sample generated around 35 million paired reads .",
"Read quality was assessed by FastQC software ( Andrews , 2010 ) .",
"Read length and distribution were optimized and adapter sequences were trimmed , using Trimmomatic software ( Bolger et al . , 2014 ) .",
"Reads were aligned and mapped to the Arabidopsis genome ( TAIR10 annotation ) , using splice site-guided HISAT2 alignment software ( John Hopkins University , second iteration of ( Kim et al . , 2015 ) ) .",
"For all samples , more than 95% of reads could successfully be mapped once or more onto the Arabidopsis genome .",
"Number of reads per gene were quantified with the Python package HTseq ( Anders et al . , 2015 ) .",
"Differential expression analysis was performed using the DESeq2 R package , which applies a negative binomial generalized linear model to estimate mean and dispersion of gene read counts from the average expression strength between samples ( Love et al . , 2014 ) .",
"Prior to principal component analysis ( PCA ) by the plotPCA function , gene read counts were subjected to regularized logarithmic transformation , using the rlog function ( Love et al . , 2014 ) .",
"Likelihood ratio tests of variance within a three-factorial linear model for epigenotype , treatment , time-point and interactions thereof were used to identify genes showing differences in expression across one or more factors ( Love et al . , 2014 ) .",
"Differentially expressed genes ( DEGs ) were subjected to hierarchical clustering ( Ward method ) and presented as a heat map , using the pheatmap R package ( Kolde , 2015 ) .",
"For each gene , rlog-normalized read counts of each sample were subtracted from the mean of all samples , and divided by the standard deviation to facilitate heatmap visualisation ( z-score ) .",
"To identify DEGs between two treatment/time-point/epi-genotype combinations , pair-wise comparisons ( Wald test; q < 0 . 05 ) were performed with the DEGs selection obtained by the lrt test , using the selection criteria illustrated in Figure 2—figure supplement 2a .",
"All Hpa-inducible genes in the Wt and/or epiRILs were selected for elevated expression in the more resistant epiRILs during Hpa infection .",
"Subsequently , these genes were divided between two groups based on their expression profile .",
"Group 1 genes were selected for constitutively enhanced expression in the epiRIL ( s ) relative to the Wt ( Figure 2—figure supplements 2 and 3 ) ; Group 2 genes were selected for enhanced levels of Hpa-induced expression in the epiRIL ( s ) relative to the Wt ( Figure 2—figure supplements 2 and 4 ) .",
"To determine the number of Group 2 genes that show a statistically significant interaction between epigenotype x Hpa treatment ( all 16 , 009 genes significant for this interaction were selected from the three-factorial linear model , using the contrast function , and cross-referenced against Group 2 genes .",
"Gene ontology ( GO ) term enrichment analysis was performed with the Plant GSEA toolkit ( Yi et al . , 2013 ) .",
"GO terms were checked for significant enrichment against the whole genome background , using a hypergeometric test and Benjamini-Hochberg false discovery rate correction ( q < 0 . 05 ) .",
"Lists of enriched GO terms in each treatment were analysed by the GO Trimming 2 . 0 algorithm ( Jantzen et al . , 2011 ) to remove redundancy of terms , applying a soft trimming threshold of 0 . 40 .",
"The output list from GO Trimming 2 . 0 was run through GOSlim Viewer ( AgBase ) to reduce GO terms according to GO slim ontologies ( GO consortium ) .",
"Enrichment was quantified as the percentage of GO term-annotated genes within a certain selection relative to the total number of Arabidopsis genes in that GO term .",
"For each line , three independent biological replicates were collected , consisting of pooled leaves from six plants of the same developmental stage .",
"High-quality genomic DNA was extracted from leaves of 5-week-old plants , using the GenElute Plant Genomic DNA Miniprep Kit ( Sigma-Aldrich ) .",
"Bisulfite sequencing was performed by GATC Biotech ( UK ) .",
"After quality trimming of read sequences , adapter sequences were removed , and reads were filtered by Cutadapt ( version 1 . 9; Pair end-mode; phred score = 20 , min . length = 40 ) .",
"Reads were mapped to an index genome , using of BS-Seeker2 ( version 2 . 0 . 10 , mismatch = 0 . 05 , maximum insert size = 1000 bp ) .",
"Bowtie2 ( version 2 . 2 . 2 ) was used for alignment of reads , as described previously ( Lauss et al . , 2018 ) .",
"Differential methylation for promoter regions ( −2 kb ) , gene bodies , and downstream regions ( +1 kb ) relative to the Wt was called using methylkit ( version 1 . 0 . 0; minimum coverage = 5 x , q = 0 . 05 ) .",
"Differentially methylated states were visualised as a heat map , using the ‘pheatmap’ R package ( version 1 . 0 . 8 ) ( Kolde , 2015 ) .",
"To differentiate Wt methylation states of all epiQTL-based genes in Group 2 ( see above ) , gene bodies of all nuclear genes were categorised between un-methylated , gene body methylated ( gbM; CG context only ) or TE-like methylated ( teM; CHG and/or CHH with or without CG ) .",
"For each gene containing 20 or more cytosines , methylated and un-methylated cytosine base calls in each context were extracted from the sequence read alignments .",
"Positions with less than 4x coverage were ignored .",
"Methylation patterns were categorised as TE-like if methylated read calls relative to un-methylated read calls in CHG and/or CHH contexts showed a statistically significant increase over average methylation rates of all genes across the genome in the respective context , using the ‘binom . test’ function in R ( FDR-adjusted p<0 . 01 ) .",
"The remaining genes were classified either as gbM if the same test revealed a statistically significant increase in CG context , or as un-methylated if no statistically significant increase in DNA methylation could be detected in any sequence context .",
"Correlations between augmented expression ratio of Group 2 genes ( see Transcriptome analysis ) and DNA hypomethylation ( CG ) , were determined by plotting augmented gene ratios at 48 hpi against average hypomethylation compared to Wt ( % ) across promoter region , gene body , and downstream region ( see Methylome analysis ) .",
"To determine which type of DNA hypomethylation correlates with augmented expression in the epiRILs , hypomethylation at gene bodies of Group 2 genes were divided between teM and gbM and plotted against the corresponding expression ratios at 48 hpi .",
"If hypomethylation occurred at CG context only , genes were classified as being reduced in gene body methylation ( gbM ) ; if hypomethylation occurred all three sequence contexts ( CG , CHG , CHH ) , genes were classified as being reduced in TE methylation ( teM ) .",
"Values of gbM hypomethylation were expressed as percentage reduction in GC methylation relative to the Wt; values of teM hypomethylation were expressed as percentage reduction in all sequence contexts .",
"Linear regression analyses were performed using R software ( v . 3 . 5 . 1 ) .",
"HiC sequence libraries SRR1504819 and SRR150482464 were downloaded from NCBI SRA .",
"Sequences were pre-processed and aligned to the TAIR10 Arabidopsis nuclear genome sequence ( Berardini et al . , 2015 ) , using HiCUP ( 0 . 5 . 9 ) ( Wingett et al . , 2015 ) and Bowtie2 ( Langmead and Salzberg , 2012 ) ( 2 . 2 . 6 ) .",
"Alignments were filtered and de-duplicated as part of the processing by HiCUP , before being further processed in HOMER ( Heinz et al . , 2010 ) ( 4 . 9 . 1 ) at 5 kb resolution .",
"Differential interactions were assessed reciprocally , using each sample as background ( analyzeHiC-ped ) .",
"Interactions were determined to be potentially dependent on genotype if the absolute z-score of the primary versus the secondary experiment was more than 1 .",
"Visualisations were generated using Circos ( Krzywinski et al . , 2009 ) ( 0 . 69–5 ) , based on bundled links ( -max_gap 10001 ) .",
"Transcriptome sequencing and bisulfite sequencing reads are available from the European Nucleotide Archive ( ENA ) under accession code PRJEB26953 ."
]
] | [
"Variation in DNA methylation enables plants to inherit traits independently of changes to DNA sequence .",
"Here , we have screened an Arabidopsis population of epigenetic recombinant inbred lines ( epiRILs ) for resistance against Hyaloperonospora arabidopsidis ( Hpa ) .",
"These lines share the same genetic background , but show variation in heritable patterns of DNA methylation .",
"We identified four epigenetic quantitative trait loci ( epiQTLs ) that provide quantitative resistance without reducing plant growth or resistance to other ( a ) biotic stresses .",
"Phenotypic characterisation and RNA-sequencing analysis revealed that Hpa-resistant epiRILs are primed to activate defence responses at the relatively early stages of infection .",
"Collectively , our results show that hypomethylation at selected pericentromeric regions is sufficient to provide quantitative disease resistance , which is associated with genome-wide priming of defence-related genes .",
"Based on comparisons of global gene expression and DNA methylation between the wild-type and resistant epiRILs , we discuss mechanisms by which the pericentromeric epiQTLs could regulate the defence-related transcriptome ."
] | [
"In plants , animals and microbes genetic information is encoded by DNA , which are made up of sequences of building blocks , called nucleotide bases .",
"These sequences can be separated into sections known as genes that each encode specific traits .",
"It was previously thought that only changes to the sequence of bases in a DNA molecule could alter the traits passed on to future generations .",
"However , it has recently become clear that some traits can also be inherited through modifications to the DNA that do not alter its sequence .",
"One such modification is to attach a tag , known as a methyl group , to a nucleotide base known as cytosine .",
"These methyl tags can be added to , or removed from , DNA to create different patterns of methylation .",
"Previous studies have shown that plants whose DNA is less methylated than normal ( ‘hypo-methylated’ ) are more resistant to plant diseases .",
"However , the location and identity of the hypo-methylated DNA regions controlling this resistance remained unknown .",
"To address this problem , Furci , Jain et al . studied how DNA methylation in a small weed known as Arabidopsis thaliana affects how well the plants can resist a disease known as downy mildew .",
"Furci , Jain et al . studied a population of over 100 A . thaliana lines that have the same DNA sequences but different patterns of DNA methylation .",
"The experiments identified four DNA locations that were less methylated in lines with enhanced resistance to downy mildew .",
"Importantly , this form of resistance did not appear to reduce how well the plants grew , or make them less able to resist other diseases or environmental stresses .",
"The results of further experiments suggested that reduced methylation at the four DNA regions prime the plant’s immune system , enabling a faster and stronger activation of a multitude of defence genes across the genome after attack by downy mildew .",
"The next steps following on from this work are to investigate exactly how the four DNA regions with reduced methylation can prime so many different defence genes in the plant .",
"Further research is also needed to determine whether it is possible to breed crop plants with lower levels of methylation at specific DNA locations to improve disease resistance , but without decreasing the amount and quality of food produced ."
] | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"structural biology and molecular biophysics"
] | The isolated voltage sensing domain of the Shaker potassium channel forms a voltage-gated cation channel | elife-18130-v2 | [
[
"Voltage-gated ion channels play a fundamental role in a wide range of physiological processes , including the generation and propagation of electrical signals in excitable cells and muscle tissues , neurotransmitter release in pre-synaptic nerve endings , and maintaining cell homeostasis ( Hille , 1978 ) .",
"They comprise four subunits of each six transmembrane segments ( S1–S6 ) , which are assembled into two functionally distinct parts: a central ion-selective pore formed by the combined S5 and S6 segments of four domains , surrounded by four voltage-sensing domains ( VSDs ) formed by the four helices S1–S4 .",
"S4 is considered the principal component of voltage sensing because it contains positively charged residues that are periodically aligned at every third position .",
"The first four arginines ( R1-R4 ) of S4 sense and interact with the surrounding electric field , and move the S4 outward during depolarization and inward during hyperpolarization in a combination of translation , rotation and tilt ( Faure et al . , 2012; Li et al . , 2014a , 2014b; Vargas et al . , 2012; Yarov-Yarovoy et al . , 2012 ) .",
"These changes are mechanically transmitted to the pore domain via the S4–S5 linker and the C-terminal S6 of the same and neighboring subunit , leading to gating of the central pore ( Batulan et al . , 2010; Wall-Lacelle et al . , 2011; Haddad and Blunck , 2011; Muroi et al . , 2010; Long et al . , 2005b; Catterall , 2010; Bezanilla , 2005; Lu et al . , 2001 , 2002; Labro et al . , 2008 ) .",
"Opening of the pore has been shown to occur in at least two steps ( Del Camino et al . , 2005; Kalstrup and Blunck , 2013 ) .",
"In addition to the coupling in this region , a direct structural and functional interaction between the VSD and pore has been demonstrated ( Lee et al . , 2009a; Li-Smerin et al . , 2000; Petitjean et al . , 2015 ) .",
"The topology and structure of the Kv channels suggest that the VSD and pore domain make up modules that can be adapted to different scenarios ( Blunck and Batulan , 2012; Kim et al . , 2014 ) .",
"Chimeras of VSD of one and pore domain of another protein were able to gate in a voltage-dependent fashion ( Lu et al . , 2001; Arrigoni et al . , 2013 ) .",
"The notion that the VSD acts as an independent module is further supported by proteins where the VSD is linked not to a pore domain but instead to a cytosolic enzymatic domain such as the voltage-sensitive phosphatase of Ciona intestinalis ( Ci-VSP ) .",
"It was possible to carry out structural studies on isolated voltage sensor domains ( Li et al . , 2014a; Chakrapani et al . , 2008; Jiang et al . , 2003 ) , and the VSD of Ci-VSP has been shown to function in the absence of the phosphatase ( Labro et al . , 2012; Murata et al . , 2005 ) .",
"Finally , the voltage-gated proton channels ( Hv ) lack a separate pore domain altogether such that its VSD is directly responsible for proton permeation ( Lee et al . , 2009b; Li et al . , 2015; Ramsey et al . , 2006; Sasaki et al . , 2006 ) .",
"In view of the modular nature of voltage-gated ion channels , VSDs have been considered in structure function studies largely as their own entity with energetic coupling to the pore domain , and they have been directly compared to other VSD-containing proteins such as Ci-VSP and Hv channels .",
"We do not know , however , how possible restraint forces from the pore domain may have obscured conformational changes during VSD activation .",
"Prolonged depolarization has been shown to reconfigure VSDs of numerous voltage-gated proteins into a stable ‘relaxed’ state , resulting in a hyperpolarizing shift of the voltage dependence of both pore closure and return of the VSD to its resting position ( Haddad and Blunck , 2011; Piper et al . , 2003; Tan et al . , 2012; Olcese et al . , 1997; Kuzmenkin et al . , 2004; Bruening-Wright and Larsson , 2007; Villalba-Galea et al . , 2008 ) .",
"Relaxation or mode shift has been shown to be influenced by the state of the pore domain; it was observed to be correlated with slow C-type inactivation ( Olcese et al . , 1997; Cuello et al . , 2010; Männikkö et al . , 2005 ) and pore stabilization in the open state ( Haddad and Blunck , 2011 ) .",
"On the other hand , mode shift also developed in Ci-VSP , which does not possess any pore domain , suggesting relaxation to be an intrinsic property of the VSD ( Labro et al . , 2012; Villalba-Galea et al . , 2008 ) .",
"Recent studies have suggested that the N-terminal tail ( Tan et al . , 2012 ) and S3-S4 linker length ( Priest et al . , 2013 ) may affect the VSD relaxation in hERG and Shaker channels , respectively .",
"However , the conformational changes related to relaxation remain unknown .",
"To gain insights into the gating mechanisms of the Shaker potassium channel VSD as an independent structural and functional unit , we generated a Shaker channel pore deletion mutant , the Shaker isolated voltage sensor domain ( Shaker-iVSD ) .",
"In Shaker-iVSD any conformational changes would not be affected any longer by the energetic load or by structural constraints imposed by the pore domain .",
"Here , we report , for the first time , the gating currents and conformational changes monitored via fluorescence of Shaker-iVSD ."
],
[
"In the pore-deletion mutant Shaker-iVSD , the pore domain ( S5-S6 ) after position I384 and almost the complete C-terminus were removed and a cysteine at position A359 in the S3-S4 linker was introduced to follow conformational changes ( Figure 1a , bottom , for details see Materials and methods ) .",
"Shaker-iVSD mutants were transiently expressed in Xenopus laevis oocytes , and their electrophysiological properties examined using the cut-open oocyte voltage-clamp technique ( Batulan et al . , 2010; Taglialatela et al . , 1992 ) .",
"Because this deletion mutant does no longer contain the ion conducting pore domain , we used gating current measurements and voltage-clamp fluorometry as an indicator for trafficking to the plasma membrane and responsiveness to changes in membrane potential .",
"Shaker-iVSD was site-directed fluorescently labeled by attaching an extrinsic fluorescent probe ( tetramethyl-rhodamine maleimide , TMRM ) to a cysteine introduced at position A359C in the S3–S4 linker just N-terminal to S4 .",
"As a reference , we used Shaker-A359C-W434F ( Shaker-W434F ) , in which a mutation rendering the channel non-conducting ( W434F ) and A359C were introduced into background Shaker H4-IR ( Figure 1a ) . 10 . 7554/eLife . 18130 . 003Figure 1 . Expression of Shaker-iVSD lacking pore domains is sufficient to reconstitute voltage-dependent channel activity .",
"( a ) Top: topology of Shaker K+ channel .",
"Amino acid positions relevant to the present study are highlighted in yellow .",
"Middle: Shaker channels constructs ( Shaker-W434F and Shaker-iVSD ) used in the present study .",
"Thiol-reactive fluorophores are attached to a cysteine at position A359C in the S3–S4 linker .",
"The mutation W434F in the pore region produces an instantly C-type inactivated Shaker channel .",
"Shaker-iVSD is a deletion mutant truncated after the S4 helix .",
"Bottom: Sequence of the C-terminus of Shaker-iVSD",
"( b ) Gating currents recorded from oocytes expressing Shaker-iVSD ( top ) and Shaker-W434F ( bottom ) using cut-open oocyte voltage clamp .",
"Leak , background , and capacitive currents were subtracted from the current traces using a P/4 protocol .",
"Inset shows the current step protocol .",
"Gating currents were elicited by test pulses from −100 mV to +120 mV at a holding potential of −90 mV .",
"Zero level was indicated by the red dashed line .",
"( c and",
"d ) Representative ionic currents recorded by cut-open oocyte voltage-clamp from oocytes expressing Shaker-iVSD and Shaker-W434F .",
"At depolarized ( 0 mV , top ) or hyperpolarized ( −90 mV , bottom ) holding potentials , currents were elicited by voltage pluses at a range between −180 and +140 mV in 10 mV increments without leak substraction .",
"The interval between test pulses was 5 s to allow complete recovery of the channels .",
"An inset on top of current traces shows the corresponding voltage protocols .",
"Zero level was indicated by the red dashed line .",
"NMDG+-based solutions were used as the external and internal solutions , pHin/pHout 7 . 35/7 . 35 .",
"( e ) Representative ionic currents recorded from HEK293 cells expressing Shaker-iVSD using the whole-cell configuration of the patch-clamp technique .",
"Cells were depolarized for 500 ms to potentials ranging from –140 mV to +100 mV ( at a holding potential of 0 mV , top ) , or from −140 mV to +80 mV ( at a holding potential of −90 mV , bottom ) in 10 mV increments .",
"An inset on top of current traces shows the corresponding voltage protocols .",
"Zero level was indicated by the red dashed line .",
"To compare with the ionic current recorded from oocytes as shown in c , NMDG+-based solutions were also used as the extracellular and intracellular solutions , pHin/pHout 7 . 35/7 . 35 .",
"( f ) I-V relations of currents recorded from Shaker-iVSD-injected oocytes at holding potentials of 0 mV ( black triangle , n = 14 ) or −90 mV ( red triangle , n = 13 ) , and H2O-injected oocytes at holding potentials of 0 mV ( blue triangle , n = 5 ) using the cut-open oocyte voltage clamp technique ( see protocol in the upper inset of",
"c ) .",
"The mean steady-state currents during the last 50 ms of the command pulses were plotted versus voltage .",
"( g ) I-V relations of ionic currents recorded from HEK293 cells transfected with Shaker-iVSD at holding potentials of 0 mV ( black triangle , n = 8 ) or −90 mV ( red triangle , n = 4 ) using the whole-cell patch clamp technique ( see protocol in the upper inset of",
"e ) .",
"The mean steady-state currents during the last 50 ms of the command pulses were plotted versus voltage . DOI: http://dx . doi . org/10 . 7554/eLife . 18130 . 003 Gating currents arising from Shaker-iVSD were detected 4–5 days after cRNA injection using the cut-open oocyte technique ( Figure 1b , top ) .",
"Our initial objective was to obtain the gating charge-voltage ( QV ) relation of Shaker-iVSD .",
"However , unlike those observed in Shaker-W434F ( Figure 1b bottom ) , the gating currents of Shaker-iVSD could not be estimated reliably because of substantial ionic currents in particular when holding the potential at −90 mV .",
"The observed ionic currents in response to a series of 250 ms test pulses applied in 10 mV steps exhibited a weak voltage dependence ( Figure 1c ) .",
"In contrast , at the depolarized holding potential of 0 mV , the currents exhibited strong rectification with inward currents at hyperpolarized potentials .",
"The phenotype was consistent with an ion channel , conducting at negative membrane potentials and very slow activation and deactivation kinetics ( Figure 1c and",
"f ) .",
"We confirmed that no similar currents developed for Shaker-W434F at similar expression level ( Figure 1d ) and that endogenous currents were negligible in H2O-injected oocytes ( Figure 1f ) .",
"In order to further verify that the currents are not caused by Shaker-iVSD assembling with or inducing expression of endogenous channels in Xenopus oocytes , we expressed Shaker-iVSD in HEK293 cells .",
"The resulting currents in whole-cell patch-clamp recordings show the same ionic currents and voltage dependence as observed in Xenopus oocytes ( Figure 1e ) .",
"Holding the membrane potential polarized ( −90 mV ) led , as in Xenopus oocytes , to constitutively open channels , whereas holding at 0 mV led to hyperpolarization-activated ionic currents with slightly less rectification than in Xenopus oocytes ( Figure 1g ) .",
"Since the inward ionic current was also observed with NMDG+ ( N-methyl-D-glucamine ) as the dominant ion in the extracellular solution , we tested the proton selectivity of the Shaker-iVSD-induced current .",
"The proton driving force was altered by shifting the proton reversal potential , E ( H+ ) , through alterations of the transmembrane pH gradient .",
"Oocytes were bathed in 70 mM NMDG+ solutions with extracellular pH modulated to 9 . 5 , 7 . 5 and 4 . 5 and intracellular pH maintained at 7 . 5 .",
"Consistent with a proton selective pore , the inward currents increased and E ( H+ ) shifted to more positive potentials when decreasing external pH , although the reversal potential did not reach E ( H+ ) completely ( 12 . 5 ± 1 . 6 mV for pH 4 . 5 and −5 . 4 ± 1 . 8 mV for pH 9 . 5; Figure 2a ) . 10 . 7554/eLife . 18130 . 004Figure 2 . Selectivity of Shaker-iVSD-induced current in high buffer ( HB ) solutions or NMDG+-based solutions .",
"( a ) Top .",
"Normalized IV relationships of Shaker-iVSD induced currents under different external pH conditions with HB solutions .",
"Tail currents were evoked by 250 ms voltage pulses ranging from −180 to +120 mV following a 200 ms hyperpolarization prepulse to −120 mV using cut-open oocyte technique ( see inset for protocol ) .",
"External HB solution contained ( in mM ) 70 NMDG+ , and either 180 acetic acid ( for pH 4 . 5 ) , 180 HEPES ( for pH 7 . 5 ) , or 136 CHES with 44 D-sorbitol ( for pH 9 . 5 ) .",
"The composition of the HB internal solution was the same as the HB internal solution at pH 7 . 5 .",
"Bottom .",
"Same normalized IV relationships , but zoomed in to emphasize the changes of Vrev .",
"At pHin/pHout 7 . 5/7 . 5 , Vrev = −0 . 14 ± 0 . 18 mV ( black triangle , n = 56 ) ; at pHin/pHout 7 . 5/4 . 5 , Vrev = 12 . 5 . 2 ± 1 . 6 mV ( blue triangle , n = 5 ) ; at pHin/pHout 7 . 5/9 . 5 , Vrev = −5 . 4 ± 1 . 76 mV ( red triangle , n = 5 ) .",
"( b ) Top .",
"Normalized IV relations of currents with NMDG+in/NMDG+out 115 mM/10 mM or 115 mM/115 mM , at pHin/pHout 7 . 35/7 . 35 .",
"NMDG+-based external and internal solutions contained 115 mM NMDG-MeSO3 were the same as in Figure 1b–d .",
"To test the permeability of Shaker-iVSD-induced currents to NMDG+ ions , 115 mM NMDG-MeSO3 in the external solution was replaced by 10 mM NMDG-MeSO3 and 210 mM D-sorbitol , at pH 7 . 35 .",
"Currents were elicited by the voltage pulse protocol shown in the inset .",
"Bottom .",
"Same normalized IV relationships , but zoomed in to emphasize the changes of Vrev .",
"At NMDG+in/NMDG+out 115 mM/10 mM , Vrev = −62 . 6 ± 2 . 5 nmV ( red triangle , n = 4 ) ; at NMDG+in/NMDG+out 115 mM/115 mM , Vrev = −0 . 8 ± 1 . 1 ( black triangle , n = 4 ) .",
"( c ) Top .",
"Normalized IV relationships of currents recorded from HEK293 cells transfected with Shaker-iVSD cDNA with HB solutions .",
"Tail currents were elicited from a holding potential of 0 mV by 250 ms voltage pulses ranging from –140 to +100 mV in 10 mV increments , following a 200 ms hyperpolarization prepulse to −120 mV using whole-cell patch clamp technique ( see inset for protocol ) .",
"The intracellular and extracellular HB solutions contained ( in mM ) 10 NMDG+ , and either 24 HEPES ( for PH 7 . 5 ) , 56 MES ( for pH5 . 5 ) , or 35 acetic acid ( for pH4 . 5 ) .",
"Osmolarity was adjusted to 300~320 mOsm by D-sorbitol .",
"Bottom .",
"Same normalized IV relationships , but zoomed in to emphasize the changes of Vrev .",
"Vrev values were 0 . 5 ± 0 . 2 mV at pHin/pHout 7 . 5/7 . 5 ( black triangle , n = 9 ) , −7 . 0 ± 1 . 1 mV at pHin/pHout 5 . 5/7 . 5 ( red triangle , n = 6 ) , and −34 . 9 ± 1 . 8 mV at pHin/pHout 4 . 5/7 . 5 ( blue triangle , n = 10 ) .",
"( d–e )",
"Comparison of conductance of Shaker-iVSD for external NMDG+ ( black triangles , n = 12 ) , K+ ( f , red triangles , n = 7 ) and Na+ ( g , blue triangles , n = 7 ) using cut-open oocyte technique .",
"The NMDG+-based external solution and internal solutions were the same as in Figure 1b–d at pHin/pHout 7 . 35/7 . 35 .",
"Na+-based or K+-based external solutions were the same as the NMDG+-based external solution except that 115 mM NMDG- MeSO3 was replaced by the same concentration of NaOH- MeSO3 or KOH- MeSO3 .",
"Currents were elicited by the voltage pulse protocol shown in the inset of Figure 1c , and currents were normalized relative to the maximum NMDG+ inward currents and plotted versus voltage .",
"( f ) Replacing MeSO3 with chloride ( green triangle , n = 7 ) in the external NMDG+-based solution did not exhibit significantly difference from the currents recorded in external NMDG-MeSO3 solution , indicating that it is likely not carried by anions . DOI: http://dx . doi . org/10 . 7554/eLife . 18130 . 004 The discrepancy between Vrev and E ( H+ ) may be due to the imperfect control of the local proton concentration near the membrane , or due to permeation of other ions through the channel .",
"To distinguish between these two possibilities , we measured the reversal potential Vrev after reducing the ionic strength by D-sorbitol dilution while keeping the pH constant .",
"Reducing the ionic strength should not alter the reversal potential if the current was perfectly proton selective since the proton concentration remains constant while all other ion concentrations were reduced ( DeCoursey , 2013 ) .",
"Substitution of NMDG+ with D-sorbitol in the external solution to a final concentration of 10 mM ( versus 115 mM cytosolic ) substantially decreased the normalized inward current and shifted the Vrev from −1 ± 1 mV to −62 . 5 ± 2 . 5 mV at symmetric pH = 7 . 35 , suggesting that even NMDG+ ions are to a certain extent permeant charge carriers of Shaker-iVSD-induced currents ( Figure 2b ) .",
"A proton gradient of pHin/pHout5 . 5/7 . 5 and 4 . 5/7 . 5 at a low NMDG+ concentration ( 10 mM ) and high buffer concentrations in both external and internal solutions resulted in a Vrev −7 . 0 ± 1 . 1 mV and −34 . 9 ± 1 . 8 mV , respectively ( Figure 2c ) .",
"These experiments were performed on whole-cell patches from HEK293 cells .",
"The relative permeability for protons over NMDG+ was PH/PNMDG ~ 1400 according to the Goldman-Hodgkin-Katz equation .",
"The experiments also excluded any significant permeability for anions ( symmetric 10 mM ) .",
"To identify the relative selectivity of Shaker-iVSD to different cations , we performed ionic substitution experiments in oocytes using the cut-open oocytes technique .",
"Normalized current amplitudes of Shaker-iVSD recorded in external solutions containing either Na+ or K+ as the predominant extracellular cation were 1 . 35 and 1 . 54 times higher than in NMDG+ containing external solution ( Figure 2d–e ) .",
"The order of permeation efficiency was P ( H+ ) >> P ( K+ ) ≈ P ( Na+ ) > P ( NMDG+ ) .",
"Replacement of the anion mesylate ( MeSO3− ) with chloride in the external solution ( NMDG-MeSO3 vs NMDG-Cl ) did not alter the Shaker-iVSD-induced current , confirming that the current is not carried by anions ( Figure 2f ) .",
"Ionic currents through the VSD of the Shaker channel have been described for protons and other cations upon mutations along the S4 ( Starace and Bezanilla , 2004; Moreau et al . , 2015; Tombola et al . , 2005 ) , and the transmembrane domain of Hv1 proton channels consist of only S1-S4 ( Ramsey et al . , 2006; Sasaki et al . , 2006; Takeshita et al . , 2014 ) .",
"We thus expected that the cations follow a similar path through the VSD .",
"Since Hv channels are blocked by Zn2+ in the micromolar range ( Ramsey et al . , 2006; Sasaki et al . , 2006 ) , we tested Shaker-iVSD’s sensitivity to the application of external Zn2+ .",
"We found that with increasing concentrations of Zn2+ ( 0–20 µM ) , the ionic current of Shaker-iVSD continuously reduced ( Figure 3a and",
"c ) . 10 . 7554/eLife . 18130 . 005Figure 3 . Inhibition of Shaker-iVSD-induced ionic currents by external ZnCl2 .",
"( a ) Ionic currents and normalized fluorescence traces recorded from oocytes expressing Shaker-iVSD before and after the application of 2~20 µM ZnCl2 using cut-open technique ( see inset for protocol ) .",
"Zero level is indicated by the grey dashed line .",
"To illustrate the effects of ZnCl2 on kinetics , the same ionic currents were normalized to the value at the end of the 250 ms pulse ( see lower inset ) .",
"( b ) Fluorescence traces to the recordings in a obtained from fluorescent labeling at position A359C atop S4 .",
"( c ) IV-relations of Shaker-iVSD at increasing concentrations of ZnCl2 .",
"( for protocol see",
"a ) .",
"Currents were normalized to the maximal current amplitude in the absence of Zn2+ .",
"( d ) Normalized FV relations in the absence ( black ) and presence of external 2 μM ( red ) or 10 μM ( green ) ZnCl2 .",
"Fluorescence curves were generated using the protocol shown as inset .",
"Smooth curves are fits to a Boltzmann function yielding the following V1/2 and dV values: −88 . 2 ± 1 . 2 and 32 . 2 ± 1 . 7 mV for Shaker-iVSD control ( n = 6 ) ; −89 . 6 ± 2 . 3 mV and 32 . 8 ± 2 . 6 mV for 2 μM ZnCl2 ( n = 6 ) ; −98 . 4 ± 2 . 9 mV and 37 . 5 ± 2 . 0 mV for 10 μM ZnCl2 ( n = 6 ) .",
"Each curve was normalized to its own maximal relative change dF/F .",
"( e ) Effect of Zn2+ on relative fluorescence change ( dF/F , grey ) and ionic current .",
"The relative fluorescence change remains constant even during bleaching ( Blunck et al . , 2004 ) , assuming not too high background fluorescence .",
"A reduction of the relative fluorescence change is thus correlated with immobilization of the voltage sensors .",
"The current values are the maximal current amplitudes of c normalized to the value in the absence of Zn2+ .",
"( f ) Position of H140 ( S2 ) and H193 ( S3-S4 linker; numbering according to human Hv1 ) in the crystal structure of Hv1 ( PDB: 3WKV ) in the entry to the gating pore .",
"Missing residues in the crystal structure were modeled using Modeller ( Sali and Blundell , 1993 ) .",
"( g ) Ionic current traces recorded from oocytes expressing Shaker-iVSD D277H in the absence ( black ) and presence ( red ) 0 . 1 µM ZnCl2 using cut-open technique ( see inset in a for protocol ) .",
"( h ) Normalized FV relations in the absence ( black triangle , n = 5 ) and presence ( red triangle , n = 5 ) of 0 . 1 µM ZnCl2 .",
"Fluorescence signals were generated using the protocol shown as inset in d .",
"Smooth curves are Boltzmann fits to the data with the following V1/2 and dV values: −99 . 0 ± 2 . 5 mV and 25 . 9 ± 1 . 3 for control; −110 . 4 ± 0 . 2 mV and 25 . 7 ± 1 . 9 mV for ZnCl2 .",
"( i ) Dose-response curves of inhibition by external ZnCl2 for Shaker-iVSD and Shaker-iVSD D277H ionic currents .",
"Data points correspond to normalized amplitudes of ionic currents elicited by step hyperpolarization to −150 mV ( As shown in",
"a ) , plotted as a function of ZnCl2 concentration , and fitted with the Hill equation .",
"The IC50 value of ZnCl2 for the Shaker-iVSD D277H inward currents was 158 . 4 nM ( maximum inhibition 79% , n = 5 ) , which was significantly lower than the IC50 value obtained from Shaker-iVSD currents 6 . 04 µM ( maximum inhibition 80% , n = 6 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 18130 . 00510 . 7554/eLife . 18130 . 006Figure 3—figure supplement 1 . Effect of Zn2+ on fluorescence ( left ) and gating currents ( right ) .",
"Fluorescence changes reduced significantly upon addition of Zn2+ and recover upon washout .",
"Gating currents were inhibited upon addition of 5 µM Zn2+ .",
"At 5 µM Zn2+ , tail currents at hyperpolarized potentials are still visible . DOI: http://dx . doi . org/10 . 7554/eLife . 18130 . 006 While we were not able to record gating currents reliably , we were able to trace the conformational changes occurring in Shaker-iVSD using voltage-clamp fluorometry .",
"Attaching TMRM at position A359C reports conformational changes related to the gating currents ( Haddad and Blunck , 2011 ) .",
"Oocytes , expressing Shaker-iVSD and labeled with TMRM , exhibited clear fluorescence signals two days after injection ( Figure 3b and",
"d ) .",
"The time course of the normalized fluorescence change did not alter during activation in the presence of Zn2+ but deactivation was slowed ( Figure 3b and",
"d ) .",
"Accordingly , also the fluorescence voltage ( FV ) relation remained unaltered .",
"While the activation kinetics and FV remained unaltered , the relative fluorescence change was diminished with increasing Zn2+-concentration similar to the decrease in current amplitude ( Figure 3b and",
"e ) .",
"A reduced relative fluorescence change indicates that immobilization of the voltage sensor by Zn2+ caused the current decrease .",
"Fluorophores attached to voltage sensors immobilized by Zn2+ would still contribute to the total fluorescence intensity ( F ) but would no longer be displaced and thus not display a voltage dependent change ( dF ) .",
"Accordingly , the relative fluorescence change dF/F would be lower .",
"This was further validated by the reduction in gating currents with addition of Zn2+ ( Figure 3—figure supplement 1 ) .",
"In Hv1 channels , histidines at position H140 and H193 have been shown to be responsible for the Zn2+ sensitivity ( Ramsey et al . , 2006; Takeshita et al . , 2014 ) .",
"These histidines are positioned at the entry to the gating pore , right above the positively charged arginines of the S4 ( Figure 3f ) and would thus prevent proton conduction through the pore .",
"While the histidines are not conserved in Shaker-iVSD , H140 and H193 align with F280 and E335 , respectively .",
"F280 unlikely coordinates a Zn2+ , this role is more likely taken over by either D277 or E283 .",
"The lower affinity of glutamate and aspartate might be responsible for the lower sensitivity of Shaker-iVSD to Zn2+ , as we find ~40% of the current still at 20 µM , at which Hv1 is already fully blocked ( Takeshita et al . , 2014 ) .",
"Wild type Shaker channels are stabilized in the closed state by external ( 300 µM ) Zn2+ ( Boland et al . , 1994 ) .",
"In Shaker-iVSD , this would reflect the conducting state .",
"However , the addition of external Zn2+ to Shaker-iVSD did reduce and not increase the current at −90 mV , suggesting that either binding of Zn2+ caused the voltage sensing domain to adopt its ‘native’ conformation ( i . e . the non-conducting one in the full Shaker channel ) or that the Zn2+ directly blocks the ion permeation pathway .",
"The position of D277/E283 and E335 – corresponding to H140 and H194 – support the notion that the ion permeation follows the pathway through the gating pore like in Hv1 and ω-currents .",
"To corroborate this finding , we substituted D277 with histidine ( D277H ) and tested the Zn2+-sensitivity .",
"( Unfortunately , the double mutant D277H-E335H did not express sufficiently . )",
"The voltage dependence of opening of Shaker-iVSD-D277H was shifted to more hyperpolarized potentials ( V½ = −99 mV ) .",
"While the kinetics were very similar ( Figure 3g–h ) .",
"When blocking the currents with Zn2+ , we found that the channel showed a 40-times higher affinity to Zn2+ with an IC50 of 0 . 15 µM versus 6 µM for Shaker-iVSD ( Figure 3i ) , confirming the pathway through the gating pore in the central voltage sensor domain .",
"The development of ionic current in the absence of a pore domain shows that the Shaker-iVSD adopts a different conformation than the voltage sensing domain in the complete channel protein .",
"This raises the question as to how the absence of the pore influences the gating kinetics .",
"To analyze the gating kinetics of Shaker-iVSD in detail , we elicited fluorescence traces in response to 250 ms voltage clamp pulses to a range of potentials from −180 mV to +160 mV .",
"The resulting fluorescence kinetics of Shaker-iVSD were slower than Shaker-W434F ( Figure 4a ) .",
"At a holding potential of −90 mV , the channels were already in the open state , and they closed ( or deactivated ) very slowly upon depolarization .",
"The corresponding fluorescence changes did not seem to correlate to the ionic current . 10 . 7554/eLife . 18130 . 007Figure 4 . Conformational changes in Shaker-iVSD . Shaker-iVSD displays slow voltage-dependent fluorescence quenching in oocytes under voltage-clamp using cut-open oocyte technique .",
"( a ) Typical fluorescence signals are shown for Shaker-iVSD and Shaker-W434F for 250 ms pulses between −180 and +160 mV from holding potentials of −90 mV ( left ) , 0 mV ( center ) , and +90 mV ( right ) .",
"The inset shows the corresponding clamp protocol .",
"( b ) Scaled and overlaid representative ionic current ( black ) and fluorescence traces ( red ) in response to a voltage step from 0 mV to −150 , –130 , −110 and −90 mV for Shaker-iVSD ( see inset for protocol ) .",
"Note that the fluorescence signal has been inverted for a direct kinetic comparison with the current traces .",
"( c ) The activation kinetics of the Shaker-iVSD ionic currents ( as shown on the top of Figure 1c ) and fluorescence traces during channel activation ( as shown in",
"a ) were fitted by two exponential components , Ifast and Islow for ionic currents ( n = 7 ) , Ffast and Fslow for fluorescence traces ( n = 7 ) .",
"The fitted time constants were plotted as a function of test potential .",
"( d ) FV relationships of Shaker-iVSD and Shaker-W434F at holding potential of −90 mV calculated by normalizing the mean ΔF values during the last 50 ms of the command pulse to the maximum fluorescence change , and plotted against voltage .",
"Smooth curves were fits to a Boltzmann function yielding the following V1/2 and dV values: −38 . 8 ± 3 . 4 and 49 . 3 ± 2 . 3 mV for Shaker-iVSD ( n = 12 ) , −49 . 2 ± 1 . 3 and 8 . 6 ± 0 . 4 mV for Shaker-W434F ( n = 12 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 18130 . 007 At a holding resting potential of 0 mV , it became more feasible to compare the slower development of the ionic currents ( Figure 1c top ) to the corresponding fluorescence changes ( Figure 4a , center ) .",
"The fluorescence developed slower than at −90 mV , and superposition of ionic current and fluorescence changes from simultaneous measurements showed that both current and fluorescence developed with similar time constants when pulsing to strongly hyperpolarized potentials ( Figure 4b ) .",
"The fact that the signals coincided during hyperpolarization ( holding at 0 mV ) but not during depolarization ( holding at −90 mV ) shows that fluorescence change and conduction are two independent steps .",
"The step developing conduction is rate limiting only when holding at 0 mV .",
"From a holding potential of −90 mV , the iVSD first undergoes the fast conformational change , reported by the fluorescence that corresponds to the gating currents ( Haddad and Blunck , 2011 ) , before the channels close slowly .",
"Conversely , when holding at 0 mV , iVSD first opens the channel ( slow , conduction ) before undergoing the normal gating movement ( fast , fluorescence ) .",
"The fluorescence now follows the rate-limiting slow channel opening .",
"In a more in-depth analysis activation kinetics of ionic current traces ( as shown on the top left of Figure 1c ) and fluorescence signals ( as shown on the top of Figure 4 ) followed a bi-exponential function with a fast and a slow time constant ( τfast and τslow , respectively , Figure 4c ) .",
"The time constants were voltage-dependent and showed a typical bell shape with maxima at about −100 mV for fluorescence signals and −90 ~ −70 mV for ionic currents .",
"The time constants of current and fluorescence signal correlated well in the range where most of the ionic current developed ( <−120 mV ) whereas the ionic current developed slower at more depolarized potentials .",
"Although we found the same time constants in current and fluorescence , the time courses did not superimpose directly ( Figure 4b ) , indicating that the processes associated to both time constants did not contribute equally to the fluorescence amplitudes .",
"In the fluorescence voltage-relation , the midpoint of activation was not significantly shifted in Shaker-iVSD compared to wild type , but the voltage dependence was much shallower , so much so that even at the extreme potentials the FV relation were not yet fully saturated ( Figure 4d ) .",
"However , we have shown above that the kinetics of the fluorescence changes differed significantly when holding at −90 mV as compared to holding at 0 mV ( Figure 4a ) .",
"The differences also stretched towards the corresponding voltage dependences of the fluorescence voltage relations , which shifted by ~50 mV when holding at both voltages ( Figure 5a ) .",
"Such a hysteresis was earlier identified as ‘relaxation’ or 'mode shift' ( Bruening-Wright and Larsson , 2007; Villalba-Galea et al . , 2008 ) .",
"We previously described that the pore domain contributes to the development of the mode shift ( Haddad and Blunck , 2011 ) and it had been related to C-type inactivation ( Olcese et al . , 1997 ) .",
"However , mode shift , or ‘relaxation’ , was also observed in the voltage-gated phosphatase of C . intestinalis ( Ci-VSP ) , which is devoid of a pore domain ( Labro et al . , 2012; Villalba-Galea et al . , 2008 ) , suggesting that it is an intrinsic property of voltage sensing domains .",
"Our finding here resolves the issue since also Shaker-iVSD follows the hysteresis pattern . 10 . 7554/eLife . 18130 . 008Figure 5 . Mode shift in Shaker-iVSD .",
"( a ) FV relationships at 7 different holding potentials of Shaker-iVSD ( left ) and Shaker-W434F ( right ) .",
"Smooth curves were fits to a Boltzmann function yielding the following V1/2 and dV values: for Shaker-iVSD , −38 . 8 ± 3 . 4 and 49 . 3 ± 2 . 3 mV at HP −90 mV ( n = 12 ) , −58 . 5 ± 2 . 5 and 57 . 4 ± 2 . 3 mV at HP −60 mV ( n = 24 ) , −77 . 0 ± 2 . 7 and 44 . 9 ± 1 . 3 mV at HP −30 mV ( n = 20 ) , −88 . 7 ± 1 . 4 and 33 . 6 ± 1 . 5 mV at HP 0 mV ( n = 24 ) , −98 . 2 ± 2 . 4 and 32 . 0¸± 2 . 0 mV at HP + 30 mV ( n = 9 ) , −107 . 1 ± 3 . 7 and 37 . 2 ± 2 . 0 mV at HP + 60 mV ( n = 11 ) , −133 . 7 ± 4 . 2 and 40 . 6 ± 1 . 9 mV at HP + 90 mV ( n = 7 ) ; for Shaker-W434F , −49 . 2 ± 1 . 3 and 8 . 6 ± 0 . 4 mV at HP −90 mV ( n = 12 ) , −5 . 5 ± 3 . 0 and 14 . 2 ± 0 . 9 mV at HP −60 mV ( n = 14 ) , −75 . 4 ± 2 . 4 and 11 . 6 ± 1 . 1 mV at HP −30 mV ( n = 14 ) , −77 . 1 ± 2 . 0 and 11 . 2 ± 0 . 9 mV at HP 0 mV ( n = 7 ) , −77 . 7 ± 2 . 1 and 11 . 7 ± 0 . 8 mV at HP + 30 mV ( n = 7 ) , −78 . 9 ± 2 . 0 and 11 . 2 ± 0 . 8 mV at HP + 60 mV ( n = 8 ) , −80 . 3 ± 1 . 8 and 10 . 8 ± 0 . 5 mV at HP + 90 mV ( n = 6 ) .",
"( b ) V1/2 values from a plotted versus holding potential .",
"( c–d )",
"Normalized FV relations of Shaker-iVSD and Shaker-W434F from a holding potential of 0 mV to a pre-pulse of −90 mV for variable duration ( 5–5000 ms ) followed by a series of test pulses of −180 to +160 mV in steps of 10 mV ( see inset for protocol ) .",
"For Shaker-iVSD , smooth curves are fits to a Boltzmann function with V1/2 values shown in e as a function of pre-pulse duration .",
"For Shaker-W434F , the resulting distribution is a superposition of two Boltzmann curves representing the FV0 mV and the mode-shifted FV−90 mV .",
"Smooth curves are fits of the data to a double Boltzmann function , and the fractional amplitudes of FV−90 mV component are shown in e as a function of pre-pulse duration .",
"( e ) .",
"Data points of Shaker-iVSD ( red circle , right ordinate ) were fitted by a single-exponential function with a time constant for entering the mode shift of 452 ms . Data points of Shaker-W434F ( black circle , left ordinate ) were fitted by a double-exponential function having time constants for entering the mode shift of 36 ms and 279 ms .",
"( f ) Comparision the time course for entering mode-shift with the development of the ionic current of Shaker-iVSD .",
"The red data points are V1/2 values recorded from one oocyte plotted as a function of pre-pulse duration ( 5–1000 ms ) ( see protocol in",
"c ) , and fitted by a single-exponential function ( red curve ) .",
"Ionic current recorded from the same oocyte ( black ) was scaled and overlaid with the red curve . DOI: http://dx . doi . org/10 . 7554/eLife . 18130 . 008 Still , the conformational changes associated with relaxation remain unknown; we therefore investigated whether , in Shaker-iVSD , the development of an ion conducting pore is related to relaxation .",
"We determined the FV relationships for Shaker-iVSD for holding potentials between −90 mV and +90 mV ( Figure 5a ) .",
"Just like wild type Shaker channels , the FV relations were shifted to more hyperpolarized potentials when holding at depolarized potentials .",
"The shift of the FV relations was more pronounced in Shaker-iVSD , shifting by −95 mV compared to −31 mV shift of Shaker-W434F ( Figure 5b ) .",
"The FV relations of Shaker-iVSD were continuously shifted to more hyperpolarized potentials at more depolarized holding potentials and did not saturate within the tested holding potentials .",
"Unfortunately , holding at more extreme potentials did not yield reliable data since the oocytes did not remain stable for sufficiently long time .",
"In Shaker-W434F , the shift already saturated at a holding potential of −30 mV .",
"The much more pronounced mode shift in the isolated voltage sensor versus the complete Shaker channel indicated that the Shaker-iVSD requires much stronger hyperpolarization to return to its resting position when lacking the pore domain .",
"We were interested in comparing the time course for entering the mode shift of Shaker-iVSD with the development of the ionic current .",
"We applied pre-pulses of increasing duration from a holding potential of 0 mV to −90 mV , followed by a series of test pulses , determining the relative fluorescence change .",
"For Shaker-iVSD , the resulting curve was mono-exponential with a time constant of 452 ms , whereas recovery from relaxation occurred in double-exponential fashion with time constants of 36 ms and 279 ms for Shaker-W434F ( Figure 5c–e ) .",
"The mode shift coincided temporally with the onset of the ionic current in Shaker-iVSD ( Figure 5f ) , indicating that the permeation pathway , although open in both resting and activated state , closes during relaxation of the voltage sensing domain ."
],
[
"The voltage sensing domain of the Shaker Kv channel altered its behaviour significantly when expressed in the absence of the pore domain .",
"The voltage dependence is much shallower than the voltage dependence of Shaker-WT and it is evident from the development of cation conduction that the voltage sensing domain assumes a different structural conformation than in the full-length channel .",
"The shallower voltage dependence is probably related to a dispersion of the electric field in the voltage sensor; with the 'hydrophobic plug' or 'gating charge transfer center' ( Tao et al . , 2010 ) no longer intact , the electric field will be less focussed and thus diminish the effective valence of the gating transition .",
"There are two possible explanations for the rearrangements ,",
"( i ) the missing interaction with the pore domain leads to a different , energetically more favourable state of the entire voltage sensing domain , or",
"( ii ) uncoupling from the pore domain allows the voltage sensor to continue its normal gating movement and enter a deeper resting state not accessible in the wild type .",
"If the second case were true , the deactivation kinetics ( i . e . here: return to 0 mV ) should remain unaltered , but we observed significant slowing of deactivation ( Figure 4a ) .",
"More importantly , activation of the voltage sensor , monitored by the fast fluorescence change , did not close the ion conduction pathway: the activation kinetics when holding at −90 mV ( resting ) , measured via fluorescence , were fast ( Figure 4a ) , and the current did not reduce during a 250 ms-pulse ( Figure 1c ) .",
"iVSD thus leaves the conducting state only very slowly , whereas entering is significantly faster albeit slower than the gating kinetics of wild type Shaker channels .",
"The effects can be explained by a simple model for entering the relaxed state ( Figure 5g ) , where both the resting and activated state are conducting , whereas the relaxed state is non-conducting .",
"We can assign the fluorescence changes to the fast transition from resting to the activated state .",
"The current vanishes when entering the relaxed state , but this transition is silent in the fluorescence changes .",
"This model is supported by the temporal correlation between relaxation ( mode-shifting ) and the opening of the iVSD-channel from a holding potential of 0 mV ( Figure 5e ) .",
"Our data do not exclude the possibility that additional states exist .",
"Channel closing and relaxation , although linked , do not necessarily have to be identical transitions , or an activated-relaxed state as suggested by Bezanilla and co-workers is possible ( Labro et al . , 2012; Villalba-Galea et al . , 2008 ) .",
"The permeation path is likely to be similar to those described for the ω-currents and Hv1 ( Starace and Bezanilla , 2004; Tombola et al . , 2005; Takeshita et al . , 2014; Tombola et al . , 2007; Musset et al . , 2011 ) .",
"This notion is supported by the block through Zn2+ ( Figure 3 ) as the location of the involved residues are positioned at the entrance to the gating pore .",
"In the case of the proton and ω-currents passing through the Shaker voltage sensing domain , it is suggested that the cations follow the path normally taken up by the arginines in S4 responsible for the gating charges .",
"In both cases , the arginines were replaced either with histidines or cysteines .",
"In the activated wild type Shaker , these arginines are coordinated by acidic residues ( Li et al . , 2014a; Vargas et al . , 2012; Long et al . , 2005a ) ; more specifically , the second , third and fourth cationic residues in S4 , R3 ( R368 ) , R4 ( R371 ) and K5 ( K374 ) , are coordinated by the acidic residues E247 , E283 and E293 , respectively , according to the crystal structure ( Long et al . , 2007 ) .",
"This coordination is likely transferred to R1 ( R362 ) and R2 ( R365 ) in the transition to the resting state when the S4 assumes its ‘down’ position ( Li et al . , 2014a; Vargas et al . , 2012; Starace and Bezanilla , 2004 ) .",
"In Hv1 channels in resting position , similarly , R3 ( R208 ) is coordinated by the aspartate D112 , responsible for the proton selectivity in these proton channels ( Takeshita et al . , 2014 ) .",
"The major difference between the voltage sensors of Shaker and Hv1 are an abundance of basic and acidic residues pointing towards the common interface , respectively .",
"In Shaker , we can therefore assume that every acidic residue in the core of the VSD remains coordinated to an arginine whereas , in Hv1 , D112 likely becomes uncoordinated in the activated position because the fifth positive charge in S4 is missing .",
"D112 can then act as the selectivity filter facilitating proton transport ( Takeshita et al . , 2014; Musset et al . , 2011 ) .",
"In Shaker-iVSD , no residue in the core of the VSD has been mutated , indicating that the salt bridges between R1-R6 and the acidic residues of S1-S3 must have been broken by a different conformational change .",
"At this time , it would be purely speculative to specify which salt bridge would be responsible without further information or a high-resolution structure .",
"We can , however , link gating of the Shaker-iVSD channel to relaxation of the VSD .",
"Relaxation or mode shift is the displacement of the voltage dependence of charge movement and conductance to more hyperpolarized potentials upon prolonged holding at hyperpolarized potentials .",
"Relaxation has been linked to C-type inactivation , stabilization of the open state , length of the S3-S4 linker and N-terminus in different constructs ( Haddad and Blunck , 2011; Piper et al . , 2003; Tan et al . , 2012; Olcese et al . , 1997; Kuzmenkin et al . , 2004; Bruening-Wright and Larsson , 2007; Villalba-Galea et al . , 2008; Priest et al . , 2013 ) .",
"While the S3-S4 linker and the N-terminus ( T1 domain ) rest intact and may still influence relaxation , in the absence of the pore , neither C-type inactivation nor open state stabilization may have an influence .",
"In the resting and activated state , Shaker-iVSD forms a cation-selective pore whereas this pore is closed in the relaxed state , indicating that the acidic residues responsible for the cation pore are no longer accessible either by reorientation or coordination with a basic residue .",
"Alternatively , relaxation might stabilize the ‘native’ conformation of the voltage sensing domain .",
"The conformational changes of the voltage sensing domain in the absence of the pore domain might be minor , but the development of a cation pore and the much slower kinetics in spite of the same primary sequence indicates that the stabilizing effects of the pore domain are essential for the native function .",
"Stabilization might include direct protein-protein contacts between the voltage sensing domain and S5 ( Li-Smerin et al . , 2000 ) , in the coupling region ( Batulan et al . , 2010; Wall-Lacelle et al . , 2011; Haddad and Blunck , 2011; Muroi et al . , 2010; Long et al . , 2005b; Catterall , 2010; Bezanilla , 2005; Lu et al . , 2001 , 2002; Labro et al . , 2008 ) or between voltage sensing domain and pore at the outer surface ( Lee et al . , 2009a; Petitjean et al . , 2015; Lainé et al . , 2003 ) .",
"Stabilization might also be evoked by the energetic interaction between the domains for instance the missing coupling or cooperativity between subunits .",
"In the construct used here , we removed the pore region and the C-terminus whereas the N-terminal tetramerization ( T1 ) -domain ( Shen et al . , 1993; Li et al . , 1992 ) is still present .",
"The T1-domain is responsible for the correct assembly of Kv tetramers and can tetramerize in the isolated form ( Kreusch et al . , 1998 ) .",
"It is therefore possible that the Shaker-iVSD are held together in the cytosol by the T1-domain .",
"While this leaves the possibility that the voltage sensors interact with each other , the higher sensitivity of D277H to zinc block indicates that this interaction is not responsible for the formation of the permeation pathway .",
"The position of D277H suggests that the ions pass through the gating pore in the center of the VSD as discussed above .",
"The system as a whole will have a different energy landscape than the isolated domains , which becomes evident in the very different voltage ranges causing relaxation in Shaker-W434F and –iVSD .",
"In most cases , these effects cannot be strictly separated .",
"Although interpretation is facilitated when domains of macromolecules are considered separate entities with energetic interactions , it is essential when interpreting macromolecular structure function relations or structures of isolated domains to keep in mind that the final assembly does not necessarily behave like the sum of its components and might adopt a different conformation .",
"The fact that simply removing the pore domain of the Shaker K+ channel leads to a voltage-gated cation channel suggests that the structural link between Kv and Hv channels might be closer than previously thought .",
"If the phenomenon of a cation leak for the isolated voltage sensor is conserved also in other voltage-gated ion channels , it might be responsible for disease development in truncation mutants downstream of S4 ( Cox et al . , 2006 ) ."
],
[
"The wild-type background Shaker H4 channel cDNA with inactivation peptide removed ( IR , Δ6–46 ) in a pBSTA vector was used as a template for all mutants ( Batulan et al . , 2010; Choi et al . , 1991 ) .",
"A cysteine was inserted into the S3-S4 linker at position A359C for simultaneous fluorescence measurements ( Mannuzzu et al . , 1996; Cha and Bezanilla , 1997 ) .",
"This mutation was present in all constructs and is not specifically mentioned .",
"A W434F mutation was inserted in the pore region ( Perozo et al . , 1993 ) , rendering the channel non-conducting for gating current measurements .",
"The Shaker-iVSD construct was generated by removing all residues between I384 and the C-terminus in the Shaker-W434F-A359C background using two BglII restriction sites .",
"The resulting C-terminus became 373 . .",
". FKLSRHSKGLQIWLRYH* ( S4 underlined ) .",
"Site-directed mutagenesis and deletion mutagenesis were done with QuickChangeTM site-directed mutagenesis kits from Stratagene .",
"All of the cDNA clones were sequenced to verify mutations .",
"Oocytes from Xenopus laevis were surgically obtained .",
"Follicular membrane was removed with collagenase type 1 A ( C9891 Sigma , Oakville , Canada ) in a Ca2+-free solution ( 1 mg/ml ) .",
"cRNA was in vitro transcribed ( mMachine T7; Invitrogen ) , and 27–32 nl were injected into each oocyte with a concentration of 1 μg/μl using a ‘nano-injector’ ( Drummond Scientific Co . , Broomall , PA ) .",
"As control , we injected oocytes with the same volume of H2O .",
"After injection , oocytes were incubated at 18°C in modified Barth solution to allow the translation , processing and embedding of the proteins into the cell membrane .",
"The modified Barth solution contained ( mM ) : 90 NaCl , 3 KCl , 0 . 41 MgSO4 , 0 . 41 CaCl2 , 0 . 33 Ca ( NO3 ) , 5 HEPES , 2 . 5 sodium pyruvate , 100 U/ml pen-strep , 5% horse serum , adjusted to pH 7 . 6 with NaOH .",
"If not specified otherwise , electrophysiological recordings were performed 2–3 days after injection of cRNA .",
"N is stated in each experiment; data were obtained from at least 3 independent oocyte preparations .",
"HEK293 cells ( RRID: CVCL_0045 ) were cultured using standard tissue culture conditions ( 5% CO2 , 37°C ) in high glucose DMEM supplemented with FBS ( 10% ) , L-glutamine ( 2 mM ) , penicillin ( 100 U/ml ) , and streptomycin ( 10 mg/ml ) ( Gibco BRL Life Technologies , Burlington , ON , Canada ) .",
"Cells were generally grown to 50% confluency prior to transfection , and were transiently transfected with cDNA encoding Shaker-iVSD-A359C and a Zeocin-resistance-eGFP fusion protein by calcium phosphate precipitation .",
"Cells were plated on glass coverslips pre-treated with poly-D-lysine 6 hr post-transfection .",
"Whole-cell patch clamp experiments were performed 24~48 hr after transfection .",
"Transfected cells expressing GFP were identified under a fluorescent microscope ( Eclipse Ti , Nikon ) .",
"All experiments used Cl−-free solutions to minimize the contamination with endogenous oocyte Cl− currents , unless stated otherwise .",
"All electrophysiology and fluorescence recordings were carried out at room temperature ( ~20°C ) using the cut-open oocyte voltage-clamp , voltage-clamp fluorometry , and excised inside-out patch technique .",
"Cut-open oocyte voltage-clamp ( Taglialatela et al . , 1992 ) was performed with a CA-1B amplifier ( Dagan Corp . ) using GPatch software ( Department of Anesthesiology , University of California , Los Angeles , Los Angeles , CA ) as described earlier ( Batulan et al . , 2010; Haddad and Blunck , 2011 ) .",
"Oocytes were placed in an apparatus , comprising three electrically isolated chambers containing the external solution , and then permeabilized by adding 0 . 2% saponin to the external solution in the bottom chamber , giving electrical access to the cytosol .",
"Saponin was washed out and the bottom chamber filled with internal solution .",
"For experiments characterizing the gating current , the voltage-dependent fluorescence changes , the amplitude and the voltage dependence of ionic currents , the external solution contained ( mM ) 115 NMDG-MeSO3 , 10 HEPES , and 2 Ca ( OH ) 2 at pH 7 . 35 , and the internal solution contained 115 NMDG-MeSO3 , 10 HEPES , and 2 EDTA at pH 7 . 35 .",
"Solutions used to characterize the permeability of Shaker-iVSD-induced ionic current to different ions ( e . g . , NMDG+ , H+ , Na+ , K+ , and Cl− ) are described in the legend of Figure 2 .",
"The voltage electrode was filled with 3 M KCl .",
"Fluorometry was performed simultaneously with cut-open oocyte voltage-clamp .",
"For fluorescent labeling , Xenopus oocytes were incubated in a depolarizing solution ( 115 mM K-MeSO3 , 2 mM Ca ( OH ) 2 , 10 mM HEPES , at pH 7 . 1 ) containing 5 µM tetramethylrhodamine- 5-maleimide ( TMRM ) at room temperature for 20 min .",
"For fluorescence measurements , an upright fluorescence microscope ( Axioskop 2FS; Zeiss ) and a Photomax 200 photodetection system ( Dagan Corp . ) were used .",
"Voltage-dependent fluorescence changes were measured from TMRM fluorophores attached to the cysteine at position A359C , located at the extracellular end of the S4 segment .",
"For whole-cell patch clamp experiments , ionic currents from transfected HEK293 cells were recorded using pCLAMP software 10 . 2 and an Axopatch 200B amplifier ( Molecular Devices , Sunnyvale , CA , USA ) .",
"Patch electrodes were fashioned from borosilicate glass ( Corning 8161 ) with resistance about 1 . 5 MΩ when filled with intracellular solution .",
"Whole-cell currents were low-pass filtered at 2 kHz , digitized at a sampling rate of 100 µs during acquisition , and stored on a microcomputer equipped with an AD converter ( Axon Digidata 1322 A , Molecular Devices , Sunnyvale , CA , USA ) .",
"The pipette solution contained ( in mM ) 140 NMDG-MeSO3 , 10 EDTA , 10 HEPES at pH 7 . 35 .",
"The extracellular solution contained ( in mM ) 140 NMDG-MeSO3 , 1 Ca ( OH ) 2 , 1 Mg ( OH ) 2 , 10 HEPES , 10 Glucose at pH 7 . 35 .",
"High-buffer ( HB ) solutions were also used for isolated characterization of Shaker-iVSD-induced currents requiring manipulation of the pH gradient .",
"for clarity , the compositions hb solutions used to characterize the permeability of Shaker-iVSD-induced ionic current are described in the legend of Figure 2 .",
"for selectivity determination in oocytes , the nonspecific currents from mock ( H2O ) -injected oocytes exposed to the same ionic conditions as the experimental group was subtracted from steady-state Shaker-iVSD-induced currents , although they were small and negligible ( <2% of the total currents recorded from Shaker-iVSD-injected oocytes ) .",
"Fluorescence-voltage ( FV ) relations were calculated based on the peak change in emission of the total trace .",
"Most FV relationships were fit with a single Boltzmann function:dFF=11+exp ( ( V1/2−V ) dV ) where dF/F is the change in fluorescence amplitude normalized to maximal fluorescence amplitude , V1/2 is the half-activation potential , V is the membrane potential , and dV is the slope factor .",
"For the analysis of the non-conducting Shaker-W434F mutation fluorescence , some data were best fit with the sum of two Boltzmann functions:dFF=a11+exp ( V1/2 , 1−VdV1 ) + ( 1−a ) 11+exp ( ( V1/2 , 2−V ) dV2 ) where symbols are as described above , a refers to the fraction of each Boltzmann component .",
"The kinetics of the ionic and fluorescence traces during channel activation were fitted with a double-exponential function of the form:y ( t ) =Afast⋅ ( 1−e−tτfast ) +Aslow⋅ ( 1−e−tτslow ) where Afast and Aslow correspond to the amplitudes and τfast and τslow to the time constants of the fast and slow components , respectively , and t is time .",
"To determine the speed of recovery from mode shift ( relaxation ) , we measured fluorescence changes of Shaker-iVSD and –W434F by pulsing from 0 mV to −90 mV for various durations ( 5–5000 ms ) before applying a series of test pulses using the cut-open oocyte voltage-clamp technique .",
"For Shaker-iVSD , the V1/2 values of the FV curves as a function of the pre-pulse duration to −90 mV were obtained by fitting each curve to a single Boltzmann function .",
"For Shaker-W434F , FV curves were fitted with double Boltzmann functions , and the resulting distribution was a superposition of two Boltzmann curves representing the FV relation at a holding potential of 0 mV ( FV0 mV ) and the mode-shifted FV relationship when pre-pulse duration at −90 mV ≥ 1000 ms ( FV−90 mV ) .",
"The fractional amplitudes of FV−90 mV component are shown as a function of pre-pulse duration .",
"All the fluorescence , gating , and ionic current measurements were analyzed using a combination of Analysis software ( Department of Anesthesiology , University of California , Los Angeles ) and SigmaPlot for Windows version 12 . 5 ( SPS , Chicago , IL , USA ) .",
"Data reported throughout the text and figures are reported as mean ± SEM ."
]
] | [
"Domains in macromolecular complexes are often considered structurally and functionally conserved while energetically coupled to each other .",
"In the modular voltage-gated ion channels the central ion-conducting pore is surrounded by four voltage sensing domains ( VSDs ) .",
"Here , the energetic coupling is mediated by interactions between the S4-S5 linker , covalently linking the domains , and the proximal C-terminus .",
"In order to characterize the intrinsic gating of the voltage sensing domain in the absence of the pore domain , the Shaker Kv channel was truncated after the fourth transmembrane helix S4 ( Shaker-iVSD ) .",
"Shaker-iVSD showed significantly altered gating kinetics and formed a cation-selective ion channel with a strong preference for protons .",
"Ion conduction in Shaker-iVSD developed despite identical primary sequence , indicating an allosteric influence of the pore domain .",
"Shaker-iVSD also displays pronounced 'relaxation' .",
"Closing of the pore correlates with entry into relaxation suggesting that the two processes are energetically related ."
] | [
"Cells in the heart and other muscles rely on electrical signals to coordinate their activity .",
"They generate these electrical signals by controlling the movement of ions across the membrane that surrounds each cell .",
"Proteins called ion channels in this membrane form pores that allow particular types of ions to pass through .",
"The opening and closing of the pores is tightly controlled so that electrical signals are only generated at specific times .",
"The Shaker Kv channel is an ion channel that allows potassium ions to pass through the membrane .",
"This protein is made of several modules including one called the voltage sensing domain , which senses changes in the electrical voltage across the membrane to open or close the pore module .",
"However , it is not clear how voltage sensing domain and pore influence each other’s structure and behaviour .",
"To address this question , Zhao and Blunck investigated how the voltage sensing domain behaves in animal cells when the pore module is absent .",
"Zhao and Blunck show that , in the absence of the pore module , the voltage sensing domain becomes an ion channel that allows protons and other positively-charged ions to pass through the membrane .",
"Further experiments reveal this new channel opens at a voltage when the main Shaker Kv channel is usually closed .",
"This may result in small leaks of ions across the membrane that cause long-lasting changes in the timing , intensity and number of electrical signals in cells and organs .",
"The findings of Zhao and Blunck suggest that different modules of ion channels influence each other more than previously thought .",
"The next steps following on from this work are to explore how the voltage sensing domain channels open and close in more detail and to examine how the pore module influences this behaviour .",
"Investigating whether similar leaks occur in the voltage sensing domains of other ion channels may aid research into some inherited heart or neurological disorders where the channels are cut short between the voltage sensing domain and the pore module ."
] | 2016 |
[
"Introduction",
"Results and discussion",
"Materials and methods"
] | [
"cell biology",
"tools and resources",
"neuroscience"
] | Reconstruction of genetically identified neurons imaged by serial-section electron microscopy | elife-15015-v2 | [
[
"Relating neural circuits to the computations they perform requires mapping patterns of structural and functional connectivity among neurons .",
"Innovative light microscopic methods such as GRASP , trans-synaptic viral tracing and super-resolution imaging enable visualization of synapses made on or by identified neurons ( Wickersham and Feinberg , 2012; Tønnesen and Nägerl , 2013 ) .",
"At present , however , only electron microscopy ( EM ) provides sufficient resolution to visualize the complete complement of synapses that neurons form and receive .",
"Indeed , large-scale reconstructions from serial sections have provided deep insights into neuronal circuit principles ( Briggman et al . , 2011; Bock et al . , 2011; Takemura et al . , 2013; Morgan et al . , 2016 ) .",
"The optimal strategy is to collect serial sections containing all circuit elements and image them at nanometer resolution .",
"Processes in the imaged volumes are then segmented to reconstruct the neurons ( or parts of neurons ) they contain .",
"Advances in sectioning , imaging and segmentation methods make so-called 'saturated' reconstruction of volumes around 1000 μm3 feasible ( Kasthuri et al . , 2015 ) .",
"Even these modest volumes remain challenging , however , and when multiple samples must be compared -e . g . , controls vs . mutant or treated vs . untreated animals - this approach is currently out of reach ( Plaza et al . , 2014 ) .",
"An attractive alternative is 'sparse' reconstruction of specific cells within a fully imaged volume .",
"For example , neuronal activity can be monitored using calcium indicators , then neurons with particular patterns of activity can be relocated in thin sections and reconstructed ( Briggman et al . , 2011; Bock et al . , 2011 ) .",
"This method is , however , technically demanding and infeasible in many tissues .",
"We therefore devised an alternative approach to sparse reconstruction that relies on marking specific cells with an electron-dense tracer .",
"We then exploit the high contrast provided by the tracer to speed up imaging and reconstruction , which are currently the rate-limiting steps in connectomic analysis .",
"Our pipeline includes the following series of steps:",
"( a ) tagging a specific cell type with a genetically encoded EM tracer ,",
"( b ) enhancing the electron-density of the stain without compromising ultrastructure of the surrounding tissue ,",
"( c ) imaging the cell rapidly at relatively low resolution ,",
"( d ) re-imaging selected small volumes at higher resolution to map connectivity and",
"( e ) segmenting the cell using a novel algorithm that is reliable , fast and does not require computationally intense pre-training .",
"Together , the gains from targeted reimaging and unsupervised segmentation decrease the time required for reconstruction by over two orders of magnitude .",
"We call the method ARTEMIS for Assisted Reconstruction Technique for Electron Microscopic Interrogation of Structure ."
],
[
"A classical ultrastructural tracer is horseradish peroxidase ( HRP ) , which catalyzes the formation of a 3 , 3’-diaminobenzidine ( DAB ) polymer; the polymer binds osmium and is thereby rendered electron-dense .",
"Recombinant HRP is enzymatically inactive in the cytosol because it fails to form disulfide bonds or become glycosylated , but this limitation can be overcome by directing the protein to topologically extracellular compartments such as vesicles ( Li et al . , 2010; Atasoy et al . , 2014; Schikorski et al . , 2007 ) .",
"We therefore generated an HRP variant that was codon-optimized , mutated to increase activity , and fused to an endoplasmic reticulum targeting sequence ( erHRP ) .",
"We also tested derivatives of plant ascorbate peroxidases called APX and APEX2 , which are active in the cytosol ( Martell et al . , 2012; Lam et al . , 2015 ) .",
"Initial studies using cultured HEK293 cells confirmed that all three constructs generated active peroxidase in the transfected cells ( Figure 1a , b and data not shown ) . 10 . 7554/eLife . 15015 . 003Figure 1 . Enhanced staining of genetically encoded tags for EM .",
"( a–b )",
"Bright-field images of HEK-cells rendered photon-dense by DAB polymerization , catalyzed either by APEX2 tagged with a nuclear export signal ( APEX2NES )",
"( a ) or endoplasmic reticulum tagged HRP ( erHRP )",
"( b ) .",
"( c–d )",
"Bright-field images of direction-selective retinal ganglion cells ( ooDSGC ) expressing APEX2NES",
"( c ) or erHRP",
"( d ) and rendered photon-dense as in",
"( a ) .",
"( e ) Enhanced staining strategy .",
"( f–g )",
"EM-micrographs of HEK-cells rendered electron-dense after DAB-polymerization as in",
"( a ) .",
"A standard EM staining protocol showed no detectable cytosolic DAB-polymer staining",
"( f ) , whereas the addition of a reduction step dramatically enhanced DAB-polymer staining",
"( g ) .",
"( h ) Ribbon synapses in the outer plexiform layer; ultrastructure is well preserved after reduction .",
"( i ) EM-micrograph of an ooDSGC soma ( arrowhead ) rendered electron dense after tissue reduction next to a non-expressing RGC ( asterisk ) .",
"( j ) Close-up of cytosolic APEX2 staining .",
"( k ) Dendritic processes expressing APEX2 ( asterisk ) contacted by a presynaptic partner ( arrowhead ) in the inner plexiform layer .",
"( l ) Axonal long-range projections of an APEX2-expressing ooDSGC in the superior colliculus ( asterisk ) with a postsynaptic target ( arrowhead ) .",
"( m ) erHRP-expressing ( arrowhead ) RGC next to a non-expressing RGC ( asterisk ) .",
"( n , o )",
"Close-up of erHRP staining of a J-RGC soma",
"( n ) and dendrite",
"( o ) ( arrowhead point to presynaptic partners ) .",
"( c , d , i-l ) are from Cart-cre mice; ( m–o ) from Jam-B-creER mice .",
"Scale bars: ( a–d ) :25 μm; ( f–g ) : 10 μm; I , m : 5 μm; h , j−l , n , o : 500 nm;DOI: http://dx . doi . org/10 . 7554/eLife . 15015 . 00310 . 7554/eLife . 15015 . 004Figure 1—figure supplement 1 . DAB-polymer enhancement .",
"( a ) EM-micrograph of an APEX2NES expressing process .",
"Long DAB reaction times ( 5 hr ) allowed visualizing the DAB-polymer using a standard EM-staining protocol , but severely deteriorated the quality of the ultrastructure .",
"( b ) EM-micrograph of an RGC using a fixation and staining protocol optimized to preserve good ultrastructure .",
"Under these conditions , DAB-polymer signals were absent or very weak , as exemplified by the weakly stained soma ( encircled with asterisk ) and its corresponding unstained process ( encircled ) .",
"( c–f )",
"EM-micrograph of APEX2NES expressing HEK-cells fixed with the same protocol used for retinas and stained for EM after the DAB reaction .",
"Four conditions were tested: the osmium only ( c , d ) or rOTO stainings ( e , f ) with the standard and reduction protocol ( see Materials and methods ) .",
"Using the standard protocol , no or very weak signal was detected using the osmium",
"( c ) or the rOTO",
"( e ) staining , respectively .",
"Asterisks in",
"( e ) depict weakly stained HEK-cell .",
"In contrast , reducing the tissue dramatically enhanced the signal in both conditions ( d–f ) .",
"( g–h )",
"Bright-field images of erythrocytes in retinal tissue after DAB-polymerization ( arrowheads ) before",
"( g ) and after the reduction step",
"( h ) .",
"The heme content in erythrocytes catalyzes the DAB reaction producing a strong precipitate that is enhanced by a reduction step .",
"( i–j )",
"EM-micrograph of erythrocytes using the rOTO staining protocol in standard",
"( i ) and reduced",
"( j ) conditions .",
"The reduction enhances the DAB-polymer’s affinity to osmium .",
"Scale bars:",
"( a ) : 1 μm;",
"( b ) : 5 μm; c–f: 10 μm; g–h : 20 μm; i–j : 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 15015 . 00410 . 7554/eLife . 15015 . 005Figure 1—figure supplement 2 . Cytosolic APEX2 . EM-micrograph of a mouse direction selective RGC soma rendered electron dense after tissue reduction ( arrowhead ) .",
"Note the small process visible in the inner plexiform layer ( boxes , enlarged image on the right ) and the strongest signal of the erythrocytes ( asterisk ) .",
"Scale bar: 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 15015 . 00510 . 7554/eLife . 15015 . 006Figure 1—figure supplement 3 . Endoplasmic reticulum tagged HRP ( erHRP ) .",
"( a ) Bright-field images of Cre-dependent virally infected mouse direction-selective retinal ganglion cell ( ooDSGC ) expressing erHRP and rendered photon-dense .",
"( b ) EM-micrograph of a vertical retinal cross-section with an erHRP expressing RGC .",
"Asterisk depicts erHRP-expressing soma .",
"GCL: ganglion cell layer , IPL: inner plexiform layer , INL , inner nuclear layer , OPL , outer plexiform layer , ONL , outer nuclear layer , PL: photoreceptor layer .",
"( c ) erHRP expressing soma .",
"( d–e ) erHRP expressing dendritic compartments in the IPL .",
"( f ) Micrograph of ribbon synapses in the IPL ( arrowheads ) .",
"Ultra-structural quality is preserved .",
"Scale bars: a :20 μm; b:10 μm; c:5 μm; d : 1 μm; e–f : 500 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 15015 . 00610 . 7554/eLife . 15015 . 007Figure 1—figure supplement 4 . Chemical tissue reduction improves contrast-to-noise ratio between membrane and cytosol .",
"( a–b ) .",
"EM-micrographs of the inner plexiform layer of mouse retinas not treated with DAB and stained with the standard",
"( a ) or reduction protocol",
"( b ) .",
"Note the enhancement of the membrane and synaptic density stain in",
"( b ) compared to",
"( a ) .",
"( c–d )",
"Intensity histograms of manually selected membrane or cytosol pixels from standard and reduced EM micrographs .",
"The reduction shifts the distribution of the membrane pixels ( black traces ) by 24 intensity values to a lower mean value ( μ ) compared to the cytosolic distribution ( gray traces; Intensity values: 0 = dark; 255 = white ) .",
"Thus , the reduction step increases the electron density of membranes specifically .",
"Orange traces: Gaussian fits .",
"The contrast-to-noise value is defined as: CNR= μ2−μ1σ22+σ12 Contrast-to-noise improves by 62% by reducing the tissue and therefore could be a valuable signal enhancing step to improve automatic segmentation results that are based on machine-learning approaches .",
"( e ) Blind-to-condition selection of synaptic quality based on randomized pairwise comparisons .",
"Scale bar: 1 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 15015 . 00710 . 7554/eLife . 15015 . 008Figure 1—figure supplement 5 . Synaptic contacts received and made by peroxidase expressing cells .",
"( a ) EM-micrographs of the inner plexiform layer of mouse retinas expressing APEX2 in ooDS ganglion cells contacted by presynaptic partners ( arrowheads ) .",
"( b ) EM-micrographs of the upper layers of the superior colliculus including axonal termini of APEX2 in ooDS ganglion cells contacting postsynaptic partners ( arrowheads ) .",
"( c ) EM-micrographs of the inner plexiform layer of retinas expressing erHRP in ganglion cells ( asterisks ) contacted by presynaptic partners ( arrowheads ) .",
"Scale bars: 500 nmDOI: http://dx . doi . org/10 . 7554/eLife . 15015 . 008 For selective expression in molecularly-identified cells , we generated adeno-associated viral ( AAV ) vectors in which expression of erHRP , APX or APEX2 required Cre-dependent recombination .",
"These were used to infect retinas of transgenic mice in which specific retinal ganglion cell ( RGC ) types expressed Cre recombinase ( ooDSGCs in Cart-cre [Kay et al . , 2011] ) or tamoxifen-activated Cre ( J-RGCs in JAM-B-creER [Kim et al . , 2008; Joesch and Meister , 2016] ) .",
"Two to four weeks after infection , retinas were reacted with DAB and H2O2 and examined light-microscopically , revealing intense labeling of RGCs ( Figure 1c , d ) .",
"Retinas were then processed for EM ( see Methods ) .",
"Unexpectedly , levels of electron-dense precipitate were so low that stained processes could not be traced reliably ( Figure 1—figure supplement 1a , b ) .",
"Numerous alterations to balance peroxidase activity and ultrastructural quality failed to improve matters: when ultrastructure was adequately preserved , staining for peroxidase was poor , and when reaction product was adequate , synaptic structures were poorly preserved .",
"This difficulty may have been less apparent in previous studies using injected native HRP , which has substantially higher activity than the recombinant proteins we use .",
"The reason for the difference between light and electron microscopic results is likely that the opacity and electron density of the DAB polymer arise in different ways: its polycyclic structure renders it photon absorbent , but its electron-density results from redox reactions with osmium ( Bahr , 1954 ) .",
"We reasoned that radicals produced during the long peroxidase reaction might oxidize the relevant functional groups in the polymer , leaving it photon-absorbent but inert to osmium .",
"If this were true , reduction of functional groups on the polymer could restore reactivity to osmium ( Figure 1e ) .",
"We tested this hypothesis in transfected HEK cells .",
"When cells were treated with the protocol we had used for retina , the precipitate was clearly visible by light but not electron microscopy .",
"However , when the HEK cells were treated with a mild reducing agent ( 5 mM sodium hydrosulfite ) between the peroxidase reaction and osmication , they were highly electron-dense ( Figure 1f , g ) .",
"This was the case using either conventional osmium staining or an enhanced 'double osmium' staining protocol ( rOTO ) , although the latter showed a slight improvement probably due to the change in the redox state of osmium tetroxide ( Figure 1—figure supplement 1c–f; see Methods ) .",
"A similar effect was observed in erythrocytes , in which endogenous heme catalyzes the DAB reaction ( Figure 1—figure supplement 1g–j ) .",
"When the reduction protocol was applied to retinas , we were able to visualize RGCs that had been tagged with APX , APEX2NES ( APEX2 fused to a nuclear export signal ) or erHRP ( Figure 1i–o , Figure 1—figure supplement 2 and 3 ) .",
"As expected , APX and APEX2NES labeled the cytoplasm diffusely ( Figure 1j ) while erHRP labeled membrane-bound intracellular compartments ( Figure 1n ) .",
"Thus they could be used as orthogonal labels , although we have not yet pursued this application .",
"The strength of the signal allowed us to identify small stained dendritic profiles ( Figure 1k , o ) and to view the terminals of RGCs in the superior colliculus , approximately 1 cm from the somata ( Figure 1l ) .",
"Remarkably , the reduction protocol actually improved the visualization of the ultrastructure irrespective of peroxidase expression or DAB treatment ( Figure 1h ) .",
"Part of the improvement resulted from an increase in the reactivity of membranes to osmium , thereby enhancing membrane-cytoplasm contrast ( Figure 1—figure supplement 4 ) .",
"This improvement is also visible in the staining strength of synaptic densities ( Figure 1—figure supplement 4a , b ) .",
"When we asked blind-to-condition observers to judge the quality of synapses between both conditions , the reduced tissue was selected ~3 times more frequently than unreduced tissue ( Figure 1—figure supplement 4e ) .",
"Thus , rather than sacrificing ultrastructure for reactivity or vice versa , this protocol improved both .",
"For the approach to be useful , it is essential that peroxidase expression does not affect synapse formation and that synaptic partners of peroxidase expressing cells can be identified .",
"When analyzing our datasets , we could not detect any structural differences between tissues expressing either of the peroxidases .",
"The number of synapses counted in peroxidase expressing and control sections was also similar , 0 . 32 and 0 . 34 synapses / μm2 , respectively .",
"To test whether the electron dense precipitate hinders reliable synaptic identification , we characterized the synaptic connections received and made by APEX2 or erHRP expressing retinal ganglion cells ( Figure 1k , o; Figure 1— figure supplement 5 ) .",
"Cells presynaptic to APEX2 or erHRP could be clearly identified based on ultrastructural details ( Figure 1—figure supplement 5a , c ) .",
"Postsynaptic partners of APEX2-expressing cells could also be identified .",
"Moreover , synaptic vesicles of APEX2-expressing neurons could be detected in presynaptic terminals because they were unstained , and thereby contrasted with the electron-dense cytosol .",
"However , the endoplasmic reticulum did not regularly extend to axonal terminals , making it difficult to identify postsynaptic partners of erHRP expressing ganglion cells .",
"To test the generality of the method we expressed APEX fused to GFP in direction-selective tangential cells of Drosophila melanogaster ( Joesch et al . , 2008 ) ( DB331-Gal4 → UAS-APEX-GFP; see Methods ) .",
"This driver line expresses mainly in 6 vertically sensitive and 3 horizontally sensitive tangential cells .",
"This expression pattern was apparent using either GFP fluorescence or the polymerized DAB to mark the cells ( Figure 2a , b ) .",
"We were also able to visualize the electron dense precipitate of these processes in the fly’s optic lobes ( Figure 2c , d ) .",
"Although our method was optimized for mammalian tissue , ultrastructural detail was reasonable ( Figure 2e ) and allowed the identification of synaptic contacts made by and onto APEX expressing cells ( Figure 2f–h ) . 10 . 7554/eLife . 15015 . 009Figure 2 . APEX expressing interneurons in Drosophila melanogaster .",
"( a ) Fluorescent image of a brain of Drosophila melanogaster ( DB331-Gal4 → UAS-APEX-GFP ) expressing APEX in direction-selective lobula plate tangential cells ( LPTCs ) .",
"( b ) Bright field image of a similar plane from another ( DB331-Gal4 → UAS-APEX-GFP ) fly , labeled with DAB .",
"( c ) Electron micrograph of a frontal brain section ( 30 nm thickness ) containing electron dense staining in axonal processes of LPTCs ( arrowhead and framed box ) .",
"( d ) Enlarged view of the framed region in",
"( c ) .",
"Arrows point to electron dense processes .",
"( e ) Unstained synaptic terminal , showing quality of ultrastructural detail .",
"( f ) Synaptic nerve terminal in an APEX-positive process , identifiable by contrast-reversed vesicles .",
"( g , h )",
"APEX-positive postsynaptic processes , identifiable by the presence of adjacent vesicle-laden , T-bar-containing terminals .",
"Scale bars: a–c :100 μm; d :10 μm; e–g : 500 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 15015 . 009 To implement sparse reconstruction , we used the ATUM ( Hayworth et al . , 2014 ) ( automated tape-collecting ultra-microtome ) to serially section retinas containing either APEX2NES-expressing retinal ganglion cells ( J-RGCs ) or retinal interneurons ( starburst amacrine cells ( SACs ) ) labeled using Choline Acetyltransferase-Cre ( Rossi et al . , 2011 ) .",
"Because we could identify small peroxidase-stained neurites at overview resolution ( 20–30 nm per pixel ) , we tested the minimal requirements to reconstruct both cell types at these resolutions .",
"Rapidly imaging every 10th section ( 270 nm separation ) , at 30 nm resolution was sufficient for mapping a J-RGC dendrite ( 144 sections covering > 1 × 107 µm3 , imaged in 22 hr; Figure 3a , b ) .",
"By comparison , imaging the same volume at high resolution ( 4 nm/pixel ) would have taken ~2500 hr on the same microscope .",
"Manual reconstruction of the J-RGC was straightforward and took 2 worker-hours ( Figure 3c ) .",
"To extend this result to another cell type , we imaged all 1260 sections from a bloc with APEX2NES-positive SACs and reconstructed SAC processes in this volume ( Figure 3d–f ) .",
"Thus , we can reliably find and reconstruct genetically identified cells . 10 . 7554/eLife . 15015 . 010Figure 3 . Targeted interrogation of serial-section EM volumes in mouse retina .",
"( a ) .",
"Overview-resolution EM volume containing J-RGCs expressing APEX2NES .",
"Every 10th section was imaged at 30 nm/pixel .",
"( b ) Two consecutive sections containing APEX2NES expressing J-RGC processes ( arrows ) .",
"( c ) Reconstructed J-RGC .",
"Note the reduced complexity of the dendritic field due to the subsampling of the overview dataset .",
"( d ) Overview-resolution EM volume that contains SACs expressing APEX2NES .",
"The sections were imaged at 20 nm/pixel .",
"( e ) Two consecutive sections showing a SAC-SAC contact ( arrowheads ) .",
"( f ) Reconstruction of the SAC-SAC interaction in",
"( e ) ; arrowhead indicates contact shown in",
"e . ( g–h ) .",
"Comparison of ultrastructural detail visible at 20 nm/pix",
"( g ) or 4 nm/pix",
"( h ) .",
"Although both images contain ribbon synapses , the low-resolution image",
"( g ) lacks the required resolution to identify them .",
"Arrowheads point to ribbons .",
"( i ) High-resolution ( 4 nm/pixel ) EM subvolume selected from",
"( d ) .",
"( j ) Two high-resolution EM-micrographs containing SAC-processes expressing APEX2NES receiving synaptic contacts ( arrowheads ) .",
"( k ) Reconstruction of the SAC plexus with its characteristic dendritic fasciculation and net-like structure .",
"Asterisk indicates contact and arrowheads the synaptic inputs shown in j ( top panel ) .",
"Scale bars: b: 5 μm; c: 50 μm; e , j: 1 μm; g–h: 200 nm . DOI: http://dx . doi . org/10 . 7554/eLife . 15015 . 010 Although 20 nm resolution is sufficient for viewing neurites , it does not allow optimal visualization of synaptic contacts ( Figure 3g–h ) .",
"However , sections generated using ATUM are collected on wafers , and can be re-imaged; a program , Wafer-Mapper ( Hayworth et al . , 2014 ) , facilitates returning to the same place on a given section with micron-level precision .",
"This feature allows targeted imaging at high resolution of areas chosen from the rapidly acquired , lower resolution reconstruction , thereby substantially reducing imaging time .",
"To test this multi-scale approach , we focused on the SACs , whose dendrites can be less than 100 nm in diameter .",
"We selected a heavily-labeled ~5 × 105 µm3 volume from the overview-resolution reconstruction of ~2 × 107 µm3 and acquired a high-resolution ( 4 nm/pixel ) data set in 250 hr ( Figure 3i ) .",
"Compared to a completely high-resolution approach , this amounts to a 40-fold reduction in imaging time .",
"More importantly , it expedited reconstruction , the current time-limiting step in connectomics ( Plaza et al . , 2014 ) , by constraining efforts to defined regions of interest .",
"We manually traced and reconstructed the SAC plexus in this volume , observing multiple SAC-SAC interactions , dendritic fasciculation in the SAC plexus and dendritic branching ( Figure 3j , k and data not shown ) .",
"Finally , to further expedite the reconstruction pipeline , we developed an algorithm that detects and segments APEX2-positive processes , taking advantage of the high contrast rendered by the DAB polymer .",
"Our algorithm is fast , parameterized and does not rely on the supervised machine learning training regimes currently required for lower contrast material ( Kasthuri et al . , 2015 ) ( Figure 4a , Materials and methods ) .",
"We tested the algorithm using 401 sections from the high-resolution dataset ( Figure 3i ) .",
"We hand-segmented a set of 444 APEX2 positive segments , and then compared these results to those obtained computationally .",
"We calculated a recall statistic of 91 . 8% for the 2D segmentation portion of the algorithm ( Figure 4b , c ) .",
"All missed segments were small ( on average 45 × 42 pixels ) , and were seldom necessary to establish connectivity .",
"Consistent with human inferred segmentation , the analysis of segment adjacency in local neighborhoods can make connections in regions with small gaps .",
"To test the accuracy of the algorithm for longer ( >50 μm ) processes , we segmented and reconstructed a dendritic branch of a starburst amacrine cell .",
"Our algorithm could reconstruct the main characteristics of the dendritic processes and recapitulated most of the manually segmented details ( Figure 4d , e ) .",
"The primary advantage of this approach is processing time , being two orders of magnitude faster than approaches based on convolutional neural networks for membrane classification ( Kasthuri et al . , 2015; Kaynig et al . , 2015 ) .",
"Training regimes in the neural network approaches require adjusting at least thousands of parameters separately for each new tissue; in contrast , our algorithm incorporates unsupervised components with a small fixed set of tunable parameters ( see Methods and Supplementary Code ) .",
"Importantly , the high contrast-to-noise ratio of the peroxidase-labeled cells is the enabling factor for the efficacy of this algorithm , which means that it could be used for reconstruction of DAB-stained structures in any tissue .",
"Thus , although detailed synaptic properties may be obscured by the electron-dense stain , our methods are well-suited for rapid reconstruction of targeted neuron morphology and connectivity . 10 . 7554/eLife . 15015 . 011Figure 4 . Automatic segmentation and reconstruction algorithm for APEX2 positive processes .",
"( a ) The algorithm performs unsupervised 2D segmentation and 3D reconstruction without the need of pre-training .",
"The procedure follows these steps:",
"1 . Clustering-based image thresholding over pixel intensities from known contrast-enhanced ranges , modulated via quality assessment;",
"2 . 2D segmentation based on unsupervised clustering;",
"3 . Calculation of geometric properties for each identified segment , followed by noise and artifact pruning based on these properties;",
"4 . Identification of large segments based on segment properties;",
"5 . Graph-based segment search for reconstruction and labeling in 3D , followed by a final merging procedure to enforce consistency across the volume .",
"( b ) Unsupervised automatic segmentation of mouse SAC processes .",
"Top: manual segmentation , ground-truth; bottom: automatic segmentation .",
"( c ) .",
"Reconstructions of the dendritic process in",
"( b ) , comparing manual ( top ) and automatic reconstructions .",
"( d ) Manual reconstruction of a starburst amacrine cell process .",
"En face ( top ) and side view ( bottom ) .",
"( e ) Automatic reconstruction of the same process shown in",
"d . En face ( top ) and side view ( bottom ) .",
"Scale bar in b: 1 μm; d , e: 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 15015 . 011 In summary , we have assembled a set of tools that enables rapid reconstruction of genetically identified neurons , so that their shapes and connections can be mapped at high resolution in much less time than required for conventional imaging and segmentation protocols .",
"By expediting the analysis of neural motifs , ARTEMIS renders the interrogation of diverse samples feasible and holds a clear promise to unravel mechanisms ranging from neuronal development to computations ."
],
[
"Animals were used in accordance with NIH guidelines and protocols approved by Institutional Animal Use and Care Committee at Harvard University .",
"JAM-B-creER mice ( Kim et al . , 2008 ) were generated in our laboratory .",
"ChaT-cre mice ( Rossi et al . , 2011 ) , and Cart-cre mice ( Madisen et al . , 2010 ) were obtained from Jackson Laboratories .",
"In the Chat-cre line , the Cre recombinase gene was targeted to the endogenous Chat gene; this line expresses Cre in starburst amacrine cells ( SACs ) .",
"In the Cart-cre transgenic line , Cre expression is controlled by regulatory elements from the Cartpt gene .",
"In this line , Cre marks ON-OFF direction selective retinal ganglion cells .",
"Mice were maintained on a C57/BL6J background .",
"Both male and female mice were used in this study .",
"Animals were 60 to 100 days old at the time of euthanasia .",
"Flies were raised on standard cornmeal-agar medium .",
"The DB331 Gal4-line ( Joesch et al . , 2008 ) was kindly provided by Vivek Jayaraman ( Janelia Research Campus ) and the UAS-APEX-GFP ( Chen et al . , 2015 ) line was generated and kindly provided by Chiao-Lin Chen and Norbert Perrimon ( Harvard Medical School and HHMI ) .",
"The endoplasmic reticulum-targeted HRP ( erHRP ) was designed as follows .",
"First , to improve expression in mammals , the nucleotide sequence of HRP ( horseradish peroxidase from a plant , Armoracia rusticana ) was codon-optimized ( DNA2 . 0 , Menlo Park , CA ) .",
"Second , to regulate protein trafficking a signal secretion sequence from the human immunoglobulin kappa chain ( from pDisplay , Invitrogen , Carlsbad , CA ) as well as an endoplasmic reticulum ( ER ) -retention signal ( -KDEL ) were appended at the N- and C-termini , respectively .",
"Finally , the N175S mutation was introduced to confer heat stability and resistance to H2O2 ( Morawski et al . , 2001 ) .",
"The sequence of this cDNA is available from GenBank #KU504630 .",
"Ascorbate peroxidase ( APX , dimeric ) from pea ( Martell et al . , 2012 ) , and the enhanced monomeric version APEX2 ( derived from soybean APEX ) ( Lam et al . , 2015 ) were codon-optimized for better expression in mammals .",
"In APEX2NES , the nuclear export signal ( NES ) was appended to APEX2 .",
"The erHRP , APX , and APEX2NES plasmids under CMV promoter were transfected to HEK293T cells ( ATCC ) using the DMRIE-C transfection reagent ( Life Technologies ) .",
"Subsequently , the cDNAs were cloned into a plasmid encoding a Cre-dependent Adeno-associated virus ( AAV ) backbone with the CAG ( CMV-beta actin promoter + beta-globin leader ) promoter , woodchuck post-transcriptional element ( WPRE ) , and the FLEX switch ( Atasoy et al . , 2008 ) ( Supplementary file 1 ) .",
"AAV viruses were generated by transfecting these plasmids together with appropriate helper plasmids , and prepared using a chemical precipitation method ( Guo et al . , 2012 ) .",
"Plasmids encoding the viral vectors will be sent to AddgenePlasmids described in this paper are available from Addgene ( www . addgene . org ) .",
"For viral-mediated gene transfer , adult Cre-mice were anaesthetized with ketamine/xylazine by intraperitoneal injection .",
"A 30 1/2G needle was used to make a small hole in the temporal eye , below the cornea .",
"1 μl of vitreous fluid was withdrawn and then 1 μl of rAAV2 or rAAV2/9 Cre-dependent viruses ( a titre of ~1 × 1011–12 genome copies per ml ) was injected into the subretinal space with a Hamilton syringe and 33G blunt-ended needle .",
"Animals were euthanized and retinas were dissected 4–6 weeks following injection .",
"Mouse retinas were dissected from eyecups in oxygenated Ames’ medium ( Sigma ) with constant bubbling ( 95% O2 , 5% CO2 ) at room temperature .",
"Four incisions were made to flat-mount the retina with ganglion cells facing up onto nitrocellulose filter paper .",
"The tissue was drop-fixed , with 2% PFA and 2 . 5% glutaraldehyde followed by 2 . 5% glutaraldehyde ( times specified in Supplementary file 2 ) then washed .",
"HEK-cells were fixed with 2% PFA and 2 . 5% glutaraldehyde for 15 min followed by a 45 min fix in 2 . 5% glutaraldehyde .",
"Flies were decapitated and dissected in oxygenated Ringer solution .",
"A small incision was made on the back of the head and the posterior cuticle was separated from the head .",
"This ensured that the fixative and staining solutions could penetrate into the brain while the rest of the cuticle protected brain tissue during processing .",
"Flies were fixed with 2% PFA and 2 . 5% glutaraldehyde for 15 min followed by a 45 min fix in 2 . 5% glutaraldehyde .",
"Following aldehyde fixation , cells and tissues were washed , reacted with DAB to reveal sites of peroxidase activity , washed again , reduced with 50 mM sodium hydrosulfite and stained with osmium .",
"Osmium treatments included 2% aqueous osmium tetroxide ( used only in HEK cell micrographs of Figure 1—figure supplement 1 ) or the reduced osmium tetroxide-thiocarbohydrazide ( TCH ) -osmium ( 'rOTO' ) ( Willingham and Rutherford , 1984; Hua et al . , 2015; Tapia et al . , 2012 ) protocol ( all other Figures ) .",
"Due to the two consecutive osmication steps , the 'rOTO' protocol improves the signal of membranes compared to the aqueous osmium .",
"This enables reasonable signal-to-noise ratios at high scanning , an essential requirement for our approach .",
"Finally , the stained tissue was dehydrated and infiltrated with Durcupan resin .",
"Sodium-cacodylate ( cat . no . 12300 ) , glutaraldehyde ( 16220 and 16120 ) , paraformaldehyde ( 15710 ) , osmium tetraoxide ( 19190 ) , maleic acid ( 18150 ) , acetone ( glass distilled; 10015 ) and uranyl acetate ( 22400 ) were purchased at Electron Microscopy Sciences ( EMS ) ; AMES medium ( A1420 ) , 3 , 3’-diaminobenzidine ( DAB; D5905 ) , potassium hexacyanoferrate ( II ) ( P9387 ) , thiocarbohydrazide ( 88535 ) , sodium hydrosulfite ( 157953 ) and durcopan resin ( 44610 ) were purchased at Sigma-Aldrich .",
"Concentrations and incubations times , along with details on reagents are provided in Supplementary files 2 and 3 .",
"For the image in Figure 2a , the fly brain was fixed in 2% paraformaldehyde in PBS for 30 min on ice , washed with PBS and blocked with 3% goat serum/1% Triton X-100/PBS .",
"For staining , tissue was incubated with 3% goat serum/1% Triton X-100/PBS and rabbit anti-GFP Alexa Fluor 488 conjugate ( dilution 1:1000 , Invitrogen , A-21311 ) at 4°C for 1 days and washed with PBS .",
"Brains were mounted on Vectashield mounting medium ( Vectorlabs ) and imaged in a confocal microscope ( Olympus FVA ) .",
"For the image in Figure 2b , brains were prepared for electron microscopic and imaged before osmication .",
"The cured blocks were trimmed to a 2 × 3 mm rectangle and a depth of 400 µm and then readied for automated serial sectioning .",
"The automated , unattended collection of ~ 30 nm serial sections was accomplished using a custom tape collection device ( ATUM ) ( Hayworth et al . , 2014 ) attached to a commercial ultramicrotome .",
"The sections were collected on plasma-treated carbon-coated polyamide ( Kapton , Sheldahl ) 8-mm-wide tape .",
"Sections were post-stained with 1% uranyl acetate in maleate buffer for 30 s and with 3% Lead Citrate ( Ultrostain II; Leica - cat . no . 16707235 ) for 30 s .",
"An automated protocol to locate and image sections on the wafers was used ( Hayworth et al . , 2014 ) with a Sigma scanning electron microscope ( Carl Zeiss ) , equipped with the ATLAS software ( Fibics ) .",
"Images were acquired using secondary electron detection .",
"For the medium- and high-resolution data sets , non-affine alignment was accomplished through the FijiBento alignment package ( https://github . com/Rhoana/FijiBento ) that enables the alignment of large data sets in a relatively short period of time .",
"To this end , the alignment was performed on the Odyssey cluster supported by the FAS Division of Science , Research Computing Group at Harvard University .",
"The aligned images were then manually segmented using a custom volume annotation and segmentation tool ( VAST; http://openconnecto . me/Kasthurietal2014/Code/VAST ) .",
"The segmented images were processed for data analysis and 3D modeling with Matlab scripts , and Persistence of Vision Raytracer ( http://www . povray . org/ ) for rendering steps .",
"Full resolution EM images ( 100000 × 50000 pixels ) underwent contrast adjustment via histogram equalization to normalize image intensities across slices .",
"For each normalized image , a global threshold τ was computed via clustering-based image analysis ( Bankman , 2008 ) , modulated by the known contrast-enhanced pixel ranges of the ARTEMIS markers for a data set ( e . g . , for Suppl . Figure 4b: if τ < 0 . 6 , τ = τ * 0 . 82 , else τ = τ * 0 . 86 ) .",
"The normalized images were then converted to a binary representation , and clustering-based image thresholding was applied again , assuming two classes ( positive and negative ) , to gather 2D candidate segments .",
"To remove artifacts such as speckle noise , and holes in the tissue , only segments that satisfy the following constraints were stored: 3 pixels < area < 130 , 000 pixels and major axis length < 900 pixels .",
"The remaining segments were assigned a unique label , and stored in a MySQL database , along with their coordinates and other geometric properties Based on the expected morphological progression of each process , irregularly large imaging artifacts that appear between adjacent segments in the volume were pruned .",
"Graph-based segment search over the database entries established the connectivity between segments , including those with gaps between them , by finding the minimum distance between centroid points from all pairs of segments in a local neighborhood using a k-Nearest Neighbors-like ( Hastie et al . , 2009 ) algorithm ( k = 2 ) .",
"Using the graph as a guide , 2D segments were merged into the final 3D reconstruction .",
"We included the source code in the supplementary information , packaged as a Matlab live script , with example images and an animation .",
"All relevant data is available on request .",
"The electron microscopic data set of Figure 3k , which includes the segmented processes used for the reconstructions , has been deposited to Dyrad .",
"Doi:10 . 5061/dryad . h67t6 ."
]
] | [
"Resolving patterns of synaptic connectivity in neural circuits currently requires serial section electron microscopy .",
"However , complete circuit reconstruction is prohibitively slow and may not be necessary for many purposes such as comparing neuronal structure and connectivity among multiple animals .",
"Here , we present an alternative strategy , targeted reconstruction of specific neuronal types .",
"We used viral vectors to deliver peroxidase derivatives , which catalyze production of an electron-dense tracer , to genetically identify neurons , and developed a protocol that enhances the electron-density of the labeled cells while retaining the quality of the ultrastructure .",
"The high contrast of the marked neurons enabled two innovations that speed data acquisition: targeted high-resolution reimaging of regions selected from rapidly-acquired lower resolution reconstruction , and an unsupervised segmentation algorithm .",
"This pipeline reduces imaging and reconstruction times by two orders of magnitude , facilitating directed inquiry of circuit motifs ."
] | [
"Neurons connect with each other to form complex circuits that underlie mental activities .",
"Mapping these connections to obtain a so-called wiring diagram is an essential step in learning how the brain works .",
"The only way to do this precisely enough is by using electron microscopy .",
"However , this technique is so time-consuming that thousands of hours of work are typically required to image even the smallest of tissue samples .",
"Electron microscopes fire beams of electrons at tissue samples , and detect the scattering of the electrons .",
"Stains are used to make specific neurons less permeable to electrons , or more “electron dense” .",
"Labeled cells scatter more electrons , which increases the contrast of the images .",
"In an approach called serial-section electron microscopy , a tissue sample is first cut into extremely thin sections .",
"These are imaged individually , and the images are then pieced together to reconstruct the sample .",
"Joesch et al . have now developed a new procedure – named ARTEMIS – that uses a combination of multiple techniques to speed up the mapping of neurons and their connections .",
"ARTEMIS makes use of genetic engineering , serial-scanning electron microscopy , an enhanced chemical staining procedure and a new image processing approach .",
"First , gene technology is used to selectively stain specific types of neurons in mice and flies .",
"Then , a tissue sample is collected and treated with a chemical that enhances the electron density of the stained neurons , without disrupting the tissue’s structure .",
"Next , a labeled target neuron is imaged at relatively low resolution to reveal its overall structure .",
"Small areas of that neuron are then re-imaged at higher resolution to map the connections between neurons .",
"Lastly , an algorithm pieces together the individual images to produce a reconstruction of the cell .",
"This pipeline of steps reduces the time required to map the shapes and connectivity of neurons with electron microscopy by some two orders of magnitude .",
"This should enable neuroscientists to obtain more rapid insights into the roles of specific neural circuits in the brains of healthy animals , and to identify cases where this wiring goes awry and leads to disease ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"cell biology"
] | Lipid-mediated regulation of SKN-1/Nrf in response to germ cell absence | elife-07836-v3 | [
[
"The nematode Caenorhabditis elegans has been invaluable for identifying mechanisms that slow aging and may prevent chronic disease ( Kenyon , 2010 ) .",
"An intriguing finding that was first made in this organism is that when germline stem cells ( GSCs ) are ablated , mechanisms are activated in somatic tissues that protect against stress and increase lifespan ( Hsin and Kenyon , 1999; Kenyon , 2010; Antebi , 2013; Hansen et al . , 2013 ) .",
"GSC loss also increases lifespan in Drosophila melanogaster ( Flatt et al . , 2008 ) , and castration has been associated with longevity in men ( Min et al . , 2012 ) , suggesting that this relationship might be conserved .",
"These beneficial effects of GSC removal may have evolved to maximize reproductive fitness under adversity ( Partridge et al . , 2005; Kenyon , 2010 ) .",
"This relationship provides paradigms for how tissue non-autonomous signals influence aging ( Kenyon , 2010 ) , and how a stem cell population communicates with the ‘niche’ that sustains it ( Jones and Wagers , 2008 ) .",
"In C . elegans , the effects of GSC absence have been studied by laser ablation of GSC precursors , which results in a complete loss of GSCs , or by analysis of genetic mutants in which GSC proliferation is inhibited so that the GSC number is very low , and mature germ cells are not formed ( Hsin and Kenyon , 1999; Arantes-Oliveira et al . , 2002; Kenyon , 2010 ) .",
"For simplicity , we will refer to each of these types of animals as GSC ( − ) animals .",
"The lifespan extension seen in GSC ( − ) animals ( GSC ( − ) longevity ) requires the action of several conserved transcription factors in the intestine , the counterpart of the mammalian liver , digestive system , and adipose tissue ( Kenyon , 2010; Antebi , 2013; Hansen et al . , 2013 ) .",
"DAF-16/FOXO is needed for longevity from GSC ablation or reduced insulin/IGF-1 signaling ( IIS ) but is regulated differently by each pathway ( Lin et al . , 2001; Libina et al . , 2003; Kenyon , 2010 ) .",
"GSC ( − ) longevity also requires HLH-30/TFEB , PHA-4/FOXA , and the nuclear receptors DAF-12/FXR , NHR-80/HNF4 , and NHR-49/PPARα ( Hsin and Kenyon , 1999; Goudeau et al . , 2011; Lapierre et al . , 2011; O'Rourke and Ruvkun , 2013; Ratnappan et al . , 2014 ) .",
"Under most conditions , GSC ( − ) longevity also depends upon a hormonal signal from the somatic gonad that activates DAF-12 ( Kenyon , 2010; Antebi , 2013 ) .",
"Aside from the identification of mechanisms required for DAF-16 function , we understand little about how GSCs influence these transcription factors ( Kenyon , 2010; Antebi , 2013 ) .",
"One hallmark of GSC ( − ) animals is enhancement of both proteostasis and stress resistance .",
"During aging , GSC ( − ) animals maintain more robust responses to thermal and proteotoxic stress ( Ben-Zvi et al . , 2009 ) .",
"They also exhibit a striking daf-16-dependent increase in activity of the proteasome ( Vilchez et al . , 2012 ) , a multisubunit complex which degrades proteins that the ubiquitylation system has tagged for decay ( Glickman and Ciechanover , 2002; Goldberg , 2003 ) .",
"In addition , GSC removal enhances immunity ( Alper et al . , 2010 ) and boosts oxidative stress resistance through an undetermined DAF-16-independent mechanism ( Libina et al . , 2003 ) .",
"Another notable characteristic of GSC ( − ) animals is that many aspects of lipid metabolism are altered .",
"Expression of particular fatty acid ( FA ) oxidation , FA desaturation , and triglyceride lipase genes is increased , as is total lipase activity ( Wang et al . , 2008; Goudeau et al . , 2011; Lapierre et al . , 2011; McCormick et al . , 2012; Ratnappan et al . , 2014 ) .",
"Given that lipid catabolism is elevated , it seems paradoxical that GSC ( − ) animals also exhibit dramatically increased fat accumulation ( O'Rourke et al . , 2009 ) .",
"Interestingly , GSC ( − ) longevity seems to depend upon particular lipid metabolism processes .",
"Production of the unsaturated free FA ( FFA ) oleic acid ( OA ) is required ( Goudeau et al . , 2011 ) , as are the triglyceride lipases LIPL-4/LIPA and FARD-1/FAR2 ( Wang et al . , 2008; McCormick et al . , 2012 ) .",
"It is of intense interest to determine whether the fat accumulation seen with GSC ablation might derive from production and storage of particular beneficial fats , or a salutary overall balance of lipid metabolism that is consistent with longevity ( Ackerman and Gems , 2012; Hansen et al . , 2013 ) .",
"The C . elegans transcription factor SKN-1 controls a broad detoxification response to oxidative and xenobiotic stress and is orthologous to the mammalian Nrf1/2/3 ( NF-E2-related factor ) proteins ( An and Blackwell , 2003; Oliveira et al . , 2009; Park et al . , 2009 ) .",
"SKN-1/Nrf proteins have been implicated in longevity from C . elegans to rodents ( An and Blackwell , 2003; Bishop and Guarente , 2007; Leiser and Miller , 2010; Sykiotis and Bohmann , 2010; Steinbaugh et al . , 2012; Ewald et al . , 2015 ) .",
"Recent findings raise the question of whether these transcription regulators might also have important functions in lipid homeostasis .",
"SKN-1/Nrf proteins influence expression of lipid metabolism genes ( Oliveira et al . , 2009; Paek et al . , 2012; Hayes and Dinkova-Kostova , 2014; Tsujita et al . , 2014 ) , and SKN-1 has been linked to fat mobilization under particular starvation or dietary conditions ( Paek et al . , 2012; Pang et al . , 2014 ) .",
"Mice that lack Nrf1 in the liver develop non-alcoholic fatty liver disease ( NAFLD ) that progresses to non-alcoholic steatohepatitis ( NASH ) , and Nrf2−/− mice develop NASH on a high-fat diet ( Xu et al . , 2005; Okada et al . , 2013; Tsujita et al . , 2014 ) .",
"However , reduced Nrf protein function is thought to predispose to NASH by impairing hepatic stress resistance ( Xu et al . , 2005; Lee et al . , 2013 ) .",
"An understanding of NAFLD is a high priority , because its incidence is increasing as a sequella of metabolic syndrome ( Cohen et al . , 2011 ) .",
"Here , we examined the role of SKN-1 in the effects of GSC absence on lifespan , stress resistance , and lipid metabolism .",
"Genetic inhibition of GSCs activates SKN-1 , thereby increasing lifespan and stress resistance .",
"Expression profiling revealed that GSC ( − ) animals upregulate stress defense , extracellular matrix , and lipid metabolism genes , in many cases dependent upon skn-1 .",
"SKN-1 is also required for GSC inhibition to increase proteasome activity .",
"SKN-1 is needed for GSC ( − ) longevity but reduces lipid storage , arguing against the idea that GSC ( − ) animals simply accumulate beneficial fat .",
"Instead , these high-fat levels appear to derive from unconsumed yolk that was produced for reproduction .",
"Unexpectedly , in GSC ( − ) animals , SKN-1 appears to be activated by specific FA signals , defining a new mechanism of SKN-1/Nrf protein regulation and GSC-to-soma communication .",
"This homeostatic function of SKN-1 in lipid metabolism suggests that Nrf proteins have a similar role in preventing NASH ."
],
[
"To investigate the importance of skn-1 in GSC ( − ) animals , we analyzed temperature-sensitive ( ts ) mutations in glp-1/Notch , which is required for GSC proliferation ( Kimble and Crittenden , 2005 ) .",
"glp-1 ( ts ) mutants that undergo larval development at the non-permissive temperature of 25°C ( GSC ( − ) animals ) are sterile , exhibit a markedly reduced number of GSCs , and live considerably longer than wild type ( WT ) controls ( Arantes-Oliveira et al . , 2002 ) ( Figure 1A , B ) .",
"By contrast , this lifespan extension was blocked in a skn-1 mutant background ( Figure 1A , B ) .",
"Lack of skn-1 also impaired lifespan extension when glp-1 ( ts ) animals were downshifted to 20°C after development was complete ( Table 1 ) .",
"Similar results were obtained with or without 5-fluoro-2′-deoxyuridine ( FUdR ) , which inhibits offspring formation in the control ( Table 1 ) .",
"Consistent with these findings , in an earlier experiment in which glp-1 ( ts ) extended lifespan by less than 7% , skn-1 knockdown by RNA interference ( RNAi ) prevented this increase ( Vilchez et al . , 2012 ) .",
"In contrast to daf-16 , skn-1 was also required for GSC inhibition to increase oxidative stress resistance ( Figure 1C–F; Table 2 ) . 10 . 7554/eLife . 07836 . 003Figure 1 . SKN-1 promotes longevity and stress resistance in germline stem cell ( GSC ) ( − ) animals .",
"( A , B )",
"Wild type , skn-1 ( zu135 ) , glp-1 ( bn18ts ) , and glp-1 ( bn18ts ) ;skn-1 ( zu135 ) double mutants were assayed for lifespan at 25°C .",
"skn-1 ( zu135 ) is a presumed null that is used throughout the study .",
"Unless otherwise specified , glp-1 ( ts ) refers to glp-1 ( bn18ts ) .",
"( A ) Composite survival curve .",
"( B ) Graph of mean lifespans .",
"( C–F ) glp-1 ( ts ) mutants require skn-1 for oxidative stress resistance .",
"Day-3 adult glp-1 ( ts ) and control worms treated with skn-1 RNAi or empty vector were exposed to ( C , D ) 5 mM sodium arsenite ( AS ) or ( E , F ) 15 . 4 mM tert-butyl hydroperoxide ( TBHP ) .",
"Data are represented as mean ± SEM .",
"p < 0 . 001*** .",
"The interaction between glp-1 and skn-1 was significant for both lifespan and stress resistance ( p < 0 . 001 ) .",
"Statistical analysis and replicates are in Tables 1 , 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 00310 . 7554/eLife . 07836 . 004Table 1 . LifespansDOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 004SetStrainMean lifespan ± SEM ( days ) Median lifespan ( days ) 75th % ( days ) N% Mean lifespan Ext . p valueLifespan at 25°C Composite lifespan at 25°C with FUdR ( from 2 replicates ) C1N215 . 40 ± 0 . 21617133/148––skn-1 ( zu135 ) 14 . 66 ± 0 . 21516158/167––glp-1 ( bn18ts ) 19 . 94 ± 0 . 32022164/17129 . 48<0 . 0001*glp-1 ( bn18ts ) ;skn-1 ( zu135 ) 15 . 23 ± 0 . 21518149/1703 . 89<0 . 0001†two-way ANOVA glp-1 ( ts ) and skn-1 interaction<0 . 0001 Replicate lifespans at 25°C with FUdR #1N213 . 99 ± 0 . 4141740/55––skn-1 ( zu135 ) 12 . 54 ± 0 . 3131441/50––glp-1 ( bn18ts ) 22 . 46 ± 0 . 8252750/5560 . 54<0 . 0001*glp-1 ( bn18ts ) ;skn-1 ( zu135 ) 13 . 29 ± 0 . 4121453/706 . 00<0 . 0001†glp-1 ( ts ) and skn-1 interaction<0 . 0001 #2N216 . 07 ± 0 . 2161793/93––skn-1 ( zu135 ) 15 . 42 ± 0 . 21517117/117––glp-1 ( bn18ts ) 18 . 86 ± 0 . 31921114/11617 . 36<0 . 0001*glp-1 ( bn18ts ) ;skn-1 ( zu135 ) 16 . 42 ± 0 . 2171896/1006 . 49<0 . 0001†glp-1 ( ts ) and skn-1 interaction0 . 0002Lifespan at 20°C ( 25°C during development , downshifted to 20°C at D1 adulthood ) Lifespan at 20°C without FUdR #3N220 . 36 ± 0 . 6182142/50––skn-1 ( zu135 ) 18 . 39 ± 0 . 5181827/47––glp-1 ( bn18ts ) 25 . 87 ± 1 . 3243335/6627 . 060 . 0002*glp-1 ( bn18ts ) ;skn-1 ( zu135 ) 20 . 02 ± 0 . 6182434/558 . 86<0 . 0001†glp-1 ( ts ) and skn-1 interaction0 . 0230 Composite lifespan at 20°C with FUdR ( from 2 replicates ) C2N220 . 51 ± 0 . 5212486/90––skn-1 ( zu135 ) 17 . 78 ± 0 . 4192088/94––glp-1 ( bn18ts ) 24 . 52 ± 0 . 6252881/10419 . 55<0 . 0001*glp-1 ( bn18ts ) ;skn-1 ( zu135 ) 20 . 47 ± 0 . 5202491/9815 . 13<0 . 0001†glp-1 ( ts ) and skn-1 interaction0 . 1892 Replicate lifespans at 20°C with FUdR #4N218 . 09 ± 0 . 4171933/37––skn-1 ( zu135 ) 14 . 78 ± 0 . 5141731/35––glp-1 ( bn18ts ) 21 . 79 ± 1 . 0212828/5020 . 450 . 0002*glp-1 ( bn18ts ) ;skn-1 ( zu135 ) 16 . 83 ± 0 . 3171836/4313 . 87<0 . 0001†glp-1 ( ts ) and skn-1 interaction0 . 1491 #5N222 . 00 ± 0 . 6232553/53––skn-1 ( zu135 ) 19 . 40 ± 0 . 4202157/59––glp-1 ( bn18ts ) 25 . 91 ± 0 . 7253053/5417 . 77<0 . 0001*glp-1 ( bn18ts ) ;skn-1 ( zu135 ) 22 . 80 ± 0 . 6242655/5517 . 53<0 . 0001†glp-1 ( ts ) and skn-1 interaction0 . 6607Percent lifespan extension refers to glp-1 ( ts ) vs wild type or skn-1 control .",
"p values were calculated by log-rank test .",
"Symbols denote effect relative to N2* or glp-1 ( ts ) † .",
"The interaction effect of glp-1 ( ts ) and skn-1 was calculated by two-way ANOVA using mean lifespan .",
"The last p value reflects the specific requirement of skn-1 for glp-1 ( ts ) lifespan , as opposed to its effect on lifespan in general .",
"Homozygous skn-1 mutants produce eggs that do not hatch because of a catastrophic defect in developmental patterning but do not exhibit known defects in the germline itself ( Bowerman et al . , 1992 ) . 10 . 7554/eLife . 07836 . 005Table 2 . Stress resistance assaysDOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 005SetStrainMean survival ± SEM ( hrs ) Median survival ( hrs ) 75th % survival ( hrs ) N% Mean survival Ext . p valueAS ( day 1 adulthood ) 25°C continuous; RNAi from L1 #1N2 + vector RNAi20 . 20 ± 1 . 022 . 522 . 5122/122––N2 + rme-2 RNAi39 . 51 ± 1 . 147 . 547 . 5117/11795 . 59<0 . 0001* #2N2 + vector RNAi26 . 82 ± 1 . 228 . 028 . 057/57––N2 + rme-2 RNAi53 . 21 ± 0 . 952 . 571 . 5252/25298 . 44<0 . 0001*glp-1 ( bn18ts ) + vector RNA53 . 06 ± 1 . 052 . 571 . 5216/21697 . 85<0 . 0001* #3N2 + vector RNAi ( 20°C ) 38 . 59 ± 0 . 647 . 547 . 5281/281––N2 + rme-2 RNAi63 . 00 ± 0 . 771 . 571 . 5287/28763 . 25<0 . 0001* #4N2 + vector RNAi ( 20°C ) 49 . 10 ± 0 . 846 . 064 . 0306/306––N2 + vector/skn-1 mix RNAi32 . 03 ± 0 . 640 . 040 . 0271/271––N2 + vector/rme-2 mix RNAi65 . 38 ± 1 . 164 . 070 . 0361/36133 . 16<0 . 0001*N2 + rme-2/skn-1 mix RNAi33 . 76 ± 0 . 540 . 040 . 0409/4095 . 40<0 . 0001‡two-way ANOVA rme-2 and skn-1 interaction<0 . 0001 #5N2 + vector RNAi23 . 18 ± 0 . 522 . 522 . 5125/125––N2 + lipl-3 RNAi23 . 04 ± 0 . 622 . 528 . 0139/139––N2 + sbp-1 RNAi11 . 01 ± 0 . 67 . 522 . 5163/163––N2 + skn-1 RNAi20 . 88 ± 0 . 722 . 528 . 0115/115––glp-1 ( bn18ts ) + vector RNAi40 . 58 ± 1 . 147 . 547 . 5105/10575 . 05<0 . 0001*glp-1 ( bn18ts ) + lipl-3 RNAi28 . 52 ± 1 . 028 . 032 . 5178/17823 . 77<0 . 0001†glp-1 ( bn18ts ) + sbp-1 RNAi7 . 50 ± 0 . 56 . 07 . 5138/138−31 . 88<0 . 0001†glp-1 ( bn18ts ) + skn-1 RNAi12 . 90 ± 0 . 99 . 022 . 5129/129−38 . 21<0 . 0001†glp-1 ( ts ) and lipl-3 interaction<0 . 0001glp-1 ( ts ) and sbp-1 interaction<0 . 0001glp-1 ( ts ) and skn-1 interaction<0 . 0001 #6N2 + vector RNAi24 . 36 ± 0 . 822 . 528 . 0121/121––N2 + fat-6/7 mix RNAi15 . 56 ± 0 . 822 . 522 . 5159/159––N2 + skn-1 RNAi20 . 94 ± 0 . 622 . 522 . 5124/124––glp-1 ( bn18ts ) + vector RNAi38 . 49 ± 1 . 547 . 547 . 598/9858 . 03<0 . 0001*glp-1 ( bn18ts ) + fat-6/7 mix RNAi12 . 04 ± 0 . 79 . 022 . 5153/153−22 . 66<0 . 0001†glp-1 ( bn18ts ) + skn-1 RNAi23 . 69 ± 0 . 928 . 032 . 5116/11613 . 13<0 . 0001†glp-1 ( ts ) and fat-6/7 interaction<0 . 0001glp-1 ( ts ) and skn-1 interaction<0 . 0001AS ( day 3 adulthood ) 25°C during development , 20°C from D1; RNAi from D1 #7N2 + vector RNAi6 . 55 ± 0 . 37 . 08 . 0104/104––N2 + skn-1 RNAi5 . 08 ± 0 . 25 . 07 . 0103/103––glp-1 ( bn18ts ) + vector RNAi19 . 36 ± 0 . 119 . 020 . 097/97195 . 57<0 . 0001*glp-1 ( bn18ts ) + skn-1 RNAi7 . 86 ± 0 . 48 . 012 . 0100/10054 . 72<0 . 0001†glp-1 ( ts ) and skn-1 interaction<0 . 0001 #8N2 + vector RNAi8 . 31 ± 0 . 49 . 010 . 098/98––N2 + skn-1 RNAi6 . 89 ± 0 . 38 . 09 . 090/90––glp-1 ( bn18ts ) + vector RNAi16 . 95 ± 1 . 020 . 026 . 081/81103 . 97<0 . 0001*glp-1 ( bn18ts ) + skn-1 RNAi9 . 90 ± 0 . 410 . 012 . 091/9143 . 69<0 . 0001†glp-1 ( ts ) and skn-1 interaction<0 . 0001TBHP ( day 3 adulthood ) 25°C during development , 20°C from D1; RNAi from D1 #9N2 + vector RNAi9 . 02 ± 0 . 39 . 011 . 084/108––N2 + skn-1 RNAi6 . 16 ± 0 . 26 . 07 . 063/98––glp-1 ( bn18ts ) + vector RNAi11 . 62 ± 0 . 311 . 013 . 061/6128 . 82<0 . 0001*glp-1 ( bn18ts ) + skn-1 RNAi6 . 48 ± 0 . 18 . 07 . 063/655 . 19<0 . 0001†glp-1 ( ts ) and skn-1 interaction<0 . 0001 #10N24 . 29 ± 0 . 24 . 04 . 065/94––skn-1 ( zu135 ) 4 . 51 ± 0 . 15 . 03 . 073/74––glp-1 ( bn18ts ) 6 . 25 ± 0 . 26 . 04 . 073/7445 . 69<0 . 0001*glp-1 ( bn18ts ) ;skn-1 ( zu135 ) 4 . 78 ± 0 . 15 . 04 . 073/755 . 99<0 . 0001†glp-1 ( ts ) and skn-1 interaction<0 . 0001Survival after sodium arsenite ( AS ) or tert-butyl hydroperoxide ( TBHP ) treatment was assayed in adult animals .",
"The increase in oxidative stress resistance of glp-1 ( ts ) germline stem cell ( GSC ( − ) ) animals was impaired by loss of fat-6/7 , lipl-3 , sbp-1 , and skn-1 .",
"Representative assays are shown .",
"Percent survival extension refers to glp-1 ( ts ) or rme-2 RNAi vs the matching wild type or skn-1 control .",
"p values were calculated by log-rank test .",
"Symbols denote effect relative to N2* , glp-1 ( ts ) † , or rme-2 RNAi‡ .",
"The interaction effect of GSC ( − ) or rme-2 with skn-1 , or fat-6/7 , lipl-3 , and sbp-1 were calculated by two-way ANOVA using mean lifespan .",
"The last p value reflects the specific requirement of each gene for GSC ( − ) or rme-2 stress resistance as opposed to its effect on stress resistance in general .",
"We investigated whether the benefits of GSC absence simply require that SKN-1 be present or involve activation of SKN-1 .",
"SKN-1 accumulates in intestinal nuclei in response to certain stresses , or inhibition of mechanisms that include IIS , mTORC2 , glycogen synthase kinase-3 , translation elongation , and the ubiquitin ligase WDR-23 ( An and Blackwell , 2003; Tullet et al . , 2008; Choe et al . , 2009; Park et al . , 2009; Li et al . , 2011; Robida-Stubbs et al . , 2012 ) .",
"The levels of a SKN-1::GFP ( green fluorescent protein ) fusion in intestinal nuclei were also notably elevated in GSC ( − ) animals ( Figure 2A , B ) .",
"This was associated with increased expression of direct SKN-1 target genes , apparently through activation of their intestinal expression ( Figure 2C–E ) .",
"The KRI-1/KRIT1 ankyrin repeat protein and the TCER-1/TCERG1 transcription factor are required for GSC absence to induce DAF-16 nuclear accumulation and extend lifespan ( Berman and Kenyon , 2006; Ghazi et al . , 2009 ) .",
"In contrast , in GSC ( − ) animals SKN-1 nuclear accumulation was only partially or minimally affected by loss of kri-1 or tcer-1 , respectively , but was abolished by knockdown of the pmk-1/p38 kinase ( Figure 2F , G ) , which phosphorylates SKN-1 and under most circumstances is required for SKN-1 nuclear accumulation ( Inoue et al . , 2005 ) .",
"In GSC ( − ) animals , DAF-12 is needed for DAF-16 nuclear accumulation and activity ( Berman and Kenyon , 2006; Kenyon , 2010; Antebi , 2013 ) , and induces expression of the microRNAs mir-84 and mir-241 , which target inhibitors of DAF-16 ( Shen et al . , 2012 ) .",
"In GSC ( − ) animals , daf-12 knockdown only mildly affected SKN-1::GFP accumulation ( Figure 2G ) , and SKN-1 target gene induction was generally not impaired by daf-12 or mir-241;mir-84 mutations ( Figure 2H ) .",
"The absence of GSCs therefore activates SKN-1 in the intestine but through a different mechanism from DAF-16 . 10 . 7554/eLife . 07836 . 006Figure 2 . GSCs inhibit SKN-1 activity in the intestine .",
"( A ) Representative images of SKN-1::green fluorescent protein ( GFP ) in intestinal nuclei; GFP channel ( top ) , bright field ( BF; bottom ) .",
"( B ) Accumulation of SKN-1::GFP in intestinal nuclei in GSC ( − ) animals .",
"( C ) skn-1-dependent activation of direct SKN-1 target genes ( Robida-Stubbs et al . , 2012 ) in response to GSC absence , detected by qRT-PCR .",
"( D , E )",
"Increased expression of gst-4p::GFP in the intestine of glp-1 ( ts ) animals .",
"Hypodermal gst-4p::GFP expression appeared to be unaffected .",
"( D ) Representative 10× images .",
"( E ) Intestinal gst-4p::GFP quantification .",
"( F–H )",
"GSCs regulate SKN-1 parallel to DAF-16 and DAF-12 .",
"In ( H ) , SKN-1 target genes are assayed by qRT-PCR .",
"glp-1 ( ts ) refers to glp-1 ( e2141ts ) , and horizontal black lines indicate strains lacking GSCs .",
"( C , H )",
"Data are represented as mean ± SEM .",
"n = 3 for qRT-PCR samples .",
"( B , E–G )",
"GFP quantification with high , medium , low scoring .",
"Numbers above bars denote sample size .",
"p < 0 . 05*; p < 0 . 01**; p < 0 . 001*** .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 006 To investigate how SKN-1 promotes longevity and stress resistance upon GSC loss , we used RNA sequencing ( RNA-seq ) to identify genes that are ( 1 ) expressed at higher levels in adult somatic tissues when germ cells are largely absent and ( 2 ) dependent upon SKN-1 ( Figure 3A ) .",
"We compared intact glp-1 ( ts ) ( GSC ( − ) ) animals to wild-type GSC ( + ) controls at the non-permissive temperature of 25° , analyzing day-1 adults in which development was complete and performing differential expression analyses on the normalized RNA-seq data for 12 , 595 expressed genes ( Figure 3B ) .",
"We detected similar expression levels of SKN-1 upregulated targets in qRT-PCR analyses of the samples used for sequencing , giving us confidence in our RNA-seq results ( Figure 3—figure supplement 1A ) .",
"Moreover , in the GSC ( − ) gene set , the canonical DAF-16 targets mtl-1 and sod-3 ( Murphy et al . , 2003; Kenyon , 2010 ) were upregulated ( Supplementary file 1a ) , and functional groups that are characteristic of germline-specific genes were under-represented ( Figure 3—figure supplement 1B ) . 10 . 7554/eLife . 07836 . 007Figure 3 . Effects of GSC absence and skn-1 on gene expression .",
"( A ) RNA-seq experiment setup .",
"For each condition , three biological replicates were obtained from synchronized intact day-1 adults at 25°C .",
"Arrows indicate comparisons that were made , and GSC ( − ) refers to glp-1 ( ts ) .",
"( B ) Heatmap of all genes evaluated , showing biological replicates .",
"( C ) DAVID functional annotation analysis of GSC ( − ) -upregulated genes .",
"( D ) Heatmap of genes upregulated in glp-1 ( ts ) in a skn-1-dependent manner ( 87 genes; fold change ( FC ) > 4 in GSC ( − ) ; FC < 0 . 67 with skn-1 RNAi in GSC ( − ) ) .",
"( E ) Functional annotation of the genes shown in ( D ) .",
"Genes and additional statistics are provided in Supplementary file 1a , d . DOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 00710 . 7554/eLife . 07836 . 008Figure 3—figure supplement 1 . Gene expression changes following GSC inhibition .",
"( A ) qRT-PCR validation of RNA samples used for RNA-seq .",
"gst-4 and nit-1 are direct SKN-1 targets ( Robida-Stubbs et al . , 2012 ) .",
"Data are represented as mean ± SEM .",
"n = 3; p < 0 . 05* .",
"Analysis of a different RNA sample set is shown in Figure 2D .",
"( B ) DAVID analysis of genes that were downregulated in glp-1 ( ts ) .",
"Processes related to GSC maintenance and reproduction were highly represented , as expected .",
"( C ) Altered abundance of tissue-specific genes in GSC ( − ) animals .",
"Adult hermaphrodite worms have 959 somatic cells and ∼2000 GSCs ( Kimble and Crittenden , 2005 ) .",
"A representative somatic-specific gene would therefore be predicted to be present at higher relative abundance in GSC ( − ) samples after normalization to either total RNA or reference housekeeping genes .",
"Accordingly , representative somatic tissue-specific genes ( Richmond , 2005; Moerman and Williams , 2006; Chikina et al . , 2009 ) were present at threefold to fourfold higher relative levels in the GSC ( − ) samples .",
"By contrast , a germline-specific gene ( efl-1 ) was underrepresented ( ∼4× ) in GSC ( − ) samples .",
"Reference genes that are ubiquitously expressed in all tissues and commonly used for qRT-PCR normalization ( Hoogewijs et al . , 2008 ) do not have altered relative abundance in the GSC ( − ) samples .",
"( D , E )",
"Frequency distribution plots of mRNA levels in glp-1 ( ts ) worms relative to wild type .",
"A cutoff of FC > 4 denotes 1 , 306 out of 12 , 595 genes sequenced ( 10 . 4% ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 008 No previous studies have globally profiled genes that are upregulated in the soma in response to germ cell loss .",
"mRNAs that are present at higher relative levels in GSC ( − ) samples compared to WT would include not only those genes , but also genes that are expressed only in somatic cells , because the germline accounts for about two-thirds of all adult nuclei ( Kimble and Crittenden , 2005 ) .",
"To gauge the maximal extent of this background , we examined mRNAs that are expressed specifically in somatic tissues .",
"These somatic-specific mRNAs were enriched threefold to fourfold in the GSC ( − ) samples , approximately , the level predicted from the 2:1 proportion of germline to somatic nuclei ( Figure 3—figure supplement 1C ) .",
"Accordingly , if an mRNA that is not somatic specific was present at a fourfold-elevated level in GSC ( − ) samples , we considered this mRNA to be upregulated in the soma in response to GSC absence , although we expect that this stringent cutoff would miss many upregulated genes .",
"In GSC ( − ) animals , 1306 and 615 genes were upregulated more than fourfold and fivefold , respectively , indicating a broad remodeling of transcription ( Figure 3—figure supplement 1D , E; Supplementary file 1a ) .",
"In the latter set , which is more amenable to functional annotation analysis because of its smaller size , the most prominently overrepresented category was collagen ( Figure 3C; Supplementary file 1a ) .",
"Although collagens may be expressed primarily in the soma , this overrepresentation is likely to be meaningful because of the extent to which these genes were upregulated ( Supplementary file 1a ) , and because collagens are generally overrepresented in other longevity-associated gene sets , with certain collagens being critical for lifespan extension ( Ewald et al . , 2015 ) .",
"Also , notably upregulated in GSC ( − ) animals were genes involved in detoxification , immunity ( C-type lectin and galectin ) , and metabolism , particularly FA oxidation and other lipid metabolism processes ( Figure 3C; Supplementary file 1a ) .",
"We used RNAi to investigate the contribution of skn-1 to gene expression in GSC ( − ) animals ( Figure 3A ) .",
"A previous microarray analysis of skn-1 RNAi-treated L4 larvae at 20°C found that in the absence of acute stress , SKN-1 upregulates genes involved in processes that include detoxification , lipid metabolism , immunity , and proteostasis ( Oliveira et al . , 2009; Li et al . , 2011 ) .",
"Similar processes were prominent in the sets of genes for which skn-1 RNAi reduced expression at 25°C in day-1 WT adults ( ‘SKN-1 upregulated in WT genes’; ≥33% reduction , p < 0 . 05 ) ( Supplementary file 1b ) or day-1 GSC ( − ) animals ( ‘SKN-1-upregulated in GSC ( − ) genes’; ≥33% reduction , p < 0 . 05 ) ( Supplementary file 1c ) .",
"Our finding that SKN-1 nuclear occupancy is increased in GSC ( − ) animals ( Figure",
"2 ) predicts that GSC inhibition would induce SKN-1 to activate genes .",
"Accordingly , skn-1 RNAi reduced expression of 87 genes that were upregulated at least fourfold in glp-1 ( ts ) compared to WT ( Figure 3D , E; Supplementary file 1d ) .",
"This number probably underestimates the full contribution of SKN-1 , because RNAi only partially reduces its activity .",
"In addition to detoxification and lipid metabolism genes , these 87 genes included many involved in extracellular matrices ( ECMs ) , as expected from the skn-1-dependence of many ECM genes that are upregulated in other long-lived C . elegans ( Ewald et al . , 2015 ) .",
"Many of these skn-1-dependent GSC ( − ) genes appear to be direct SKN-1 targets , as predicted by presence of SKN-1 binding sites in their upstream regions and direct binding of SKN-1 in genome-wide chromatin immunoprecipitation ( ChIP ) surveys ( Supplementary file 1d ) .",
"In summary , SKN-1 upregulates numerous genes that are associated with phenotypes seen in GSC-ablated animals , including increased stress resistance , immunity , and longevity , as well as alterations in lipid metabolism .",
"A previous RNAi experiment suggested that SKN-1 is dispensable for the elevated proteasome activity seen in GSC ( − ) animals ( Vilchez et al . , 2012 ) .",
"We re-examined this question because SKN-1 maintains proteasome gene expression and intestinal proteasome activity in WT C . elegans ( Li et al . , 2011 ) , and because proteasome genes were prominent in the SKN-1-upregulated GSC ( − ) gene set ( Supplementary file 1c ) .",
"The proteasome holocomplex consists of a 20S barrel-like structure in which proteins are degraded , and a 19S regulatory cap that directs ubiquitylated proteins into this structure ( Glickman and Ciechanover , 2002; Goldberg , 2003 ) .",
"In general , and consistent with previous findings ( Vilchez et al . , 2012 ) , the relative levels of proteasome subunit mRNAs were lower in GSC ( − ) animals ( Figure 4—figure supplement 1A ) , possibly because of the lack of germ cells .",
"In both WT and GSC ( − ) animals , skn-1 knockdown comparably decreased expression of 19S and 20S proteasome subunit genes ( Figure 4A and Figure 4—figure supplement 1A ) , the majority of which appear to be direct transcriptional targets of SKN-1 ( Figure 4B ) .",
"As these findings would predict , in GSC ( − ) animals , the lack of skn-1 dramatically reduced proteasome activity at days 1 and 5 of adulthood ( Figure 4C , D , and Figure 4—figure supplement 1B–G ) .",
"It is possible that in the earlier analysis ( Vilchez et al . , 2012 ) , RNAi might not have inhibited skn-1 expression sufficiently to detect its importance for proteasome activity in GSC ( − ) animals . 10 . 7554/eLife . 07836 . 009Figure 4 . SKN-1 increases proteasome activity in response to GSC absence .",
"( A ) Reduction in relative proteasome gene subunit mRNA levels by skn-1 RNAi , detected by RNA-seq .",
"( B ) Venn diagram indicating the number of proteasome subunit genes ( pas , pbs , rpn , rpt families ) that have SKN-1::GFP ChIP-seq peak hits near the transcription start site at the indicated larval stage ( Niu et al . , 2011 ) .",
"( C , D )",
"SKN-1-dependence of increased chymotrypsin-related proteasome activity in GSC-ablated worms .",
"The slopes from ( C ) are graphed in ( D ) .",
"Additional experiments are in Figure 4—figure supplement 1 .",
"( E ) SKN-1-dependence of rpn-6 . 1 upregulation .",
"Data are represented as mean ± SEM .",
"n = 3 for all experiments .",
"p < 0 . 05*; p < 0 . 01**; p < 0 . 001*** .",
"( F ) Direct binding of SKN-1 and DAF-16 to the rpn-6 . 1 gene , indicated by TRANSFAC transcription factor binding prediction and modENCODE GFP ChIP-seq analyses .",
"Both the predicted promoter and first intron of rpn-6 . 1 are highly enriched for SKN-1 and DAF-16 binding . DOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 00910 . 7554/eLife . 07836 . 010Figure 4—figure supplement 1 . SKN-1-dependence of the increased proteasome activity in GSC ( − ) animals .",
"( A ) GSC absence reduces relative proteasome subunit gene mRNA abundance , detected by RNA-seq .",
"( B , C )",
"The skn-1 ( zu135 ) mutation suppresses the increase in 26S proteasome activity seen in day-1 adult glp-1 ( ts ) animals .",
"( D , E ) skn-1 RNAi administered from the egg stage suppresses proteasome activity in day-1 adult glp-1 ( ts ) animals .",
"( F , G ) skn-1 RNAi administered post-developmentally , starting at day-1 adulthood , significantly reduces proteasome activity in day-5 adult glp-1 ( ts ) animals .",
"( B , D , F )",
"Kinetic curves of chymotrypsin-like proteasome activity .",
"( C , E , G )",
"Graphs of proteasome activity slopes .",
"Data are represented as mean ± SEM .",
"n = 3; p < 0 . 001*** .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 010 The increased proteasome activity of GSC ( − ) animals is thought to derive from DAF-16-dependent transcriptional upregulation of the RPN-6 . 1/PSMD11 subunit , which connects the 19S and 20S proteasome particles ( Vilchez et al . , 2012 ) .",
"rpn-6 . 1 appears to be unique among proteasome subunit genes , in that its mRNA levels are proportionally higher in GSC ( − ) animals ( Vilchez et al . , 2012 ) ( Figure 4E ) .",
"skn-1 was required for this increased rpn-6 . 1 expression ( Figure 4E ) , and binding site and ChIP studies suggested that rpn-6 . 1 is upregulated directly by both SKN-1 and DAF-16 ( Figure 4F ) .",
"We conclude that by promoting expression of multiple proteasome subunit genes , including rpn-6 . 1 , SKN-1 plays a central role in the increased proteasome activity that results from GSC loss .",
"Genetic GSC inhibition increased expression of lipid metabolism genes that represent a wide range of processes , including FFA formation from triglyceride lipolysis , as well as FA oxidation , desaturation , and elongation ( Figure 5A; Supplementary file 1a ) .",
"Many of these genes were also upregulated by SKN-1 ( Figure 5A; Supplementary file 1a–c ) .",
"Of particular note , the high-confidence GSC ( − ) and SKN-1-upregulated gene set included the conserved lysosomal triglyceride lipase lipl-3 , which increases C . elegans lifespan when overexpressed and is normally induced by fasting ( O'Rourke and Ruvkun , 2013 ) .",
"This gene set also included the FA oxidation genes acs-10 ( acyl-CoA synthetase ) , cpt-3 ( carnitine palmitoyltransferase ) , and ech-9 ( enoyl-CoA hydratase ) ( Figure 5A; Supplementary file 1d ) .",
"This suggested that SKN-1 might have a major role in lipid metabolism , and how it is influenced by GSC absence . 10 . 7554/eLife . 07836 . 011Figure 5 . SKN-1 regulates lipid metabolism in GSC ( − ) animals .",
"( A ) Functional map of lipid metabolism gene expression .",
"Left columns show the effects of GSC absence ( GSC ( − ) vs WT ) and right columns the effects of skn-1 RNAi in GSC ( − ) animals .",
"SKN-1 regulates genes involved in fatty acid ( FA ) oxidation , breakdown of triacylglycerols ( TAG ) to free FAs , production of mono- and poly-unsaturated FAs ( MUFA , PUFA ) , and FA transport .",
"Color coding reflects relative representation in RNA-seq data , with blue and yellow indicating increased and decreased expression , respectively .",
"( B–E )",
"Increased fat levels in glp-1 ( ts ) and skn-1 mutants but not daf-16 mutants .",
"( D , E ) glp-1 ( ts ) refers to glp-1 ( e2141ts ) .",
"Representative 40× differential interference contrast ( DIC ) images of fixed ORO-stained worms are shown in ( B , D ) , with quantification provided in ( C , E ) .",
"Additional images and quantification are provided in Figure 5—figure supplement 1 .",
"Data are represented as mean ± SEM .",
"Numbers above bars denote sample size .",
"p < 0 . 001*** .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 01110 . 7554/eLife . 07836 . 012Figure 5—figure supplement 1 . Representative ORO staining images with quantification . Representative images divided into approximate quintiles , ordered by mean pixel intensity , are shown for each strain .",
"BF images are presented on the left and background subtracted images with quantification on the right .",
"( A ) glp-1 ( bn18ts ) and skn-1 ( zu135 ) mutants .",
"( B ) glp-1 ( ts ) mutants treated with skn-1 and sbp-1 RNAi .",
"Increased staining was observed both with skn-1 mutants and skn-1 RNAi .",
"RNAi against sbp-1 , which is required for lipogenesis ( Yang et al . , 2006 ) , decreases ORO staining in both WT and glp-1 ( ts ) genetic backgrounds .",
"( C ) glp-1 ( e2141ts ) and daf-16 ( mu86 ) mutants .",
"Numbers indicate mean pixel intensity above background ( see ‘Materials and methods’ for additional details ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 01210 . 7554/eLife . 07836 . 013Figure 5—figure supplement 2 . Analysis of the intestinal lipid droplet marker DHS-3::GFP , and TAG levels . skn-1 RNAi increases DHS-3::GFP intensity in both wild-type and GSC ( − ) animals , whereas sbp-1 RNAi decreases DHS-3::GFP intensity .",
"( A ) 10× slide-mounted DHS-3::GFP images .",
"White arrows indicate intestine-specific expression .",
"( B , C )",
"COPAS Biosorter quantification of DHS-3::GFP in live day-1 adult worms .",
"( B ) Representative 10× inverted scope images of worms suspended in M9 buffer used for COPAS scoring .",
"( C ) Graph of mean DHS-3::GFP fluorescence , assayed by COPAS .",
"Numbers above bars denote sample size .",
"Asterisks directly above bars indicate p values relative to WT or RNAi control .",
"Asterisks above black lines denote effect of RNAi in glp-1 ( ts ) background .",
"RNAi was started from egg stage , and animals were raised at 25°C .",
"( D ) TAG levels are significantly elevated in skn-1 mutants .",
"Data are represented as mean ± SEM .",
"p < 0 . 01**; p < 0 . 001*** .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 01310 . 7554/eLife . 07836 . 014Figure 5—figure supplement 3 . RNA-seq counts of select lipid metabolism and yolk transporter genes . Expression of the known SKN-1 target genes gst-4 and nit-1 is elevated in GSC ( − ) animals in a skn-1-dependent manner .",
"The TAG lipase lipl-3 and FABP lbp-8 are similarly elevated in GSC ( − ) animals in a skn-1-dependent manner , but expression of sbp-1 , dhs-3 , and yolk protein vitellogenins ( VIT genes ) are not affected by skn-1 RNAi .",
"rme-2 expression is germline enriched but regulated independently of skn-1 .",
"Open circles denote replicates .",
"Vertical lines indicate mean counts per million ( CPM ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 014 Given that SKN-1 increases both lifespan and stress resistance in GSC ( − ) animals , its effects on lipid metabolism should also be beneficial .",
"If the elevated fat storage in GSC ( − ) animals reflects simply elevated production and storage of ‘good’ lipids , we might expect skn-1 to support this fat production .",
"We investigated whether SKN-1 affects fat storage in WT and GSC ( − ) animals by oil red O ( ORO ) staining of fixed animals , a method that reliably indicates fat accumulation ( O'Rourke et al . , 2009 ) .",
"Remarkably , ablation of skn-1 by either mutation or RNAi significantly increased lipid levels in either WT or GSC ( − ) day-1 adults , so that glp-1 ( ts ) ;skn-1 ( − ) animals exhibited markedly high levels of ORO staining ( Figure 5B , C , and Figure 5—figure supplement 1A , B ) .",
"As an independent method of assessing fat accumulation in the intestine , we examined levels of the predicted short-chain FA dehydrogenase DHS-3 ( Zhang et al . , 2012 ) .",
"Proteomic and microscopy analyses have shown that DHS-3 localizes almost exclusively to intestinal lipid droplets ( Figure 5—figure supplement 2A ) and marks the vast majority of these lipid droplets in vivo ( Zhang et al . , 2012; Na et al . , 2015 ) .",
"Consistent with ORO staining , lack of skn-1 increased accumulation of a DHS-3::GFP fusion in the intestine in WT and GSC ( − ) animals , without affecting expression of the dhs-3 mRNA ( Figure 5—figure supplements 2B , C , 3 ) .",
"An analysis of total triglyceride levels also indicated that SKN-1 reduces the overall level of fat accumulation ( Figure 5—figure supplement 2D ) .",
"DAF-16 increases lipid accumulation in response to reduced IIS , and influences expression of some lipid metabolism genes in response to GSC removal ( Wang et al . , 2008; McCormick et al . , 2012 ) .",
"However , consistent with a previous study ( O'Rourke et al . , 2009 ) , we found that loss of daf-16 did not substantially affect overall fat storage in GSC ( − ) animals ( Figure 5D , E , and Figure 5—figure supplement 1C ) .",
"Together , our data indicate that SKN-1 is required to prevent excess fat accumulation under normal feeding conditions , and that SKN-1 but not DAF-16 reduces the lipid load that accumulates in response to GSC loss .",
"Given that SKN-1 inhibits fat storage , we considered whether the SKN-1 activation seen in GSC ( − ) animals might be triggered by lipid accumulation .",
"It is possible that GSC loss simply increases production of certain fats .",
"However , GSC ablation or inhibition prevents formation of oocytes , which endocytose lipid-rich yolk that is synthesized in the intestine ( Grant and Hirsh , 1999 ) .",
"Fat storage might therefore be increased indirectly by GSC loss , through accumulation of unused yolk lipids .",
"We tested a key prediction of this model by examining yolk accumulation and distribution , which can be visualized with GFP-tagged vitellogenin ( YP170/VIT-2::GFP ) , a major yolk lipoprotein ( Grant and Hirsh , 1999 ) .",
"VIT-2::GFP was visible primarily in oocytes and embryos in WT day-1 adults but accumulated to extremely high levels throughout the body cavity in the absence of GSCs ( Figure 6A , B , and Figure 6—figure supplement 1 ) .",
"Apparently , yolk production was not slowed sufficiently to compensate for the lack of gametogenesis .",
"The failure to consume yolk-associated lipid could account for the increase in overall fat storage seen in GSC ( − ) animals . 10 . 7554/eLife . 07836 . 015Figure 6 . GSC absence activates SKN-1 through FA signaling .",
"( A ) Accumulation of yolk transporter VIT-2::GFP in the soma of GSC ( − ) animals .",
"Detailed higher magnification images are provided in Figure 6—figure supplement 1 .",
"( B ) COPAS quantification of VIT-2::GFP .",
"Data are represented as mean ± SEM .",
"( C ) Knockdown of the oocyte-specific yolk receptor rme-2 promotes somatic VIT-2 accumulation .",
"( D–F ) rme-2 RNAi activates SKN-1 in the intestine .",
"( D , E )",
"SKN-1::GFP accumulates in intestinal nuclei in rme-2 RNAi-treated worms .",
"( F ) In rme-2 RNAi-treated worms , gst-4p::GFP levels in the intestine are increased at the L4 stage and increased further by day-1 adulthood .",
"( G ) rme-2 RNAi enhances resistance to AS , in a skn-1-dependent manner ( see replicates in Table 2 ) .",
"( H , I )",
"An oleic acid ( OA ) -dependent signal is required for SKN-1 to be activated by GSC inhibition but not oxidative stress .",
"In GSC ( − ) animals , SKN-1 nuclear accumulation is abolished by sbp-1 RNAi and rescued by OA supplementation .",
"SKN-1 remains capable of responding to oxidative stress ( 30 min AS exposure ) after sbp-1 RNAi in GSC ( − ) ( H , I ) or WT ( Figure 6—figure supplement 2A ) worms .",
"( J ) Dependence of SKN-1::GFP accumulation in GSC ( − ) animals on FAT-6/7-mediated FA desaturation , and proteins that generate free unsaturated FAs ( LIPL-1/-3 lipases ) , or transport them to the nucleus ( LBP-6/7/8 ) .",
"( K ) OA and coconut oil ( CO ) increase skn-1-dependent gst-4p::GFP expression in the intestine .",
"lbp-8 RNAi reduces induction by OA .",
"( A , C )",
"Representative 10× GFP images .",
"( D , H )",
"Representative 40× GFP images of day-1 adults .",
"( E , F , I–K )",
"GFP quantification with high , medium , low scoring .",
"Numbers above bars denote sample size .",
"p < 0 . 001*** .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 01510 . 7554/eLife . 07836 . 016Figure 6—figure supplement 1 . Enlarged VIT-2::GFP images . 40× GFP and BF images of ( A ) GSC ( + ) and ( B ) GSC ( − ) animals are shown .",
"Note that glp-1 ( ts ) ;VIT-2::GFP is presented with 5× lower exposure times due to increased VIT-2::GFP intensity in GSC ( − ) animals .",
"All worms shown are day-1 adults raised at 25°C .",
"Arrowheads indicate the normal distribution of VIT-2::GFP in the intestine and oocytes , and arrows indicate the ectopic accumulation of VIT-2::GFP seen in GSC ( − ) animals .",
"Numbers superimposed on images denote GFP exposure times . DOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 01610 . 7554/eLife . 07836 . 017Figure 6—figure supplement 2 . SKN-1 is activated in response to FA signaling . Accumulation of SKN-1 in intestinal nuclei in ( A ) response to arsenite exposure for 30 min or ( B ) daf-2 mutants is not impaired by sbp-1 RNAi .",
"( C ) Effects of OA and CO doses on intestinal gst-4p::GFP expression , compared to AS treatment .",
"( D ) Larval development and ( E ) egg laying rate are not affected by OA or CO treatment .",
"CO supplementation induces intestinal ( F ) SKN-1 nuclear accumulation but not ( G ) DAF-16 accumulation .",
"( H , I )",
"DAF-16 accumulation in glp-1 ( e2141ts ) is unaffected by sbp-1 RNAi .",
"( H ) Representative 40× DIC images of day-1 adults .",
"( J ) PMK-1 ( p38 kinase ) phosphorylation is not affected by GSC removal , consistent with a previous report ( Alper et al . , 2010 ) .",
"PMK-1 phosphorylation is increased dramatically by AS oxidative stress and reflects activation of its kinase activity ( Inoue et al . , 2005 ) .",
"( A–C , F , G , I )",
"GFP quantification with high , medium , low scoring .",
"Numbers above bars denote sample size .",
"p < 0 . 05*; p < 0 . 01**; p < 0 . 001*** .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 01710 . 7554/eLife . 07836 . 018Figure 6—figure supplement 3 . FA desaturation is required for GSC ( − ) stress resistance . glp-1 ( ts ) and control day-1 adult worms treated with lipl-3 , sbp-1 , fat-6/7 mix , skn-1 , or empty vector RNAi were exposed to 5 mM AS .",
"Knockdown of lipl-3 ( A , B ) , and either sbp-1 ( A , B ) or fat-6/7 ( C , D ) abolished the increase in AS resistance seen in glp-1 ( ts ) animals .",
"( B , D )",
"Data are represented as mean ± SEM .",
"p < 0 . 05*; p < 0 . 01**; p < 0 . 001*** .",
"The interaction between glp-1 ( ts ) and fat-6/7 , lipl-3 , sbp-1 , and skn-1 were significant ( p < 0 . 001 ) .",
"Additional information and statistics are provided in Table 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 018 We investigated whether accumulation of yolk-associated lipids might induce SKN-1 to mount a protective response .",
"Supporting this idea , when the oocyte-specific yolk receptor rme-2 is knocked down , yolk accumulates to high levels ( Grant and Hirsh , 1999 ) , and in the intestine , SKN-1 accumulates in nuclei and its target gene gst-4 is activated ( Figure 6C–F ) .",
"Additionally , rme-2 RNAi increased stress resistance in a skn-1-dependent manner ( Figure 6G and Table 2 ) .",
"When de novo lipogenesis was prevented by knockdown of the SREBP1 ortholog sbp-1 ( Yang et al . , 2006 ) , SKN-1::GFP failed to accumulate in intestinal nuclei in response to GSC inhibition ( Figure 6H , I ) , but not oxidative stress ( Figure 6—figure supplement 2A ) or reduced IIS ( daf-2 mutants , Figure 6—figure supplement 2B ) .",
"The sbp-1 lipogenesis defect can be rescued by supplementation with 600 µM OA ( Yang et al . , 2006 ) , which is the most abundant FA in olive oil , chicken egg yolk , and human adipose tissue ( National Research Council , 1976; Kokatnur et al . , 1979 ) .",
"In C . elegans , the abundance of OA is increased in GSC ( − ) animals , and its synthesis by the FA desaturases , FAT-6 and FAT-7 ( SCD orthologs ) , is required for GSC ( − ) lifespan extension ( Goudeau et al . , 2011 ) .",
"fat-6/7 were also required for SKN-1 to accumulate in nuclei after GSC inhibition ( Figure 6J ) .",
"Moreover , in GSC ( − ) animals subjected to sbp-1 RNAi , SKN-1 nuclear accumulation was fully restored by OA supplementation ( Figure 6H , I ) .",
"Consistent with their importance for SKN-1 function , sbp-1 and fat-6/7 were required for GSC absence to increase stress resistance ( Figure 6—figure supplement 3 ) .",
"Together , our data suggest that certain unsaturated lipids are required for SKN-1 to be activated in response to GSC loss , but not necessarily by other stimuli , and therefore , that lipid accumulation per se might activate SKN-1 .",
"Accordingly , feeding of either OA- or coconut oil ( CO ) -activated gst-4 in WT animals in a skn-1-dependent manner without impairing development or reproduction ( Figure 6—figure supplement 2C–E ) .",
"Under these conditions , CO feeding provided OA ( 300 µM with 0 . 1% CO ) along with multiple saturated FAs .",
"CO feeding strongly induced nuclear accumulation of SKN-1 but not DAF-16 ( Figure 6—figure supplement 2F , G ) , and sbp-1 RNAi did not impair DAF-16 nuclear accumulation in GSC ( − ) animals ( Figure 6—figure supplement 2H , I ) , supporting the notion that GSCs regulate SKN-1 and DAF-16 differently .",
"The lipid overload that results from reproductive failure might induce stress that activates SKN-1 .",
"Oxidative stress induced by sodium arsenite ( AS ) robustly activates the p38/PMK-1 kinase through phosphorylation , leading in turn to SKN-1 activation ( Inoue et al . , 2005 ) .",
"GSC loss induced SKN-1 nuclear accumulation at least as dramatically as AS treatment ( Figure 2B and Figure 6—figure supplement 2A ) but did not detectably increase PMK-1/p38 activity ( Figure 6—figure supplement 2J ) , suggesting that any stress arising from the lack of GSCs might not be sufficient on its own to explain SKN-1 activation .",
"By breaking down triglycerides , the lysosomal lipases LIPL-1/3 and LIPL-4 enable production of specific unsaturated FFAs that promote autophagy and longevity ( Lapierre et al . , 2011; O'Rourke et al . , 2013; O'Rourke and Ruvkun , 2013; Folick et al . , 2015 ) .",
"Some of these FAs are escorted from the lysosome to the nucleus by the conserved lipid-binding protein LBP-8/FABP1 ( Folick et al . , 2015; Han and Brunet , 2015 ) .",
"In GSC ( − ) animals , SKN-1 nuclear accumulation was inhibited modestly by lipl-4 RNAi but more strongly by lipl-1/3 double knockdown ( Figure 6J ) .",
"Furthermore , lipl-3 RNAi reduced stress resistance in GSC ( − ) but not WT animals ( Figure 6—figure supplement 3A , B ) .",
"Given that fat storage is increased in lipl-1/3 mutants ( O'Rourke and Ruvkun , 2013 ) , our data suggest that in GSC ( − ) animals , SKN-1 activity may depend upon particular lipl-1/3-dependent products , not lipid levels per se .",
"Knockdown of lbp-8 or other LBPs also interfered with SKN-1::GFP nuclear accumulation in GSC ( − ) animals , and lbp-8 RNAi impaired SKN-1-dependent gst-4 activation by OA , indicating involvement of FA transport ( Figure 6J , K ) .",
"Together , our data suggest that in GSC ( − ) animals , excessive lipid levels lead to production of OA- and LIPL-1/3-dependent FAs that activate SKN-1 , possibly through FA-based signaling ( Figure 7 ) . 10 . 7554/eLife . 07836 . 019Figure 7 . SKN-1 regulation in the GSC longevity pathway . GSC absence results in activation of transcription factors in the intestine , with SKN-1 being regulated in parallel to DAF-12 and DAF-16 .",
"Yolk transport to oocytes is disrupted by GSC loss , resulting in lipid accumulation in the intestine and body cavity .",
"The resulting SKN-1 activation requires OA , the FAT-6/7 FA desaturases , and the lysosomal lipases LIPL-1/3 .",
"This lipid-based signaling to SKN-1 depends partially upon LBP-8 , which transports FAs from the lysosome to the nucleus .",
"SKN-1 induces transcription of genes involved in stress resistance , detoxification , proteasome maintenance , extracellular matrix , and lipid metabolism , thereby reducing fat storage and increasing stress resistance and lifespan .",
"Magenta denotes processes that are active in the presence of GSCs . DOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 019"
],
[
"The question of how events in one tissue can influence aging in others is of fundamental importance .",
"The effects of GSC loss in C . elegans provide a paradigm for investigating this problem , as well as interactions between a stem cell population and its environment .",
"Here , we determined that GSC inhibition leads to a broad transcriptional reprogramming in somatic tissues that involves activation of SKN-1 , and that SKN-1 is required for many beneficial effects of GSC absence , including lifespan extension .",
"Previous work showed that SKN-1 is required for lifespan to be extended by reduced insulin/IGF-1 , mTORC1 , or mTORC2 signaling , by dietary restriction and by low-level mitochondrial reactive oxygen species ( ROS ) production ( Bishop and Guarente , 2007; Tullet et al . , 2008; Robida-Stubbs et al . , 2012; Zarse et al . , 2012; Schmeisser et al . , 2013; Mizunuma et al . , 2014; Moroz et al . , 2014; Ewald et al . , 2015 ) .",
"Our new data further support the idea that SKN-1/Nrf proteins are broadly important for longevity assurance .",
"One major role of SKN-1 in GSC ( − ) animals is to increase proteasome activity ( Figure 4C , D , and Figure 4—figure supplement 1 ) .",
"In WT animals , SKN-1 activates most proteasome subunit genes when the proteasome is inhibited ( Li et al . , 2011 ) .",
"This compensatory function is conserved in its mammalian ortholog Nrf1 , which is cleaved and activated when proteasome activity is low ( Radhakrishnan et al . , 2010; Steffen et al . , 2010; Radhakrishnan et al . , 2014; Sha and Goldberg , 2014 ) .",
"By contrast , in GSC ( − ) animals , proteasome activity is elevated ( Figure 4C , D ) ( Vilchez et al . , 2012 ) , suggesting that additional mechanisms influence SKN-1/Nrf regulation of proteasome genes .",
"In GSC ( − ) animals , most if not all proteasome subunit genes are dependent upon SKN-1 for their expression ( Figure 4A ) , and SKN-1 and DAF-16 together activate the proteasome subunit gene rpn-6 . 1 ( Figure 4E , F ) , the levels of which are rate limiting for proteasome activity ( Vilchez et al . , 2012 ) .",
"Overexpression of either rpn-6 . 1 or the 20S proteasome subunit pbs-5 increases C . elegans lifespan , and in the latter case , lifespan extension was shown to be skn-1-dependent ( Vilchez et al . , 2012; Chondrogianni et al . , 2015 ) , suggesting that enhancement of proteasome activity may be an important mechanism through which SKN-1/Nrf promotes longevity .",
"The evidence that GSC ( − ) longevity is associated with fat accumulation , and altered lipid metabolism has raised the intriguing possibility that GSC absence induces production of lipids that promote health and longevity ( see ‘Introduction’ ) .",
"Our results are consistent with aspects of this model but suggest an important modification .",
"GSC ( − ) animals accumulate dramatically high levels of yolk lipoproteins by the first day of adulthood ( Figure 6A , B ) , providing a likely reason that they accumulate so much lipid .",
"Moreover , SKN-1 acts to reduce fat accumulation but is critical for the benefits of GSC loss ( Figure 5B , C , and Figure 5—figure supplement 1A , B ) , suggesting that GSC ( − ) animals do not simply overproduce healthful lipids .",
"Finally , excess yolk accumulation induced by another method ( rme-2 RNAi ) leads to increased SKN-1 nuclear accumulation and target gene activation , and skn-1-dependent stress resistance ( Figure 6D–G ) .",
"Taken together , our data suggest that GSC ( − ) animals accumulate excess fat because they cannot stop production of fat that would otherwise support reproduction ( Figure 7 ) .",
"Importantly , however , in responding to and metabolizing this fat they produce specific lipids that activate SKN-1 and other regulators , which in turn increase lifespan and may promote a more healthy balance of lipids ( Figure 7 ) .",
"Several lines of evidence support this model .",
"GSC inhibition induces SKN-1 and NHR-49 to upregulate largely distinct sets of FA oxidation genes ( Figure 5A ) ( Ratnappan et al . , 2014 ) .",
"This effect of SKN-1 could account for its inhibitory role in fat accumulation ( Figure 5B , C ) .",
"A need to metabolize excess fat could also explain the importance of lipophagy in GSC ( − ) longevity ( Lapierre et al . , 2011; Hansen et al . , 2013 ) .",
"With respect to signaling lipids , GSC ( − ) longevity requires the triglyceride lipase LIPL-4 ( Wang et al . , 2008 ) , which generates unsaturated FFAs that promote longevity ( O'Rourke et al . , 2013; Folick et al . , 2015 ) .",
"While LIPL-4-dependent FAs act through NHR-49 and NHR-80 ( Lapierre et al . , 2011; Folick et al . , 2015 ) , and possibly not SKN-1 ( Figure 6J ) , in GSC ( − ) animals , SKN-1 activation involves the LIPL-1/3 lipases ( Figure 6J ) , which also promote longevity ( O'Rourke and Ruvkun , 2013 ) .",
"This elevated SKN-1 activity also depends upon lipid transfer proteins , as well as OA ( Figure 6H–J and Figure 6—figure supplement 1C ) .",
"OA is required for GSC ( − ) longevity ( Goudeau et al . , 2011 ) and is a precursor to unsaturated FAs that have signaling functions ( O'Rourke et al . , 2013; Folick et al . , 2015 ) .",
"Finally , SKN-1 upregulates lipl-3 and lbp-8 in WT and GSC ( − ) animals ( Figure 5A and Figure 5—figure supplement 3; Supplementary file 1a , c ) , suggesting that it may function both downstream and upstream of lipid signals .",
"The idea that SKN-1 can be activated by lipids that arise from prevention of reproduction and yolk consumption should be considered in evaluation of genetic or pharmacological interventions that increase SKN-1 activity .",
"SKN-1 is activated by lipids and regulates lipid metabolism gene expression not only in the high-fat GSC ( − ) model but also in WT animals under normal feeding conditions ( Figures 5 , 6; Supplementary file 1b–d ) .",
"Moreover , SKN-1 profoundly reduced fat storage by the beginning of adult life in healthy , reproductively active animals that have not begun to age .",
"Taken together , our findings suggest that SKN-1 plays an integral and direct role in maintaining lipid homeostasis .",
"Our results predict that insufficient mammalian Nrf function does not lead to NAFLD/NASH simply by increasing chronic hepatic stress ( Xu et al . , 2005; Lee et al . , 2013 ) , and that a protective function of Nrf proteins in fat metabolism is likely to be involved .",
"Nrf proteins therefore may provide an important line of defense against metabolic disease .",
"Development of NAFLD is a growing obesity-related public health issue ( Cohen et al . , 2011 ) .",
"Our data suggest that analysis of Nrf proteins could be a promising underexplored direction for investigating causes and prevention of NAFLD , and that SKN-1 and C . elegans provide a genetically tractable model that will be valuable in this effort .",
"Our evidence that GSCs activate SKN-1 through lipid-based signaling suggests a new mechanism through which GSCs influence the soma .",
"The response to GSC loss therefore involves metabolic signals that reflect the altered nutritional balance within the organism .",
"Similar interactions could be important in other stem cell contexts .",
"For example , in the mammalian bone marrow microenvironment , adipose tissue profoundly influences the function of hematopoietic and mesenchymal stem cells ( Adler et al . , 2014; Muruganandan and Sinal , 2014 ) .",
"In addition to the mechanisms we have described here , GSC ( − ) longevity involves endocrine signals from the somatic gonad and depends upon absence of GSCs per se , not simply reproductive cessation ( Kenyon , 2010; Antebi , 2013 ) .",
"It will now be of interest to determine how these mechanisms , as well as other transcription factors that are required for GSC ( − ) longevity ( see ‘Introduction’ ) , interface with SKN-1 and its regulation .",
"Signaling lipids from endogenous , dietary , or microbiota sources constitute an area of considerable excitement , because these signals can induce beneficial effects such as anti-inflammatory protection , enhanced insulin sensitivity , protection against metabolic disease , and increased lifespan in C . elegans ( Wang et al . , 2008; Kniazeva and Han , 2013; Lim et al . , 2013; O'Rourke and Ruvkun , 2013; Folick et al . , 2015; Han and Brunet , 2015 ) .",
"Signals derived from OA are of particular interest in this regard , because of its dietary availability in olive oil .",
"Mechanisms through which lipid signals are known to act on transcription networks include binding to nuclear receptors , and OA-induced protein kinase A activation that ultimately leads to FA oxidation ( Fu et al . , 2003; Lim et al . , 2013; Folick et al . , 2015 ) .",
"By revealing SKN-1 as a new regulator of metabolism and stress defenses that is activated in response to lipids , our data suggest the exciting possibility that this might also be the case for mammalian Nrf proteins .",
"They also suggest that a ‘lipohormesis’ pathway in which signaling lipids confer health benefits by activating SKN-1/Nrf may not only be a characteristic of GSC-ablated animals but also might be more broadly applicable for enhancing health- and possibly lifespan ."
],
[
"Worms were maintained on nematode growth medium ( NGM ) plates seeded with Escherichia coli ( OP50 ) at 15°C , using standard techniques ( Brenner , 1974 ) .",
"In all experiments , glp-1 ( ts ) mutants were matched with the wild-type N2 strain used for outcrossing .",
"The C . elegans strains used in this study are detailed in Table 3 . 10 . 7554/eLife . 07836 . 020Table 3 . C .",
"elegans strains used in this studyDOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 020CodeGenetic backgroundTransgeneReferenceAA003daf-12 ( rh61rh411 ) X– ( Shen et al . , 2012 ) AA983glp-1 ( e2141ts ) III; daf-12 ( rh61rh411 ) X– ( Shen et al . , 2012 ) AA1049mir-241 ( n4315 ) V; mir-84 ( n4037 ) X– ( Shen et al . , 2012 ) AA1709glp-1 ( e2141ts ) III; mir-241 ( n4315 ) V; mir-84 ( n4037 ) X– ( Shen et al . , 2012 ) AA2735glp-1 ( e2141ts ) III– ( Shen et al . , 2012 ) CF1903glp-1 ( e2141ts ) III– ( Berman and Kenyon , 2006 ) CF1935daf-16 ( mu86 ) I; glp-1 ( e2141ts ) IIImuIs109[daf-16p::GFP:: DAF-16 + odr-1p::RFP] X ( Berman and Kenyon , 2006 ) CL2166N2dvIs19[pAF15 ( gst-4p::GFP::NLS ) ] III ( Link and Johnson , 2002 ) DH1033sqt-1 ( sc103 ) IIbIs1[vit-2p::VIT-2:: GFP + rol-6 ( su1006 ) ] X ( Grant and Hirsh , 1999 ) EU31skn-1 ( zu135 ) IV– ( Bowerman et al . , 1992 ) LD001N2ldIs7[SKN-1b/c:: GFP + rol-6 ( su1006 ) ] ( An and Blackwell , 2003 ) LD002N2ldIs1[SKN-1b/c:: GFP + rol-6 ( su1006 ) ] ( An and Blackwell , 2003 ) LD1025daf-2 ( e1370 ) IIIldIs7[SKN-1b/c:: GFP + rol-6 ( su1006 ) ] ( Tullet et al . , 2008 ) LD1425glp-1 ( bn18ts ) IIIldIs1[SKN-1b/c:: GFP + rol-6 ( su1006 ) ]This studyLD1434glp-1 ( bn18ts ) III; skn-1 ( zu135 ) IV–This studyLD1473kri-1 ( ok1251 ) I; glp-1 ( bn18ts ) IIIldIs1[SKN-1b/c:: GFP + rol-6 ( su1006 ) ]This studyLD1474tcer-1 ( tm1452 ) II; glp-1 ( bn18ts ) IIIldIs1[SKN-1b/c:: GFP + rol-6 ( su1006 ) ]This studyLD1548N2Is[dhs-3p::DHS-3:: GFP] I ( Zhang et al . , 2012 ) LD1549glp-1 ( bn18ts ) IIIIs[dhs-3p::DHS-3:: GFP] IThis studyLD1644sqt-1 ( sc103 ) II; glp-1 ( bn18ts ) IIIbIs1[vit-2p::VIT-2:: GFP + rol-6 ( su1006 ) ] XThis studyLD1653glp-1 ( bn18ts ) III– ( Dorsett et al . , 2009 ) Outcrossed from DG2389LD1744glp-1 ( bn18ts ) IIIldEx119[pAF15 ( gst-4p::GFP::NLS ) + rol-6 ( su1006 ) ]This studyTJ356N2zIs356[daf-16p:: DAF-16a/b::GFP + rol-6] IV ( Henderson and Johnson , 2001 ) Feeding RNAi was performed using tetracycline-resistant HT115 bacteria carrying the pL4440 plasmid with ampicillin/carbenicillin resistance ( Kamath et al . , 2001 ) .",
"RNAi cultures were grown overnight in 50 ml conical tubes at 37°C with shaking at 220 RPM in 5 ml LB medium containing 50 µg/ml carbenicillin and 12 . 5 µg/ml tetracycline .",
"Cultures were diluted 1:5 the following day in LB containing carbenicillin and tetracycline to allow for re-entry into the logarithmic growth phase , grown to an OD600 of 1 . 5 ( ∼6 hr ) .",
"Cultures were centrifuged at 4500 RPM for 10 min , concentrated to a volume of 5 ml , and then induced with 1 mM IPTG prior to plating .",
"Bacterial cultures were seeded onto standard NGM plates containing 50 µg/ml carbenicillin , 12 . 5 µg/ml tetracycline , and 0 . 4 mM IPTG .",
"Worms were synchronized by timed egg lay , upshifted to 25°C at the L2 stage , then scored for lifespan at 25°C or 20°C , as previously described ( Arantes-Oliveira et al . , 2002; Robida-Stubbs et al . , 2012 ) .",
"For analyses of glp-1 ( ts ) at 20°C , worms were downshifted from 25°C upon reaching adulthood .",
"Animals were transferred at the first day of adulthood to fresh plates containing FUdR ( ACROS Organics/Thermo Fisher Scientific , Geel , Belgium ) at a concentration of 100 µg/ml to inhibit progeny development ( Mitchell et al . , 1979 ) , unless otherwise indicated .",
"RNAi-treated worms were placed on RNAi feeding plates starting at the first day of adulthood .",
"Worms were maintained at a density of 30 worms per 6 cm plate on live bacteria and scored every other day .",
"Animals that crawled off the plate , ruptured , or died from internal hatching were censored .",
"Lifespans were graphed as Kaplan–Meier survival curves with JMP Pro 12 ( SAS Institute , Middleton , MA ) .",
"p values for survival curve analysis were generated using log-rank test .",
"Additional statistical analysis was performed with GraphPad Prism 6 ( GraphPad Software , La Jolla , CA ) .",
"p values for mean lifespan analysis were calculated by two-way ANOVA with post hoc Holm-Šídák correction .",
"Synchronized animals were incubated at 25°C during development then scored for survival hourly beginning at either days 1 or 3 of adulthood .",
"Feeding RNAi was started at the L1 stage for day-1 stress assays or post-developmentally at day-1 adulthood for stress assays performed at day-3 adulthood .",
"For the arsenite stress assay , worms were incubated in M9 buffer containing 5 mM AS ( Riedel-de Haën , Seelze , Germany ) .",
"For the tert-butyl hydroperoxide ( TBHP ) stress assay , worms were placed on solid NGM plates containing 15 . 4 mM TBHP ( Sigma–Aldrich , St . Louis , MO ) .",
"TBHP plates were freshly prepared on the day of the experiment .",
"Survival assays were graphed as Kaplan–Meier survival curves with JMP Pro 12 .",
"p values for survival curve analysis were generated using log-rank test .",
"Additional statistical analysis was performed with GraphPad Prism 6 .",
"p values for mean survival analysis were calculated by two-way ANOVA with post hoc Holm-Šídák correction .",
"Animals were anaesthetized for 5 min in 0 . 06% tetramisole/M9 buffer , mounted on 2% agarose pads on glass slides under coverslips , and imaged with ZEN 2012 software on an Axio Imager . M2 microscope ( Zeiss , Jena , Germany ) .",
"Intestinal SKN-1::GFP nuclear localization and gst-4p::GFP::NLS expression were scored as ‘high’ , ‘medium’ , or ‘low’ as previously described ( An and Blackwell , 2003; Ewald et al . , 2015 ) .",
"‘High’ denotes strong intensity in all intestinal nuclei; ‘medium’ indicates relatively lower intensity or distribution in approximately half of intestinal nuclei; ‘low’ denotes weak or no visible GFP intensity in intestinal nuclei .",
"p values were calculated by two-sided χ2 test .",
"Intestinal DAF-16::GFP nuclear localization was scored as ‘high’ , ‘medium’ , or ‘low’ as previously described ( Henderson and Johnson , 2001; Berman and Kenyon , 2006; Curran and Ruvkun , 2007 ) .",
"‘High’ denotes more DAF-16::GFP observed in the nucleus compared to the cytoplasm; ‘medium’ indicates animals with noticeable DAF-16::GFP in the nucleus but higher levels in the cytoplasm; ‘low’ denotes entirely cytoplasmic DAF-16::GFP .",
"SKN-1::GFP color isolation was performed to reduce gut granule autofluorescence using selective color matching against rgb ( 99 , 159 , 94 ) with a fuzziness setting of 125 and auto contrast in Adobe Photoshop CC 2014 ( Adobe , San Jose , CA ) .",
"DAF-16::GFP color isolation was similarly performed using selective color matching against rgb ( 0 , 255 , 111 ) with a fuzziness setting of 100 .",
"Samples were prepared from ∼200 day-3 adult worms synchronized by timed egg lay .",
"RNA was extracted using TRIzol ( Thermo Fisher , Waltham , MA ) -based phenol-chloroform extraction and purified with RNA Clean and Concentrator-5 spin columns ( Zymo Research , Irvine , CA ) .",
"RNA concentration and quality was assessed with a NanoDrop 1000 spectrophotometer ( Thermo Fisher ) .",
"cDNAs were prepared using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR ( Thermo Fisher ) .",
"mRNA levels were quantified from biological triplicates and technical duplicates using SYBR Green ( Thermo Fisher ) fluorescence on a 384-well format Real-Time PCR 7900 ( Applied Biosystems , Foster City , CA ) .",
"After an initial denaturation step ( 95°C for 10 min ) , amplification was performed using 40 cycles of denaturation ( 95°C for 15 s ) and annealing ( 60°C for 1 min ) .",
"Samples were analyzed by the standard curve method , with normalization to the reference genes cdc-42 and Y45F10D . 4 ( Hoogewijs et al . , 2008 ) .",
"p values were calculated by two-sided Student's t-test with post hoc Holm-Šídák correction in GraphPad Prism 6 .",
"The primers used in this study are provided in Table 4 . 10 . 7554/eLife . 07836 . 021Table 4 . qRT-PCR primers used in this studyDOI: http://dx . doi . org/10 . 7554/eLife . 07836 . 021GeneSequenceAnnotationPrimer pairgst-4K08F4 . 7Glutathione S-transferaseFWD: CCCATTTTACAAGTCGATGG REV: CTTCCTCTGCAGTTTTTCCAF20D6 . 11F20D6 . 11Flavin-adenine dinucleotide ( FAD ) -binding oxidoreductaseFWD: GGAAATTCTCGGTAGAATCGAA REV: ACGATCACGAACTTCGAACAnit-1ZK1058 . 6NitrilaseFWD: AATCCTCCGACTATCCCTTG REV: AGCGAATCGTTTCTTTTGTGrpn-6 . 1F57B9 . 1019S non-ATPase subunitFWD: AATATTGGAAAAGCACCTGAAATGT REV: TTTGATGTGGAAGTGAAGTCATTGTlipl-3R11G11 . 14Lysosomal triglyceride lipaseFWD: ATGGGCAGGCAAATCCACCA REV: AGTTGTTCTGCGCAATTATA*cdc-42R07G3 . 1Housekeeping geneFWD: CTGCTGGACAGGAAGATTACG REV: CTCGGACATTCTCGAATGAAG*Y45F10D . 4Y45F10D . 4Housekeeping geneFWD: GTCGCTTCAAATCAGTTCAG CREV: GTTCTTGTCAAGTGATCCGACASelect primer sequences were obtained from previous publications ( Robida-Stubbs et al . , 2012; Vilchez et al . , 2012; O'Rourke and Ruvkun , 2013 ) .",
"Samples were prepared from ∼5000 synchronized , L1 arrested day-1 adult animals cultured at 25°C .",
"Worms were synchronized by sodium hypochlorite ( bleach ) treatment , as previously described ( Porta-de-la-Riva et al . , 2012 ) .",
"Bleach solution ( 9 ml ddH2O; 1 ml 1 N NaOH; 4 ml Clorox bleach ) was freshly prepared before each experiment .",
"Worms were bleached for 5 min , washed 5× in M9 , and arrested at the L1 stage at 25°C in M9 containing 10 µg/ml cholesterol .",
"Feeding RNAi was started at the L1 stage .",
"This approach only partially reduces skn-1 function but allows analysis of larger samples than would be feasible with skn-1 mutants , which are sterile ( Bowerman et al . , 1992 ) .",
"Because these animals were not treated with FUdR , the WT adults contained an intact germline and eggs .",
"As is explained in the ‘Results’ section , we therefore confined our analysis to genes that were overrepresented in glp-1 ( ts ) animals , which lack eggs and most of the germline , and established a high-confidence cutoff for genes that were upregulated by GSC absence as opposed to simply being expressed specifically in somatic tissues .",
"RNA was extracted using the same protocol for qRT-PCR samples .",
"Purified RNA samples were DNase treated and assigned an RNA Integrity Number ( RIN ) quality score using a Bioanalyzer 2100 ( Agilent Technologies , Santa Clara , CA ) .",
"Only matched samples with high RIN scores were sent for sequencing .",
"Single read 50 bp RNA-seq with poly ( A ) enrichment was performed at the Dana-Farber Cancer Institute Center for Computational Biology using a HiSeq 2000 ( Illumina , San Diego , CA ) .",
"FASTQ output files were aligned to the WBcel235 ( February 2014 ) C . elegans reference genome using STAR ( Dobin et al . , 2013 ) .",
"These files have been deposited at the Gene Expression Omnibus ( GEO ) with the accession number GSE63075 .",
"Samples averaged 75% mapping of sequence reads to the reference genome .",
"Differential expression analysis was performed using a custom R and Bioconductor RNA-seq pipeline ( http://bioinf . wehi . edu . au/RNAseqCaseStudy/ ) ( Gentleman et al . , 2004; Anders et al . , 2013; R Core Team , 2014 ) .",
"Quantification of mapped reads in the aligned SAM output files was performed using featureCounts , part of the Subread package ( Liao et al . , 2013 , 2014 ) .",
"We filtered out transcripts that didn’t have at least one count per million reads in at least two samples .",
"Quantile normalization and estimation of the mean–variance relationship of the log counts was performed by voom ( Law et al . , 2014 ) .",
"Linear model fitting , empirical Bayes analysis , and differential expression analysis were then conducted using limma ( Smyth , 2005 ) .",
"To identify genes that are upregulated in a SKN-1-dependent manner by GSC loss , we sought genes for which glp-1 ( ts ) expression was higher than WT , and for which glp-1 ( ts ) ;skn-1 ( − ) expression was reduced relative to glp-1 ( ts ) .",
"To test for this pattern , if a gene's expression change was higher in the comparison of glp-1 ( ts ) vs WT and lower in the comparison of glp-1 ( ts ) ;skn-1 ( − ) vs glp-1 ( ts ) , then we calculated the minimum ( in absolute value ) of the t-statistics from these two comparisons , and assessed the significance of this statistic by comparing to a null distribution derived by applying this procedure to randomly generated t-statistics .",
"We corrected for multiple testing in this and the differential expression analysis using the false discovery rate ( Benjamini and Hochberg , 1995 ) .",
"Heatmaps were generated using heatmap . 2 in the gplots package ( Warnes et al . , 2014 ) .",
"Functional annotations and phenotypes were obtained from WormBase build WS246 .",
"SKN-1 transcription factor binding site analysis of hits was conducted with biomaRt , GenomicFeatures , JASPAR , MotifDb , motifStack , MotIV , and Rsamtools ( Sandelin et al . , 2004; Durinck et al . , 2005 , 2009; Lawrence et al . , 2013; Ou et al . , 2013; Mercier and Gottardo , 2014; Shannon , 2014 ) .",
"JASPAR analysis was performed with the SKN-1 matrix MA0547 . 1 using 2 kb upstream sequences obtained from Ensembl WBcel235 ( Staab et al . , 2013a ) .",
"modENCODE SKN-1::GFP ChIP-seq analysis of hits was performed using biomaRt , ChIPpeakAnno , IRanges , and multtest ( Durinck et al . , 2005 , 2009; Gerstein et al . , 2010; Zhu et al . , 2010; Niu et al . , 2011; Lawrence et al . , 2013 ) .",
"SKN-1::GFP ChIP-seq peaks were generated by Michael Snyder's lab .",
"We used the peak data generated from the first 3 larval stages: L1 ( modENCODE_2622; GSE25810 ) , L2 ( modENCODE_3369 ) , and L3 ( modENCODE_3838; GSE48710 ) .",
"Human ortholog matching was performed using WormBase , Ensembl , and OrthoList ( Shaye and Greenwald , 2011 ) .",
"Gene lists were evaluated for functional classification and statistical overrepresentation with Database for Annotation , Visualization , and Integrated Discovery ( DAVID ) version 6 . 7 ( Dennis et al . , 2003 ) .",
"SKN-1 and DAF-16 binding peaks within the first intron and the promoter of rpn-6 . 1 were previously identified by the modENCODE project ( Furuyama et al . , 2000; Gerstein et al . , 2010; Niu et al . , 2011 ) .",
"We identified multiple hits with the consensus binding sequence ATCAT in the TRANSFAC matrices N$SKN1_01 and N$SKN1_02 using MATCH ( Biobase , Wolfenbüttel , Germany ) that overlap with the SKN-1::GFP ChIP-seq binding peaks within the first intron and the promoter of rpn-6 . 1 ( Kel et al . , 2003; Matys et al . , 2006 ) .",
"Our analysis also confirmed a previously identified hit ( Vilchez et al . , 2012 ) in the N$DAF16_01 matrix with the consensus binding sequence TGTTT that overlaps with the DAF-16::GFP ChIP-seq peak within the first intron .",
"No putative DAF-16 binding sites were identified in the rpn-6 . 1 promoter in the TRANSFAC MATCH analysis .",
"In vitro chymotrypsin-like proteasome activity assays were performed as previously described ( Kisselev and Goldberg , 2005; Vilchez et al . , 2012 ) .",
"Worms were bleach synchronized and maintained at 25°C from egg stage , then lysed at day 1 of adulthood , unless otherwise noted , in freshly prepared proteasome activity assay buffer ( 50 mM Tris–HCl , pH 7 , 250 mM sucrose , 5 mM MgCl2 , 0 . 5 mM EDTA , 2 mM ATP , and 1 mM dithiothreitol ) using a Branson digital sonifier at 4°C .",
"Lysates were centrifuged at 10 , 000×g for 15 min at 4°C .",
"25 µg of protein , calculated using the BCA protein assay ( #23225; Pierce Biotechnology/Thermo Fisher , Rockford , IL ) , was transferred to a flat 96-well microtiter plate ( Nunc , Roskilde , Denmark ) .",
"Samples were incubated at 25°C , and fluorogenic chymotrypsin substrate ( #230914; Calbiochem/EMD Millipore , San Diego , CA ) was added to the plate immediately before analysis .",
"Fluorescence ( 380 nm excitation; 460 nm emission ) was measured every 3 min for 1 hr at 25°C using a Synergy MX microplate reader with Gen5 software ( Bio-Tek , Winooski , VT ) .",
"Lysates were assayed in triplicate .",
"p values were calculated by two-sided Student's t-test in GraphPad Prism 6 .",
"ORO staining was performed on fixed animals , essentially as described ( O'Rourke et al . , 2009; Yen et al . , 2010 ) , with some modifications .",
"200–300 day-1 adult worms synchronized by timed egg lay were washed three times with phosphate-buffered saline ( PBS ) then snap frozen in a dry ice/ethanol bath .",
"Upon thawing , worms were treated with PBS containing 2% paraformaldehyde ( PFA ) ( #15713; Electron Microscopy Services , Hatfield , PA ) , using three freeze thaw cycles with dry ice/ethanol to permeabilize the cuticle .",
"Worms were then washed with PBS to remove the PFA .",
"Filtered ORO solution ( 0 . 5 g of ORO powder [#O0625; Sigma–Aldrich] in 100 ml of 60% isopropanol ) was prepared freshly before each experiment .",
"Worms were stained for 3 hr in a round bottom 96 well plate in ORO solution at room temperature with gentle shaking .",
"Longer staining periods , such as overnight incubation ( O'Rourke et al . , 2009 ) , saturated ORO staining in glp-1 ( ts ) animals to a level that rendered glp-1 ( ts ) and glp-1 ( ts ) ;skn-1 strains indistinguishable .",
"Animals were imaged at 40× using differential interference contrast microscopy .",
"Quantification of ORO staining was performed on the upper intestine , directly below the pharynx .",
"Since ORO absorbs light at 510 nm ( green channel ) , we performed background subtraction of the red channel from the green channel in Adobe Photoshop CC ( Adobe ) to specifically isolate the ORO staining , as previously described ( Yen et al . , 2010 ) .",
"Quantification of mean intensity over background for each animal was performed using Fiji ( http://fiji . sc ) .",
"Statistical analysis was performed with GraphPad Prism 6 .",
"p values were calculated by one-way ANOVA with post hoc Holm-Šídák correction .",
"Using a COPAS Biosort ( Union Biometrica , Holliston , MA ) ( Pulak , 2006 ) , bleach-synchronized day-1 adult worms were scored for GFP fluorescence .",
"RNAi was initiated after L1 arrest .",
"The COPAS was used to record three attributes for each individual nematode: time of flight ( TOF ) , which corresponds to nematode length; extinction ( EXT ) , which corresponds to the optical density; and GFP fluorescence intensity .",
"TOF and EXT measurements are related to the size and age of the nematode; both increase with development .",
"These parameters were used to specifically gate adult worms .",
"GFP fluorescence was normalized to worm size as a ratio of GFP/TOF values .",
"Representative GFP images of each strain were captured at 4× using an Olympus IX51 inverted microscope ( Olympus , New Orleans , LA ) .",
"p values were calculated by one-way ANOVA with post-hoc Holm-Šídák correction in GraphPad Prism 6 .",
"Triglyceride ( TAG ) levels were measured with the Triglyceride Colorimetric Assay Kit ( #10010303; Cayman Chemical , Ann Arbor , MI ) .",
"Samples were run according to the manufacturer's protocol in triplicate .",
"TAG concentrations were normalized relative to protein concentration using the BCA protein assay ( Pierce Biotechnology ) ."
]
] | [
"In Caenorhabditis elegans , ablation of germline stem cells ( GSCs ) extends lifespan , but also increases fat accumulation and alters lipid metabolism , raising the intriguing question of how these effects might be related .",
"Here , we show that a lack of GSCs results in a broad transcriptional reprogramming in which the conserved detoxification regulator SKN-1/Nrf increases stress resistance , proteasome activity , and longevity .",
"SKN-1 also activates diverse lipid metabolism genes and reduces fat storage , thereby alleviating the increased fat accumulation caused by GSC absence .",
"Surprisingly , SKN-1 is activated by signals from this fat , which appears to derive from unconsumed yolk that was produced for reproduction .",
"We conclude that SKN-1 plays a direct role in maintaining lipid homeostasis in which it is activated by lipids .",
"This SKN-1 function may explain the importance of mammalian Nrf proteins in fatty liver disease and suggest that particular endogenous or dietary lipids might promote health through SKN-1/Nrf ."
] | [
"Understanding how animals age may help us to prevent age-related or chronic diseases , such as type 2 diabetes and cancer .",
"The tiny nematode worm known as C . elegans is widely used as a model to study aging and has enabled researchers to identify factors that can slow down the aging process .",
"Like other animals , these worms contain female and male sex cells that originate from cells called germline stem cells .",
"The normal lifespan of C . elegans is less than three weeks , but when the germline stem cells are removed , the worms can live for much longer .",
"Reproduction requires a lot of energy , which is typically ‘stored’ in molecules of fat .",
"Animals utilize their fat reserves and release this energy by breaking the fat molecules down into smaller molecules as part of their ‘metabolism’ .",
"Worms that have had their germline stem cells removed have altered fat metabolism , and it is thought that this may contribute to their increased lifespan .",
"These worms have increased levels of a protein called SKN-1 , which alters fat metabolism and helps to protect cells from toxic molecules and other stresses .",
"SKN-1 works by regulating the activity ( or ‘expression’ ) of many genes in cells , but it is not clear how this increases the lifespan of the worms .",
"Steinbaugh et al . studied mutant worms that were lacking SKN-1 .",
"Unlike normal worms , when the germline stem cells were removed from the mutants , their lifespan did not increase .",
"Further experiments analyzed the genes that are switched on by SKN-1 , and identified many that are involved in fat metabolism , in degrading other proteins , and in detoxifying harmful molecules .",
"The experiments also found that SKN-1 reduces the overall amount of fat stored in the body .",
"Next , Steinbaugh et al . investigated how SKN-1 stops fat from being stored .",
"During reproduction , cells in the gut produce yolk—which is rich in fats—that will be provided to germ cells to nourish the developing embryo .",
"Worms lacking germline stem cells are not able to reproduce , but they continue to make yolk .",
"Steinbaugh et al . found that the build up of the yolk activates SKN-1 , which in turn inhibits the further accumulation of fats .",
"Steinbaugh et al . 's findings show that SKN-1 can be activated by fat molecules and plays a direct role in controlling the amount of fat stored in the body of the worms .",
"A future challenge will be to identify the specific fat molecules that activate SKN-1 , which could provide a model for understanding how specific fats in human diets could have wide-ranging health benefits ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"neuroscience"
] | Low FoxO expression in Drosophila somatosensory neurons protects dendrite growth under nutrient restriction | elife-53351-v2 | [
[
"Proper animal development requires coordinated growth of various organs to achieve the correct relative organ proportions in mature individuals .",
"However , when developing animals face adverse conditions , such as limited availability of nutrients , they reallocate essential resources to favor growth of vital organs at the expense of other organs .",
"This phenomenon of ‘organ sparing’ is exemplified by the preferential growth of the brain in human fetuses experiencing intrauterine growth restriction , resulting in undersized newborns with disproportionately large heads ( Gruenwald , 1963 ) .",
"Although changes of circulation are known to enhance oxygen and blood supply towards the brain under nutrient deprivation in mammals ( Severi et al . , 2000; Cohen et al . , 2015 ) , how systemic growth control is altered at the molecular level to favor brain development remains poorly understood .",
"A well-characterized example of brain sparing occurs in Drosophila larvae experiencing nutrient deprivation .",
"Systemic larval body growth of Drosophila is controlled by the conserved insulin/insulin like growth factor ( IGF ) pathway ( Rulifson et al . , 2002 ) .",
"Drosophila insulin-like peptides ( Dilps ) secreted by the insulin producing cells ( IPCs ) in the larval brain promote cell proliferation and growth of peripheral tissues by activating the insulin receptor ( InR ) and the downstream signaling components phosphatidylinositol 3-kinase ( PI3K ) and Akt ( PKB ) ( Verdu et al . , 1999; Brogiolo et al . , 2001; Ikeya et al . , 2002; Oldham et al . , 2002 ) .",
"Nutrient restriction suppresses insulin secretion through an intricate nutrient sensing mechanism involving inter-organ communications between the fat body and IPCs ( Ikeya et al . , 2002; Géminard et al . , 2009; Rajan and Perrimon , 2012 ) , and consequently , curbs the growth of most peripheral tissues .",
"However , the larval brain is protected against nutrient deprivation and exhibits continuous neurogenesis ( Cheng et al . , 2011 ) .",
"This protection is mediated by the glia-derived Jelly belly ( Jeb ) ligand that activates the Anaplastic lymphoma kinase ( Alk ) receptor on neural stem cells ( NSCs ) to turn on the downstream PI3K pathway independent of nutrition ( Cheng et al . , 2011 ) .",
"Although cell proliferation of the nervous system is spared under nutrient deprivation , whether other aspects of neural development are also subject to organ sparing is unknown .",
"The arbor growth of post-mitotic neurons is achieved by cell expansion rather than cell number increase and therefore represents a different type of neural growth from cell proliferation .",
"Following innervation of the target field , the dendritic or axonal arbor of the neuron expands in coordination with the tissue it innervates .",
"For example , the dendritic arbors of Drosophila somatosensory neurons called dendritic arborization ( da ) neurons are known to scale with the body wall during normal larval development ( Parrish et al . , 2009 ) .",
"This scaling involves synchronous expansion of body wall epidermal cells and of da dendritic arbors , such that neurons maintain the same coverage of the sensory fields while the body surface area expands exponentially ( Jiang et al . , 2014 ) .",
"Da neurons are categorized into four classes that differ in their dendrite morphology and transcription factor expression ( Grueber et al . , 2002; Hattori et al . , 2013 ) .",
"Recently , class IV da ( C4da ) neurons , which completely cover the body surface and thus are called ‘space-filling’ neurons ( Grueber et al . , 2002; Grueber et al . , 2003 ) , were found to elaborate more dendrite branches when larvae develop on a low-nutrient diet ( Watanabe et al . , 2017 ) , suggesting that dendritic scaling of C4da neurons is regulated by the nutrient state .",
"However , whether this dendritic hyperarborization is related to organ sparing and how nutrient stress promotes dendrite growth are unclear .",
"The conserved PI3K-Akt-mechanistic target of rapamycin ( mTOR ) pathway promotes dendrite growth in both insects and mammals ( Jaworski et al . , 2005; Kumar et al . , 2005; Parrish et al . , 2009; Skalecka et al . , 2016 ) .",
"Receiving signaling inputs from membrane receptor tyrosine kinases ( RTKs ) , notably InR ( Sancak et al . , 2007; Vander Haar et al . , 2007; Wang et al . , 2007 ) , this pathway enhances translation in most cells by mTOR kinase-mediated phosphorylation of S6 protein kinase ( S6K ) and 4E-binding protein ( 4E-BP ) ( Burnett et al . , 1998 ) .",
"At the center of this pathway , mTOR activity is also influenced by the cellular state , including nutrient availability , cellular energy levels , and stress factors ( Zoncu et al . , 2011 ) .",
"In particular , cellular nutrient starvation suppresses mTOR and consequently induces autophagy ( Ganley et al . , 2009; Hosokawa et al . , 2009; Jung et al . , 2009 ) , the self-eating process that helps to conserve and recycle vital cellular building blocks .",
"mTOR regulates autophagy in part through the transcription factor EB ( TFEB ) , which promotes autophagosome biogenesis but is suppressed by mTOR-mediated phosphorylation ( Jung et al . , 2009; Martina et al . , 2012; Roczniak-Ferguson et al . , 2012 ) .",
"Among the cellular stress sensors that inhibit mTOR activity , the forkhead box O ( FoxO ) family of transcription factors can be activated by a variety of stress signals and respond by suppressing cell growth and inducing autophagy ( Eijkelenboom and Burgering , 2013 ) .",
"Although the regulation of mTOR activity by cellular stress has been extensively investigated in many cell types , how mTOR signaling is modulated by the nutrient state to impact neuronal arbor growth has not been examined .",
"Furthermore , although FoxO members have been found to enhance dendritic space-filling of C4da neurons in Drosophila ( Sears and Broihier , 2016 ) and to regulate dendrite branching and spine morphology of adult-generated neurons in mice ( Schäffner et al . , 2018 ) , whether they also influence neuronal arbor growth in response to nutrient stress is unclear .",
"In this study , we demonstrate that dynamically growing Drosophila da neurons exhibit organ sparing at the level of individual cells , with dendrites growing preferentially at the expense of other non-neural tissues under nutrient stress .",
"Mechanistically , the amplitude of Tor signaling in da neurons is attenuated less dramatically by nutrient stress than in non-neural tissues like epidermal cells , muting the induction of autophagy in neurons .",
"The distinct sensitivities of da neurons and epidermal cells to nutrient stress are at least partly due to their differential FoxO expression levels: Foxo is lowly expressed in neurons and hence has minimal effect on dendrite growth , while it is highly expressed in epidermal cells , resulting in suppression of cell growth only under nutrient restriction .",
"Functionally , preferential dendrite growth of da neurons increases the sensory acuity , allowing larvae to respond more nimbly to environmental stimuli ."
],
[
"A recent study by Watanabe et al . , 2017 reported that C4da neurons were hyperarborized when Drosophila larvae developed on a low yeast diet that restricts the availability of lipids and amino acids , an interesting and surprising finding that agrees with our independent observation .",
"In our experiments , we examined larvae reared in high yeast ( HY , 8% yeast ) and low yeast ( LY , 1% yeast ) media that otherwise contained only glucose as a carbon source .",
"In wandering third instar larvae , C4da neurons in the HY condition showed sparse dendrites with gaps of dendritic coverage between neighboring neurons ( Figure 1A ) .",
"In contrast , C4da neurons in the LY condition completely covered the body wall with dense dendrites ( Figure 1B ) .",
"This apparent dendrite overgrowth in the LY condition ( Figure 1D ) was due to 60% more total branches and 59% more terminal branches but not increase of terminal branch length as compared to the HY condition ( Figure 1—figure supplement 1 ) .",
"The increased density of epidermal innervation by C4da dendrites suggests that nutrient restriction differentially affected growth of C4da neurons and the body wall .",
"To investigate this possibility , we monitored growth of C4da neurons and the body wall simultaneously during larval development in HY and LY media .",
"We measured various parameters of the larval body wall and dendrite growth every 24 hr ( hrs ) starting from 48 hr after egg laying ( AEL ) to the wandering 3rd instar stage ( Figure 1 and Figure 1—figure supplement 2 ) .",
"The body wall was measured at the levels of the whole body ( body length ) , the body segments ( segment width ) , and individual epidermal cells ( average cell width as visualized by the septate junction marker Nrg-GFP ) .",
"We found that all three parameters correlated with one another in both HY and LY conditions ( Figure 1—figure supplement 2G and H ) .",
"We have therefore taken the segment width as an indicator of the larval body size .",
"Compared to the HY condition , LY caused a significant delay in larval body growth , with animals in LY taking 2 . 2 times longer ( 264 hr compared to 120 hr ) to pupariate .",
"Notably , larvae reared in HY and LY reached a nearly identical maximum segment width of ~600 μm before pupariation ( Figure 1A–C ) .",
"These observations verify that the LY medium caused the larvae to experience nutrient stress and developmental delay .",
"The fact that the pupariation occurs at the same maximum segment width regardless of the rate of growth suggests that the segment width is not only a good proxy for body size , but it also provides a good indication of the developmental stage .",
"C4da neurons also grew slower in LY than in HY , as indicated by smaller increases of the total dendrite length during each 24 hr period ( Figure 1D ) .",
"However , because LY larvae had more time to develop , their neurons ultimately outgrew those of HY larvae , resulting in 1 . 39-fold longer total dendrites at the end of the larval period ( Figure 1D ) .",
"To make more meaningful comparisons of dendrite growth in animals of similar developmental stages prior to pupariation , we plotted the dendrite length against the segment width ( Figure 1E ) , as the segment width better indicates the developmental stage than the age of the larva .",
"This plot shows that C4da dendrites grow 57% faster ( based on the slopes of the linear fits ) in LY than in HY when normalized by the segment width .",
"Interestingly , the dendrite density ( dendrite length/dendrite field size ) was always higher in LY when plotted against the segment width ( Figure 1F ) , even though the dendrite length was not greater in LY when the segment width was below 200 μm ( Figure 1E ) .",
"Likely contributing to this discrepancy between dendrite density and dendrite length is that larvae reared in LY were thinner than those in HY ( Figure 1—figure supplement 2I and J ) and consequently had smaller body wall areas to be covered by C4da dendrites .",
"The above data suggest that C4da neurons preferentially grow compared to epidermal cells when nutrients are limited .",
"To determine if C4da neurons still have a growth advantage under starvation , we transferred larvae reared in HY media to agar-only media at 84 hr AEL , a time when larvae had reached the critical weight ( Beadle et al . , 1938; Bakker , 1959 ) .",
"Interestingly , C4da neurons showed significant dendrite growth as measured by the dendrite length normalized to the segment width ( abbreviated as normalized dendrite length ) over a 24 hr period ( Figure 1—figure supplement 3A ) , even though the larval body size did not change in the same period ( Figure 1—figure supplement 3B ) .",
"Collectively , these results show that while nutrient restriction delays overall larval growth , it differentially affects growth of C4da neurons and epidermal cells , such that C4da neurons exhibit a growth advantage , or are spared , under nutrient stress .",
"Moreover , in the absence of nutrient intake , larvae mobilize existing nutrient storage to support the growth of C4da neurons at the expense of non-neural tissues like epidermal cells .",
"Nutrient-mediated systemic control of larval growth depends on InR and the Drosophila mTOR homolog Tor ( Boulan et al . , 2015 ) .",
"To examine effects of the InR/Tor pathway on dendrite growth during nutrient stress , we selectively inactivated InR or Tor in C4da neurons .",
"For these assays , we measured normalized dendrite length when larvae reached a segment width of 500–550 μm , a size at which C4da neurons in control larvae exhibit a significant increase in dendrite growth in response to nutrient stress ( Figure 1E ) .",
"Downregulation of the InR/Tor pathway in neurons by InR knockdown ( InR RNAi ) , InRDN ( dominant negative ) overexpression ( InR DN ) , Tor knockdown ( Tor RNAi ) , or TorDN overexpression ( Tor DN ) in the HY condition caused mild or statistically non-significant dendrite reduction compared to the control ( Figure 2A–C and G , and Figure 2—figure supplement 1A–1C ) .",
"In contrast , the same genetic manipulations caused pronounced dendritic reduction in the LY condition , generating dendrite patterns resembling those of wildtype neurons in the HY condition ( Figure 2D–F and G , and Figure 2—figure supplement 1D–1F ) .",
"Statistical analyses suggest that the effects of the manipulations are nutrient-dependent , that is , much greater reduction of normalized dendrite length in the LY condition .",
"The ratio of the average normalized dendrite length between LY and HY thus drops from 1 . 44 for control neurons to values closer to one for neurons in which InR or Tor is suppressed ( Figure 2H ) .",
"These results suggest that neuronal InR/Tor signaling is responsible for the preferential dendrite growth observed under nutrient stress .",
"To test whether reducing the rate of epidermal growth , as is seen under nutrient stress , can lead to excessive dendrite growth , we inhibited InR and Tor in epidermal cells under the HY condition .",
"The efficacy of genetic manipulations in attenuating epidermal growth was assessed using Gal4R16D01 , which is expressed in a stripe of epidermal cells in the middle of each segment ( Poe et al . , 2017; Figure 2—figure supplement 1G ) , allowing comparison of Gal4-expressing epidermal cells to neighboring wildtype cells .",
"UAS-driven transgenes were then expressed by a pan-epidermal driver Gal4R38F11 ( Figure 2—figure supplement 1H ) to inhibit InR or Tor in the entire epidermal sheet .",
"Downregulating InR or Tor effectively reduced the epidermal cell size as expected ( Figure 2—figure supplement 1I–1M ) , reducing the ratio between the sizes of Gal4-positive ( Gal4 ) and neighboring Gal4-negative ( WT ) cells ( Figure 2—figure supplement 1N ) .",
"Suppressing InR or Tor function throughout the epidermal sheet delayed epidermal growth such that larvae took 6–30 extra hours to reach the segment width of 500–550 μm , at which size C4da neurons exhibited 22–61% greater normalized dendrite length than controls ( Figure 2I–L and Figure 2—figure supplement 1O–1Q ) .",
"Taken together , the above results demonstrate that changes in the relative strengths of the InR/Tor signaling in neurons and epidermal cells can alter dendritic scaling: Reducing the throughput of the pathway in neurons can cause dendrite reduction , while suppression of InR/Tor in the epidermis can lead to dendrite overgrowth .",
"To determine how nutrient levels modulate InR/Tor signaling in neurons and epidermal cells , we examined Tor activity by immunostaining phosphorylated Ribosomal protein S6 ( pRpS6 ) , a substrate of S6K and a faithful indicator of Tor activity in Drosophila tissues ( Ruvinsky and Meyuhas , 2006; Kim et al . , 2017 ) .",
"In HY , the cytoplasm of epidermal cells exhibited high and uniformly distributed pRpS6 signals , while the soma and primary dendrites of C4da neurons showed comparatively low pRpS6 intensities ( Figure 2M ) .",
"As a result , the ratios of pRpS6 intensity ( average intensity within regions of interest ) between the neuronal compartments and epidermal cells are much lower than 1 ( Figure 2P ) .",
"Under nutrient stress , the overall pRpS6 staining on the larval body wall dramatically decreased ( Figure 2O ) , consistent with the notion that nutrient stress reduces InR/Tor signaling in peripheral tissues ( Géminard et al . , 2009 ) .",
"However , in these animals , the pRpS6 signals were brighter and more even in C4da cell bodies and dendrites than in the epidermal cells ( Figure 2N ) , causing the ratios of pRpS6 intensity between the neuronal compartments and epidermal cells to be larger than 1 ( Figure 2P ) .",
"These data suggest that nutrient stress switches the relative strength of the InR/Tor signaling in C4da neurons and epidermal cells such that the neurons gain a growth advantage over epidermal cells .",
"Nutrient stress suppresses mTOR activity to induce many cellular responses , including autophagy ( He and Klionsky , 2009 ) .",
"We therefore tested if autophagy is also differentially regulated by nutrient levels in C4da neurons and epidermal cells .",
"To monitor autophagy levels , we used an mCherry-Atg8a reporter under the control of the endogenous Atg8a regulatory sequence , which labels autophagic structures ( Hegedűs et al . , 2016 ) .",
"As expected , autophagosome levels in epidermal cells were low under the HY condition ( Figure 3A ) but increased nearly 5-fold in the LY diet ( Figure 3B and C ) .",
"In contrast , autophagosomes in C4da cell bodies were present at low levels under both HY and LY conditions ( Figure 3A–C ) .",
"Similarly , epidermal expression of Lamp-mCherry , a lysosomal and autolysosomal marker driven by the endogenous Lamp1 promoter ( Hegedűs et al . , 2016 ) , increased by 4 . 8-fold under nutrient stress ( Figure 3—figure supplement 1A–1C ) .",
"In contrast , the same reporter exhibited a much lower baseline labeling in C4da cell bodies ( 12 . 6% of that in epidermal cells ) under HY and non-significant increase under LY ( Figure 3—figure supplement 1A–1C ) .",
"To examine the levels of autophagic flux , we overexpressed in epidermal cells and neurons a tandem fluorescent marker GFP-mCherry-Atg8a , which is converted from GFP + mCherry dual fluorescence to mCherry alone when autophagosomes mature ( Kimura et al . , 2007; Nezis et al . , 2010 ) .",
"Thus , increased autophagic flux results in a reduction of cytosolic GFP and an increase of vesicular mCherry , indicated by an increased ratio of mCherry area over GFP intensity .",
"Using this marker , we found that although nutrient stress enhanced autophagic flux in both epidermal cells and C4da neuron cell bodies , the autophagic flux level was always higher in epidermal cells ( Figure 3—figure supplement 1D–1I ) .",
"These data suggest that C4da neurons maintain low levels of autophagy even under nutrient stress , despite an increase of autophagic flux .",
"To examine the effects of autophagy on the growth of epidermal cells and neurons , we knocked down Atg8a to suppress autophagy .",
"Epidermal Atg8a knockdown using Gal4R16D01 had no effect on cell size under the HY condition ( Figures 3D , E and H ) , consistent with the low autophagy level in these cells .",
"However , the same manipulation caused a 16% increase of the epidermal cell size under LY ( Figure 3F and G , and 3H ) , supporting the idea that autophagy suppresses epidermal cell growth under nutrient stress .",
"In comparison , neuronal Atg8a knockdown in both HY and LY conditions resulted in similar increases of normalized dendrite length ( 13% and 11% increases , respectively ) ( Figure 3I and Figure 3—figure supplement 2A–2D ) , suggesting that C4da neurons maintain a constant level of autophagy regardless of nutrient availability to mildly suppresses dendrite growth .",
"Nutrient restriction upregulates expression of autophagy-related genes through the transcription factor TFEB , a substrate of the Tor kinase ( Füllgrabe et al . , 2016 ) .",
"To understand why nutrient stress fails to induce autophagy in neurons , we examine the expression of Mitf-GFPnls , a transcription reporter for the Drosophila TFEB homolog Mitf ( Zhang et al . , 2015 ) .",
"Mitf-GFPnls showed nutrient-independent expression in epidermal nuclei but its expression could not be detected in da neurons in either HY or LY condition ( Figure 3—figure supplement 2E–G ) , suggesting that Mitf transcription may be low in da neurons .",
"We next investigated the effects of inducing autophagy on dendrite growth by overexpressing Mitf in C4da neurons , as overexpression of TFEB/Mitf is sufficient to dominantly induce autophagy in cells ( Settembre et al . , 2011; Zhang et al . , 2015 ) .",
"This manipulation strongly reduced the normalized dendrite length under LY ( Figure 3J–L ) , suggesting that high autophagy levels suppress dendrite growth .",
"These data together suggest that nutrient restriction upregulates autophagy in epidermal cells but not in C4da neurons , and that the lack of autophagy induction likely protects neurons from growth suppression under nutrient stress .",
"To further understand the mechanisms responsible for the differential growth regulations of epidermal cells and C4da neurons by nutrients , we first examined three genes known to inhibit mTor signaling under stress conditions , including cryptocephal ( crc ) /ATF4 ( Kang et al . , 2017 ) , Sestrin ( Sesn ) ( Lee et al . , 2016 ) , and Sirtuin 2 ( Sirt2 ) ( personal communications with Hening Lin ) .",
"Anticipating that inhibiting the responsible genes in epidermal cells would relieve growth suppression under nutrient restriction , we knocked down the candidate genes in epidermal cells using Gal4R16D01 .",
"However , knockdown of none of these genes in epidermal cells yielded a statistically significant increase of the cell size under either HY or LY condition ( Figure 4—figure supplement 1A ) , indicating either that these genes do not suppress epidermal cell growth or that the RNAi lines were not effective .",
"We also examined dendrite growth in a Sirt2 null mutant and noticed nutrient-independent increases of normalized dendrite length compared to the control ( Figure 4—figure supplement 1B ) .",
"We next examined neuronal roles of two other components in the Tor pathway: Ras homolog enriched in brain ( Rheb ) , a GTPase involved in Tor kinase activation ( Inoki et al . , 2003; Saucedo et al . , 2003; Tee et al . , 2003; Zhang et al . , 2003 ) , and Slimfast ( Slif ) , an amino acid transporter that maintains the cellular amino acid level necessary for Tor activation ( Colombani et al . , 2003 ) .",
"Interestingly , knocking down Rheb in C4da neurons using a validated RNAi line ( Francis and Ghabrial , 2015 ) caused a mild ( 11% ) increase of dendrites in HY condition and a weak ( 11% ) reduction in LY condition ( Figure 4—figure supplement 1C , D , F , G and I ) , suggesting that Rheb suppresses dendrite growth under nutrient abundance but promotes dendrite growth under nutrient restriction .",
"On the other hand , slif knockdown caused severe dendrite reduction in both HY ( 40% ) and LY ( 74% ) conditions ( Figure 4—figure supplement 1E , H and I ) , suggesting that slif is required for proper dendrite growth regardless of the nutrient state .",
"The fact that dendrite reduction in LY food is greater suggests that dendrite growth under nutrient restriction may rely more on the availability of amino acid transporters .",
"We further asked why epidermal cells exhibit more pronounced growth suppression than C4da neurons under nutrient stress by investigating the role of FoxO , because FoxO is an important cellular stress sensor that can impact mTor signaling ( Eijkelenboom and Burgering , 2013 ) .",
"We first examined FoxO expression in neurons and epidermal cells using a foxo-GFP transgenic line that carries a 77 kb genomic fragment containing the full foxo locus with a C-terminal GFP tag and therefore should mimic the endogenous FoxO expression .",
"In epidermal cells , FoxO-GFP showed cytoplasmic distribution under normoxia but translocated to epidermal nuclei within several minutes of hypoxia ( Figure 4—figure supplement 2A and B ) , consistent with its known role as a transcription factor responsive to cellular stress ( Eijkelenboom and Burgering , 2013 ) .",
"FoxO-GFP was expressed at similar levels in epidermal cells in both HY and LY conditions ( Figure 4A–C ) but could not be detected above the background noise level in C4da cell bodies ( Figure 4A–C ) .",
"To confirm these FoxO expression patterns , we stained larval body walls using a validated anti-FoxO antibody ( Slaidina et al . , 2009 ) , which also showed much higher signals in epidermal cells than in C4da neurons ( Figure 4D and Figure 4—figure supplement 2C and D ) .",
"FoxO staining signals overlapped with those of FoxO-GFP in epidermal cells ( Figure 4—figure supplement 2E ) and were markedly reduced upon foxo knockdown ( Figure 4—figure supplement 2F ) .",
"Suspecting that the lack of nuclear FoxO-GFP in epidermal cells and neurons in LY media may be due to insufficient cellular stress , we subjected larvae to starvation or knocked down slif in all neurons to create nutrient stress .",
"Within 4 hr of starvation , FoxO-GFP was drastically enriched in epidermal nuclei ( Figure 4—figure supplement 2H and J ) .",
"In the same experiment , FoxO-GFP remained undetectable in da neurons of 75% of larvae ( Figure 4—figure supplement 2H and J ) but increased to moderate levels in nuclei of da neurons of the rest of larvae ( Figure 4—figure supplement 2I and J ) .",
"In comparison , pan-neural slif knockdown severely impaired larval locomotion and caused nuclear accumulation of FoxO-GFP in epidermal cells ( Figure 4—figure supplement 2K–2M ) .",
"However , FoxO-GFP still could not be detected in nuclei of da neurons in these animals ( Figure 4—figure supplement 2K–2M ) .",
"These results together suggest that FoxO is differentially expressed and reacts differently to cellular stress in epidermal cells and C4da neurons .",
"To further investigate FoxO expression patterns , we converted a MiMIC insertion line of foxo into a foxo-Gal4 using the Trojan exon technique ( Diao et al . , 2015 ) , so that the Gal4 is under the same transcription regulation as the endogenous foxo ( Figure 4—figure supplement 3A ) .",
"This foxo-Gal4 drove uniform epidermal expression of a UAS-tdTom reporter under HY , and the expression was enhanced 2 . 9 folds by nutrient stress ( Figure 4E–G ) .",
"In contrast , foxo-Gal4 showed minimal activity in C4da neurons under both HY and LY conditions ( Figure 4E–G ) , confirming the results obtained by FoxO-GFP and FoxO staining .",
"Interestingly , foxo-Gal4 is expressed in a subset of neurons and non-neural cells in the larval brain and ventral nerve cord ( Figure 4—figure supplement 3B and C ) , suggesting cell-type-specific expression in the CNS as well .",
"Prior loss-of-function ( LOF ) and gain-of-function ( GOF ) studies of foxo in C4da neurons showed that FoxO plays a role in enhancing dendritic space-filling by stabilizing dendritic microtubule ( Sears and Broihier , 2016 ) .",
"However , whether FoxO contributes to nutrient regulation of dendrite growth is unclear .",
"Because foxoΔ94 , a null mutation of foxo ( Slack et al . , 2011 ) , caused larval lethality before the 3rd instar in both HY and LY media , we chose to knock down or overexpress foxo in C4da neurons .",
"foxo knockdown in epidermal cells effectively eliminated UAS-FoxO-GFP expression ( Figure 4—figure supplement 3D–3F ) , confirming the efficacy of this RNAi line .",
"We found that knocking down foxo in C4da neurons had no effect on normalized dendrite length in either condition ( Figures 4H , I , K , L and N ) , but caused 13% reduction of dendrite density in LY medium .",
"This decrease in dendrite density was caused in part by an expansion of dendritic fields of ppk >foxo RNAi neurons , which occupied a larger 2-dimensional area under LY conditions , despite exhibiting total dendrite lengths comparable to controls .",
"On the other hand , overexpressing FoxO in C4da neurons caused mild ( 11 . 7% ) and strong ( 49 . 2% ) reduction of normalized dendrite length in HY and LY conditions , respectively ( Figures 4J , M and N ) .",
"Lastly , we examined the role of FoxO in epidermal cell growth by knocking down foxo using Gal4R16D01 .",
"The knockdown had no effect in HY but increased the epidermal cell size by 34% under the LY condition ( Figure 4O–S ) .",
"The above results collectively suggest that FoxO differentially affects the growth of C4da neurons and epidermal cells .",
"In neurons , although high FoxO levels are growth-inhibitory , endogenous FoxO is likely expressed at levels too low to directly affect dendrite growth; in epidermal cells , FoxO is more highly expressed , but inhibits cell growth only under nutrient stress .",
"Our results also suggest that among genes known to inhibit mTor signaling under nutrient stress , foxo plays a more significant role in nutrient-dependent dendrite overgrowth of somatosensory neurons .",
"We therefore chose to focus our further analysis on how FoxO overexpression suppresses dendrite growth .",
"Because the relative strength of Tor signaling in C4da neurons as compared to epidermal cells determines the extent of dendrite innervation in the epidermis ( Figure 2 ) , we used pRpS6 staining to examine whether overexpressed FoxO affects dendrite growth through suppressing Tor signaling .",
"In HY , neuronal FoxO overexpression did not cause detectable changes in the ratio of pRpS6 levels between neuronal compartments and epidermal cells ( Figures 2M , 5A , C and D ) .",
"However , in LY , while pRpS6 levels in the somas of FoxO-overexpressing neurons were still higher than in epidermal cells , the dendritic pRpS6 signal was reduced to levels lower than those of epidermal cells ( Figure 5B–D ) .",
"These data suggest that high levels of FoxO specifically suppress Tor signaling in dendrites in a nutrient-dependent manner .",
"We further examined the autophagy level of FoxO-overexpressing neurons using mCherry-Atg8a , as higher autophagy levels suppress the growth of both C4da neurons and epidermal cells ( Figure 3 ) .",
"While the autophagosome level in C4da cell bodies was not altered by FoxO-expression in HY , it increased 4 . 9 folds in LY ( Figure 4E–G ) .",
"The above results together suggest that nutrient stress enables overexpressed FoxO to suppress dendritic Tor signaling and to induce autophagy in neurons .",
"The lack of high FoxO expression in wildtype neurons thus ensures preferential dendrite growth in nutrient stress by protecting dendritic Tor signaling and suppressing autophagy .",
"C4da neurons are polymodal nociceptive neurons that respond to noxious thermal , mechanical , chemical , and light stimuli ( Tracey et al . , 2003; Hwang et al . , 2007; Xiang et al . , 2010 ) .",
"To test whether the preferential dendrite growth under nutrient stress is physiologically relevant , we examined whether nutrient stress influences larval nociception using an established heat-response assay ( Babcock et al . , 2009 ) .",
"In this assay , a temperature-controlled heat probe is used to deliver noxious thermal stimuli that elicit C4da-dependent larval rolling behavior .",
"We first examined wildtype larvae reared in HY and LY media .",
"Over a range of temperatures ( from 40°C to 46°C ) , we found that a larger percentage of LY larvae exhibited nocifensive rolling responses to thermal stimuli than HY larvae ( Figure 6A ) .",
"The temperatures required to induce response in a similar percentage of larvae were approximately 2°C lower for the LY condition than for the HY condition .",
"At temperatures above 46°C the vast majority of larvae in both conditions exhibited nocifensive rolling .",
"We additionally monitored response latency and found that significantly more larvae in LY responded within 5 s ( fast response ) of heat stimulus over a temperature range from 43°C to 48°C ( Figure 6B ) .",
"When we examined response latency at 46°C with higher temporal resolution , we found that the majority of LY larvae responded within 6 s while HY responses were distributed over a broader range of durations ( Figure 6C ) .",
"These data suggest that nutrient stress sensitizes larvae so that they react more acutely to noxious heat .",
"We next examined the effects of FoxO overexpression in C4da neurons by stimulating the larvae at 46°C .",
"While FoxO overexpression did not significantly change the rolling behaviors of HY larvae , it significantly reduced the percentages of LY larvae that showed fast responses and overall response ( Figure 6D and E ) .",
"This decrease of nociceptive response thus correlates with the strong dendrite reduction of FoxO-overexpressing neurons under nutrient restriction , even though we cannot rule out the possibility that FoxO overexpression impairs neuronal function through other means .",
"Among the four classes of da neurons , C4da neurons have dendritic arbors that exhibit space-filling and are highly dynamic ( Grueber et al . , 2003; Poe et al . , 2017 ) , while C1da and C2da neurons have simple arbors and sparse dendrites occupying defined territories and grow mostly by scaled expansion of the existing dendritic arbors established in late embryogenesis ( Grueber et al . , 2002 ) .",
"C3da neurons grow more complex dendritic arbors and are characterized by numerous short terminal branches called dendritic spikes , which are highly dynamics during larval growth ( Grueber et al . , 2002; Nagel et al . , 2012 ) .",
"We asked whether nutrient stress also impacts dendritic growth of C1da and C3da neurons .",
"Consistent with the previous report by Watanabe et al . , 2017 , we found that the yeast concentration did not have obvious effects on the total dendrite length of C1da neuron ddaE ( Figure 7A–C ) .",
"However , nutrient stress stimulated the dendritic growth of C3da neurons ddaA and ddaF: the total dendrite length increased by 40% and 32% , respectively; the total terminal dendrite length increased by 61% and 46% , respectively; the terminal branch numbers increased by 47% and 58% , respectively ( Figure 7D–H ) .",
"These data suggest that nutrient stress promotes overbranching of complex dendritic arbors of C3da and C4da neurons but not simple arbors of C1da neurons .",
"We then examined whether C1da and C3da neurons are subjected to the same regulation of autophagy as C4da neurons under nutrient stress .",
"Interestingly , the cell bodies of these neurons showed variable and cell-specific baseline autophagy levels in HY , as indicated by mCherry-Atg8a , but these levels did not appear to be altered by the nutrient level ( Figure 7I–M ) .",
"Similarly , C3da neurons showed low and nutrient-independent lysosomal level ( Figures 7O , Q and R ) .",
"Interestingly , C1 ddaE neurons showed a much higher level of the lysosomal marker Lamp-mCherry than other da classes in HY , and this level was enhanced 2 . 7 folds by nutrient stress ( Figures 7N , P and R ) , suggesting that C1da neurons have uniquely high and nutrient-dependent lysosomal system .",
"Lastly , we examined foxo expression in C1da and C3da neurons using foxo-GFP and foxo-Gal4 .",
"Similar to C4da neurons , C1da and C3da neurons showed only background-noise levels of FoxO-GFP and foxo >tdTom signals ( Figure 7M and N ) .",
"These data suggest that , similar to C4da neurons , C1da and C3da neurons exhibit low FoxO expression and nutrient-independent autophagy levels , while C3da neurons , but not C1da neurons , show nutrient stress-induced dendrite overgrowth ."
],
[
"In this study , we show that Drosophila C3da and C4da neurons exhibit a growth advantage over neighboring epidermal cells under nutrient restriction , resulting in dendrite overgrowth .",
"This tissue-specific growth regulation by nutrient stress is at least partially determined by FoxO expression level ( Figure 8 ) .",
"In non-neural tissues like epidermal cells , the stress sensor FoxO is expressed at sufficient levels to allow epidermal cells to respond robustly to nutrient stress .",
"In these tissues , high nutrition elevates InR/Tor signaling and suppresses FoxO activity and autophagy , leading to a high growth rate; in low nutrients , the reduction in InR/Tor signaling combined with high FoxO activity stimulates autophagy and greatly slows down cell size increase .",
"In contrast , PNS neurons express FoxO at much lower levels and exhibit low basal levels of autophagy .",
"As a result , dendritic InR/Tor signaling is not further suppressed by FoxO when the systemic insulin level is low .",
"Therefore , the low FoxO expression and the lack of autophagy induction in PNS neurons protect dendrite growth from nutrient stress .",
"Interestingly , Rheb in neurons mildly suppresses dendrite growth in high nutrition but enhances dendrite growth under nutrient stress .",
"Whether these effects are mediated by Tor remains to be determined .",
"Brain sparing has been recognized in both mammals and insects as an important means to protect the developing nervous system from nutrient deficiency .",
"The preferential dendrite growth of da neurons constitutes another form of nervous system sparing that bears important distinctions from Drosophila brain sparing .",
"First , while proliferating neural stem cells in the CNS is protected against starvation ( Cheng et al . , 2011 ) , individual post-mitotic da neurons are spared at the level of neuronal arbor growth .",
"Second , unlike CNS neuroblasts , which rely on a special extrinsic factor ( the glial-derived Jeb ) to sustain neurogenesis , PNS neurons possess a unique intrinsic genetic program that endows them the resistance to nutrient stress .",
"Lastly , the CNS sparing is independent of InR and Tor , made possible by the alternative Jeb/Alk/PI3K pathway , while the PNS protection still relies on InR/Tor signaling .",
"Therefore , our work reveals a novel mechanism of neural protection under nutrient stress .",
"In the mammalian brain , FoxO members are highly expressed in NSCs and are required for the long-term maintenance of the adult NSC pool critical for adult neurogenesis ( Paik et al . , 2009; Renault et al . , 2009; Yeo et al . , 2013 ) .",
"Recently , FoxOs were also found to regulate dendrite branching and spine density of adult-born hippocampal neurons ( Schäffner et al . , 2018 ) .",
"Interestingly , FoxOs in these neurons suppress mTor signaling and maintain a level of autophagic flux that is necessary for the normal morphogenesis of the neurons .",
"In addition , FoxOs were found to be upregulated in aged brains and function to delay aging‐related axonal tract degeneration by suppressing mTor activity ( Hwang et al . , 2018 ) .",
"In Drosophila C4da neurons , FoxO was previously found to promote dendrite space-filling and to mediate polyQ-induced neuronal toxicity ( Sears and Broihier , 2016; Kwon et al . , 2018 ) .",
"While these prior studies established key roles for FoxO proteins in nervous system development , the relationship between nutrition and neuronal FoxO function was previously unexplored .",
"Likewise , FoxO expression patterns in neural and non-neural tissues have not been compared .",
"Our results demonstrate that FoxO is expressed at a much lower level in da neurons than in non-neural larval tissues .",
"Consequently , inhibiting foxo in neurons does not directly affect dendrite growth , even though overexpressing FoxO in neurons inhibits dendrite growth .",
"Nevertheless , our results support that neuronal FoxO mildly promotes dendritic space-filling of C4da neurons only under nutrient restriction by reducing the size of the dendrite field though an unknown mechanism .",
"More importantly , the lack of high FoxO expression makes neurons insensitive to nutrient stress , giving them a growth advantage over non-neural tissues that express higher levels of FoxO .",
"Interestingly , this FoxO-dosage-dependent nutrient-insensitivity has been previously described in the adult genitalia ( Tang et al . , 2011 ) .",
"While adult wings and maxillary palps become small on poor diets , the size of genital arches is affected much less; these differential responses are linked to tissue-intrinsic levels of FoxO expression .",
"However , in this example and previously described FoxO functions in growth regulation , FoxO primarily regulates cell numbers but not cell size ( Jünger et al . , 2003; Puig et al . , 2003 ) .",
"Therefore , our study reveals a novel function for FoxO in environmental regulation of neural development .",
"It is worth noting that although FoxO is minimally expressed in da neurons , it is expressed at a higher level in a subpopulation of CNS neurons , raising the possibility that the arbor growth of these neurons may be differentially regulated by nutrient availability .",
"Lastly , our results reveal a level of neuronal diversity in the response to nutrient stress .",
"Although all da neurons we examined show similarly low FoxO expression and the lack of autophagy induction under nutrient restriction , only class III and IV but not class I neurons display preferential dendrite growth .",
"This distinction may be related to their arbor growth mechanisms and neuronal functions .",
"As proprioceptive neurons that detect body surface folds during locomotion ( He et al . , 2019; Vaadia et al . , 2019 ) , C1da neurons have simple arbors covering defined territories on the larval body wall .",
"Their dendritic arbors grow mainly by expanding the shafts of the dendritic branches established during embryogenesis .",
"Their functional demands may require a tighter growth coupling with the epidermis but not with environmental nutrition availability .",
"In contrast , C3da and C4da neurons have highly dynamic high-order branches that grow also by branching and tip extension .",
"The lack of growth suppression therefore leads to dendrite hyperarborization .",
"As these neurons sense mild to extreme levels of external stimuli , the heightened sensations allowed by their dendrite overgrowth may bestow the larva a survival advantage in an unfavorable environment ."
],
[
"Animals were raised at 25°C in density-controlled vials containing between 50 and 70 embryos collected in a 3 hr time window .",
"To achieve optimum embryo densities , approximately 50 virgin females were aged 5 days on molasses food with yeast paste , crossed with approximately 15–20 males , and then allowed to mate for 1–2 days on molasses food with yeast paste .",
"Embryo collections were then performed in a 3 hr time window on both 1% and 8% yeast food .",
"Third instar larvae at 86 hr AEL on 8% yeast or 216 hr AEL on 1% yeast were mounted in glycerol and imaged with a Leica SP8 confocal .",
"The A2-A3 segments of 8–10 larvae were imaged for each genotype using a 20X oil objective .",
"To image larvae younger than 72 hr AEL , larvae were anesthetized by isoflurane for 2 min and then mounted in halocarbon oil .",
"To image FoxO-GFP under normoxia , larvae were handled with care and imaged within 2 min after mounting on slides .",
"For imaging under hypoxia , larvae were left on the slides for 5 min before imaging .",
"Fly food was prepared using the following recipes ( for the dispersal of ~12 mL into 20 vials ) .",
"Ingredients:1% yeast ( LY ) 8% yeast ( HY ) Distilled H2O240 mL234 mLAgar2 . 4 g ( 12 g/L ) 2 g ( 10 g/L ) Glucose20 g20 gInactive yeast2 . 5 g20 gAcid mix ( phosphoric acid + propionic acid ) 2 mL2 mLTarget final solution volume250 mL250 mL Acid Mix was made by preparing Solution A ( 41 . 5 ml Phosphoric Acid mixed with 458 . 5 ml distilled water ) and Solution B ( 418 ml Propionic Acid mixed with 82 ml distilled water ) separately and mixing Solution A and Solution B together .",
"The strains used in this study are listed in the Key Resources Table .",
"We used the following neuronal markers to label specific classes of da neurons: ppk-CD4-tdGFP , ppk-Gal4 , UAS-CD4-tdGFP and UAS-CD4-tdTom for C4da ( Han et al . , 2011; Han et al . , 2012 ) ; R10D05-CD4-tdTom for C1da; NompC-LexA::p65 LexAop-CD4-tdTom and Gal419-12 UAS-CD4-tdGFP repo-Gal80 for C3da ( Awasaki et al . , 2008; Rumpf et al . , 2011 ) ; RabX4-Gal4 UAS- mIFP-2A-HO1 for all classes of da neurons .",
"ppk-Gal4 was used for RNAi knockdown and overexpression in C4da neurons .",
"RabX4-Gal4 was used for RNAi knockdown in all neurons .",
"Gal4R38F11 and Gal4R16D01 were used for RNAi knockdown and overexpression in all or stripes of epidermal cells , respectively .",
"foxo-Gal4[MI00786] was generated using Trojan-MiMIC system ( Diao et al . , 2015 ) .",
"MiMIC line MI00786 was crossed to flies with the triplet Trojan donor construct .",
"The progeny of this cross were then crossed to females expressing Cre recombinase and ΦC31 integrase in the germline which allow the Trojan exons to replace the MiMIC attP cassettes .",
"Progeny were then crossed to UAS-GFP for screening Gal4 expression by fluorescence microscopy .",
"Adults positive for GFP expression were used to establish the line .",
"The 2A-Gal4 insertions from established lines were sequenced to confirm the accuracy of the site and the reading frame .",
"nsyb-tdGFP ( Poe et al . , 2019 ) was used to visualize neuronal cell bodies in the larval brain when foxo-Gal4 expression was examined .",
"Antibody staining was done as previously described ( Poe et al . , 2017 ) .",
"Briefly , third instar larvae were dissected in cold PBS , fixed in 4% formaldehyde/PBS for 20 min at room temperature , and stained with the proper primary antibodies and subsequent secondary antibodies , each for 2 hr at room temperature .",
"The details of the antibodies used are in the Key Resources Table .",
"For statistical comparison of two samples ( e . g . WT vs . KD ) and comparison of two conditions ( HY vs . LY ) in each tissue , Student’s t-test was used .",
"For results with one independent variable ( e . g . genotype ) , one-way analysis of variance ( ANOVA ) with Tukey’s HSD test was used .",
"For results with two variables ( e . g . genotype and nutrient condition ) , the data were analyzed using two way ANOVA with genotype , nutrient condition and their interaction term .",
"Posthoc contrasts with a Dunnett correction for multiple comparisons were used .",
"For results of interaction terms ( genotype: nutrient condition ) , two-way ANOVA was used .",
"For additional information on number of samples , see figure legends .",
"R studio was used for all statistical analyses .",
"Larval heat-induced pain response was measured as described previously ( Babcock et al . , 2009 ) .",
"Wandering third larvae were scooped out of the food and gently cleaned with water , then transferred into a small petri dish with water drops to keep the animals moist .",
"A temperature-controlled heat probe ( ProDev Engineering , TX ) was used to apply the heat onto the larval body surface .",
"The stimulus was delivered by gently touching the animals laterally on segment A4 .",
"Each animal can only be tested once .",
"The response latency was measured from the start of touch on the animal until it initiated a complete 360° roll ."
]
] | [
"During prolonged nutrient restriction , developing animals redistribute vital nutrients to favor brain growth at the expense of other organs .",
"In Drosophila , such brain sparing relies on a glia-derived growth factor to sustain proliferation of neural stem cells .",
"However , whether other aspects of neural development are also spared under nutrient restriction is unknown .",
"Here we show that dynamically growing somatosensory neurons in the Drosophila peripheral nervous system exhibit organ sparing at the level of arbor growth: Under nutrient stress , sensory dendrites preferentially grow as compared to neighboring non-neural tissues , resulting in dendrite overgrowth .",
"These neurons express lower levels of the stress sensor FoxO than neighboring epidermal cells , and hence exhibit no marked induction of autophagy and a milder suppression of Tor signaling under nutrient stress .",
"Preferential dendrite growth allows for heightened animal responses to sensory stimuli , indicative of a potential survival advantage under environmental challenges ."
] | [
"The organs of a young animal develop in a carefully controlled way to reach the right size relative to each other .",
"However , if the animal’s diet does not contain the right amount of nutrients — a condition known as malnutrition – the body prioritizes the needs of the brain and other vital organs .",
"This means that certain organs keep on growing while others stop .",
"The brain is at the center of the nervous system , which is formed of networks of nerve cells ( or neurons ) that rapidly carry messages around the body .",
"In the larvae of malnourished fruit flies , a molecular signal allows the nervous system to continue making new neurons as other parts of the body slow down their growth .",
"During development , neurons also connect to each other by growing tree-like structures known as dendrites .",
"However , it remained unclear whether the growth of dendrites was also protected during episodes of malnutrition .",
"To address this question , Poe , Xu et al . performed experiments in the larvae of fruit flies , focusing on a type of neuron whose dendrites extend into the skin .",
"When nutrients were scarce , the neurons grew more rapidly than the surrounding skin cells , resulting in dendrite overgrowth .",
"Compared to neurons , the skin cells had higher levels of a stress sensor known as FoxO , which stops cell growth when nutrients are scarce .",
"Conversely , low quantities of FoxO in neurons allow these cells to keep on growing dendrites , which ultimately helps the starved animals to better react to their environment .",
"These results suggest that the growth of neurons and their connecting structures is preserved during malnutrition .",
"Ultimately , dissecting how organisms prioritize resources can help to develop new approaches to treat human conditions that emerge during malnutrition ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"microbiology and infectious disease"
] | Dengue viruses cleave STING in humans but not in nonhuman primates, their presumed natural reservoir | elife-31919-v1 | [
[
"Dengue viruses cause clinical disease in approximately 100 million individuals each year and are found in over 100 countries ( Bhatt et al . , 2013 ) .",
"Yet , to date no vaccine exists that conveys cross-protection against all human dengue viruses ( Scherwitzl et al . , 2017 ) .",
"Dengue viruses are positive sense RNA viruses in the family Flaviviridae , and are related to yellow fever virus , Zika virus , and West Nile virus ( Best , 2016 ) .",
"These viruses are primarily transmitted between humans in highly populated areas by Aedes aegypti and Aedes albopictus mosquitoes , in what are referred to as human ( or ‘urban’ ) transmission cycles ( Diamond and Pierson , 2015; Hanley et al . , 2013; Vasilakis et al . , 2011 ) .",
"Sylvatic ( i . e . forest ) dengue virus transmission cycles , which are separate from the human transmission cycles , exist in Asia and Africa and involve nonhuman primates and forest-dwelling Aedes mosquitos ( Vasilakis et al . , 2011; Wang et al . , 2000; Rico-Hesse , 1990 ) .",
"While the exact nonhuman primate species that serve as the sustaining natural reservoirs for sylvatic dengue viruses are unknown , the global distribution of both dengue viruses and their transmitting mosquitoes could be consistent with a significant number of primate species being involved ( Figure 1—figure supplement 1 ) ( Hanley et al . , 2013; Vasilakis et al . , 2011 ) .",
"Primarily , dengue viruses have been associated with monkeys ( rather than apes ) found in Africa and Asia ( Figure 1 ) .",
"Human dengue viruses cluster into four phylogenetically distinct clades referred to as DENV1 , 2 , 3 , and 4 ( Vasilakis and Weaver , 2008 ) .",
"These clades have sylvatic dengue virus isolates at their bases , supporting zoonotic origins of the four dengue viruses that now circulate in humans ( Wang et al . , 2000; Pyke et al . , 2016; Weaver and Vasilakis , 2009 ) .",
"Human dengue viruses have now become uncoupled from the sylvatic reservoir and require only humans and mosquitoes to be sustained ( Mayer et al . , 2017 ) .",
"In side-by-side experiments , sylvatic and human dengue viruses replicate similarly in human cells ( Vasilakis et al . , 2007; Vasilakis et al . , 2008 ) .",
"These results have been interpreted to mean that there is little or no adaptive barrier for the emergence of sylvatic dengue viruses into human populations , and the view that dengue viruses are generalists capable of infecting a wide range of primate species including humans .",
"Thus , a paradox exists in understanding why human dengue viruses are so difficult to model in nonhuman primates .",
"Chimpanzees ( Pan troglodytes ) ( Scherer et al . , 1978 ) , rhesus macaques ( Macaca mulatta ) ( Halstead et al . , 1973; Hickey et al . , 2013 ) , marmosets ( multiple Callithrix species ) ( Moi et al . , 2013; Ferreira et al . , 2014 ) , and other nonhuman primate species ( Althouse et al . , 2014 ) have been explored as possible primate models for studying dengue virus pathogenesis and for vaccine challenge .",
"In general , it has been observed that dengue does not replicate to high titers in these models , and little or no overt disease pathology is observed ( Cassetti et al . , 2010; Zompi and Harris , 2012 ) .",
"If human and sylvatic viruses are the same in their properties , we speculated that there must instead be something special about the replication of these viruses in the human host .",
"STING is a multi-pass transmembrane protein found in the endoplasmic reticulum , and functions as a critical component in the innate immune sensing pathway for intracellular pathogens ( Ishikawa and Barber , 2008; Zhong et al . , 2008; Ishikawa et al . , 2009; Jin et al . , 2008; Sun et al . , 2009; Burdette and Vance , 2013 ) .",
"Although originally described as part of the response to cytosolic DNA sensing ( Zhang et al . , 2011 ) , STING is also activated upon RNA virus infection ( Holm et al . , 2016 ) .",
"Underscoring this , several RNA viruses encode proteins that antagonize or degrade STING ( Sun et al . , 2012; Nitta et al . , 2013; Ding et al . , 2013; Aguirre et al . , 2012; Yu et al . , 2012 ) .",
"For instance , the NS2B3 protease of one human dengue virus , DENV2 , has been shown to target human STING for cleavage ( Aguirre et al . , 2012; Yu et al . , 2012 ) .",
"Through the cleavage of STING , DENV2 renders the host unable to induce the phosphorylation of Interferon Regulatory Factor 3 ( IRF3 ) , therefore decreasing production of type I interferon and increasing viral titers ( Green et al . , 2014 ) .",
"Mouse STING is resistant to cleavage by the DENV2 protease ( Aguirre et al . , 2012; Yu et al . , 2012 ) .",
"This at least partially explains why mice mount an effective immune response against dengue viruses , protecting them against infection and compromising their utility as model organisms ( Cassetti et al . , 2010; Zompi and Harris , 2012; Ashour et al . , 2010 ) .",
"Dengue viruses are known to mute the host interferon response in other ways as well , with the other predominant mechanism being the degradation of STAT2 ( Ashour et al . , 2009; Jones et al . , 2005; Mazzon et al . , 2009; Best , 2017; Morrison et al . , 2012 ) .",
"In this study , we show that the NS2B3 proteases of human ( DENV1-4 ) and sylvatic dengue viruses universally cleave human STING .",
"However , none of these proteases can cleave the STING proteins of chimpanzees , macaques , or marmosets , three primate species that have been pursued as model organisms .",
"We show that an ‘RG’ motif at positions 78/79 of STING is critical for susceptibility to cleavage , and conversion of these residues to ‘RG’ renders all nonhuman primate STING proteins tested , as well as mouse STING , sensitive to dengue virus proteases .",
"Out of the entire Genbank database , along with our sequencing of STING from 16 additional primate species , we identify only a small number of apes ( gorillas , orangutans , and gibbons ) , and three small rodent species ( chinchillas , naked mole rats , and desert woodrats ) as encoding a functional dengue virus cleavage determinant in STING .",
"This may , in part , explain why modeling dengue virus in animal models has been so difficult ."
],
[
"To begin , we cloned STING from chimpanzee ( Pan troglodytes , Genbank XM_016953921 ) , rhesus macaque ( Macaca mulatta , Genbank MF622060 ) , and the common marmoset ( Callithrix jacchus , Genbank MF622061 ) .",
"These species have been explored as animal models of dengue infection , and also represent the three major clades of simian primates: apes ( represented by chimpanzee ) , Old World monkeys ( represented by macaque ) , and New World monkeys ( represented by marmoset; Figure 1A ) .",
"Most suspected dengue virus reservoir hosts belong to the Old World monkey clade ( red and green type in Figure 1A ) .",
"On the other hand , New World monkeys ( such as marmosets ) , which reside exclusively in the Americas , have presumably never been exposed to sylvatic dengue viruses since sylvatic cycles do not exist in the New World .",
"We also included human ( Genbank MF622062 ) and mouse ( Mus musculus , Genbank MF622063 ) STING in our studies as positive and negative controls , since it was previously shown that human but not mouse STING is sensitive to DENV2 NS2B3 cleavage ( Aguirre et al . , 2012; Yu et al . , 2012 ) .",
"The dengue virus NS2B3 protease complex is composed of the viral non-structural proteins NS2B and NS3 ( Preugschat et al . , 1990; Zhang et al . , 1992; Falgout et al . , 1991 ) .",
"In the dengue virus genome , the NS2B and NS3 genes sit adjacent and are cotranslated as part of a single long viral polyprotein ( Perera and Kuhn , 2008; Chambers et al . , 1990 ) .",
"When the NS2B - NS3 region is expressed from a plasmid , the region is translated into a small polyprotein that then auto-cleaves itself to become the functional protease complex ( Yusof et al . , 2000; Bera et al . , 2007 ) .",
"We used a plasmid expressing the NS2B-NS3 region , including a 3x Flag tag at the C-terminus of NS3 , from the New Guinea C isolate of DENV2 ( see methods ) .",
"As a control , a mutation was created at the active-site serine , changing it to an alanine ( S135A ) , which renders the protease inactive ( Rodriguez-Madoz et al . , 2010 ) .",
"We then used a previously established cotransfection assay ( Aguirre et al . , 2012; Yu et al . , 2012 ) to determine if the dengue virus protease could cleave primate STING orthologs .",
"Plasmids encoding primate or mouse STING , and either active or S135A ( dead ) NS2B3 dengue proteases , were cotransfected into 293T cells .",
"STING cleavage was assessed 24 hr later by western blot .",
"The inactivity of the S135A protease can be seen in the anti-Flag blot , where the NS2B-NS3 polyprotein does not self-cleave when this mutation is present ( Figure 1B ) .",
"We see only a fraction of the human STING being cleaved , but this is consistent with previous publications and is presumably exacerbated by the overexpression of STING achieved in transfection experiments ( Aguirre et al . , 2012; Yu et al . , 2012 ) .",
"Unexpectedly , none of the nonhuman primate STINGs tested were susceptible to cleavage ( Figure 1B ) .",
"Remarkably , the DENV2 protease could not even cleave chimpanzee STING , which differs from human STING at only three amino acid positions .",
"The dengue virus cleavage site in STING was previously mapped to between the 95th and 96th residues ( Aguirre et al . , 2012; Yu et al . , 2012 ) .",
"Some uncertainty existed , though , because in the previous studies it was noted that the human residues around 95/96 were not sufficient to convey cleavage susceptibility to mouse STING .",
"Indeed , human and chimpanzee STING proteins have the exact same amino acid sequence at these positions ( Figure 2A ) .",
"Human and chimpanzee STING differ at only three amino acid positions , residues 78 , 230 , and 232 .",
"We found that mutating the human STING to encode the chimpanzee residue at site 78 ( 78W ) caused it to become resistant to cleavage by DENV NS2B3 ( Figure 2B ) .",
"Likewise , mutating the chimpanzee STING at residue 78 to the human amino acid ( 78R ) rendered the chimpanzee STING susceptible to cleavage ( Figure 2B ) .",
"We saw no effect of mutations at a second site , 230 , either alone or in combination with residue 78 ( Figure 2B ) .",
"Previously , it had been shown that STING site 78 may be important for retention in the endoplasmic reticulum ( ER ) ( Sun et al . , 2009 ) .",
"To ensure that ER retention was not disrupted by the mutations that we tested , we disrupted both copies of STING in A549 cells using CRISPR-Cas9 targeting , and then stably re-complemented them with wildtype or 78W ( cleavage resistant ) human STING ( Figure 2—figure supplement 1 ) .",
"Both the wildtype and mutant STING similarly localized to the ER ( Figure 2—figure supplement 2 ) .",
"It is logical that the 78W substitution would not affect ER-localization of STING , since 78W is naturally occurring in the chimpanzee STING protein .",
"Therefore , we can conclude that position 78R is a critical determinant for dengue virus cleavage , either as a binding site or a cleavage site for the dengue protease .",
"It was previously estimated that STING is cleaved in a way that divides the protein into approximately 25% and 75% of its original molecular weight , with the N-terminus of the protein representing the smaller portion ( Yu et al . , 2012 ) .",
"This would place the cleavage site in the vicinity of the 78th residue .",
"In addition , the 78/79 ‘RG’ motif is in good agreement with what is known about the preferences of NS2B3 , where glycine ( G ) often lies directly downstream of the peptide cleavage site , and an arginine ( R ) directly upstream ( Li et al . , 2005a ) .",
"Next we wished to ensure that the cleavage of STING alters its ability to signal in the interferon induction pathway .",
"Transfection of plasmids encoding STING into cells is sufficient to activate the interferon induction pathway ( Ishikawa and Barber , 2008 ) .",
"We again performed cotransfection of plasmids encoding STING and the dengue virus protease .",
"48 hr after transfection , cell lysates were probed in western blots for phosphorylated IRF3 ( pIRF3 ) and for total IRF3 .",
"We found that pIRF3 was reduced when human or chimpanzee STING was susceptible to NS2B3 cleavage , and not reduced when STING was resistant to cleavage ( Figure 2C , bottom ) .",
"We also monitored the activation of the interferon-beta ( IFNb ) promoter .",
"We performed an identical cotransfection assay with plasmids encoding STING and NS2B3 , only in triplicate , and with two additional plasmids: one encoding a firefly luciferase reporter gene downstream of the IFNb promoter , and another encoding a renilla luciferase reporter gene downstream of a CMV promoter ( used to normalize transfection efficiencies between samples , by taking the ratio of firefly:renilla luciferase ) .",
"With human STING and the version of chimpanzee STING rendered sensitive to cleavage ( 78R ) , there was a significant reduction in firefly luciferase production in the presence of active NS2B3 , in comparison to the catalytically dead version of the protease ( Figure 2C , top ) .",
"This reduction is not observed with chimpanzee STING , or with human STING rendered resistant to cleavage by the 78W mutation .",
"We then verified these results with infection experiments .",
"We stably re-complemented our A549 STING knockout cells , using retroviral transduction , to express various forms of STING: chimpanzee or human 78W ( both cleavage resistant ) , human or chimpanzee 78R ( both cleavage susceptible ) , or cells were complemented with an empty vector ( Figure 2—figure supplement 1 ) .",
"These cells were infected with dengue virus 2 ( strain 16681 ) at MOI 0 . 3 .",
"At 24 and 48 hr post infection , supernatant was harvested and viral content was quantified by plaque assay on BHK21 cells , and at the same time cells were harvested and lysed for western blot .",
"We found that A549 cells re-complemented with STING , regardless of the version , produced less dengue virus than the STING knockout cell line that was not re-complemented ( Figure 3 ) .",
"However , cells re-complemented with a cleavage-resistant STING produced less virus than those re-complemented with a cleavage-susceptible STING ( Figure 3 ) .",
"In fact , cell lines in this experiment that differ by only a single amino acid in STING demonstrate as much as a 176-fold change in infectious virus produced at 24 hr post-infection , according to the titration experiments ( human versus human 78W STING ) .",
"The difference remains significant at 48 hr post-infection .",
"This suggests that cleavage of STING is critically important for dengue virus replication , and has a large impact on viral titers .",
"The STING cleavage product was not visible in the western blots performed during these experiments .",
"This cleavage product is typically only detectable when cells are treated with MG132 proteasome inhibitor for several hours before cell lysis .",
"While our transfection-based cleavage assays typically incorporate MG132 treatment ( see Materials and methods ) , it was not used in the infection experiments shown here in order to not perturb infectious virus produced .",
"In a separate experiment performed in the presence of MG132 , we do see the cleavage of STING during infections ( Figure 3—figure supplement 1 ) .",
"Further , the cleavage of endogenous STING during dengue infection was previously demonstrated under other conditions ( Aguirre et al . , 2012; Yu et al . , 2012 ) .",
"We next wanted to determine if our newly identified cleavage determinant could explain the resistance to STING cleavage seen in other species .",
"The dengue protease also cannot cleave rhesus macaque , marmoset , or mouse STING ( Figure 1B ) , all of which deviate from the ‘RG’ motif found in human STING ( highlighted green in Figure 4A ) .",
"We next performed site-directed mutagenesis to alter either the 78th or 79th residue in STING of these species .",
"We found that , in all cases , mutations that restored this motif to the human ‘RG’ restored susceptibility to cleavage ( Figure 4B ) .",
"Consistent with previous studies ( Aguirre et al . , 2012; Yu et al . , 2012 ) , mutation of residues 93–96 in mouse STING to match the human ‘LRRG’ did not confer susceptibility to cleavage by NS2B3 ( Figure 4B ) .",
"Overall , these results further support the conclusion that sites 78 and 79 are critical determinants for cleavage by the DENV NS2B3 protease .",
"An ‘RG’ motif at these two positions is both necessary and sufficient to make primate and rodent STING susceptible to cleavage by the DENV2 protease .",
"To test whether these results are generalizable to other dengue viruses endemic in humans , we cloned the region encoding the NS2B3 protease complex from three additional DENV isolates ( one from each endemic human virus ) : DENV1 ( Hawaii ) , DENV3 ( Philippines/H887/1956 ) , and DENV4 ( H241 ) .",
"While some of these proteases expressed better than others , all were able to cleave wildtype human STING far more efficiently than human STING bearing the 78W mutation ( Figure 5A ) .",
"This data indicates that the 78/79 RG motif of STING is recognized ( i . e . bound or cleaved ) by the NS2B3 proteases of all endemic human dengue viruses .",
"If residues 78/79 in fact constitute the actual cleavage site for the protease , this would be in line with biochemical studies showing that the proteases of all four endemic human dengue viruses have similar cleavage motif preferences ( Li et al . , 2005a ) .",
"We next cloned the NS2B3 protease from a sylvatic dengue strain ( DakAr-141069 ) .",
"This virus was first isolated from an Ae .",
"luteocephalus mosquito in Senegal in 1999 ( Vasilakis et al . , 2008 ) .",
"We find that this viral protease also cleaves human STING , but not the STING of chimpanzee , rhesus macaque , or marmoset ( Figure 5B ) .",
"Further , the restoration of the ‘RG’ motif at positions 78/79 again renders all of these STING proteins susceptible to cleavage ( Figure 5B ) , indicating that the sylvatic protease is targeting ( i . e . binding or cleaving ) the same cleavage determinant as the proteases from human dengue viruses .",
"This is consistent with the high degree of similarity between human and sylvatic proteases , as can be seen in alignment of the two ( Figure 5—figure supplement 1 ) .",
"It is curious to find that a sylvatic dengue virus does not cleave nonhuman primate STING .",
"Since we don’t know the exact species that constitute the viral reservoir , we next considered the question of whether any nonhuman primates encode the correct cleavage determinant at positions 78/79 in STING .",
"To address this , we harvested mRNA from cell lines derived from 16 different nonhuman primate species ( see Materials and methods ) .",
"From these mRNA pools , we made cDNA libraries and sequenced the STING cDNA using Sanger sequencing .",
"We also gathered STING sequence for 14 additional primate species from Genbank .",
"An alignment of the eight amino acid region in STING surrounding the 78/79 cleavage determinant ( downward arrow in Figure 6A ) is shown for all 30 of these primate species ( a full alignment of primate STING sequences is provided in Supplementary file 1 ) .",
"With the exception of chimpanzees and bonobos , all apes encode the same amino acids as human in this motif , constituting the correct cleavage determinant for dengue virus .",
"In contrast , no monkey species encodes an ‘RG’ at positions 78/79 .",
"Instead , Old World monkeys all encode ‘RD , ’ which is the same motif found in the macaque clone that we have tested .",
"Also , no monkeys from the Americas encode an ‘RG’ at these residues , and instead these species encode a ‘QG’ at positions 78/79 , just like the marmoset clone tested herein .",
"Finally , we queried the entire Genbank database for STING sequences available from placental mammals .",
"Mice and pigs , two current models for dengue virus infection ( Cassetti et al . , 2010 ) , also do not have the correct RG residues at STING 78/79 ( Figure 6A ) .",
"Out of the entire database , only two other mammals were identified that share the exact same sequence as humans in the eight amino acid region surrounding the newly identified dengue virus cleavage determinant in STING: chinchilla and naked mole rat , both of which are rodents ( Figure 6A ) .",
"A third rodent species , the desert woodrat , has the RG at positions 78/79 , but encodes two amino acid substitutions just downstream of these residues , compared to human STING ( Figure 6A ) .",
"The fact that only a small handful of mammals encode an RG at position 78/79 , out of the entire database , may in part explain why modeling dengue viruses in animals has been so difficult .",
"We next wished to determine if STING is in fact cleaved by dengue in these rodent species , since all three already serve as animal models for biological research ( Keane et al . , 2014; Nathaniel et al . , 2013; Shimoyama et al . , 2016; Campbell et al . , 2016; Skopec et al . , 2013 ) .",
"We synthesized HA-tagged STING genes for the rodent species discussed in Figure 6A , as well as an additional rodent ( 13-lined ground squirrel ) which does not have the ‘RG’ motif at STING 78/79 , as a negative control .",
"Cleavage assays were performed using the co-transfection assay described previously .",
"We see that the STING of naked mole rat and desert woodrat is clearly cleaved by the DENV2 protease , in that the cleavage product is evident ( Figure 6B ) .",
"We do not see a cleavage product for chinchilla STING , even under long exposure , but we do see the STING band disappear .",
"It’s possible that , in this case , the cleavage product is too unstable to be detected .",
"The identification of animal models encoding STING proteins that can be cleaved by dengue might be important; the advantage to using such species as models is that , unlike in STING knockout mice , the STING pathway would be intact in these animals ."
],
[
"In humans cells , sylvatic and human dengue viruses replicate similarly ( Vasilakis et al . , 2007 , 2008 ) .",
"These results have been interpreted to mean that there is little or no adaptive barrier for the emergence of sylvatic dengue viruses into human populations .",
"Our data agree with , but add a new element to , this model .",
"Rather than there being critical differences between human and sylvatic viruses , our data suggest that there are critical differences between human and monkey hosts .",
"This difference tracks , at least in part , to STING , revealing one way in which dengue viruses are reaching higher titers in humans than in monkey models .",
"Collectively our data suggest that all dengue viruses cleave human STING , but not the versions of STING found in most other mammals .",
"We have used the STING proteins of closely related primate species to map the determinant of cleavage in STING .",
"We find that the viral protease is recognizing ( i . e . cutting or binding ) residues around positions 78/79 of STING .",
"We show that an ‘RG’ motif at these two residues is necessary and sufficient for cleavage by the proteases of all four human epidemic dengue viruses , and one sylvatic dengue virus .",
"Yet , only some apes and three rodent species , out of all of the mammalian STING sequences in Genbank , encode an RG at positions 78/79 .",
"Why do dengue viruses universally cleave human but not monkey STING ?",
"It’s possible that what we have uncovered is a brilliant method for balancing alternate host species , one of which is dense and abundant ( humans ) , versus others that are spare and exist in smaller populations ( primates in nature ) .",
"In this scenario , dengue viruses have evolved to suppress innate immunity in humans in order to increase viral titers and spread , even though this trait comes at the cost of increased pathogenicity in some individuals .",
"This might be a good strategy in our abundant and dense host population , where the fitness cost of severe disease in a fraction of individuals would be outweighed by excellent spread .",
"Remarkably , though , dengue viruses have achieved this by evolving to recognize a portion of human STING that is not conserved in the STING of the wild and more rare animals that serve as their sustaining reservoir in nature , allowing the viruses to maintain decreased pathogenicity in these species .",
"The evolution of the viral proteases to cleave human STING and simultaneously to avoid cleavage of monkey STING would be expected to reduce virus titers in monkeys , as the interferon pathway would be at least partially enabled .",
"This may be beneficial for many reasons , one of which is that the production of a low-level innate immune response may allow the virus to replicate in reservoir host species without inducing high titers and strong adaptive immune responses .",
"Alternately , a second possibility is that sylvatic dengue viruses do cleave the STING of monkeys , but that the sylvatic virus ( DakAr-141069 ) that we tested is not representative .",
"However , we find this unlikely .",
"Because DENV1-4 also cannot cleave monkey STING , and all derive from the sylvatic reservoir , this supports the finding that sylvatic viruses do not cleave monkey STING .",
"Third , a final possibility is that apes are critical reservoirs for dengue viruses in nature .",
"Gorillas encode ‘RG’ at 78/79 and are found in Africa , while orangutans and gibbons are found in Asia and also encode the correct cleavage motif for the dengue virus protease .",
"In fact , wild orangutans have previously been found to have neutralizing antibodies against dengue virus ( Wolfe et al . , 2001 ) .",
"While apes could be playing a role as sylvatic hosts , the highly endangered and rare status of most apes makes it hard to believe that they are playing a major role in sustaining sylvatic dengue virus currently ( Geissmann , 2007; Walsh et al . , 2003 ) .",
"The specific primate species that serve as the sustaining reservoir for sylvatic dengue viruses in nature are unknown ( Vasilakis et al . , 2011 ) .",
"Various Old World monkey species in both Asia and Africa are suspected hosts ( Vasilakis et al . , 2011; Rodhain , 1991; Diallo et al . , 2003 ) .",
"For instance , sylvatic dengue viruses have been isolated directly from macaques ( Macaca fascicularis ) and leaf monkeys ( Presbytis obscura ) that were placed as sentinels in forest canopies ( Rudnick , 1986 ) .",
"Other primates have been shown to have antibodies to dengue virus , including macaques ( Macaca fascicularis and Macaca nemestrina ) , leaf monkeys ( Presbytis cristata ) ( Rudnick , 1965 ) , African green monkeys ( Chlorocebus sabaeus ) ( Diallo et al . , 2003 ) , and one ape species , the Bornean orangutan ( Pongo pygmaeus ) ( Wolfe et al . , 2001 ) .",
"But these results are not definitive due to the cross-reactivity of antibodies directed against various flaviviruses ( Calisher et al . , 1989; Tesh et al . , 2002; Mansfield et al . , 2011 ) , and the possibility that some primates might be accidental , rather than sustaining reservoir hosts ( Vasilakis et al . , 2011 ) .",
"Instead of being directly isolated from primates , most sylvatic dengue viruses have been obtained from forest mosquitoes ( Diallo et al . , 2003; Rudnick , 1986 ) , or from humans that contracted the virus in the forest ( Pyke et al . , 2016; Cardosa et al . , 2009; Franco et al . , 2011 ) .",
"Therefore , there are many deficiencies in our understanding of the natural reservoir for dengue viruses .",
"Interestingly , though , we have not identified any monkey species with an ‘RG’ at positions 78/79 in STING .",
"Our results would indicate that dengue viruses , in general , cannot cleave the STING of monkey hosts .",
"Our data suggests that even a sylvatic dengue virus , which we find targets the same residues in STING , would not be able to cleave STING of monkeys .",
"There are also implications of these findings to our understanding of dengue virus model organisms .",
"If dengue proteases do not cleave most nonhuman forms of STING , this may at least partially explain why it has been so difficult to model dengue infection in immune-competent animals .",
"Nonhuman primates infected with dengue virus generally don’t develop clinical signs of disease , consistent with enhanced control of the virus compared to humans ( Cassetti et al . , 2010 ) .",
"In fact , when human dengue viruses have been observed to replicate robustly in primate cell lines , these experiments have typically been done in cells such as Vero ( Vasilakis et al . , 2008; Rossi et al . , 2012; Vasilakis et al . , 2009 ) which are deficient in the type I interferon response ( Osada et al . , 2014; Desmyter et al . , 1968 ) .",
"Human dengue viruses also cannot cleave mouse STING ( ( Aguirre et al . , 2012; Yu et al . , 2012 ) and herein ) , consistent with the heightened control of this virus in mice as well ( Cassetti et al . , 2010 ) .",
"Dengue virus will replicate to high titers in mice lacking key genes important for the interferon response , but for many reasons it is desirable to develop animal models in immune competent hosts ( Cassetti et al . , 2010 ) .",
"STING now adds to a growing list of host proteins that regulate viral infection differently even in closely related host species ( for example , [Stabell et al . , 2016; Lou et al . , 2016; Rowley et al . , 2016; Kerr et al . , 2015; Meyerson et al . , 2015; Demogines et al . , 2012; Stremlau et al . , 2004; Demogines et al . , 2013; Ng et al . , 2015; Hueffer et al . , 2003; Radoshitzky et al . , 2008; Patel et al . , 2012; Elde et al . , 2009; Martin et al . , 2013; Miller et al . , 2012; Sawyer and Elde , 2012; Meyerson et al . , 2017] ) .",
"The identification of such genes is critical to our understanding of viral adaptation during host switching , and to the development of animal models in which to study human viruses .",
"It is possible that the identification of small mammals that have a cleavage-susceptible STING would facilitate the development of better animal models for studying dengue virus .",
"Our work suggests the identity of three such species: the naked mole rat , the common chinchilla , and the desert woodrat .",
"All three of these small rodents are already used as animal models in biomedical research , and the genomes of all three have been sequenced ( Keane et al . , 2014; Nathaniel et al . , 2013; Shimoyama et al . , 2016; Campbell et al . , 2016; Skopec et al . , 2013 ) .",
"These species could be superior to STING knockout mice , in that the STING pathway would be intact and the cleavage of STING by the virus would be naturally modeled rather than just bypassed .",
"These species may also be superior to future models where mouse ( Mus musculus ) STING would be replaced with human STING in transgenic animals .",
"In this case , it is unknown if human STING would perform all of its functions the same in mouse as it does in humans .",
"The advantage of using a rodent model with a STING that is naturally susceptible to dengue virus cleavage would be that the STING pathways would all be fully functional and intact .",
"It is important to point out that , in addition to cleaving STING , dengue viruses modulate the interferon response in other ways as well .",
"For instance , dengue viruses also bypass the type I interferon response by binding and degrading host STAT2 via the viral NS5 protein ( Ashour et al . , 2009; Jones et al . , 2005; Mazzon et al . , 2009; Best , 2017 ) .",
"Ideally , human dengue viruses would also be able to bind and degrade STAT2 in newly developed models , as they do in humans .",
"Further , dengue viruses neutralize both the type I and type II interferon responses in other ways as well ( Perry et al . , 2011; Shresta et al . , 2004; Aguirre et al . , 2017; Aguirre and Fernandez-Sesma , 2017 ) .",
"Other known host-virus interactions would also need to be characterized in any potential new model organism .",
"It is notable that chimpanzees and bonobos encode STINGs that are resistant to cleavage , while STINGs of all other apes are susceptible .",
"These two species differ from other apes in encoding a ‘WG’ at 78/79 of STING rather than the ‘RG’ encoded by all other apes ( Figure 6 ) .",
"Remarkably , it was previously found that the ‘W’ at position 78 , destroying the dengue cleavage determinant , was fixed by positive natural selection in wild chimpanzee populations ( Mozzi et al . , 2015 ) .",
"Chimpanzees are not one of the suspected natural reservoirs of dengue virus , but chimpanzee ranges do co-occur with known human outbreaks and with sylvatic cycles ( Figure 1—figure supplement 1 ) .",
"One model is that , as dengue virus spread through Africa , a SNP in chimpanzee STING ( or the STING of the chimpanzee/bonobo ancestor ) at position 78 started to experience strong selection because it provided protection against cleavage by dengue viruses .",
"This would have driven a selective sweep in chimpanzee populations , causing this species to become less susceptible to viral infection .",
"It has previously been proposed that evolutionary pressure imposed by flavivirus proteases can drive selection at cleavage sites .",
"For examples , the hepatitis C protease cleaves MAVS , another host signaling protein in the interferon induction cascade ( Li et al . , 2005b; Meylan et al . , 2005 ) .",
"MAVS has experienced positive selection at a residue in the cleavage site for the hepatitis C virus protease ( Patel et al . , 2012 ) .",
"The authors of this study speculated that ancient viruses may have exerted selective pressure on primate genomes to acquire mutations in the cleavage site .",
"Like other viruses , dengue viruses remodel their host cellular environment in numerous ways , including the cleavage of STING and degradation of STAT2 .",
"Using the rich information that exists on how dengue viruses accomplish this , the genetic susceptibility of both suspected reservoir hosts , and potential new animal models , can be systematically assessed .",
"Characterizing how host-virus interactions play out uniquely in different host species will help us to understand dengue virus in critical ways .",
"For instance , it will reveal how dengue viruses do ( or do not ) need to evolve their genomes as they transmit to humans from nonhuman primates in nature .",
"Also , understanding the genetics of host tropism will help identify better laboratory animals that can be used to study dengue virus pathogenesis and to develop drugs and vaccines ."
],
[
"DENV2 NS2B3 , expressed from the pCR3 . 1 plasmid , was a gift from Yi-Ling Lin .",
"This plasmid , and all DENV1-4 protease-expressing plasmids described below , include a 3x FLAG tag at the C-terminus of NS3 .",
"For the experiment where proteases from human dengue viruses DENV1-4 are compared , primers were designed at the 5’ end of NS2B ( DENV1: taagcaAAGCTTcaccATGAGTTGGCCCCTC , DENV2: taagcaAAGCTTcaccATGAGCTGGCCACTAAATGA , DENV3: taagcaAAGCTTcaccATGAGCTGGCCACTG , DENV4: taagcaAAGCTTcaccATGTCTTGGCCCCTTAAC ) and the 3’ end of NS3 ( DENV1: TGCTTAgtcgacaTCTTCTTCCTGCTGCAAACTCTTTAAACTC , DENV2: TGCTTAgtcgacaCTTTCTTCCAGCTGCAAACTCCTTG , DENV3: TGCTTAgtcgacaCTTTCTGCCAGCTGCAAAATCCTTG , DENV4: TGCTTAgtcgacaCTTTCTTCCACTGGCAAACTCCTTG ) to amplify the NS2B + NS3 genomic region in one fragment .",
"In this experiment , the protease from DENV2 was re-cloned so that the structure of the four protease clones was identical in all four cases .",
"The PCR templates were cDNAs created from RNA obtained through the World Reference Center for Emerging Viruses and Arboviruses ( WRCEVA ) ( Cat# NR-32847 ) .",
"DENV1 ( Hawaii , NR-4287 ) , DENV2 ( New Guinea C , NR-4288 ) , DENV3 ( Philippines/H87/1956 , NR-2771 ) , and DENV4 ( H241 , NR-4289 ) .",
"The PCR products , and the plasmid containing the DENV2 protease mentioned above ( gift from Yi-Ling Lin ) , were both digested with HindIII and Sal1 .",
"The PCR products were ligated into this plasmid and transformed into DH5α chemically competent E . coli .",
"The sylvatic NS2B3 ( DakAr141069 ) was synthesized ( without an epitope tag ) using the sequence information deposited on NCBI ( Genbank accession EF105389 ) .",
"STING genes used for functional analysis were amplified from cDNA libraries constructed from the following cell lines: human ( A549 ) , chimpanzee/bonobo ( STING sequence identical for these two species , clone amplified from Coriell , PR00748 ) , rhesus macaque ( Mm265-95 , a gift from Welkin Johnson ) , marmoset ( Coriell , PR07404 ) , and mouse ( generated from RNA extracted from whole liver ) .",
"Either an HA or 3xFlag tag were engineered onto the 3' end of the gene sequences , separated from the coding sequence by a 3xGlycine-Alanine ( GAGAGA ) linker region ( nucleotide sequence = GGTGCTGGTGCTGGTGCT ) .",
"These sequences were cloned into the pcDNA3 . 1 expression vector with a 5' Kozak sequence ( GCCACC ) .",
"Rodent STING constructs were synthesized ( Quintarabio ) to include a Kozak sequence , C-terminal HA-tag , and flanking linkers that were used for Gibson cloning into the pLPCX mammalian expression plasmid .",
"293 T cells ( mycoplasma negative ) were grown at 37°C in DMEM supplemented with 10% FBS , Pen/Strep , and L-glutamine .",
"24 hr prior to transfection , cells were plated at a density of 4 . 5 × 105 cells per well in a 12-well dish in antibiotic free media .",
"Wells were transfected with 800 ng plasmid encoding STING and 800 ng plasmid encoding NS2B3 using TransIT 293 reagent ( Mirus MIR 2704 ) .",
"For most experiments ( Figures 1B , 3B , 5A and B ) , cells were treated with 10uM MG132 for 8 hr prior to harvesting for western blot .",
"Cells were lysed in RIPA buffer supplemented with protease inhibitor ( Roche , 4693159001 ) .",
"Protein concentration was calculated using the Bradford method .",
"10% 37 . 5:1 Acrylamide/Bisacrylamide gels were used to run 30 ug of whole cell lysate for each sample .",
"Protein was transferred overnight at 30 volts onto a polyvinyl membrane .",
"Blocking was performed with a 10% milk solution in tris-buffered saline supplemented with 0 . 1% TWEEN20 .",
"Primary antibodies used were used against HA ( 3f10 clone Sigma 11867423001 ) , Flag ( M2 clone Sigma F3165 ) , GAPDH ( CellSignaling 14C10 ) , STING ( Abcam 92605 ) , actin ( Santa Cruz Biotech Sc47778 ) , and dengue virus NS3 ( mouse polyclonal antibody raised against purified full-length NS3 from dengue 2 strain 16681 [Heaton et al . , 2010] ) .",
"Secondary antibodies used were goat-anti-mouse-HRP ( Thermo 62–6520 ) and goat-anti-rabbit ( Thermo 65–6120 ) .",
"Blots were developed using ECL Prime ( Amersham RPN2232 ) and imaged using ImagQuant LAS 4000 ( Amersham 28-9558-10 ) .",
"A549 cells ( mycoplasma negative ) were transfected with the pSPCAS9 ( BB ) -P2A-eGFP ( PX458 ) with the guide RNA sequence 5’ AGAGCACACTCTCCGGTACC 3’ .",
"GFP-positive cells were single-cell sorted into a 96-well dish and colonies were grown up .",
"Cloned A549 cells were screened for homozygous mutations that disrupted the coding sequence of STING as follows .",
"10 , 000 cells were used to prep whole genomic DNA .",
"The region surrounding the guide RNA was amplified using the following primers: 5’ GTCCCCAAGGGTTCTTGGTT 3’ and 5’ AACCAGTCCCACTCCCAGTA 3’ .",
"Amplified genomic DNA was Sanger sequenced to determine the nature of the CRISPR-CAS9-mediated genomic disruption .",
"A cell line with confirmed homozygous disruption of STING ( Figure 2—figure supplement 1 ) was then re-complemented with primate orthologs of STING .",
"Four different C-terminally HA-tagged versions of STING were cloned into the pLPCX retroviral vector: wildtype human STING , R78W human STING , wildtype chimpanzee STING , and W78R chimpanzee STING .",
"These were packaged into retroviral particles by cotransfecting into 293T cells ( mycoplasma negative ) each pLPCX-STING construct with plasmids expressing NB-tropic murine leukemia virus ( MLV ) Gag-Pol and VSV-G .",
"As a control , we also made virus to complement with an empty pLPCX vector .",
"Supernatants were collected and used to transduce 10^5 A549 cells in the presence of 10 ug/mL polybrene .",
"24 hr post transduction , cells were selected in 0 . 75 ug/mL puromycin .",
"24 hr after plating , cells were fixed with 4% paraformaldehyde and permeabilized with 1% TritonX100 in PBS .",
"Blocking was performed with 3% BSA solution in PBS .",
"Primary antibodies used were rabbit-anti-GRP78 ( BiP ) ( Abcam ab21685 ) and mouse-anti-HA ( clone 16B12 abcam ab130275 ) .",
"Secondary antibodies used were donkey-anti-rabbit conjugated to AlexaFluor594 ( Invitrogen A21207 ) and donkey-anti-mouse conjugated to AlexaFluor488 ( Invitrogen A21202 ) .",
"Cells were mounted using VECTASHIELD hardset mounting media ( VectorLabs H-1400 ) .",
"The indicated STING knockout and re-complemented cell lines were plated out in F-12K media with 10% FBS , after 24 hr the cells were counted .",
"An MOI of 0 . 3 was calculated for each well and dengue virus 2 ( 16681 ) was allowed to attach to cells for 1 hr at room temperature .",
"Unattached virus was then removed from cells , 2%serum in F-12K media was added to cells and they were maintained at 37°C with 5% CO2 .",
"After 24 and 48 hr the virus supernatant was removed for downstream titration on BHK21 cells .",
"At the same time , cells were removed for downstream western blotting .",
"The following STING sequences were collected from GenBank: chimpanzee ( Pan troglodytes , XM_016953921 . 1 ) , gorilla ( Gorilla gorilla gorilla , XM_004042612 . 1 ) , Sumatran orangutan ( Pongo abelii , XM_002815952 . 2 ) , golden snub-nosed monkey ( Rhinopithecus roxellana , XM_010388119 . 1 ) , black snub-nosed monkey ( Rhinopithecus bieti , XM_017895026 . 1 ) , African green monkey ( Chlorocebus sabaeus , XM_008014636 . 1 ) , pigtail macaque ( Macaca nemestrina , XM_011716377 . 1 ) , rhesus macaque ( Macaca mulatta , XM_015141010 . 1 ) , sooty mangabey ( Cercocebus atys , XM_012090448 . 1 ) , drill ( Mandrillus leucophaeus , XM_011997224 . 1 ) , marmoset ( Callithrix jacchus , XM_00898588 . 2 ) , capuchin monkey ( Cebus capucinus imitator , XM_017536735 . 1 ) , black-capped squirrel monkey ( Saimiri boliviensis , XP_003933962 . 1 ) .",
"The remaining STING gene sequences were obtained by direct sequencing of cDNA libraries produced from the following primary or immortalized primate fibroblast cell lines: Bonobo ( Pan paniscus , Coriell PR00748 ) , Bornean orangutan ( Pongo pygmaeus , Coriell PR00650 ) , white-handed gibbon ( Hylobates lar , Coriell PR01131 ) , agile gibbon ( Hylobates agilis , Coriell PR00773 ) , siamang ( Symphalagus syndactylus , Coriell PR00722 ) , white-cheeked gibbon ( Nomascus leucogenys , Coriell PR01037 ) , leaf monkey ( Trachypithecus francoisi , Coriell PR01099 ) , colobus monkey ( Colobus guereza , Coriell PR00980 ) , Wolf’s guenon ( Cercopithecus wolfi , Coriell PR01241 ) , talapoin ( Miopithecus talapoin , Coriell PR00716 ) , crab-eating macaque ( Macaca fasicularis , 103–06 , gift from Welkin Johnson ) , olive baboon ( Papio anubis , Coriell PR00978 ) , grey-cheeked mangabey ( Lophocebus albigena , Coriell PR01215 ) , Bolivian red howler monkey ( Alouatta sara , Coriell PR00708 ) , red titi monkey ( Callicebus ( or Plecturocebus ) cupreus , Coriell PR00793 ) , common squirrel monkey ( Saimiri sciureus , Coriell PR00603 ) .",
"Briefly , cells were grown in DMEM ( Cellgro ) supplemented with 15% FBS ( Gibco ) at 37°C and 5% CO2 .",
"RNA was extracted using the AllPrep DNA/RNA extraction kit ( QIAGEN ) .",
"cDNA libraries were generated using SuperScript III first strand synthesis kit ( Invitrogen ) .",
"PCR was performed using PCR SuperMix High Fidelity ( Invitrogen ) .",
"PCR products were directly sequenced .",
"Each primate sequence was used as a query to search the human genome , and human STING gene was returned as the top hit .",
"STING gene sequences generated in this study have been deposited in GenBank ( accession numbers MF616339-MF616355 ) ."
]
] | [
"Human dengue viruses emerged from primate reservoirs , yet paradoxically dengue does not reach high titers in primate models .",
"This presents a unique opportunity to examine the genetics of spillover versus reservoir hosts .",
"The dengue virus 2 ( DENV2 ) - encoded protease cleaves human STING , reducing type I interferon production and boosting viral titers in humans .",
"We find that both human and sylvatic ( reservoir ) dengue viruses universally cleave human STING , but not the STING of primates implicated as reservoir species .",
"The special ability of dengue to cleave STING is thus specific to humans and a few closely related ape species .",
"Conversion of residues 78/79 to the human-encoded ‘RG’ renders all primate ( and mouse ) STINGs sensitive to viral cleavage .",
"Dengue viruses may have evolved to increase viral titers in the dense and vast human population , while maintaining decreased titers and pathogenicity in the more rare animals that serve as their sustaining reservoir in nature ."
] | [
"Dengue viruses are found in over 100 countries and cause the tropical disease known as dengue fever .",
"Dengue viruses affect around 100 million people per year and can – in severe cases – lead to death .",
"Unlike many other deadly diseases , there is currently no vaccine that completely prevents dengue fever .",
"It is thought that dengue viruses that circulate in human populations were derived from monkey versions of that same virus .",
"However , research suggests that both human and primate variations of dengue viruses appear to multiply much better in humans than in other species .",
"Scientists believe that this is because some animals , including primates , have defense mechanisms that are ineffective in humans .",
"To explore this idea , Stabell et al . looked at a protein called STING in humans and in three different primates: the chimpanzee , the rhesus macaque , and the common marmoset .",
"STING plays an important role in the immune system and helps to fight infections caused by viruses and other microbes .",
"During replication – the process by which a virus spreads through an organism’s cells – the dengue virus cuts and inactivates the human STING protein , and so helps the virus spread .",
"Stabell et al . discovered that in most primates , dengue viruses cannot inactivate STING .",
"This was found to be reliant on a small region in the STING protein that differed between humans and primates .",
"This small difference may , in part , explain why dengue viruses replicate better in humans than other primates .",
"Stabell et al . then searched for other animals whose STING protein would be susceptible to dengue virus inactivation .",
"Using a database with genetic information of over 5 , 000 mammals , Stabell et al . identified STING proteins of three types of apes and three types of rodents that could also be deactivated by dengue viruses .",
"To develop a vaccine or antiviral drug scientists generally need to study the disease in living animals .",
"Since dengue viruses replicate more successfully in humans than they do in other animal models , it makes it more challenging to find an effective treatment .",
"The results from Stabell et al . may help to identify animals that could be strong candidates for future research into dengue viruses , potentially paving the way for further therapeutic development ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | QIL1 is a novel mitochondrial protein required for MICOS complex stability and cristae morphology | elife-06265-v1 | [
[
"Mitochondria exhibit a complex topology encompassing two membranes that create distinct internal compartments .",
"The inner membrane ( IM ) runs parallel to the outer membrane ( OM ) at regions called inner boundary membranes ( IBM ) and invaginates into the mitochondrial matrix forming the cristae structures which connect to the inter membrane space ( IMS ) through narrow openings ( Freya and Mannellab , 2000 ) .",
"This intricate architecture is maintained by structures called cristae junctions ( CJs ) and contact sites ( CSs ) and has been shown to be essential for numerous mitochondrial pathways such as protein import , oxidative phosphorylation , and apoptosis ( Freya and Mannellab , 2000; Vogel et al . , 2006; Yang et al . , 2012; Cogliati et al . , 2013 ) .",
"It has been reported that changes in physiological energetic states and oxygen availability affect the morphology of cristae and CJs ( Lorente et al . , 2002; Walker and Benzer , 2004 ) , suggesting that the formation of CJs is a dynamic process that promotes cristae remodeling as an adaptive mechanism to the different needs and metabolic states of the cell .",
"Moreover , several mitochondrial disorders in humans are often accompanied by alterations of mitochondrial ultrastructure including MERRF syndrome ( Myoclonic epilepsy with ragged red fibers ) , BTHS ( Barth syndrome ) , fatal neonatal lactic acidosis , mtDNA defects and hypertrophic cardiomyopathy and loss of CJs with the formation of concentric stacks of cristae membrane are some of the hallmarks observed in mitochondria from such patients ( Silva-Oropeza et al . , 2004; Acehan et al . , 2007; Roels et al . , 2009; Götz et al . , 2012 ) .",
"As such , deciphering how CJs and CSs are formed and regulated is crucial for the understanding of cristae dynamics and maintenance in health and disease .",
"Despite its critical importance , only recently have components of a protein complex that localizes at CJs and CSs and are responsible for mitochondrial cristae architecture been identified ( van der Laan et al . , 2012 ) .",
"Recent studies in both yeast and human cells led to the identification of various subunits of a highly conserved heterooligomeric protein complex of approximately ∼700 kDa , referred to as the mitochondrial contact site and cristae junction organizing system ( MICOS ) , which controls the formation of CJs and CSs and IM morphology ( Gieffers et al . , 1997; John et al . , 2005; Xie et al . , 2007; Rabl et al . , 2009; Darshi et al . , 2011; Harner et al . , 2011; Hoppins et al . , 2011; von der Malsburg et al . , 2011; Alkhaja et al . , 2012; An et al . , 2012; Bohnert et al . , 2012; Jans et al . , 2013; Korner et al . , 2012; Ott et al . , 2012; Pfanner et al . , 2014; Weber , 2013 ) .",
"A recent study has proposed to unify the nomenclature for MICOS , with subunits of this complex termed MIC10 to MIC60 ( with subunit mass indicated by the numbers ) ( Pfanner et al . , 2014 ) .",
"To date , 7 bona fide subunits have been described in yeast ( Mic10 , Mic12 , Mic19 , Mic25 , Mic26 , Mic27 , Mic60 ) , 6 of which have obvious human orthologs ( MIC10/MINOS1 , MIC19/CHCHD3 , MIC25/CHCHD6 , MIC27/APOOL , MIC26/APOO and MIC60/Mitofilin ) ( Icho et al . , 1994; Odgren et al . , 1996; Gieffers et al . , 1997; John et al . , 2005; Xie et al . , 2007; Rabl et al . , 2009; Mun et al . , 2010; Darshi et al . , 2011; Harner et al . , 2011; Head et al . , 2011; Hoppins et al . , 2011; von der Malsburg et al . , 2011; Alkhaja et al . , 2012; An et al . , 2012; Bohnert et al . , 2012; Korner et al . , 2012; Ott et al . , 2012; Itoh et al . , 2013; Jans et al . , 2013; Weber , 2013 ) .",
"The MIC10-MIC19-MIC25-MIC26-MIC27-MIC60 complex is thought to reside in the IM at the site of CJs .",
"Genetic removal of several MICOS subunits causes mitochondrial fragmentation , reduced respiration , CJs loss , and the formation of concentric stacks of IM disconnected from the IBM ( John et al . , 2005; Rabl et al . , 2009; Darshi et al . , 2011; Harner et al . , 2011; Hoppins et al . , 2011; Alkhaja et al . , 2012; Ott et al . , 2012; van der Laan et al . , 2012; Weber , 2013 ) .",
"These phenotypes strikingly resemble cristae abnormalities observed in patients with the aforementioned mitochondrial disorders .",
"Moreover , alterations of several MICOS subunit protein levels , mutations and protein modifications have been linked to a variety of human diseases , including diabetic cardiomyopathy , epilepsy , Parkinson's disease , and cancer ( Zerbes et al . , 2012 ) .",
"One study has employed proteomic analysis of MIC10/MINOS1 protein interactors in human cells to identify further components of the complex , and in addition to the IM core subunits , this work also identified the OM proteins SAMM50 , MTX1 , MTX2 , and the chaperone DNAJC11 ( Alkhaja et al . , 2012 ) .",
"A role for these proteins ( Xie et al . , 2007 ) and in particular the sorting and assembly machinery protein SAMM50 in CJ assembly was also found independently ( Ott et al . , 2012 ) , suggesting a role for β-barrel insertion into the OM in CJ formation or maintenance .",
"Interestingly , mutations in DNAJC11 in the mouse result in mitochondrial disruption , cristae abnormalities , and motor neuron pathology ( Ioakeimidis et al . , 2014 ) .",
"Given the crucial role that the MICOS complex plays in CJ architecture and mitochondrial homeostasis ( Zerbes et al . , 2012 ) , a further understanding of its constituents and regulatory mechanisms is needed .",
"To develop a comprehensive understanding of MICOS composition and regulation , we performed proteomic IP-MS analysis of the MICOS complex .",
"In addition to core MICOS subunits , we identified several new candidate MICOS-associated proteins .",
"One of these—QIL1—is a previously unstudied IM protein conserved from human to Drosophila .",
"Using quantitative proteomics coupled with native gel analysis , we show that QIL1 is a component of a ∼700 KDa MICOS complex and its depletion from cells results in loss of MIC10 , MIC26 , and MIC27 from the complex and a reduction in the abundance of these proteins in mitochondria .",
"Thus , QIL1 appears to be required for incorporation of MIC10 , MIC26 , and MIC27 into the MICOS complex .",
"Depletion of QIL1 in human cells results in several mitochondrial phenotypes including loss of CJs , cristae rearrangement into stacks of concentric membranes , and reduced respiration .",
"In Drosophila muscle and neuronal cells , QIL1 depletion leads to analogous morphological phenotypes .",
"This study provides the most comprehensive protein interaction network map of the MICOS complex to date and will serve as a resource for further elucidation of MICOS assembly and regulation ."
],
[
"In order to examine the composition of the MICOS complex using interaction proteomics , open reading frames for MIC27 , MIC19 , MIC25 , MIC60 , MTX2 , and DNAJC11 ( Figure 1—figure supplement 1A shows a schematic representation of the MICOS complex where the MICOS subunits and interactors used for our IP-MS approach are depicted in red ) were C-terminally tagged with an HA-FLAG epitope in a lentiviral vector and expressed stably in 293T and HeLa cells ( Figure 1—figure supplement 1B ) .",
"Confocal microscopy after immunostaining with α-HA and α-TOMM20 verified that each protein was targeted to mitochondria in HeLa cells ( Figure 1A ) .",
"To identify high confidence interacting proteins ( HCIPs ) , we employed a modified version of the ComPASS platform ( Sowa et al . , 2009 ) .",
"This method uses a large collection of parallel AP-MS experiments to generate a database populated with peptide spectral matches , allowing the frequency , abundance , and reproducibility of interacting proteins to be determined .",
"To enhance detection of membrane-associated proteins , we employed 1% digitonin , and proteins were purified using α-FLAG beads .",
"After extensive washing , complexes were trypsinized prior to proteomic analysis .",
"As a validation approach , three of the baits ( MIC60 , MTX2 , and MIC19 ) were also expressed in HCT116 cells and immunopurified with a different antibody ( α-HA ) .",
"Interaction data are summarized in Figure 1B ( Figure 1—figure supplement 2 contains the entire data set ) .",
"Overall , the interaction network contained 26 proteins and 97 interactions ( edges ) after filtering as described in the ‘Materials and methods’ .",
"The six baits analyzed showed extensive reciprocal connectivity ( Figure 1B ) .",
"Confirming previously reported data , several core subunits of the MICOS complex ( MIC19 , MIC25 , MIC60 , MIC26 , MIC27 ) also associated with known interactors at the OM ( SAMM50 , MTX1 and MTX2 ) , indicating that our method is able to retrieve nearly all known subunits and interactors of the MICOS complex , located at the IM , IMS , and OM with high confidence .",
"In addition to known interactors , our map also revealed potential novel interacting partners , associated with one or more MICOS subunits .",
"These include two OM proteins , the MUL1 E3 ubiquitin ligase and the RHOT2 GTPase involved in mitochondrial trafficking ( Figure 1B ) .",
"RHOT2 has been shown to co-fractionate with SAMM50 in correlation profiling proteomic experiments ( Havugimana et al . , 2012 ) .",
"In addition , we identified TMEM11 as a protein associated with multiple MICOS subunits and capable of associating with MIC60 endogenously ( Figure 1B , F ) .",
"A TMEM11 ortholog in Drosophila has been shown genetically to be required for cristae organization and biogenesis , but the mechanisms involved are unknown ( Rival et al . , 2011; Macchi et al . , 2013 ) .",
"Our results indicate that TMEM11 may function in these processes in association with the MICOS complex .",
"Components of the MICOS complex were not detected in GFP-FLAG immune complexes prepared similarly ( Figure 1—figure supplement 2 ) , pointing the specificity of the interactions observed . 10 . 7554/eLife . 06265 . 003Figure 1 . Interaction proteomics of the MICOS complex reveals QIL1 as a novel interactor .",
"( A ) Immunofluorescence analysis of the subcellular localization of tagged proteins .",
"Bars , 20 µm .",
"( B ) Overview of the MICOS interaction network obtained from IP-MS analysis .",
"( C ) Validation of QIL1 protein interactions by IP-MS analysis .",
"Figure 1—figure supplement 1 shows a schematic representation of the MICOS complex highlighting the subunits and interactors analyzed by IP-MS in red and the expression levels of C-terminally tagged proteins compared to the endogenous version .",
"Figure 1—figure supplement 2 contains the entire IP-MS data set .",
"( D ) Immunofluorescence analysis of the subcellular localization of C-terminally tagged QIL1 .",
"Bars , 20 µm .",
"Endogenous QIL1 ( E ) , MIC60 ( F ) , or MIC10 ( G ) was immunopurified from crude mitochondria isolated from 293T cells .",
"Endogenous interactions with MIC60 , MIC10 , AFG3L2 , TMEM11 , or QIL1 were assessed .",
"( H ) Alignment of QIL1 orthologs using the ClustalW software .",
"‘H’ indicates amino acids present within a conserved predicted transmembrane region ( TMPred algorithm ) .",
"( I ) Cytoplasmic and mitochondrial fractions were separated in 293T lysates .",
"Soluble and membrane fractions were separated by alkaline extraction .",
"S indicates soluble , P indicates pellet .",
"( J ) Alkaline extraction was performed at increasing pH ( pH11 , pH11 . 5 , pH12 ) .",
"( K ) Proteinase K ( PK ) and increasing concentrations of digitonin were used to assess QIL1 sub-mitochondrial localization .",
"( L ) Proteinase K ( PK ) , osmotic shock ( OS ) , and Triton X-100 ( TX100 ) were used to assess QIL1 sub-mitochondrial localization . DOI: http://dx . doi . org/10 . 7554/eLife . 06265 . 00310 . 7554/eLife . 06265 . 004Figure 1—figure supplement 1 . Expression levels of C-terminally tagged MICOS subunits and related proteins analyzed by IP-MS in this study compared to the endogenous version .",
"( A ) Schematic representation of the MICOS complex and its interactors .",
"Proteins C-terminally tagged and subjected to IP-MS analysis are represented in red .",
"( B ) Immunoblot analysis was performed to examine the protein levels of the overexpressed C-terminally tagged proteins compared to the endogenous levels in each stable cell line . DOI: http://dx . doi . org/10 . 7554/eLife . 06265 . 00410 . 7554/eLife . 06265 . 005Figure 1—figure supplement 2 . Mass spectrometry analysis of immunopurified protein interactors for QIL1 , MIC27 , MIC19 , MIC25 , MIC60 , MTX2 , DNAJC11 and GFP in 293T and/or HCT116 cells . Spectra search with Sequest , target-decoy peptide filtering , and linear discriminant analysis ( Huttlin et al . , 2010 ) was performed on raw data obtained from technical duplicate runs on either an Thermo LTQ or a LTQ Orbitrap Elite mass spectrometer .",
"Protein Assembler was used to convert spectral counts to APSMs .",
"Peptide data ( APSMs ) were uploaded into the CompPASS algorithm housed within the CORE environment .",
"Interaction confidence was ranked according to the NWD scores ( Sowa et al . , 2009 ) .",
"The results obtained from the CompPASS analysis were filtered to determine proteins with a NWD score>1 or high confidence interactors ( HCIPs ) .",
"HCIPs were then filtered for mitochondrial proteins based on the proteins present in Mitocarta ( Pagliarini et al . , 2008 ) .",
"For the 293T data set , HCIPs with 2 or more ASPMs were included . DOI: http://dx . doi . org/10 . 7554/eLife . 06265 . 005 Our attention was drawn to a previously uncharacterized protein with unknown function—C19orf70 ( also called QIL1 ) —which was detected in association with MIC19 , MIC60 , and MTX2 in both 293T FLAG IPs and HCT116 HA IPs and with MIC27 additionally in 293T ( Figure 1B ) .",
"As an initial approach for validating the interactions , C-terminally tagged QIL1 was subjected to IP-MS analysis .",
"The result elicited the generation of an interaction map containing 13 nodes and 20 edges ( interactions ) ( Figure 1C ) , wherein we identified 5 core MICOS subunits ( MIC60 , MIC19 , MIC25 , MIC26 and MIC27 ) , 3 OM known interactors ( SAMM50 , MTX1 and MTX2 ) as well as the chaperone DNAJC11 and TMEM11 in association with QIL1 .",
"In a further attempt to validate these interactions , we first employed antibodies directed at endogenous QIL1 for immunoprecipitation ( IP ) and detected endogenous MIC60 , but not the abundant mitochondrial IM protein AFG3L2 protein , after western blot analysis ( Figure 1E ) .",
"Reciprocally , endogenous QIL1 co-precipitated with immunopurified endogenous MIC60 ( Figure 1F ) .",
"The transmembrane protein MIC10 was the only known MICOS subunit that was not detected by our proteomics approach , possibly due to its small size ( 78 residues ) and predominantly hydrophobic peptides .",
"To address if QIL1 also interacted with MIC10 , we immunopurified endogenous MIC10 in isolated mitochondria from 293T cells and assessed binding to QIL1 by western blot analysis ( Figure 1G ) .",
"In addition to detecting MIC60 in MIC10 immune complex ( Alkhaja et al . , 2012 ) , we also detected QIL1 .",
"Thus , QIL1 endogenously associates with components of the MICOS complex .",
"We then examined the sub-cellular localization of QIL1 .",
"First , QIL1-HA-FLAG was found to co-localize with endogenous TOMM20 in HeLa cells , ( Figure 1D ) .",
"Second , endogenous QIL1 was detected in purified mitochondria from 293T cells by western blotting , with a level of enrichment similar to that found with other mitochondrial proteins ( Figure 1I ) , confirming that QIL1 is a mitochondrial protein .",
"Our bioinformatics analysis identified QIL1 orthologs across metazoans , including Drosophila ( Figure 1H ) .",
"Additionally , primary sequence analysis using the TMpred algorithm revealed that QIL1 contains one possible conserved transmembrane helix ( Figure 1H ) .",
"To investigate whether QIL1 is a transmembrane protein , we performed carbonate extraction on mitochondria isolated from 293T cells .",
"As expected , the transmembrane proteins TOMM70A and TIMM23 were resistant to alkaline extraction regardless of the increase in pH , whereas DLD was efficiently released into the soluble fraction at higher pH ( Figure 1I , J ) .",
"Most bona fide MICOS subunits described so far are transmembrane proteins , with the exception of MIC19 and MIC25 ( Pfanner et al . , 2014 ) .",
"Interestingly , MIC19 has been shown to be myristoylated at the N-terminus allowing its docking onto SAMM50 , which is embedded in the OM , and possesses a CHCH domain at its C-terminus that binds to MIC60 , present in the IM ( Darshi et al . , 2012 ) ( see Figure 1—figure supplement 1A ) .",
"MIC19 was only partially extracted from the membrane fraction with increasing pH ( Figure 1J ) .",
"Similarly , while entirely present in the membrane fraction at lower pH , QIL1 was partially extracted from the membrane fraction at higher pH ( Figure 1I , J ) .",
"We did not identify a predicted myristoylation site or a CHCH domain in QIL1 .",
"These results suggest that QIL1 may indeed possess membrane anchoring activity , being possibly anchored by a single conserved membrane spanning helix as predicted by the TMPred algorithm ( Hofmann and Stoffel , 1993 ) .",
"To examine further the topology of QIL1 in the IM , we employed digitonin permeabilization of isolated mitochondria combined with proteinase K digestion ( Figure 1K ) as previously described ( Sancak et al . , 2013 ) .",
"Mitochondria were isolated from HCT116 cells and incubated with increasing concentrations of digitonin in the presence of proteinase K ( PK ) .",
"Subsequently , samples were analyzed by immunoblotting for the OM protein TOMM70A , IM proteins TIMM23 and MIC60 , and matrix protein CLPP ( Figure 1K ) .",
"These experiments showed that the topology of QIL1 is similar to the topology of the IM proteins TIMM23 and MIC60 ( Figure 1K ) .",
"To confirm the topology of QIL1 , we performed Proteinase K digestion with and without osmotic shock in the presence or absence of Triton X-100 ( Figure 1L ) as described previously ( Harner et al . , 2014 ) .",
"QIL1 displayed resistance to Proteinase K treatment in isolated mitochondria , but became accessible to protease digestion when the OM was disrupted by osmotic shock ( Figure 1L ) , as also found with the IM proteins TIMM23 and MIC60 ( Figure 1L ) .",
"Importantly , the matrix protein CLPP was only accessible to Proteinase K digestion when mitochondria were solubilized with 1% Triton X-100 , demonstrating the integrity of the IM .",
"Thus , we concluded that QIL1 behaves like an IM protein .",
"QIL1 interaction partners suggested an association with CJs .",
"To examine QIL1 localization directly , we employed immunogold labeling followed by transmission electron microscopy analysis ( Figure 2A–C ) and measured the distance between each gold particle and the nearest CJ in nanometers as described previously ( Jans et al . , 2013 ) .",
"QIL1-HA was predominantly located within 50 nm of CJs ( Figure 2A ) , being therefore enriched at CJs to a similar degree as MIC25-HA ( Figure 2B ) , used as a positive control .",
"In contrast , NDUFA13-HA , a Complex I subunit , was distributed along cristae membranes ( Figure 2C ) . 10 . 7554/eLife . 06265 . 006Figure 2 . QIL1 localizes at cristae junctions and is present in the mature MICOS complex .",
"( A–C )",
"Immunogold labeling of 293T cells overexpressing C-terminally tagged QIL1 ( A ) , MIC25 ( B ) , or NDUFA13 ( C ) using an α-HA antibody coupled to 10-nm gold particles .",
"Bars , 100 nm .",
"Representative mitochondria are shown .",
"The arrows point to the position of a gold particle .",
"The distance in nanometers between the gold particles and the nearest CJ was measured using the ImageJ software .",
"The histograms show the fraction of gold particles within the indicated distance to the crista junction in nanometers in QIL1-HA ( n = 231 gold particles ) , MIC25-HA ( n = 192 gold particles ) , and NDUFA13-HA ( n = 309 gold particles ) expressing cells .",
"( D ) Mitochondria isolated from stable 293T cell lines expressing C-terminally tagged QIL1 , MIC60 , MIC19 , or MIC25 were lysed in 1% digitonin , subjected to BN-PAGE followed by immunotransfer to nitrocellulose membranes and probing with α-HA antibody .",
"The mature ∼700 kDa MICOS complex is highlighted with an asterisk .",
"MIC60 , MIC19 , and MIC25 were also detected in a sub-complex of ∼500 kDa ( two asterisks ) .",
"( E ) Two-dimensional blue native electrophoresis of 293T mitochondrial lysates .",
"Endogenous QIL1 is present in the mature ∼700 kDa MICOS complex ( asterisk ) .",
"MIC60 , MIC19 , and MIC25 were also present in a smaller sub-complex ( two asterisks ) .",
"( F–G )",
"C-terminally tagged ( F ) or endogenous QIL ( G ) was immunopurified from mitochondria lysed in 1% digitonin or 1% Triton X-100 ( TX100 ) .",
"Immunoblot analysis was performed to detect interaction with other MICOS subunits . DOI: http://dx . doi . org/10 . 7554/eLife . 06265 . 006 The enrichment of QIL1 at CJs and its physical interaction with multiple MICOS subunits strongly supports the notion that QIL1 is a subunit of the MICOS complex .",
"Thus , we asked whether QIL1 is present in the ∼700 kDa MICOS complex found in mitochondria from human cells ( Ott et al . , 2012 ) .",
"To address this question , mitochondria isolated from 293T cells stably expressing C-terminally tagged QIL1 , MIC60 , MIC19 , or MIC25 ( Figure 1—figure supplement 1B ) were lysed in 1% digitonin and subjected to BN-PAGE followed by immunoblot analysis ( Figure 2D ) .",
"QIL1 was found to be predominantly localized at ∼700 kDa ( asterisks ) .",
"Similarly , MIC60 , MIC19 , and MIC25 were found in a ∼700 kDa complex as expected .",
"Additionally , MIC60 , MIC19 , and MIC25 were also present in a smaller complex of ∼500 kDa ( two asterisks ) suggesting the existence of an assembly intermediate or sub-complex containing these three proteins .",
"Using 2-dimensional BN-PAGE electrophoresis , we found that endogenous QIL1 was present within the mature MICOS complex migrating at ∼700 kDa ( Figure 2E ) .",
"Confirming our findings in one-dimensional BN-PAGE immunoblotting , MIC60 , MIC19 , and MIC25 were found in the ∼700 kDa complex ( asterisk ) and in the smaller sub-complex ( two asterisks ) , while MIC10 and QIL1 were primarily found in the ∼700 kDa complex .",
"To address whether QIL1 transiently associates with the dissociated MICOS subunits , we solubilized mitochondria with 1% Triton X-100 , which leads to the dissociation of the MICOS complex into smaller complexes as previously described ( Harner et al . , 2014 ) .",
"While C-terminally tagged QIL1 efficiently co-purified with endogenous MIC60 , MIC19 , MIC27 , and MIC10 when mitochondria were solubilized with 1% digitonin , no specific binding was detected when mitochondria were solubilized with Triton X-100 , consistent with an association of QIL1 with the mature MICOS complex ( Figure 2F ) .",
"These findings were confirmed at the endogenous level recapitulating an interaction between QIL1 and MIC60 , as well as MIC10 , in mitochondria lysed with 1% digitonin but not with 1% Triton X-100 ( Figure 2G ) .",
"We next examined mitochondrial cristae morphology upon RNAi-mediated QIL1 depletion using transmission electron microscopy ( Figure 3A–H ) .",
"In agreement with previously published data , transient depletion of MIC60 as a control ( Figure 3I ) resulted in dramatic changes in cristae organization .",
"In particular , IM structures composed of one to several layers of concentric rings , resembling ‘onion-like’ structures , were found , with no connection to the IBM due to loss of CJs as reported previously ( John et al . , 2005 ) ( Figure 3B ) .",
"Similarly , QIL1 depletion in HeLa ( Figure 3J , C–D ) and HCT116 ( Figure 3K , F–H ) cells resulted in analogous rearrangement of cristae structures .",
"Quantification of electron microscopy images revealed a dramatic increase in the number of mitochondria containing swirls upon MIC60 or QIL1 depletion ( Figure 3L ) . 10 . 7554/eLife . 06265 . 007Figure 3 . QIL1 deletion alters CJs formation , cristae morphology , and mitochondrial respiration . Electron microscopy of HeLa cells transfected with Control siRNA ( A ) , MIC60 siRNA ( B ) , two independent QIL1 siRNAs ( C and D ) , FF2 shRNA ( E ) , or QIL1 shRNAs ( F–H ) .",
"Bars , 100 nm .",
"( I–K )",
"Knock-down levels are shown by immunoblot analysis .",
"( L ) Quantification of the number of mitochondria containing membrane swirls based on electron microscopy images .",
"( M ) Oxygen consumption rate ( pmoles/min ) was measured at baseline conditions , after oligomycin , FCCP , and antimycin A injections as indicated by arrowheads in HeLa cells transfected with control or QIL1 siRNAs . DOI: http://dx . doi . org/10 . 7554/eLife . 06265 . 007 As seen previously with other MICOS subunits ( Darshi et al . , 2011; Weber , 2013 ) , depletion of QIL1 resulted in a substantial reduction in respiration , suggesting an important role for QIL1 in mitochondrial homeostasis through CJ formation ( Figure 3M ) .",
"Our bioinformatics analysis identified an apparent QIL1 ortholog ( CG7603 ) in Drosophila ( Figure 1H ) .",
"Muscle tissue is rich in mitochondria and relies strongly on mitochondrial function .",
"Using the UAS/Gal4 system , and specifically the Dmef2-Dcr-Gal4 driver to express UAS-ControlRNAi or UAS-QIL1RNAi , we achieved significant depletion of QIL1 mRNA in Drosophila third larval instar bodywall muscle ( Figure 4A ) .",
"To address whether QIL1 depletion resulted in the same ultrastructural defects in mitochondrial cristae morphology as those observed in human cell lines , we dissected muscles from crawling third instar larvae and submitted the tissues to transmission electron microscopy analysis ( Figure 4B–C ) .",
"Strikingly , upon QIL1 depletion , we observed a significant increase in the number of abnormal mitochondria , with many mitochondria showing loss of CJs and concentric stacks of IM inside the matrix compartment ( Figure 4C ) .",
"Quantification revealed an ∼10-fold increase in the number of mitochondria containing IM swirls ( Figure 4D ) .",
"Moreover , we observed an increase in mitochondrial fragmentation and sphericity in the Drosophila muscle upon QIL1 depletion as shown by our confocal microscopy analysis ( Figure 4E–J ) .",
"Similarly , silencing of QIL1 specifically in neurons using the Elav-Dcr-Gal4 driver to express UAS-ControlRNAi or UAS-QIL1RNAi ( Figure 4K ) led to loss of CJs and the formation of concentric stacks of IM inside the matrix compartment in neurons at neuromuscular junctions ( Figure 4L–M ) .",
"Quantification of the number of mitochondria containing IM swirls in neurons revealed a significant increase upon QIL1 knockdown ( Figure 4N ) .",
"Together , these results demonstrate that QIL1 loss destabilizes CJs and alters cristae morphology in different cell types in vivo in a cell autonomous fashion . 10 . 7554/eLife . 06265 . 008Figure 4 . Drosophila QIL1 is required for mitochondrial homeostasis and CJ formation in vivo .",
"( A ) DMef2-driven QIL1 knock-down efficiency in three-instar larvae muscles was assessed by qPCR .",
"Ultrastructural analysis of Control ( B ) vs QIL1 RNAi ( C ) third instar larval bodywall muscles in transversal sections .",
"Bars , 100 nm .",
"( D ) The number of mitochondria containing IM swirls was quantified .",
"Immunofluorescence analysis of muscles dissected from Control ( E ) or QIL1 RNAi ( F ) third instar larvae stained with Phalloidin ( red ) and α-ATP5A ( green ) .",
"Bars , 10 µm .",
"( I–J )",
"Mitochondrial length ( I ) and sphericity ( G-H , J ) of each individual mitochondria were measured .",
"( K ) Elav-driven QIL1 knock-down efficiency in three-instar larvae neurons was assessed by qPCR .",
"Ultrastructural analysis of Control ( L ) vs QIL1 RNAi ( M ) third instar larval neuromuscular junctions in longitudinal sections .",
"Bars , 500 nm .",
"( N ) The number of mitochondria containing IM swirls was quantified . DOI: http://dx . doi . org/10 . 7554/eLife . 06265 . 008 To gain insight into the molecular mechanism by which QIL1 causes morphological changes in cristae membrane organization , we coupled SILAC ( stable isotopic labeling with amino acids in culture ) with size-based fractionation of mitochondria purified from cells with or without QIL1 to investigate whether QIL1 is required for MICOS complex formation ( Figure 5A ) .",
"Briefly , SILAC-labeled cells with and without QIL1 depletion were mixed 1:1 , mitochondria isolated , subjected to BN-PAGE , and gel slices across the entire mass range of the BN-PAGE were subjected to LC-MS2 .",
"We analyzed the relative abundance of all subunits of the MICOS complex , with the exception of MIC10 , which was not quantified by mass spectrometry perhaps due to its small size and the unfavorable properties of its peptides following tryptic digestion as discussed previously .",
"Quantitative proteomic analysis revealed a marked reduction of MICOS subunits at ∼700 kDa ( Figure 5A; H:L ratio <1 , green ) , corresponding to the mature heterooligomeric complex ( Harner et al . , 2011; Ott et al . , 2012 ) and concomitant accumulation of MIC19 , MIC25 , and MIC60 in a smaller ∼500 kDa sub-complex and below in response to QIL1 depletion ( Figure 5A; H:L ratio >1 , red ) .",
"Moreover , when we analyzed the estimated relative protein abundance across all fractions , the total protein abundance for MIC26 and MIC27 were reduced upon QIL1 depletion ( Figure 5B ) . 10 . 7554/eLife . 06265 . 009Figure 5 . QIL1 deficiency impairs MICOS assembly .",
"( A ) Light-labeled ( K0 ) shFF2 and heavy ( K8 ) -labeled shQIL1 stable cell lines were mixed at a 1:1 ratio and mitochondria subsequently isolated and lysed with 1% Digitonin .",
"Mitochondrial protein complexes were separated using BN-PAGE and sliced in 20 gel pieces ranging from >1 MDa to <60 kDa .",
"Proteins were subjected to tryptic digestion and analyzed by quantitative mass spectrometry .",
"Heavy to light ( H:L ) ratios were calculated for MICOS components .",
"The ratios were plotted in heatmaps where values <1 are represented in green and values >1 are represented in red .",
"( B ) Ratios from the summed heavy and light intensities for each peptide separately across all BN-PAGE fractions from Figure 4A .",
"( C ) BN-PAGE followed by immunotransfer to nitrocellulose membranes .",
"QIL1 knockdown lead to a decrease in MIC60 , MIC19 , and MIC10 in the ∼700 kDa mature MICOS complex ( asterisk ) and accumulation of MIC60 and MIC19 in a smaller ∼500 kDa sub-complex ( two asterisks ) .",
"( D ) Immunoblot analysis of MICOS subunits .",
"( E ) Densitometry analysis was performed using ImageJ .",
"( F ) qPCR analysis .",
"( G ) Endogenous MIC60 was immunopurified from crude mitochondria isolated from HCT116 cells stably expressing FF2 or QIL1 shRNA .",
"Endogenous interactions with MIC19 , MIC25 , SAMM50 , MIC26 , MIC10 , MIC27 , AFG3L2 , or QIL1 were assessed .",
"( H ) Densitometry analysis was performed using ImageJ . DOI: http://dx . doi . org/10 . 7554/eLife . 06265 . 009 To confirm these findings , we turned to western blot analysis .",
"Mitochondria were isolated from HCT116 cells stably expressing FF2 shRNA or three different shRNAs-targeting QIL1 .",
"Mitochondria were lysed in 1% digitonin and subjected to BN-PAGE followed by immunoblot analysis ( Figure 5C ) .",
"Antibodies against MIC60 , MIC19 , and MIC10 were used to assess MICOS assembly .",
"ATP5A antibody was used as a control , and QIL1 knockdown was confirmed by submitting an input sample to SDS-PAGE .",
"Again , we observed a decrease in the abundance of the ∼700 kDa complex with all subunits tested .",
"Moreover , MIC10 , which was not detected by mass spectrometry , was regulated in a similar fashion as the other MICOS subunits ( Figure 5C ) .",
"Interestingly , there was an accumulation of MIC60 and MIC19 at lower molecular weights ( ∼500 kDa ) , as was also observed in the SILAC experiment for MIC60 , MIC19 , and MIC25 , at ∼500 kDa and below ( Figure 5C ) , suggesting the accumulation of an assembly intermediate containing these proteins in the absence of QIL1 .",
"We analyzed protein levels for several MICOS subunits as well as the OM interactor , SAMM50 , by SDS-PAGE ( Figure 5D ) .",
"This analysis revealed that , while most proteins remained unchanged after QIL1 knockdown , MIC27 , MIC26 , and MIC10 levels were significantly reduced ( Figure 5D–E ) , confirming our quantitative proteomics experiment .",
"Reduced levels of the MIC26 , MIC27 and MIC10 in response to QIL1 depletion were post-transcriptional , as determined by qPCR ( Figure 5F ) .",
"Thus , QIL1 appears to be important for maintaining the levels of several MICOS complex subunits .",
"To further strengthen our conclusions regarding MICOS complex formation , we immunopurified endogenous MIC60 from HCT116 cells stably expressing FF2 control or QIL1 shRNAs ( Figure 5G ) .",
"While MIC60 efficiently bound to MIC19 and MIC25 in QIL1-depleted mitochondria , the abundance of MIC10 , MIC26 , and MIC27 was significantly reduced in these immune complexes , confirming the existence of a stable sub-complex containing MIC60 , MIC19 , and MIC25 that lacks MIC10 , MIC26 , and MIC27 in QIL1-depleted cells ( Figure 5G , H ) .",
"Interestingly , MIC60 retained its ability to interact with the OM protein SAMM50 ( Figure 5G , H ) , indicating that the MIC60 sub-complex could act in association with the OM independently of MIC10 , MIC26 , and MIC27 .",
"Importantly , ectopic QIL1 expression rescued cristae morphology defects ( Figure 6A–E ) , MICOS disassembly ( Figure 6F ) and downregulation of MIC10 and MIC27 protein levels ( Figure 6G ) that resulted from silencing QIL1 with an shRNA targeting the 3′ untranslated region .",
"Collectively , these data attest to the specificity of the observed loss of function phenotype . 10 . 7554/eLife . 06265 . 010Figure 6 . Cristae morphology defects , MICOS disassembly and MIC10 and MIC26 degradation can be rescued by QIL1 overexpression . Electron microscopy of HCT116 cells transfected with FF2 shRNA and empty vector ( A ) FF2 shRNA and C-terminally tagged QIL1 ( B ) , a QIL1 shRNA targeting the 3′ untranslated region and empty vector ( C ) or QIL1 shRNA and C-terminally tagged QIL1 ( D ) .",
"Bars , 100 nm .",
"( E ) Quantification of the number of mitochondria containing membrane swirls based on electron microscopy images .",
"( F ) BN-PAGE followed by immunotransfer to nitrocellulose membranes .",
"Overexpression of C-terminally tagged QIL1 in cells expressing a QIL1 shRNA targeting the 3′ untranslated region restored the levels of MIC10 , MIC19 , and MIC60 in the mature ∼700 kDa MICOS complex and reversed the accumulation of MIC19 and MIC60 in the ∼500 kDa assembly intermediate sub-complex ( two asterisks ) .",
"It also restored MIC10 and MIC26 protein levels as observed in immunoblot analysis of total cell lysates ( G ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 06265 . 010 Our quantitative proteomics and BN-PAGE data suggested that QIL1 depletion resulted in a reduction in the abundance of the ∼700 kDa mature MICOS complex , as revealed by examining core subunits .",
"Moreover , we observed a concomitant accumulation of MIC60 , MIC19 , and MIC25 in a smaller sub-complex of ∼500 kDa .",
"These data agree with the presence of both overexpressed and endogenous MIC60 , MIC19 , and MIC25 in this sub-complex ( Figure 2D–E ) .",
"We hypothesized that QIL1 promotes the maturation of the MICOS complex by acting as a scaffold at the IM , bridging a sub-complex containing MIC60 , MIC19 , and MIC25 to other subunits including MIC10 , MIC26 , and MIC27 .",
"We hypothesized that when QIL1 is depleted , MIC10 , MIC26 , and MIC27 fail to integrate into the complex and are degraded .",
"To test this , we asked whether overexpressed MIC10 could physically interact with other MICOS subunits and be incorporated into the mature MICOS complex in cells where QIL1 had been silenced .",
"Thus , we transiently expressed C-terminally tagged MIC10 in cells expressing FF2 shRNA or a shRNA-targeting QIL1 and assessed the presence of the overexpressed protein in the mature MICOS complex at ∼700 kDa by BN-PAGE followed by immunoblotting ( Figure 7A ) .",
"C-terminally tagged MIC10 was more efficiently assembled into the MICOS complex in control cells compared to those lacking QIL1 even though MIC10 was expressed at similar levels ( Figure 7A–B ) and was properly imported into mitochondria ( Figure 7B–C ) in both , control and QIL1-depleted cell lines .",
"In agreement with these findings , MIC10 binding to MIC60 , MIC19 , and SAMM50 was significantly reduced in cells expressing QIL1 shRNA ( Figure 7D–E ) . 10 . 7554/eLife . 06265 . 011Figure 7 . QIL1 is required for the binding of MIC10 to the MICOS complex .",
"( A ) BN-PAGE followed by immunotransfer to nitrocellulose membranes .",
"Incorporation of C-terminally tagged MIC10 into the mature ∼700 kDa MICOS complex ( asterisk ) was decreased in cells expressing QIL1 shRNA compared to those expressing FF2 shRNA .",
"( B ) Cytoplasmic and mitochondrial fractions were separated in lysates obtained from HCT116 cells stably expressing FF2 or QIL1 shRNA and transiently expressing empty vector or C-terminally tagged MIC10 .",
"( C ) Immunofluorescence analysis of the subcellular localization of C-terminally tagged MIC10 .",
"Bars , 20 µm .",
"( D ) C-terminally tagged MIC10 was transiently expressed in HCT116 cell lines expressing FF2 shRNA or QIL1 shRNA and immunopurified from mitochondrial lysates .",
"Immunoblot analysis was performed to detect interaction with other MICOS subunits .",
"( E ) Densitometry analysis was performed using ImageJ .",
"Figure 7—figure supplement 1 shows the analysis of cardiolipin content and species distribution by LC-MS/MS in mitochondria obtained from cells stably expressing FF2 or 3 different shRNAs targeting QIL1 . DOI: http://dx . doi . org/10 . 7554/eLife . 06265 . 01110 . 7554/eLife . 06265 . 012Figure 7—figure supplement 1 . Analysis of cardiolipin content and species distribution by LC-MS/MS in mitochondria obtained from cells stably expressing FF2 shRNA or 3 different shRNAs targeting QIL1 . ( A–B ) Typical spectra of cardiolipin ( CL ) obtained from mitochondria isolated from HCT116 cells expressing control ( A ) or QIL1 shRNA ( B ) .",
"( C ) Bar graph showing the quantification of cardiolipin ( CL ) content in pmol/mg protein by LC-MS/MS .",
"( D ) Acyl chain distribution of cardiolipin .",
"First numbers represent the sum of the carbon atoms in the four acyl chains; the second numbers indicate the total number of double bonds . DOI: http://dx . doi . org/10 . 7554/eLife . 06265 . 012 Interestingly , we found that MIC10 overexpression promoted an increase in the incorporation of MIC60 and MIC19 in the mature MICOS complex ( ∼700 kDa , asterisk ) and a decrease in the smaller sub-complex ( ∼500 kDa , two asterisks ) in the presence of QIL1 , consistent with an increase in MICOS assembly ( Figure 7A ) .",
"Altered levels of cardiolipin have been shown to promote severe cristae morphological defects that resemble those caused by loss of MICOS ( Xu et al . , 2006; Acehan et al . , 2007 ) .",
"Thus , we investigated whether QIL1 knockdown had an effect on cardiolipin content by mass spectrometry ( Figure 7—figure supplement 1 ) .",
"Our results showed no apparent differences in cardiolipin content or species distribution upon QIL1 knockdown , using three different shRNAs .",
"These results exclude the possibility that the effect of QIL1 on MICOS is an indirect mechanism through regulation of cardiolipin levels and reinforces a model in which QIL1 is a structural component of MICOS required for its assembly ."
],
[
"In this work , we report a systematic proteomic analysis of the MICOS complex and identify QIL1 as a new subunit required for CJ formation , MICOS complex maturation , and mitochondrial homeostasis .",
"QIL1 associates with the core IM MICOS complex , the cardiolipin binding subunits ( MIC26 and MIC27 ) , as well as the SAMM50-MTX1-MTX2 OM complex ( Figure 1C ) .",
"Moreover , QIL1 is enriched at CJs to an extent similar to that of the MICOS subunit MIC25 and is present in a ∼700 kDa complex , as observed by BN-PAGE ( Figure 2 ) .",
"Thus , QIL1 may be a central component of CJs and the machinery that connects inner and outer membranes ( Figure 8 ) .",
"Indeed , depletion of QIL1 leads to MICOS-like defects in cristae morphology , with loss of CJs and formation of swirls of cristae membranes inside mitochondria ( Figure 3A–H ) .",
"QIL1 is conserved in Drosophila where its depletion also causes mitochondrial phenotypes ( Figure 4 ) .",
"Moreover , QIL1 knockdown resulted in MICOS disassembly , accumulation of a MIC60-MIC19-MIC25 sub-complex and loss of MIC10 , MIC26 , and MIC27 from the MICOS complex , as determined by quantitative proteomics and immunoblot analysis ( Figure 5A–D ) . 10 . 7554/eLife . 06265 . 013Figure 8 . QIL1 is a novel MICOS subunit required for MICOS assembly . Model for QIL1 incorporation into the MICOS complex and the effect of loss of QIL1 on the composition of MICOS subunits .",
"We propose that MIC10 , MIC26 , and MIC27 fail to assemble into a stable MICOS sub-complex containing MIC60 , MIC19 , and MIC25 and are degraded when QIL1 is depleted leading to MICOS disassembly , loss of CJs and cristae morphology defects . DOI: http://dx . doi . org/10 . 7554/eLife . 06265 . 013 Previous studies indicate that loss of MIC10 , MIC26 , and MIC27 resulted in cristae remodeling analogous to that found upon QIL1 depletion ( Harner et al . , 2011; Head et al . , 2011; Alkhaja et al . , 2012; Weber , 2013 ) .",
"The molecular mechanism through which these subunits regulate CJs stability is yet to be determined , but it is possible that the phenotype observed in cells after QIL1 depletion is a result of changes in MIC10 , MIC26 , or MIC27 protein levels .",
"The physical properties of cardiolipins are particularly important at areas of the IM with high membrane curvature , such as CJs and at the tip of the cristae ( Ortiz et al . , 1999 ) .",
"In fact , cardiolipins have been shown to be enriched at those regions .",
"In Barth syndrome patients , the levels of the enzyme Tafazzin , required for the remodeling of acyl chains within cardiolipins , are decreased , resulting in reduced cardiolipin levels .",
"This leads to major alterations of mitochondrial membranes morphology and curvature , with the appearance of collapsed cristae packaged in multiple concentric layers ( Acehan et al . , 2007 ) .",
"Interestingly , Tafazzin mutations in flies generated a Barth syndrome-related phenotype characterized by decreased cardiolipin levels , mitochondria showing cristae membrane swirls and motor defects ( Xu et al . , 2006 ) .",
"Tafazzin has been shown to be regulated by the MICOS interactor Aim24 in yeast ( Harner et al . , 2014 ) and MIC27 in mammals ( Weber , 2013 ) has been shown to bind cardiolipin .",
"Due to its ability to bind cardiolipin , the presence of MIC27 at CJs is particularly important ( Weber , 2013 ) .",
"Thus , alterations in mitochondrial cardiolipin levels after QIL1 depletion could potentially offer an alternative , indirect explanation for the observed phenotypes .",
"To test the possibility that QIL1 regulates mitochondrial cardiolipin levels , we measured cardiolipin content and species distribution in QIL1-depleted cells compared to control cells expressing FF2 shRNA .",
"We observed that the cardiolipin profiles of cells expressing FF2 or three different QIL1 shRNAs were similar ( Figure 7—figure supplement 1 ) , excluding an indirect effect of QIL1 through regulation of cardiolipin levels .",
"This observation is in agreement with data published previously , showing that cardiolipin composition was not substantially changed by the lack of the central MICOS subunits MIC60 or MIC10 ( Bohnert et al . , 2012 ) .",
"These data support a more direct structural role for QIL1 in MICOS stability .",
"Our results demonstrate that MIC60 forms a stable MICOS sub-complex with MIC19 and MIC25 ( Figure2D–E ) and retains the ability to interact with the OM component SAMM50 , independently of MIC10 , MIC26 , and MIC27 ( Figure 5G , H ) .",
"These data suggest that MIC60-MIC19-MIC25 could perform other functions in association with the OM , independently of MIC10 , MIC26 , and MIC27 .",
"This is not surprising given that it has been shown previously that a fraction of MIC60 molecules , but not MIC10 , functions in biogenesis of β-barrel proteins of the OM ( Bohnert et al . , 2012 ) independently of other subunits .",
"Restoration of MIC10 levels by overexpression in cells depleted for QIL1 failed to significantly rescue its interaction with other MICOS subunits ( Figure 7D–E ) and incorporation into the ∼700 kDa complex ( Figure 7A ) as compared to wild-type cells even though MIC10 was efficiently imported into mitochondria and expressed at similar levels in both conditions ( Figure 7B–C ) .",
"This indicates that QIL1 is required for the binding of MIC10 to the MIC60-MIC19-MIC25 sub-complex .",
"Thus , we propose a stratified model of MICOS assembly , where the stable MIC60-MIC19-MIC25 sub-complex at the IM binds to MIC10 , MIC26 , and MIC27 to generate a mature MICOS complex in a step facilitated by QIL1 ( Figure 8 ) .",
"Further studies are required to fully understand the regulation of CJ assembly and maintenance .",
"Interestingly , in flies exposed to hyperoxia to induce oxygen stress , the cristae lose their orderly structure , generating a swirl within the muscle mitochondrion and the IM forms concentric stacks inside the matrix ( Walker and Benzer , 2004 ) , reminiscent of the phenotype observed after MICOS depletion .",
"Intriguingly , the number of mitochondria containing swirls increases slowly with aging ( Walker and Benzer , 2004 ) .",
"In contrast , giant mitochondria with honeycomb-like cristae were observed in animals exposed to hypoxia ( Lorente et al . , 2002; Perkins and Hsiao , 2012 ) .",
"These findings demonstrate the importance of cristae remodeling as a mechanism of adaptation to different metabolic and pathological states in vivo .",
"It still remains to be addressed whether regulation of the MICOS complex takes place in these scenarios .",
"The MICOS protein interaction network described here provides a resource for additional studies into the regulation of CJ assembly .",
"Indeed , TMEM11 appears to be a novel interactor of the MICOS complex .",
"We identified TMEM11 in association with MIC19 , MIC27 , MIC60 , and MTX2 , and endogenous MIC60 associated with endogenous TMEM11 by co-immunoprecipitation ( Figure 1B , F ) .",
"TMEM11 contains 2 transmembrane domains .",
"In Drosophila , TMEM11 was shown to localize to the IM and regulate cristae morphology , length , and biogenesis ( Rival et al . , 2011; Macchi et al . , 2013 ) , but the mechanism through which it was involved remained unknown .",
"Our results suggest that it contributes to the MICOS complex .",
"Additional novel interacting proteins identified here ( Figure 1B ) may also be involved in MICOS function .",
"Taken together , our work provides a comprehensive overview of the MICOS complex interactome and defines a role for the novel IM component QIL1 in MICOS assembly ."
],
[
"HCT116 , HeLa , HEK293T cells were grown in Dulbecco's modified Eagle's medium ( DMEM ) supplemented with 10% fetal calf serum and maintained in a 5% CO2 incubator at 37°C .",
"Drosophila stocks were maintained at room temperature on a standard cornmeal agar diet .",
"UAS-ControlRNAi; UAS-QIL1RNAi , and mef2-GAL4 lines were obtained from the Bloomington stock center .",
"QIL1 knockdown was achieved by crossing virgin mef2-GAL4 females with UAS-QIL1RNAi males .",
"As a control virgin mef2-GAL4 females were crossed with UAS-ControlRNAi males .",
"All experiments were conducted at 25°C .",
"For plasmid transfection , Lipofectamine 2000 ( Invitrogen , Carlsbad , USA ) was used for transfection of HeLa cells and TransIT-293 ( Mirus , Madison , USA ) or Lipofectamine 2000 ( Invitrogen ) was used for transfection of HEK293T cells according to manufacturer's specifications .",
"Transfected cells were harvested ∼48 hr post-transfection for further analysis .",
"Viral particles were generated in HEK293T cells through the transfection of a pHAGE lentiviral vector ( Murphy et al . , 2006 ) , containing the gene of interest with a C-terminal HA-Flag tag and four helper vectors ( VSVG , Tat1b , Mgpm2 , and CMV-Rev ) ( Wilson , 2008 ) .",
"Virus-containing supernatants were used to infect HeLa , HCT116 , or HEK293T cells .",
"Cells stably expressing the tagged proteins were selected with Puromycin ( Invitrogen ) for at least one week .",
"For the generation of stable knock-down cell lines , we used miR-30-based shRNA constructs and VSVG and gag-pol helper vectors .",
"Cells were selected for at least one week in Puromycin .",
"For siRNA transfection , Lipofectamine RNAiMAX was used to transfect 20 nM of indicated siRNA into indicated cell lines .",
"Transfected cells were analyzed ∼48 hr after transfection .",
"The siRNA and shRNA sequences used in the study were: QIL1 siRNA_1: 5′-CCAAGGAGGGCUGGGAGUA-3′; QIL1 siRNA_2: 5′-GAUGUCAGCUCUGUCGGUG-3′; QIL1 shRNA_1: GCAGGGCTGCCACTGACCTGAA; QIL1 shRNA_2: CCGGCCTTGCCGGCCCAATAAA; QIL1 shRNA_3: GGCCCAATAAAGGACTTCAGAA .",
"Cells were lysed in lysis buffer ( 50 mM Tris–HCl [pH 7 . 5] , 150 mM NaCl , 1% Digitonin [Calbiochem , Billerica , USA] , and supplemented with protease inhibitors [Roche , Basel , Switzerland] ) for 30 min on ice to obtain whole cell extracts .",
"Lysates were cleared by centrifugation at 4°C for 15 min at maximum speed .",
"Approximately 0 . 5 mg of mitochondrial lysate was used for endogenous IP .",
"Lysates were incubated with 1 μg of the indicated antibody or control IgG overnight at 4°C .",
"Protein A resin ( 10 μl ) was then added to the IP reaction and incubated further for 2 hr at 4°C .",
"Beads were washed 3 times with lysis buffer .",
"After washing , 2× SDS loading buffer was added and the samples were boiled for 5 min .",
"Samples were separated on a SDS-PAGE gel prior to immunoblot analysis .",
"For proteins with similar molecular weights ( e . g . , QIL1 and MIC10 ) , separate gels were run for immunoblotting .",
"For IPs of ectopically expressed proteins , agarose beads conjugated with HA or Flag antibodies ( Sigma , St . Louis , USA ) were used .",
"IP-MS and CompPASS analysis were performed as described previously ( Sowa et al . , 2009; Behrends et al . , 2010 ) .",
"Briefly , cells ( 107 ) were lysed for IP-MS using α-HA beads or α-Flag .",
"After washing , proteins were eluted with HA or Flag peptide , subjected to trichloroacetic acid precipitation , and trypsinized prior to passage through StageTips .",
"Samples were ran in technical duplicate on either an Thermo LTQ mass spectrometer or an LTQ-Orbitrap Elite , and spectra search with Sequest prior to target-decoy peptide filtering , and linear discriminant analysis ( Huttlin et al . , 2010 ) .",
"Protein Assembler was used to convert spectral counts to average peptide spectral matches ( APSMs ) , which takes into account peptides , which match more than one protein in the database .",
"Peptides were identified with a false discovery rate of <1 . 0% , and the protein false discovery rate was <2 . 0% ( Figure 1—figure supplement 2 ) .",
"Peptide data ( APSMs ) were uploaded into the CompPASS algorithm housed within the CORE environment .",
"For CompPASS analysis of HCT116 cells , we employed a stats table of 214 unrelated bait proteins analyzed in an analogous manner .",
"For analysis of 293T cells , a database of 48 baits was used .",
"The CompPASS system identifies HCIPs based on the WDN-score , which incorporates the frequency with which they identified within the stats table , the abundance ( APSMs ) when found , and the reproducibility of identification in technical replicates , and also determines a z-score based on APSMs ( Sowa et al . , 2009; Behrends et al . , 2010 ) .",
"Proteins with WDN-scores >1 . 0 are considered HCIPs , although we also note that some proteins that may be bona fide-interacting proteins may not reach the strict threshold set by a WDN-score of >1 . 0 ( Figure 1—figure supplement 2 ) .",
"Candidate proteins not known to be localized to mitochondria based on Mitocarta ( Pagliarini et al . , 2008 ) were omitted from the interaction maps .",
"Antibodies used in this work include: α-QIL1 ( Sigma SAB1102836 ) , α-MINOS1 ( Aviva , San Diego , USA , ARP44801-P050 ) , α-CHCHD3 ( Aviva ARP57040-P050 ) , α-CHCHD6 ( Proteintech , Chicago , USA , 20639-1-AP ) , α-IMMT ( Abcam , Cambridge , UK , ab110329 ) , α-TIMM23 ( BD-Biosciences , Franklin Lakes , USA , 611222 ) , α-SAMM50 ( Proteintech 20824-1-AP ) , α-APOOL ( Aviva OAAF03292 ) , α-PCNA ( Santa Cruz , Dallas , USA , sc-56 ) , α-HSP90 ( Epitomics , Burlingame , USA , 3363-1 ) , α-TOMM70A ( Epitomics T1677 ) , α-Flag ( Sigma SLB6631 ) , α-HA ( Covance , Princeton , USA , D13CF00834 ) , α-ATP5A ( Abcam ab14748 ) , α-DLD ( Santa Cruz sc-271569 ) , α-TMEM11 ( Proteintech 16564-1-AP ) , α-Cytochrome C ( Cell Signaling , Danvers , USA , 4272S ) , α-TOMM20 ( Santa Cruz sc-11415 ) , Phalloidin ( Invitrogen A22287 ) , and α-APOO ( Novus Bio , Littleton , USA , NBP1-28870 ) .",
"Mitochondria were isolated by differential centrifugation .",
"Cells were resuspended in mito-isolation buffer ( 250 mM sucrose , 1 mM EDTA , 10 mM MOPS-KOH , pH 7 . 2 ) and ruptured with 30 s sonication in the lowest setting .",
"The homogenized cellular extract was then centrifuged at 600×g to obtain a post-nuclear supernatant .",
"Mitochondria were pelleted by centrifugation at 8 , 000×g for 10 min and washed twice in isolation buffer .",
"HCT116 stable cell lines expressing FF2 shRNA or QIL1 shRNA were grown in light ( K0 ) or heavy media ( K8 ) and an equal number of cells mixed , prior to purification of mitochondria .",
"Mitochondria were lysed with 1% Digitonin and protein complexes fractionated by blue native-polyacrylamide gel electrophoresis ( BN-PAGE ) .",
"Gel bands were excised and proteins subjected to reduction , alkylation , and trypsinization .",
"Tryptic peptides were analyzed using a Q Exactive mass spectrometer ( Thermo Fisher Scientific , San Jose , USA ) coupled with a Famos Autosampler ( LC Packings ) and an Accela600 liquid chromatography ( LC ) pump ( Thermo Fisher Scientific ) .",
"Peptides were separated on a 100-μm inner diameter microcapillary column packed with ∼0 . 25 cm of Magic C4 resin ( 5 μm , 100 Å , Michrom Bioresources , Billerica , USA ) followed by ∼18 cm of Accucore C18 resin ( 2 . 6 μm , 150 Å , Thermo Fisher Scientific ) .",
"For each analysis , we loaded ∼1 μg onto the column .",
"Peptides were separated using a 90 gradient of 5–28% acetonitrile in 0 . 125% formic acid with a flow rate of ∼300 nl/min .",
"The scan sequence began with an Orbitrap MS1 spectrum with the following parameters: resolution 70 , 000 , scan range 300–1500 Th , automatic gain control ( AGC ) target 1 × 106 , maximum injection time 250 ms , and centroid spectrum data type .",
"We selected the top 20 precursors for MS2 analysis which consisted of higher energy collision dissociation ( HCD ) with the following parameters: resolution 17 , 500 , AGC 1 × 105 , maximum injection time 60 ms , isolation window 2 Th , normalized collision energy 25 , and centroid spectrum data type .",
"The underfill ratio was set at 9% , which corresponds to a 1 . 5 × 105 intensity threshold .",
"In addition , unassigned and singly charged species were excluded from MS2 analysis and dynamic exclusion was set to automatic .",
"Peptides were identified and quantified using MaxQuant software ( Cox and Mann , 2008 ) .",
"Total RNA was obtained using NucleoSpin RNA II ( Macherey–Nagel , Germany ) RNA Kit according to manufacturer's protocol .",
"The extracted RNA was then used for reverse transcription using High-Capacity cDNA Reverse Transcription Kit ( Applied Biosystems , Waltham , USA ) .",
"The cDNA obtained was used for qPCR with gene-specific primers and SYBR-green for detection on a LightCycler 480 system ( Roche ) .",
"Primers specific to TUBB were used for normalization .",
"Primer sequences are as follows: TUBB_F: CTGGACCGCATCTCTGTGTA; TUBB_R: CCCAGGTTCTAGATCCACCA; QIL1_F: GCCAGTACGTGTGTCAGCAG; QIL1_R: GGAGTCACGGATGGGAAAGT; MINOS1_F: CGGATGCGGTCGTGAAGATA; MINOS1_R: ATCCCATGCCAGAACCGAAG; APOO_F: GCTGGCCTTATTGGACTCCT; APOO_R: ACACGATGGCTTGTTGTGGA; APOOL_F: CACCACCGCTCCAGTCTAAA; APOOL_R: CAGTGCGGATGGAAGCAAAG; ACT5C_F: TACTCTTTCACCACCACCGC; ACT5C_R: GGCCATCTCCTGCTCAAAGT; and CG7603_F: CGCAACCGTCTACTACACACA; CG7603_R: CGTTGTACAGCTTGTCCGTC .",
"Mitochondria were purified as described above and lysed in 1% Digitonin prior to separation by 4–16% BN-PAGE as previously described ( McKenzie et al . , 2006 ) .",
"Proteins were transferred to PVDF membrane , and proteins were detected using the indicated antibodies .",
"Oxygen consumption rate ( OCR ) was measured using an XF24 extracellular analyzer ( Seahorse Bioscience , North Billerica , USA ) .",
"HeLa cells transiently transfected with control or QIL1 siRNAs were seeded in 24-well assay plates ( BD Bioscience ) at a concentration of 20 , 000 cells per well .",
"After 24 hr , cells were loaded into the instrument for O2 concentration determinations .",
"Cells were sequentially exposed to oligomycin ( 1 μM ) , carbonylcyanide p-trifluoromethoxyphenylhydrazone ( FCCP; 150 nM ) , and Antimycin A ( 10 µM ) .",
"After each injection , OCR was measured for 3 min , the medium was mixed and again measured for 3 min twice .",
"HeLA cells transfected with control siRNA or 2 different siRNAs targeting QIL1 and HCT116 stable cell lines expressing FF2 shRNA or 3 independent shRNAs targeting QIL1 were grown to 60% confluency in 6-cm culture dishes and fixed with 1 . 25% paraformaldehyde , 2 . 5% glutaraldehyde , 0 . 03% picric acid followed by osmication and uranyl acetate staining , dehydration in alcohols and embedded in Taab 812 Resin ( Marivac Ltd , Nova Scotia , Canada ) .",
"Sections were cut with Leica ultracut microtome , picked up on formvar/carbon-coated copper slot grids , stained with 0 . 2% Lead Citrate , and imaged under the Phillips Tecnai BioTwin Spirit transmission electron microscope .",
"For the in vivo analysis , Drosophila muscle tissues from third instar larvae were dissected in Ca2+ free buffer , fixed overnight at 4°C and processed as described above .",
"Cells were fixed in 4% paraformaldehyde and 0 . 1% glutaraldehyde in 0 . 1 M Sodium Phosphate buffer , pH 7 . 4 for 2 hr at room temperature .",
"The cells were subsequently washed in PBS , infiltrated with 2 . 3 M sucrose in PBS containing 0 . 2 M glycine .",
"Frozen samples were sectioned at −120°C , and the sections were transferred to formvar carbon-coated copper grids .",
"Immunogold labeling was subsequently carried out at room temperature .",
"Both α-HA antibody and protein A gold were diluted in 1% BSA in PBS .",
"The diluted antibody solution was centrifuged 1 min at 14 , 000 rpm prior to labeling to avoid possible aggregates .",
"Grids were floated on drops of 1% BSA for 10 min to block for unspecific labeling , transferred to 5 µl drops of primary antibody and incubated for 30 min .",
"The grids were then washed in 4 drops of PBS for a total of 15 min , transferred to 5 µl drops of Protein-A gold for 20 min , washed in 4 drops of PBS for 15 min and 6 drops of double distilled water .",
"Contrasting/embedding of the labeled grids was carried out on ice in 0 . 3% uranyl acetete in 2% methyl cellulose for 10 min .",
"The grids were examined in a JEOL 1200EX Trans or a TecnaiG² Spirit BioTWIN mission electron microscope and images were recorded with an AMT 2k CCD camera .",
"Mitochondria were isolated and subjected to alkaline extraction in freshly prepared 0 . 1 M Na2CO3 ( pH 11 , 11 . 5 , or 12 ) .",
"Membranes were pelleted at 100 , 000×g for 30 min at 4°C , and supernatants were precipitated with the addition of 1/5 volume of 72% trichloroacetic acid and washed 4 times with cold acetone .",
"After treatments , soluble ( S , supernatant ) and insoluble ( P , pellet ) fractions were subjected to SDS-PAGE and western blotting analysis using antibodies against TOMM70A , TIMM23 , Cytochrome C , DLD , CHCHD3 , and QIL1 .",
"Isolated mitochondria from HCT116 cells were subjected to proteinase K ( 50 µg/ml ) proteolysis to digest exposed proteins .",
"Osmotic shock ( 25 mM sucrose , 10 mM MOPS-KOH , pH 7 . 2 ) was used to disrupt the outer mitochondrial membrane .",
"After treatments as indicated , Proteinase K activity was blocked with PMSF ( 2 mM ) and fractions were subjected to SDS-PAGE and western blotting analysis using antibodies against TOMM70A , TIMM23 , DLD , Cytochrome C , and QIL1 .",
"Cells grown on 15-mm glass coverslips or 384-well clear bottom plates were fixed with PBS , 4% PFA , and 4% Sucrose for 10 min , permeabilized with PBS 0 . 1% Triton X-100 for 3 min , blocked with PBS 1% BSA for 30 min , and incubated with α-TOMM20 ( 1:100; Santa Cruz ) and α-HA ( 1:200; Covance ) in PBS 1% BSA for one hour at room temperature .",
"After extensive washing , fixed cells were incubated with Alexa488-chicken anti-rabbit and Alexa594-goat anti-mouse ( 1:10000 ) secondary antibodies for 1 hr at room temperature .",
"For the in vivo analysis , Drosophila muscle tissue was dissected in PBS , fixed for 30 min in 4% paraformaldehyde , washed , permeabilized for 2 hr in 0 . 5% Triton X-100 in PBS , and blocked with normal goat serum in 0 . 1% Tween in PBS ( PBST ) .",
"α-ATP5A primary antibody ( 1:300; Abcam ) was diluted in blocking buffer and incubated overnight at 4°C .",
"After extensive washing with PBST , tissues were incubated with the secondary antibody ( Alexa488-chicken anti-rabbit , 1:10000 ) and Phalloidin ( 1:10000 , Invitrogen ) for 2 hr .",
"Tissues were washed and mounted in SlowFade Gold antifade reagent ( Invitrogen ) .",
"Images were acquired with a Nikon confocal microscope with a 100× oil objective .",
"We used the Imaris software to create 3D reconstructions of Drosophila muscle mitochondria from z-stack images .",
"Mitochondrial length and sphericity were determined using the Filament and Surface applications of the software .",
"Statistical significance was calculated using the paired t-test analysis; p-values <0 . 05 were considered significant .",
"Asterisks represent p values <0 . 05 .",
"Error bars ( ± s . e . m ) show the mean of 3 or 4 biological replicates .",
"Cardiolipin was extracted from mitochondrial extracts according to 50 μg of protein by chloroform/methanol ( 2:1 ) extraction .",
"Dried lipid extract from cholroform/methanol extractions was dissolved in choloroform/methanol ( 2:1 ) and injected into the HPLC for analysis .",
"Lipid extracts were separated on an Accucore C18 column ( 2 . 1 × 150 mm , 2 . 6 µm; Thermo ) connected to an Ultimate 3000 HPLC ( Thermo ) .",
"A binary solvent system was used ( mobile phase A: 50:50 ACN/H20 , 10 mM NH4HCO2 , 0 . 1% HCO2H; mobile phase B: 10:88:2 AcN/IPA/H2O , 2 mM NH4HCO2 , 0 . 02% HCO2H ) in a 28 min gradient at a flow rate of 400 μl/min with the column temperature kept constant at 35°C .",
"The HPLC was connected on-line to a Q-exactive mass spectrometer equipped with an electrospray ionization source ( ESI; Thermo ) .",
"Mass spectra were acquired in a data-dependent mode to automatically switch between full scan MS and up to 15 data-dependent MS/MS scans .",
"The maximum injection time for full scans was 75 ms , with a target value of 1 , 000 , 000 at a resolution of 70 , 000 at m/z 200 and a mass range of 150–1800 m/z in negative mode .",
"The 15 most intense ions from the survey scan were selected and fragmented with HCD with stepped normalized collision energies of 30 , 60 , and 100 .",
"Target values for MS/MS were set at 100 , 000 with a maximum injection time of 120 ms at a resolution of 17 , 500 at m/z 200 .",
"To avoid repetitive sequencing , the dynamic exclusion of sequenced peptides was set at 10 s .",
"Peaks were analyzed using the Lipid Search algorithm ( MKI , Tokyo , Japan ) .",
"Peaks were defined through raw files , product ion and precursor ion accurate masses .",
"Candidate molecular species were identified by database ( >1 , 000 , 000 entries ) search of negative ion adducts .",
"Mass tolerance was set to 5 ppm for the precursor mass .",
"Samples were aligned within a time window and results combined in a single report .",
"An internal standard for cardiolipin ( CL 14:1/14:1/14:1/15:1 ) spiked in prior to extraction was used for normalization and calculation of the amounts of lipids in pmol/mg protein ."
]
] | [
"The mitochondrial contact site and cristae junction ( CJ ) organizing system ( MICOS ) dynamically regulate mitochondrial membrane architecture .",
"Through systematic proteomic analysis of human MICOS , we identified QIL1 ( C19orf70 ) as a novel conserved MICOS subunit .",
"QIL1 depletion disrupted CJ structure in cultured human cells and in Drosophila muscle and neuronal cells in vivo .",
"In human cells , mitochondrial disruption correlated with impaired respiration .",
"Moreover , increased mitochondrial fragmentation was observed upon QIL1 depletion in flies .",
"Using quantitative proteomics , we show that loss of QIL1 resulted in MICOS disassembly with the accumulation of a MIC60-MIC19-MIC25 sub-complex and degradation of MIC10 , MIC26 , and MIC27 .",
"Additionally , we demonstrated that in QIL1-depleted cells , overexpressed MIC10 fails to significantly restore its interaction with other MICOS subunits and SAMM50 .",
"Collectively , our work uncovers a previously unrecognized subunit of the MICOS complex , necessary for CJ integrity , cristae morphology , and mitochondrial function and provides a resource for further analysis of MICOS architecture ."
] | [
"Mitochondria are the cell's power plants , and churn out molecules that provide a portable energy source throughout the cell .",
"To do this efficiently , the mitochondria have a double membrane .",
"The inner membrane is ruffled , which provides a large surface area for energy-producing reactions to occur on .",
"Structures called cristae junctions and contact sites hold the folds of the inner membrane in place .",
"As mitochondria are found in every cell in the body , mitochondrial diseases can produce a wide range of symptoms , but they commonly affect the muscles .",
"In some forms of these diseases , the inner membrane of a mitochondrion is no longer folded; instead , the membrane may form concentric rings like the layers of an onion .",
"Knowing how the folding of the inner membrane is regulated may therefore help scientists to better understand mitochondrial diseases .",
"Scientists already know that several proteins join together to form a complex that anchors the mitochondrion's inner membrane to its outer membrane at cristae junctions .",
"To learn more about the proteins involved in these complexes , Guarani et al . systematically screened for proteins that associate with cristae junctions and found a previously unknown protein called QIL1 .",
"Next , Guarani et al . conducted a series of experiments to determine what role QIL1 plays at the cristae junctions .",
"The experiments showed that QIL1 is needed to bind a protein called MIC10 into the protein complex that anchors the cristae junctions to the outer membrane .",
"In human and fruit fly cells without QIL1 , this protein complex falls apart and is not repaired if extra MIC10 is added into the cells .",
"Furthermore , in human cells lacking QIL1 , the inner mitochondrial membrane forms the same onion-like rings seen in the cells of humans with mitochondrial diseases .",
"Future studies are necessary to understand how the structure of the QIL1 complex is organized and to work out how the complex is capable of causing the mitochondrial inner membrane to curve ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Munc18-1 is a dynamically regulated PKC target during short-term enhancement of transmitter release | elife-01715-v1 | [
[
"Vesicle fusion and transmitter release at synapses is a fundamentally important signaling mechanism that guarantees fast information transfer between neurons ( Südhof , 2004 ) .",
"Interestingly , transmitter release is not static , but the amount of quanta released with each presynaptic action potential ( AP ) can vary dynamically , depending on the recent history of presynaptic activity .",
"During high-frequency activity , short-term depression leads to a decrease of release in many synapses , but following recovery from depression , transmitter release can overshoot , giving rise to short-term enhancement of release .",
"Various forms of short-term enhancement , like facilitation , augmentation and post-tetanic potentiation ( PTP ) , have been observed and discriminated based on their duration ( Magleby and Zengel , 1975; Zucker and Regehr , 2002 ) .",
"There is good evidence that the longer-lasting of these , augmentation and PTP , depend on the activation of intracellular second messengers in the nerve terminal , which in turn influence the release machinery .",
"A second messenger with a centrally important role in presynaptic plasticity is diacylglycerol ( DAG ) , which can activate both protein kinase-C and Munc13-1 ( Lackner et al . , 1999; Rhee et al . , 2002; Wierda et al . , 2007 ) .",
"During augmentation , activation of the presynaptic protein Munc13-1 either by DAG or by Ca2+/Calmodulin results in increased vesicle priming ( Rosenmund et al . , 2002; Junge et al . , 2004 ) .",
"The other common form of short-term enhancement , PTP , has been observed at excitatory synapses in hippocampus ( McNaughton , 1982; Lee et al . , 2007 ) , cerebellum ( Beierlein et al . , 2007 ) , calyx of Held brainstem synapses ( Habets and Borst , 2005; Korogod et al . , 2005 ) and at the neuromuscular junction ( Magleby and Zengel , 1975 ) .",
"PTP has been shown to be sensitive to pharmacological inhibition of PKC ( Alle et al . , 2001; Brager et al . , 2003; Korogod et al . , 2007 ) , and deletion of the PKC α and β genes in mice suppressed PTP at the calyx synapse ( Fioravante et al . , 2011 ) .",
"Therefore , PTP is likely caused by a presynaptic PKC phosphorylation step , but the target protein of PKC during PTP has remained unknown .",
"Munc18-1 is a member of the Sec1/Munc18 family of proteins essential for membrane fusion from yeast to mammals ( Verhage et al . , 2000; Südhof and Rothman , 2009 ) .",
"Munc18-1 has two consensus sites for PKC phosphorylation ( Fujita et al . , 1996 ) which become phosphorylated during depolarization of synaptosomes ( de Vries et al . , 2000 ) , and which are necessary for phorbol ester potentiation of transmitter release in cultured neurons ( Wierda et al . , 2007 ) .",
"Therefore , Munc18-1 is a candidate for PKC phosphorylation during PTP .",
"Nevertheless , there are other presynaptic proteins which might act as PKC targets .",
"First , SNAP-25 and Synaptotagmin-1 ( Syt1 ) have PKC consensus sites ( Shimazaki et al . , 1996 , Hilfiker et al . , 1999 ) .",
"However , no evidence was found for an involvement of SNAP-25 in the phorbol ester potentiation of synaptic transmission in hippocampal neurons ( Finley et al . , 2003 ) , and the PKC/CaM kinase consensus site in Syt1 is not conserved in Syt2 ( Nagy et al . , 2006 ) .",
"Since robust PTP is also observed at synapses which express Syt2 as their main Ca2+ sensor , like the calyx of Held and neuromuscular synapses ( Pang et al . , 2006 ) , Syt1 is unlikely a general phosphorylation target for the induction of PTP .",
"Second , ion channels like voltage-gated K+ channels and Ca2+ channels are targets of PKC phosphorylation ( Zamponi et al . , 1997; Song et al . , 2005 ) .",
"However , studies at the calyx synapse have shown only marginal changes in the presynaptic AP waveform or presynaptic Ca2+ influx during PTP ( Habets and Borst , 2006; Korogod et al . , 2007 ) , or during phorbol ester mediated potentiation of transmitter release ( Lou et al . , 2005 , 2008 ) .",
"These findings argue against a major role of ion channel phosphorylation during PKC-dependent short-term enhancement of release .",
"Therefore , it is attractive to hypothesize that a protein of the release machinery is a PKC target during short-term enhancement , and Munc18-1 is an interesting candidate for this role .",
"Here , we wished to study the role of Munc18-1 phosphorylation during PTP .",
"Since PTP has been observed mainly at synapses of acute preparations but not in cultured synapses , we developed an in vivo gene replacement strategy at the calyx of Held synapse , a large CNS model synapse at which PTP measurements are well established ( see references above ) .",
"We used a floxed mouse line in which exon 2 of the Munc18-1 coding gene Stxbp1 is flanked by loxP sites ( called Munc18-1lox/lox mice; Heeroma et al . , 2004 ) , combined with in vivo virus-mediated protein expression ( Wimmer et al . , 2004 ) to recombine the floxed allele , and to re-express mutant or wild-type Munc18-1 protein .",
"Using these approaches , we show that a transient PKC phosphorylation of Munc18-1 causes the increased transmitter release that underlies PTP .",
"These results identify Munc18-1 as a PKC target protein during PTP , and suggest that Munc18-1 , besides its essential role in catalyzing membrane fusion , can mediate a second-messenger modulation of the release machinery during presynaptic plasticity ."
],
[
"Previous studies have found evidence for a role of PKC during PTP , a form of short-term enhancement of release ( Alle et al . , 2001; Brager et al . , 2003; Korogod et al . , 2007; Fioravante et al . , 2011 ) .",
"However , it remains possible that the requirement for PKC merely represents a background PKC activity permissive for the induction of PTP ( see discussion in Korogod et al . , 2007 ) .",
"We hypothesized that if PTP is caused by a dynamic phosphorylation/de-phosphorylation cycle of a presynaptic protein , phosphatase blockers should prolong the duration of PTP .",
"We studied PTP at the calyx of Held synapse in a slice preparation , by first testing baseline synaptic strength with double stimuli ( interval , 10 ms ) repeated every 10 s .",
"PTP was induced every 5–7 min using 4 s 100 Hz trains of afferent fiber stimuli ( Figure 1A , arrowheads ) .",
"PTP induction trains under control conditions led to ∼twofold PTP which decayed nearly completely over the next 3 min , similarly as shown previously ( Korogod et al . , 2005 ) .",
"Acute application of calyculin ( 1 µM ) , an inhibitor of phosphatases PP1 and PP2A ( Ishihara et al . , 1989 ) , strongly prolonged the decay of PTP ( Figure 1A ) .",
"We estimated the decay rate of PTP by line fits ( Figure 1A , grey and red line ) , and found that the PTP decay rate was slowed from 16 . 0 ± 2 . 6%/min to 4 . 04 ± 1 . 7%/min ( Figure 1B; n = 7 cells; p<0 . 01 ) .",
"Calyculin acted without changing the baseline synaptic strength ( Figure 1A , C; p=0 . 96 ) , nor the peak PTP amplitude ( 219 ± 17% and 227 ± 17% , in control and calyculin respectively; p=0 . 7 ) .",
"Following removal of calyculin , PTP gradually reversed to its normal decay kinetics ( Figure 1A ) . 10 . 7554/eLife . 01715 . 003Figure 1 . A phosphatase terminates the increased transmitter release which underlies PTP .",
"( A ) Time course of EPSC amplitude during the repetitive inductions of PTP ( arrowheads , HFS at 100 Hz for 4",
"s ) , demonstrating the slowing of PTP decay upon acute application of phosphatase inhibitor Calyculin A . Line fits ( grey and red lines ) were used to estimate the decay rates of PTP .",
"Upper inset shows example EPSCs induced by double stimuli ( interval , 10 ms ) before and after HFS , both for control condition ( left ) and following calyculin application ( right ) .",
"( B and C )",
"Quantifications of PTP decay rates for control and calyculin ( B and Figure 1—source data 1A ) and basal EPSC amplitudes for the two conditions ( C and Figure 1—source data 1B ) .",
"( D ) Average time courses of normalized EPSC amplitudes ( top , PTP ) and normalized paired-pulse ratio ( EPSC2/EPSC1 , bottom ) in control conditions ( black symbols ) and in the presence of Calyculin A ( red symbols ) , obtained in the same recordings ( n = 7 cells ) .",
"( E ) Time course of evoked EPSCs ( top ) and mEPSC frequency ( bottom , line trace ) and scatter plot of individual mEPSC amplitudes ( bottom , gray data points ) as a function of experiment time , from a different example recording as the one shown in ( A ) .",
"Example mEPSC traces are shown for the time points indicated by arrows .",
"( F ) Quantification of average mEPSC amplitudes before PTP induction stimuli ( sampled from at least five 10 s long mEPSC traces , left bars ) , and during a single 10 s interval immediately following PTP induction ( right bars ) .",
"Data for both control conditions ( left ) and in the presence of calyculin ( right ) are shown ( see Figure 1—source data 1C ) .",
"( G ) Quantification of the mEPSC frequency late after induction of PTP , both under control conditions , and in the subsequent presence of calyculin ( 1 µM ) in the same cell .",
"Note that in the presence of calyculin , the steady state mEPSC frequency was persistently increased ( see Figure1—source data 1D ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01715 . 00310 . 7554/eLife . 01715 . 004Figure 1—source data 1 . ( A ) Data values and statistics underlying Figure 1B .",
"( B ) Data values and statistics underlying Figure 1C .",
"( C ) Data values and statistics underlying Figure 1F .",
"( D ) Data values and statistics underlying Figure 1G . DOI: http://dx . doi . org/10 . 7554/eLife . 01715 . 004 It is conceivable that pharmacological block of phosphatases interferes with postsynaptic plasticity mechanisms , and thereby causes an apparent prolongation of PTP .",
"To distinguish between pre- and postsynaptic sites of action of calyculin , we first analyzed the paired-pulse ratio ( EPSC2/EPSC1 ) .",
"Under control conditions , the paired-pulse ratio was decreased during the peak of PTP , and then recovered when PTP decayed back to baseline ( Figure 1D , black data points ) .",
"The decreased paired-pulse ratio confirms the view that PTP is presynaptic in origin , and mediated by an increased transmitter release ( Habets and Borst , 2005; Korogod et al . , 2005 ) .",
"In the presence of calyculin both the decay of PTP as well as the recovery of the paired pulse ratio were slowed , revealing a parallel regulation of both the synaptic strength and the paired-pulse ratio by calyculin ( Figure 1D; red data points; n = 7 cells ) .",
"Thus , in the presence of a phosphatase inhibitor , the increased transmitter release during PTP decays more slowly .",
"We further analyzed the quantal mechanism of PTP in the absence and presence of calyculin by analyzing the spontaneous ( miniature ) EPSCs ( mEPSCs ) , sampled in 10 s intervals in-between evoked EPSCs .",
"Figure 1E shows plots of the evoked EPSC amplitude ( top ) , and of the individual mEPSC amplitudes and time-averaged mEPSC frequency ( bottom ) , both in control conditions , and following application of calyculin ( 1 µM ) .",
"Following the PTP induction trains , we found that the mEPSC frequency was increased and then relaxed back to baseline value over several tens of seconds , as shown before ( Korogod et al . , 2005 ) .",
"Under control conditions , the mEPSC amplitude was increased during the first 10 s interval following PTP induction ( by 23% on average; Figure 1F; p<0 . 05 ) .",
"However , the increase in mEPSC amplitudes did not reach statistical significance in the other data sets of this study ( see below , Figure 2—figure supplement 1 , Figure 4—figure supplement 1 ) .",
"These findings are consistent with the view that PTP largely represents an increase in the amount of released quanta ( Korogod et al . , 2005 ) , and that a significant increase in mEPSC amplitude , maybe caused by compound fusion , is only observed with stronger PTP induction stimuli ( He et al . , 2009; Xue and Wu , 2010 ) .",
"Interestingly , in the presence of calyculin , the elevated mEPSC frequency following PTP induction trains did not recover completely , but rather , persisted at 2–3-fold elevated levels ( Figure 1E , bottom ) .",
"This effect was observed in all cells in which mEPSC frequency was analyzed ( n = 5; p<0 . 001; Figure 1G ) .",
"Thus , the persistently enhanced transmitter release following PTP induction trains under phosphatase block is mirrored by an enhanced spontaneous release rate .",
"Phosphatases reverse the action of many serine–threonine kinases ( Hunter , 1995 ) .",
"We therefore wanted to find further evidence that the action of a phosphatase is related to an initial PKC phosphorylation step .",
"Furthermore , we wanted to distinguish pharmacologically whether ‘novel’ or ‘conventional’ PKCs initiate PTP ( Newton , 2001 ) .",
"For this purpose , we made use of cell-permeable inhibitory peptides directed either against the conventional PKC isoforms α and βΙ−ΙΙ ( called PKCi ) , or against protein kinase A in a control experiment ( called PKAi ) .",
"We measured PTP in slices pre-incubated either with PKAi or with PKCi at 10 µM each ( Figure 2A; see ‘Materials and methods’ for procedures of drug application ) .",
"PKCi strongly suppressed PTP ( Figure 2A , B; p<0 . 001 ) , whereas PTP was normal in the presence of PKAi ( Figure 2A , B ) .",
"The baseline synaptic strength was similar under both conditions ( Figure 2C; p=0 . 92 ) .",
"Since large EPSCs usually show smaller ( relative ) PTP at the calyx synapse ( Korogod et al . , 2005 ) , we plotted the amount of PTP vs EPSC amplitude to control for a possible sampling bias with respect to baseline EPSC amplitudes .",
"This revealed that PKCi also reduced PTP in recordings with small baseline EPSCs ( Figure 2D ) .",
"Therefore , the smaller average PTP in the presence of PKCi was not caused by a sampling bias towards large baseline EPSCs . 10 . 7554/eLife . 01715 . 005Figure 2 . Conventional PKCs but not phospholipase-C initiate PTP .",
"( A ) Average time courses of normalized EPSC amplitude ( PTP plots ) in the presence of PKC inhibitory peptide ( PKCi; red symbols , n = 8 cells ) or PKA inhibitory peptide ( PKAi; black symbols , n = 9 cells ) .",
"Note the significant suppression of PTP by PKCi .",
"( B and C )",
"Quantifications of peak PTP ( B and Figure 2—source data 1A ) and of baseline EPSC amplitude ( C and Figure 2—source data 1B ) in the presence of PKCi ( red symbols ) and PKAi ( black ) .",
"( D ) Plot of peak PTP amplitudes vs the basal EPSC amplitudes .",
"Note that PTP was strongly reduced also when baseline EPSC amplitude was small .",
"The correlation coefficients",
"( r ) are indicated .",
"( E ) Average PTP plots in neurons recorded after pre-incubation with the PLC blocker U73122 ( red symbols; n = 10 cells ) , and under control conditions with 0 . 1% DMSO ( black symbols; n = 7 cells ) .",
"( F and G )",
"Quantification of average peak PTP under control conditions and in the presence of U73122 ( F , and Figure 2—source data 1C ) , and plot of peak PTP amplitude vs baseline EPSC amplitude ( G , black and red symbols , respectively ) .",
"Note the absence of an effect of phospholipase-C inhibition on PTP . DOI: http://dx . doi . org/10 . 7554/eLife . 01715 . 00510 . 7554/eLife . 01715 . 006Figure 2—source data 1 . ( A ) Data values and statistics underlying Figure 2B .",
"( B ) Data values and statistics underlying Figure 2C .",
"( C ) Data values and statistics underlying Figure 2F .",
"( D ) Data values and statistics underlying Figure 2—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 01715 . 00610 . 7554/eLife . 01715 . 007Figure 2—figure supplement 1 . mEPSC amplitude before and after PTP induction protocols is unchanged for the PTP data sets in Figure 2 . Shown are quantifications of average mEPSC amplitudes ( individual cells and group averages ) for the PTP data sets shown in Figure 2A–D ( in the presence of PKAi and PKCi ) , and for the data set shown in Figure 2E–G ( control and following pre-incubation with U73122 ) .",
"In all cases , the difference between mEPSC amplitude before and after the high frequency train ( HFS ) to induce PTP was not significantly different ( ‘n . s . ’; p>0 . 05; see also Figure 2—source data 1D ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01715 . 00710 . 7554/eLife . 01715 . 008Figure 2—figure supplement 2 . PTP is insensitive to the PLC inhibitor Neomycin . Left: PTP was first induced under control conditions ( left arrowhead ) .",
"Following that , 10 μM Neomycin was added to the slice preparation , and a second PTP induction train was applied .",
"Note a strong decrease in the baseline EPSC amplitude , which might be due to a partial block of P/Q-type Ca2+ channels by Neomycin ( Pichler et al . , 1996 ) .",
"In relative terms , however , the second PTP induction under Neomycin also caused an ∼twofold PTP .",
"Right: relative PTP ( average of n = 4 cells ) appears normal in the presence of Neomycin .",
"Since we showed previously that lowering extracellular [Ca2+] ( from 2 to 1 mM ) caused a reduction in baseline EPSC ( similar to the Neomycin effect ) , but did not change the relative PTP ( Korogod et al . , 2007 ) , we assume that PTP is insensitive to Neomycin . DOI: http://dx . doi . org/10 . 7554/eLife . 01715 . 008 The experiments with the PKCi inhibitory peptide suggest that conventional PKCs ( PKCα and -β ) are involved in PTP at the calyx synapse , in agreement with recent genetic evidence ( Fioravante et al . , 2011 ) .",
"An earlier study had shown , however , that the novel PKC isoform PKCε becomes translocated in the calyx of Held nerve terminal upon phorbol ester stimulation ( Saitoh et al . , 2001 ) .",
"If novel PKCs are involved in PTP , one might expect a contribution of upstream phospholipase C signaling which produces DAG , since novel PKCs are activated by DAG but not by Ca2+ ( Newton , 2001 ) .",
"Therefore , we tested the role of the phospholipase C inhibitor U73122 ( 3 µM ) , but we could not find significant effects on PTP ( Figure 2E–G ) .",
"Similarly , another PLC inhibitor Neomycin ( 10 µM ) did not suppress PTP ( Figure 2—figure supplement 2 ) , despite an immediate effect of Neomycin on transmitter release , which was probably caused by inhibition of presynaptic P/Q-type Ca2+ channels ( Pichler et al . , 1996 ) .",
"Taken together , the experiments with the phosphatase inhibitor calyculin and with the PKC inhibitory peptide strongly suggest that a dynamic phosphorylation/de-phosphorylation cycle , initiated by conventional PKC isoforms , determines the time course of PTP at the calyx of Held .",
"PTP was insensitive to PLC blockers , consistent with the view that conventional , but not novel PKCs initiate the phosphorylation of a presynaptic target protein during PTP .",
"We next wished to study the role of Munc18-1 phosphorylation for PTP at the calyx of Held .",
"Munc18-1 is an essential protein for vesicle fusion and membrane trafficking ( Südhof and Rothman , 2009 ) , which has two PKC phosphorylation sites ( Fujita et al . , 1996; de Vries et al . , 2000; Wierda et al . , 2007 ) .",
"To investigate the role of Munc18-1 in PTP , it was necessary to develop a gene replacement strategy in which endogenous Munc18-1 protein is replaced with a PKC-insensitive mutant .",
"Since constitutive genetic deletion of Munc18-1 in mice is lethal at the late embryo stage ( Verhage et al . , 2000 ) , we used Munc18-1lox/lox mice ( Heeroma et al . , 2004 ) and recombined the loxP sites using virus-mediated Cre expression .",
"In addition , we wished to re-express phosphorylation-deficient Munc18-1 , or wild-type Munc18-1 using the same viral construct .",
"This required the simultaneous expression of three proteins:",
"( i ) Munc18-1 , in the wild-type or mutant form ,",
"( ii ) Cre-recombinase , and",
"( iii ) GFP to label successfully transduced calyces of Held .",
"We used an adenovirus vector which allows the use of two independent expression cassettes ( Young and Neher , 2009 ) ; one of these carried an internal ribosome entry site ( IRES ) to allow the expression of a third protein ( Figure 3A3 ) .",
"Several additional constructs were prepared for control experiments ( Figure 3A1 , A2 ) .",
"We injected the adenovirus into the ventral cochlear nucleus ( VCN ) of Munc18-1lox/lox mice early postnatally , to allow for sufficient time for protein replacement ( injection , P1; recordings 8–10 days later; see ‘Materials and methods’ , Figure 3—figure supplements 1 , 2 ) . 10 . 7554/eLife . 01715 . 009Figure 3 . Endogenous floxed Munc18-1 can be removed and replaced by recombinant protein in vivo at the calyx of Held .",
"( A ) Scheme of the adenoviral DNA constructs .",
"A1: control vector driving the expression of GFP alone; A2: control vector driving the expression of GFP-IRES-Cre; A3: three-protein expression vector , which drives the expression of Munc18-1 ( either wild-type or myc-tagged ) , Cre-recombinase , and GFP from an additional cassette in the viral genomic backbone ( see ‘Materials and methods’ ) .",
"( B ) Results from paired recordings from calyx synapses in Munc18-1lox/lox mice expressing either GFP alone ( B1 ) , GFP-IRES-Cre ( B2 ) , or M18WT-IRES-Cre and GFP ( B3 ) .",
"Shown are the corresponding GFP-positive calyces ( top ) , the presynaptic voltage-clamp protocols and presynaptic Ca2+ currents ( middle ) , and the resulting postsynaptic EPSCs ( bottom ) .",
"Note the abolishment of release when Cre recombinase is expressed alone ( B2 – note remaining quantal release in grey trace with enhanced scale ) , and the rescue of release when Cre-recombinase is expressed together with M18WT protein ( B3 ) .",
"These measurements were done following virus injection at P1 , which we found necessary for efficient elimination of endogenous Munc18-1 protein in Cre expressing calyces .",
"( C and D )",
"Summary of EPSC amplitudes recorded in response to 50 ms presynaptic depolarization ( C; see B ) , for the following conditions: expression of GFP alone ( left bar , black data points ) , expression of Cre-recombinase ( middle; grey data points ) , and expression of Cre-recombinase together with M18WT ( right; red data points and see Figure3—source data 1A ) .",
"The presynaptic Ca2+ current amplitudes were unaffected by genetic removal of Munc18-1 ( D and Figure3—source data 1B ) .",
"( E ) Adenovirus-mediated expression of myc-Munc18-1 in E2T packaging cells analyzed by SDS-PAGE and western blotting shows strong expression of recombinant protein ( myc-Munc18-1 , 67 kDa , actin , 43 kDa ) .",
"( F ) Immunohistochemistry of calyces of Held expressing the myc-tagged M18 construct in a P11 Munc18lox/lox mouse after injection at P1 .",
"Antibodies against GFP ( left , green channel ) and c-myc ( middle , red channel ) were used; the overlay image is shown on the right .",
"Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01715 . 00910 . 7554/eLife . 01715 . 010Figure 3—source data 1 . ( A ) Data values and statistics underlying Figure 3C .",
"( B ) Data values and statistics underlying Figure 3D . DOI: http://dx . doi . org/10 . 7554/eLife . 01715 . 01010 . 7554/eLife . 01715 . 011Figure 3—figure supplement 1 . Early postnatal Cre expression was necessary for complete removal of endogenous Munc18-1 . We tested the influence of the postnatal time of Cre expression on the efficiency of conditional removal of the endogenous ( floxed ) Munc18-1 protein .",
"Munc18-1lox/lox mice were injected with GFP-IRES-Cre ( except the rightmost column ) ; the stereotactic injection time point was varied between postnatal day ( P ) 1 and P6 as indicated .",
"In these experiments , slices were prepared 7–8 days later .",
"Examples of paired recordings from GFP-expressing calyx of Held terminals and their postsynaptic neuron are shown .",
"Note that only injection at P1 leads to a clear abrogation of transmitter release ( leftmost column ) , whereas later injections caused a significant persistence of depolarization-evoked release ( middle columns ) .",
"A control recording from a P9 mouse injected at P1 with an adenovirus expressing GFP alone is shown in the rightmost column . DOI: http://dx . doi . org/10 . 7554/eLife . 01715 . 01110 . 7554/eLife . 01715 . 012Figure 3—figure supplement 2 . Early postnatal Cre expression was necessary for complete removal of endogenous Munc18-1: summary . Summary plot of EPSC amplitudes vs the time point of virus injection .",
"The most robust and efficient Munc18-1 removal , as judged from the postsynaptic responses in paired recordings , was found for the early injections ( at P1 ) .",
"We attribute this to the fact that the calyces strongly increase in size at ∼P2–P4 ( Hoffpauir et al . , 2006 ) .",
"Thus , early recombination of the floxed Munc18-1 gene likely curtails protein synthesis at a time when new proteins are most needed for the growing calyx ( the estimated onset time for protein expression from the adenovirus is ∼24 hr; unpublished observations ) .",
"In contrast , removal of Munc18-1 protein at later times is expected to depend more strongly on the Munc18-1 protein turnover rate in the calyx . DOI: http://dx . doi . org/10 . 7554/eLife . 01715 . 012 We first tested the feasibility of the Munc18-1 gene replacement strategy in a series of control experiments .",
"When we expressed Cre recombinase in VCN neurons of Munc18-1lox/lox mice using a GFP-IRES-Cre expression cassette ( Figure 3A2 ) , depolarization-evoked release at the calyx of Held synapse was essentially abolished ( Figure 3B2 ) .",
"In contrast , control experiments with a GFP expressing virus ( Figure 3A1 ) in Munc18-1lox/lox mice showed normal depolarization-evoked EPSCs ( Figure 3B1 ) .",
"On average , EPSCs were 0 . 251 ± 0 . 199 nA ( n = 6 cells ) and 11 . 2 ± 1 . 0 nA ( n = 3 cells ) when presynaptic neurons expressed GFP-IRES-Cre and GFP respectively ( Figure 3C; p<0 . 01 ) .",
"The amplitude of presynaptic Ca2+ currents was unchanged under both conditions ( 1 . 07 ± 0 . 20 and 1 . 08 ± 0 . 08 nA for GFP-IRES-Cre and GFP respectively; see also Figure 3D ) .",
"Thus , Cre recombinase was active , and 8–10 days following virus injection , all functionally relevant copies of Munc18-1 proteins had disappeared from calyx terminals .",
"Additional experiments showed that virus injections early postnatally at P1 were necessary for complete removal of Munc18-1 function ( Figure 3—Figure supplements 1 , 2 ) .",
"We next wished to test whether the simultaneous re-expression of Munc18-1 protein , together with the expression of Cre recombinase and GFP in the three-protein expression construct ( Figure 3A3 ) , would lead to efficient rescue of the Munc18-1 k . o . release phenotype .",
"Indeed , depolarization-evoked release responses were nearly completely rescued ( 6 . 7 ± 2 . 3 nA , n = 5; Figure 3B3 , C ) .",
"In all cases , presynaptic Ca2+ currents were unchanged ( Figure 3B , D; p>0 . 05 ) , which suggests that Munc18-1 is necessary for vesicle fusion , but not for presynaptic Ca2+ channel function .",
"In a third line of control experiments , we used a Myc-tag labeled Munc18-1 construct in the three protein expression construct ( Figure 3A3 , bottom ) .",
"Western blotting and immunohistochemistry with an anti-Myc antibody showed that recombinant Munc18-1 protein was produced in cell lines ( Figure 3E ) , and transported to the calyx of Held nerve terminals following in vivo expression in the VCN ( Figure 3F ) .",
"Together , these experiments suggest that simultaneous expression of Cre-recombinase , and a rescue Munc18-1 construct in VCN neurons of Munc18-1lox/lox mice leads to the exchange of the endogenous Munc18-1 by the recombinant protein .",
"Having established an in vivo gene replacement approach for Munc18-1 , we next studied the role of PKC phosphorylation of Munc18-1 during presynaptic plasticity , by replacing the endogenous Munc18-1 with a PKC-insensitive mutant .",
"The PKC-insensitive Munc18-1 mutant carried alanine substitutions at the two PKC consensus sites ( S306A , S313A ) as well as at a third serine residue ( S312A; called M18SA mutant here; Wierda et al . , 2007 ) .",
"As a control , we used the M18WT construct ( Figure 4A ) .",
"Following injection of either one of the two protein constructs into the VCN of Munc18-1lox/lox mice at P1 , EPSCs and presynaptic plasticity were measured in slices made 8–10 days later .",
"The mutant and wild-type forms of Munc18-1 produced baseline EPSCs of similar amplitudes ( 2 . 87 ± 0 . 59 nA and 2 . 91 ± 0 . 61 nA respectively; p=0 . 96; Figure 4B1–D1 , E ) .",
"However , when we applied brief high-frequency trains of afferent fiber stimulation ( 100 Hz , 4",
"s ) , we observed a striking difference in presynaptic plasticity between synapses rescued with M18WT , and with the M18SA mutant .",
"While M18WT-synapses showed normal PTP of 221 ± 15% of baseline EPSC amplitude ( n = 12; see Figure 4B1 for an example ) , synapses rescued with the Munc18SA mutant showed significantly smaller PTP ( 147 ± 13% of baseline; n = 15; p<0 . 001; Figure 4B1–D1 , F ) .",
"In some cases , PTP was abolished completely ( Figure 4C1 ) , whereas in other cases PTP was smaller and lasted for shorter times ( Figure 4D1 ) .",
"To analyze the total amount of potentiation independent of its duration , we calculated the cumulative amount of PTP ( Figure 4B1–D1 bottom; Figure 4H ) .",
"The cumulative PTP attained at 150 s was 5 . 06 ± 1 . 17 and 0 . 83 ± 0 . 85 ( unit , fold change −1 ) in M18WT and M18SA-rescued synapses , respectively ( n = 12 and 13 , respectively; p<0 . 01 ) .",
"A plot of the peak PTP amplitude vs baseline EPSC amplitude showed that PTP was reduced over the entire range of baseline EPSC amplitudes in the sample ( Figure 4I ) . 10 . 7554/eLife . 01715 . 013Figure 4 . The PKC phosphorylation sites of Munc18-1 are necessary for the expression of post-tetanic potentiation , PTP .",
"( A ) Scheme of the three-protein expression constructs used for the experiments shown in this figure .",
"Either wild-type Munc18-1 ( M18WT ) or the PKC-phosphorylation site triple mutation ( S306A , S312A , S313A; M18SA ) were expressed together with Cre-recombinase and GFP .",
"( B-D )",
"PTP from three example cells , one rescued with wild-type Munc18-1 ( M18WT , B1 , B2 ) , the other two rescued with the PKC-phosphorylation site deficient mutant ( M18SA; C1 , C2 and D1 , D2 ) .",
"From top to bottom , individual EPSC traces before ( left ) and after ( right ) PTP; plots of relative EPSC amplitudes vs time ( PTP plot ) , and plots of cumulative PTP vs time .",
"Note that PTP was absent ( C1 ) or smaller in synapses rescued with M18SA; when substantial PTP remained , it decayed more rapidly ( D1 , middle ) .",
"The panels in ( B2–D2 ) plot the mEPSC frequency for the corresponding cells on the same time scale .",
"( E and F )",
"Summary plots of baseline EPSC amplitudes ( E and Figure 4—source data 1A ) and of peak PTP ( F and Figure 4—source data 1B ) for synapses rescued with wild-type Munc18-1 ( M18WT; black symbols ) and with the mutant form ( M18SA; red symbols ) .",
"( G ) Average PTP plots for synapses rescued with M18WT ( black symbols ) and M18SA mutant ( red; n = 12 and n = 15 cells , respectively ) .",
"( H ) Average cumulative PTP for synapses rescued with M18WT ( black symbols ) and M18SA mutant ( red; n = 12 and n = 13 cells , respectively ) .",
"( I ) Plot of peak PTP amplitudes vs the basal EPSC amplitudes shows that PTP was reduced over the entire range of basal EPSC amplitudes .",
"The correlation coefficients",
"( r ) are indicated .",
"( J ) Normalized average mEPSC frequency following PTP induction trains , recorded at synapses rescued with M18WT ( black ) and Munc18SA ( red ) .",
"In the range of 0–60 s , significance was tested by paired t-test .",
"Two data points at around 30–40 s were found to be significantly different between the two conditions ( p<0 . 05; see star symbols ) .",
"Thus , Munc18-1 phosphorylation was not necessary for late release in the first 10 s interval after PTP induction , but probably supported some late enhanced mEPSC frequency , in agreement with the calyculin data in Figure 1E . DOI: http://dx . doi . org/10 . 7554/eLife . 01715 . 01310 . 7554/eLife . 01715 . 014Figure 4—source data 1 . ( A ) Data values and statistics underlying Figure 4E .",
"( B ) Data values and statistics underlying Figure 4F .",
"( C ) Data values and statistics underlying Figure 4—figure supplement 1 .",
"( D ) Data values and statistics underlying Figure 4—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 01715 . 01410 . 7554/eLife . 01715 . 015Figure 4—figure supplement 1 . mEPSC amplitude before and after PTP induction is unchanged in rescue experiments with wild-type or PKC-insensitive Munc18-1 mutant . Shown are quantifications of average mEPSC amplitudes ( individual cells and group averages ) for the PTP data sets shown in Figure 4 ( synapses of Munc18-1 floxed mice rescued either with M18WT or with the M18SA mutant ) .",
"There was no significant difference between mEPSC amplitude before and after the high frequency train ( HFS ) to induce PTP ( ‘n . s . ’; p>0 . 05; see also Figure 4—source data 1C ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01715 . 01510 . 7554/eLife . 01715 . 016Figure 4—figure supplement 2 . PTP is blocked in synapses rescued by a phosphomimetic Munc18-1 mutant . Left , Average PTP plot ( n = 9 cells ) for synapses of Munc18-1 floxed mice rescued with the phosphomimetic mutant Munc18-1SD .",
"Note the absence of PTP; inset shows example EPSC traces .",
"Right , quantifications of paired-pulse ratio and baseline EPSC amplitude for synapses rescued with M18WT ( black symbols; same data set as shown in Figure 4 ) , and with the M18SD mutant .",
"Note that paired-pulse ratio was not significantly changed ( p>0 . 05 ) , but baseline EPSC amplitude and rescue efficiency were lower with the M18SD mutant ( p<0 . 05 ) .",
"See also Figure4—source data 1D . DOI: http://dx . doi . org/10 . 7554/eLife . 01715 . 016 Previous studies showed that following high-frequency stimulation to induce PTP , the mEPSC frequency is increased and this post-tetanic late release correlated with elevated residual [Ca2+]i in the nerve terminal ( Habets and Borst , 2005; Korogod et al . , 2005 ) .",
"We analyzed the late post-tetanic release ( Figure 4B2–D2 ) to investigate a possible role of Munc18-1 phosphorylation in this component of release .",
"During the first 10 s interval following PTP induction trains , a strongly increased mEPSC frequency was observed both in synapses rescued with M18WT and M18SA ( Figure 4B2–D2 ) .",
"This post-tetanic late release seemed to decay back to baseline faster in M18SA synapses than in M18WT synapses ( Figure 4B2–D2 ) .",
"Indeed , we found that for a few sample points at intermediate times ( at around 20–30 s following the PTP induction train ) , asynchronous release was reduced in M18SA synapses as compared to M18WT ( Figure 4J , star symbols; p<0 . 05 ) .",
"In addition , the mEPSC amplitudes did not show a significant increase following PTP induction ( Figure 4—figure supplement 1 ) , which shows again that PTP under these stimulation conditions largely represents an increase in the amount of released quanta .",
"Taken together , replacing endogenous Munc18-1 protein by a PKC-deficient mutant selectively impaired the potentiation of evoked release during PTP , whereas the baseline transmitter release was unaffected , and late asynchronous release depended only marginally on PKC phosphorylation of Munc18-1 .",
"These experiments show that Munc18-1 is an important target protein for PKC phosphorylation during PTP .",
"We next wished to test whether the PKC phosphorylation sites of Munc18-1 were also important for the phorbol ester-mediated potentiation of evoked and spontaneous release .",
"The phorbol ester PDBu ( 1 µM ) still potentiated EPSCs in calyx synapses rescued with M18SA , but the potentiation was significantly , about twofold smaller than with Munc18WT ( 157 ± 10% of control and 202 ± 18% of control , respectively; p<0 . 05; Figure 5A–D ) .",
"Similarly , the frequency of spontaneous mEPSCs was potentiated by phorbol ester , but the potentiation was again smaller in synapses rescued with M18SA as compared to synapses expressing M18WT ( 307 ± 41% vs 518 ± 50% , respectively; p<0 . 05 , Figure 5H ) .",
"At first inspection , the baseline mEPSC frequency seemed to be higher in synapses rescued with M18SA as compared to M18WT synapses ( Figure 5G ) .",
"However , the difference did not reach statistical significance ( p=0 . 50 ) , and was caused by an outlier in the M18SA data set ( Figure 5G , pink data point ) .",
"When removing this data point , the average baseline mEPSC frequency was unchanged between M18WT and M18SA synapses ( 0 . 65 ± 0 . 22 and 0 . 48 ± 0 . 19 Hz , respectively; Figure 5G; p=0 . 54 ) .",
"Therefore , the reduced potentiation of mEPSC frequency by phorbol ester in M18SA synapses was not the result of a higher baseline mEPSC frequency ( Figure 5G ) .",
"We conclude that PKC phosphorylation of Munc18-1 accounts for part of the potentiation of evoked- and spontaneous release by phorbol esters . 10 . 7554/eLife . 01715 . 017Figure 5 . About half of the phorbol ester potentiation of evoked and spontaneous EPSC depends on PKC phosphorylation of Munc18-1 . ( A and B ) Evoked EPSC traces ( top ) and spontaneous EPSCs ( middle ) are shown both before ( left ) and after ( right ) application of 1 µM PDBu to the slice .",
"The bottom panels show time plots of evoked EPSC amplitudes and their potentiation by PDBu .",
"Data for a M18WT rescued synapse ( A ) and for a M18SA rescued synapse ( B ) are shown .",
"( C ) Average time courses of normalized EPSC amplitudes during PDBu potentiation for synapses rescued with M18SA ( red symbols ) and M18WT ( black , n = 7 and n = 6 cells , respectively ) .",
"( D and E )",
"Quantifications of the average and individual values for EPSC potentiation ( D , Figure 5—source data 1A ) and for the baseline EPSC amplitudes ( E , Figure 5—source data 1B ) in synapses rescued with M18SA ( red ) and with M18WT ( black ) .",
"Note that ∼half of the potentiation of evoked EPSC amplitudes depended on an intact Munc18-1 phosphorylation site .",
"( F ) Average time courses of normalized spontaneous EPSC frequency before and after PDBu application , both for synapses rescued with Munc18SA ( red symbols ) and with M18WT ( black; n = 4 and 6 , respectively ) .",
"( G ) Quantification of absolute mEPSC frequencies before and after PDBu application , for synapses rescued with M18WT and M18SA .",
"For the M18SA data , an outlier data point with an unusually high baseline frequency ( 4 Hz; pink symbols ) was removed when calculating the average absolute mEPSC frequencies ( see Figure5—source data 1C ) .",
"( H ) Average relative potentiation of mEPSC frequency under both conditions .",
"Note that about half of the potentiation of spontaneous release depends on the PKC phosphorylation of Munc18-1 ( see Figure5—source data 1D ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01715 . 01710 . 7554/eLife . 01715 . 018Figure 5—source data 1 . ( A ) Data values and statistics underlying Figure 5D .",
"( B ) Data values and statistics underlying Figure 5E .",
"( C ) Data values and statistics underlying Figure 5G .",
"( D ) Data values and statistics underlying Figure 5H . DOI: http://dx . doi . org/10 . 7554/eLife . 01715 . 01810 . 7554/eLife . 01715 . 019Figure 5—figure supplement 1 . Model of Munc18-1 PKC phosphorylation and de-phosphorylation and its effect on presynaptic plasticity . Following elevation of residual Ca2+ , conventional PKC will be activated , and phosphorylate Munc18-1 .",
"Munc18-1 is de-phosphorylated during the decay of PTP by a phosphatase ( PP ) .",
"We assume that Munc18-1 is bound to the partially formed SNARE complex of docked and readily releasable vesicles , and that PKC phosphorylation of Munc18-1 represents a switch towards a higher release probability of readily releasable vesicles .",
"Note that phorbol esters , which are DAG analogues , activate a wider range of presynaptic C1 domain proteins including PKC and Munc13-1 .",
"See also ‘Discussion’ .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 01715 . 019"
],
[
"Using a novel in vivo gene replacement strategy at a large CNS synapse , the calyx of Held , we show that PKC phosphorylation of the presynaptic protein Munc18-1 is critically important for PTP , a form of short-term enhancement observed at many CNS synapses .",
"This identifies Munc18-1 as a presynaptic PKC target during PTP .",
"Pharmacological experiments suggested that the PKC action during PTP is dynamic , since blocking phosphatases led to a marked prolongation of PTP ( Figure 1 ) .",
"Together , these results show that a dynamic phosphorylation/de-phosphorylation cycle of Munc18-1 , initiated by PKC activity , causes PTP .",
"A membrane-permeable peptide inhibitor of conventional PKC isoforms α and β suppressed PTP , whereas a similar PKA inhibitor had no effect ( Figure 2 ) .",
"The peptide inhibitors have a more defined PKC isoform specificity than the synthetic PKC inhibitors used earlier ( Brager et al . , 2003; Korogod et al . , 2007 ) .",
"This , together with recent findings from knock-out mouse lines ( Fioravante et al . , 2011 ) , allows us to conclude that during PTP induction stimuli , conventional PKC isoforms are activated .",
"A previous study evaluated the effects of BIS-like PKC inhibitors on PTP as non-specific ( Lee et al . , 2008 ) .",
"However , the genetic evidence for an involvement of conventional PKCs ( Fioravante et al . , 2011 ) and for the PKC phosphorylation sites of Munc18-1 in PTP ( present study ) , as well as our pharmacological evidence with specific peptide inhibitors , firmly establishes the role of PKC and Munc18-1 phosphorylation in PTP .",
"An earlier study found that phorbol esters translocates PKCε at the calyx of Held ( Saitoh et al . , 2001 ) .",
"Although we cannot exclude that PKCε contributes to phorbol ester potentiation of release , it seems unlikely that novel PKCs , including PKCε , play a major role during PTP , a conclusion which agrees with the inefficiency of PLC inhibitors ( Figure 2 ) .",
"The inefficiency of PLC blockers in PTP further contrasts this form of short-term enhancement with augmentation , since the latter is sensitive to PLC blockers , but not affected by PKC inhibitors ( Rosenmund et al . , 2002 ) .",
"We developed a novel gene replacement approach for the calyx of Held synapse , which makes use of a floxed mouse line , combined with virus-mediated expression of three proteins:",
"( i ) Cre-recombinase;",
"( ii ) the protein of interest in mutated , or wild-type form , and",
"( iii ) the reporter gene GFP .",
"We used adenoviral vectors , which have a high cloning capacity and which allow the use of two expression cassettes ( Young and Neher , 2009 ) .",
"The additional use of an IRES site allowed us to drive the expression of a third protein; the Cre-recombinase placed downstream of the IRES site was expressed efficiently as shown in control experiments ( Figure 3A2 , B2 ) .",
"Using this approach , we showed that replacing the endogenous ( floxed ) Munc18-1 with a Munc18-1 mutant resistant to PKC phosphorylation strongly suppressed PTP .",
"These genetic experiments at the calyx of Held show that Munc18-1 is a necessary target for PKC during short-term enhancement of release .",
"Perturbing presynaptic PKC signaling with various methods , including PKC inhibition ( Figure 2; see also Korogod et al . , 2007 ) , phosphatase block ( Figure 1 ) , and introducing a PKC insensitive Munc18-1 mutant ( Figure 4 ) , did not lead to changes of the baseline synaptic strength .",
"This indicates that Munc18-1 is not phosphorylated in the nerve terminal under baseline conditions; a conclusion which agrees with previous work using a synaptosome preparation ( de Vries et al . , 2000 ) .",
"Therefore , it seems likely that during , or shortly after high-frequency activity , Munc18-1 is phosphorylated by a conventional PKC isoform , which induces short-term enhancement of release .",
"About a minute later , a phosphatase de-phosphorylates Munc18-1 , which terminates the enhanced evoked release during PTP .",
"Thus , a dynamic regulation of the PKC phosphorylation state of Munc18-1 underlies the rise and decay of PTP .",
"Using the gene replacement approach , we also studied the potentiation of transmitter release by phorbol esters .",
"We found that the Munc18-1 phosphorylation sites were necessary for ∼half of the phorbol ester-mediated potentiation of evoked and spontaneous release ( Figure 5 ) .",
"Phorbol esters , analogues of the membrane lipid product DAG , can activate several proteins including novel and conventional PKCs , Munc13-1 , and other C1 domain containing proteins ( Newton , 2001; Brose and Rosenmund , 2002; Rhee et al . , 2002 ) .",
"Therefore , it is expected that phorbol ester activation extends beyond the conventional , Ca2+-dependent PKCs that are activated during PTP ( see Discussion above ) .",
"Indeed , we have shown previously that ∼half of the phorbol ester potentiation of spontaneous release at the calyx synapse depended on an intact DAG binding site of Munc13-1 ( Lou et al . , 2008 ) .",
"Thus , phorbol ester activates at least two parallel signaling pathways at the release machinery: on the one hand , Munc13-1 via direct binding to its C1 domain ( Betz et al . , 1998; Rhee et al . , 2002; Lou et al . , 2008 ) , and on the other hand PKC which then phosphorylates Munc18-1 ( Figure 5; Wierda et al . , 2007; see Figure 5—figure supplement 1 ) .",
"The specificity of activation of conventional PKCs and phosphatases during PTP likely indicates the existence of signaling subcompartments in the presynaptic nerve terminal .",
"Indeed , a recent proteomics study showed that various protein phosphatases and conventional PKCs are enriched at short distances to P/Q-type and N-type Ca2+ channels ( Müller et al . , 2010 ) , indicating a PKC–protein phosphatase signaling complex at the active zone .",
"How might phosphorylated Munc18-1 increase transmitter release during PTP ?",
"Munc18-1 was first described as a protein which tightly binds to Syntaxin ( Pevsner et al . , 1994; Dulubova et al . , 1999 ) .",
"The binding of Munc18-1 to Syntaxin is thought to maintain the latter in a closed state not amenable to SNARE complex formation .",
"An early study showed that the phosphorylated Munc18-1 has a reduced binding affinity for Syntaxin ( Fujita et al . , 1996 ) ; thus , PKC phosphorylation could aid in the transition from a binary Munc18-1/Syntaxin complex to incorporation into SNARE complexes .",
"However , it was shown later that Munc18-1 also binds to the SNARE complex ( Dulubova et al . , 2007 ) , and stimulates SNARE-mediated vesicle fusion in a reconstituted system , by binding to the partially assembled SNARE complex ( Shen et al . , 2007 ) .",
"Therefore , Munc18-1 likely remains present at the partially formed SNARE complexes of docked and readily releasable vesicles; interestingly , Munc13-1 is also present in this complex ( Ma et al . , 2013 ) .",
"In this model , PKC phosphorylation of Munc18-1 would then increase the release probability of readily releasable vesicles , in what appears a ‘post-priming’ regulatory step of vesicle fusion ( see schematic in Figure 5—figure supplement 1 ) .",
"A ‘post-priming’ regulation of the fusion competence of docked vesicles during PTP is consistent with previous Ca2+ uncaging data at the calyx synapse .",
"It was shown that phorbol esters induce a shift and a decreased slope in the dose–response curve between transmitter release rate and presynaptic Ca2+ concentration ( Lou et al . , 2005 ) ; this modulation was reduced by PKC inhibitors ( Korogod et al . , 2007 ) .",
"We could not apply Ca2+ uncaging to study PTP , since PTP is not observed during presynaptic whole-cell recordings ( Korogod et al . , 2005; Lee et al . , 2010 ) .",
"To gain further insights into the mechanism of release modulation following Munc18-1 phosphorylation , we made use of a phosphomimetic mutation of Munc18-1 , in which the S306 and S313 sites were changed to aspartate residues ( called M18SD mutant ) .",
"With this mutation , however , functionally rescued synapses were difficult to obtain , and synapses which were rescued showed significantly smaller EPSCs as compared to synapses rescued with M18WT and M18SA constructs ( p<0 . 05; Figure 4—figure supplement 2 ) .",
"The M18SD mutant led to a block of PTP consistent with the role of PKC phosphorylation in PTP , but the paired-pulse ratio was not changed significantly ( Figure 4—figure supplement 2 ) .",
"Thus , while phosphorylation of Munc18-1 is required for PTP , mimicking its phosphorylation is not sufficient to cause a constitutive increase in release probability .",
"We cannot rule out alternative explanations , like homeostatic plasticity ( Davis , 2013 ) which could have reverted the increased release probability , or incorrect folding of the M18SD mutant protein; the latter might also be the reason for the lower rescue efficiency .",
"Taken together , the M18SD mutant data do not allow us to further extend our model of how Munc18-1 phosphorylation by PKC leads to an increased transmitter release .",
"Munc18-1 is essential for vesicle fusion at synapses ( Figure 3; Verhage et al . , 2000 ) , and Munc18-1 is expressed widely in the brain ( Website , 2012; Lein et al . , 2007 ) .",
"However , PTP is probably not observed at all synapses in the brain , and target-cell specific differences in PTP mechanisms have been observed for hippocampal mossy fiber synapses ( Lee et al . , 2007 ) .",
"A long-lasting residual Ca2+ signal , caused by mitochondrial Ca2+ uptake and release mechanisms , is important for short-term enhancement of release including PTP ( Kamiya and Zucker , 1994; Regehr et al . , 1994; Tang and Zucker , 1997; Habets and Borst , 2005; Korogod et al . , 2005; Lee et al . , 2007 ) .",
"It is possible that differences in Ca2+ extrusion mechanisms , as well as differential subcellular localization of members of the signaling complex downstream of Ca2+ , like conventional PKCs , determine the expression of PTP at a given synaptic connection .",
"Together with previous work ( Wierda et al . , 2007 ) , our study implies that besides having an essential function for vesicle fusion , Munc18-1 can also modulate the release process following PKC phosphorylation .",
"Our study identifies an important physiological context , post-tetanic potentiation , for this presynaptic regulatory pathway .",
"We hypothesize that Munc18-1 is present at the partially formed SNARE complex of readily releasable vesicles , where it would exert a modulatory role on the fusion process , depending on its phosphorylation state .",
"Future studies could investigate the role of Munc18-1 dependent presynaptic plasticity for information processing in neuronal networks , by making use of the PKC-insensitive mutation in transgenic mouse approaches ."
],
[
"Protocols of animal experiments with mice and rats were approved by the Veterinary Office of the Canton of Vaud , Switzerland .",
"For the experiments shown in Figures 3–5 , Figure 3—figure supplements 1 , 2 , Figure 4—figure supplements 1 , 2 , we used floxed Munc18-1 mice ( Heeroma et al . , 2004 ) that were generated by insertion of loxP sites flanking exon 2 of the mouse Stxbp1 gene by homologous recombination in embryonic stem cells ( Heeroma et al . , 2004 ) .",
"Floxed Stxbp1 mice were bred to homozygosity and referred to as Munc18-1lox/lox mice .",
"Munc18-1lox/lox mice were injected at postnatal day 1 ( P1 ) with adenovirus unilaterally into the ventral cochlear nucleus ( VCN ) under isoflurane anesthesia , following a subcutaneous lidocaine injection .",
"In general , mice were used for brainstem slice preparation 8–10 days following virus injection .",
"In some experiments with Cre-recombinase alone , stereotactic injections were performed at different ages ( P1–P6 ) to test the efficiency of Munc18-1 removal depending on postnatal injection time .",
"In these initial experiments , slices were made at 7–8 days following injection ( Figure 3—figure supplements 1 , 2 ) .",
"The stereotactic coordinates were adjusted from the previously established VCN injections at P6 mice ( Kochubey and Schneggenburger , 2011 ) .",
"Lambda was located through the skin , still transparent at P1 , and the mouse head was aligned in a model 900 stereotactic instrument ( Kopf Instruments , Tujunga , CA ) for the lambda , and the point 3 . 7 mm anterior from lambda , being in one horizontal plane and lying on the longitudinal axis of the instrument .",
"The skin and the skull were co-punctured at two points which were 0 . 3 and 0 . 9 mm posterior , and 1 . 57 mm lateral from lambda .",
"The virus ( 0 . 2 µl per site ) was injected with a 35G stainless steel needle ( Coopers Needle Works , Birmingham , UK ) inserted through the punctures , at three sites vertically spaced at 150 µm from each other ( maximal depth , 4 . 0 mm from surface ) .",
"The injection rate was 80 nl/min , using a SP120PZ syringe pump ( WPI , Berlin , Germany ) and a 10 µl syringe ( Hamilton , Bonaduz , Switzerland ) .",
"The mice recovered from anesthesia in 10–20 min and were brought back to the mother .",
"In order to replace the endogenous Munc18-1 protein of Munc18-1lox/lox mice , we developed adenovirus vectors which , for the final experiments , were capable to drive the expression of three proteins: Munc18-1 ( either in wild-type , or mutated form ) ; Cre-recombinase; and GFP to label calyces of successfully transduced presynaptic neurons in the VCN ( see Figure 3A1–A3 ) .",
"The constructs were designed as follows .",
"We used an open reading frame of a codon-optimized Cre recombinase ( Gradinaru et al . , 2010 ) which was placed downstream of an IRES sequence .",
"The IRES sequence was preceded either by the GFP sequence ( see Figure 3A2 ) , or by the Munc18-1 sequence ( splice variant b ) , in wild-type or mutated form ( Figure 3A3 , Figure 4A ) .",
"In the phosphorylation deficient mutant ( M18SA ) , three serine residues 306 , 312 and 313 were changed to alanine ( Wierda et al . , 2007 ) .",
"In the phosphomimetic mutant , the serine residues S306 and S313 were changed to aspartate ( called M18SD mutant; see Figure 4—figure supplement 2; ‘Discussion’ ) .",
"All expression cassettes were under control of the neuron-specific human synapsin1 promoter ( Kügler et al . , 2003; Young and Neher , 2009 ) , were preceded by the Kozak sequence GCCACC , and followed by the SV40 polyadenylation signal .",
"A second-generation serotype 5 adenovirus system was used to deliver and drive the expression of the constructs in vivo .",
"A shuttle vector pDC511 ( Microbix Biosystems , Ontario , Canada ) encoding eGFP-IRES-CreT cassette was used in combination with a custom-modified pBHGfrtΔ1 , 3FLP adenovirus backbone ( Microbix Biosystems ) in which the gene 2a was also deleted ( Zhou and Beaudet , 2000; Young and Neher , 2009 ) .",
"To enable simultaneous eGFP expression in rescue experiments , the shuttle vectors encoding for Munc18WT/SA-IRES-Cre cassettes were combined with a backbone carrying an additional hsyn:eGFP expression cassette ( Young and Neher , 2009 ) .",
"Adenovirus was propagated and purified as previously described ( Kochubey and Schneggenburger , 2011 ) using the E2T packaging cell line ( Zhou and Beaudet , 2000 ) , including the plaque purification step .",
"Final purification was done from the total lysate of 5 × 15 cm cell culture plates using Adenopack 100 RT kit ( Sartorius , Aubagne , France ) with the final buffer containing ( in mM ) 250 sucrose , 10 HEPES , 1 MgCl2 , pH 7 . 4 , which typically resulted in ∼750 µl of injection-ready virus stock with 1–2·1012 particles/ml titer ( OD260 ) .",
"For the experiments shown in Figures 1 and 2 , transverse slices of brainstem ( 200 µm ) containing the medial nucleus of the trapezoid body ( MNTB ) were prepared from Wistar rats at P8–P10 .",
"For the experiments in Figures 3–5 , Munc18-1lox/lox mice that had undergone stereotaxic surgery at P1 , to express one of the above described expression constructs in the VCN , were used at P9–P12 .",
"We used the eGFP-fluorescence of calyces of Held to select for presynaptically transduced neurons using a monochromator ( TILL Photonics; Gräfelfing , Germany ) at an excitation wavelength of 470 nm , a filter set with a 470/30 bandpass excitation filter , Q495LP dichroic mirror , and dual-band eGFP+IR emission filters ( AHF Analysentechnik , Tübingen , Germany ) .",
"Whole-cell patch-clamp recordings from MNTB neurons , or from pairs of calyces of Held and MNTB neurons were made at room temperature ( 21–24°C ) using an EPC-9/2 double patch-clamp amplifier ( HEKA Elektronik , Lambrecht , Germany ) .",
"The microscope set-up was a BX-51WI upright microscope ( Olympus , Tokyo , Japan ) , equipped with a 60×/0 . 9 NA water-immersion objective ( Olympus ) , IR-DIC illumination system ( Olympus ) and an Andor iXon 885 EM-CCD camera ( Andor Technology , Belfast , UK ) .",
"In case of fiber stimulation experiments , afferent axonal fibers were stimulated with a custom-made platinum-iridium bipolar electrode , which was placed close to the midline of the brainstem slice .",
"In these cases , MNTB neurons that were amenable to successful midline stimulation were pre-selected , by measuring the action current generated at the calyx of Held synapse in response to afferent stimulation ( Borst et al . , 1995 ) .",
"It is possible that by this procedure , we also selected for calyces of Held in which Munc18-1 function was rescued successfully , since calyces with little rescue might not respond to the afferent fiber test .",
"During whole-cell recordings of postsynaptic MNTB neurons , the series resistance ( Rs; 3–10 MΩ ) was compensated by up to 90%; the remaining Rs error in postsynaptic currents was corrected offline ( Meyer et al . , 2001 ) .",
"In presynaptic recordings , Rs was 8–20 MΩ and was compensated by ∼ 50% .",
"The slice incubation solution contained ( in mM ) : 125 NaCl , 25 NaHCO3 , 2 . 5 KCl , 1 . 25 NaH2PO4 , 1 MgCl2 , 2 CaCl2 , 25 glucose , 0 . 4 ascorbic acid , 3 myo-inositol , 2 Na-pyruvate , pH 7 . 4 when bubbled with 95% O2/5% CO2 at 37°C .",
"The solution for slice patch-clamp recording was similar , but contained in addition 2 µM strychnine and 10 µM bicuculline for the fiber stimulation experiments ( Figures 1 , 2 , 4 and 5; Figure 2—figure supplements 1 , 2; Figure 4—figure supplements 1 , 2 ) .",
"For the paired pre- and post-synaptic recordings ( Figure 3 , Figure 3—figure supplements 1 , 2 ) , this solution contained , in addition , 10 mM tetraethylammonium chloride ( TEA-Cl ) , 1 μM tetrodotoxin ( TTX ) , 50 μM D-2-Amino-5-phosphonopentanoic acid ( D-AP5 ) , 2 mM γ-D-glutamylglycine ( γ-DGG ) , 100 μM cyclothiazide ( CTZ ) .",
"The patch-pipette solutions contained ( in mM ) : 140 Cs-gluconate , 20 TEA-Cl , 10 HEPES , 5 Na2Phosphocreatine , 4 Mg2ATP , 0 . 3 Na2GTP , pH 7 . 2 .",
"This solution was supplemented with 5 or 0 . 1 mM Cs-EGTA for post- or presynaptic recordings , respectively .",
"TTX , D-AP5 , CTZ and γ-DGG were from BIOTREND ( Wangen , Switzerland ) .",
"All the other chemicals were from Sigma Aldrich/Fluka ( Buchs , Switzerland ) unless indicated .",
"N-myristoylated cell-permeable PKC and PKA inhibitor peptides with specific pseudosubstrate sequences ( myr-FARKGALRQ-amide , and myr-GRTGRRNAI-amide , respectively ) were purchased from Merck ( Darmstadt , Germany ) , reconstituted in H2O at 10 mM , and kept as frozen stocks .",
"For the experiments , one slice at a time was pre-incubated in the recording chamber by circulating 10 ml of extracellular solution containing 10 µM of inhibitor peptide for 30 min at 32–36°C as set by a temperature control unit ( Warner Instruments , Hamden , CT ) .",
"Thereafter , the perfusion solution was allowed to cool to room temperature , and fiber-stimulation experiments were done in the continuous presence of the inhibitor peptide .",
"Calyculin A ( Millipore , Zug , Switzerland; 1 µM final concentration ) , phorbol-12 , 13-dibutyrate ( Merck; 1 µM ) and Neomycin ( Sigma; 10 µM ) were acutely applied via a gravity-driven bath perfusion .",
"U73122 ( Merck ) was dissolved in DMSO ( Sigma ) , and slices were incubated at the final concentration of 3 µM U73122 , or in 0 . 1% DMSO vehicle for at least 45 min prior to electrophysiology experiments , using a small slice keeping chamber containing ∼20 ml of O2/CO2 equilibrated keeping solution ( see above ) .",
"For analysis of Munc18-1 expression in calyces of Held , mice were deeply anesthetized 8–10 days after virus injection by pentobarbital ( Streuli , Uznach , Switzerland; 100–200 mg/kg peritoneally ) , and transcardially perfused with 4% paraformaldehyde ( PFA ) in phosphate buffered saline ( PBS ) .",
"After post-fixation in 4% PFA and dehydration in 30% sucrose in PBS , transverse brainstem sections containing the MNTB were cut frozen at 40 μm on a sliding microtome Hyrax S30 ( Carl Zeiss , Oberkochen , Germany ) .",
"The sections were processed as described ( Felmy and Schneggenburger , 2004 ) .",
"We used chicken anti-GFP ( 1:1000; 13 , 970; Abcam , Cambridge , UK ) and mouse anti-myc antibodies ( 1:50; 9B11; Cell Signaling Technology , Boston , MA ) , and secondary Alexa488 goat anti-chicken ( A11039 ) and Alexa647 donkey anti-mouse ( A31571 ) antibodies ( from Life Technologies , Carlsbad , CA ) , which were applied overnight at 4°C , at 1:200 dilutions .",
"The sections were mounted in DAKO fluorescence mounting medium ( Dako , Glostrup , Denmark ) and imaged on an inverted SP2 confocal microscope ( Leica Microsystems ) with a 40× oil immersion objective using 488 and 633 nm laser lines .",
"Data were analyzed using custom-written routines in IgorPro 6 . 2 ( Wavemetrics , Lake Oswego , OR ) .",
"Spontaneous mEPSCs were detected by a semi-automated analysis routine in IgorPro , implementing template-matching detection algorithm ( Clements and Bekkers , 1997 ) .",
"Detected mEPSC events were visually inspected before acceptance .",
"The mEPSC frequency time courses were constructed by splitting the 9 . 7 s long mEPSC traces in two time intervals ( Figures 1E , 4B2–D2 ) or by using the entire interval as one time bin ( Figure 5F ) .",
"Statistical significance was assessed by unpaired two-tailed Student’s t test .",
"When appropriate , a paired Student’s t test was employed ( Figure 1B , C , F , G , 5G , Figure 2—figure supplements 1 , 2 , Figure 4—figure supplement 1 ) .",
"Error bars report the standard error of the mean ( SEM ) , and the statistical significance is indicated as follows: n . s . , not significant ( p≥0 . 05 ) ; *p<0 . 05; **p<0 . 01; ***p<0 . 001 ."
]
] | [
"Transmitter release at synapses is regulated by preceding neuronal activity , which can give rise to short-term enhancement of release like post-tetanic potentiation ( PTP ) .",
"Diacylglycerol ( DAG ) and Protein-kinase C ( PKC ) signaling in the nerve terminal have been widely implicated in the short-term modulation of transmitter release , but the target protein of PKC phosphorylation during short-term enhancement has remained unknown .",
"Here , we use a gene-replacement strategy at the calyx of Held , a large CNS model synapse that expresses robust PTP , to study the molecular mechanisms of PTP .",
"We find that two PKC phosphorylation sites of Munc18-1 are critically important for PTP , which identifies the presynaptic target protein for the action of PKC during PTP .",
"Pharmacological experiments show that a phosphatase normally limits the duration of PTP , and that PTP is initiated by the action of a ‘conventional’ PKC isoform .",
"Thus , a dynamic PKC phosphorylation/de-phosphorylation cycle of Munc18-1 drives short-term enhancement of transmitter release during PTP ."
] | [
"Brain function depends on the rapid transfer of information from one brain cell to the next at junctions known as synapses .",
"Small packages called vesicles play an important role in this process .",
"The arrival of an electrical action potential at the nerve terminal of the first cell causes some vesicles in the cell to fuse with the cell membrane , and this leads to the neurotransmitters inside the vesicles being released into the synapse .",
"The neurotransmitters then bind to receptors on the second cell , which leads to an electrical signal in the second cell .",
"A protein called Munc18-1 has a central role in the fusion of the vesicle at the cell membrane .",
"The strength of a synapse—that is , how easily the first brain cell can impact the electrical behaviour of the second—can change , and this ‘synaptic plasticity’ is thought to underlie learning and memory .",
"Long-term changes in synaptic strength require additional receptors to be inserted into the membrane of the second cell .",
"However , synapses can also be temporarily strengthened: the arrival of a burst of action potentials—a tetanus—causes some synapses to increase the amount of neurotransmitter they release in response to any subsequent , single , action potential .",
"This temporary increase in synaptic strength , which is known as post-tetanic potentiation , requires an enzyme called protein kinase C; the role of this enzyme is to phosphorylate specific target proteins ( i . e . , to add phosphate groups to them ) .",
"Now , Genç et al . have genetically modified a mouse synapse in vivo and shown that protein kinase C brings about post-tetanic potentiation by phosphorylating Munc18-1 .",
"Furthermore , pharmacological experiments show that proteins called phosphatases , which de-phosphorylate proteins , normally terminate the post-tetanic potentiation after about one minute .",
"Taken together , the study identifies a target protein which is phosphorylated by protein kinase C during post-tetanic potentiation .",
"The study also suggests that in addition to its fundamental role in vesicle fusion , the phosphorylation state of Munc18-1 can change the probability of vesicle fusion in a more subtle way , thereby contributing to synaptic plasticity ."
] | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | Mitochondrial flashes regulate ATP homeostasis in the heart | elife-23908-v1 | [
[
"ATP is the most important energy currency and mitochondria constitute the largest cellular powerhouse , supplying ATP at a rate of ~6 kg/day in the human heart ( Neubauer , 2007 ) .",
"More than an energy currency , ATP also plays diverse roles in physiological processes including signal transduction , ion channel regulation , cytoskeleton remodelling , and gene transcription ( Rolfe and Brown , 1997 ) .",
"The regulation of intracellular ATP concentration is therefore a vital homeostatic function shared by all tissues and eukaryotic cells .",
"An exemplary case is found in mammalian myocardium - the rate of energy consumption and supply ( i . e . , ATP flux ) augments by 5–10 folds in situations of fight-or-flight or exercise , but the ATP concentration ( i . e . , ATP set-point ) remains remarkably constant ( Balaban et al . , 1986; Neely et al . , 1973; Matthews et al . , 1981; Allue et al . , 1996 ) .",
"The mechanism underlying such exquisite ATP homeostatic regulation has been extensively pursued over the last several decades .",
"ADP , Pi and Ca2+ have been suggested to be regulators of ATP homeostasis , but challenges exist because of the relative constancy of ADP and Pi ( Katz et al . , 1989; Yaniv et al . , 2010; Balaban , 2012 ) and the extremely low activity of the mitochondrial Ca2+ uniporter ( MCU ) in cardiac myocytes ( Fieni et al . , 2012; Williams et al . , 2013 ) .",
"Moreover , recent findings in MCU knockout mice suggest that MCU is dispensable for energy metabolism except under extreme physiological stress ( Pan et al . , 2013 ) .",
"Myocardial overexpression of the dominant negative MCU increases , rather than decreases , mitochondrial oxygen consumption due in part to secondary effect on cytosol Ca2+ homeostasis ( Rasmussen et al . , 2015 ) .",
"To date , deciphering the exact mechanism underlying ATP set-point regulation in the heart remains an unmet challenge .",
"Mitochondrial flash ( mitoflash ) represents a recently discovered dynamic activity of the mitochondria ( Wang et al . , 2008 ) and has been ubiquitously detected in all eukaryotic species examined , from C . elegans to zebrafish and to rodents and humans ( Wang et al . , 2008; Hou et al . , 2014; Wang et al . , 2016a; Shen et al . , 2014; Zhang et al . , 2015 ) .",
"Individual mitoflash consists of multiple signal components including bursting superoxide production , transient matrix alkalization , oxidative redox shift , transient depletion of the electron donors NADH and FADH2 , and mitochondrial membrane potential depolarization ( Wang et al . , 2008 , 2016b ) .",
"The generation of mitoflashes in intact cells requires the integrity of the electron transfer chain ( ETC ) ( Wang et al . , 2008 ) ; and mitoflash frequency is highly regulated over a wide dynamic range by factors including metabolic state , thus the mitoflash activity is considered a biomarker for mitochondrial energy metabolism under certain conditions ( Wei et al . , 2011; Pouvreau , 2010; Fang et al . , 2011; Gong et al . , 2015 ) .",
"Most recently , we have shown that protons produced by photolysis or electroneutral proton ionophores act as a powerful mitoflash trigger ( Wang et al . , 2016b ) .",
"This finding is instructive because , in the Mitchell chemiosmotic theory of ATP synthesis ( Nicholls and Ferguson , 2002; Mitchell , 1961 ) , proton gradients , vectorial proton movement , and proton motive force ( ΔμH ) across the inner mitochondrial membrane are quintessential for energy metabolism .",
"Thus , the mitoflash biogenesis may be mechanistically and functionally intertwined with energy metabolism at multiple levels .",
"Emboldened by these recent advances , we revisited the fundamental question of ATP set-point regulation .",
"Our central hypothesis to be tested is that mitoflashes might provide the long-sought regulatory mechanism for ATP homeostasis in the mammalian heart .",
"In particular , we sought to determine whether mitoflash activity is able to regulate mitochondrial ATP production , how mitoflash responds to altered ATP supply and expenditure , and whether manipulation of mitoflashes can reset the level at which the cellular ATP concentration is maintained stable .",
"In the scenario that mitoflash emerges as the ATP set-point regulator , we also attempted to identify a possible physiological trigger that couples the mitoflash activity with the regulation of ATP homeostasis .",
"Our findings indicate that , by sensing and counteracting the ATP supply-and-demand imbalance , mitoflashes appear to constitute a digital auto-regulator for the maintenance of ATP homeostasis in the heart ."
],
[
"To interrogate a possible role of mitoflashes in the regulation of ATP homeostasis , we first determined whether mitoflashes exert any direct effect on mitochondrial metabolic activity and , in particular , ATP production .",
"To circumvent complicated homeostatic regulation at the cellular level , we opted to use isolated cardiac mitochondria freshly prepared from mt-cpYFP-transgenic mouse hearts ( Wang et al . , 2008 ) .",
"While the mitochondrial respiration was supported by the presence of succinate , ADP and phosphate , mitoflash activity was visualized with confocal imaging , and the rate of ATP production was measured with the luciferin luminescence method ( Jouaville et al . , 1999 ) .",
"We found that mitoflash events occurred randomly at a rate of 26 ± 2 events / ( 1000 µm2·100s ) ( Figure 1A and B ) and individual mitoflashes exhibited an average rise time of 7 s , peak amplitude of 0 . 5 ( ΔF/F0 ) , and duration of 22 s , comparable with those in intact cardiac myocytes ( Figure 1—figure supplement 1 ) .",
"Treatments using the mitochondria-targeted antioxidants , SS31 and mitoTEMPO , or the mitochondrial permeability transition pore ( mPTP ) inhibitor cyclosporin A ( CsA ) decreased the mitoflash frequency by 44% , 31% , and 47% ( Figure 1A and B ) , whereas the amplitudes and time courses of mitoflashes were largely unchanged ( Figure 1—figure supplement 1 ) .",
"Parallel luciferin luminescence measurement showed that , along with the repression of mitoflash activity , the rate of ATP production , assessed as the cumulative ATP formation over a 5 min time window , was enhanced by 25% , 24% , and 22% , respectively ( Figure 1C ) .",
"This result suggests that mitoflashes negatively regulate ATP production at the organelle level .",
"In other words , mitoflash activity may also be directly involved in the regulation of energy metabolism , in addition to serving a biomarker of respiratory activity ( Gong et al . , 2015 ) . 10 . 7554/eLife . 23908 . 003Figure 1 . Mitoflashes negatively regulate ATP production in isolated cardiac mitochondria .",
"( A ) Inhibition of mitoflashes by mitochondrial targeted antioxidants , mitoTEMPO ( 1 μM ) or SS31 ( 50 μM ) , and the mPTP inhibitor CsA ( 2 μM ) .",
"Surface plots show mitoflashes registered in 60 s periods overlaying the respective confocal images .",
"A spike indicates a mitoflash event .",
"Scale bar: 10 μm .",
"( B ) Statistics of mitoflash frequency .",
"n = 9–22 image files per group; **p<0 . 01 versus control .",
"( C ) Mitochondrial ATP production measured with the luciferin luminescence assay .",
"Data are expressed as fold-change relative to control group .",
"n = 6 experiments per group; *p<0 . 05; **p<0 . 01 versus control . DOI: http://dx . doi . org/10 . 7554/eLife . 23908 . 00310 . 7554/eLife . 23908 . 004Figure 1—source data 1 . Source data for Figure 1 . This file contains raw source data used to make the graphs presented in Figure 1B , Figure 1C and Figure 1—figure supplement 1 .",
"Excel software was used to graph all the quantitative data and perform statistical analyses .",
"Student’s t-test was applied to determine the statistical significance . DOI: http://dx . doi . org/10 . 7554/eLife . 23908 . 00410 . 7554/eLife . 23908 . 005Figure 1—figure supplement 1 . Averaged traces of mitoflashes aligned by onset . Data were from isolated cardiac mitochondria treated with mitochondrial targeted antioxidants , SS31 ( 50 μM ) or mitoTEMPO ( 1 μM ) , or the mitochondrial permeability transition pore inhibitor CsA ( 2 μM ) .",
"n = 117–169 events per group .",
"Note that no significant changes in amplitude or kinetic properties of individual mitoflashes were found . DOI: http://dx . doi . org/10 . 7554/eLife . 23908 . 005 To investigate whether or not mitoflash activity responds to altered energy metabolism , we first tipped the ATP supply-and-demand balance by altering mitochondrial substrate supply in intact cardiac myocytes .",
"As shown in Figure 2A , all metabolites examined , including glucose , free fatty acid and pyruvate , dose-dependently increased the mitoflash frequency , consistent with previous reports ( Fang et al . , 2011; Pouvreau , 2010; Wei et al . , 2011; Gong et al . , 2015 ) .",
"Strikingly , 10 mM pyruvate , the immediate metabolite that enters mitochondria and feeds the tricarboxylic cycle , elicited the most vigorous mitoflash activity , increasing the mitoflash frequency by 12 folds comparing to 5 . 6 mM glucose ( Figure 2A ) .",
"This stimulatory effect on mitoflash activity was fully reversed upon pyruvate washout , and was largely prevented by α-cyano-4-hydroxycinnamic acid , an inhibitor of the mitochondrial pyruvate transporter ( Figure 2A ) .",
"Likewise , a large and robust mitoflash response was obtained in beating hearts under Langendorff perfusion: switching from 5 . 6 mM glucose to 10 mM pyruvate augmented the mitoflash frequency from 1 . 2 ± 0 . 3 to 11 . 5 ± 2 . 8 mitoflashes / ( 1000 µm2·100s ) ( Figure 2B ) . 10 . 7554/eLife . 23908 . 006Figure 2 . Mitoflash and ATP responses to metabolic stimulation in adult cardiac myocytes .",
"( A ) Mitoflash frequencies in the presence of different metabolic substrates .",
"n = 14–39 cells per group .",
"*p<0 . 05; **p<0 . 01 versus substrate-free group; ##p <0 . 01 versus 10 mM pyruvate group .",
"FFA , free fatty-acid .",
"Here we used 0 . 6 mM palmitate .",
"( B ) Pyruvate ( 10 mM ) stimulation of mitoflashes in perfused heart .",
"Left: Confocal image of epimyocardium in an mt-cpYFP transgenic mouse heart under Langendorff perfusion .",
"Scale bar , 50 μm .",
"Right: statistics .",
"n = 13 cells from three hearts .",
"**p<0 . 01 versus 5 . 6 mM glucose group .",
"( C ) Time-resolved ATP/ADP ratio ( top , indexed by PercevalHR signal change , n = 39 cells ) , ATP content ( middle , indexed by luciferin luminescence , n = 26 cells ) , and mitoflash frequency ( bottom ) in response to pyruvate stimulation ( 10 mM ) .",
"Arrow heads indicate the time of adding pyruvate .",
"a . u . , arbitrary units .",
"The pH-corrected PercevalHR signal is reported as the normalized fluorescence ratio .",
"The smooth curve overlaying the time-course of the mitoflash frequency was drawn by eye .",
"n = 7 cells per datum point; **p<0 . 01 versus basal conditions .",
"( D ) Changes of oxygen consumption rate ( OCR ) upon pyruvate stimulation ( 10 mM ) .",
"n = 5 independent experiments .",
"**p<0 . 01 versus 5 . 6 mM glucose group .",
"( E ) Changes in NADH content .",
"As a positive control , inhibition of complex I , which oxidizes NADH into NAD+ , by 1 μM rotenone increased NADH autofluorescence .",
"n = 54–67 cells per group .",
"**p<0 . 01 versus 5 . 6 mM glucose group .",
"a . u . arbitrary units .",
"( F ) FAD autofluorescence upon 10 mM pyruvate stimulation .",
"The trace is the averaged data from 16 cells .",
"Arrow heads indicate the time of adding pyruvate or FCCP .",
"Note that a decrease of FAD indicates an increase of FADH2 . DOI: http://dx . doi . org/10 . 7554/eLife . 23908 . 00610 . 7554/eLife . 23908 . 007Figure 2—source data 1 . Source data for Figure 2 . This file contains raw source data used to make the graphs presented in Figure 2A–2F .",
"Excel software was used to graph all the quantitative data and perform statistical analyses .",
"Student’s t-test was applied to determine the statistical significance . DOI: http://dx . doi . org/10 . 7554/eLife . 23908 . 007 Parallel measurements revealed that the intracellular ATP content ( measured with the luciferin luminescence method ) and the ATP/ADP ratio ( measured with the indicator PercevalHR [Tantama et al . , 2013] ) exhibited an initial overshoot upon the glucose-to-pyruvate switch and returned to its baseline in about 100 s ( Figure 2C ) , indicating a transient escape of homeostatic control of the ATP level .",
"Interestingly , the time-course response of mitoflashes showed that the significant rise of mitoflash activity occurred concomitantly with a decline in the ATP transient ( Figure 2C ) .",
"Along with the high activity of mitoflashes at the steady state , the rate of oxygen consumption and the cellular levels of NADH and FADH2 were all increased despite the constancy of ATP ( Figure 2D–2F ) , indicative of decreased efficiency of ATP synthesis and substantiating the conclusion that mitoflashes negatively regulate ATP production .",
"Thus , the higher mitoflash activity in response to superfluous substrate supply tends to dampen ATP production and restore the ATP concentration to its set-point .",
"In the second set of experiments , we aimed to investigate the response of mitoflashes to ATP supply-and-demand imbalance caused by altering energy expenditure .",
"In cardiac myocytes , a single heartbeat consumes ~2% of the cellular ATP ( Jacobus , 1985 ) .",
"The higher the heart rate , the greater is the rate of ATP expenditure and replenishment .",
"We therefore assessed the mitoflash response to electrical pacing at various frequencies .",
"To minimize interference from cell shortening , image acquisition in contracting cells was synchronized with the electrical pacing ( See Materials and methods ) .",
"The mitoflash activity in pyruvate-treated cells declined precipitously at the onset of electrical pacing , alongside the onset of intracellular Ca2+ transients and cell shortenings; it was readily restored at the offset of pacing , with the cessation of Ca2+ and contractile responses ( Figure 3A and Figure 3—figure supplement 3 ) .",
"Quantitatively , mitoflash frequency was diminished by 45% at 1 Hz , 69% at 2 Hz , and 80% at 5 Hz pacing ( Figure 3B ) .",
"Similar results were found in cardiomyocytes exposed to physiological 5 . 6 mM glucose in Tyrode’s solution , i . e . , increasing the workload inhibited rather than stimulated mitoflash activity ( Figure 3C ) .",
"Remarkably , the cellular ATP content and ATP/ADP ratio were held constant regardless of pacing at different frequencies ( Figure 3D ) , substantiating the tightness of the ATP set-point regulation . 10 . 7554/eLife . 23908 . 008Figure 3 . Mitoflash and ATP responses to workload changes in adult cardiac myocytes .",
"( A ) Electrical pacing suppressed mitoflash activity .",
"Top: Space-time plots of mitoflashes right before , during , and right after 1 Hz electrical pacing in a cardiac myocyte in the presence of 10 mM pyruvate .",
"Each plot shows events registered in 10 s periods .",
"Scale bar , 10 μm .",
"Middle: Continuous recordings of cell length and Ca2+ transients .",
"Downward and upward deflections indicate cell shortenings and Ca2+ transients reported by Rhod-4 fluorescence , respectively .",
"Bottom: Raster plots of mitoflash incidence in three representative cells subject to the pacing protocol .",
"( B ) Inverse relationship between the incidence of mitoflashes during pacing and the pacing frequency at 10 mM pyruvate .",
"n = 12–17 cells per group .",
"**p<0 . 01 versus resting group .",
"( C ) Inverse relationship between the mitoflash frequency during pacing and the pacing frequency at 5 . 6 mM glucose .",
"n = 8–27 cells per group .",
"*p<0 . 05; **p<0 . 01 versus resting group .",
"( D ) Real-time measurement of cellular ATP content with the luciferin luminescence assay ( n = 37 cells ) and ATP/ADP ratio with PercevalHR ( n = 8 cells ) during pacing at different frequencies .",
"a . u . , arbitrary units .",
"The pH-corrected PercevalHR signal is reported as the normalized fluorescence ratio .",
"( E–G )",
"Effects of β-adrenergic stimulation and EGTA treatment on mitoflash activity ( E ) , ATP content ( measured with Mg2+-Green ) ( F ) , and ATP/ADP ratio ( G ) .",
"Isoproterenol ( ISO; 1 μM ) or 5 mM EGTA in 0 Ca2+ Tyrode’s solution was used to alter Ca2+ cycling and hence cellular energy expenditure in quiescent cardiac myocytes in 10 mM pyruvate .",
"For panel D , n = 13–19 cells per group .",
"*p<0 . 05; **p<0 . 01 versus control .",
"For panel E , n = 7–15 cells per group .",
"For panel F , n = 6–9 cells per group . DOI: http://dx . doi . org/10 . 7554/eLife . 23908 . 00810 . 7554/eLife . 23908 . 009Figure 3—source data 1 . Source data for Figure 3 . This file contains raw source data used to make the graphs presented in Figure 3A–3G .",
"Excel software was used to graph all the quantitative data and perform statistical analyses .",
"Student’s t-test was applied to determine the statistical significance . DOI: http://dx . doi . org/10 . 7554/eLife . 23908 . 00910 . 7554/eLife . 23908 . 010Figure 3—figure supplement 1 . Ca2+ transients and cell shortenings elicited by electrical pacing . Data from Figure 3A are shown on expanded scales . DOI: http://dx . doi . org/10 . 7554/eLife . 23908 . 010 Because the oxidative phosphorylation ( OXPHOS ) activity is expected to be elevated upon increasing workloads , together with the results in Figure 2 , it is clear that the mitoflash frequency is not a unique function of mitochondrial respiration .",
"This finding reinforces the notion that mitoflash plays an active role on its own .",
"To this end , we noticed that the rapid and reversible mitigation of mitoflash activity in response to pacing and the inverse relation between mitoflash activity and pacing frequency fit naturally the cell logic that the mitoflash acts as a rapid and sensitive responder to altered ATP demand .",
"For instance , when the ATP demand is augmented at the onset of pacing , the mitoflash activity is downregulated to alleviate its inhibition on ATP production ( Figure 1 ) , leading to increased ATP supply .",
"Similar reasoning is applicable to explain the equally fast mitoflash changes on cessation of electrical pacing .",
"Taken together , we propose a unifying interpretation - mitoflash acts as a reporter of ATP supply-and-demand imbalance and an ATP homeostasis regulator through negative regulation of ATP production .",
"That mitoflash can serve as the biomarker of mitochondrial respiration is merely a special case when the substrate supply is changed and at the same time the ATP demand is held constant .",
"Paradoxically , pacing-induced suppression of mitoflashes occurred concurrently with the onset of large cyclic intracellular Ca2+ transients , opposite to the prediction of Ca2+-dependent activation of mitoflashes in different cell types including cardiac myocytes ( Jian et al . , 2014; Gong et al . , 2014 ) .",
"This discrepancy is only apparent , because cardiac mitochondria are relatively inert in terms of Ca2+ uptake ( Fieni et al . , 2012; Williams et al . , 2013; Chen et al . , 2011a ) and a prolonged pacing protocol elicited only a moderate mitoflash-stimulatory effect as an after-effect ( Gong et al . , 2014 ) .",
"Thus , the bioenergetics-dependent mechanism might be more powerful than the Ca2+-dependent mechanism , thus dominating the mitoflash response in the present experimental conditions .",
"Next , we extended our studies to other situations of altered ATP demand .",
"Even in quiescence , it takes a significant portion of energy expenditure for a cardiac myocyte to maintain physiological Ca2+ gradients across different compartments .",
"In this regard , we demonstrated that β-adrenergic stimulation with isoproterenol ( ISO , 1 μM ) , to enhance Ca2+ cycling and thus to increase ATP demand , inhibited mitoflash frequency by 70% in pyruvate-treated cells ( Figure 3E ) .",
"In contrast , brief removal of extracellular Ca2+ ( 5 mM EGTA in nominal 0 Ca2+ solution ) increased mitoflash activity by 48% ( Figure 3E ) .",
"These results provide additional lines of evidence that mitoflash regulation is dominated by bioenergetics - rather than Ca2+-dependent mechanism in the present experimental settings .",
"Notably , the ATP content and ATP/ADP ratio were kept constant under either ISO stimulation or Ca2+ manipulation ( Figure 3F and G ) .",
"Taken together , both sets of experiments unveil a unifying interpretation of the mitoflash responses: mitoflash activity waxes and wanes in accordance with the ATP supply-and-demand imbalance , with higher activity occurring in response to over-exuberant ATP supply or diminished ATP demand , and vice versa .",
"The above results indicate that mitoflashes responding to altered energy supply and demand safeguard the ATP-set-point .",
"The question arises whether tuning mitoflash activity can shift the ATP set-point when both energy expenditure and substrate supply are held unchanged .",
"To answer this question , we inhibited mitoflashes with mitochondria-targeted antioxidants SS31 or mitoTEMPO , or overexpression of SOD2 in intact cardiac myocytes .",
"As shown in Figure 4A and B , all these manipulations decreased mitoflash frequency and increased the ATP content even in intact cardiac myocytes .",
"Quantitatively , the ATP set-point was elevated by ~60% while the mitoflash activity was mitigated by ~40% ( Figure 4A and B ) .",
"Linear regression revealed a strong inverse correlation between the mitoflash activity and the cellular ATP content ( r = −0 . 96 , p=0 . 009 ) ( Figure 4B ) .",
"A similar trend was seen in cells under physiological condition ( at 5 . 6 mM glucose ) displaying a lower basal mitoflash incidence ( r = −0 . 79 , p=0 . 11 ) ( Figure 4C and D ) .",
"Together with the above findings that mitoflashes inhibits ATP production , this result indicates that mitoflash activity is a potent ATP set-point regulator in cardiac myocytes . 10 . 7554/eLife . 23908 . 011Figure 4 . Tuning mitoflash frequency shifts cellular atp set-point .",
"( A ) In the presence of 10 mM pyruvate , mitoflash activity was manipulated by mitoTEMPO ( 1 μM ) , SS31 ( 100 μM ) , or SOD2 overexpression ( SOD2 OE ) .",
"n = 17–51 cells per group for mitoflash detection .",
"n = 169–488 cells per group for ATP measurement with the luciferin luminescence assay .",
"*p<0 . 05; **p<0 . 01 versus control .",
"For SOD2 OE , Lac Z overexpression ( Lac Z ) was used as the control .",
"Data are expressed as fold-change relative to respective control group .",
"Inset shows a representative western-blot of SOD2 .",
"( B ) Inverse relation between ATP content and mitoflash frequency .",
"Dashed line shows the linear regression yielding .",
"r = −0 . 96 , p=0 . 009 .",
"( C ) As in ( A ) , except that data were obtained in 5 . 6 mM glucose .",
"n = 15–36 cells per group for mitoflash detection and 98–531 cells per group for ATP measurement .",
"*p<0 . 05; **p<0 . 01 versus control or LacZ .",
"( D ) Linear regression analysis of ( C ) ( r = −0 . 79 , p=0 . 11 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 23908 . 01110 . 7554/eLife . 23908 . 012Figure 4—source data 1 . Source data for Figure 4 . This file contains raw source data used to make the graphs presented in Figure",
"4 . Excel software was used to graph all the quantitative data and perform statistical analyses .",
"Student’s t-test was applied to determine the statistical significance .",
"Prism linear regression analysis was used to investigate the correlation between mitoflash activity and ATP content .",
"The coefficient of determination ( R2 ) was used to evaluate the goodness of fit of the linear regression model . DOI: http://dx . doi . org/10 . 7554/eLife . 23908 . 012 The above results indicate that mitoflashes regulate the cellular ATP homeostasis in a digital and frequency-modulatory manner .",
"Then , what is the specific mechanism that senses the ATP supply-and-demand imbalance and regulates mitoflash activity ?",
"To date , three mitochondrial signals have been shown to be effective triggers or activators of mitoflashes , i . e . , matrix Ca2+ , basal ROS , and protons ( at nanodomains of the inner mitochondrial membrane ) ( Hou et al . , 2013; Jian et al . , 2014; Wang et al . , 2016b ) .",
"Mitochondrial Ca2+ is unlikely the candidate signal for the bioenergetics-dependent mitoflash activity , because no apparent Ca2+ changes were detected upon pyruvate-stimulated mitoflash activation ( Figure 5—figure supplement 1A ) and electrical pacing elicited opposite mitoflash and Ca2+ responses .",
"Moreover , inhibiting MCU with Ru360 did not show any significant effect on mitoflash frequency ( Figure 5—figure supplement 2 ) .",
"The lack of significant effect of Ru360 on mitoflashes in cardiomyocytes is expected because pyruvate treatment did not induce any changes in cytosolic Ca2+ and thus mitochondrial Ca2+ uptake ( Figure 5—figure supplement 1A ) .",
"However , mitoflash is sensitive to MCU inhibition in situations when it is induced by elevations of mitochondrial Ca2+ , e . g . , in HeLa cells under hyperosmotic stress or high cytosolic Ca2+ stimulation ( in permeabilized cells ) ( Hou et al . , 2013; Jian et al . , 2014 ) .",
"Further , electrical pacing did not alter ( Figure 5—figure supplement 1C ) and pyruvate stimulation caused a slight decrease of mitochondrial ROS ( Figure 5—figure supplement 1B ) , indicating that ROS are unlikely a major player , either .",
"TMRM measurement revealed little effects of these manipulations on ΔΨm , excluding changes in ΔΨm as a possible cause of the mitoflash response ( Figure 5—figure supplement 1D ) .",
"Given that protons act as a potent trigger of mitoflashes ( Wang et al . , 2016b ) , we reckoned that ATP synthesis-uncoupled proton entry might be a logic candidate for the trigger of mitoflashes to counteract the ATP supply-and-demand imbalance .",
"While proton entry at the F1Fo-ATP synthase drives ATP production , there exist multiple routes for ATP synthesis-uncoupled futile proton entry , and varying the proportion of the coupled and uncoupled proton entry would effectively alter the metabolic efficiency .",
"We therefore determined the proton leakage by measuring the ATP-synthesis uncoupled oxygen consumption rate ( OCR ) .",
"Interestingly , we found that the proton leakage associated OCR was augmented by 28% in response to increasing ATP supply , i . e . 10 mM pyruvate stimulation ( Figure 5—figure supplement 3 ) .",
"Increasing workloads by ISO treatment attenuated the proton leakage associated OCR by 11% while decreasing energy expenditure by brief removal of extracellular Ca2+ ( 5 mM EGTA in nominal 0 Ca2+ solution ) elevated the proton leakage associated OCR by 14% ( Figure 5—figure supplement 3 ) .",
"This result indicates that the ATP-synthesis uncoupled proton leakage varies in accordance with the changes of ATP supply and demand , consistent with its role in sensing the ATP supply-and-demand imbalance and triggering mitoflashes .",
"In search for the specific uncoupled proton leak for the triggering of mitoflashes , we examined possible involvement of proton leakage through mitochondrial uncoupling proteins ( UCPs ) .",
"Inhibition of UCP2 , the dominant isoform of UCPs in heart ( Ricquier and Bouillaud , 2000 ) ( Figure 5—figure supplement 4 ) with either genipin ( Zhang et al . , 2006 ) or RNA interference , slightly increased , rather than decreased , the mitoflash incidence ( Figure 5A–5C and Figure 5—figure supplement 4 ) .",
"This result excludes UCPs as the source of mitoflash-triggering proton flux .",
"Recently , a Bcl-xL-regulated proton leakage through F1Fo-ATPase has been demonstrated with multiple approaches , including patch-clamping of sub-mitochondrial vesicles , proton flux measurement , and immunochemical demonstration of a Bcl-xL interaction with the β-subunit of ATP synthase in hippocampal neuronal mitochondria ( Alavian et al . , 2011; Chen et al . , 2011b ) .",
"We therefore turned our attention to this F1Fo-ATPase-mediated proton leakage with its hallmark sensitivity to Bcl-xL regulation .",
"Indeed , we found that Bcl-xL overexpression , to inhibit the uncoupled proton flux , markedly decreased mitoflash incidence , whereas knockdown of Bcl-xL with siRNAs ( Figure 5—figure supplement 5 ) , increased mitoflashes at both 5 . 6 mM glucose and 10 mM pyruvate ( Figure 5D and E ) .",
"Likewise , treatment with ABT-737 , a mimetic of BH3-only proteins that inhibits Bcl-xL ( Oltersdorf et al . , 2005 ) , also elevated mitoflash production ( Figure 5D ) .",
"Furthermore , the mitoflash responses were accompanied by a 19% upward shift of the ATP set-point with Bcl-xL overexpression , and an 11% downward shift with ABT-737 treatment ( Figure 5F and G ) .",
"These lines of evidence support the notion that proton leakage through ATP synthase is an important physiological trigger of mitoflashes , coupling the mitoflash biogenesis with auto-regulation of ATP homeostasis in cardiac myocytes . 10 . 7554/eLife . 23908 . 013Figure 5 . Proton leakage through F1Fo-ATPase , not UCP2 , triggers mitoflashes .",
"( A–C )",
"Effect of UCP2 knockdown and inhibition on mitoflashes .",
"( A ) Slight change of mitoflash frequency after UCP2 inhibition with genipin ( 50 μM ) .",
"n = 12 adult cardiac myocytes per group .",
"**p<0 . 01 versus control .",
"( B ) Representative western-blot for UCP2 knockdown in neonatal cardiac myocytes .",
"( C ) Effects of UCP2 knockdown or genipin treatment ( 50 μM ) on mitoflash activity in neonatal cardiac myocytes .",
"n = 28–42 cells per group .",
"*p<0 . 05; **p<0 . 01 versus NC .",
"( D ) Mitoflash activity altered by Bcl-xL inhibition or overexpression ( OE ) in adult cardiac myocytes .",
"Bcl-xL was inhibited by ABT-737 ( ABT , 10 μM ) .",
"Inset shows representative western-blot for Bcl-xL overexpression .",
"n = 33–37 cells per group .",
"*p<0 . 05; **p<0 . 01 versus control .",
"( E ) Activation of mitoflashes by Bcl-xL knockdown in neonatal cardiac myocytes .",
"Three double-strand small RNAs ( siRNA-1 , siRNA-2 , siRNA-3 ) targeted to Bcl-xL or a negative control siRNA ( control ) were transfected .",
"Inset shows representative western-blot analysis for Bcl-xL knockdown .",
"n = 22–60 cells per group .",
"*p<0 . 05; **p<0 . 01 versus control .",
"( F ) Effects of Bcl-xL inhibition or overexpression on ATP content .",
"Adult cardiac myocytes were in 10 mM pyruvate containing solution .",
"n = 203–363 cells per group .",
"**p<0 . 01 versus control .",
"( G ) Inverse relation between ATP content and mitoflash frequency in the presence of 10 mM pyruvate . DOI: http://dx . doi . org/10 . 7554/eLife . 23908 . 01310 . 7554/eLife . 23908 . 014Figure 5—source data 1 . Source data for Figure 5 . This file contains raw source data used to make the graphs presented in Figure 5A , Figure 5C–5G , Figure 5—figure supplement 1A–D , Figure 5—figure supplement 2A–C , Figure 5—figure supplement 3 , Figure 5—figure supplement 4A–C , and Figure 5—figure supplement",
"5 . Excel software was used to graph all the quantitative data and perform statistical analyses .",
"Student’s t-test was applied to determine the statistical significance . DOI: http://dx . doi . org/10 . 7554/eLife . 23908 . 01410 . 7554/eLife . 23908 . 015Figure 5—figure supplement 1 . Ca2+ , basal ros and mitochondrial membrane potential in adult cardiac myocytes responding to pyruvate stimulation or workload alterations .",
"( A ) Cytosolic Ca2+ response to 10 mM pyruvate stimulation .",
"The cytosolic Ca2+ was measured with Rhod-4 .",
"Arrow indicates the time of adding pyruvate .",
"The trace shows the average result from 8 cells .",
"( B ) Effect of 10 mM pyruvate stimulation on mitochondrial basal ROS measured with mitoSOX .",
"n = 22 cells per group .",
"( C ) Effect of electrical pacing on mitochondrial basal ROS .",
"n = 20 cells .",
"( D ) TMRM measurements of ΔΨm in different experimental conditions .",
"n = 66–79 cells per group . DOI: http://dx . doi . org/10 . 7554/eLife . 23908 . 01510 . 7554/eLife . 23908 . 016Figure 5—figure supplement 2 . Effect of inhibiting MCU on mitoflashes in neonatal cardiac myocytes . Ru360 ( 5 μM ) was used to inhibit MCU and mitochondrial Ca2+ uptake was measured with mitochondrial targeted GCaMP5 ( mt-GCaMP5 ) .",
"( A ) Averaged traces of Ca2+ transients stimulated by 10 mM caffeine .",
"Arrow indicates the time of caffeine application .",
"n = 25 cells for control group and n = 93 cells for Ru360 group .",
"( B ) Statistics for the amplitude of Ca2+ transients in ( A ) .",
"*p<0 . 05 versus control .",
"( C ) Lack of effect of Ru360 on mitoflashes .",
"n = 25–31 cells per group .",
"**p<0 . 01 versus glucose group . DOI: http://dx . doi . org/10 . 7554/eLife . 23908 . 01610 . 7554/eLife . 23908 . 017Figure 5—figure supplement 3 . Changes of uncoupled proton leakage by altering ATP supply and demand . Mitochondrial proton leakage was indexed by the ATP synthesis-uncoupled oxygen consumption rate ( OCR ) in mouse adult cardiac myocytes .",
"10 mM pyruvate stimulation was used to increase ATP supply .",
"Isoproterenol ( ISO , 1 μM ) and 5 mM EGTA in nominal 0 Ca2+ solution ( EGTA ) were used to increase and decrease ATP expenditure , respectively .",
"n = 15–21 experiments per group .",
"*p<0 . 05; **p<0 . 01 . DOI: http://dx . doi . org/10 . 7554/eLife . 23908 . 01710 . 7554/eLife . 23908 . 018Figure 5—figure supplement 4 . Expression of UCP isoforms in cardiac myocytes .",
"( A , B )",
"Quantitative real-time PCR analysis for the expression of UCP2 and UCP3 in adult ( A ) and neonatal cardiac myocytes ( B ) .",
"Two pairs of primers were designed for either UCP2 ( UCP2-p1 and UCP2-p2 ) or UCP3 ( UCP3-p1 and UCP3-p2 ) .",
"n = 6 experiments .",
"Note that UCP2 is the dominant isoform expressed in the heart .",
"( C ) Western-blot analysis for siRNA knockdown of UCP2 .",
"Neonatal cardiac myocytes were transfected with one of the three double-stranded small RNAs ( siRNA-1 , siRNA-2 , or siRNA-3 ) targeted to UCP2 or a negative control siRNA ( NC ) and cultured for 72 hr . n = 3 experiments .",
"*p<0 . 05 versus NC . DOI: http://dx . doi . org/10 . 7554/eLife . 23908 . 01810 . 7554/eLife . 23908 . 019Figure 5—figure supplement 5 . Western-blot analysis of Bcl-xL Knockdown . Neonatal cardiac myocytes were transfected with three double-strand small RNAs ( siRNA-1 , siRNA-2 , siRNA-3 ) targeted to Bcl-xL or a negative control siRNA ( NC ) and cultured for 72 hr . n = 3 experiments .",
"*p<0 . 05; **p<0 . 01 versus NC . DOI: http://dx . doi . org/10 . 7554/eLife . 23908 . 019"
],
[
"It has been established for decades that myocardial ATP level remains constant in the face of large fluctuations in the rate of ATP consumption and production .",
"Indeed , the present results show that the ATP content and ATP/ADP ratio are held constant regardless of electrical pacing , altered substrate supply , receptor stimulation , and Ca2+ manipulations in isolated single cardiac myocytes , attesting to the exquisiteness of the ATP set-point regulation .",
"Ironically , as ADP , Pi and Ca2+ have been dismissed as the major players , the exact mechanism underlying ATP set-point regulation remains an unsolved enigma .",
"From the design principle of control systems , at least three criteria should be met for any candidate mechanism to be qualified as an ATP set-point regulator .",
"First , it should have an impact on mitochondrial ATP production through the OXPHOS process .",
"Second , it should respond to cellular ATP supply-demand imbalance .",
"More specifically , its direction of response should counteract the imbalance and its dynamic range as well as kinetics of response should be robust and fast enough to cope with 10-fold fluctuations of ATP supply and demand on a beat-to-beat basis .",
"Third , actively tuning this regulatory mechanism should be able to reset the ATP level in situations when the ATP supply and demand are held unaltered .",
"Additional mechanism is also required to sense the state of ATP supply-demand imbalance and to couple the regulator’s response with the OXPHOS process .",
"In this scenario , the major finding of the present study is that the recently-discovered , digital mitoflash activity meets all these criteria at once .",
"First , the current study provides direct evidence that mitoflashes negatively regulate ATP production in isolated cardiac mitochondria .",
"Inhibiting mitoflash activity can increase the rate of ATP production by 25% .",
"Multiple mechanisms could contribute to the mitoflash-mediated inhibition of ATP production .",
"First of all , multifaceted energy-consuming processes occur in a mitoflash , including diversion of electrons from energy metabolism to bursting superoxide production , dissipation of ΔΨm , and loss of membrane permeability due to flickering mPTP opening ( Wang et al . , 2008 , 2016b ) .",
"Albeit the ETC activity is accelerated as indicated by transient matrix alkalization and depletion of the electron-donor pools of NADH and FADH2 , ΔμH for ATP synthesis is dissipated because of loss of ΔΨm .",
"All these events indicate that mitochondria undergoing a mitoflash are at a state of futile respiration .",
"That is , intermittent mitoflashes of the ATP-generating organelles are analogous to discharges of the safety-valve of a working steam engine .",
"Second , as ΔμH dissipates , the Complex V could operate in the reverse mode , switching on its ATPase activity and consuming ATP in the flashing mitochondria .",
"Third , mitoflashes may activate some yet-unknown downstream pathways to impose prolonged inhibition of OXPHOS activity .",
"Regardless of specific mechanism of action , the finding that mitoflashes negatively regulate ATP production sheds new light on possible bioenergetics role of this digital activity built-in in the powerhouse of the cell .",
"Second , based on results from numerous maneuvers purported to perturb the ATP supply-and-demand balance , a unifying pattern emerges in which mitoflashes counteract the imbalance between ATP supply and expenditure , whereby safeguarding the ATP set-point .",
"Specifically , the mitoflash frequency increases with either superfluous substrate supply or diminished energy demand ( e . g . , offset of electrical pacing , removal of extracellular Ca2+ ) ; conversely , it mitigates upon substrate washout or with enhanced workload and energy expenditure ( e . g . , onset of electrical pacing , β-adrenergic stimulation ) .",
"Remarkably , the dynamic range of mitoflash frequency spans over an order of magnitude and its kinetics are fast enough to cope with sudden changes occurring in the pacing protocol , allowing for only a transient escape of ATP from its tightly controlled constant level .",
"Thus , compared to the interpretation of mitoflashes as a biomarker of mitochondrial respiration ( Gong et al . , 2015 ) , which holds true in certain conditions ( e . g . , varying substrate supply without altering the ATP demand ) , we re-interpret mitoflashes as a reporter of ATP supply-and-demand imbalance and an ATP set-point regulator through its ability to regulate ATP production .",
"Third , not only that the mitoflash stabilizes the ATP level in fluctuations of energy expenditure and substrate supply , but also it participates in determining the specific level at which cellular ATP is held stable .",
"When the mitoflash activity is manipulated independently of changes in ATP supply and demand , e . g . , by ROS scavenging , SOD2 overexpression , and Bcl-xL manipulation , it can tune the ATP set-point upwardly or downwardly: the higher the mitoflash activity is , the lower the ATP set-point becomes .",
"The negative correlation between mitoflash activity and ATP level attained is in remarkable contrast to the constancy of ATP level in situations of electrical pacing , receptor stimulation and calcium manipulation , and testifies the robustness of this mitoflash-mediated ATP set-point regulation .",
"It is of interest to note that mitoflash regulation of the ATP set-point bears prominent features of a digital , distributed control system .",
"Individual mitoflashes occur only intermittently , and are confined to single mitochondria .",
"They are digital and operate in the frequency-modulatory mode , i . e , the average amplitude and duration of mitoflashes are relatively constant in different conditions .",
"At the single-mitochondrion level , a mitoflash is a dramatic event blanketing the entire organelle and interrupting the ATP synthesis .",
"In a cardiac myocyte typically containing 5 , 000–8 , 000 mitochondria , only a tiny portion of mitochondria undergo the flashing activity at any moment; No central control mechanism is identifiable in this control system , because stochastic mitoflash events are evenly distributed among the population of mitochondria , with their frequency rapidly and tightly regulated in accordance with the ATP supply-and-demand imbalance .",
"Keeping in mind that ATP diffuses freely in the cytosol , such local , stochastic ATP-negating events afford a novel mechanism to finely regulate ATP homeostasis at the cellular level .",
"Taken together , we conclude that mitoflashes act as the long-sought ATP set-point regulator in the mammalian heart .",
"In search for possible mechanism that couples metabolism and mitoflashes in the context of ATP set-point regulation , we demonstrate that the proton leakage through F1Fo-ATP synthase regulated by Bcl-xL ( Chen et al . , 2011b; Alavian et al . , 2011 ) provides a physiological proton trigger of mitoflashes for the regulation of metabolic efficiency .",
"Our recent studies have shown that protons at the nanodomains of inner mitochondrial membrane trigger mitoflashes ( Wang et al . , 2016b ) , suggesting a possible link between mitoflash biogenesis and the OXPHOS process at the deepest mechanistic level .",
"In the present study , we first show that the ATP synthesis- uncoupled proton leakage varies in accordance with alterations in ATP supply or expenditure , increasing when the supply is superfluous and decreasing when the ATP demand is high .",
"More importantly , we then provide direct evidence that bidirectional manipulations of Bcl-xL-sensitive proton leakage , but not those of UCP2 , cause robust , bidirectional changes in the mitoflash frequency and ATP content .",
"By analogy of local Ca2+ signalling ( Cheng and Lederer , 2008 ) , we have envisaged a ‘local control model’ for proton triggering of mitoflashes ( Wang et al . , 2016b ) in order to explain the finding that protons from different sources differ in terms of mitoflash triggering efficiency .",
"Recent numerical analysis has revealed that free protons are short-lived ( lifetime ~1 . 4 ns ) and diffuse only over a nanometre scale ( ~2 . 1 nm ) in the matrix environment ( Wang et al . , 2016b ) .",
"Furthermore , free protons are extremely scarce in an alkaline mitochondrial matrix ( ~0 . 4 protons in a mitochondrion of 2 μm length and 200 nm diameter at pH 8 . 0 ) , and matrix pH buffering capacity is estimated to be about 5 mM per pH unit at pH 8 . 0 ( i . e . , 1:500 , 000 for free: bound proton ratio ) ( Poburko et al . , 2011 ) .",
"Thus , we envision that only protons in the nanoscopic vicinity of the putative trigger sites can effectively serve their role as the mitoflash trigger .",
"Consistent with this model , super-resolution microscopy has shown , in the mitochondria from neurons , nanoscale protein partition of UCP4 and F1Fo-ATP synthase , residing in the inner boundary membrane and the cristae membrane , respectively ( Klotzsch et al . , 2015 ) .",
"Several groups have independently proposed that F1Fo-ATP synthase is of the same molecular identity as that of mPTP ( Giorgio et al . , 2013; Alavian et al . , 2014; Bonora et al . , 2013; Bernardi et al . , 2015 ) , whose flickering openings signify the ignition of mitoflashes ( Wang et al . , 2008; Hou et al . , 2014 ) .",
"If this picture turns out to be true , both proton leakage and mitoflash triggering would occur within the same macrocomplex protein machinery , thus conferring high selectivity among different proton sources .",
"In summary , we have presented a set of novel and cohesive findings on mitoflashes as a digital auto-regulator of ATP homeostasis in the heart .",
"First , mitoflashes respond rapidly and robustly to ATP supply-and-demand imbalance , increasing with a superfluous supply and mitigating with a greater demand .",
"Because mitoflashes negatively regulate mitochondrial ATP production , such bioenergetics-dependent mitoflash activity acts as an auto-regulator of the ATP homeostasis ( mode I , Figure 6 ) , akin to the safety-valve of a steam engine .",
"Second , tuning the mitoflash activity is able to shift the level at which ATP is held constant ( mode II , Figure 6 ) , revealing that mitoflash activity is an important , heretofore unappreciated determinant of the ATP set-point .",
"Third , we demonstrate that the Bcl-xL-sensitive proton leakage through F1Fo-ATP synthase constitutes a physiological proton trigger of mitoflashes ( Figure 6 ) .",
"These findings not only mark mitoflashes as the putative auto-regulator for cellular ATP homeostasis , but also uncover compelling cell logic for the biogenesis of mitoflashes in the heart . 10 . 7554/eLife . 23908 . 020Figure 6 . Schematic of mitoflash regulation of ATP set-point . In mode I , mitoflash activity , which is triggered by ATP synthesis-uncoupled proton leakage , waxes and wanes in accordance with fluctuations in ATP supply or demand , and whereby stabilizes the ATP set-point at a constant level .",
"In mode II , selective tuning of the mitoflash activity ( e . g . , Bcl-xL-regulated proton leakage through F1Fo-ATP synthase , or antioxidant repression of mitoflash activity ) suffices to shift the ATP set-point upward ( at decreased mitoflash activity ) or downward ( at increased mitoflash activity ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 23908 . 020"
],
[
"Luciferin , carbonyl cyanide 4- ( trifluoromethoxy ) phenylhydrazone ( FCCP ) , pyruvate , palmitate , α-cyano-4-hydroxycinnamic acid ( α-CCA ) , isoproterenol ( ISO ) , blebbistatin , cyclosporin A ( CsA ) , antimycin A , and genipin were from Sigma .",
"Ethylene glycol tetraacetic acid ( EGTA ) was from Amresco .",
"ABT-737 ( ABT ) was from Selleck Chemicals ( Houston , Texas ) .",
"Rotenone was from Calbiochem .",
"MitoTEMPO was from Enzo Life Sciences .",
"Mg2+-Green AM , Rhod-4 AM , BCECF AM , and mitoSOX were from Invitrogen ( Eugene , Oregon ) .",
"The tetrapeptide SS31 ( D-arg-dmt-lys-phe-NH2 ) was synthesized as described ( Zhao et al . , 2004 ) .",
"All procedures were carried out according to the rules of the American Association for the Accreditation of Laboratory Animal Care International and approved by the Animal Care Committee of Peking University accredited by AAALAC International ( IMM-ChengHP-1 ) .",
"This investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health ( NIH Publication No . 85–23 , revised 1996 ) .",
"Single ventricular myocytes were enzymatically isolated from the hearts of adult male Sprague–Dawley rats ( 200–250 g ) or adult C57BL-6 mice ( 25–35 g ) , as described previously ( Cheng et al . , 1993; Shang et al . , 2016 ) .",
"Freshly-isolated cardiac myocytes were plated on culture dishes coated with laminin ( Sigma ) for 1 hr and then the attached cells were cultured in M199 medium ( Invitrogen , Carksbad , California ) along with ( in mM ) 5 creatine , 2 L-carnitine , 5 taurine , and 25 HEPES ( all from Sigma ) .",
"Cells were then infected with adenovirus carrying mt-cpYFP , firefly luciferase , Bcl-xL , or the SOD2 gene at an m . o . i . of 20 and experiments were performed after 60–72 hr in culture .",
"Ventricular myocytes were isolated from 1-day-old Sprague-Dawley rats , as described previously ( Shen et al . , 2007 ) .",
"Myocytes were plated at 5 × 105 cells/cm2 in DMEM ( Invitrogen ) supplemented with 10% FBS ( Hyclone ) in the presence of 0 . 1 mM 5-bromo-2-deoxyuridine ( Sigma ) .",
"Adenovirus infection or siRNA transfection was implemented after 24 hr quiescence in serum-free DMEM following 48–72 hr culture in DMEM containing 10% FBS .",
"For adenovirus infection , cells were infected with adenovirus carrying mt-cpYFP , mt-GCaMP5 or the Bcl-xL gene at an m . o . i . of 20 .",
"For siRNA transfection , 100 nM siRNA was transiently transfected using Lipofectamine RNAiMax ( Invitrogen ) according to the manufacturer’s instructions .",
"The knockdown efficiency was assessed by western-blot .",
"The mitochondria were isolated from mouse hearts as previously reported ( Zhao et al . , 2012 ) .",
"Briefly , mouse hearts were washed with ice-cold isolation buffer ( 210 mM mannitol , 70 mM sucrose , 5 mM HEPES ( pH 7 . 4 ) , 1 mM EGTA and 0 . 5 mg/ml BSA ) , minced , and homogenized .",
"The homogenate was centrifuged at 4°C for 10 min at 1000 g , and the supernatant was collected and further centrifuged at 4°C for 10 min at 12000 g .",
"The pellet was re-suspended for functional assessment .",
"An inverted confocal microscope ( Zeiss LSM 710 ) with a 40× , 1 . 3 NA oil-immersion objective was used for imaging .",
"When acquiring the mt-cpYFP signal , images were captured by exciting alternately at 488 and 405 nm , and collecting the emission at >505 nm .",
"For mitoSOX measurement , the indicator ( 5 μM ) was loaded at 37°C for 20 min followed by washing three times .",
"The mitoSOX fluorescence was reported by exciting at 514 nm and collecting the emission at 559–740 nm .",
"In typical time-series recordings of mitoflashes , 100 frames of 900 × 256 ( for adult cardiac myocytes ) or 512 × 512 pixels ( for neonatal cardiac myocytes ) were collected at 0 . 10–0 . 14 μm/pixel in bidirectional scanning mode .",
"The frame rate was 30–60 frames/min , and the axial resolution was set to 1 . 0 μm .",
"All experiments were performed at room temperature ( 22–26°C ) unless specified otherwise .",
"For ex vivo imaging of mitoflashes , the heart was excised from mt-cpYFP transgenic mice ( 10–14 weeks old ) ( Wang et al . , 2008 ) , and the ascending aorta was cannulated with a customized needle .",
"The heart was perfused in the Langendorff configuration under constant perfusion pressure ( ~90 mmHg ) with oxygenated ( 100% O2 ) Tyrode’s solution consisting of ( in mM ) 137 NaCl , 5 . 4 KCl , 1 . 2 MgCl2 , 1 . 2 NaH2PO4 , 1 . 8 CaCl2 , 5 . 6 glucose or 10 pyruvate , and 20 HEPES ( pH 7 . 35 , adjusted with NaOH ) at 37°C .",
"The image acquisition plane was focused ~30 μm deep into the epimyocardium and the motion artefacts due to spontaneous beating of the heart were minimized with 10 μM blebbistatin ( Sigma ) .",
"For imaging mitoflashes in isolated cardiac mitochondria , the mitochondria were suspended in a solution consisting of ( in mM ) 100 potassium aspartate , 1 . 0 MgCl2 , 20 KCl , 0 . 5 EGTA , 10 glutathione , 20 HEPES , 8% dextran ( MW 35 , 000–45 , 000 ) , 10 KH2PO4 , 2 . 5 succinate , and 0 . 2 ADP ( pH 7 . 2 ) .",
"Typically , 100 frames of 512 × 512 pixels were collected at the rate of 60 frames/min .",
"In a subset of experiments , electrical field stimulation ( 5 ms square-wave pulses at 2× threshold voltage ) was applied via a pair of platinum electrodes , and cells were perfused with Tyrode’s solution at a rate of 3 ml/min .",
"Cells displaying healthy contractility were chosen for mitoflash measurement and those near the anode , where electrolysis can produce reactive oxygen species locally ( Jackson et al . , 1986 ) , were avoided .",
"These cautious measures were necessary because we have recently shown that ROS generated by electrolysis of Tyrode’s solution at the anode can cause significant increase in mitoflash as well as mitochondrial Ca2+ ( Zhang et al . , 2014 ) .",
"To minimize interference from cell shortening , image acquisition in contracting cells was at 1 s/frame and synchronized with the electrical pacing , such that it was phase-locked with cell contraction .",
"In parallel experiments , cell length and cytosolic Ca2+ transients were monitored continuously by placing the scan line along the long axis of the cell .",
"The cytosolic Ca2+ transients were measured by Rhod-4 with excitation at 543 nm and emission collection at 548–646 nm .",
"Fluorescent images of the line were acquired every 3 . 78 ms and the time-course of cell shortening was extracted from the line-scan images by an edge-detection algorithm .",
"Cellular ATP content was measured directly with the firefly luciferase-catalysed chemiluminescence method ( Jouaville et al . , 1999 ) and indirectly with the small-molecule indicator Mg2+-Green ( Campanella et al . , 2008 ) .",
"For chemiluminescence measurement , cultured cardiac myocytes expressing firefly luciferase were bathed in Tyrode’s solution with 1 mM luciferin ( Sigma ) .",
"The luminescence was recorded with an electron-multiplying charge-coupled device camera ( Andor iXon DU-897D-C00-#BV , Andor Technology , South Windsor , CT ) and analysed by AndoriQ software ( version 1 . 0 ) .",
"To measure the ATP content with Mg2+-Green , cells were loaded with 5 μM Mg2+-Green AM and the fluorescence was recorded with excitation at 488 nm and emission at >560 nm .",
"The cellular ATP/ADP ratio was measured by expressing PercevalHR ( Tantama et al . , 2013 ) in cardiac myocytes .",
"Briefly , the PercevalHR fluorescence was acquired by alternate excitation at 488 nm and 405 nm and emission collection at 506–702 nm .",
"To remove the pH bias of PercevalHR , we adopted the procedure developed previously ( Tantama et al . , 2013 ) .",
"Specifically , the changes of BCECF fluorescence and PercevalHR fluorescence upon pH alterations were recorded and an empirical linear correlation between the PercevalHR signal and the BCECF signal was established for calibration .",
"The pH bias in the PercevalHR signal was corrected by subtracting the estimated pH component based on the pH calibration of PercevalHR and parallel BCECF measurement .",
"For measuring ATP production in isolated cardiac mitochondria , the isolated mitochondria were suspended in respiration solution containing ( in mM ) 220 mannitol , 70 sucrose , 5 KH2PO4 , 2 . 5 MgCl2 , 0 . 5 EDTA , 2 . 5 succinate , 0 . 2 ADP , 2 HEPES ( pH7 . 4 ) , and 0 . 1% BSA .",
"The ATP content was measured with the luciferase assay ( Promega , Madison , Wisconsin ) .",
"Oxygen consumption rate in intact adult cardiac myocytes was measured with a Clark-type oxygen electrode ( Strathkelvin 782 2-Channel Oxygen System version 1 . 0; Strathkelvin Instruments , Motherwell , UK ) .",
"Briefly , 1 × 105 cells were suspended in Tyrode’s solution containing 5 . 6 mM glucose or 10 mM pyruvate and the oxygen consumption was measured over 10 min with the Strathkelvin System .",
"To measure ATP synthesis-uncoupled oxygen consumption rate , the mouse cardiac myocytes were cultured in XF24 cell-culture microplates ( Seahorse Bioscience ) .",
"The cells were suspended in different medium: Tyrode’s solution with 5 . 6 mM glucose , Tyrode’s solution with 10 mM pyruvate , Tyrode’s solution with 10 mM pyruvate and 1 μM ISO , nominal 0 Ca2+ Tyrode’s solution with 10 mM pyruvate and 5 mM EGTA .",
"Bioenergetics analyses were performed in an XF24 Extracellular Flux Analyzer ( Seahorse Bioscience ) with the injection of oligomycin ( 1 μM ) , FCCP ( 1 μM ) , and rotenone ( 1 μM ) and antimycin A ( 1 μM ) sequentially .",
"The uncoupled oxygen consumption rate was calculated as the oligomycin-insensitive percentage relative to basal oxygen consumption .",
"Cell lysates were separated by 4–12% NuePAGE ( Invitrogen ) and transferred to nitrocellulose membranes ( Millipore ) .",
"Membranes were blocked with 5% non-fat dry milk and incubated with primary antibody overnight at 4°C .",
"Monoclonal anti-UCP2 antibody ( Santa Cruz Biotechnology , California ) , monoclonal anti-Bcl-xL ( Sigma ) , polyclonal anti-SOD2 ( Abcam , RRID:AB_300434 ) , and anti-α-tubulin antibody ( Sigma , RRID:AB_477593 ) were used .",
"Blots were visualized using secondary antibodies conjugated with IRDye ( LI-COR , Lincoln , Nebraska ) and an Odyssey imaging system ( LI-COR ) .",
"Total RNA was extracted from adult or neonatal cardiac myocytes using Trizol reagent ( Invitrogen ) and converted to cDNA using M-MLV reverse transcriptase ( TaKaRa , Japan ) with oligo-dT primers .",
"Quantitative real-time PCR was performed using Trans Start Green qPCR Super Mix ( TransGen Biotech , China ) with primers designed with the NCBI/Primer-BLAST tool ( see the following table for the primer sequences ) .",
"The Ct values were obtained using CFX Manager Quantification software ( BioRad , Hercules , California ) and the relative expression of target genes was analysed by comparison with 18S rRNA .",
"Confocal images were analysed using custom-developed programs written in Interactive Data Language ( IDL , ITT ) .",
"Cell-motion artefacts and background fluorescence changes were corrected by image processing and individual mitoflashes were located with the aid of FlashSniper ( Li et al . , 2012; LJHIS007 , 2017; https://github . com/ljhis007/flashsniper . A copy is archived at https://github . com/elifesciences-publications/flashsniper ) .",
"Data are expressed as mean ± SEM .",
"When appropriate , Student’s t-test was applied to determine the statistical significance , and a simple linear regression model was used to investigate the correlation between mitoflash activity and ATP content .",
"The coefficient of determination ( R2 ) was used to evaluate the goodness of fit of the model .",
"Prism linear regression analysis was used for the statistical analysis .",
"p<0 . 05 was considered statistically significant .",
"The number of samples chosen for each comparison was determined based on past similar experiments ."
]
] | [
"The maintenance of a constant ATP level ( ‘set-point’ ) is a vital homeostatic function shared by eukaryotic cells .",
"In particular , mammalian myocardium exquisitely safeguards its ATP set-point despite 10-fold fluctuations in cardiac workload .",
"However , the exact mechanisms underlying this regulation of ATP homeostasis remain elusive .",
"Here we show mitochondrial flashes ( mitoflashes ) , recently discovered dynamic activity of mitochondria , play an essential role for the auto-regulation of ATP set-point in the heart .",
"Specifically , mitoflashes negatively regulate ATP production in isolated respiring mitochondria and , their activity waxes and wanes to counteract the ATP supply-demand imbalance caused by superfluous substrate and altered workload in cardiomyocytes .",
"Moreover , manipulating mitoflash activity is sufficient to inversely shift the otherwise stable ATP set-point .",
"Mechanistically , the Bcl-xL-regulated proton leakage through F1Fo-ATP synthase appears to mediate the coupling between mitoflash production and ATP set-point regulation .",
"These findings indicate mitoflashes appear to constitute a digital auto-regulator for ATP homeostasis in the heart ."
] | [
"A small molecule called ATP is often referred to as the primary “energy currency” of living cells .",
"It is required to power tasks as diverse as the general housekeeping processes that keep all cells alive to the programmed cell death response that dismantles any cells that are no longer needed .",
"It is also crucial that cells maintain a constant level of ATP at all times , even when the supply of and demand for ATP fluctuate .",
"This control is particularly important in the mammalian heart where the rates of ATP production and consumption change ten-fold during intense exercise .",
"Despite intensive research over the past decades , it was still not known how cells keep ATP levels constant .",
"In many cell types , including heart muscle cells , ATP is mainly produced inside compartments called mitochondria .",
"Each heart muscle cell contains between 5 , 000 and 8 , 000 mitochondria .",
"Recent experiments have shown that ATP production in mitochondria is interrupted by ten-second bursts called “mitochondrial flashes” ( or mitoflashes for short ) , during which the mitochondria release chemicals called reactive oxygen species .",
"The mitoflashes are tightly linked with energy usage , and Wang , Zhang , Wu et al . have now explored if and how mitoflashes regulate ATP levels in the heart .",
"Experiments on isolated mitochondria from mouse heart muscle cells showed that mitoflashes inhibit the production of ATP .",
"When the intact heart muscle cells were given excess of the building blocks needed to produce ATP – mitoflashes occurred more often .",
"Conversely , when the cells were forced to contract more quickly , which increased demand for ATP , the mitoflashes occurred less often .",
"Importantly , the level of ATP inside the cells actually remained constant in the experiments .",
"Furthermore , inhibiting mitoflashes with antioxidants increased the ATP concentration in heart muscle cells .",
"Lastly , Wang et al . demonstrated that mitoflashes could be triggered under certain conditions .",
"Overall , these experiments uncovered a way in which highly active cells can maintain a constant level of ATP .",
"Future studies are needed to understand exactly how mitoflashes are initiated and how they in turn inhibit ATP production .",
"A better understanding of these processes might uncover molecules that could be targeted by drugs to the control of the rate of ATP production to treat heart failure ."
] | 2017 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"plant biology"
] | Irreversible fate commitment in the Arabidopsis stomatal lineage requires a FAMA and RETINOBLASTOMA-RELATED module | elife-03271-v2 | [
[
"Plants exhibit remarkable developmental plasticity and their cells are typically considered totipotent , in that a complete plant can be regenerated from nearly any isolated individual cell .",
"In intact plants , however , distinct cell lineages emerge and terminal fates are stable .",
"A prime example of a specialized lineage is in the Arabidopsis leaf epidermis ( Figure 1A ) where asymmetric divisions of protodermal cells generate meristemoid mother cells ( MMC ) and meristemoids ( M ) , self-renewing cells akin to transit amplifying cells in mammalian stem cell lineages ( Lau and Bergmann , 2012; Pillitteri and Dong , 2013 ) .",
"At the end of their renewing stages , these meristemoids differentiate into guard mother cells ( GMCs ) , which undergo a single symmetric division to generate the paired guard cells ( GCs ) of the mature stomata .",
"GCs and each of the intermediate stages leading to their formation are characterized by distinct morphologies and unique gene expression profiles , allowing experimental dissection of lineage progression in intact , developing organs ( Lau and Bergmann , 2012; Pillitteri and Dong , 2013 ) . 10 . 7554/eLife . 03271 . 003Figure 1 . FAMA and RBR physically interact and regulate guard cell division and differentiation .",
"( A ) Schematic of key stages in stomatal development mediated by bHLHs SPCH , MUTE and FAMA .",
"Cell types are labeled as: meristemoid mother cell ( MMC ) , meristemoid ( M ) , stomatal lineage ground cell ( SLGC ) , guard mother cell ( GMC ) , guard cell ( GC ) , pavement cell ( PC ) .",
"( B and C )",
"Expression of FAMA and RBR in GMCs and GCs .",
"Confocal images of 5-days post germination ( dpg ) cotyledon of FAMAp:GFP-FAMA ( B , in green ) and RBRp:RBR-CFP ( C , in green ) .",
"Inset in ( B ) is a fama mutant GMC at 10-dpg .",
"Cell outlines ( purple ) were visualized with propidium iodide .",
"( D and E )",
"Reduction in RBR level leads to extra divisions in GCs .",
"Confocal images of a co-suppressed RBRp:RBR-CFP line ( D ) and FAMAp:amiRBR expressing a CDKA1;1 reporter ( green ) ( E ) .",
"Yellow arrowheads in ( D ) indicate ectopic cell divisions .",
"Cell outlines ( white ) were visualized with propidium iodide .",
"( F ) ClustalW2-based protein alignment of the LxCxE motif among FAMA relatives .",
"( G and H )",
"FAMA interacts with RBR in vivo and in vitro through its LxCxE motif .",
"Representative images ( G , left ) and quantified data ( G , right; rep: replicate ) of Bimolecular Fluorescence Complementation ( BIFC ) analysis between FAMA and RBR .",
"Pairs of CYCD/CYCDLGK-RBR and FAMA-bHLH93 ( Ohashi-Ito and Bergmann , 2006 ) were used as controls .",
"( H ) Yeast two-hybrid interaction assays between FAMA and RBR .",
"( I ) Complementation of seedling lethality in fama mutants by FAMALGK ( FAMAp:FAMALGK; fama ) .",
"( J–L )",
"Diversity of GC defects in adaxial cotyledon epidermis of 12-dpg FAMALGK .",
"DIC images of a mature GC showing strong phenotype ( J , false red colors indicate different GC units within another ) and a broader view of GCs with different defects ( K ) .",
"Key: ectopic asymmetric divisions ( arrowheads ) , new GC units ( asterisks ) , properly spaced divisions and GC units ( brackets ) .",
"Inset shows a lobed GC .",
"( L ) Quantitation of different classes of GC defects ( cartoons on Y-axis ) in FAMALGK at 6 , 9 and 12-dpg .",
"Bars represent the percentages of each class over all GCs on adaxial cotyledons .",
"All images are at the same magnification ( including insets in B and K ) .",
"Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03271 . 00310 . 7554/eLife . 03271 . 004Figure 1—figure supplement 1 . Additional images of FAMA promoter-driven expression of amiRBR and of FAMALGK-YFP .",
"( A ) Reduction of RBR levels under the FAMA promoter ( FAMAp:amiRBR ) drives ectopic cells divisions exclusively in guard cells ( black arrowheads ) .",
"( B–C )",
"Expression pattern of YFP-tagged FAMALGK ( FAMAp:FAMALGK-YFP , green ) rescuing the fama mutant is indistinguishable from the wild type in guard cells ( GCs ) of 6-dpg cotyledons; it first appears in GMCs ( single green nuclei ) , persists into young GCs ( pairs of green nuclei ) , but disappears before GCs mature and make full pores .",
"( D ) When ectopic GCs divisions appear , FAMALGK-YFP is absent from most divisions but only appears in cells ( top left ) that are likely new GMCs based on morphology .",
"Cell outlines ( purple ) were visualized with propidium iodide .",
"Scale bar in A , 50 μm , scale bars in B–D , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03271 . 00410 . 7554/eLife . 03271 . 005Figure 1—figure supplement 2 . Categorization of guard cell ( GC ) defects and increase in severity over time in FAMALGK .",
"( A ) DIC images of distinguishable phenotypic defects in GCs in FAMALGK plants ( FAMAp:FAMALGK; fama ) .",
"For quantification analysis , 13 distinct phenotypes ( 1–13 ) were identified at appreciable frequencies and grouped into 5 phenotypic classes ( box ) .",
"( B ) GC defects of a second , independent FAMALGK line from the one characterized in Figure 1L at 6 , 9 and 12-dpg; note similar results between the two lines .",
"Cartoons of GCs on the Y-axis indicate the 5 phenotypic classes scored .",
"Bars represent the average percentages of each phenotypic class over total number of GCs ( ± SEM ) in 0 . 320 mm−2 DIC images of adaxial cotyledons at the indicated age . DOI: http://dx . doi . org/10 . 7554/eLife . 03271 . 005 The basic helix-loop-helix ( bHLH ) transcription factor FAMA is a master regulator of guard cell identity; it is necessary and sufficient for GC fate acquisition and its epidermal expression is limited to GMCs and young GCs ( Ohashi-Ito and Bergmann , 2006 ) and ( Figure 1B ) .",
"GMCs are made in fama mutants , but they fail to progress into GCs and instead continue dividing while maintaining expression of earlier fate markers ( Ohashi-Ito and Bergmann , 2006 ) and ( Figure 1B , inset ) ; this failure to make GCs results in seedling lethality ( Ohashi-Ito and Bergmann , 2006 ) and ( Figure 1I ) .",
"Overexpression of FAMA reprograms other cells into GC identity , while simultaneously repressing cell division to yield single-celled stomata ( Ohashi-Ito and Bergmann , 2006 ) .",
"The mechanisms by which FAMA regulates cell division and terminal differentiation are not known , but FAMA's direct targets include cell cycle regulators and genes associated with mature guard cell function ( Hachez et al . , 2011 ) .",
"FAMA has been shown to act as a transcriptional activator ( Ohashi-Ito and Bergmann , 2006 ) but can also participate in repression of certain cell cycle targets ( Hachez et al . , 2011 ) .",
"Here we show that FAMA is required for the irreversible differentiation of GCs and that it fulfills this role through recruitment of the Arabidopsis Retinoblastoma homologue , RETINOBLASTOMA-RELATED ( RBR ) .",
"Point mutations that disrupt FAMA-RBR interactions render FAMA capable of promoting initial GC identity , but unable to maintain commitment .",
"By demonstrating FAMA-promoted binding of RBR to the regulatory regions of stomatal regulators whose genomic regions contain repressive chromatin marks , we define a molecular mechanism by which the ubiquitously expressed RBR is recruited to specific genomic contexts at specific times to regulate key developmental events ."
],
[
"RBR is broadly expressed in Arabidopsis development and reduction of RBR activity has been correlated with excess division and loss of cell identity in many different contexts , including the early stomatal lineage ( Borghi et al . , 2010 ) .",
"In the epidermis of actively dividing young leaves , RBRp:RBR-CFP ( Cruz-Ramirez et al . , 2012 ) is expressed in all cell nuclei; as the leaf matures , expression becomes restricted to stomatal lineage cells ( Figure 1C ) .",
"Mosaic co-suppression of the RBRp:RBR-CFP transgene leads to loss of fluorescence and concomitant excessive divisions in the CFP-minus sectors , suggesting that RBR represses cell divisions in both the early lineage and the terminally differentiated GCs ( Figure 1D ) .",
"To examine RBR's role specifically in the GCs , we drove expression of artificial microRNAs ( amiRNAs ) against RBR by the FAMA promoter .",
"FAMAp:amiRNA-RBR GCs underwent inappropriate extra divisions oriented transverse to the long axis of the cells , while other epidermal cells were not affected , confirming a direct requirement for RBR in GCs ( Figure 1E and Figure 1—figure supplement 1A ) and confirming phenotypes reported using different amiRNAs directed against RBR ( Lee et al . , 2014a ) .",
"FAMA encodes a canonical RBR binding motif ( LxCxE ) ( Burkhart and Sage , 2008 ) that is conserved among dicot FAMA orthologs , but not in FAMA's closest paralogs SPEECHLESS ( SPCH ) and MUTE ( Figure 1F ) .",
"LxCxE-dependent physical interaction between FAMA and RBR was tested by in planta Bimolecular Fluorescence Complementation ( BiFC ) ( Figure 1G ) and yeast two-hybrid ( Figure 1H ) assays .",
"In both assays , WT FAMA , but not a version bearing point mutations changing the Cysteine ( C ) and Glutamate ( E ) in the LxCxE motif to Glycine ( G ) and Lysine ( K ) ( FAMALGK ) could interact with RBR .",
"Importantly , FAMALGK could still interact with its dimerization partner bHLH93 ( Ohashi-Ito and Bergmann , 2006 ) ( Figure 1G ) , indicating that the FAMALGK variant maintains its overall structural integrity .",
"We then asked whether physical interaction with RBR was required for FAMA function in the context of normal leaf development .",
"FAMAp:FAMALGK was tested for its ability to complement fama lethality and defects in GC differentiation , and FAMAp:FAMALGK-YFP was monitored to determine whether the LCE→LGK modification altered FAMA's expression , stability or subcellular localization .",
"In young cotyledons and leaves , FAMAp:FAMALGK-YFP was exclusively nuclear .",
"Like G/YFP-tagged versions of FAMA published previously ( Ohashi-Ito and Bergmann , 2006; Pillitteri et al . , 2007; Lee et al . , 2014a ) , FAMAp:FAMALGK-YFP is first apparent in GMCs , remains highly expressed as the GMCs undergo cell division , and is downregulated as GCs mature such that stomata with clearly defined pores express the protein at low levels or not at all ( Figure 1B and Figure 1—figure supplement 1B–C ) .",
"Plants of genotype fama;FAMApro:FAMALGK ( hereafter referred to as FAMALGK plants ) were recovered and were moderately healthy and fertile , though smaller than wild type , indicating that FAMALGK was sufficient to rescue fama lethality ( Figure 1I ) .",
"In the GCs of rescued FAMALGK plants , however , we observed excessive cell divisions , changes in cell morphology , and , most strikingly , production of paired GCs inside of existing GCs ( Figure 1J–L and Figure 1—figure supplement 2 , phenotypic classes 6–11 ) .",
"Phenotypes conferred by FAMALGK and by manipulating RBR in the late stomatal lineage both involved increased cell division , but were not identical .",
"To improve phenotypic resolution , we characterized the expression patterns of cell fate and cell cycle markers in FAMALGK and FAMAp:amiRBR plants ( Figure 2 and Figure 2—figure supplement 1 ) .",
"This detailed analysis revealed clear phenotypic differences between reducing RBR levels in GMCs and reducing RBR's interaction with FAMA ( Figure 2—figure supplement 1A–B ) .",
"Notably , the FAMALGK phenotype results , not from chaotic or uncontrolled divisions and fate changes , but rather an orderly reiteration of stomatal lineage progression .",
"This manifested itself as a progressive increase in phenotypic severity with age ( Figure 1L ) and by the appearance of stomatal lineage markers in patterns suggesting that the GCs reverted to MMC identity and proceeded through the intermediate stages of the pathway normally ( Figure 2 ) .",
"Expression of stomatal-promoting transcription factors ( SPCH , MUTE , FAMA , Figure 2A–C and Figure 2—figure supplement 2 ) , stomatal-restricting signaling elements ( TMM , EPF1 , EPF2 , Figure 3A–D ) , and general division reporters ( CDKA1;1 , Figure 2B–C ) followed the normal temporal patterns , and ectopic GC divisions appeared to follow early lineage division rules .",
"For example , when a ‘reprogrammed’ GC produced two stomata , they were separated by a non-stomatal cell , indicating that spacing divisions occurred .",
"Distinct cell orientations characteristic of amplifying divisions were also visible ( Figure 2B and Figure 2—figure supplement 3 ) .",
"Further evidence for normal asymmetric divisions is polarized localization of BASL ( Dong et al . , 2009 ) in the larger daughter of a GC division ( Figure 3E ) .",
"Based on the lack of expression of stomatal lineage markers ( Figure 2D ) , we interpret the lobed GCs we observe at low , but significant , frequencies in FAMALGK plants ( Figure 1K , inset , and Figure 1L ) as cells that are transdifferentiating into an epidermal pavement cell identity . 10 . 7554/eLife . 03271 . 006Figure 2 . Disruption of FAMA-RBR interaction leads to failure of terminal differentiation and reiteration of stomatal lineage divisions and gene expression programs .",
"( A ) Diagram of stages of stomatal development ( abbreviated and color-coded as in Figure 1A ) and expression window of bHLH transcription factors SPCH , MUTE and FAMA .",
"( B ) Characterization of GC defects in FAMALGK plants accompanied by key stomatal reporters .",
"Wild type-looking GCs of FAMALGK plants re-iterate the stomatal developmental pathway , undergo further divisions and exhibit correct orderly expression of stage-specific stomatal regulators and cell cycle genes .",
"Each column from left to right represents a stage in the progression of the stomatal lineage ( abbreviated as in Figure 1A ) .",
"Rows from top to bottom show expression patterns of plasma membrane ( PM ) marker ( row 1 ) , and reporters of SPCH ( row 2 , beige ) , MUTE ( row 3 , orange ) , FAMALGK ( row 4 , red ) and CDKA1;1 ( row 5 ) .",
"Images are of independent GCs of adaxial cotyledons at 6 , 9 or 12-dpg .",
"( C ) Expression of each marker ( rows 1 to 5 ) in GCs that underwent amplifying or spacing divisions .",
"( D ) Guard cells exhibiting pavement cell-like lobed growth with no divisions or expression of stomatal and cell cycle reporters .",
"Cell outlines ( purple ) were visualized with propidium iodide or ML1p:mCherry-RCI2A .",
"Autofluorescence of chloroplasts ( blue spheres ) may be visible in some images .",
"All images are at the same magnification .",
"Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03271 . 00610 . 7554/eLife . 03271 . 007Figure 2—figure supplement 1 . Expression of cell cycle and stomatal reporters in FAMALGK plants ( FAMAp:FAMALGK;fama ) and amiRBR ( FAMAp:amiRBR ) mutants and examples of timelapse images for SPCH and MUTE markers .",
"( A ) GUS staining of transcriptional reporters for cell cycle genes CDKB1;1 and CDC6 ( rows ) in WT Col ( left column ) , amiRBR ( middle ) , and FAMALGK plants ( right column ) .",
"Note that the pattern and levels of expression differ between the RBR knockdown line ( amiRBR ) and when the interaction between FAMA and RBR is disrupted ( FAMALGK ) .",
"In the amiRBR line , CDKB1;1 and CDC6 are strongly expressed in both GCs , each of which displays ectopic cell divisions ( outline in left guard cell and arrowheads in the right guard cell ) .",
"Broad expression of CDKB1;1 and CDC6 is consistent with RBR's function as a direct repressor of the transcription factor E2F and its cell cycle target genes required for the G1 to S-phase transition ( Gutzat et al . , 2012 ) .",
"In FAMALGK plants , however , CDKB1;1 and CDC6 are restricted to only some stomatal cell divisions ( stars mark new GCs and arrows mark amplifying ACDs ) .",
"Expression of CDKB1;1 and CDC6 are likely consequences of regulated cell divisions as the mutant GCs progress through the stomatal lineage .",
"( B ) Confocal images of stomatal lineage reporters in GCs of amiRBR .",
"Cell outlines are visualized with propidium iodide ( purple ) .",
"Arrowheads correspond to ectopic cell divisions .",
"Of the reporters tested , SPCH and MUTE are weakly and infrequently seen ( <20% of GCs ) and only in GCs with many ectopic divisions ( green asterisks ) .",
"TMM and EPF2 , however , were not detectable .",
"Small blue disks visible in these cells are chloroplasts .",
"Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03271 . 00710 . 7554/eLife . 03271 . 008Figure 2—figure supplement 2 . Timelapse imaging of cell fate reporters in FAMALGK lines .",
"( A–B )",
"Examples of MUTE expression in reprogrammed guard cells .",
"MUTE expression always appears after an asymmetric division and before a symmetric division to create a new guard cell pair .",
"( C–D )",
"Examples of SPCH expression .",
"SPCH appears before divisions , persists after division in both daughters , but then becomes undetectable in one daughter ( white arrowheads track expressing cells in C ) .",
"SPCH expression has disappeared before cells undergo symmetric divisions to create the new guard cell pair .",
"All images are from abaxial cotyledons placed in the timelapse imaging chamber at 6 days post germination .",
"Time relative to first panel image in hours:minutes is indicated in the bottom right corner of each image .",
"Because development is asynchronous , T0 is a different absolute time for each montage . DOI: http://dx . doi . org/10 . 7554/eLife . 03271 . 00810 . 7554/eLife . 03271 . 009Figure 2—figure supplement 3 . Guard cells in FAMALGK plants reiterate the stomatal developmental pathway and undergo stereotypic stomatal asymmetric cell divisions that generate the diversity in phenotype .",
"( A ) Diagram of three types of asymmetric cell division ( ACD ) in the stomata lineage .",
"Entry division ( black ) of a meristemoid mother cell ( MMC ) initiates the lineage and results in the formation of a meristemoid ( M ) and a stomatal lineage ground cell ( SLGC ) .",
"Amplifying division ( green ) denotes a subsequent ACD of a meristemoid .",
"Spacing division ( blue ) is the ACD of a SLGC , where the newly formed M is spaced away from existing M , guard mother cell ( GMC ) or guard cells ( GCs ) .",
"( B ) GCs of FAMALGK plants re-enter the stomatal lineage and can undergo the three types of stomatal ACD .",
"DIC images of GCs from FAMALGK plants illustrating the entry ( black ) , amplifying ( green ) and spacing ( blue ) divisions and their subsequent progression in the lineage ( left to right ) .",
"Images were false colored to indicate MMC ( light blue ) , M ( beige ) , GMC ( orange ) , GC ( red ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 03271 . 00910 . 7554/eLife . 03271 . 010Figure 3 . Reprogrammed FAMALGK guard cells re-express early stomatal signaling components and polarity regulators .",
"( A ) Diagram of stages of stomatal development ( abbreviated as in Figure 1A ) and expression window of signaling and polarity regulators indicated as bars spanning lineage stages .",
"Re-expression of TMMp:TMM-YFP ( B ) , EPF1p:YFPnuc ( C ) , EPF2p:YFPnuc ( D ) , and BASLp:YFP-BASL ( E ) in GCs from adaxial cotyledons of 6-dpg FAMALGK seedlings .",
"Arrowhead in ( E ) indicates the polarized crescent characteristic of BASL in asymmetrically dividing cells .",
"Cell outlines ( purple ) were visualized with propidium iodide .",
"Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03271 . 010 In FAMAp:amiRBR lines , by contrast , excessive GC division was accompanied by elevation of cell cycle gene expression throughout the GCs ( CDKA1;1 , Figure 1E , CDKB1;1 and CDC6 , Figure 2—figure supplement 1A ) , but only rarely by misexpression of early stomatal lineage markers ( Figure 2—figure supplement 2B ) .",
"Consistent with gene expression behaviors , spacing and amplifying divisions were not seen in FAMAp:amiRBR cotyledon GCs at any appreciable frequency ( <1/1000 GCs ) in 6–12 day old plants .",
"Expression of an additional copy of tagged RBR ( RBR-CFP ) , however , does not alter divisions in the stomatal lineage; we observed neither arrested cells nor hyperproliferating cells ( Figure 4A–B ) .",
"In FAMALGK plants , GCs that undergo extra divisions re-express RBR as would be expected from RBR's normal expression pattern in the early stomatal lineage ( Figure 4C–G ) . 10 . 7554/eLife . 03271 . 011Figure 4 . Expression of RBRp:RBR-CFP reappears in reprogramed FAMALGK guard cells . RBRp:RBR-CFP ( green ) is expressed in GMCs and young GCs , but expression in WT does not confer any guard cell phenotype at 6-dpg ( A ) or 12-dpg ( B ) .",
"Reprogrammed guard cells in FAMALGK plants re-express RBR in specific cells ( green ) as they recapitulate the stomatal development pathway and undergo precursor divisions .",
"Meristemoid mother cell ( MMC ) and meristemoid ( M ) divisions ( asterisks ) captured at 6-dpg ( C ) and GMC and spacing asymmetric cell division ( ACD ) captured at 12-dpg ( D–G ) .",
"Cell outlines in confocal images are visualized with propidium iodide ( purple ) .",
"Small disks visible in color in these cells are chloroplasts .",
"Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03271 . 011 Expression of early stomatal markers indicated that FAMALGK GCs re-acquired stomatal precursor identities , but did these cells return to an even earlier stem-cell or embryonic identity ?",
"Moreover , was a change in identity tied to failure of FAMALGK to activate its normal downstream targets ?",
"We addressed these questions by monitoring gene expression in isolated 12-dpg cotyledons of WT ( Col ) and FAMALGK plants ( Figure 5A ) .",
"Analysis of genes shown in Figures 2 and 3 to be inappropriately up-regulated in FAMALGK verified that a qRT-PCR-based approach could accurately assess gene expression changes ( Figure 5A , bracket indicating stomatal precursor genes ) .",
"There was a dramatic increase in expression levels for the stomatal precursor genes , but variable change in expression of mature GC genes , consistent with a situation in which the overproduction of GCs through repeated re-entry is balanced by the loss of identity of older GCs ( Figure 5A , mature GC genes ) .",
"FAMALGK was also still capable of up-regulating several , but not all , of the direct targets reported in ( Hachez et al . , 2011 ) ( Figure 5A , FAMA direct targets ) .",
"When expression of shoot meristem ( SHOOT MERISTEMLESS , STM ) , root meristem ( WUS HOMEOBOX , WOX5 ) or embryo genes ( WOX9 , WOX2 , FUSCA3 ( FUS3 ) , LEC1 ) ( Breuninger et al . , 2008; De Smet et al . , 2010 ) was monitored in FAMALGK plants , however , we found no evidence that cells were being reprogrammed into embryonic or other stem-cell-like fates ( Figure 5A and Figure 5—figure supplement 1 ) .",
"Taken together , the gene expression data indicate that disruption of FAMA-RBR interaction via the FAMALGK modification leads to a stomatal lineage-specific loss of terminal commitment . 10 . 7554/eLife . 03271 . 012Figure 5 . Terminal differentiation of guard cells may be mediated by FAMA-guided recruitment of RBR to suppress stomatal regulatory genes .",
"( A ) Expression analysis in mature cotyledons ( 12-dpg ) of FAMALGK and wild type ( Col ) by quantitative RT-PCR .",
"Signals were normalized to ACTIN2 and then to Col . Values shown are means ± SEM .",
"UD , undetected .",
"Asterisks indicate significant difference ( Student's t test , * p < 0 . 05 ) .",
"( B–D )",
"Binding of FAMA and RBR to regulatory regions of stomatal genes .",
"ChIP assays were performed with FAMAp:FAMA-MYC in fama ( B ) , FAMAp:RBR-MYC in Col ( C ) , and FAMAp:RBR-MYC in FAMALGK ( FAMAp:FAMALGK;fama ) ( D ) using an anti-Myc antibody as in ( Lau et al . , 2014 ) .",
"ChIPed DNA was quantified by qPCR with primers specific to the indicated gene promoters or the negative control region , IR1 ( Cruz-Ramirez et al . , 2012 ) .",
"Input-adjusted signals were normalized to Col . Values are means ± SEM .",
"( E ) FAMA promoter-driven expression of SPCH in wild type is not sufficient to reprogram guard cells to FAMALGK phenotype .",
"Confocal image of FAMAp:SPCH-YFP ( green ) in 12-dpg cotyledon visualized with propidium iodide ( purple ) .",
"Scale bar , 10 μm .",
"( F ) The stomatal lineage represents a stem-cell like lineage that is distinct from other stem-cell like compartments in the shoot , root or embryo .",
"The FAMA-RBR module maintains terminal differentiation of guard cells ( GCs ) through repression of the early stomatal lineage genes , likely made permanent by chromatin modification .",
"In FAMALGK plants , RBR is no longer recruited to SPCH and other stomatal lineage gene promoters allowing inappropriate re-expression of these genes and subsequent reiteration of the stomatal development pathway . DOI: http://dx . doi . org/10 . 7554/eLife . 03271 . 01210 . 7554/eLife . 03271 . 013Figure 5—figure supplement 1 . Validation of primers for the stem cell markers FUS3 , LEC1 , STM and WOX9 . RT-PCR reactions for RNA extracted from immature siliques of Arabidopsis .",
"Target size of the amplified products is indicated in Supplementary file 1 .",
"Lanes: molecular weight DNA ladder ( MW ) , independent RNA samples ( 1 , 2 , 3 ) , negative controls ( − ) .",
"Gel was stained with ethidium bromide . DOI: http://dx . doi . org/10 . 7554/eLife . 03271 . 01310 . 7554/eLife . 03271 . 014Figure 5—figure supplement 2 . Generation of transgenic lines expressing Myc-tagged RBR driven by FAMA promoter , in vivo immunoprecipitation of the RBR-Myc protein and phenotypic analysis of the transgenic lines .",
"( A ) Western detection of RBR-Myc in transgenic plants harboring the FAMAp:RBR-MYC construct in either Col ( lines 1–4 ) or FAMALGK ( lines 1–3 ) backgrounds .",
"Total protein was extracted from 5-dpg seedlings of the indicated genotypes and probed with α-Myc antibody .",
"Recombinant RBR-Myc has a calculated M . W . of 128 . 5 kDa .",
"( B ) In vivo pull-down assay of stomatal lineage expressed RBR-MYC from transgenic plants .",
"Total soluble protein from Col and FAMAp:RBR-MYC ( in Col ) was incubated with an anti-Myc antibody .",
"Precipitated samples were probed with the same antibody in Western analysis .",
"IB: Immunoblot , IP: Immunoprecipitation .",
"( C–F )",
"Confirmation that expression of FAMAp:RBR-MYC does not alter guard cell development .",
"( C–F )",
"Confocal images of 6-dpg cotyledons and DIC images of 12-dpg cotyledons of wild type and FAMAp:RBR-MYC plants .",
"Transgenic plants harboring the FAMAp:RBR-MYC construct ( E–F ) do not exhibit changes in GC divisions ( neither fewer , nor more divisions are found ) and are indistinguishable from wild type ( C–D ) at 6 and 12-dpg .",
"Cell outlines ( purple ) in confocal images were visualized with propidium iodide .",
"All images are at the same magnification .",
"Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 03271 . 01410 . 7554/eLife . 03271 . 015Figure 5—figure supplement 3 . Biological replicates for ChIP experiments in Figure 5 . ChIP assays were performed with FAMAp:FAMA-MYC in Col ( A ) , FAMAp:RBR-MYC in Col ( B ) , and FAMAp:RBR-MYC in FAMALGK plants ( C ) using an anti-Myc antibody as in ( Lau et al . , 2014 ) .",
"ChIPed DNA was quantified by qPCR with primers specific to the indicated gene promoters or the negative control region , IR1 or RB45 ( Cruz-Ramirez et al . , 2012 ) and ( Weimer et al . , 2012 ) .",
"Input-adjusted signals were normalized to Col . Values are means ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 03271 . 01510 . 7554/eLife . 03271 . 016Figure 5—figure supplement 4 . Dissection of FAMA and RBR binding on stomatal target genes .",
"( A and D )",
"Map of the SPCH ( A ) and EPF1 ( D ) loci .",
"Arrow indicates orientation of the gene and the transcription start site .",
"Genome coordinate is indicated above the gene structure .",
"Black bars indicate genomic region probed by ChIP-qPCR assays .",
"Key: U , upstream; P , promoter; D , downstream .",
"( B , C , E and F )",
"ChIP assays were performed with FAMAp:FAMA-MYC in fama ( B and E ) and FAMAp:RBR-MYC in Col ( C and F ) , using an anti-Myc antibody as in ( Lau et al . , 2014 ) .",
"ChIPed DNA was quantified by qPCR with primers specific to the indicated genomic regions relative to SPCH ( B and C ) and EPF1 ( E and F ) or the negative control region , RB45 ( Weimer et al . , 2012 ) .",
"Input-adjusted signals were normalized to Col . Values are means ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 03271 . 016 By what molecular mechanism might this specific loss of commitment take place ?",
"Analysis of chromatin states in maturing leaves revealed H3K27me3 ( a chromatin mark associated with transcriptional repression ) in the genomic regions of SPCH , MUTE , FAMA , EPF1 and other stomatal genes ( Lafos et al . , 2011 ) , and a recent report showed that manipulation of a member of the POLYCOMB REPRESSIVE COMPLEX 2 ( PRC2 ) can alter developmental regulation of H3K27me3 deposition at SPCH and MUTE loci ( Lee et al . , 2014b ) .",
"Animal and plant Rb/RBR proteins can serve as interaction bridges between chromatin modifying enzymes and specific genomic contexts ( Burkhart and Sage , 2008; Gutzat et al . , 2012 ) and RBR was previously found to be associated with SPCH regulatory regions via ChIP in whole seedlings ( Weimer et al . , 2012 ) .",
"Therefore it is plausible that , as the final master regulator bHLH in the stomatal pathway , FAMA ( with RBR ) ensures terminal differentiation of GCs by facilitating stable repression of early stomatal lineage genes .",
"To test this model , we assayed the co-association of FAMA and RBR with regulatory regions of three key stomatal lineage genes that have significant H3K27me3 coverage ( SPCH , FAMA and EPF1 ) and , as specificity controls , two cell cycle genes known to be RBR targets ( Nowack et al . , 2012 ) .",
"Because RBR is essential and expressed in nearly all cells , to accurately assay its role as a potential partner of FAMA in the stomatal lineage , we generated a Myc-tagged version of RBR expressed under the FAMA promoter ( FAMAp:RBR-MYC ) .",
"We confirmed that expression of this transgene did not alter stomatal development and that we could effectively immunoprecipitate it from plants ( Figure 5—figure supplement 2 ) .",
"In ChIP assays , SPCH , FAMA and EPF1 were all targets of FAMA and of stomatal lineage-expressed RBR ( Figure 5B–C and Figure 5—figure supplement 3 ) .",
"Further dissection of the binding regions at SPCH and EPF1 indicates that FAMA and RBR are enriched in the same pattern , suggesting that they bind as part of the same complex ( Figure 5—figure supplement 4 ) .",
"We then tested the key prediction of our model–that association of RBR with a stomatal target gene is dependent on its interaction with FAMA .",
"ChIPs of FAMAp:RBR-MYC in a FAMALGK background showed that RBR enrichment at the promoters of SPCH and FAMA , but not of the general RBR target gene PCNA , was significantly reduced ( Figure 5D and Figure 5—figure supplement 4 ) , consistent with our recruitment model .",
"RBR enrichment at the promoter of a negative regulator of stomatal development , EPF1 , was more variable in our assays , sometimes showing little change ( Figure 5D ) , but failing to associate with RBR in other replicates ( Figure 5—figure supplement 3C ) .",
"The association of FAMA and RBR with the SPCH locus is intriguing , as in normal development SPCH is required for initiation of the stomatal lineage and is essential for robust expression of all stomatal genes so far reported ( MacAlister et al . , 2007; Pillitteri et al . , 2007; Kanaoka et al . , 2008 ) .",
"In theory , failure of FAMALGK to stably repress SPCH expression could , by itself , be sufficient to reinitiate the stomatal lineage program .",
"If this were true , ectopic expression of SPCH in GCs should recapitulate the FAMALGK phenotype .",
"Expression of FAMAp:SPCH-YFP ( or its hyperactive variants FAMAp:SPCH1-4A or FAMAp:SPCH2-4A [Lampard et al . , 2008] ) in an otherwise WT background , however , did not mimic FAMALGK ( Figure 5E ) .",
"This suggests that competence to reinitiate the stomatal pathway requires more than expression of a single ‘trigger’ gene , but rather a more generally permissive expression state , a fact supporting a broader chromatin regulating role for the FAMA-RBR complex ( Figure 5F ) ."
],
[
"Recently , physical associations between RBR and the Arabidopsis transcription factor SCARECROW ( SCR ) were found to be essential for modulating stem-cell behavior in the root ( Cruz-Ramirez et al . , 2012 , 2013 ) .",
"Both SCR and FAMA bind to RBR via an LxCxE motif , yet the consequences of these transcription factor/RBR interactions are different; RBR antagonizes SCR function in asymmetric division in the root stem cell compartment ( Cruz-Ramirez et al . , 2012 , 2013 ) , whereas RBR and FAMA have similar functions promoting differentiation at the terminal stage of stomatal development .",
"Yet , while different RBR/transcription factor complexes may be customized for unique developmental contexts , the underlying molecular mechanisms of gene regulation might be similar .",
"As with FAMA targets , RBR is required for repression of SCR target genes and can associate with their promoter regions , but there have been no experiments addressing whether disrupting association of SCR and RBR affects either proteins' chromatin association .",
"In this study , we provide key data in support of a specific molecular mechanism for transcriptional repression utilizing RBR in combination with cell-type specific transcription factors: first , we demonstrate , through cell-type specific ChIPs , that RBR is associated with the promoter of the stomatal lineage initiator SPCH in cells as they are committing to terminal fates and second , we show that this binding is reduced when RBR's interaction with FAMA is disrupted .",
"Thus , our data provide strong support for RBR being recruited by cell-type specific transcription factors to lead to transcriptional repression of their targets ( Figure 5F ) .",
"We observed significant changes in RBR association with SPCH and FAMA regulatory regions in FAMALGK lines; however , RBR was still associated with the EPF1 locus in some experimental replicates .",
"This could indicate that there are FAMA-independent ways to recruit RBR to this site , or that our FAMALGK manipulation does not eliminate all FAMA-RBR interactions in the endogenous complex .",
"It is interesting , however , that EPF1 differs from SPCH and FAMA in being a repressor of stomatal development and thus alleviation of the repression of EPF1 would be expected to antagonize reprogramming to a stomatal precursor identity .",
"The role of PRC2 complex protein CURLY LEAF ( CLF ) was recently investigated in connection to stomatal lineage termination and was found to promote the accumulation of H3K27me3 marks on early stomatal lineage genes ( Lee et al . , 2014b ) .",
"These data complement ours in connecting chromatin modification to stable acquisition of terminal cell identities in the stomatal lineage .",
"Additionally , ( Lee et al . , 2014b ) report phenotypes , similar to , but milder than those seen in FAMALGK , caused by prolonged expression of a C-terminal GFP-tagged version of FAMA ( FAMAtrans ) .",
"In a timepoint and tissue common to their data and this study ( 12 day old cotyledons ) , FAMALGK plants display reprogramming of ∼80% guard cells ( Figure 1L ) compared with 10% of FAMAtrans .",
"Lee et al . interpret reinitiation of divisions in guard cells as resulting from a gain of FAMA function , however , this interpretation is at odds with previously published data that FAMA overexpression limits cell division ( Ohashi-Ito and Bergmann , 2006; Hachez et al . , 2011 ) and is difficult to reconcile with most models of chromatin-mediated repression of transcription .",
"In light of our data showing that a loss of a specific FAMA activity ( RBR binding ) produces strong lineage reprogramming , we think a more parsimonious explanation of the FAMAtrans-induced phenotype is that blocking of the FAMA C-terminus by addition of GFP creates a protein that dominantly interferes with FAMA-RBR interactions .",
"By independently manipulating RBR levels and RBR-FAMA interactions in terminally differentiating GCs , we could uncouple division and fate modulating roles of RBR .",
"Notably , depletion of RBR in many contexts leads to hyperproliferation and derepression of cell cycle promoting genes ( Figure 1E and Figure 2—figure supplement 1 and Borghi et al . , 2010; Wachsman et al . , 2011; Weimer et al . , 2012 ) .",
"This is in contrast to phenotypes that dominate when its association with FAMA is disrupted; namely that specific cell fates and division behaviors and their accompanying gene expression patterns re-emerge in an orderly pattern .",
"RBRp:RBR-CFP itself becomes ectopically expressed in re-dividing FAMALGK GCs .",
"Were RBR to be playing its cell-cycle repressive role in this context , one would expect this ectopic expression might completely halt divisions .",
"That the opposite occurs , however , suggests a qualitatively different role for RBR in combination with FAMA in these cells .",
"Because RBR is expressed in all cells of the plant , it is difficult to measure whether our cell-type specific amiRNA completely eliminated RBR in guard cells , but our data suggest that RBR's cell-cycle repression activity is more sensitive to dosage than its activity modulating stomatal gene expression .",
"Only by retaining RBR levels but disrupting the FAMA-dependent activity was a clear role for RBR in terminal differentiation unmasked .",
"In the balance between proliferation and differentiation , between developmental flexibility and robust fate commitment , several decades of cell culture and animal genetic knockout studies have placed Rb family proteins in key , if disputed , roles ( Chinnam and Goodrich , 2011; Gu et al . , 1993; Sage , 2012 ) .",
"Regulation of the stomatal lineage can parallel that of stem cell populations in animals at cellular , developmental and molecular levels .",
"Both plant stomatal and mammalian myogenic lineages , for example , employ series of paralogous bHLHs during fate specification and differentiation , and activities of these bHLHs are regulated through conserved upstream kinases and by association with Rb/RBR ( reviewed in Matos and Bergmann , 2014 ) .",
"Bound by immobile cell walls , stomatal lineage cells leave a record of their fate and division history in their marker expression and spatial arrangement on the leaf surface .",
"This plant model , therefore , provides a unique opportunity to dissect cell division and cell fate activities of Rb and other conserved proteins during programming and reprogramming and is a powerful comparative system for future discoveries of fundamental regulatory mechanisms of stem cell initiation , maintenance and termination ."
],
[
"Arabidopsis thaliana Columbia-0 ( Col ) was used as wild type in all experiments .",
"All mutants and transgenic lines tested are in this ecotype .",
"The following previously described mutants and reporter lines were used in this study: fama-1 and FAMAp:GFP-FAMA ( Ohashi-Ito and Bergmann , 2006 ) ; MUTEp:MUTE-GFP ( Pillitteri et al . , 2007 ) ; KAT1p:GUS ( Nakamura et al . , 1995 ) ; CDKB1;1p:GUS ( Boudolf et al . , 2004 ) ; CDC6p:GUS ( Castellano et al . , 2001 ) , RBRp:RBR-CFP ( Cruz-Ramirez et al . , 2012 ) and CDKA;1p:YFP-DB ( Jakoby et al . , 2006 ) .",
"The CDKA;1 reporter contains a destruction box ( DB ) within the YFP fusion to ensure that reporter expression does not persist after a cell division .",
"Versions of reporters previously published with different fluorescent proteins include: SPCHp:SPCH-YFP ( MacAlister et al . , 2007 ) , TMMp:TMM-YFP ( Nadeau and Sack , 2002 ) , EPF2p:YFPnuc ( Hara et al . , 2009 ) , EPF1p:YFPnuc ( Hara et al . , 2007 ) , ML1p:mCherry-RCI2A ( Roeder et al . , 2010 ) , and BASLp:YFP-BASL ( Dong et al . , 2009 ) .",
"Seedlings were grown on 0 . 5 Murashige and Skoog ( MS ) medium at 22°C under 16 hr-light/8 hr-dark cycles and were examined at the indicated time .",
"The FAMA promoter ( 2 . 5 kb , [Ohashi-Ito and Bergmann , 2006] ) and full-length cDNAs of FAMA and RBR were cloned into Gateway compatible entry vectors , typically pENTR/D-TOPO ( Life Technologies , Carlsbad , CA ) , to facilitate subsequent cloning into plant binary vectors .",
"To mutate the LxCxE motif of FAMA to LxGxK , site directed mutagenesis was performed using the QuikChange II Kit ( Agilent , Santa Clara , CA ) .",
"Gateway entry vectors containing the FAMA promoter and FAMALGK cDNA were recombined into the plant binary destination vectors pHGY ( Kubo et al . , 2005 ) and pGWBI ( Nakagawa et al . , 2007 ) to generate YFP-tagged and untagged versions of FAMAp:FAMALGK , respectively .",
"For the FAMAp:amiRBR construct , the artificial microRNA sequence was designed with the Web MicroRNA Designer platform ( http://wmd3 . weigelworld . org ) .",
"The microRNA sequence was engineered using the pRS300 plasmid as template , and together with the FAMA promoter , was subcloned into the destination vector pGWBI ( Nakagawa et al . , 2007 ) .",
"The constructs FAMAp:FAMA-MYC and FAMAp:RBR-MYC were generated with the tripartite recombination of the plant binary vector R4pGWB419 ( Nakagawa et al . , 2008 ) , with the Gateway entry clones of the FAMA promoter and cDNAs of FAMA or RBR .",
"The constructs FAMAp:SPCH-YFP , FAMAp:SPCH1-4A and FAMAp:SPCH2-4A were generated with the plant binary vector R4pGWB430 ( Nakagawa et al . , 2008 ) , the FAMA promoter and the respective SPCH coding sequences described in Lampard et al . ( 2008 ) .",
"Primer sequences used for each construct are provided in Supplementary file 1 .",
"Transgenic plants were generated by Agrobacterium–mediated transformation ( Clough , 2005 ) and transgenic seedlings were selected by growth on 0 . 5 MS plates supplemented with 50 mg/l hygromycin ( pHGY and pGWB1 based constructs ) or kanamycin 100 mg/l ( pGWB419 and pGWB430 based constructs ) .",
"The constructs FAMAp:FAMALGK and FAMAp:FAMALGK-YFP were transformed into the fama/+ segregating line and homozygous lines for fama ( as detected by PCR genotyping ) were recovered in subsequent generations .",
"FAMAp:FAMALGK;fama−/− is referred to as FAMALGK throughout the study .",
"FAMAp:RBR-MYC was transformed into Col and FAMAp:FAMALGK-YFP;fama lines .",
"All other constructs were transformed into Col . Seedlings from two independent and homozygous lines of FAMAp:FAMALGK;fama were collected at 6 , 9 and 12 dpg .",
"Samples were cleared in 7:1 ethanol:acetic acid , treated 30 min with 1 N potassium hydroxide , rinsed in water , and mounted in Hoyer's medium .",
"Differential contrast interference ( DIC ) images were obtained from the middle region of adaxial epidermis of cotyledons at 20× ( 0 . 32 mm−2 field of view ) on a Leica DM2500 microscope .",
"For quantification , 13 different guard cell phenotypes were counted and grouped into 5 classes ( Figure 1L and Figure 1—figure supplement 2 ) .",
"Results are shown as mean percentages of each phenotypic class divided by the total number of guard cells per field view ± SEM ( n = 30 ) .",
"To analyze reporter expression in FAMALGK , FAMAp:amiRBR and fama , all transcriptional and translational reporters were introgressed into the mutant backgrounds and homozygous lines ( as determined by PCR-based genotyping and segregation ratios ) in subsequent generations were recovered for analysis .",
"For confocal microscopy , images were taken with a Leica SP5 microscope and processed in ImageJ .",
"Cell outlines were visualized by either 0 . 1 mg/ml propidium iodide in water ( Molecular Probes , P3566 ) or the plasma membrane marker ML1p:mCherry-RCI2A .",
"GUS staining of transcriptional reporters was performed as described in Scarpella et al . ( 2004 ) and seedlings were mounted in Hoyer's and visualized with DIC microscopy as described above .",
"After 6 days of growth on half strength MS media , seedlings were transferred to a sterilized perfusion chamber similar to that described in Robinson et al . ( 2011 ) for imaging on a Leica SP5 Confocal microscope .",
"The chamber was perfused with ¼ strength 0 . 75% ( wt/vol ) sucrose liquid MS growth media ( pH 5 . 8 ) at a rate of 2 ml/hr .",
"Z-stacks through the epidermis of the reporter lines were captured with Leica software every 20 min ( SPCH ) or 2 hr ( MUTE ) and then processed with Fiji/ImageJ ( NIH ) .",
"Full-length ORFs with no stop codon of each test candidate ( FAMA , FAMALGK , bHLH93 , RBR , CYCD and CYCDLGK ) were cloned into BiFC vectors ( Walter et al . , 2004 ) to generate fusion proteins with either N or C terminal half of the yellow fluorescence protein ( YFP ) fused to the C-terminus of the test candidate .",
"FAMA and bHLH93 constructs were reported in ( Ohashi-Ito and Bergmann , 2006 ) .",
"Assays were performed in Nicotiana benthamiana leaves as described in Ohashi-Ito and Bergmann ( 2006 ) .",
"BiFC signals were visualized on a Leica DM5000 fluorescence microscope and quantified as percentage of YFP-positive nuclei over total number of pavement cells in a field of view ( centered on the injection site ) .",
"Results from three experiments are presented in Figure 1G .",
"Full-length ORFs containing stop codons for each test candidate ( FAMA , FAMALGK , bHLH93 , BASL , RBR , CYCD , CYCDLGK ) were cloned into pENTR/D-TOPO ( Life Technologies ) and then recombined into the yeast vectors pGADT7 ( Clontech , Mountain View , CA ) and pXDGATcy86 ( Ding et al . , 2007 ) Yeast stain AH109 was transformed using the Yeastmaker yeast transformation system ( Clontech ) according to manufacture's instructions .",
"Pairwise interactions were tested based on growth complementation on nutritional selective media .",
"Cotyledons from 10 FAMALGK or Col seedlings were harvested at 12 dpg and RNA was extracted using the RNeasy plant mini kit ( QIAGEN , Valencia , CA ) with on-column DNAse digestion .",
"700 ng of total RNA was used for cDNA synthesis using oligo ( dT ) primers and the Supercript III First-strand cDNA synthesis kit ( Life Technologies ) .",
"qPCR reactions were performed on a CFX96 Real-Time PCR detection system ( Bio-Rad ) with the Ssofast EvaGreen Supermix ( Bio-Rad , Hercules , CA ) .",
"Three technical replicates were performed on each of two biological replicates .",
"Expression values were normalized to the reference gene ACTIN2 using the ΔCT method and relative expression of a target was calculated from the ratio of FAMALGK to Col . All data are presented as mean ± SEM .",
"The significance of difference between the mean values was determined using two-tailed unpaired Student's t test .",
"Statistical analysis was applied to normalized ΔCT values .",
"p < 0 . 05 was considered statistically significant .",
"All calculations were performed using GraphPad Prism software .",
"Primer sequences are listed in Supplementary file 1 .",
"Since expression of the embryonic genes WOX9 , LEC1 , FUS3 and the shoot apical meristem gene STM were not detectable in cotyledons of either Col nor FAMALGK , we confirmed that primers were functional by testing them in RT-PCRs with RNA from immature siliques , as described in Onate-Sanchez and Vicente-Carbajosa ( 2008 ) .",
"STM , WOX9 , LEC1 and FUS3 were all detectable in these assays ( Figure 5—figure supplement 1 ) .",
"ChIP experiments were carried out based on standard protocols ( Gendrel et al . , 2005 ) or with adaptations as described in Lau et al . ( 2014 ) .",
"Briefly , for ChIPs of FAMA , ∼5 g of 5-day-old whole seedlings of FAMAp:FAMA-MYC ( in Col or in fama ) and Col ( control ) were used as starting materials .",
"For ChIPs of RBR , ∼25 g of 5-day-old whole seedlings of Col ( control ) and FAMAp:RBR-MYC in Col or in FAMApro::FAMALGK-YFP;fama were used in the assays .",
"For RBR ChIP , input materials were processed in standard-sized aliquots during nuclei isolation and DNA fragmentation steps before combining for immunoprecipitation .",
"Expression and pull-down of the cell-type specific RBR-Myc were verified by Western and immunoprecipitation experiments ( Figure 5—figure supplement 2A–B ) .",
"Chromatin was fragmented by a Bioruptor ( Diagenode ) programed at high intensity for 3 × 7 . 5 min ( cycles of 30 s on and 30 s off ) at 4°C .",
"Immunoprecipitation was carried out with a monoclonal anti-Myc antibody ( 71D10; Cell Signaling Technology ) , followed by incubation with magnetic beads ( Dynabeads Protein A; Invitrogen ) .",
"ChIPed DNA was purified by the ChIP DNA Clean & Concentrator ( Zymo ) .",
"For real-time qPCR , reactions were performed using SsoFast EvaGreen or SsoAdvanced Universal SYBR Green Supermix ( Bio-Rad ) , according to manufacturer's recommended conditions , with primers targeted to the indicated region of selected genes ( Supplementary file 1 ) on a CFX96 Real-Time PCR detection system ( Bio-Rad ) .",
"CT values were obtained for sonicated chromatin taken before ( input ) and after immunoprecipitation ( ChIP ) .",
"Three technical replicates were assayed for each sample .",
"CT values for ChIP DNA were normalized to mean of CT values for input DNA ( CT ChIP—μCT Input ) .",
"Fold enrichment was calculated by dividing the normalized value of Myc-tagged with that of untagged Col . All data are presented as mean ± SEM .",
"Two biological replicates were assayed for each ChIP-qPCR experiment ."
]
] | [
"The presumed totipotency of plant cells leads to questions about how specific stem cell lineages and terminal fates could be established .",
"In the Arabidopsis stomatal lineage , a transient self-renewing phase creates precursors that differentiate into one of two epidermal cell types , guard cells or pavement cells .",
"We found that irreversible differentiation of guard cells involves RETINOBLASTOMA-RELATED ( RBR ) recruitment to regulatory regions of master regulators of stomatal initiation , facilitated through interaction with a terminal stomatal lineage transcription factor , FAMA .",
"Disrupting physical interactions between FAMA and RBR preferentially reveals the role of RBR in enforcing fate commitment over its role in cell-cycle control in this developmental context .",
"Analysis of the phenotypes linked to the modulation of FAMA and RBR sheds new light on the way iterative divisions and terminal differentiation are coordinately regulated in a plant stem-cell lineage ."
] | [
"Stem cells in animals and plants help to make and replenish the tissues of the body by dividing and becoming specialized types of cells .",
"Once specialized for a certain function , it is important that a cell keeps that function .",
"In plant leaves , one type of stem cell makes two different types of specialized cells: pavement cells and stomatal guard cells .",
"Pavement cells lock together to form a waterproof barrier to the outside , while guard cells surround the small pores that open and close to allow the plant to exchange water , oxygen and carbon dioxide with the atmosphere .",
"Once a cell becomes a pavement cell or a guard cell , it does not change its identity again .",
"However , if a single cell is removed from a plant , it can revert to a stem cell and a whole new plant can be grown from it .",
"This poses the question of how , in intact plants , specialized cells like pavement cells and guard cells are prevented from reverting to stem cells .",
"In Arabidopsis thaliana , a small flowering plant that is widely used as a model organism in research , a protein called FAMA is responsible for controlling a set of genes that turn stem cells into guard cells .",
"Matos et al . have now found that FAMA needs to bind to another protein called RBR to control this process .",
"It seems that these two proteins make the transition from stem cell to guard cell permanent by changing the structure of DNA in regions that control stem cell genes .",
"RBR is similar to a human protein called Retinoblastoma that helps prevent tumors and regulate stem cells , but how it actually performs these functions in humans is still debated .",
"Because stem cells and guard cells are displayed on the surface of plant leaves and leave behind clues of their past , Matos et al . were able to watch stem cells grow up to be mature guard cells .",
"When the partnership between FAMA and RBR was broken , it was possible to watch those same guard cells revert backwards into stem cells .",
"Seeing development ‘rewind’ could provide useful insights into the way in which cell identity is controlled in both plants and animals ."
] | 2014 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology"
] | Mia40 is a trans-site receptor that drives protein import into the mitochondrial intermembrane space by hydrophobic substrate binding | elife-16177-v2 | [
[
"In most cellular compartments , cysteine residues are predominantly present in the reduced state .",
"In contrast , the bacterial periplasm , the endoplasmic reticulum ( ER ) and the IMS of mitochondria contain dedicated oxidation machineries ( disulfide relays ) to introduce disulfide bonds into a broad range of substrate proteins ( Riemer et al . , 2009; Kadokura et al . , 2003; Bulleid and Ellgaard , 2011; Stojanovski et al . , 2012; Modjtahedi et al . , 2016; Riemer et al . , 2015 ) .",
"At least in the case of the periplasm and the ER , disulfide bond formation presumably serves the function of stabilizing the structures of ( secretory ) proteins .",
"While oxidative protein folding in the periplasm and the ER is well characterized , the details of the mitochondrial disulfide bond formation are still elusive .",
"Mitochondria consist of about 600 ( yeast ) to 1500 ( humans ) nuclear encoded proteins ( Pagliarini et al . , 2008; Vögtle et al . , 2009 ) .",
"Following their synthesis on cytosolic ribosomes , these proteins are recognized by receptors on the mitochondrial surface and threaded through mitochondrial protein translocases ( Schulz et al . , 2015; Harbauer et al . , 2014; Endo et al . , 2011; Chacinska et al . , 2009; Neupert and Herrmann , 2007 ) .",
"Proteins destined for the matrix of mitochondria contain presequences ( or matrix-targeting signals ) at their N termini that target these proteins through the protein-conducting channels of the translocase of the outer membrane ( TOM complex ) and the inner membrane ( TIM23 complex ) .",
"After import into the mitochondria the presequences are proteolytically removed by the matrix processing peptidase MPP ( von Heijne , 1986; Vögtle et al . , 2009 ) .",
"The targeting of proteins into the IMS , the compartment between the outer and the inner membrane of mitochondria , is less well understood .",
"Some IMS proteins also contain N-terminal signals in the form of bipartite presequences which consist of a matrix-targeting signal followed by a hydrophobic stop-transfer domain .",
"These proteins are arrested at the inner membrane before their mature domains are released into the IMS by proteolytic cleavage ( Glick et al . , 1992; Herlan et al . , 2004; Rojo et al . , 1998 ) .",
"The cytochrome b2 protein of Saccharomyces cerevisiae is the best studied example for this stop-transfer targeting ( Glick et al . , 1992; Hartl et al . , 1987; Gärtner et al . , 1995 ) .",
"Most IMS proteins do not contain N-terminal targeting signals but instead contain patterns of cysteine residues within their mature sequence that serve as targeting signals ( Koehler , 2004; Sideris and Tokatlidis , 2007; Sideris et al . , 2009; Milenkovic et al . , 2009 ) .",
"In most cases , these proteins contain two pairs of cysteine residues that are either spaced by three or nine amino acid residues , therefore referred to as ’twin Cx3C’ and ’twin Cx9C’ proteins , respectively .",
"In yeast , five ’twin Cx3C’ ( also called small Tim proteins ) play a role as chaperones for carrier proteins in the IMS ( Curran et al . , 2002b; 2002a; Koehler et al . , 1998; Sirrenberg et al . , 1998; Luciano et al . , 2001; Vial et al . , 2002 ) , and 13 ’twin Cx9C’ proteins contribute to the stabilization and assembly of inner membrane proteins ( Potting et al . , 2010; Vögtle et al . , 2012; Longen et al . , 2009; Modjtahedi et al . , 2016; Horn et al . , 2010; Bode et al . , 2015 ) .",
"But recently also proteins with different disulfide configurations were discovered ( Okamoto et al . , 2014; Wrobel et al . , 2013; Varabyova et al . , 2013; Hangen et al . , 2015; Klöppel et al . , 2011; Wrobel et al . , 2016 ) .",
"The import of these proteins into mitochondria relies on the mitochondrial disulfide relay ( also called MIA pathway ) which employs two conserved , essential proteins , Erv1 and Mia40 .",
"Erv1 is an FAD-binding sulfhydryl oxidase which can ‘generate’ disulfide bonds de novo thereby transferring electrons either directly to molecular oxygen or to cytochrome c of the respiratory chain ( Lee et al . , 2000; Dabir et al . , 2007; Ang and Lu , 2009; Tienson et al . , 2009; Bien et al . , 2010; Mesecke et al . , 2005; Allen et al . , 2005; Bihlmaier et al . , 2007; Fass , 2008 ) .",
"The oxidoreductase Mia40 contains a redox-active cysteine-proline-cysteine ( CPC ) motif ( Terziyska et al . , 2009; Chacinska et al . , 2004; Banci et al . , 2009; 2010; Kawano et al . , 2009 ) .",
"Erv1 maintains this motif in an oxidized conformation which then permits the transfer of disulfide bonds to Mia40 substrates during their translocation into the IMS ( von der Malsburg et al . , 2011 ) .",
"Mia40 substrates are often small proteins that are unstructured in the reduced form but very stable as soon as they are oxidized ( Morgan et al . , 2009; Curran et al . , 2004; 2002a; Baker et al . , 2012; Banci et al . , 2009; 2012 ) .",
"It was proposed that oxidation traps the incoming polypeptides in the IMS so that oxidation-induced folding drives their net translocation into the IMS ( ‘folding trap model’ ) ( Lutz et al . , 2003; Koehler , 2004 ) .",
"Mechanistically , this would be very different from protein translocation into the periplasm and the ER where the translocation of proteins is driven in ATP-dependent reactions by SecA or BiP , respectively , prior to and independent of their oxidative folding ( Matlack et al . , 1999; Economou and Wickner , 1994; Taufik et al . , 2013 ) .",
"Moreover , the formation of mixed disulfides of Mia40 with incoming polypeptides was suggested to serve as a crucial reaction in the translocation reaction ( von der Malsburg et al . , 2011; Longen et al . , 2014; Bien et al . , 2010 ) that is critical to avoid the mistargeting of reduced IMS proteins to the cytosol ( Bragoszewski et al . , 2015; Wrobel et al . , 2015 ) .",
"Structural analysis revealed the presence of a hydrophobic substrate-binding pocket on the surface of Mia40 ( Figure 1A ) ; like the CPC motif this substrate-binding region of Mia40 is essential for its function in the import and folding of IMS proteins and mutants were shown to be inviable ( Banci et al . , 2009 , 2010; Kawano et al . , 2009; Weckbecker et al . , 2012 ) .",
"This region is essential for the binding and hence the oxidation of Mia40 substrates as well as for binding to Erv1 .",
"Mia40 substrates contain internal signals , known as MISS or ITS sequences , which specifically dock onto this binding region thereby selecting cysteine residues for interaction with the redox-active cysteine pair in Mia40 ( Koch and Schmid , 2014b; Sideris and Tokatlidis , 2007; Sideris et al . , 2009; Peleh et al . , 2014; Milenkovic et al . , 2009 ) . 10 . 7554/eLife . 16177 . 003Figure 1 . Generation of Mia40 mutants lacking either the oxidoreductase or substrate-binding activity .",
"( A ) Schematic representation of the Mia40 structure .",
"( B ) Structure of the Mia40 variants used for complementation studies .",
"Molecular masses of the matured ( MPP-cleaved ) variants are indicated .",
"( C , D )",
"The indicated variants were expressed in GAL-Mia40 strains .",
"Cells were grown in synthetic lactic acid-based medium containing 0 . 5% galactose ( ↑ ) or glucose ( ↓ ) to induce or repress the GAL promoter , respectively .",
"The expression levels of the indicated Mia40 variants were assessed by Western blotting .",
"Due to the increased net charge , Mia40-FE shows a reduced migration on SDS-PAGE .",
"Mia40* depicts the endogenous full-length Mia40 .",
"( E ) To monitor the redox state of the Mia40 variants , proteins of the indicated strains were TCA-precipitated , denatured in SDS , treated with the reducing agent TCEP and the alkylating compound mmPEG24 and visualized by SDS-PAGE and Western blotting .",
"Mdj1 is a matrix chaperone with 10 reduced cysteine residues which was used for control .",
"Green arrows depict fully oxidized Mia40 species , yellow arrows the wild type Mia40 with the CPC reduced and the twin Cx9C structure oxidized , and the red arrow a Mia40 protein in which the twin Cx9C structure does not contain disulfide bonds .",
"( F ) The redox state of the Mia40-STOP variant was analyzed as described for E with the exception that mmPEG12 was used for alkylation .",
"The arrowheads depict different redox states of the protein suggesting that Mia40-STOP is partially oxidized . DOI: http://dx . doi . org/10 . 7554/eLife . 16177 . 003 In this study , we analyzed the relevance of both structural elements of Mia40 in more detail .",
"To this end , we generated Mia40 mutants which lack either the redox-active cysteine pair ( Mia40-SPS ) or the substrate-binding pocket ( Mia40-FE and Mia40-STOP ) .",
"Interestingly , the Mia40-SPS mutant still mediates protein import of Mia40 substrates with high efficiency .",
"It also allows the accumulation of Mia40 substrates in mitochondria , albeit at reduced levels .",
"Our observations suggest that trapping activity of Mia40 is essential since Mia40 serves as a ‘trans-site receptor’ whose hydrophobic binding to incoming polypeptides drives protein import into the mitochondrial IMS .",
"Hence , protein oxidation in the IMS is not directly linked to translocation , and mechanistically is independent of the transport reaction , similar to the situation in the ER and the periplasm ."
],
[
"Studies on the structure of Mia40 ( Kawano et al . , 2009; Banci et al . , 2009; 2010 ) revealed the presence of two conserved functional elements , an N-terminal redox-active CPC motif and a C-terminal hydrophobic substrate-binding pocket ( Figure 1A ) .",
"We constructed Mia40 variants in which either the CPC motif was mutated to a redox-inactive SPS motif ( Figure 1B , Mia40-SPS ) or the hydrophobic binding region was compromised by replacement of two conserved phenylalanine residues at positions 315 and 318 by glutamate residues ( Figure 1B , Mia40-FE ) .",
"In order to distinguish these versions from the endogenous Mia40 , we deleted the functionally irrelevant residues 211 to 283 from the membrane anchor in Mia40-SPS and Mia40-FE ( Terziyska et al . , 2009 ) .",
"Moreover , we constructed a Mia40 version in which the entire substrate-binding pocket ( i . e . the twin Cx9C domain ) was replaced by a hemagglutinin tag ( Figure 1B , Mia40-STOP ) .",
"All variants were expressed from single copy plasmids under control of the endogenous MIA40 promoter in a GAL-Mia40 mutant in which the chromosomal MIA40 gene is under control of a regulatable GAL10 promoter ( Mesecke et al . , 2005 ) .",
"Growth of these strains on galactose or glucose caused the induction or repression , respectively , of chromosomal MIA40 but did not affect the levels of the Mia40 variants expressed from the plasmids ( Figure 1C , D ) .",
"Next we tested whether the Mia40 mutants still contain the two structural disulfide bonds , which are crucial for the functionality of the substrate-binding pocket ( Terziyska et al . , 2009 ) .",
"We isolated mitochondria from the different mutants after depletion of the endogenous Mia40 ( GAL-Mia40↓ ) , precipitated proteins with trichloroacetic acid ( TCA ) to denature them and to preserve their redox state , and incubated them with mmPEG24 ( Figure 1E ) or mmPEG12 ( Figure 1F ) .",
"These maleimide-based alkylating agents lead to mass shifts of about 1 . 2 and 0 . 7 kDa per alkylated thiol group , respectively .",
"For Mia40 two species were observed both containing the two structural disulfides of the substrate-binding domain but differing in the redox state of the CPC motif ( Figure 1E , left panel: green arrow , CPC oxidized; yellow arrow , CPC reduced ) .",
"Whereas the Mia40-SPS mutant contained a properly oxidized substrate-binding domain ( Figure 1E , middle panel: green arrow ) , the structural disulfides were not formed in the Mia40-FE mutant ( Figure 1E , right panel: red arrow ) indicating that the negative charges prevented the folding of the substrate-binding domain .",
"In the Mia40-STOP variant , different redox states of the CPC motif were observed ( Figure 1F ) indicating that its cysteine residues are partially oxidized even in the complete absence of the substrate-binding domain ( cf . Figure 1B ) .",
"We conclude that the different Mia40 variants can be expressed in the IMS of mitochondria and either lack the redox-active cysteine pair ( but contain the correctly folded substrate-binding domain ) or lack a functional substrate-binding domain ( but contain the CPC motif ) .",
"Next we tested if the Mia40 mutants can functionally replace the endogenous Mia40 .",
"To this end , we followed three different strategies .",
"First , we followed a plasmid-shuffling approach in which we tested whether a Mia40-expressing URA3 plasmid can be functionally replaced by plasmids that express the mutated Mia40 variants ( Figure 2A ) .",
"Second , we introduced the Mia40-expressing plasmids in a GAL-Mia40 strain and repressed the endogenous Mia40 ( Figure 2B ) .",
"And third , we tested complementation in a temperature-sensitive mia40 mutant ( Chacinska et al . , 2004 ) at a restrictive temperature ( mia40-3 , Figure 2C ) .",
"Although the different Mia40 variants were efficiently expressed in these strains and correctly targeted to the mitochondrial IMS ( Figure 2D , E and data not shown ) , neither Mia40-SPS , nor Mia40-FE rescued the loss of the endogenous Mia40 .",
"The endogenous Mia40 could only be functionally replaced by an expression of wildtype Mia40 . 10 . 7554/eLife . 16177 . 004Figure 2 . Mia40 mutants lacking either the oxidoreductase or the substrate-binding activity cannot cross-complement each other .",
"( A ) Δmia40 cells containing MIA40 on a URA3 plasmid were transformed with plasmids for expression of the indicated Mia40 variants .",
"After growth on uracil-containing medium , loss of the Mia40-containing URA3 plasmid was tested by growth on 5-fluoroorotic acid ( 5´FOA , which is converted to the toxic 5-fluorouracil in the presence of Ura3 ) .",
"Loss of the URA3 plasmid was lethal except upon expression of wild type Mia40 , indicating that none of the mutated Mia40 variants rescued the Δmia40 mutants , even if expressed in combinations .",
"( B ) Expression of wild type Mia40 but not of the Mia40 variants rescued the inability of GAL-Mia40 mutants to grow on glycerol medium .",
"( C ) Wild type cells and the temperature-sensitive mia40-3 mutant were transformed with plasmids expressing full-length Mia40 ( Mia40f ) ( cf . Figure 1A ) or the indicated Mia40 variants ( cf . Figure 1B ) .",
"The mutated Mia40 variants did not allow growth at restrictive temperature .",
"( D , E )",
"Protein levels were analyzed by Western blotting showing that the different Mia40 variants were well expressed in the mia40-3 mutant .",
"Please note that only the expression of wild type Mia40 resulted in the efficient accumulation of Mia40 substrates such as Cmc1 and Atp23 .",
"However , small amounts of Cmc1 and Atp23 were also observed in the Mia40-SPS but not in Mia40-FE or the Mia40-STOP cells . DOI: http://dx . doi . org/10 . 7554/eLife . 16177 . 004 It should be noted that Mia40 remained essential even upon simultaneous co-expression of Mia40-SPS and Mia40-FE or of Mia40-SPS and Mia40-STOP .",
"Hence , Mia40 variants lacking either the redox-active cysteine pair or the substrate-binding pocket cannot cross-complement each other and obviously both elements need to be present in close proximity in the same protein .",
"Cross-complementation of Mia40-SPS and Mia40-FE might have failed because the redox-active disulfide and the substrate-binding pocket are not in close proximity ( at the necessary concentrations ) required to allow efficient substrate oxidation .",
"We therefore tested whether the addition of a chemical oxidizer can support the growth of the Mia40-SPS mutant .",
"Diamide is a thiol-specific chemical oxidant that induces disulfide bond formation in proteins ( Frand and Kaiser , 1998; Hansen et al . , 2009 ) and which was shown to efficiently oxidize proteins of the mitochondrial IMS when added to the growth medium of yeast cells ( Kojer et al . , 2012; 2015 ) .",
"We transformed GAL-Mia40 cells with plasmids for the individual or simultaneous expression of Mia40-SPS and Mia40-FE , grew the cells on glucose medium and placed a diamide-containing filter onto the plates ( Figure 3A ) .",
"Interestingly , we observed a ring of growing colonies around the diamide-containing filter in cells expressing Mia40-SPS but not in cells expressing Mia40-FE .",
"We conclude that diamide is able to partially rescue the growth defect of the oxidation-deficient Mia40 mutant .",
"A similar growth stimulation by diamide was also observed in liquid cultures ( Figure 3B ) .",
"When GAL-Mia40 cells are grown in lactic acid-based medium containing 0 . 5% glucose they are unable to proliferate until the glucose concentration is strongly reduced by metabolism which takes about 24 hr under the conditions we used ( Figure 3B , left panels ) .",
"However , upon expression of the Mia40 variant the cells start growing already after a short lag phase ( Figure 3B , middle panels ) .",
"Short lag phases were likewise observed upon expression of Mia40-SPS , however , only in the presence of sublethal concentrations of diamide ( Figure 3B , right panels ) .",
"Thus , in the presence of a chemical oxidizer , the hydrophobic binding pocket of Mia40 is apparently sufficient – and still essential - for viability . 10 . 7554/eLife . 16177 . 005Figure 3 . Diamide partially rescues the growth defect of the Mia40-SPS mutant .",
"( A ) The indicated strains were grown in synthetic lactic acid-based medium containing 0 . 5% glucose for three days to deplete endogenous Mia40 and spread on glucose plates .",
"10 µl water or 1 M diamide was applied onto a filter dish in the middle of the plate .",
"Note the ring-like growth of the Mia40-SPS mutant around diamide-containing filter .",
"( B ) GAL-Mia40 mutants lacking or containing plasmids for expression of the Mia40 or the Mia40-SPS variant were grown for 72 hr in synthetic lactic acid-based medium supplemented with 0 . 5% glucose and diluted to OD 0 . 1 in the same medium lacking or containing 2 . 4 mM diamide .",
"Growth at 30°C under constant shaking was then analyzed continuously in a multiwell absorption reader ( BioTek ELx808 , BMG Labtech ) .",
"Error bars represent SD with n = 4 .",
"( C ) Mia40-SPS- and Mia40-FE-containing mitochondria were incubated in import buffer for 5 min in the presence of the indicated concentrations of diamide .",
"Then the radiolabeled cysteine-less Atp23 variant 10CS was added .",
"After incubation for 15 min at 25°C , non-imported protein was removed by protease treatment .",
"Mitochondria were washed and subjected to SDS-PAGE and autoradiography .",
"( D ) Mitochondria were isolated from glucose-grown GAL-Mia40 cells expressing the indicated Mia40 variants .",
"They were treated with or without 5 mM diamide for 10 min at 30°C , reisolated , resuspended in non-reducing sample buffer and analyzed by Western blotting .",
"Many disulfide-linked adducts are observed with wild type Mia40 , particularly after incubation with diamide , but not with Mia40-SPS , not even when Erv1 is overexpressed . DOI: http://dx . doi . org/10 . 7554/eLife . 16177 . 005 Why does diamide suppress the growth defect of Mia40-SPS mutants ?",
"Either it supports the functionality of the Mia40-SPS protein ( e . g . , by the formation of the structural disulfides in the twin Cx9C domain of this protein ) , or it might act downstream in oxidizing proteins that were initially imported by Mia40-SPS .",
"To test whether diamide directly supports the functionality of the Mia40-SPS protein , we isolated mitochondria containing either Mia40-SPS or Mia40-FE , pretreated them with increasing amounts of diamide and then added a radiolabeled cysteine-less model substrate of Mia40 , 10CS .",
"This protein represents a mutant version of the IMS protease Atp23 in which all ten cysteine residues were replaced by serine residues ( Weckbecker et al . , 2012 ) .",
"The import of this protein into the mitochondria was assessed by analysis of the protease-protected 10CS fraction after 15 min of incubation ( Figure 3C ) .",
"Interestingly , Mia40-SPS was able to efficiently promote the import of this protein whereas 10CS was not taken up by Mia40-FE mitochondria .",
"However , diamide did not affect the import into either mutant suggesting that it has no effect on the functionality of Mia40-SPS or Mia40-FE .",
"Western blotting of extracts of diamide-treated mitochondria suggests that diamide increases the number of disulfide-linked adducts on Mia40 , but not on Mia40-SPS , even in the presence of overexpressed Erv1 ( Figure 3D ) .",
"Hence , diamide does not induce non-canonical disulfides between Mia40-SPS and imported proteins but presumably acts on the imported substrates after their translocation into the mitochondria thereby stabilizing their structure .",
"In summary , we conclude that the redox-inactive Mia40-SPS mutant is still functional in the import of a cysteine-free model protein and sufficient for cell survival in the presence of the chemical oxidizer diamide .",
"The observation that Mia40-SPS efficiently drives the import reaction of the 10CS protein inspired us to test whether this variant likewise supports the import of cysteine-containing Mia40 substrates .",
"To this end , we imported radiolabeled Tim9 and Atp23 , two proteins that are imported in a strictly Mia40-dependent reaction ( Chacinska et al . , 2004; Naoé et al . , 2004; Weckbecker et al . , 2012 ) .",
"Mia40-SPS was fully sufficient to drive the import reaction of both proteins ( Figure 4A–D ) .",
"We conclude that Mia40-driven oxidation is not critical for protein translocation into mitochondria .",
"This was very surprising since the oxidative folding by Mia40 was proposed to drive the import of its substrates across the outer membrane ( 'folding trap model' ) ( Lutz et al . , 2003; Koehler , 2004; Bihlmaier et al . , 2007 ) .",
"Whereas the redox-active cysteines were dispensable for protein import , the hydrophobic binding domain was essential since no import of Mia40 substrates was observed with the Mia40-STOP mutant ( Figure 4B , E–G ) . 10 . 7554/eLife . 16177 . 006Figure 4 . Substrate-binding by Mia40 is necessary and sufficient for protein translocation into the IMS .",
"( A ) Depletion of Mia40 blocks import of Tim9 .",
"Radiolabeled Tim9 was incubated at 25°C for the times indicated with mitochondria isolated from Mia40-depleted cells lacking or containing a plasmid for expression of the Mia40 variant .",
"After treatment with proteinase K ( PK ) mitochondria were reisolated , washed and subjected to SDS-PAGE and autoradiography .",
"10% of the radiolabeled protein used per import sample is shown for comparison .",
"( B–I )",
"In vitro import reactions with radiolabeled Tim9 , Atp23 and Cmc1 into the mitochondria of the indicated strains . DOI: http://dx . doi . org/10 . 7554/eLife . 16177 . 006 It is conceivable that the redox-active cysteine pair in Mia40 is not crucial because Erv1 might directly oxidize these proteins in the Mia40-SPS mutant .",
"Indeed , it was reported that overexpression of Erv1 partially suppresses the growth defect of some Mia40 mutants ( Kawano et al . , 2009 ) .",
"However , when we overexpressed Erv1 in the Mia40-SPS background , we did not observe a stimulation , but rather a considerable reduction of the import of Atp23 and Cmc1 into the IMS ( Figure 4H , I ) .",
"This reduction is most likely the consequence of a competitive binding of Erv1 and the incoming polypeptides to the substrate-binding pocket of Mia40 ( Banci et al . , 2011 ) .",
"When Mia40-SPS was isolated by immunoprecipitation during the import reactions , considerable fractions of Tim9 and Cmc1 were co-isolated ( Figure 5A , B ) , albeit not covalently bound via disulfides ( Figure 5C , D ) .",
"This indicates a tight association of the incoming proteins with the substrate-binding pocket of Mia40 .",
"In contrast , Mia40 substrates ( Cox19 , Tim9 , Atp23 ) were not co-isolated with the Mia40-STOP variant ( Figure 5—figure supplements 1 and 2 ) .",
"Tim9 and Cmc1 were also not coisolated with Erv1 , not even when Erv1 was overexpressed in the Mia40-SPS background ( Figure 5—figure supplement 3 ) , indicating that Erv1 does not interact with these proteins directly or the interaction is too transient or weak to be detected .",
"In summary , we conclude that the substrate-binding pocket of Mia40 is necessary and sufficient for protein import of Mia40 substrates whereas the redox activity of Mia40 is apparently dispensable . 10 . 7554/eLife . 16177 . 007Figure 5 . Mia40-SPS binds to import intermediates of Tim9 and Cmc1 . ( A , B ) Radiolabeled Tim9 and Cmc1 were incubated for 2 min with isolated mitochondria containing Mia40 or Mia40-SPS .",
"Mitochondria were reisolated and lysed with 1% SDS .",
"The extract was used for immunoprecipitation with Tim9- , Cmc1- or Mia40-specific antibodies or with preimmune serum ( PIS ) .",
"Disulfide bonds were reduced with DTT .",
"Radioactive proteins were visualized by SDS-PAGE and autoradiography .",
"Total samples contain 10% of the material used per immunoprecipitation reaction .",
"Arrows depict radiolabeled proteins pulled down with Mia40 and Mia40-SPS .",
"( C , D )",
"Mia40- or Mia40-SPS-containing mitochondria were treated for 10 min with 5 mM DTT at 30°C , tenfold diluted and incubated with radiolabeled Tim9 or Cmc1 for 2 min .",
"Reisolated mitochondria were lysed with 1% SDS before the extract was used for immunoprecipitation using Mia40-specific antibodies or preimmune serum for control .",
"Reducing ( + ß-mercaptoethanol , ß-ME ) or non-reducing samples were analyzed by SDS-PAGE and autoradiography .",
"See figure supplements for additional panels . DOI: http://dx . doi . org/10 . 7554/eLife . 16177 . 00710 . 7554/eLife . 16177 . 008Figure 5—figure supplement 1 . The substrate-binding cleft but not the redox-active CPC motif of Mia40 is sufficient for the import of Tim9 and Cox19 . ( A , B ) Radiolabeled Tim9 and Cox19 were imported for 2 min into isolated mitochondria containing Mia40-SPS or Mia40-STOP .",
"Mitochondria were reisolated and lysed under native conditions with lysis buffer N ( 30 mM Tris/HCl pH 8 , 100 mM NaCl , 0 . 1% Triton X-100 , 2 mM PMSF ) , followed by a clarifying spin .",
"The preimmune serum or antibodies against Tim9 , Cox19 , Mia40 or hemagglutinin were coupled to protein A-sepharose beads .",
"The beads were incubated with the mitochondrial extract for 1 hr at 4°C , washed twice with lysis buffer N and once with 20 mM Tris pH 8 , resuspended in reducing SDS-sample buffer and boiled for 5 min at 96°C .",
"Samples were analyzed by SDS-PAGE and autoradiography .",
"Total samples contain 10% of the material used per immunoprecipitation reaction . DOI: http://dx . doi . org/10 . 7554/eLife . 16177 . 00810 . 7554/eLife . 16177 . 009Figure 5—figure supplement 2 . Cysteines in the CPC motif of Mia40 are dispensable for the import of Tim9 and Atp23 . ( A , B ) Radiolabeled Tim9 and Atp23 were imported for 2 min into isolated mitochondria containing Mia40-STOP .",
"Mitochondria were reisolated and lysed with 1% SDS .",
"The extract was used for immunoprecipitation with Tim9- , Atp23- or hemagglutinin-specific antibodies or with preimmune serum .",
"Samples were loaded reducing or non-reducing as indicated , and analyzed by SDS-PAGE and autoradiography .",
"Total samples contain 10% of the material used per immunoprecipitation reaction . DOI: http://dx . doi . org/10 . 7554/eLife . 16177 . 00910 . 7554/eLife . 16177 . 010Figure 5—figure supplement 3 . Tim9 and Cmc1 form mixed disulfides with Mia40 but not with Mia40-SPS or Mia40-STOP .",
"( A , B )",
"Radiolabeled Tim9 or ( C , D ) Cmc1 were imported for 2 min into isolated mitochondria containing Mia40 , Mia40-STOP , Mia40-SPS or Mia40-SPS+Erv1↑ .",
"Mitochondria were reisolated and lysed with 1% SDS , diluted in Triton X100 buffer and subjected to a clarifying spin .",
"The preimmune serum or antibodies against Mia40 , Erv1 or hemagglutinin were coupled to protein A-sepharose beads .",
"The beads were incubated with the mitochondrial extracts over night at 4°C , washed , resuspended in reducing SDS-sample buffer and boiled for 5 min at 96°C .",
"Samples were analyzed by SDS-PAGE and autoradiography .",
"Total samples contain 10% of the material used per immunoprecipitation reaction . DOI: http://dx . doi . org/10 . 7554/eLife . 16177 . 010 Next , we tested whether Mia40-SPS can also mediate protein targeting to the IMS in vivo .",
"The depletion of Mia40 from mitochondria results in the concomitant depletion of its substrates as shown in many previous studies ( Chacinska et al . , 2004; Naoé et al . , 2004; Mesecke et al . , 2005; Allen et al . , 2005 ) .",
"However , when Mia40-SPS was expressed in Mia40-depleted cells , we observed that the levels of Mia40 substrates were partially restored ( Figure 6A ) .",
"When we tested the redox states of the Mia40 substrate Tim10 in the Mia40-SPS mutant we found , that both disulfides in Tim10 were properly formed ( Figure 6B , C ) .",
"This suggests that the redox-active CPC motif in Mia40 accelerates substrate oxidation , but it is not absolutely essential for protein oxidation in the IMS . 10 . 7554/eLife . 16177 . 011Figure 6 . Expression of Mia40-SPS leads to the accumulation of Mia40 substrates in mitochondria .",
"( A ) Mitochondria were isolated from Mia40-depleted cells ( ↓ ) which expressed Mia40 or Mia40-SPS .",
"The steady state levels of Mia40 substrates and control proteins were analyzed by Western blotting .",
"( B , C )",
"To determine the redox state of Tim10 , proteins of Mia40- and Mia40-SPS-containing mitochondria were TCA-precipitated , denatured in SDS , treated with or without the reducing agent TCEP and the alkylating compounds mmPEG24 ( B ) or AMS ( C ) and visualized by SDS-PAGE and Western blotting .",
"An inverse shift was achieved by blocking reduced thiols with N-ethylmaleimide ( NEM ) prior to TCA precipitation .",
"Green arrows depict fully oxidized Tim10 , red arrows the matrix chaperone Mdj1 which does not contain disulfide bonds .",
"( D ) Western blots of mitochondrial extracts of the indicated strains .",
"Please note that expression of Mia40-SPS but not of Mia40-STOP leads to accumulation of low levels of Atp23 and Cmc1 in mitochondria .",
"( E ) Wild type and mia40-4 cells were transformed with a Cox19-HA-expressing plasmid ( Bode et al . , 2015 ) allowing detection of Cox19 with hemagglutinin ( HA ) antibodies in whole cell extracts .",
"Levels of Mia40 substrates and other mitochondrial proteins were analyzed by Western blotting in the indicated cells after growth at 34°C .",
"( F ) Western blot analysis of protein levels in wild type and GAL-Mia40 cells expressing Mia40-SPS variant and high levels of Erv1 .",
"Arrows up and down indicate the expression or depletion of Mia40 in this strain , respectively .",
"( G , H )",
"To follow the oxidation of Cox19 in vivo , cells of the indicated strains were pulse-labeled for 3 min with [35S]-methionine and chased with cold methionine for different times ( Kojer et al . , 2015 ) .",
"TCA-precipitated protein extracts were treated with mmPEG24 , immunoprecipitated with hemagglutinin-antibodies and analyzed by SDS-PAGE and autoradiography . DOI: http://dx . doi . org/10 . 7554/eLife . 16177 . 011 The presence of a functional substrate-binding pocket in Mia40 was critical for the accumulation of Mia40 substrates in vivo as Atp23 and Cmc1 were not detectable in Mia40-STOP mitochondria unless Mia40-SPS was co-expressed ( Figure 6D ) .",
"The ability of Mia40-SPS to partially restore the levels of Cox19 , Atp23 , and Cmc1 was also observed in cells of temperature-sensitive mia40 mutant that were grown under restrictive conditions ( Figure 6E ) , again indicating that the substrate-binding pocket is sufficient for protein import .",
"Overexpression of Erv1 did not further increase the levels of IMS proteins in the Mia40-SPS mutant but rather strongly diminished them ( Figure 6F ) .",
"This again points to a competitive binding of Erv1 and Mia40 substrates to the crucial substrate-binding domain of Mia40-SPS .",
"Next , we followed the oxidation of newly synthesized Cox19-HA in a pulse chase experiment in wild type and mia40-4 cells ( Figure 6G , H ) .",
"To this end , translation products were radiolabeled in yeast cells for 3 min .",
"Then , the labeling was stopped by washing the cells and by the addition of an excess of non-radioactive methionine .",
"After different times of incubation , samples were taken and subjected to alkylation with mmPEG24 .",
"Then , Cox19-HA was immunoprecipitated and analyzed by SDS-PAGE .",
"In the wild type , Cox19-HA was rapidly oxidized and within less than 2 min the two disulfide bonds were formed .",
"In the temperature-sensitive mia40-4 mutant , the oxidation of Cox19-HA was considerably slower and only after 4 to 8 min , half of the protein was oxidized .",
"The oxidation was not accelerated by expression of Mia40-SPS .",
"This confirms that the positive effect on the import and the accumulation of IMS proteins in the Mia40-SPS mutant is independent of their oxidation .",
"From the partial restoration of the IMS import by Mia40-SPS we conclude that the primary function of Mia40 is a role as a trans-site receptor that drives the translocation of proteins across the outer membrane .",
"In addition , it also facilitates protein oxidation , however , this function appears to be important only after the translocation reaction , presumably to fold IMS proteins into a functional and protease-resistant confirmation .",
"The observation that the depletion of Mia40 leads to a rapid concomitant depletion of Mia40 substrates inspired us to test whether – on the contrary - overexpression of Mia40 increases the levels of Mia40 substrates .",
"As shown in Figure 7A , overexpression of Mia40 leads indeed to much higher cellular levels of Mia40 substrates , such as Atp23 , Tim10 and Cmc1 .",
"Thus , the endogenous levels of Mia40 are obviously rate-limiting under physiological conditions , suggesting that only a fraction of the Mia40 substrates that are initially synthesized in the cytosol finally accumulate as stable proteins in vivo and that overexpression of Mia40 increases this fraction .",
"This was also obvious when isolated mitochondria of these strains were analyzed ( Figure 7B ) .",
"We even observed increased amounts of cytochrome b2 , a protein that is imported into the IMS on a stop-transfer pathway ( Glick et al . , 1992; Gärtner et al . , 1995; Esaki et al . , 1999 ) and that was not expected to be influenced by the presence of Mia40 .",
"However , it was previously proposed that in Candida albicans , the homolog of cytochrome b2 might be imported in a Mia40-driven reaction ( Hewitt et al . , 2012 ) . 10 . 7554/eLife . 16177 . 012Figure 7 . Mia40 is rate-limiting for the import of IMS proteins .",
"( A ) Western blot analysis of mitochondrial protein levels in galactose-grown wild type ( WT ) cells and in GAL-Mia40 cells grown in lactic acid-based media containing 0 . 5% glucose ( ↓ ) or galactose ( ↑ ) .",
"Overexpression of Mia40 leads to strongly increased steady-state levels of its substrates such as Atp23 , Tim10 and Cmc1 .",
"( B ) Mitochondria were isolated from WT and GAL-Mia40 cells cultured as described in A . Steady state levels of IMS proteins were analyzed by Western blotting .",
"Please note the increased level of cytochrome b2 upon overexpression of Mia40 .",
"( C ) Quantifications of the amounts of imported radiolabeled proteins from in vitro import experiments with the Mia40 substrates Cmc1 , Atp23 and Tim9 .",
"Error bars correspond to SEM with n = 4 .",
"Radioactive signals of one representative experiment are shown .",
"( D ) In vitro import reaction with radiolabeled cytochrome b2 into mitochondria of the indicated strains .",
"A shorter ( ‘pseudomature’ ) form of cytochrome b2 that is formed by initiation at the second ATG codon is indicated by an asterisk .",
"( E–G )",
"Schematic representations of the ‘folding trap model’ and the ‘trans-site receptor’ models .",
"According to the folding trap model , protein translocation into the IMS is driven by the formation of disulfides which prevents back-translocation into the cytosol .",
"The ‘trans-site receptor model' suggests that translocation is driven by the affinity of incoming proteins to the substrate-binding pocket of Mia40 .",
"It was suggested that the disulfide-mediated trapping is essential for the translocation reaction .",
"However , our results shown here suggest that the trapping via hydrophobic binding of the incoming polypeptide to Mia40 is essential and sufficient for the translocation reaction .",
"Oxidative folding is only a subsequent reaction which can facilitate the release from Mia40 and increase the proteolytic stability of imported proteins .",
"See discussion for details . DOI: http://dx . doi . org/10 . 7554/eLife . 16177 . 012 We next tested the import efficiencies of different Mia40 substrates with wild type and Mia40-upregulated mitochondria ( Figure 7C ) .",
"We found consistently , that the upregulation of Mia40 considerably improved the import of Cmc1 , Atp23 and Tim9 .",
"This confirms a rate-limiting function of Mia40 during the import of IMS proteins .",
"In contrast , upregulation of Mia40 did not increase , but rather reduced , the import of cytochrome b2 ( Figure 7D ) , a well-characterized substrate of the presequence pathway ( Hartl et al . , 1986 ) .",
"Depletion of Mia40 did hardly affect the import of cytochrome b2 , however , it impaired its processing .",
"This is presumably explained by the fact that the Mia40 substrate Som1 is a component of the inner membrane protease complex that mediates the maturation of cytochrome b2 ( Jan et al . , 2000; Nunnari et al . , 1993 ) .",
"Immunoprecipitation experiments did not indicate a direct binding of Mia40 to import intermediates of cytochrome b2 ( not shown ) and we assume that the up- and down-regulation of Mia40 regulates the levels of cytochrome b2 indirectly .",
"Thus , the strong rate-limiting function of Mia40 on the import of its substrates is obviously highly relevant to the protein composition of the IMS in general and critical even for proteins which are imported in a Mia40-independent fashion ."
],
[
"Mia40 is a highly conserved IMS protein which consists of two functionally and structurally distinct elements .",
"Here we show that both the redox-active cysteine pair and the substrate-binding region of Mia40 are essential and mutants lacking individual elements cannot cross-complement each other .",
"However , we found that cysteines in the CPC motif of Mia40 are dispensable for the import reaction of all Mia40 substrates we tested .",
"This was unexpected because it was suggested that the mitochondrial disulfide relay operates as a folding trap ( Figure 7E ) ( Lutz et al . , 2003; Koehler , 2004 ) .",
"This model postulated that substrates would enter the IMS via the TOM pore in a reversible reaction .",
"The reversible nature of the outer membrane translocation of Mia40 substrates is experimentally well documented ( Lutz et al . , 2003; Bragoszewski et al . , 2015 ) .",
"According to the folding trap hypothesis , the directivity of the import reaction is driven by the introduction of disulfide bonds thereby locking proteins in a folded confirmation which traps them in the IMS ( Mesecke et al . , 2005; Bihlmaier et al . , 2007 ) .",
"Based on the identification of the MISS/ITS signal as specific recognition sites in Mia40 substrates ( Koch and Schmid , 2014b; Sideris and Tokatlidis , 2007; Sideris et al . , 2009; Peleh et al . , 2014; Milenkovic et al . , 2009 ) and the observation of Mia40-linked import intermediates that span the TOM complex ( von der Malsburg et al . , 2011 ) it was suggested that Mia40 serves as an IMS-located receptor protein critical for outer membrane translocation of its substrates ( Figure 7F ) .",
"According to both models , disulfide bond formation by Mia40 is the critical feature for its role in protein translocation .",
"However , our observation that the Mia40-SPS variant can efficiently drive the import reaction suggests that the affinity to the substrate-binding pocket provides the driving force for the translocation reaction rather than protein oxidation .",
"Hence , we propose that Mia40 serves as a 'trans-site receptor' on the IMS site of the TOM complex that mediates protein translocation across the outer membrane ( Figure 7G ) .",
"At present it is not known whether the trapping function of Mia40 is restricted to disulfide-containing proteins or plays a broader , more general role in mitochondrial protein import .",
"However , the recent identification of non-canonical substrates of Mia40 ( Petrungaro et al . , 2015; Okamoto et al . , 2014; Wrobel et al . , 2013; Hangen et al . , 2015 ) might indeed suggest a much more general role of Mia40 in protein translocation across the outer membrane than previously expected .",
"The oxidoreductase activity of Mia40 is obviously important , but it apparently plays a role after translocation .",
"This is consistent with a recent study which showed that mutants of small Tim proteins , in which all cysteine residues were replaced by serine residues , accumulated to wild type levels in the IMS as soon as the iAAA protease Yme1 was deleted ( Baker et al . , 2012 ) .",
"Surprisingly , the presence of diamide can partially suppress the growth defect of Mia40-SPS cells suggesting that the substrate-specificity of the mitochondrial disulfide relay is not absolutely necessary .",
"The observation of oxidized Tim10 in the Mia40-SPS mutant indicates that the CPC of Mia40 is not absolutely essential for protein oxidation in the IMS .",
"Whether Erv1 can directly oxidize IMS proteins or whether other factors might serve as oxidoreductases here is not known , but it will be interesting to test the relevance of the recently identified thioredoxins and glutaredoxins in the IMS in this process ( Vögtle et al . , 2012; Kojer et al . , 2015 ) .",
"However , it should be noted that , even in the presence of diamide , Mia40-SPS cells were very sick and protein oxidation in the IMS is obviously still an essential process .",
"Our observations indicate that Mia40 is rate-limiting for the import into mitochondria as overexpression of Mia40 leads to a considerable increase of the levels of many Mia40 substrates .",
"Apparently , a fraction of the Mia40 substrates that are initially synthesized in the cytosol are degraded in the cytosol or the IMS , an assumption that is supported by pulse labeling experiments in whole cells ( Wrobel et al . , 2015; Longen et al . , 2014; Kojer et al . , 2015; Fischer et al . , 2013 ) .",
"Increased levels of Mia40 might reduce the degradation either by accelerating the import or the folding reaction .",
"In contrast , up- or down-regulation of the levels of Erv1 had no positive effect on the steady state levels of IMS proteins again supporting the idea that the trapping rather than the oxidase function of Mia40 is decisive ( Bien et al . , 2010; Mesecke et al . , 2005 ) .",
"The strong increase in Mia40 substrates in Mia40-overexpressing cells suggests that more Mia40 leads to more import sites for Mia40 substrates .",
"It is conceivable that mitochondria contain specific import sites for different types of preproteins which differ at the level of the 'trans-site receptors' in the IMS ( Figure 7G ) .",
"Thus , matrix-targeted proteins might specifically encounter TOM complexes that are associated to Tim50 ( Lytovchenko et al . , 2013; Marom et al . , 2011 ) whereas IMS proteins might use TOM complexes that are in proximity to Mia40 .",
"This hypothesis is supported by studies on the Mic60 subunit of the MICOS complex which presumably tethers Mia40 to a subpopulation of the TOM complex ( von der Malsburg et al . , 2011; Herrmann , 2011; Varabyova et al . , 2013 ) .",
"The existence of a discrete translocation route into the IMS was recently proposed for Mia40 substrates on the basis of in organello competition import experiments ( Gornicka et al . , 2014 ) and , even much earlier , for the most abundant IMS protein , cytochrome c ( Wiedemann et al . , 2003; Diekert et al . , 2001; Mayer et al . , 1995 ) .",
"The bacterial periplasm , the ER and the mitochondrial IMS are the three compartments in which dedicated machineries mediate the oxidative folding of a broad range of proteins ( Riemer et al . , 2009 ) .",
"Members of the thioredoxin protein family mediate protein oxidation in the periplasm ( DsbA ) and the ER ( PDI ) .",
"Apparently , the thioredoxin-based oxidation system of the periplasm of the endosymbiont that served as the ancestor of mitochondria was replaced during evolution by the Mia40 system .",
"This replacement during evolution was hardly driven to increase protein oxidation in the IMS since Mia40 is a poor catalyst: in its oxidizing power , it is weaker than DsbA , and in forming correct disulfides , it is slower than PDI ( Koch and Schmid , 2014b; 2014a; Hudson and Thorpe , 2015; Tienson et al . , 2009 ) .",
"However , Mia40 binds much stronger and for much longer interaction times to its substrates than DsbA or PDIs ( Koch and Schmid , 2014b; Mesecke et al . , 2005; Naoé et al . , 2004; Chacinska et al . , 2004 ) .",
"It therefore appears likely that the ability of Mia40 to trap incoming preproteins efficiently at the trans-site of the TOM complex made Mia40 the superior oxidoreductase to mediate the translocation and oxidation of IMS proteins ."
],
[
"All yeast strains used in this study were based on the wild type strain YPH499 ( Sikorski and Hieter , 1989 ) including the regulatable GAL-Mia40 strain ( Mesecke et al . , 2005 #5543 ) and the temperature-sensitive mia40-3 and mia40-4 strains ( Chacinska et al . , 2004 #5355 ) .",
"To inactivate Mia40 in the temperature-sensitive mia40 strains , the culture was shifted to 37°C for 17 hr .",
"Yeast strains were either grown on synthetic media containing 2% galactose , in synthetic lactic acid-based medium ( containing 2% lactic acid and 0 . 5% galactose or glucose ) or in YP ( 1% yeast extract , 2% peptone ) medium containing 2% galactose , glucose or glycerol ( Peleh et al . , 2014 #7089 ) .",
"For expression of the different Mia40 variants ( Mia40 , Mia40-SPS , and Mia40-FE ) the MIA40 promoter and sequences corresponding to the protein sequence of residues 1–70 and 284–403 were cloned into the single-copy plasmids pRS314 , pRS315 or pRS316 .",
"Point mutations within MIA40 were introduced by site-directed mutagenesis ( Weckbecker et al . , 2012 ) .",
"The resulting mutations were confirmed by sequencing .",
"For expression of the Mia40-STOP variant the MIA40 promoter and the sequence corresponding to amino acid residues 1–306 were cloned into pRS315 .",
"Mitochondria were isolated as described ( Peleh et al . , 2015 ) .",
"To analyze the redox state of cysteine residues , mitochondrial proteins were TCA precipitated and treated as described ( Peleh et al . , 2014 ) .",
"For modification , final concentrations of 15 mM mmPEG24 , mmPEG12 and AMS were used to modify reduced thiols .",
"The pulse-chase labeling of Cox19 was performed as described ( Kojer et al . , 2015 ) .",
"The import reactions were performed as described ( Mesecke et al . , 2005 ) in the following import buffer: 1 M sorbitol , 100 mM Hepes pH 7 . 4 , 160 mM KCl , 20 mM magnesium acetate , 4 mM KH2PO4 .",
"In addition 2 mM ATP , 2 mM NADH , 10 mM creatine phosphate , 100 µg/ml creatine kinase , 2 mM malate , and 2 mM succinate were supplied to energize the mitochondria .",
"To keep the radiolabeled proteins in the reduced unfolded state 5 mM GSH and 5 mM EDTA were added to the import mix .",
"To immunoprecipitate import intermediates with Mia40 or Erv1 , import reactions were carried out for 2 min at 25°C .",
"Mitochondria were reisolated by centrifugation ( 20 , 000 xg for 20 min at 4°C ) and resuspended in lysis buffer I ( 30 mM Tris/HCl pH 8 , 100 mM NaCl , 1% SDS , 2 mM PMSF ) .",
"The extract was diluted tenfold in lysis buffer II ( 30 mM Tris/HCl pH 8 , 100 mM NaCl , 1% Triton X-100 , 2 mM PMSF ) .",
"After incubation for 10 min at 4°C , the extract was cleared by a clarifying spin ( 20 , 000 xg for 10 min at 4°C ) .",
"Antibodies against Mia40 , Erv1 , Atp23 , Tim9 , Cmc1 , Cox19 or hemagglutinin were coupled to protein A-sepharose beads .",
"The beads were incubated with the mitochondrial extract at 4°C , washed three times with the lysis buffer II ( 2 , 000 xg for 2 min at 4°C ) , resuspended in SDS-sample buffer and boiled for 5 min at 96°C .",
"Samples were analyzed by SDS-PAGE and autoradiography ."
]
] | [
"Many proteins of the mitochondrial IMS contain conserved cysteines that are oxidized to disulfide bonds during their import .",
"The conserved IMS protein Mia40 is essential for the oxidation and import of these proteins .",
"Mia40 consists of two functional elements: an N-terminal cysteine-proline-cysteine motif conferring substrate oxidation , and a C-terminal hydrophobic pocket for substrate binding .",
"In this study , we generated yeast mutants to dissect both Mia40 activities genetically and biochemically .",
"Thereby we show that the substrate-binding domain of Mia40 is both necessary and sufficient to promote protein import , indicating that trapping by Mia40 drives protein translocation .",
"An oxidase-deficient Mia40 mutant is inviable , but can be partially rescued by the addition of the chemical oxidant diamide .",
"Our results indicate that Mia40 predominantly serves as a trans-site receptor of mitochondria that binds incoming proteins via hydrophobic interactions thereby mediating protein translocation across the outer membrane by a ‘holding trap’ rather than a ‘folding trap’ mechanism ."
] | [
"Human , yeast and other eukaryotic cells contain compartments called mitochondria that perform several vital tasks , including supplying the cell with energy .",
"Each mitochondrion is surrounded by an inner and an outer membrane , which are separated by an intermembrane space that contains a host of molecules , including proteins .",
"Intermembrane space proteins are made in the cytosol before being transported into the intermembrane space through pores in the mitochondrion’s outer membrane .",
"Many of these proteins have the ability to form disulfide bonds within their structures , which help the proteins to fold and assemble correctly , but they only acquire these bonds once they have entered the intermembrane space .",
"An enzyme called Mia40 sits inside the intermembrane space and helps other proteins to fold correctly .",
"This Mia40-induced folding had been suggested to help proteins to move into the intermembrane space .",
"Mia40 contains two important regions: one region acts as an enzyme and adds disulfide bonds to other proteins , and the other region binds to the intermembrane space proteins .",
"Peleh et al . have now generated versions of Mia40 that lack one or the other of these regions in yeast cells , and then tested to see if these mutants could drive proteins across the outer membrane of mitochondria .",
"The results show that it is the ability of Mia40 to bind proteins – and not its enzyme activity – that is essential for importing proteins into the intermembrane space .",
"As disulfide bond formation is not critical for importing proteins into the intermembrane space , future studies could test whether Mia40 also helps to transport proteins that cannot form disulfide bonds .",
"Presumably , Mia40 has a much broader relevance for importing mitochondrial proteins than was previously thought ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"evolutionary biology"
] | Metabolic co-dependence drives the evolutionarily ancient Hydra–Chlorella symbiosis | elife-35122-v2 | [
[
"Symbiosis has been a prevailing force throughout the evolution of life , driving the diversification of organisms and facilitating rapid adaptation of species to divergent new niches ( Moran , 2007; Joy , 2013; McFall-Ngai et al . , 2013 ) .",
"In particular , symbiosis with photosynthetic symbionts is observed in many species of cnidarians such as corals , jellyfish , sea anemones and hydra , contributing to the ecological success of these sessile or planktonic animals ( Douglas , 1994; Davy et al . , 2012 ) .",
"Among the many animals dependent on algal symbionts , inter-species interactions between green hydra Hydra viridissima and endosymbiotic unicellular green algae of the genus Chlorella have been a subject of interest for decades ( Muscatine and Lenhoff , 1963; Roffman and Lenhoff , 1969 ) .",
"Such studies not only provide insights into the basic ‘tool kit’ necessary to establish symbiotic interactions , but are also of relevance in understanding the resulting evolutionary selective processes ( Muscatine and Lenhoff , 1965a; 1965b; Thorington and Margulis , 1981 ) .",
"The symbionts are enclosed in the host endodermal epithelial cells within perialgal vacuoles called ‘symbiosomes’ .",
"The interactions at play here are clearly metabolic: the algae depend on nutrients that are derived from the host or from the environment surrounding the host , while in return the host receives a significant amount of photosynthetically fixed carbon from the algae .",
"Previous studies have provided evidence that the photosynthetic symbionts provide their host with maltose , enabling H . viridissima to survive periods of starvation ( Muscatine and Lenhoff , 1963; Muscatine , 1965; Roffman and Lenhoff , 1969; Cook and Kelty , 1982; Huss et al . , 1994 ) .",
"Chlorella-to-Hydra translocation of photosynthates is critical for polyps to grow ( Muscatine and Lenhoff , 1965b; Mews , 1980; Douglas and Smith , 1983; 1984 ) .",
"Presence of symbiotic algae also has a profound impact on hydra´s fitness by promoting oogenesis ( Habetha et al . , 2003; Habetha and Bosch , 2005 ) .",
"Pioneering studies performed in the 1980 s ( McAuley and Smith , 1982; Rahat and Reich , 1984 ) showed that there is a great deal of adaptation and specificity in this symbiotic relationship .",
"All endosymbiotic algae found in a single host polyp are clonal and proliferation of symbiont and host is tightly correlated ( Bossert and Dunn , 1986; McAuley , 1986a ) .",
"Although it is not yet known how Hydra controls cell division in symbiotic Chlorella , Chlorella strain A99 is unable to grow outside its polyp host and is transmitted vertically to the next generation of Hydra , indicating loss of autonomy during establishment of its symbiotic relationship with this host ( Muscatine and McAuley , 1982; Campbell , 1990; Habetha et al . , 2003 ) .",
"Molecular phylogenetic analyses suggest that H . viridissima is the most basal species in the genus Hydra and that symbiosis with Chlorella was established in the ancestral viridissima group after their divergence from non-symbiotic Hydra groups ( Martínez et al . , 2010; Schwentner and Bosch , 2015 ) .",
"A recent phylogenetic analysis of different strains of green hydra resulted in a phylogenetic tree that is topologically equivalent to that of their symbiotic algae ( Kawaida et al . , 2013 ) , suggesting these species co-evolved as a result of their symbiotic relationship .",
"Although our understanding of the factors that promote symbiotic relationships in cnidarians has increased ( Shinzato et al . , 2011; Davy et al . , 2012; Lehnert et al . , 2014; Baumgarten et al . , 2015; Ishikawa et al . , 2016 ) , very little is known about the molecular mechanisms allowing this partnership to persist over millions of years .",
"Recent advances in transcriptome and genome analysis allowed us to identify the metabolic interactions and genomic evolution involved in achieving the Hydra-Chlorella symbiotic relationship .",
"We present here the first characterization , to our knowledge , of genetic complementarity between green Hydra and Chlorella algae that explains the emergence and/or maintenance of a stable symbiosis .",
"We also provide here the first report of the complete genome sequence from an obligate intracellular Chlorella symbiont .",
"Together , our results show that exchange of nutrients is the primary driving force for the symbiosis between Chlorella and Hydra .",
"Subsequently , reduction of metabolic pathways may have further strengthened their codependency .",
"Our findings provide a framework for understanding the evolution of a highly codependent symbiotic partnership in an early emerging metazoan ."
],
[
"As tool for our study we used the green hydra H . viridissima ( Figure 1A ) colonized with symbiotic Chlorella sp .",
"strain A99 ( abbreviated here as Hv_Sym ) , aposymbiotic H . viridissima from which the symbiotic Chlorella were removed ( Hv_Apo ) , as well as aposymbiotic H . viridissima , which have been artificially infected with Chlorella variabilis NC64A ( Hv_NC64A ) .",
"The latter is symbiotic to the single-cellular protist Paramecium ( Karakashian and Karakashian , 1965 ) .",
"Although an association between H . viridissima and Chlorella NC64A can be maintained for some time , both their growth rate ( Figure 1B ) and the number of NC64A algae per Hydra cell ( Figure 1—figure supplement",
"1 ) are significantly reduced compared to the symbiosis with native symbiotic Chlorella A99 .",
"H . H . viridissima genes involved in the symbiosis with Chlorella algae were identified by microarray based on the contigs of H . viridissima A99 transcriptome ( NCBI GEO Platform ID: GPL23280 ) .",
"For the microarray analysis , total RNA was extracted from the polyps after light exposure for six hours .",
"By comparing the transcriptomes of Hv_Sym and Hv_Apo , we identified 423 contigs that are up-regulated and 256 contigs that are down-regulated in presence of Chlorella A99 ( Figure 1C ) .",
"To exclude genes involved in oogenesis and embryogenesis , only contigs differently expressed with similar patterns in both sexual and asexual Hv_Sym were recorded .",
"Interestingly , contigs whose predicted products had no discernible homologs in other organisms including other Hydra species were overrepresented in these differentially expressed contigs ( Chi-squared test p<0 . 001 ) ( Figure 1—figure supplement 2 ) .",
"Such taxonomically restricted genes ( TRGs ) are thought to play important roles in the development of evolutionary novelties and morphological diversity within a given taxonomic group ( Khalturin et al . , 2009; Tautz and Domazet-Lošo , 2011 ) .",
"We further characterized functions of the differentially expressed Hydra genes by Gene Ontology ( GO ) terms ( Ashburner et al . , 2000 ) and found the GO term ‘localization’ overrepresented among up-regulated contigs ( Hv_Sym > Hv_Apo ) , whereas the GO term ‘metabolic process’ was enriched among down-regulated contigs ( Hv_Sym < Hv_Apo ) ( Figure 1D ) .",
"More specifically , the up-regulated contigs included many genes related to ‘transmembrane transporter activity’ , ‘transmembrane transport’ , ‘transposition’ , ‘cilium’ and ‘protein binding , bridging’ ( Figure 1E ) .",
"In the down-regulated contig set , the GO classes ‘cellular amino acid metabolic process’ , ‘cell wall organization or biogenesis’ and ‘peptidase activity’ were overrepresented ( Figure 1E ) .",
"These results suggest that the Chlorella symbiont affects core metabolic processes and pathways in Hydra .",
"Particularly , carrier proteins and active membrane transport appear to play a prominent role in the symbiosis .",
"As next step , we used GO terms , domain search and similarity search to further analyze the differentially expressed contigs between Hv_Sym and Hv_Apo ( Supplementary file 1 ) .",
"As the genes with GO terms related to localization and transport , we identified 27 up-regulated contigs in Hv_Sym ( Table 1 ) .",
"Interestingly , this gene set included a contig showing sequence similarity to the glucose transporter GLUT8 gene , which was previously reported to be up-regulated in the symbiotic state of the sea anemone Aiptasia ( Lehnert et al . , 2014; Sproles et al . , 2018 ) .",
"Thus , a conserved mechanism may be responsible for photosynthate transport from the symbiont into the host cytoplasm across the symbiosome membrane .",
"Further , a contig encoding a carbonic anhydrase ( CA ) enzyme was up-regulated in Hv_Sym ( Table 1 ) .",
"CA catalyzes the interconversion of HCO3 and CO2 .",
"Similar to the GLUT8 gene , carbonic anhydrase also appears to be up-regulated in symbiotic corals and anemones ( Weis et al . , 1989; Grasso et al . , 2008; Ganot et al . , 2011; Lehnert et al . , 2014 ) .",
"It appears plausible that for efficient photosynthesis in symbiotic algae , the host may need to convert CO2 to the less freely diffusing inorganic carbon ( HCO3 ) to maintain it in the symbiosome ( Lucas and Berry , 1985; Weis et al . , 1989; Barott et al . , 2015 ) .",
"We also observed up-regulation of contigs encoding proteins involved in vesicular and endosomal trafficking , such as spe-39 protein , otoferlin , protein fam194b and V-type proton ATPase 21 kda proteolipid , which may have a function in nutrition exchange between host and symbiont and maintenance of proper condition in the symbiosome .",
"Upregulated genes also include genes encoding rhamnospondin and fibrillin , known to be involved in cell adhesion and extracellular matrix , and retention of the symbiont at the proper site in the Hydra cells .",
"Photosynthesis by symbiotic algae imposes Reactive Oxygen Species ( ROS ) that can damage lipids , proteins and DNA in the host cells ( Lesser , 2006 ) .",
"Therefore , in symbiosis with photosynthetic organisms an appropriate oxidative stress response of the host is required for tolerance of the symbiont .",
"Indeed , an increase of antioxidant activities in symbiotic states of cnidarians has been reported previously ( Richier et al . , 2005 ) and it has been suggested that ROS produced by stress could be the major trigger of symbiosis breakdown during coral bleaching ( Lesser , 2006; Weis , 2008 ) .",
"To understand the oxidative stress response in green hydra , we searched the differentially expressed genes with the GO terms ‘response to oxidative stress’ , ‘oxidation-reduction process’ and ‘oxidoreductase activity’ .",
"In Hv_Sym , contigs for peroxidase , methionine-r-sulfoxide reductase/selenoprotein and glutaredoxin , which are known to be related to oxidative stress response were up-regulated ( Table 1 ) .",
"On the other hand , some contigs encoding glutathione S-transferase and ascorbate peroxidase were down-regulated in Hv_Sym .",
"In addition , two contigs encoding polycystin were down-regulated in Hv_Sym .",
"Polycystin is an intracellular calcium release channel that is inhibited by ROS ( Montalbetti et al . , 2008 ) and is also down-regulated in a different strain of symbiotic green hydra ( Ishikawa et al . , 2016 ) .",
"In addition , six contigs encoding metalloproteinases showed differential expression between Hv_Sym and Hv_Apo .",
"Although metalloproteinases have many functions such as cleavage of cell surface proteins and remodeling of the extracellular matrix , in a previous study they also were found to play a role in the oxidative stress response ( Császár et al . , 2009 ) .",
"A key antioxidant in the oxidative stress response in symbiotic cnidarians turns out to be glutathione ( Sunagawa et al . , 2009; Meyer and Weis , 2012 ) .",
"The gene encoding glutathione S-transferase was previously observed to be downregulated in corals , sea anemones , different strains of green hydra and Paramecium ( Kodama et al . , 2014; Lehnert et al . , 2014; Ishikawa et al . , 2016; Mohamed et al . , 2016 ) .",
"Our study supports this view ( Table",
"1 ) and may point to a conserved feature of oxidative stress response in algal-animal symbiosis .",
"Previous studies have suggested that during establishment of coral–algal symbiosis the host immune response may be partially suppressed ( Weis et al . , 2008; Mohamed et al . , 2016 ) .",
"Our observations in Hydra together with previous findings in corals indicate that regulation of symbiosis by innate immunity pathways indeed may be a general feature of cnidarian symbiosis .",
"Among the differentially expressed contigs we identified a number of genes involved in innate immunity and apoptosis .",
"Pattern recognition receptors ( PRRs ) and the downstream innate immunity and apoptosis pathways are thought to play important roles in various symbiotic interactions including cnidarian-dinoflagellate symbiosis ( Davy et al . , 2012 ) .",
"In Hv_Sym we found two up-regulated contigs that contain a Toll/interleukin-1 receptor ( TIR ) domain ( Table 1 ) .",
"TIR is a known PRR that is inserted in the host cell membrane and plays an important role in the innate immune system by specifically recognizing microbial-associated molecular patterns , such as flagellin , lipopolysaccharide ( LPS ) and peptidoglycan ( Hoving et al . , 2014 ) .",
"Furthermore , we found one up-regulated contig with similarity to a mannose receptor gene with C-type lectin domain ( Table 1 ) .",
"This is worth noting since C-type lectin receptors bind carbohydrates and some of them are known to function as PRRs .",
"Host lectin-algal glycan interactions have been proposed to be involved in infection and recognition of symbionts in some cnidarians including green hydra , sea anemones and corals ( Meints and Pardy , 1980; Lin et al . , 2000; Wood-Charlson et al . , 2006 ) .",
"Interestingly , up-regulation of C-type lectin genes was also observed during onset of cnidarian–dinoflagellate symbiosis ( Grasso et al . , 2008; Schwarz et al . , 2008; Sunagawa et al . , 2009; Mohamed et al . , 2016 ) .",
"Furthermore , contigs encoding chitinase enzymes also were differentially expressed between Hv_Sym and Hv_Apo ( Table 1 ) .",
"Chitinases are involved in degradation of chitin , which is a component of the exoskeleton of arthropods and the cell wall of fungi , bacteria and some Chlorella algae ( Kapaun and Reisser , 1995 ) , and also might play a role in host-defense systems for pathogens which have chitinous cell wall .",
"Chitinases are classified into two glycoside hydrolase families , GH18 and GH19 , with different structures and catalytic mechanisms .",
"In Hv_Sym two contigs encoding GH18 chitinases were up-regulated , while one contig encoding a GH19 chitinase was down-regulated , suggesting that the enzymes involved in chitin degradation are sensitive to the presence or absence of symbiotic Chlorella .",
"To narrow down the number of genes specifically affected by the presence of the native symbiont Chlorella A99 , we identified 12 contigs that are differentially expressed in symbiosis with Chlorella A99 , but not in presence of foreign Chlorella NC64A ( Figure 1C A99-specific ) .",
"Independent qPCR confirmed the differential expression pattern for 10 of these genes ( Table 2 ) .",
"The genes up-regulated by the presence of the symbiont encode a Spot_14 protein , a glutamine synthetase ( GS ) and a sodium-dependent phosphate ( Na/Pi ) transport protein in addition to a H . viridissima specific gene ( rc_12891: Sym-1 ) and a Hydra genus specific gene ( rc_13570: Sym-2 ) ( Table 2 ) .",
"Hydra genes down-regulated by the presence of Chlorella A99 were two H . viridissima-specific genes and three metabolic genes encoding histidine ammonia-lyase , acetoacetyl-CoA synthetase and 2-isopropylmalate synthase ( Table 2 ) .",
"Of the up-regulated genes , Spot_14 is described as thyroid hormone-responsive spot 14 protein reported to be induced by dietary carbohydrates and glucose in mammals ( Tao and Towle , 1986; Brown et al . , 1997 ) .",
"Na/Pi transport protein is a membrane transporter actively transporting phosphate into cells ( Murer and Biber , 1996 ) .",
"GS plays an essential role in the metabolism of nitrogen by catalyzing the reaction between glutamate and ammonia to form glutamine ( Liaw et al . , 1995 ) .",
"Interestingly , out of the three GS genes H . viridissima contains only GS-1 was found to be up-regulated by the presence of the symbiont ( Figure 1—figure supplement 3 ) .",
"The discovery of these transcriptional responses points to an intimate metabolic exchange between the partners in a species-specific manner .",
"To better understand the specificity of Hydra´s response to the presence of the foreign symbiont , we also identified the genes differentially expressed in Hydra polyps hosting a non-native Chlorella NC64A ( Hv_NC64A ) compared to both polyps hosting the obligate symbiont Chlorella A99 ( Hv_A99 ) and aposymbiotic Hydra ( Hv_Apo ) .",
"We found 19 contigs that were up-regulated and 45 contigs that were down-regulated in presence of NC64A , which strikingly did not include any genes related to immunity or oxidative stress response ( Supplementary file 1 ) .",
"Instead , the differentially expressed contigs showed similarity to methylase genes involved in ubiquinone menaquinone biosynthesis and secondary metabolite synthesis such as n- ( 5-amino-5-carboxypentanoyl ) -l-cysteinyl-d-valine synthase and non-ribosomal peptide synthase .",
"Four differentially expressed contigs specifically up-regulated in Hv_NC64A encoded ubiquitin carboxyl-terminal hydrolases , ( Table 3 ) .",
"To test whether photosynthetic activity of the symbiont is required for up-regulation of gene expression , Hv_Sym was either cultured under a standard 12 hr light/dark alternating regime or continuously in the dark for 1 to 4 days prior to RNA extraction ( Figure 2A ) .",
"Interestingly , four ( GS1 , Spot14 , Na/Pi and Sym-1 ) of five genes specifically activated by the presence of Chlorella A99 showed significant up-regulation when exposed to light ( Figure 2B ) , indicating the relevance of photosynthetic activity of Chlorella .",
"This up-regulation was strictly dependent on presence of the algae , as in aposymbiotic Hv_Apo the response was absent ( Figure 2B ) .",
"On the other hand , symbiosis-regulated Hydra genes not specific for Chlorella A99 ( Figure 1C Symbiosis-regulated , Table 4 ) appear to be not up-regulated in a light-dependent manner ( Figure 2—figure supplement 1 ) .",
"These genes are involved in Hydra´s innate immune system ( e . g . proteins containing Toll/interleukin-1 receptor domain or Death domain ) or in signal transduction ( C-type mannose receptor , ephrin receptor , proline-rich transmembrane protein 1 , ‘protein-kinase , interferon-inducible double stranded RNA dependent inhibitor , repressor of ( p58 repressor ) ’ ) .",
"That particular transcriptional changes observed in Hydra rely solely on the photosynthetic activity of Chlorella A99 was confirmed by substituting the dark incubation with selective chemical photosynthesis inhibitor DCMU ( Dichorophenyl-dimethylurea ) ( Vandermeulen et al . , 1972 ) , which resulted in a similar effect ( Figure 2C , D ) .",
"To further characterize the symbiont induced Hydra genes , we performed whole mount in situ hybridization ( Figure 3A–F ) and quantified transcripts by qPCR using templates from isolated endoderm and ectoderm ( Figure 3—figure supplement 1 ) , again comparing symbiotic and aposymbiotic polyps ( Figure 3G–I ) .",
"The GS-1 gene and the Spot14 gene are expressed both in ectoderm and in endoderm ( Figure 3A , B ) and both genes are strongly up-regulated in the presence of the symbiont ( Figure 3G , H ) .",
"In contrast , the Na/Pi gene was expressed only in the endoderm ( Figure 3C ) and there it was strongly up-regulated by the symbiont ( Figure 3I ) .",
"Since Chlorella sp .",
"A99 colonizes endodermal epithelial cells only , the impact of algae on symbiosis-dependent genes in both the ectodermal and the endodermal layer indicates that photosynthetic products can be transported across these two tissue layers or some signals can be transduced by cell-cell communication .",
"To more closely dissect the nature of the functional interaction between Hydra and Chlorella and to explore the possibility that maltose released from the algae is involved in A99-specific gene regulation , we cultured aposymbiotic polyps ( Hv_Apo ) for 2 days in medium containing various concentrations of maltose ( Figure 3J ) .",
"Of the five A99 specific genes , GS-1 and the Spot14 gene were up-regulated by maltose in a dose-dependent manner; the Na/Pi gene was only up-regulated in 100 mM maltose and the Hydra specific genes Sym-1 and Sym-2 did not show significant changes in expression by exposure to maltose ( Figure 3J ) .",
"This provides strong support for previous views that maltose excretion by symbiotic algae contributes to the stabilization of this symbiotic association ( Cernichiari et al . , 1969 ) .",
"When polyps were exposed to glucose instead of maltose , the genes of interest were also transcriptionally activated in a dose-dependent manner , while sucrose had no effect ( Figure 3—figure supplement 2A–D ) .",
"Exposure to low concentrations of galactose increased transcriptional activity but at high concentration it did not , indicating a substrate inhibitor effect for this sugar .",
"That the response to glucose is similar or even higher compared to maltose after 6 hr of treatment ( Figure 3—figure supplement 2E ) , suggests that Hydra cells transform maltose to glucose as a source of energy .",
"In animals including cnidarians , several glucose transporters have been identified ( Sproles et al . , 2018 ) , but yet no maltose transporters .",
"This is consistent with the view that maltose produced by the symbiont is digested to glucose in the symbiosome and translocated to the host cytoplasm through glucose transporters .",
"To better understand the symbiosis between H . viridissima and Chlorella and to refine our knowledge of the functions that are required in this symbiosis , we sequenced the genome of Chlorella sp .",
"strain A99 and compared it to the genomes of other green algae .",
"The genome of Chlorella sp .",
"A99 was sequenced to approximately 211-fold coverage , enabling the generation of an assembly comprising a total of 40 . 9 Mbp ( 82 scaffolds , N50 = 1 . 7 Mbp ) ( Table 5 ) .",
"Chlorella sp .",
"A99 belongs to the family Chlorellaceae ( Figure 4A ) and of the green algae whose genomes have been sequenced it is most closely related to Chlorella variabilis NC64A ( NC64A ) ( Merchant et al . , 2007; Palenik et al . , 2007; Worden et al . , 2009; Blanc et al . , 2010; Prochnik et al . , 2010; Blanc et al . , 2012; Gao et al . , 2014; Pombert et al . , 2014 ) .",
"The genome size of the total assembly in strain A99 was similar to that of strain NC64A ( 46 . 2 Mb ) ( Figure 4B ) .",
"By k-mer analysis ( k-mer = 19 ) , the genome size of A99 was estimated to be 61 Mbp ( Marçais and Kingsford , 2011 ) .",
"Its GC content of 68% , is the highest among the green algae species recorded ( Figure 4B ) .",
"In the A99 genome , 8298 gene models were predicted .",
"As shown in Figure 4C , about 80% of these predicted genes have extensive sequence similarity to plant genes , while 13% so far have no similarity to genes of any other organisms ( Figure 4C ) .",
"It is also noteworthy that 7% of the A99 genes are similar to genes of other kingdoms but not to Hydra , indicating the absence of gene transfer from Hydra to the symbiont genome ( Figure 4C ) .",
"Several independent lines of evidence demonstrate that nitrogen limitation and amino-acid metabolism have a key role in the Chlorella–Hydra symbiosis and that symbiotic Chlorella A99 depends on glutamine provided by its host ( Rees , 1986; McAuley , 1987a; 1987b; McAuley , 1991; Rees , 1991;1989 ) .",
"To identify Chlorella candidate factors for the development and maintenance of the symbiotic life style , we therefore used the available genome information to assess genes potentially involved in amino acid transport and the nitrogen metabolic pathway .",
"When performing a search for the Pfam domain ‘Aa_trans’ or ‘AA_permease’ to find amino acid transporter genes in the A99 genome , we discovered numerous genes containing the Aa_trans domain ( Table 6A ) .",
"In particular , A99 contains many orthologous genes of amino acid permease 2 and of transmembrane amino acid transporter family protein ( solute carrier family 38 , sodium-coupled neutral amino acid transporter ) , as well as NC64A ( Table 6B , Supplementary file 2 ) .",
"Both of these gene products are known to transport neutral amino acids including glutamine .",
"This observation is supporting the view that import of amino acids is an essential feature for the symbiotic way of life of Chlorella .",
"In symbiotic organisms , loss of genes often occurs due to the strictly interdependent relationship ( Ochman and Moran , 2001; Wernegreen , 2012 ) , raising the possibility that Chlorella A99 might have lost some essential genes .",
"To test this hypothesis , we searched the Chlorella A99 genome for genes conserved across free-living green algae Coccomyxa subellipsoidea C169 ( C169 ) , Chlamydomonas reinhardtii ( Cr ) and Volvox carteri ( Vc ) .",
"In a total of 9851 C169 genes , we found 5701 genes to be conserved in Cr and Vc ( Supplementary file 3 ) .",
"Of these , 238 genes did not match to any gene models and genomic regions in Chlorella A99 and thus were considered as gene loss candidates .",
"Interestingly , within these 238 candidates , genes with the GO terms ‘transport’ in biological process and ‘transporter activity’ in molecular function were overrepresented ( Figure 5 ) .",
"In particular , the 28 genes annotated to these GO terms encoded nitrate transporter , urea transporter and molybdate transporter , which are known to be involved in nitrogen metabolism ( Table 7 ) .",
"Beside ammonium , nitrate and urea are major nitrogen sources for plants , whereas molybdate is a co-factor of the nitrate reductase , an important enzyme in the nitrate assimilation pathway .",
"These transporter genes are conserved across green algae including Chlorella NC64A ( Sanz-Luque et al . , 2015; Gao et al . , 2014 ) and appear to be lost in the Chlorella A99 genome .",
"In nitrogen assimilation processes , plants usually take up nitrogen in the form of nitrate ( NO3- ) via nitrate transporters ( NRTs ) or as ammonium ( NH4+ ) via ammonium transporters ( AMT ) ( Figure 6A ) .",
"In higher plants , two types of nitrate transporters , NRT1 and NRT2 , have been identified ( Krapp et al . , 2014 ) .",
"Some NRT2 require nitrate assimilation-related component 2 ( NAR2 ) to be functional ( Quesada et al . , 1994 ) .",
"NO3- is reduced to nitrite by nitrate reductase ( NR ) , NO2- is transported to the chloroplast by nitrate assimilation-related component1 ( NAR1 ) , and NO2- is reduced to NH4+ by nitrite reductase ( NiR ) .",
"NH4+ is incorporated into glutamine ( Gln ) by glutamine synthetase ( GS ) , and Gln is incorporated into glutamate ( Glu ) by NADH-dependent glutamine amide-2-oxoglutarate aminotransferase ( GOGAT ) , also known as glutamate synthase .",
"This pathway is highly conserved among plants and all of its major components , including NRT1 and NRT2 , NAR1 and NAR2 , NR , NiR , AMT , GOGAT and GS , are present in the 10 green algae species that have been genome-sequenced so far ( with the exception of NRT1 , which is absent in Micromonas pusilla ) ( Sanz-Luque et al . , 2015 ) .",
"In Symbiodinium , the photosynthetic symbiont of marine invertebrates , all these components of the nitrogen assimilation pathway were also observed ( Supplementary file 4 ) ( Shoguchi et al . , 2013; Lin et al . , 2015; Aranda et al . , 2016; Sproles et al . , 2018 ) .",
"Based on the annotation by Sanz-Luque et al . ( Sanz-Luque et al . , 2015 ) , we searched these nitrogen assimilation genes in the Chlorella A99 genome , using ortholog grouping and a reciprocal BLAST search using the protein sequences from other green algae ( Figure 6B , Supplementary file 5 ) .",
"As expected , the Chlorella A99 genome contains many homologues of the genes involved in nitrogen assimilation in plants including genes encoding NRT1 , NAR1 , NR , AMT , GS and GOGAT ( Figure 6B ) .",
"Intriguingly , our systematic searches failed to identify representative genes for NRT2 , NAR2 and NiR in the Chlorella A99 genome ( Figure 6B ) .",
"We confirmed the absence of the NRT2 and NiR genes by PCR using primers designed for the conserved regions of these genes and which failed to produce a product with genomic DNA as a template ( Figure 6—figure supplement 1 ) .",
"Due to the weak sequence conservation of the NAR2 gene in the three algae genomes , PCR of that gene was not performed .",
"Taken together , our observations indicate that Chlorella A99 algae appears to lack NRT2 , NAR2 and NiR .",
"Since in many fungi , cyanobacteria and algae species , nitrate assimilation genes are known to act in concert and a gene cluster of NR and NiR genes is conserved between different green algae ( Sanz-Luque et al . , 2015 ) , we next investigated the level of genomic clustering of the nitrate assimilation pathway genes in the Chlorella genome .",
"Comparing the genomes of NC64A and C169 revealed the presence of a cluster of NR and NiR genes ( Figure 6C ) .",
"In NC64A , two NRT2 genes , together with genes for NAR2 , NR and NiR are clustered on scaffold 21 .",
"In C169 , one of the NR genes and NiR are clustered together , whereas the second NR gene is separate .",
"Interestingly , analysis of the sequences around the NR gene in the Chlorella A99 genome provided no evidence for the presence of a co-localized NiR gene or any other nitrate assimilation genes , nor any conserved gene synteny to NC64A and C169 ( Figure 6C ) .",
"Therefore , our comparative genomic analyses points to an incomplete and scattered nitrogen metabolic pathway in symbiotic Chlorella A99 , which lacks essential transporters and enzymes for nitrate assimilation as well as the clustered structure of nitrate assimilation genes .",
"The absence of genes essential for nitrate assimilation in the Chlorella A99 genome ( Figure 6 ) is consistent with its inability to grow outside the Hydra host cell ( Habetha and Bosch , 2005 ) and indicates that Chlorella symbionts are dependent on metabolites provided by their host .",
"We hypothesized that Chlorella is unable to use nitrite and ammonium as a nitrogen source , and that it relies on Hydra assimilating ammonium to glutamine to serve as the nitrogen source .",
"To test this hypothesis and to examine utilization of nitrogen compounds of A99 , we isolated Chlorella A99 from Hv_Sym and cultivated it in vitro using modified bold basal medium ( BBM ) ( Nichols and Bold , 1965 ) containing the same amount of nitrogen in the form of NO3- , NH4+ , Gln or casamino acids ( Figure 7 ) .",
"As controls , Chlorella variabilis NC64A ( NC64A ) isolated from Hv_NC64A and free-living C169 were used .",
"To confirm that the cultured A99 is not contamination , we amplified and sequenced the genomic region of the 18S rRNA gene by PCR ( Figure 7—figure supplement",
"1 ) and checked this against the genomic sequence of A99 .",
"Kamako et al . reported that free-living alga Chlorella vulgaris Beijerinck var .",
"vulgaris grow in media containing only inorganic nitrogen compounds as well as in media containing casamino acids as a nitrogen source , while NC64A required amino acids for growth ( Kamako et al . , 2005 ) .",
"Consistent with these observations , C169 grew in all tested media and NC64A grew in media containing casamino acids and Gln , although its growth rate was quite low in presence of NH4+ and NO3- ( Figure 7 ) .",
"Remarkably , Chlorella A99 increased in cell number for up to 8 days in media containing casamino acids and Gln ( Figure 7 ) .",
"Similar to NC64A , A99 did not grow in presence of NH4+ and NO3- .",
"The growth rates of both A99 and NC64A were higher in medium containing a mixture of amino acids ( casamino acids ) than the single amino acid Gln .",
"In contrast to NC64A , A99 could not be cultivated permanently in casamino acids or glutamine supplemented medium , indicating that additional growth factors are necessary to maintain in vitro growth of this obligate symbiont .",
"Thus , although in vitro growth of A99 can be promoted by adding Glu and amino acids to the medium , A99 cannot be cultured permanently in this enriched medium , indicating that other host derived factors remain to be uncovered ."
],
[
"Sequencing of the Chlorella A99 genome in combination with the transcriptome analyses of symbiotic , aposymbiotic and NC64A-infected H . viridissima polyps has enabled the identification of genes with specific functions in this symbiotic partnership .",
"The Hydra-Chlorella symbiosis links carbohydrate supply from the photosynthetic symbiont to glutamine synthesis by the host .",
"Characteristics of the symbiont genome obviously reflect its adaptation to this way of life , including an increase in amino acid transporters and degeneration of the nitrate assimilation pathway .",
"This conclusion is based on six observations:",
"( i ) Expression of some genes including GS-1 , Spot 14 and NaPi is specifically up-regulated in the presence of Chlorella A99 ( Figure 1C , Table 2 ) , and",
"( ii ) they are induced by both , photosynthetic activity of Chlorella and by supplying exogenous maltose or glucose ( Figures 2 and 3J , Figure 3—figure supplement 2 ) .",
"Maltose produced by the symbiont is likely to be digested to glucose in symbiosome and translocated to the host cytoplasm through glucose transporters ( Figure 8A ) .",
"Upregulation of a GLUT8 gene in the symbiotic state of green hydra may reflect activation of sugar transport ( Table 1 ) .",
"These results indicate that maltose release by photosynthesis of the symbiont enhances nutrition supply including glutamine by the host ( Figure 8A ) .",
"( iii ) Symbiotic Chlorella A99 cannot be cultivated in vitro in medium containing a single inorganic nitrogen source ( Figure 7 ) .",
"Since medium containing glutamine supports in vitro growth of A99 , this organism appears to depend on glutamine provided by the Hydra host .",
"( iv ) The genome of Chlorella A99 contains multiple amino acid transporter genes ( Table 6 ) , but lacks genes involved in nitrate assimilation ( Figure 6 ) , pointing to amino acids as main source of nitrogen and a degenerated nitrate assimilation pathway .",
"As for ammonium , which is one of the main nitrogen sources in plants , previous studies have reported the inability of symbiotic algae to take up ammonium because of the low peri-algal pH ( pH 4–5 ) that stimulates maltose release ( Douglas and Smith , 1984; Rees , 1989; McAuley , 1991; Dorling et al . , 1997 ) .",
"Since Chlorella apparently cannot use nitrite and ammonium as a nitrogen source , it seems that Hydra has to assimilate ammonium to glutamine and provides it to Chlorella A99 ( Figure 8A ) .",
"( v ) While polyps with native symbiont Chlorella A99 grew faster than aposymbiotic ones , symbiosis with foreign algae NC64A had no effect on the growth of polyps at all ( Figure 1B ) .",
"( vi ) Hydra endodermal epithelial cells host significantly fewer NC64A algae than A99 ( Figure 1—figure supplement 1 ) providing additional support for the view of a tightly regulated codependent partnership in which exchange of nutrients appears to be the primary driving force .",
"Previous studies have reported that symbiotic Chlorella in green hydra releases significantly larger amounts of maltose than NC64A ( Mews and Smith , 1982; Rees , 1989 ) .",
"In addition , Rees reported that Hydra polyps containing high maltose releasing algae had a high GS activity , whereas aposymbiotic Hydra or Hydra with a low maltose releasing algae had lower GS activity ( Rees , 1986 ) .",
"Although the underlying mechanism of how maltose secretion and transportation from Chlorella is regulated is still unclear , the amount of maltose released by the symbiont could be an important symbiont-derived driver or stabilizer of the Hydra–Chlorella symbiosis .",
"Transcriptome comparison between symbiotic and aposymbiotic H . viridissima demonstrated that symbiosis-regulated genes are involved in oxidative stress response and innate immunity .",
"The fact that PRRs and apoptosis-related genes , are also differentially expressed in a number of other symbiotic cnidarians ( Table 1 ) , suggests innate immunity as conserved mechanism involved in controlling the development and maintenance of stable symbiotic interactions .",
"Furthermore , the exchange of nitrogenous compounds and photosynthetic products between host and symbiont observed here in the Hydra-Chlorella symbiosis is also observed in marine invertebrates such as corals , sea anemones and giant clams associated with Symbiodinium algae ( Figure 8B , C ) .",
"Despite these similarities , however , there are also conspicuous differences among symbiotic cnidarians in particular with respect to the nutrients provided by the symbiont to the host .",
"For example , symbiotic Chlorella algae in green hydra , Paramecium and fresh water sponges provide their photosynthetic products in form of maltose and glucose ( Figure 8B ) ( Brown and Nielsen , 1974; Wilkinson , 1980; Kamako and Imamura , 2006 ) .",
"In contrast , Symbiodinium provides glucose , glycerol , organic acids , amino acids as well as lipids to its marine hosts ( Figure 8C ) ( Muscatine and Cernichiari , 1969; Lewis and Smith , 1971; Trench , 1971; Kellogg and Patton , 1983 ) .",
"A former transcriptome analysis of amino acid biosynthetic pathways suggested that Symbiodinium can synthesize almost all amino acids ( Shinzato et al . , 2014 ) .",
"Gene loss in cysteine synthesis pathway in the coral host Acropora digitifera seems to reflect the dependency on the amino acids provided by the Symbiodinium symbiont ( Shinzato et al . , 2011 ) .",
"In contrast to Symbiodinium which can assimilate inorganic nitrogen such as nitrate and ammonium ( Lipschultz and Cook , 2002; Grover et al . , 2003; Tanaka et al . , 2006; Yellowlees et al . , 2008 ) , the symbiotic Chlorella algae in Hydra and Paramecium can only use amino acids as a nitrogen source ( Figure",
"6 ) ( Kamako et al . , 2005 ) .",
"In efforts to explain the metabolic efficiency of nitrogen use in symbiotic organisms , two models have been proposed: the ‘nitrogen conservation’ and the ‘nitrogen recycling’ hypothesis .",
"The nitrogen conservation hypothesis suggests that photosynthetic carbon compounds from the symbiont are used preferentially by the host respiration , which reduces catabolism of nitrogenous compounds ( Rees and Ellard , 1989; Szmant et al . , 1990; Wang and Douglas , 1998 ) .",
"The ‘nitrogen recycling’ hypothesis suggests that symbionts assimilate nitrogenous waste ( ammonium ) of the host into valuable , organic compounds , which then are translocated back to the host ( Figure 8C Symbiont nitrogen assimilation ) ( Lewis and Smith , 1971; Muscatine and Porter , 1977; Falkowski et al . , 1993; Wang and Douglas , 1998 ) .",
"Our observation that in symbiotic green hydra many genes involved in amino acid metabolism are down-regulated ( Figure 1E ) is consistent with the assumption of reduction of amino acid consumption by respiration .",
"In addition to the nitrogen recycling hypothesis , it has been proposed that also corals , sea anemones , Paramecium and green hydra hosts can assimilate ammonium into amino acids ( Figure 8B , C Host nitrogen assimilation ) ( Miller and Yellowlees , 1989; Rees , 1989; Szmant et al . , 1990; Rees , 1991; Wang and Douglas , 1998; Lipschultz and Cook , 2002 ) .",
"Ammonia assimilation by the host implies that the host controls the nitrogen status to regulate metabolism of the symbionts , which may be involved in controlling the number of symbionts within the host cell .",
"For organisms such as corals living in oligotrophic sea , inorganic nitrogen assimilation and recycling may be necessary to manage the nitrogen sources efficiently .",
"In contrast , for Hydra and Paramecium living in a relatively nutrient-rich environment may be advantageous in terms of metabolic efficiency that the symbiont abandons its ability to assimilate inorganic nitrogen and specializes in the supply of photosynthetic carbohydrate to the host .",
"Metabolic dependence of symbionts on host supply occasionally results in genome reduction and gene loss .",
"For example , symbiotic Buchnera bacteria in insects are missing particular genes in essential amino acid pathways ( Shigenobu et al . , 2000; Hansen and Moran , 2011 ) .",
"The fact that the corresponding genes of the host are up-regulated in the bacteriocyte , indicates complementarity and syntrophy between host and symbiont .",
"Similarly , in Chlorella A99 the nitrogen assimilation system could have been lost as a result of continuous supply of nitrogenous amino acids provided by Hydra .",
"Compared to Chlorella NC64A , the closest relative to Chlorella A99 among the genome-sequenced algae , genome size and total number of genes in Chlorella A99 were found to be smaller ( Figure 4B ) .",
"Although both A99 and NC64A cannot be cultivated using inorganic nitrogen sources ( Figure",
"7 ) ( Kamako et al . , 2005 ) , NC64A , unlike A99 , obtains all major nitrogen assimilation genes and their cluster structure on the chromosome ( Figure",
"6 ) ( Sanz-Luque et al . , 2015 ) .",
"NR and NiR activities were found to be induced by nitrate in free-living Chlorella , but not in Chlorella NC64A , indicating mutations in the regulatory region ( Kamako et al . , 2005 ) .",
"Considering the phylogenetic position of NC64A and the symbiotic Chlorella of green hydra ( Kawaida et al . , 2013 ) , the disability of nitrate assimilation in A99 and NC64A seems to have evolved independently , suggesting convergent evolution in a similar symbiotic environment .",
"Although our findings indicate that genome reduction in Chlorella A99 is more advanced than in Chlorella NC64A , genome size and total number of genes do not differ much between the Trebouxiophyceae ( A99 , NC64A and C169 ) ( Figure 4B ) .",
"By contrast , the parasitic algae Helicosporidium and Auxochlorella have significantly smaller genome sizes and number of genes indicating extensive genome reduction ( Gao et al . , 2014; Pombert et al . , 2014 ) .",
"The apparently unchanged complexity of the Chlorella A99 genome suggests a relatively early stage of this symbiotic partnership .",
"Thus , gene loss in metabolic pathways could occur as a first step of genome reduction in symbionts caused by the adaptation to continuous nutrient supply from the host .",
"Taken together , our study suggests metabolic-codependency as the primary driving force in the evolution of symbiosis between Hydra and Chlorella ."
],
[
"Experiments were carried out with the Australian Hydra viridissima strain A99 , which was obtained from Dr . Richard Campbell , Irvine .",
"Polyps were maintained at 18°C on a 12 hr light/dark cycle and fed with Artemia two or three times a week .",
"Aposymbiotic ( algae free ) polyps were obtained by photobleaching using 5 μM DCMU ( 3- ( 3 , 4-dichlorophenyl ) −1 , 1-dimethylurea ) as described before ( Pardy , 1976; Habetha et al . , 2003 ) .",
"Experiments were carried out with polyps starved for 3–6 days .",
"Isolation of endodermal layer and ectodermal layer was performed as described by Kishimoto et al . ( Kishimoto et al . , 1996 ) .",
"Symbiotic Chlorella were isolated as described before by Muscatine and McAuley ( Muscatine , 1983; McAuley , 1986 ) .",
"Chlorella variabilis NC64A ( NIES-2541 ) , Coccomyxa subellipsoidea C-169 ( NIES-2166 ) and Chlamydomonas reinhardtii ( NIES-2235 ) were obtained from the Microbial Culture Collection at the National Institute for Environmental Studies ( Tsukuba , Japan ) .",
"Total RNA of Hydra was extracted by use of the Trizol reagent and PureLink RNA Mini Kit ( Thermo Fisher Scientific ) after lysis and removal of algae by centrifugation .",
"The genomic DNA of green algae was extracted using ISOPLANT II ( Nippon Gene , Tokyo , Japan ) following DNase I treatment to degrade contaminant DNA .",
"Quantity and quality of DNA and RNA were checked by NanoDrop ( Thermo Scientific Inc . , Madison , USA ) and BioAnalyzer ( Agilent Technologies , Santa Clara , USA ) .",
"Total RNA for synthesis of cRNA targets was extracted from about 100 green hydra for each experimental group .",
"Experiments were carried out using three biological replicates .",
"cRNA labeled with cyanine-3 were synthesized from 400 ng total Hydra RNA using a Low Input Quick Amp Labeling Kit for one color detection ( Agilent Technologies ) .",
"A set of fluorescently labeled cRNA targets was employed in a hybridization reaction with 4 × 44K Custom-Made Hydra viridissima Microarray ( Agilent Technologies ) contributing a total of 43 , 222 transcripts that was built by mRNA-seq data ( NCBI GEO Platform ID: GPL23280 ) ( Bosch et al . , 2009 ) .",
"Hybridization and washing were performed using the GE Hybridization Kit and GE Wash Pack ( Agilent Technologies ) after which the arrays were scanned on an Agilent Technologies G2565BA microarray scanner system with SureScan technology following protocols according to the manufacturer's instructions .",
"The intensity of probes was extracted from scanned microarray images using Feature Extraction 10 . 7 software ( Agilent Technologies ) .",
"All algorithms and parameters used in this analysis were used with default conditions .",
"Background-subtracted signal-intensity values ( gProcessedSignal ) generated by the Feature Extraction software were normalized using the 75th percentile signal intensity among the microarray .",
"Those genes differentially expressed between two samples were determined by average of fold change ( cut of >2 . 0 ) and Student's t-test ( p<0 . 1 ) .",
"The data series are accessible at NCBI GEO under accession number GSE97633 .",
"Total RNA was extracted from 50 green hydra polyps for each biological replicate independently .",
"For reverse transcription of total RNA First Strand cDNA Synthesis Kit ( Fermentas , Ontario , Canada ) was used .",
"Real-time PCR was performed using GoTaq qPCR Master Mix ( Promega , Madison , USA ) and ABI Prism 7300 ( Applied Biosystems , Foster City , USA ) .",
"All qPCR experiments were performed in duplicate with three biological replicates each .",
"Values were normalized using the expression of the tubulin alpha gene .",
"Primers used for these experiments are listed in Supplementary file 6A .",
"Expression patterns of specific Hydra genes were detected by whole mount in situ hybridization with digoxigenin ( DIG ) -labelled RNA probes .",
"Specimens were fixed in 4% paraformaldehyde .",
"Hybridization signal was visualized using anti-DIG antibodies conjugated to alkaline phosphatase and NBT/BCIP staining solution ( Roche ) .",
"DIG-labeled sense probes ( targeting the same sequences as the antisense probes ) were used as a control .",
"Primers used for these experiments are listed in Supplementary file 6B .",
"For genome sequencing of Chlorella sp .",
"A99 , Chlorella sp .",
"A99 was isolated from H . viridissima A99 and genomic DNA was extracted .",
"Paired-end library ( insert size: 740 bp ) and mate-pair libraries ( insert size: 2 . 2 and 15 . 2 kb ) were made using Illumina TruSeq DNA LT Sample Prep Kit and Nextera Mate Pair Sample Preparation Kit respectively ( Illumina Inc . , San Diego , USA ) , following the manufacturer's protocols .",
"Genome sequencing was performed using Illumina Miseq and Hiseq 2000 platforms .",
"Sequence reads were assembled using Newbler Assembler version 2 . 8 ( Roche , Penzberg , Germany ) and subsequent scaffolding was performed by SSPACE ( Boetzer et al . , 2011 ) .",
"Gaps inside the scaffolds were closed with the paired-end and mate-pair data using GapCloser of Short Oligonucleotide Analysis Package ( Luo et al . , 2012 ) .",
"To overcome potential assembly errors arising from tandem repeats , sequences that aligned to another sequence by more than 50% of the length using blastn ( 1e-50 ) were removed from the assembly .",
"The completeness of the genome was evaluated using CEGMA v2 . 4 ( Core Eukaryotic Genes Mapping Approach ) based on mapping of the 248 most highly conserved core eukaryotic genes ( CEGs ) on the assembled genome ( Parra et al . , 2007 ) .",
"The completeness of complete and partial CEGs in the A99 scaffolds was 80 and 88% , respectively .",
"The fraction of repetitive sequences was 12% .",
"Gene model was predicted by AUGUSTUS 3 . 0 . 1 using model parameters for NC64A ( Stanke et al . , 2006 ) .",
"This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession PCFQ00000000 ( BioProject ID: PRJNA412448 ) .",
"Genome sequences and gene models are also accessible at the website of OIST Marine Genomics Unit Genome Project ( http://marinegenomics . oist . jp/chlorellaA99/viewer/info ? project_id=65 ) .",
"Annotation of transcriptome contigs and prediction of gene models was performed by use of BLAST , Gene Ontology ( Ashburner et al . , 2000 ) and blast2go ( Conesa et al . , 2005 ) .",
"To examine the conservation of H . viridissima contigs among metazoans , homology searches by blastx ( evalue 1E-5 ) were performed using protein databases obtained from NCBI for Drosophila melanogaster and Homo sapiens , from the JGI genome portal ( http://genome . jgi . doe . gov/ ) for Branchiostoma floridae , Nematostella vectensis , from Echinobase ( http://www . echinobase . org/EchinoBase/ ) for Strongylocentrotus pupuratus , from Compagen for Hydra magnipapillata , and from the OIST marine genomics Genome browser ver . 1 . 1 ( http://marinegenomics . oist . jp/coral/viewer/info ? project_id=3 ) for Acropora digitifera .",
"For comparative analysis of gene models of Chlorella sp .",
"A99 and other algae , domain searches against the Pfam database ( Pfam-A . hmm ) were performed using HMMER ( Eddy , 1998; Finn et al . , 2016 ) , and ortholog gene grouping was done using OrthoFinder ( Emms and Kelly , 2015 ) .",
"The sequences of the reference genes and genomes were obtained from the database of the JGI genome portal for Chlorella variabilis NC64A , Coccomyxa subellipsoidea C-169 , Volvox carteri , Micromonas pusilla , and Ostreococcus tauri , from NCBI for Auxenochlorella protothecoides 0710 , from Phytozome ( http://phytozome . jgi . doe . gov/pz/portal . html ) for Chlamydomonas reinhardtii , from OIST Marine Genomics ( http://marinegenomics . oist . jp/symb/viewer/info ? project_id=21 ) for Symbiodinium minutum , Symbiodinium kawagutti genome , from Dinoflagellate Resources ( http://web . malab . cn/symka_new/ ) for Symbiodinium kawagutti and Reefgenomics ( http://reefgenomics . org/ ) for Symbiodinium microadriaticum ) ( Merchant et al . , 2007; Palenik et al . , 2007; Worden et al . , 2009; Blanc et al . , 2010; Prochnik et al . , 2010; Blanc et al . , 2012 ) Nitrogen assimilation genes in Chlorella A99 were identified by orthologous gene groups and reciprocal blast searches .",
"The number of genes for nitrate assimilation genes , glutamine synthetase and glutamate synthetase , and clustering of such genes were systematically reported by ( Sanz-Luque et al . , 2015 ) .",
"We used these data as reference for searches of nitrogen assimilation genes , and further nitrogen assimilation genes were searched by Kyoto Encyclopedia of Genes and Genomes ( KEGG ) pathway ( Kanehisa and Goto , 2000 ) .",
"JGI genome browsers of Chlorella variabilis NC64A and Coccomyxa subellipsoidea C-169 were also used for retrieving genes and checking gene order on the scaffolds .",
"For a phylogenetic tree of chlorophyte green algae , the sequences of 18S rRNA gene , ITS1 , 5 . 8S rRNA gene , ITS2 and 28S rRNA gene were obtained from scaffold20 of Chlorella A99 genome sequence , and from NCBI nucleotide database entries for Chlorella variabilis NC64A ( FM205849 . 1 ) , Auxenochlorella protothecoides 0710 ( NW_011934479 . 1 ) , Coccomyxa subellipsoidea C169 ( AGSI01000011 . 1 ) , Volvox carteri f .",
"nagariensis ( NW_003307662 . 1 ) , Chlamydomonas reinhardtii ( FR865576 . 1 ) , Ostreococcus tauri ( GQ426340 . 1 ) and Micromonas pusilla ( FN562452 . 1 ) .",
"Multiple alignments were produced with CLUSTALX ( 2 . 1 ) with gap trimming ( Larkin et al . , 2007 ) .",
"Sequences of poor quality that did not well align were deleted using BioEdit ( Hall , 1999 ) .",
"Phylogenetic analyses were performed using the Neighbor-Joining method by CLUSTALX with the default parameters ( 1000 bootstrap tests and 111 seeds ) .",
"Representative phylogenetic trees were drawn by using NJ plot ( Perrière and Gouy , 1996 ) .",
"Primers were designed based on the conserved region of the NRT2 gene , NiR and NR genes ( positive control ) identified by comparison of genes from Chlorella variabilis NC64A ( NC64A ) , Coccomyxa subellipsoidea C169 ( C169 ) , and Chlamydomonas reinhardtii ( Cr ) which belongs to Chlorophyceae class of green algae .",
"Primers for NAR2 could not be designed because of insufficient conservation .",
"As positive controls , amplicons were produced for NR of all the green algae examined and of NRT2 and NiR from NC64A , C169 and Cr , after which their sequences were checked .",
"KOD FX Neo ( TOYOBO , Tokyo , Japan ) was used under the following conditions: an initial denaturation phase ( 94°C for 120 s ) followed by 36 cycles of ( 98°C for 30 s , 69°C for 100 s ) for NiR , ( 98°C for 30 s , 58°C for 30 s and 68°C for 210 s ) for NRT2 and ( 98°C for 30 s , 59°C for 30 s and 68°C for 60 s ) for NR .",
"In each case , 10 ng gDNA was used as a template .",
"The primers used are described in Supplementary file 6C .",
"PCR products were sequenced to confirm amplification of the target genes using ABI PRISM 3100 Genetic Analyzer ( Thermo Fisher Scientific Inc . , Madison , USA ) using BigDye Terminator v3 . 1 Cycle Sequencing Kit ( Thermo Fisher Scientific ) .",
"To isolate symbiotic algae , polyps were quickly homogenized in 0 . 25% sodium dodecyl sulfate ( SDS ) solution and centrifuged at 3000 g for 1 min .",
"The pellet was resuspended in 0 . 05% SDS and centrifuged at 500 g for 5 min .",
"Isolated A99 , NC64A and C169 were washed by sterilized Bold Basal Medium ( Bischoff and Bold , 1963 ) modified by the addition of 0 . 5% glucose , 1 . 2 mg/L vitamine B1 ( Thiaminhydrochloride ) , 0 . 01 mg/L vitamine B12 ( Cyanocobalamin ) ( Supplementary file 7 ) and incubated for two days in modified Bold Basal Medium with 50 mg/l ampicillin and streptomycin .",
"The algae were cultivated in 5 ml of modified Bold Basal Medium ( BBM ) with the same amount of nitrogen ( 2 . 9 mM NaNO3 , NH4Cl , glutamine or 426 mg/l casamino acids ) and 5 mg/l Carbendazim ( anti-fungal ) with fluorescent illumination ( 12 hr light , 12 hr dark ) at 20˚C .",
"Mean numbers of algae per ml were calculated from three tubes enumerated at 4 , 8 , and 12 days after inoculation with 106 cell/sml using a hemocytometer .",
"After cultivation , gDNA was isolated from the A99 cultured in Gln-containing BBM and casamino acid-containing BBM and A99 was isolated from green hydra directly .",
"A partial genomic region of the 18S rRNA gene was amplified by PCR and sequenced to confirm absence of contamination by other algae .",
"PCR was performed using AmpliTaq Gold ( Thermo Fisher Scientific ) .",
"Sequencing was performed as described above .",
"The primers used are described in Supplementary file 6D ."
]
] | [
"Many multicellular organisms rely on symbiotic associations for support of metabolic activity , protection , or energy .",
"Understanding the mechanisms involved in controlling such interactions remains a major challenge .",
"In an unbiased approach we identified key players that control the symbiosis between Hydra viridissima and its photosynthetic symbiont Chlorella sp .",
"A99 .",
"We discovered significant up-regulation of Hydra genes encoding a phosphate transporter and glutamine synthetase suggesting regulated nutrition supply between host and symbionts .",
"Interestingly , supplementing the medium with glutamine temporarily supports in vitro growth of the otherwise obligate symbiotic Chlorella , indicating loss of autonomy and dependence on the host .",
"Genome sequencing of Chlorella sp .",
"A99 revealed a large number of amino acid transporters and a degenerated nitrate assimilation pathway , presumably as consequence of the adaptation to the host environment .",
"Our observations portray ancient symbiotic interactions as a codependent partnership in which exchange of nutrients appears to be the primary driving force ."
] | [
"All animals host microorganisms; some of which form ‘symbiotic’ relationships with their host that are mutually beneficial .",
"For instance , the human gut shelters tens of thousands of species of bacteria that break down our food for us , and corals , jellyfish or sea anemones can extract energy directly from sunlight thanks to the algae that live inside their cells .",
"Hydra , a small freshwater animal , lives in a symbiotic relationship with algae called Chlorella that it carries inside its cells .",
"Once an independent organism , Chlorella has evolved in such a way that , in nature , it cannot exist without Hydra anymore .",
"In turn , the algae produce sugars to fuel the animal when it cannot get food from the environment .",
"Yet , despite over 30 years of research , it still remains unclear how exactly the relationship between Hydra and Chlorella works , and how it came to be .",
"Understanding how these two organisms live together could help researchers to figure out the general principles that guide symbiotic interactions .",
"Nitrogen is an element that is essential for life , and organisms can extract it from various sources , such as nitrates or the amino acid glutamine .",
"Here , Hamada , Schröder et al . sequenced the entire genome of Chlorella .",
"This revealed that Chlorella has lost someof the genes required to obtain nitrates , and to process them into nitrogen .",
"However , the genetic analysis showed that the algae express genes that allow them to import amino acids .",
"In turn , analysis of the genes expressed by Hydra when it lives in symbiosis with Chlorella showed that the animal turns on genetic information needed to make glutamine .",
"It thus seems that Hydra creates glutamine which Chlorella can import; the algae then process this amino acid to obtain the nitrogen they need .",
"Hamada , Schröder et al . also discovered that if the environment was artificially enriched in glutamine , Chlorella could live on their own outside of Hydra for a while .",
"The results suggest that symbiotic relationships , such as the one between Hydra and Chlorella , were established because the organisms became dependent on each other for essential nutrients .",
"This co-dependency is strengthened if the organisms lose the ability to produce the nutrients on their own .",
"However , this partnership may be altered when the environment changes too much , especially if the balance of nutrients available gets tipped .",
"For example , if seas that are normally poor in nutrients become suddenly rich in these elements , this may disrupt the existence of symbiotic organisms such as corals ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | Optogenetic control of PRC1 reveals its role in chromosome alignment on the spindle by overlap length-dependent forces | elife-61170-v2 | [
[
"Pre-anaphase chromosome movements culminate with chromosome alignment at the spindle equator , a distinctive feature of mitosis important for the synchronous anaphase poleward movement of chromatids and proper telophase nuclear reformation ( Fonseca et al . , 2019; Maiato et al . , 2017 ) .",
"Chromosome congression to the metaphase plate , a process of directed chromosome movement from the polar regions toward the spindle equator , has been explored extensively ( Barisic et al . , 2014; Cai et al . , 2009; Kapoor et al . , 2006; Maiato et al . , 2017 ) .",
"Yet , the maintenance of chromosome alignment at the spindle equator is less understood .",
"This is a dynamic process given that the chromosomes constantly make small oscillatory excursions across the equator ( Skibbens et al . , 1993 ) .",
"Two mechanisms have been proposed to underlie the maintenance of chromosome alignment at the equator , namely length-dependent dynamics of kinetochore fiber ( k-fiber ) microtubules and polar ejection forces .",
"Pulling forces exerted by k-fiber tips on kinetochores are thought to be regulated by kinesin-8 motor proteins , which are needed for proper kinetochore alignment in various organisms from yeast to humans ( Garcia et al . , 2002; Stumpff et al . , 2008; Wargacki et al . , 2010 ) .",
"These motors can ‘measure’ microtubule length because they bind to microtubules along their length and move toward the plus end , leading to more kinesin-8 accumulated at the plus end of longer microtubules , where they promote microtubule catastrophe , that is , a switch from growth to shrinkage ( Tischer et al . , 2009; Varga et al . , 2006 ) .",
"During kinetochore movements across the spindle equator , the leading k-fiber shrinks , while the trailing one grows and accumulates kinesin-8 , resulting in microtubule catastrophe , followed by the movement of the trailing kinetochore back toward the equator .",
"Although this mechanism can explain kinetochore alignment at the spindle equator ( Klemm et al . , 2018 ) , the switching dynamics characteristic for this model , where the trailing kinetochore initiates the change of direction of motion , differs from the observations in mammalian cells where the leading kinetochore typically changes the direction before the trailing one ( Armond et al . , 2015; Civelekoglu-Scholey et al . , 2013; Wan et al . , 2012 ) .",
"In addition to the forces produced by k-fibers , polar ejection forces push chromosome arms away from the pole , powered by arm-bound chromokinesins that walk toward the plus end of microtubules ( Bajer et al . , 1982; Brouhard and Hunt , 2005 ) .",
"Because microtubule density increases toward the pole , these forces help the chromosomes to stay away from the poles , but most likely have little effect on kinetochore movements close to the spindle equator ( Cane et al . , 2013; Ke et al . , 2009 ) .",
"Thus , the current models do not provide a complete picture of kinetochore alignment at the spindle center .",
"K-fibers are surrounded by a dense network of spindle microtubules with which they have multiple interactions ( McDonald et al . , 1992; O'Toole et al . , 2020 ) , resulting in forces acting on k-fibers and thus also on kinetochores .",
"In particular , each pair of sister k-fibers is tightly linked by the bridging fiber , a bundle of antiparallel microtubules that balances the tension on sister kinetochores ( Kajtez et al . , 2016; Polak et al . , 2017; Vukušić et al . , 2017 ) .",
"However , the role of forces exerted by bridging fiber in chromosome alignment at the metaphase plate is unknown .",
"In metaphase , overlap regions within bridging fibers are crosslinked by protein regulator of cytokinesis 1 ( PRC1 ) ( Kajtez et al . , 2016; Polak et al . , 2017; Tolić , 2018 ) .",
"PRC1 , like other non-motor microtubule-associated proteins from Ase1/PRC1/MAP65 family , selectively bundles antiparallel microtubules and provides stable overlaps in vitro ( Bieling et al . , 2010b; Janson et al . , 2007; Mollinari et al . , 2002; Subramanian et al . , 2010 ) .",
"Cellular studies of its function show that PRC1 is associated with the spindle midzone in anaphase , where its activity is essential for stable microtubule organization , localization of numerous microtubule-associated proteins within this structure , and successful completion of cytokinesis , while its microtubule-binding and -bundling affinities are regulated by phosphorylation and dephosphorylation events ( Gruneberg et al . , 2006; Jiang et al . , 1998; Kurasawa et al . , 2004; Liu et al . , 2009; Mollinari et al . , 2002; Mollinari et al . , 2005; Neef et al . , 2007; Subramanian et al . , 2013; Subramanian et al . , 2010; Zhu and Jiang , 2005; Zhu et al . , 2006 ) .",
"In this work , we developed an optogenetic approach for acute and reversible removal of PRC1 from the spindle to the cell membrane , building upon ideas of dimerization or dissociation induced chemically ( Cheeseman et al . , 2013; Haruki et al . , 2008; Robinson et al . , 2010; Wordeman et al . , 2016 ) or by light ( Fielmich et al . , 2018; Guntas et al . , 2015; Okumura et al . , 2018; van Haren et al . , 2018; Yang et al . , 2013; Zhang et al . , 2017 ) to rapidly redistribute proteins .",
"By using our assay on metaphase spindles , we found that bridging fibers promote kinetochore alignment .",
"PRC1 removal resulted in partial disassembly of bridging fibers and elongation of their overlap zones .",
"Moreover , the metaphase plate widened , inter-kinetochore distance decreased , and lagging chromosomes appeared more frequently , showing that PRC1 indirectly regulates forces acting on kinetochores .",
"Kif4A/kinesin-4 and Kif18A/kinesin-8 were found to localize in the bridging fiber during metaphase and were largely lost upon PRC1 removal , with Kif4A showing a greater reduction .",
"These results , together with the finding that Kif4A or Kif18A depletion by siRNA led to elongated overlaps , suggest that these proteins regulate the overlap length of bridging microtubules .",
"PRC1 removal did not affect the localization of Kif4A on the chromosomes and Kif18A , CLASP1 , and CENP-E/kinesin-7 on the plus ends of k-fibers , arguing against perturbed polar ejection forces or molecular events at the kinetochore microtubule plus ends as origins of the observed kinetochore misalignment .",
"In conclusion , our optogenetic experiments show that bridging microtubules buffer chromosome movements , thus promoting their alignment .",
"We propose that this occurs via length-dependent forces , which depend on the antiparallel overlap length within the bridging fiber ."
],
[
"To study the role of PRC1 and the forces arising from coupling of bridging and k-fibers in chromosome alignment , we developed an optogenetic tool for fast and reversible removal of PRC1 from the spindle to the cell membrane , based on the previously designed improved light inducible dimer ( iLID ) system ( Guntas et al . , 2015 ) .",
"We attached PRC1 to the red fluorescent protein tgRFPt and the bacterial protein SspB , while the iLID , which contains the bacterial peptide SsrA and the light-oxygen-voltage ( LOV2 ) domain , is bound to the cell membrane by a short peptide , CAAX .",
"In this system , LOV2 adopts a conformation that allows dimerization of SsrA and SspB upon exposure to the blue light ( Figure 1A ) .",
"After cessation of exposure to the blue light , LOV2 adopts its initial conformation leading to decreased affinity of SsrA to SspB .",
"Therefore , exposure to the blue light should induce translocation of PRC1 from the central region of the metaphase spindle , which we will refer to as the spindle midzone , to the cell membrane , whereas cessation of exposure to blue light should restore PRC1 localization on the spindle ( Figure 1A ) .",
"To test our optogenetic approach , we used U2OS cells with stable expression of CENP-A-GFP , transient expression of PRC1-tgRFPt-SspB ( henceforth opto-PRC1 ) and iLID-CAAX ( henceforth opto cells; Figure 1B; Figure 1—video 1 ) .",
"Endogenous PRC1 was depleted 90 ± 2% ( all results are mean ± s . e . m . ) by siRNA before addition of opto-PRC1 ( Figure 1—figure supplement 1A ) .",
"Before exposure to the blue light , opto-PRC1 had normal localization on the microtubule bundles in the spindle midzone ( Figure 1B; 0:00 min ) , with the length of PRC1 streaks of 3 . 77 ± 0 . 08 µm ( n = 193 bundles , N = 30 cells ) , consistent with that of endogenous and fluorescently labeled PRC1 in metaphase ( Kajtez et al . , 2016; Polak et al . , 2017 ) , although the total signal intensity of opto-PRC1 on the spindle was higher compared to endogenous PRC1 ( Figure 1—figure supplement 1B ) .",
"Addition of opto-PRC1 did not change the duration of metaphase , as inferred from the fraction of cells that entered anaphase during image acquisition , which was similar in cells with endogenous PRC1 and cells treated with PRC1 siRNA and containing opto-PRC1 ( 79 ± 6% , N = 37 , and 71 ± 5% , N = 72 , respectively; p = 0 . 4 , Pearson’s Chi-squared test; Figure 1—figure supplement 1C ) .",
"After exposure to the blue light , opto cells were able to progress to cytokinesis ( Figure 1—figure supplement 1D ) .",
"Taken together , these data suggest that opto-PRC1 replaces the depleted endogenous PRC1 .",
"Upon exposure to the blue light , opto-PRC1 signal on the spindle decreased and its signal on the membrane increased ( Figure 1B; 0:20-20:00 min ) .",
"After the blue light was switched off , opto-PRC1 returned to the spindle midzone ( Figure 1B; 20:40-30:10 min ) .",
"To validate our system , we performed two sets of test experiments .",
"The first one was performed on the same cell line containing opto-PRC1 , but without iLID , imaged with the same imaging protocol as the opto cells , which we refer to as control throughout the paper .",
"The second test experiment was on the opto cells but without the blue light ( Figure 1—figure supplement 1E ) .",
"In both cases , opto-PRC1 remained on the spindle ( Figure 1—figure supplement 1E ) .",
"Thus , our optogenetic approach allows for acute and reversible control of PRC1 localization in metaphase .",
"To quantify the dynamics and spatial pattern of opto-PRC1 removal and return , we measured the intensity of opto-PRC1 on the metaphase spindle ( Figure 1—figure supplement 1F , G ) .",
"We found that 88 ± 3% of opto-PRC1 was removed after 20 min of exposure to the blue light with a half-time of 4 . 2 ± 0 . 1 min ( Figure 1C ) .",
"During the opto-PRC1 removal , there was simultaneous decrease in both signal intensity and length of the overlap region ( Figure 1D , left; Figure 1—figure supplement 1G , top ) , which may be due to fewer antiparallel regions being positioned laterally than in the central part of the bundle ( Mastronarde et al . , 1993 ) .",
"The signal of the outermost midzone bundles typically lasted longer than of the inner ones ( Figure 1B; 3:40-6:30 ) .",
"After the blue light was switched off , opto-PRC1 signal restored to 65 ± 1% of the initial intensity within 10 min , with return half-time being 0 . 71 ± 0 . 04 min ( Figure 1C ) .",
"The faster PRC1 return to the spindle in comparison with its removal may be due to the higher affinity difference between PRC1 binding to the spindle and to the membrane in the dark than under light .",
"During the opto-PRC1 return , it initially localized throughout the spindle , with gradual increase in intensity in the spindle midzone ( Figure 1D , right; Figure 1—figure supplement 1G , bottom ) , suggesting that PRC1 has higher unbinding rate outside than within the overlap bundles in the spindle .",
"This result is consistent with PRC1 having a life-time of several seconds on single microtubules and a 10-fold preference for overlap regions in vitro ( Subramanian et al . , 2010 ) .",
"To test whether bridging fiber has a role in the maintenance of chromosome alignment , acute optogenetic removal of PRC1 , a depletion of which is known to perturb bridging fibers ( Polak et al . , 2017 ) should affect chromosome positioning at the spindle equator ( Figure 2A ) .",
"Surprisingly , we observed that the acute removal of opto-PRC1 resulted in movement of sister kinetochore pairs away from the metaphase plate ( Figure 2B; Figure 2—video 1 ) , which is not found after long-term PRC1 depletion by siRNA ( Polak et al . , 2017 ) .",
"Upon opto-PRC1 removal , the distances of sister kinetochore midpoints from the equatorial plane increased ( dEQ , Figure 2B , C , D; Figure 2—figure supplement 1A , B ) .",
"While >95% of kinetochore pairs were found within the region of PRC1 streaks , that is , less than 2 µm away from the equatorial plane before removal of opto-PRC1 , 9 . 3 ± 2 . 7% of kinetochore pairs made excursions far outside this region after opto-PRC1 removal ( Figure 2—figure supplement 1B ) .",
"The displaced kinetochores were found more often in the inner part of the spindle , that is , close to the long spindle axis , than in the outer regions ( Figure 2E ) .",
"Kinetochores fluctuated to a similar extent in the presence and absence of opto-PRC1 , but in its absence the displaced kinetochores fluctuated within a region that was offset from the equatorial plane ( Figure 2—figure supplement 1C ) .",
"These displaced kinetochores upon opto-PRC1 removal had lower inter-kinetochore distance in comparison to non-displaced ones , suggesting that kinetochore displacement was related to a more severe reduction of tension ( Figure 2F ) .",
"On average , kinetochores remained displaced even after opto-PRC1 return ( Figure 2D ) .",
"We conclude that PRC1 has a role in keeping kinetochores in tight alignment on the metaphase plate .",
"The mean inter-kinetochore distance ( dKC , Figure 2C ) was reduced when opto-PRC1 was removed ( Figure 2G; Figure 2—figure supplement 1D ) , and the distance after 20 min of exposure to the blue light ( 0 . 79 ± 0 . 01 µm ) was closer to metaphase ( 0 . 87 ± 0 . 01 µm ) than prometaphase ( 0 . 66 ± 0 . 01 µm ) values ( see Materials and methods ) , suggesting that tension was not completely lost and that these changes were not due to kinetochore detachment from k-fibers ( Figure 2—figure supplement 1D ) .",
"In agreement with this , the fraction of cells that entered anaphase during image acquisition was similar in control and opto cells ( 71 ± 5% , N = 72 , and 60 ± 5% , N = 93 , respectively; p = 0 . 1 , Pearson’s Chi-squared test; Figure 1—figure supplement 1C ) , indicating that PRC1 removal did not prevent spindle assembly checkpoint satisfaction .",
"After opto-PRC1 return to the spindle , inter-kinetochore distance increased , implying restoration of tension , although not to the original value ( Figure 2G ) .",
"To investigate the influence of acute PRC1 removal on the orientation of sister kinetochores , we measured the angle between sister kinetochore axis and long spindle axis ( αKC , Figure 2C ) .",
"We observed that removal of opto-PRC1 caused misorientation of sister kinetochores , that is , increased αKC ( Figure 2B , H; Figure 2—figure supplement 1A , E ) .",
"Misoriented kinetochores were found at a larger distance from the equatorial plane and closer to the long spindle axis ( Figure 2—figure supplement 1F ) .",
"Interestingly , sister kinetochore pairs remained misoriented even after opto-PRC1 return ( Figure 2H ) .",
"Similarly , PRC1 bundles were misoriented upon PRC1 return ( see Materials and methods , Figure 2—figure supplement 1G ) .",
"These results suggest that when PRC1 returns to the overlaps whose geometry was perturbed by PRC1 removal , it likely confines the chromosomes in new positions and orientations .",
"The observed effects of PRC1 removal on inter-kinetochore distance , kinetochore alignment and orientation did not change when SiR-tubulin was added ( dKC p = 0 . 82 , dEQ p = 0 . 27 , αKC p = 0 . 61 , respectively; t-test ) .",
"The effects of PRC1 removal were found neither in control experiments in cells without iLID , which were stained with SiR-tubulin , nor in a different set of control experiments where cells expressing only CENP-A-GFP without SiR-tubulin were imaged with the same illumination protocol ( Figure 2D , G , H; Figure 2—figure supplement 1A , B , D , E ) .",
"Therefore , the observed effects in opto cells were not a consequence of SiR-tubulin or laser photodamage ( Douthwright and Sluder , 2017 ) .",
"As previous reports have shown that acute rapamycin-dependent protein translocation and long-term depletion by siRNA can yield different and even opposite phenotypes ( Cheeseman et al . , 2013; Wordeman et al . , 2016 ) , we compared kinetochore parameters after acute removal of PRC1 with those obtained from cells after long-term depletion of PRC1 by siRNA .",
"Strikingly , in contrast to acute removal , the long-term depletion did not cause kinetochore misalignment or misorientation ( Figure 2D , H; Figure 2—figure supplement 1H , I; Table 1 ) .",
"The two methods decreased the inter-kinetochore distance to a similar extent ( Figure 2G; Figure 2—figure supplement 1J; Table 1 ) , even though unlike acute removal , long-term removal reduced the fraction of cells that entered anaphase ( 35 ± 8% , N = 37 , and 79 ± 6% , N = 37 , for PRC1 siRNA treated and untreated , respectively; p = 0 . 046 , Pearson’s Chi-squared test; Figure 1—figure supplement 1C ) .",
"Thus , acute removal of PRC1 results in different effects in comparison with a long-term depletion .",
"To test to what extent the acute removal of PRC1 during metaphase affects chromosome segregation , we measured the frequency of lagging kinetochores .",
"We found that lagging kinetochores occurred more frequently when opto-PRC1 was being removed than in control cells ( Figure 2I , J; Figure 2—video 2 ) .",
"Similarly , long-term depletion of PRC1 by siRNA also increased the frequency of lagging kinetochores during early anaphase ( Figure 2—figure supplement 2A; Table 1 ) .",
"Opto cells that showed lagging kinetochores in anaphase had a slightly smaller inter-kinetochore distance before anaphase than opto cells without lagging kinetochores ( Figure 2—figure supplement 2B ) , suggesting that a decrease in tension may be involved in the imperfect kinetochore segregation .",
"The cells with lagging kinetochores did not have a larger average kinetochore misalignment in metaphase ( Figure 2—figure supplement 2C ) , which indicates that misalignment and lagging kinetochores are not linked on the cell level , although they may be linked locally on individual kinetochores .",
"As perturbation of the PRC1-CLASP1 interaction and the consequent absence of CLASP1 from the spindle midzone results in lagging chromosomes ( Liu et al . , 2009 ) , we inspected the localization of CLASP1 and found that it did not accumulate between segregating chromosomes in opto HeLa cells stably expressing EGFP-CLASP1 ( Figure 2—figure supplement 2D ) .",
"Thus , the observed higher occurrence of lagging kinetochores could be attributed to changes in tension during metaphase , perturbed recruitment of CLASP1 to the spindle midzone by PRC1 during early anaphase , or a combination of both effects .",
"Factors that could contribute to the altered chromosome alignment and occurrence of lagging kinetochores upon opto-PRC1 removal are changes related to ( 1 ) microtubules in the bridging fibers , ( 2 ) polar ejection forces , and/or ( 3 ) proteins that modulate the dynamics of k-fiber plus-ends ( Figure 2K ) .",
"Because PRC1 crosslinks microtubules within bridging fibers ( Kajtez et al . , 2016; Polak et al . , 2017 ) , we first tested the effects of PRC1 removal on those microtubules .",
"An important aspect of the bridging fiber that may affect chromosome alignment is the dynamics of microtubules that make up these fibers .",
"To explore their dynamics , we developed an assay to track the growing plus ends of individual microtubules in the bridging fiber by using cells expressing the plus end marker EB3 ( Stepanova et al . , 2003 ) tagged with GFP ( Figure 3 ) .",
"We followed single EB3 spots in the spindle and identified the ones belonging to a bridging fiber as the spots that move toward a kinetochore , cross the region between this kinetochore and its sister , and move beyond it toward the other spindle pole ( Figure 3A–C; Figure 3—video 1 ) .",
"We found 1 . 8 ± 0 . 2 EB3 spots per minute per bridging fiber in control cells , showing that bridging fibers are dynamically remodeled during metaphase ( Figure 3D ) .",
"This number was similar after opto-PRC1 removal , 2 . 0 ± 0 . 3 spots/min ( p = 0 . 59; t-test , Figure 3D ) , suggesting that the number of dynamic microtubules in the bridge is largely unaffected by acute PRC1 removal .",
"However , in PRC1 siRNA-treated cells fewer EB3 spots were observed to move to opposite spindle half , 1 . 5 ± 0 . 1 EB3 spots/min , compared to untreated cells , where 1 . 91 ± 0 . 09 spots/min were observed ( p = 0 . 01; t-test , Figure 3—figure supplement 1A ) , indicating that long-term PRC1 depletion slightly decreases the number of dynamic microtubules in the bridge .",
"To assess the changes in the dynamics of bridging microtubules , we followed EB3 spots in the bridge from the time when they can be distinguished from neighboring spots near the pole until they disappear in the opposite spindle half , which we interpret as the moment when the microtubule stops growing ( Maurer et al . , 2012 ) .",
"Interestingly , EB3 tracks were longer after opto-PRC1 removal than in control cells ( Figure 3B , C; Figure 3—video 1 ) , but the velocities of the EB3 spots were not affected by opto-PRC1 removal or long-term depletion when compared to untreated cells ( Figure 3E; Figure 3—figure supplement 1B ) .",
"In agreement with these results , kymographs of the central region of the spindle show that EB3 spots reach deeper into the opposite half of the spindle after opto-PRC1 removal ( Figure 3F; Figure 3—figure supplement 1C ) .",
"Thus , acute removal of PRC1 results in longer bridging microtubules , which is not a consequence of altered microtubule growth rate , but most likely due to a reduced microtubule catastrophe rate .",
"EB3 tracks allowed us to estimate the length of the overlap zone of antiparallel microtubules in the bridging fiber , which currently cannot be measured after PRC1 removal because PRC1 itself is the only available marker of antiparallel overlaps in the spindle .",
"We define the overlap half-length as the distance beyond the equatorial plane that an EB3 spot covers while moving within the bridging fiber from one spindle half into the other ( Figure 3G , left ) , where this microtubule can form antiparallel overlaps with the oppositely oriented microtubules extending from the other pole .",
"In control cell , the overlap half-length was 1 . 8 ± 0 . 2 µm ( n = 17 spots in N = 9 cells; Figure 3G , right ) , which corresponds well to the overlap half-length measured as the half-length of PRC1 streaks in this cell line , 0 . 5x ( 3 . 8 ± 0 . 4 ) µm ( N = 9 cells; Figure 3H ) , validating our method for overlap length measurement based on EB3 tracks .",
"After opto-PRC1 removal , the overlap half-length increased to 2 . 6 ± 0 . 2 µm ( n = 23 spots in N = 9 cells; Figure 3G ) .",
"In contrast to opto cells , treatment with PRC1 siRNA did not result in a change of overlap length with respect to untreated cells ( Figure 3—figure supplement 1D–F ) .",
"Thus , the antiparallel overlaps became on average 40% longer after acute , but unchanged after long-time PRC1 removal .",
"Interestingly , the overlaps were especially long in the inner part of the spindle , whereas those on the spindle periphery were similar to overlaps in control cells ( Figure 3I ) .",
"This spatial difference is correlated with our findings that PRC1 is removed faster from the inner bundles ( see Figure 1B ) and that displaced and misoriented kinetochores are found more often in the inner than in the outer spindle region ( see Figure 2D , E , H ) , suggesting a mechanistic link between the overlap length and kinetochore positioning .",
"To test to what extent PRC1 removal in metaphase affects the number of microtubules in the bridging fibers ( Figure 4A ) , we visualized microtubules by using SiR-tubulin , a far-red tubulin dye excited by red light ( Lukinavičius et al . , 2014 ) , which allowed us to observe microtubules both when the blue light is turned on and switched off .",
"Intensity profiles across the spindle midzone revealed that SiR-tubulin intensity maxima were lower upon PRC1 removal and increased after its return ( Figure 4—figure supplement 1A; Figure 4—video 1 ) .",
"Measurements of SiR-tubulin signal intensity between and lateral from sister kinetochores showed that upon PRC1 removal the tubulin signal was reduced specifically in the bridging fibers ( Figure 4A–C; Figure 4—video 1 ) .",
"As an alternative to SiR-tubulin , which is a taxol-based dye that may affect microtubule dynamics ( Lukinavičius et al . , 2014 ) , we tested YFP-tubulin , but the excitation laser for YFP also activated the optogenetic system ( Wang and Hahn , 2016; Figure 4—figure supplement 1B ) .",
"Finally , we used tubulin-GFP to determine tubulin signal intensities of the bridging fibers and k-fibers upon acute removal of PRC1 ( Figure 4A , D–H; Figure 4—figure supplement 1C–F; see Materials and methods ) .",
"Upon exposure to the blue light , tubulin signal intensity in the bridging fibers decreased ~2 . 5-fold , which corresponds to 5 . 6 ± 0 . 9 microtubules given that the average number of microtubules in the bridging fiber is 14 ( Kajtez et al . , 2016 ) .",
"Together with the finding that the number of growing microtubules in the bridging fiber is similar with and without PRC1 ( Figure 3D ) , this result implies that in the presence of PRC1 the bridge contains more microtubules with a smaller fraction of them being dynamic than without PRC1 , and that PRC1 removal leads mainly to disassembly of non-dynamic microtubules .",
"Upon PRC1 return the intensity and thus the number of microtubules remained low ( Figure 4D–G; Figure 4—figure supplement 1D ) , possibly as a consequence of the perturbed spindle architecture in the absence of PRC1 , which may be able to recover after a longer time period .",
"Importantly , the intensity of k-fiber was unaltered ( Figure 4H , see Materials and methods ) , suggesting that the k-fibers were not affected by PRC1 removal .",
"Thus , acute removal of PRC1 and its return change the number of microtubules specifically in the bridging fibers .",
"Long-term depletion of PRC1 by siRNA in HeLa cells with stable expression of tubulin-GFP and transient expression of mRFP-CENP-B resulted in a similar reduction in bridging fiber tubulin intensity as after acute PRC1 removal ( Figure 4—figure supplement 1G , H ) .",
"Long-term depletion resulted in a decreased number of microtubules in the bridge , 7 . 5 ± 1 . 1 , which was similar to the microtubule number after acute removal ( p = 0 . 21 , t-test; Figure 4—figure supplement 1I , Table 1 ) .",
"As in acute removal , the intensity of k-fibers did not change after long-term depletion ( p = 0 . 49 , t-test ) .",
"Given that the bridging fiber is under compression ( Kajtez et al . , 2016 ) , reduction of number of microtubules in the bridging fiber is expected to reduce this compression and thus to straighten the k-fibers ( Figure 4—figure supplement 1J ) .",
"Tracking of the pole-to-pole contour of the outermost k-fibers revealed that PRC1 removal indeed straightened the k-fibers and thus made the spindle diamond-shaped ( Figure 4—figure supplement 1K–M ) .",
"Similar to acute removal , long-term depletion of PRC1 caused straightening of outermost k-fibers , although to a smaller extent , whereas spindle length and width were unchanged after both treatments ( Figure 4—figure supplement 1L , N , O; Table 1 ) .",
"This result supports that compression in the bridging fibers enables the spindle to obtain a curved shape .",
"Since bridging fibers are , nevertheless , present upon PRC1 removal , we asked how the residual microtubules are bundled together .",
"Eg5/kinesin-5 , which localizes in the bridging fibers ( Kajtez et al . , 2016; Mann and Wadsworth , 2018 ) , was still detectable in these fibers after PRC1 siRNA ( Figure 4—figure supplement 1P ) .",
"Thus , we propose that microtubule crosslinkers such as Eg5 crosslink the remaining microtubules in the bridge after acute PRC1 removal .",
"To investigate the mechanism of bridging microtubule regulation via PRC1 , we analyzed the localization of major proteins that regulate spindle microtubule dynamics and/or are binding partners of PRC1: Kif4A , Kif18A , and MKLP1 ( Bieling et al . , 2010b; Bringmann et al . , 2004; Gruneberg et al . , 2006; Kurasawa et al . , 2004; Mayr et al . , 2007; Stumpff et al . , 2008; Stumpff et al . , 2012 ) before and after PRC1 removal in metaphase ( Figure 5; Figure 5—figure supplement 1; Figure 5—figure supplement 2 and Table 1 ) .",
"We first looked at the localization of these proteins in the bridging fiber .",
"Surprisingly , we found that Kif4A and Kif18A localize in the bridge during metaphase , visible as thin lines across or next to the location of sister kinetochores where PRC1-labeled bundles are found ( Polak et al . , 2017; Figure 5A; Figure 5—figure supplement 1A ) .",
"Similar localization was observed for MKLP1 , spanning the region between two sister k-fibers ( Figure 5A ) .",
"In vertically oriented spindles , whose long axis was roughly perpendicular to the imaging plane , Kif4A and Kif18A colocalized with opto-PRC1 , now visible as spots in the cross-section of the spindle ( Figure 5—figure supplement 1B ) .",
"To explore whether PRC1 removal affects the localization of Kif4A , Kif18A , and MKLP1 , we analyzed their intensities within bridging fibers .",
"Measurements of Kif4A intensity upon acute PRC1 removal and long term-depletion , in regions lateral from chromosomes which correspond to peripheral parts of bridging fibers , showed that its intensity in the bridging fibers decreased ( Figure 5A–C; Figure 5—figure supplement 1A ) .",
"Additional analysis of Kif4A intensity in HeLa cells after long-term PRC1 removal corroborated this result ( Figure 5—figure supplement 1C ) .",
"Interestingly , there was a larger reduction in Kif4A intensity after acute PRC1 removal , where it decreased by 76 ± 5% , in comparison to long-term depletion where it decreased by 52 ± 3% ( p = 1×10−4; Figure 5C; Table 1 ) .",
"Kif18A intensity in the bridging fibers also decreased upon acute PRC1 removal and long-term depletion ( Figure 5A–C; Figure 5—figure supplement 1A , C ) .",
"Yet , in contrast to Kif4A , Kif18A intensities in the bridging fiber decreased to a similar extent , that is by 43 ± 11% and 38 ± 6% , after acute and long-term PRC1 depletion , respectively ( p = 0 . 68; Figure 5C; Table 1 ) .",
"MKLP1 intensities were also similarly reduced after both approaches , 87 ± 5% after acute PRC1 removal and 83 ± 11% after long-term depletion ( p = 0 . 68; Figure 5D; Figure 5—figure supplement 1C ) .",
"Among the tested proteins , only MKLP1 was exclusively localized in the bridging fibers , co-localizing with PRC1 , both before PRC1 removal and after its return ( Figure 5A , D; Figure 5—figure supplement 1C–G ) .",
"Even though MKLP1 was removed to a large extent from the spindle by acute PRC1 removal , it was not detected at the cell membrane together with PRC1 .",
"It may be that MKLP1 binds rather weakly to PRC1 in metaphase and/or that the absence of PRC1 decreases its affinity for microtubules .",
"In addition , the ability of MKLP1 to bind along scaffold of antiparallel overlaps could depend on the role of PRC1 in dictating 35-nm-inter-microtubule spacing , proposed to be important to enable localization of specific proteins within these structures ( Kellogg et al . , 2016; Subramanian et al . , 2010 ) .",
"As Kif4A and Kif18A are known to regulate microtubule length ( Mayr et al . , 2007; Varga et al . , 2006; Wandke et al . , 2012 ) , we set out to explore their roles in overlap length regulation .",
"We hypothesized that these kinesins may regulate the length of bridging microtubules and hence their antiparallel overlaps during metaphase .",
"Indeed , removal of Kif4A or Kif18A by siRNA resulted in longer PRC1-labeled overlaps and longer spindles ( Figure 5E ) , with a small but not significant increase in the ratio of overlap length to spindle length ( Figure 5F ) .",
"However , the increase in the overlap and spindle length was significantly different after the two treatments ( Figure 5G ) .",
"Kif4A siRNA treatment increased overlap length for 0 . 9 ± 0 . 2 µm compared with control , which was similar to spindle length increase of 1 . 2 ± 0 . 3 µm ( p = 0 . 48 , t-test; Figure 5G ) .",
"In contrast , Kif18A siRNA treatment increased overlap length for 1 . 8 ± 0 . 3 µm , whereas the spindle length increased to a larger extent , for 3 . 7 ± 0 . 6 µm ( p = 0 . 004 , t-test; Figure 5G ) .",
"These results suggest that both Kif4A and Kif18A regulate the length of bridging microtubules and their overlap , but Kif18A has a greater effect on overall spindle microtubules and spindle length than Kif4A .",
"As an alternative to the changes in the bridging fiber , disruption of polar ejection forces may be the cause of kinetochore misalignment upon PRC1 removal ( Figure 2K ) , in particular because these forces are modulated by Kif4A ( Stumpff et al . , 2012 ) .",
"The most prominent Kif4A localization in metaphase is on chromosomes ( Gruneberg et al . , 2006; Kurasawa et al . , 2004; Zhu and Jiang , 2005; Figure 5A , B; Figure 5—figure supplement 1A–C; Figure 5—figure supplement 2A–C ) .",
"Neither acute nor long-term PRC1 removal had an extensive effect on the Kif4A signal on chromosome arms , although there was a slight decrease in Kif4A signal after PRC1 siRNA treatment ( Figure 5B; Figure 5—figure supplement 1A , C; Figure 5—figure supplement 2C , D; Table 1 ) .",
"In early anaphase , the amount of Kif4A on segregated chromosomes was similar in opto and control cells ( Figure 5—figure supplement 2E ) , indicating that increased occurrence of lagging kinetochores was neither due to perturbed polar ejection forces in that phase nor defects in chromosome architecture/condensation .",
"Finally , kinetochore misalignment and lagging kinetochores upon PRC1 removal may be due to disrupted localization of proteins that regulate microtubule dynamics at the plus-end of the k-fiber ( Figure 2K ) .",
"To investigate this possibility , we analyzed the k-fiber localization of the key proteins with this function , Kif18A , CLASP1 , and CENP-E ( Al-Bassam et al . , 2010; Maffini et al . , 2009; Maiato et al . , 2003; Mayr et al . , 2007; Stumpff et al . , 2008; Stumpff et al . , 2012; Yu et al . , 2016 ) .",
"These proteins were localized at the plus-ends of k-fibers before and after acute and long-term removal of PRC1 ( Figure 5A , B; Figure 5—figure supplement 1A–C; Figure 5—figure supplement 2F–I ) .",
"This indicates that kinetochore misalignment upon PRC1 removal is not caused by simultaneous removal of proteins that regulate microtubule dynamics at the k-fiber plus ends ."
],
[
"We developed an optogenetic approach that offers acute light-controlled removal of proteins from a normally formed spindle at a precise phase of mitosis .",
"The main advantages of this approach over chemically-induced protein translocation ( Cheeseman et al . , 2013; Haruki et al . , 2008; Robinson et al . , 2010; Wordeman et al . , 2016 ) are its reversibility , allowing the protein to return to its initial location within about a minute , and applicability to individual cells .",
"Unlike previous optogenetic approaches ( Fielmich et al . , 2018; Okumura et al . , 2018; van Haren et al . , 2018; Yang et al . , 2013; Zhang et al . , 2017 ) , this method allows for global loss-of-function of full-length spindle proteins , relying on simple protein tagging rather than domain splitting , with no need of chromophore addition .",
"Moreover , this method may be implemented with other optical perturbations ( Milas et al . , 2018 ) and used as ‘in vivo pull-down’ for probing protein-protein interactions in different phases of the cell cycle .",
"However , this approach depends on high turnover of the protein in comparison with the time scale of interest .",
"Acute PRC1 removal from the spindle by optogenetics and long-term PRC1 depletion by siRNA led to partially different phenotypes .",
"These differences are hard to explain by different levels of PRC1 on the spindle as both methods decreased PRC1 by ~90% .",
"It is also unlikely that the differences are caused by the interaction of the membrane-translocated PRC1 with astral microtubules because of the uniform PRC1 signal on the membrane , fast return to the spindle , and no change in spindle positioning .",
"Therefore , the generally weaker effects of siRNA in comparison with the acute optogenetic removal are most likely due to compensatory mechanisms acting during long-term depletion .",
"By overcoming temporal limitations of siRNA , our work reveals an unexpected role of PRC1 and bridging fibers in the regulation of chromosome alignment on the metaphase spindle via overlap length-dependent forces ( Figure 6A ) .",
"We propose that the interactions between k-fibers and bridging fibers regulate the movement of bi-oriented chromosomes along the pole-to-pole axis .",
"If a kinetochore pair is displaced toward one spindle pole , more motors and/or crosslinkers are expected to accumulate in the overlap facing the opposite pole because this overlap is longer , and pull the kinetochores back to the center ( Figure 6B ) .",
"As the efficiency of this type of centering depends on the relative asymmetry in the overlap length on either side , shorter overlap length leads to more precise centering of the kinetochores ( Figure 6A , B ) .",
"Our finding that acute PRC1 removal leads to chromosome misalignment and elongation of overlap zones supports this model .",
"Moreover , elongation of overlaps correlates with chromosome misalignment within spindles , as elongated overlaps and misaligned chromosomes are mostly found in the central part of the spindle rather than on the periphery .",
"In contrast to the acute PRC1 removal , long-term PRC1 depletion by siRNA leads neither to overlap elongation nor chromosome misalignment , providing further support to our model .",
"These differences between the acute and long-term depletion indicate the existence of compensatory mechanisms that regulate overlap length and hence maintain chromosome alignment during long-term PRC1 depletion .",
"How is the length of the bridging microtubules and their overlaps controlled ?",
"Interestingly , we found that Kif4A and Kif18A localize in the bridging fibers in metaphase and their intensities decreased following partial disassembly of the bridging fiber by optogenetic or siRNA-mediated PRC1 removal .",
"Both Kif4A and Kif18A suppress microtubule dynamics ( Bieling et al . , 2010b; Bringmann et al . , 2004; Stumpff et al . , 2008; Stumpff et al . , 2012 ) , and our experiments showed that depletion of either of them by siRNA leads to longer PRC1-labeled overlaps .",
"These elongated PRC1-labeled overlaps provide another line of evidence in support of our model for chromosome alignment by overlap length-dependent forces , as Kif4A and Kif18A siRNA-treated cells exhibit chromosome misalignment ( Stumpff et al . , 2008; Wandke et al . , 2012 ) .",
"Even though it was reported that Kif4A does not directly interact with PRC1 before late mitosis ( Gruneberg et al . , 2006; Kurasawa et al . , 2004; Zhu and Jiang , 2005 ) , our experiments suggest that there is a small pool of Kif4A that does so and binds to the antiparallel overlaps of bridging microtubules in metaphase .",
"Acute PRC1 removal likely results in the removal of this pool of Kif4A from the bridge .",
"Our result that Kif4A was reduced by ~76% , which is similar to the reduction of PRC1 by ~85% within the same time interval ( 12 min ) of acute removal supports this picture .",
"This reduction of Kif4A in the bridge may lead to excessive microtubule polymerization and thus longer overlap zones in metaphase , similar to the long overlaps in anaphase after Kif4A depletion ( Hu et al . , 2011; Kurasawa et al . , 2004; Zhu and Jiang , 2005 ) .",
"As the spindle length remained constant after acute PRC1 removal , we suggest that the overlap elongation is due to Kif4A acting specifically on bridging microtubules rather than overall spindle microtubules .",
"In contrast to Kif4A , Kif18A in the bridging fiber was reduced less than PRC1 within 12 min of acute PRC1 removal , ~43% compared with ~85% , respectively .",
"This difference in the behavior of Kif18A and PRC1 is consistent with the fact that Kif18A is not known to be a binding partner of PRC1 .",
"Thus , we infer that the reduction of Kif18A in the bridge was a consequence of the fewer microtubules in the bridging fiber , and that the amount of Kif18A per microtubule remained largely unaffected by PRC1 removal .",
"Finally , MKLP1 , which is a binding partner of PRC1 ( Gruneberg et al . , 2006 ) , was reduced to a similar extent as PRC1 , ~87% and ~88% within 20 min of acute PRC1 removal , respectively .",
"Based on these results , we conclude that the localization of MKLP1 and a small pool of Kif4A in the bridging fiber are directly PRC1-dependent , whereas the localization of Kif18A is not .",
"We propose that both Kif4A and Kif18A regulate the length of bridging microtubules and their overlaps under normal conditions ( Figure 6C ) , as their depletion results in longer overlaps .",
"Intriguingly , we found that Kif4A signal in the bridging fibers decreased to a larger extent after acute than after long-term PRC1 depletion , whereas Kif18A signal decreased to a similar extent by the two methods of PRC1 depletion .",
"Moreover , Kif4A depletion by siRNA resulted in an increase of ~1 µm in overlap length , which was similar to the increase observed after acute PRC1 removal , whereas Kif18A depletion by siRNA led to a larger overlap increase .",
"Thus , we suggest that the observed overlap elongation after acute PRC1 removal is a consequence of the strong reduction of Kif4A in the bridging fibers .",
"In contrast , after long-term PRC1 depletion , we speculate that a larger fraction of Kif4A is present on the microtubules during overlap formation , contributing to the compensatory mechanism maintaining normal overlap length .",
"The proteins that we found in the bridging fiber , Kif4A , Kif18A , and MKLP1 , may also slide apart and/or stabilize the bridging microtubules ( Figure 6C ) .",
"In addition to these kinesins , we observed Eg5 in the bridging fiber ( Kajtez et al . , 2016; Mann and Wadsworth , 2018 ) .",
"During early anaphase , Kif4A and Eg5 drive the sliding of antiparallel microtubules that elongates the spindle ( Vukušić et al . , 2019 ) .",
"Thus , these kinesins may have a similar role during metaphase .",
"This possibility is in agreement with previous work showing that Kif4A depletion reduces microtubule poleward flux in metaphase ( Wandke et al . , 2012 ) .",
"Similarly , Kif18A in the bridging fiber may have microtubule sliding and crosslinking activities equivalent to those of the yeast kinesin-8 ( Su et al . , 2013 ) .",
"Furthermore , MKLP1 contributes to the stabilization of bridging microtubules in early anaphase ( Vukušić et al . , 2017 ) , and we suggest that it performs a similar function in metaphase together with other motors and crosslinkers including Eg5 and PRC1 .",
"The roles of these and other proteins within bridging fibers in the regulation of microtubule dynamics and sliding will be an intriguing topic for future studies .",
"The localization of Kif4A on chromosome arms , where it is involved in polar ejection forces ( Bieling et al . , 2010a; Brouhard and Hunt , 2005 ) was preserved after acute PRC1 removal .",
"Similarly , Kif18A , CLASP1 , and CENP-E remained on plus-ends of k-fibers .",
"However , as we cannot exclude potential subtle changes in the localization of these proteins or mislocalization of other proteins , the observed chromosome misalignment and overlap elongation after acute PRC1 removal could also be promoted by the changes of the dynamics of other microtubules within the spindle consequently affecting forces produced by k-fibers and polar ejection forces .",
"The changes upon acute PRC1 removal were not spatially uniform across the spindle .",
"The most affected part was the inner part of the spindle , where the PRC1 signal disappeared faster and the bridging microtubules became longer than on the periphery of the spindle .",
"The inner bridging fibers were more severely affected by PRC1 removal possibly because they are made up of fewer microtubules than the outer bridges .",
"Severely misaligned kinetochores that moved more than 2 µm away from the equatorial plane and lagging kinetochores occurred also more often in the inner part of the spindle .",
"This local effect is in line with weak mechanical coupling between neighboring k-fibers yet strong coupling between sister k-fibers ( Elting et al . , 2017; Suresh et al . , 2020; Vladimirou et al . , 2013 ) , and indicative of a mechanistic link between the bridging fiber geometry and kinetochore alignment .",
"In conclusion , we propose that overlap length-dependent forces help to position the chromosomes at the equatorial plane of the spindle .",
"The overlap length is regulated by the PRC1-dependent Kif4A , and by Kif18A within the bridging fiber .",
"Kif4A , Eg5 , and possibly other kinesins may slide bridging microtubules apart , whereas PRC1 together with the kinesins stabilizes the overlaps .",
"Thus , in addition to the forces generated at k-fiber tips and polar ejection forces , proper chromosome alignment requires forces generated within the bridging fiber , which are transferred to the k-fiber and rely on precise regulation of the overlap region ."
],
[
"Experiments were performed using: unlabeled human osteosarcoma U2OS cell line , U2OS cells expressing CENP-A-GFP , mCherry-α-tubulin and PA-GFP-tubulin and U2OS cell line stably expressing CENP-A-GFP , used in our previous work ( Vukušić et al . , 2017 ) , which were a gift from Marin Barišić and Helder Maiato ( Institute for Molecular Cell Biology , University of Porto , Portugal ) ; U2OS cell line stably expressing 2xGFP-EB3 and mCherry-CENP-A , a gift from Julie Welburn ( University of Edinburgh , United Kingdom ) ( Kajtez et al . , 2016 ) ; HeLa-Kyoto BAC lines stably expressing MKLP1-GFP , Kif4A-GFP , Kif18A-GFP , CENP-E-GFP and PRC1-GFP were a courtesy of Ina Poser and Tony Hyman ( MPI-CBG , Dresden , Germany ) ; unlabeled HeLa-TDS cells from the High-Throughput Technology Development Studio ( MPI-CBG , Dresden ) ; HeLa-TDS cells , permanently transfected with pEGFP-α-tubulin , used in our previous work ( Kajtez et al . , 2016 ) ; HeLa cells stably expressing YFP-tubulin , a courtesy of Lars Jansen ( University of Oxford , United Kingdom ) ; HeLa cells permanently transfected with EGFP-CLASP1 , which was a gift from Helder Maiato ( Institute for Molecular Cell Biology , University of Porto , Portugal ) .",
"Cells were grown in flasks in Dulbecco’s Modified Eagle’s Medium ( DMEM; Lonza , Basel , Switzerland ) with 1 g/L D-glucose , L-glutamine , and pyruvate , supplemented with 10% of heat-inactivated Fetal Bovine Serum ( FBS; Sigma Aldrich , St . Louis , MO , USA ) , 100 IU/mL penicillin and 100 mg/mL streptomycin solution ( Lonza ) .",
"For cell lines with stable expression of fluorescently labeled proteins , 50 µg/mL geneticin ( Life Technologies , Waltham , MA , USA ) was added .",
"Cells were kept at 37°C and 5% CO2 in a Galaxy 170 R humidified incubator ( Eppendorf , Hamburg , Germany ) .",
"All used cell lines were confirmed to be mycoplasma free by using MycoAlert Mycoplasma Detection Kit ( Lonza ) .",
"To make PRC1-tgRFPt-SspB WT ( opto-PRC1 ) plasmid , PRC1 fragment was amplified from His6-PRC1 plasmid ( RRID:Addgene_69111 ) ( Nixon et al . , 2015 ) using the primers GCTAGAATTGACCGGATGAGGAGAAGTGAGGTGCTG ( FWD ) and CATGGTGGCGACCGGTAAATTCGAAGCTTGAGCTCGAGATCTGAGGGACTGGATGTTGGTTGAATTGAGG ( REV ) and inserted into plasmid tgRFPt-SspB WT ( RRID:Addgene_60415 ) ( Guntas et al . , 2015 ) using AgeI restriction site .",
"This step was performed using commercially available In-Fusion HD Cloning Kit ( Clontech , Mountain View , CA , USA ) .",
"The produced plasmid expresses PRC1 tagged with tgRFPt and SspB at the C-terminus .",
"Plasmid iLID-CAAX was purchased ( RRID:Addgene_85680 ) ( O'Neill et al . , 2016 ) .",
"Plasmids pEGFP-C1Kif4a-sires and EGFP-Kif18A were a gift from Jason Stumpff ( University of Vermont , Burlington , VT , USA ) ( Stumpff et al . , 2008; Stumpff et al . , 2012 ) .",
"Plasmid GFP-CENP-E was a gift from Marin Barišić ( Danish Cancer Society Research Center , Copenhagen , Denmark ) .",
"Plasmid mRFP-CENP-B ( pMX234; RRID:Addgene_23006 ) was provided by Linda Wordeman ( University of Washington ) .",
"For depletion of endogenous PRC1 before opto experiments , cells were transfected 72 hr ( U2OS cells ) or 24 hr ( HeLa cells ) prior to imaging with 25 µL of 20 µM Accell PRC1 siRNA ( A-019491-15-0020 , Dharmacon , Lafayette , CO , USA ) targeting 3’ UTR of PRC1 mRNA .",
"A day prior to imaging , siRNA-treated cells were transfected with corresponding plasmids in following amounts: 0 . 3 μg of iLID-CAAX , 5 . 5 μg PRC1-tgRFPt-SspB-WT ( resistant to the used RNAi ) , 0 . 5 µg pEGFP-C1Kif4a-sires , 1 µg EGFP-Kif18A , 1 µg GFP-CENP-E , and 2 . 5 µg mRFP-CENP-B .",
"In HeLa BAC lines , 24 hr prior to imaging , mock experiment cells were transfected with 100 nM Accell Non-targeting Pool ( D-001910-10-05; Dharmacon ) , PRC1 siRNA treated with 100 nM Accell PRC1 siRNA ( Dharmacon ) , Kif4A siRNA treated with 100 nM Kif4A siRNA ( sc-60888; Santa Cruz Biotechnologies , Dallas , TX , USA ) , whereas Kif18A siRNA treated with 100 nM Silencer Select Validated Kif18A siRNA ( s37882; Ambion , Austin , TX , USA ) .",
"All transfections were performed using Nucleofector Kit R with the Nucleofector 2b Device ( Lonza ) using X-001 program for U2OS and O-005 ( high viability ) program for HeLa cells .",
"Following transfection , the cells were seeded on 35 mm glass coverslip uncoated dishes with 0 . 17 mm ( 1 . 5 coverglass ) glass thickness ( MatTek Corporation , Ashland , MA , USA ) in 1 . 5 mL DMEM medium with appropriate supplements .",
"To visualize microtubules , cells were stained with silicon rhodamine ( SiR ) -tubulin ( Lukinavičius et al . , 2014; Spirochrome AG , Stein am Rhein , Switzerland ) , a far-red tubulin dye , at a concentration of 50 nM 12–16 hr prior to imaging .",
"To prevent dye efflux , verapamil , a broad-spectrum efflux-pump inhibitor ( Spirochrome Ltd . ) , was added in U2OS cells at a concentration of 1 μM .",
"To visualize chromosomes and determine the phase of the mitosis , 20 min prior to imaging SiR-DNA ( Lukinavičius et al . , 2015; Spirochrome AG , Stein am Rhein , Switzerland ) was added to a final concentration of 150 nM .",
"For experiments on U2OS cells expressing 2xGFP-EB3 and mCherry-CENP-A , the cells were synchronized by adding 20 µM of the proteasome inhibitor MG-132 ( Sigma-Aldrich ) to arrest the cells in metaphase .",
"Imaging was started 15 min after adding MG-132 .",
"Cells were fixed with ice-cold methanol for 3 min , washed three times with phosphate buffer saline ( PBS ) , followed by 15 min permeabilization with 0 . 5% Triton in PBS .",
"Cells were washed three times with PBS and blocked in 1% Normal Goat Serum ( NGS ) except in experiment for intensity of opto-PRC1 where cells were blocked in BSA in PBS for 1 hr at 4°C .",
"Cells were washed three times with PBS and then incubated in primary antibody solution in blocking buffer over night at 4°C .",
"Following primary antibodies were used: mouse anti-PRC1 monoclonal antibody ( 1:100; C-1; sc-376983 , Santa Cruz Biotechnology ) , rabbit anti-α-tubulin polyclonal antibody ( 1:100; RRID:AB_10743646; SAB4500087; Sigma-Aldrich Corporation , St . Louis , MO , USA ) , mouse monoclonal anti-Kif4A antibody ( 1:100; RRID:AB_10707683; E-8; sc-365144; Santa Cruz Biotechnology ) , rabbit polyclonal anti-MKLP1 antibody ( 1:100; RRID:AB_631959; N-19; sc-867 , Santa Cruz Biotechnology ) , mouse monoclonal anti-Eg5 antibody ( 1:100; RRID:AB_10841907; A-1; sc-365681 , Santa Cruz Biotechnology ) , rabbit polyclonal anti-Kif18A antibody ( 1:100; RRID:AB_2296551; A301-080A; Bethyl ) , rabbit polyclonal anti-Kif4A antibody ( 1:200; RRID:AB_2280904; A301-074A; Bethyl ) .",
"After washing off primary antibodies with PBS , cells were incubated in a solution of secondary antibodies in 2% NGS or BSA in PBS for 1 hr at room temperature protected from light .",
"Following secondary antibodies were used: donkey anti-mouse IgG Alexa Fluor 488 ( 1:250; ab150109 , Abcam , Cambridge , UK ) , donkey anti-rabbit IgG Alexa Fluor 594 ( 1:250; ab150064 , Abcam ) , donkey anti-rabbit IgG Alexa Fluor 405 ( 1:250; RRID:AB_2715515; ab175649 , Abcam ) , and goat anti-mouse IgG Alexa Fluor 647 ( 1:250; RRID:AB_2811129; ab150119 , Abcam ) .",
"After washing off the secondary antibodies three times in PBS , cells were incubated with a solution of 4’ , 6-diamidino-2-phenylindole ( DAPI ) ( 1:1000 ) in PBS for 20 min and washed three times in PBS or SiR-DNA ( 150 nM ) in PBS for 15 min before imaging .",
"Note that we used immunocytochemistry for PRC1 rather than Western blot analysis because the efficiency of opto-PRC1 plasmid transfection is low , and as Western blot analysis provides information about the complete cell population , these results may not be relevant for the cells used in the optogenetic experiments .",
"In contrast , by using immunocytochemistry we analyzed only the cells with a similar opto-PRC1 level and in the same phase as those in our optogenetic experiments .",
"Immunocytochemistry imaging and live imaging of unlabeled U2OS , U2OS stably expressing CENPA-GFP , HeLa-TDS pEGFP-α-tubulin and HeLa BAC CENP-E-GFP cells was performed using Bruker Opterra Multipoint Scanning Confocal Microscope ( Bruker Nano Surfaces , Middleton , WI , USA ) , described previously ( Buđa et al . , 2017 ) .",
"In brief , the system was mounted on a Nikon Ti-E inverted microscope equipped with a Nikon CFI Plan Apo VC 100x/1 . 4 numerical aperture oil objective ( Nikon , Tokyo , Japan ) .",
"During imaging , live cells were maintained at 37°C using H301-K-frame heating chamber ( Okolab , Pozzuoli , NA , Italy ) .",
"In order to obtain the optimal balance between spatial resolution and signal-to-noise ratio , 60 µm pinhole aperture was used .",
"Opterra Dichroic and Barrier Filter Set 405/488/561/640 was used to separate the excitation light from the emitted fluorescence .",
"Following emission filters were used: BL HC 525/30 , BL HC 600/37 , and BL HC 673/11 ( all from Semrock , Rochester , NY , USA ) .",
"Images were captured with an Evolve 512 Delta Electron Multiplying Charge Coupled Device ( EMCCD ) Camera ( Photometrics , Tucson , AZ , USA ) using a 200 ms exposure time .",
"Electron multiplying gain was set on 400 .",
"Camera readout mode was 20 MHz .",
"No binning was performed .",
"The xy-pixel size in the image was 83 nm .",
"The system was controlled with the Prairie View Imaging Software ( Bruker ) .",
"For kinetics experiments on U2OS cells ( Figure 1 ) , 561 and 488 nm diode laser lines were used every 10 s with 200 ms exposure time .",
"In all other optogenetic experiments , stacks were acquired using sequentially the following diode laser lines: 561 nm ( to visualize opto-PRC1 ) , 488 nm ( to activate the optogenetic system and to visualize GFP ) , and 640 nm ( to visualize SiR-tubulin or SiR-DNA , when applicable ) , with time interval between z-stacks of 60 s and with 200 ms exposure time per laser line .",
"To prevent dissociation of PRC1 from the cell membrane between acquiring two consecutive z-stacks , only blue light was turned on for 200 ms every 10 s .",
"Cells were imaged this way for 20 min in order to achieve almost complete removal of PRC1 from the spindle , after which the blue light was turned off and imaging was continued for another 10 min at 60 s intervals .",
"The total imaging time of 30 min was chosen to be close to the typical metaphase duration of 29 . 7 ± 2 . 3 min , which was measured from the metaphase plate formation until anaphase onset in U2OS cells expressing CENP-A-GFP , mCherry-α-tubulin and PA-GFP-tubulin ( N = 187 ) imaged after nuclear envelope breakdown every minute by obtaining 15 z-slices with 0 . 5 µm spacing and 150 ms exposure time .",
"After 30 min of imaging , one z-stack in each of the three channels was taken in order to visualize the spindle and kinetochores after PRC1 return .",
"In all cells except HeLa cells expressing pEGFP-α-tubulin , three focal planes with spacing between adjacent planes of 1 µm were acquired .",
"Live imaging of HeLa cells stably expressing pEGFP-α-tubulin for measurements of tubulin intensities after acute PRC1 removal was performed in the same manner as described above for optogenetic experiments .",
"Additionally , before turning the blue light on every 10 s , one z-stack was acquired using 561 , 488 , and 640 nm diode laser lines with averaging 8 , and seven focal planes with spacing between adjacent planes of 0 . 5 µm .",
"Stack was taken in the same way after 20 min of exposure to the blue light and again 10 min after the blue light was switched off .",
"The same HeLa cells were used for measurements of tubulin intensities after long-term PRC1 removal and imaged by acquiring one z-stack using 561 , 488 , and 640 nm diode laser lines with averaging 8 , and seven focal planes with spacing between adjacent planes of 0 . 5 µm .",
"Imaging of HeLa BAC CENP-E-GFP mock and PRC1 siRNA-treated cells was performed by acquiring one z-stack of 3 focal planes with spacing between adjacent planes of 1 µm .",
"For imaging of immunostained cells , five focal planes with spacing between adjacent planes of 0 . 5 µm were acquired .",
"Live imaging of U2OS cells stably expressing 2x-GFP-EB3 and mCherry-CENP-A and U2OS cells with transient expression of opto-PRC1 , iLID-CAAX and GFP-Kif4A or EGFP-Kif18A was performed on a spinning disk confocal microscope system ( Dragonfly , Andor Technology , Belfast , UK ) using 63x/1 . 47 HC PL APO glycerol objective ( Leica ) and Zyla 4 . 2P scientific complementary metal oxide semiconductor ( sCMOS ) camera ( Andor Technologies ) .",
"During imaging cells were maintained at 37° and 5% CO2 within H301-T heating chamber ( Okolab , Pozzuoli , Italy ) .",
"Images were acquired using Fusion software ( v 2 . 2 . 0 . 38 ) .",
"For live imaging of U2OS cells expressing 2x-GFP-EB3 , mCherry-CENP-A and opto-PRC1 , 488 nm and 561 nm laser lines were used for excitation to visualize GFP , and mCherry and opto-PRC1 , respectively .",
"In order to achieve PRC1 removal from the spindle , 3 z-planes with a z-spacing of 1 µm were acquired sequentially with both laser lines , every 10 s with 200 ms exposure time for 10 min .",
"This imaging protocol was followed by five minutes of faster imaging , every 1 . 5 s with both laser lines on a central z-plane in order to visualize EB3 dynamics and to prevent the opto-PRC1 return .",
"Control , mock and PRC1 siRNA-treated cells were imaged with the same imaging protocol .",
"Live imaging of U2OS cells with transient expression of opto-PRC1 , iLID-CAAX and GFP-Kif4A or EGFP-Kif18A was performed in the similar manner as described above .",
"Additionally , to better visualize the localization of the Kif4A and Kif18A , before turning the blue light on every 10 s , one z-stack was acquired using 561 , 488 and 640 nm diode laser lines with frame averaging 4 , and three focal planes with spacing between adjacent planes of 1 µm .",
"Stack was taken in the same way after 12 min of exposure to the blue light and again 5 min after the blue light was switched off .",
"Note that the exposure to the blue light was shorter than in previous experiments regarding opto-PRC1 kinetics so that the majority of the cells remained in metaphase during the experiment .",
"Live imaging of unlabeled , BAC ( except CENP-E-GFP ) , YFP-tubulin , and EGFP-CLASP1 HeLa cell lines was performed on Leica TCS SP8 X laser scanning confocal microscope with a HC PL APO 63x/1 . 4 oil immersion objective ( Leica , Wetzlar , Germany ) heated with an objective integrated heater system ( Okolab , Burlingame , CA , USA ) .",
"During imaging , cells were maintained at 37°C in Okolab stage top heating chamber ( Okolab , Burlingame , CA , USA ) .",
"The system was controlled with the Leica Application Suite X software ( LASX , 1 . 8 . 1 . 13759 , Leica , Wetzlar , Germany ) .",
"For GFP- and YFP-labeled proteins , a 488 nm and 514 nm Argon laser was used , respectively , and for SiR-DNA or SiR-tubulin , a 652 nm white light laser was used .",
"For AF-647 , a 637 nm white light laser was used .",
"GFP , and SiR-DNA , SiR-tubulin or AF-647 emissions were detected with hybrid detector .",
"For mock , PRC1 siRNA , Kif4A siRNA , and Kif18A siRNA experiments , images were acquired at 1–3 focal planes with 1 μm spacing and 0 . 05 µm pixel size .",
"In optogenetic experiments 3 z-stacks with 1 μm spacing were acquired sequentially every 10 s in the same manner as in optogenetic experiments in U2OS cells .",
"One z-stack with line averaging of 6 or 16 was acquired before system activation , 20 min after exposure to blue light and 10 min after the light was switched off .",
"Since the cells were transiently transfected with opto-PRC1 , we observed variability in PRC1 expression levels and therefore we imaged and analyzed only those metaphase spindles with PRC1 localization consistent with endogenous and fluorescently labeled PRC1 ( Kajtez et al . , 2016; Polak et al . , 2017 ) .",
"Cells were not synchronized in order to avoid additional chemical treatment of cells , and metaphase was determined by alignment of kinetochores in the equatorial plane .",
"For determination of kinetics of PRC1 removal and return ( Figure 1C ) , intensity of opto-PRC1 was measured in each time frame on one focal plane .",
"We used Polygon selection tool in Fiji ( National Institutes of Health , Bethesda , MD , USA ) to encompass the area of the spindle , Aspindle , and measure mean spindle intensity , Mspindle .",
"Mean background intensity in the cytoplasm was measured using 2 . 5 × 2 . 5 µm rectangle , Mcyto .",
"Spindle intensity was background corrected by subtracting Mcyto from Mspindle to obtain Mspindle corr .",
"In order to calculate the sum of PRC1 intensity on the spindle , Mspindle corr was multiplied with Aspindle for each timeframe .",
"The background intensity outside of the cell was negligible , thus we did not take it into account .",
"Note that for the measurements of kinetic parameters in Figure 1C , four outliers were excluded ( see Figure 1—figure supplement 1F ) .",
"The percentage of PRC1 removal was calculated from the mean value of intensity of all cells at time 20 min , that is , the last time point of the continuous exposure to the blue light .",
"The percentage of return was calculated from the mean value of intensity of all cells in the interval 25–30 min , that is , during last 5 min of PRC1 return .",
"Intensity profiles of opto-PRC1 removal and return ( Figure 1D ) were obtained on sum intensity projections of all three z-planes by drawing a pole-to-pole line along the long axis of the spindle by using Line tool in Fiji .",
"The width of the line corresponded to the width of each individual spindle .",
"Intensities were normalized to position of the poles .",
"For quantification of PRC1 knock-down by siRNA and intensity level of opto-PRC1 ( in Figure 1—figure supplement 1A , B ) PRC1 intensity on fixed cells was measured on a sum-intensity projection of five focal planes by the procedure described above , in a way that mean spindle intensity was background corrected by subtracting mean intensity in the cytoplasm .",
"For measuring opto-PRC1 intensity on the spindle , cells where PRC1 was visible on astral microtubules were not analyzed , nor imaged in live experiments .",
"For quantification of PRC1 knock-down by siRNA in HeLa BAC MKLP1-GFP cell line , PRC1 intensity was quantified in the same manner as in U2OS cells .",
"Inter-kinetochore distance was measured using Line tool in Fiji on individual or maximum intensity z-projections of up to two z-planes as a distance between centers of sister kinetochore signals .",
"Peripheral kinetochores were defined as three outermost pairs on each side of the spindle with respect to spindle long axis , while the remaining were considered as central .",
"Measurement of inter-kinetochore distances in prometaphase was performed on U2OS cells expressing CENP-A-GFP , mCherry-α-tubulin and PA-GFP-tubulin , just after nuclear envelope breakdown in one imaging plane where both sister kinetochores could be clearly distinguished .",
"For measuring kinetochore alignment and orientation , as well as orientation and length of PRC1 bundles , Multipoint tool in Fiji was used .",
"A point was placed in the center of signal of each sister kinetochore or edges of PRC1 signal for each bundle .",
"Before measuring , images were rotated in order to achieve perpendicular direction of the equatorial plane with respect to x-axis .",
"The equatorial plane was defined with two points placed between outermost pairs of kinetochores on the opposite sides of the spindle .",
"For all measurements regarding kinetochores , those located at the spindle poles were not taken into account .",
"All measurements in opto and control cells were performed in three time-points: before the blue light was switched on , after 20 min of continuous exposure to the blue light , and 10 min after the blue light was turned off .",
"In untreated and PRC1 siRNA-treated cells measurements were performed at the beginning of imaging .",
"Kymographs of kinetochore oscillations were produced by Low Light Tracking Tool ( LLTT ) , an ImageJ plugin ( Krull et al . , 2014 ) .",
"Tracking of kinetochores in x , y plane was performed on maximum intensity of 2–3 z-planes .",
"Sigma value ( standard deviation of the Gaussian used to approximate the Point Spread Function ( PSF ) of the tracked objects ) was set to Automatic .",
"EB3 spots in U2OS cells stably expressing 2x-GFP-EB3 and mCherry-CENP-A were tracked by obtaining their xy coordinates using Multipoint tool in Fiji from the frame when a spot appeared until it disappeared or was no longer clearly distinguishable from its neighbors .",
"Only spots belonging to bridging fibers were traced , which were defined as those passing between sister kinetochores or moving along PRC1 streaks .",
"Half-overlap length was measured as the distance between the last location where a tracked EB3 spot was visible and the spindle equator .",
"The number of spots in time was obtained by visually inspecting time frames where individual kinetochore pair or PRC1 bundle was visible , and dividing the total number of the observed EB3 spots in the bridging fibers by the total time of observation of individual kinetochore pairs or PRC1 bundles .",
"Kymographs were generated using KymographBuilder plugin in Fiji , and half-overlap length was measured from kymographs as the distance between a spot's trajectory end point and the mid-line between the poles corresponding to the equatorial plane .",
"These measurements were performed in the first 2 min of a fast imaging sequence , which followed after the 10 min imaging protocol required for PRC1 removal .",
"The tubulin-GFP signal intensity of a cross-section of a bridging fiber was measured by drawing a 3-pixel-thick line between and perpendicular to the tubulin signal joining the tips of sister k-fibers .",
"The same method was used in cells containing a kinetochore marker , that is , the profile intensity of the bridging fiber was extracted by looking at the tubulin channel and drawing the line between tips of k-fibers .",
"For confirmation , we subsequently checked the kinetochore channel , and observed that this line was always placed between sister kinetochores .",
"This validates our measurements of bridging fibers in opto cells using tubulin-GFP only .",
"The tubulin intensity profile was corrected by subtracting mean background signal present in the cytoplasm ( see Figure 4A and Figure 4—figure supplement 1C , G ) .",
"The signal intensity of the bridging fiber was calculated as the area under the peak using SciDavis ( Free Software Foundation Inc , Boston , MA , USA ) ( Figure 4E ) .",
"The signal intensity in the region lateral from k-fiber tip was measured in a similar manner , 0 . 882 ± 0 . 04 µm away from the k-fiber tip ( Figure 4—figure supplement 1C , G ) .",
"This intensity corresponds to the sum of the bridging and k-fiber , given that PRC1 overlap half-length is longer and EB3 spots pass further than the position where the intensity profiles were measured .",
"Therefore , the intensity of the k-fiber was calculated by subtracting the bridging fiber intensity from the corresponding sum of bridging and k-fiber intensity .",
"The profile intensity of bridging fiber and at the position lateral from k-fiber tip in the cells stained with SiR-tubulin ( Figure 4B , C ) was performed in the same manner .",
"All the measurements were performed on a single z-plane .",
"Note that mostly outermost fibers were used for these measurements because of being most easily distinguished from neighboring fibers .",
"All measurements in opto and control cells were performed at three time-points: before the blue light was switched on , after 20 min of continuous exposure to the blue light , and 10 min after the blue light was turned off .",
"Shapes of the spindle were quantified by tracking outermost k-fiber contours in the central z-slice of the spindle , or maximum intensity z-projection of two central z-slices .",
"All spindles were rotated to have horizontal long axis .",
"Pole-to-k-fiber-tip tracking was done using Multipoint tool by placing five roughly equidistant points along contour length , first point being at the pole and the last point being at the k-fiber tip .",
"First point of each contour was translated to ( 0 , 0 ) .",
"This was done for maximum of 4 trackable outermost k-fibers in the spindle .",
"Curvature of the contour was calculated by fitting a circle to the contour points of individual k-fibers and retrieving reciprocal value of its radius .",
"To test the effect of tracking errors on curvature , we introduced 1-pixel noise to the x and y values of tracked points , which did not change the result .",
"Angle between outermost k-fibers ( θ ) was calculated as the angle between lines passing through last two points along the contour of sister k-fibers .",
"Spindle length and width were measured using Line tool in Fiji .",
"For length measurements , a line was drawn from pole to pole .",
"The position of the pole was defined as the location of the strongest tubulin signal .",
"Width was measured by drawing a line between outermost kinetochore pairs on the opposite sides of the spindle , perpendicular to the spindle long axis .",
"To avoid the signal of Kif4A at the chromosomes , Kif4A bridging fiber intensity was measured on individual z-planes with 1 × 0 . 5 µm rectangles at the positions lateral from sister kinetochores and chromosomes , where no DNA ( DAPI or SiR-DNA ) was found , yet lateral parts of PRC1 streaks are present .",
"This approach was applied in cases when PRC1 was not depleted or relocated to the cell membrane , that is , in opto cells before acute PRC1 removal and untreated cells .",
"When PRC1 was not present on the spindle , that is , in opto cells after acute PRC1 removal and in PRC1 siRNA-treated cells , we placed rectangles at positions lateral from kinetochores and chromosomes , where antiparallel zones are thought to be positioned , considering their location before removal .",
"The rectangles were placed parallel to the overlap .",
"Given that bridging fiber intensity was measured mostly in the inner parts of the spindle , mean values from the spindle poles were considered as the Kif4A background and subtracted from Kif4A intensity retrieved from the positions of bridging fibers .",
"For the measurements of Kif4A in the bridging fiber , we chose only cells in which Kif4A showed previously known localization on chromosomes and at comparable levels .",
"This was important as immunostaining protocol sometimes resulted in cells with no or low chromosome staining , yet high spindle staining , and HeLa BAC Kif4A-GFP cells vary in expression levels among cells .",
"The intensity of Kif4A on chromosome arms in metaphase was measured in Kif4A channel on sum-intensity projections of all z-planes ( nine planes in immunostained , and three in opto and BAC cells ) using Polygon selection tool in Fiji by encompassing chromosomes in DAPI or SiR-DNA channel .",
"In order to avoid the Kif4A signal on the spindle microtubules , the signal was measured only on the parts of the chromosome arms protruding into cytoplasm away from the spindle .",
"For each cell , mean value of Kif4A arm intensity was calculated and corrected for background cytoplasm intensity measured in 2 . 5 × 2 . 5 µm rectangle by subtracting it from mean intensity of Kif4A .",
"In immunostained cells only those with Kif4A localized on chromosomes were taken into account as in some sessions the Kif4A signal was very strong on the whole spindle and not present on the chromosomes both in untreated and PRC1 siRNA-treated cells .",
"The intensity of GFP-Kif4A on chromosomes in anaphase was measured in GFP-Kif4A channel on sum-intensity projections of all three z-planes using Polygon selection tool in Fiji by encompassing chromosomes in SiR-DNA channel .",
"Background cytoplasm intensities in GFP-Kif4A channel were measured in 2 . 5 × 2 . 5 µm rectangle and subtracted from measured mean intensities of GFP-Kif4A .",
"Corrected intensities were divided by the number of focal planes .",
"In anaphase , measurements were performed 4 min after anaphase onset .",
"Anaphase onset was defined as the timeframe when separation of majority of sister chromatids in SiR-DNA channel occurred .",
"The mean bridging fiber intensities of Kif18A in all treatments were obtained on a single z-plane using 0 . 4 × 0 . 4 µm rectangles in Fiji covering the Kif18A signal in the bridge .",
"The rectangles of the same dimension were used to obtain the Kif18A signal from the sister k-fiber tips and the mean value for the pair was calculated .",
"Since the measurements of Kif18A were mostly obtained on the outermost fibers , these values were corrected by subtracting mean background cytoplasm intensity measured in a 2 . 5 × 2 . 5 µm rectangle .",
"The intensity of MKLP1 in the bridging fibers was measured in GFP channel on a single z-plane using 0 . 4 × 0 . 4 µm rectangle in Fiji by placing it on each bridging fiber visible in the SiR-tubulin channel .",
"These intensities were corrected for background GFP intensity in cytoplasm measured in 2 . 5 × 2 . 5 µm rectangle .",
"For quantification of PRC1 knock-down by siRNA in HeLa BAC MKLP1-GFP cell line , PRC1 intensity was quantified in the same manner as in U2OS cells: on a sum-intensity projection of five focal planes in a way that mean spindle intensity was background corrected by subtracting mean intensity in the cytoplasm , .",
"The intensity of CENP-E and CLASP1 on plus ends-of k-fibers was measured using 0 . 4 × 0 . 4 µm rectangle encompassing plus-end of k-fibers on a single z-plane .",
"Intensities were corrected for the intensity in cytoplasm measured using 2 . 5 × 2 . 5 µm rectangle in the central z-plane as this intensity was similar between different z-planes .",
"For analysis of CLASP1 and CENP-E intensity on plus-ends of k-fibers only cells with similar intensities on the spindle within each treatment were taken into account as there was a variation in expression level among cells .",
"Measurements regarding Kif4A , Kif18A , MKLP1 , CENP-E , and CLASP1 were performed in two time-points in metaphase: before the blue light was switched on and at the end of continuous exposure to the blue light .",
"The intensities were normalized on the mean value of control at each treatment , that is , before the blue light was switched on for acute PRC1 removal and untreated for long-term depletion .",
"Quantification of the signal length of PRC1-GFP in mock , Kif4A , and Kif18A siRNA-treated HeLa cells was performed by drawing a 5-pixel-thick pole-to-pole line along individual bundles and calculated as the width of the peak of the PRC1-GFP signal intensity .",
"Statistical analysis was performed in MATLAB ( RRID:SCR_001622; MathWorks , Natick , MA , USA ) and RStudio ( RRID:SCR_000432; R Foundation for Statistical Computing , Vienna , Austria ) .",
"Normality of the data was inspected by Q-Q plots and Shapiro-Wilk test of normality .",
"Two groups with normally distributed data were tested with two-tailed t-test , while more than two groups were tested with one-way ANOVA followed by Tukey HSD post hoc test .",
"Two groups with non-normally distributed data were tested with Mann-Whitney test , while more than two groups were tested with Kruskal-Wallis rank sum test followed by pairwise Wilcoxon rank sum test .",
"Used statistical analysis is noted in figure captions .",
"Proportions were statistically compared with test for equality of proportions , two-proportions z-test .",
"For data with expected count smaller than 5 , Yates's correction for continuity was used .",
"Graphs were generated in MATLAB ( MathWorks ) and RStudio ( R Foundation for Statistical Computing ) .",
"ImageJ ( RRID:SCR_003070; National Institutes of Health , Bethesda , MD , USA ) was used to crop and rotate images , and to adjust brightness and contrast to the entire image , which was applied equally to all images in the same panel .",
"The images are rotated in order for the spindle to be horizontal in every time frame .",
"To remove high-frequency noise in displayed images a Gaussian blur filter with a 0 . 5-pixel sigma ( radius ) was applied where stated .",
"Figures were assembled in Adobe Illustrator CS5 ( RRID:SCR_010279; Adobe Systems , Mountain View , CA , USA ) ."
]
] | [
"During metaphase , chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces .",
"However , the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown .",
"Here , we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment .",
"Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal , which was accompanied by misaligned and lagging kinetochores .",
"Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal , suggesting that these proteins regulate the overlap length of bridging microtubules .",
"We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promote chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers ."
] | [
"Before cells divide to create copies of themselves , they need to duplicate their genetic material .",
"To help split their DNA evenly , they build a machine called the mitotic spindle .",
"The mitotic spindle is made of fine , tube-like structures called microtubules , which catch the chromosomes containing the genetic information and line them up at the center of the spindle .",
"Microtubules push and pull the chromosomes by elongating or shortening their tips .",
"But it remains unclear how the microtubules know when the chromosomes have reached center point .",
"One way to find out is to remove proteins that accumulate in the middle of the spindle during division , such as the protein PRC1 , which helps to assemble a subset of microtubules called bridging fibers , and the proteins Kif4A and Kif18A , which work like molecular rulers , shortening long microtubules .",
"Usually , scientists would delete one of these proteins to see what impact this has .",
"However , these experiments take days , giving the cell enough time to adapt and thus making it difficult to study the role of each of the proteins .",
"Here , Jagrić , Risteski , Martinčić et al . used light to manipulate proteins at the exact moment of chromosome alignment and to move PRC1 from the spindle to the cell membrane .",
"Consequently , Kif4A and Kif18A were removed from the spindle center .",
"This caused the bridging fibers , which overlap with the microtubules that connect to the chromosomes , to become thinner .",
"Jagrić et al . discovered that without the molecular ruler proteins , the bridging fibers were also too long .",
"This increased the overlap between the microtubules in the center of the spindle , causing the chromosomes to migrate away from the center .",
"This suggests that the alignment of chromosomes in the middle of the spindle depends on the bridging microtubules , which need to be of a certain length to effectively move and keep the chromosomes at the center .",
"Thus , forces that move the chromosomes are generated both at the tips of the microtubules and along the wall of microtubules .",
"These results might inspire other researchers to reassess the role of bridging fibers in cell division .",
"The optogenetic technique described here could also help to determine the parts other proteins have to play .",
"Ultimately , this might allow researchers to identify all the proteins needed to align the chromosomes ."
] | 2021 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"biochemistry and chemical biology",
"structural biology and molecular biophysics"
] | Regulation of multispanning membrane protein topology via post-translational annealing | elife-08697-v2 | [
[
"The cotranslational integration and topogenesis of EmrE and its mutants is simulated using a recently developed CG model ( Zhang and Miller , 2012a ) , which we employ essentially unchanged from its original introduction .",
"Figure 1 illustrates the CG representation of a nascent protein and the protocol for simulating membrane integration .",
"The ribosome , translocon , and nascent protein are all composed of CG beads .",
"Each bead has a diameter of σ = 0 . 8 nm to represent approximately three amino-acid residues .",
"This bead diameter is similar to the Kuhn length of polypeptides ( Staple et al . , 2008 ) so that the nascent protein can be treated as a freely jointed chain .",
"The surrounding solvent and lipid bilayer are included implicitly , a technique that is used in other CG models of the translocon ( Rychkova and Warshel , 2013 ) .",
"The time-evolution of nascent protein configurations is calculated using Brownian dynamics with a 100 ns timestep .",
"The kinetics of the LG are modeled as stochastic transitions between a closed conformation , which prevents the nascent protein from exiting from the channel interior to the membrane , and an open conformation , which removes the barrier to membrane entry .",
"All bead positions are projected onto the plane that passes along the translocon channel axis between the helices forming the LG .",
"This off-lattice 2D approximation reflects the cylindrical geometry of the channel and is inspired by previous models of biopolymer translocation through nanopores ( Huopaniemi et al . , 2006 ) .",
"Beads representing the ribosome enclosure and translocon are placed to approximate their structures ( Van den Berg et al . , 2004; Frauenfeld et al . , 2011 ) .",
"Two negative charges are placed on a bead at the cytosolic end of the translocon LG , whereas two positive charges are placed on a bead at the periplasmic end of the translocon LG .",
"This charge distribution reflects the position of conserved charged residues ( White and von Heijne , 2004 ) near the translocon LG that have been previously shown to affect single-spanning protein topogenesis ( Goder et al . , 2004 ) .",
"Full details of the model are provided in Appendix 1 . 10 . 7554/eLife . 08697 . 003Figure 1 . Schematic of Sec-mediated cotranslational integration of EmrE and corresponding simulation representation .",
"( A ) At top , an illustration of the structural motifs in EmrE , including indication of the charged residues in the soluble loops with black circles and the transmembrane domain ( TMD ) /loop numbering scheme that is employed in the text; below , the corresponding sequence of coarse-grained ( CG ) beads that represent the EmrE amino-acid sequence .",
"TMDs and loops are assigned based on the hydropathy plot and consensus topology prediction shown in Figure 1—figure supplement 1 .",
"( B ) At top , a schematic illustration of the sequential integration of TMDs to obtain a multispanning Nperi/Cperi topology , in which both the N- and C-terminal loops are positioned in the periplasm , according to the cotranslational model; below , representative simulation snapshots of EmrE as the nascent protein grows during translation , integrates into the membrane , and exits the channel in the Nperi/Cperi multispanning topology .",
"The nascent protein is colored according to the legend at top , the ribosome is brown , and the translocon is green with translocon charges labeled explicitly . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 00310 . 7554/eLife . 08697 . 004Figure 1—figure supplement 1 . Hydropathy plot for EmrE . The amino-acid sequence is shown on the right with positive charges highlighted in red .",
"The transfer free energy according to the Wimley-White scale ( Wimley et al . , 1996 ) is plotted as a black line with a 7-residue moving average overlaid as a red line .",
"Regions predicted by TOPCONS to correspond to loops ( L1-L5 ) or TMDs ( TMD1-TMD4 ) are shaded according to the legend at right . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 00410 . 7554/eLife . 08697 . 005Figure 1—figure supplement 2 . Simulation snapshot illustrating the initial configuration comprised of 9 CG beads . Positions of several beads in the ribosome ( brown ) and translocon ( green ) are labeled , with each labeled bead indicated by a black dot .",
"Positions are labeled in units of σ .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 005 The CG model is well-suited to simulating the kinetics of cotranslational IMP integration , a process that is challenging for atomistic models ( Zhang and Miller , 2010; Gumbart et al . , 2011; Zhang and Miller , 2012b; Rychkova and Warshel , 2013 ) due to the large system size ( >100 , 000 atoms ) and the long timescale ( minutes ) of translation .",
"We note that the model does not include nascent protein secondary/tertiary structure , charged lipids , protein chaperones , or an electrostatic potential across the membrane .",
"However , the model does include explicit LG/translation dynamics , electrostatic interactions with the translocon , water/bilayer transfer free energies , and a direct mapping between the nascent protein sequence and the CG representation .",
"The model thus captures the major physicochemical features of the translocon-membrane system ( White and von Heijne , 2004 ) .",
"Moreover , the model has been shown to accurately predict features of single-spanning IMP integration and topogenesis ( Zhang and Miller , 2012a ) , including the sigmoidal dependence of stop-transfer efficiency on TMD hydrophobicity ( Hessa et al . , 2005 ) , the inversion of signal-anchor orientation during translation ( Goder and Spiess , 2003 ) , and the effect of translation rates and sequence features on signal-anchor orientation ( Goder and Spiess , 2003 ) .",
"In particular , the model has been shown ( Zhang and Miller , 2012a ) to correctly describe integration processes that are governed either by thermodynamics ( Hessa et al . , 2005 ) or kinetics ( Goder and Spiess , 2003 ) , and it has provided a means of understanding the competition between such effects .",
"The model has also been shown to correctly predict the dominant topology for a three-TMD multispanning IMP with a strong positive-inside bias ( Zhang and Miller , 2012a ) .",
"The strong agreement between simulation and experimental results presented in this work further indicates that IMP topological determinants are captured at this CG resolution ."
],
[
"For all 16 of the EmrE and nEmrE mutants with single added charges studied by Seppälä et al . ( 2010 ) , Figure 2 compares the experimentally observed IMP topologies with the prediction from the CG model .",
"Specifically , the figure compares the fraction of fully integrated proteins that adopt the Ncyto/Ccyto topology , with the remainder in the Nperi/Cperi topology .",
"The top and bottom rows show variants of EmrE and nEmrE respectively .",
"Each mutant differs only in the number and location of charges in the hydrophilic loops .",
"A schematic of each mutant drawn with the dominant topology predicted from simulations is included; positive charges are indicated as filled-in circles with additional charges relative to EmrE ( top row ) or nEmrE ( bottom row ) highlighted in red .",
"The topologies determined experimentally in Seppälä et al . ( 2010 ) are expressed as the fraction of Ncyto/Ccyto topologies by dividing the cell activity of each protein coexpressed with the EmrE ( Nperi ) mutant by the total growth of the protein coexpressed with either the EmrE ( Nperi ) or EmrE ( Ncyto ) mutant ( Seppälä et al . , 2010 ) , as described in the Experimental interpretation of EmrE topology section of the ‘Materials and methods’ . 10 . 7554/eLife . 08697 . 006Figure 2 . Topologies determined from simulations ( blue ) and compared to the experiments of Seppälä et al . ( 2010 ) ( red ) , reporting the fraction of fully integrated integral membrane protein ( IMP ) configurations in the Ncyto/Ccyto topology . Error bars indicate the standard error measured from independent blocks of simulations or taken from Seppälä et al . ( 2010 ) .",
"The dominant topology for each mutant is indicated schematically with additional positive charges relative to EmrE ( top ) or nEmrE ( bottom ) drawn as red dots . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 00610 . 7554/eLife . 08697 . 007Figure 2—figure supplement 1 . Robustness of the distribution of topologies to the trajectory termination criteria . The distribution of topologies for the EmrE , K3 , nK3 , and T28R mutants are computed using alternative trajectory termination criteria and compared to the distribution of topologies using the original protocol ( shown in Figure 2 ) .",
"At left , the plot illustrates the effect of extending each original CG trajectory by 50 s .",
"At right , the plot illustrates the effect of terminating trajectories at a distance of 20σ , rather than 4 . 5σ , from the origin .",
"The dashed line indicates perfect correlation .",
"Additional details on these calculations are included in the Robustness checks for the trajectory termination criteria section of the ‘Materials and methods’ .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 00710 . 7554/eLife . 08697 . 008Figure 2—figure supplement 2 . Correlation between the topologies determined from simulations ( x-axis ) and compared to the experiments of Seppälä et al . ( 2010 ) ( y-axis ) , reporting the fraction of fully integrated IMP configurations in the Ncyto/Ccyto topology . Mutants lying in the shaded quadrants have the same dominant topology in both the experiments and simulations .",
"The experimental and simulation measurements are linearly correlated with a Pearson correlation coefficient of r = 0 . 92 . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 008 It is clear from Figure 2 that the simulations are in excellent qualitative agreement with the experiments by correctly predicting the near 1:1 stoichiometry of wild-type EmrE and identifying the dominant topology for nearly all of the proteins considered .",
"Figure 2—figure supplement 2 illustrates that the distribution of topologies determined experimentally and the distribution of topologies measured from the simulations are linearly correlated ( Pearson correlation coefficient , r = 0 . 92 ) ; points lying in the two shaded quadrants of the graph correspond to proteins for which the simulations and experiments predict consistent topologies .",
"All mutants , with the exception of A52K , have the same dominant topology in the simulations as in the experiments within the statistical error .",
"The agreement between simulations and experiments suggests that the CG model correctly reproduces the essential molecular features of topogenesis; in the following , we analyze the ensembles of CG trajectories that give rise to these computed IMP topologies .",
"To investigate the molecular processes that govern the establishment of EmrE topology , we first examine the kinetics by which fully integrated topologies are reached .",
"As a function of time , Figure 3A shows the fraction of CG trajectories in which the studied protein reaches a fully integrated topology for several EmrE mutants and the CB mutant .",
"0 s corresponds to the end of translation and negative values of time correspond to the period that precedes the end of ribosomal translation in which the nascent protein is still attached to the ribosome .",
"Over 90% of the CB mutant trajectories reach the Ncyto/Ccyto topology within 3 s following the completion of translation and thus rapidly integrate as expected for the cotranslational model ( Blobel , 1980; Wessels and Spiess , 1988; Sadlish et al . , 2005 ) ; mechanistic features of individual TMD integration steps are discussed in the Cotranslational integration pathways section of the ‘Materials and methods’ .",
"In contrast , all variants of EmrE reach a fully integrated topology much more slowly , requiring hundreds of seconds for some CG trajectories to fully integrate ( see also Figure 3—figure supplement 2 ) . 10 . 7554/eLife . 08697 . 009Figure 3 . Kinetics of EmrE topogenesis .",
"( A ) Fraction of CG trajectories in which all TMDs are fully integrated in a multispanning topology , plotted as a function of time for several mutants .",
"( B ) Fraction of CG trajectories in which each TMD is integrated , plotted as a function of time for the cotranslationally-biased ( CB ) mutant ( top ) and EmrE ( bottom ) .",
"The snapshots show an example of a simulation in which TMD4 of EmrE does not integrate during translation .",
"In both panels , 0 s corresponds to the end of translation and negative values of time correspond to the period that precedes the end of ribosomal translation . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 00910 . 7554/eLife . 08697 . 010Figure 3—figure supplement 1 . Pathways for the cotranslational integration of TMDs into the membrane . At left , three distinct integration pathways are illustrated; the definition of each pathway is described in the Cotranslational integration pathways section of the ‘Materials and methods’ .",
"In the ‘channel-sliding’ pathway , the TMD partially enters the channel , then crosses the lateral gate ( LG ) , then fully integrates into the membrane .",
"In the ‘interface-sliding’ pathway , the TMD enters the cytoplasm through the gap between the translocon and ribosome , prior to undergoing membrane integration .",
"In the ‘in-out’ pathway , the TMD fully spans the channel prior to membrane integration .",
"At right , the fraction of cotranslational TMD integration events that exhibit each of these three pathways is presented for all four TMDs and for both the EmrE and nEmrE mutants .",
"The dominant cotranslational integration pathway is the ‘channel-sliding’ pathway for all TMDs in both mutants . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 01010 . 7554/eLife . 08697 . 011Figure 3—figure supplement 2 . Simulation time necessary for 50% , 90% , and 95% of the CG trajectories to reach fully integrated topologies for each mutant . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 01110 . 7554/eLife . 08697 . 012Figure 3—figure supplement 3 . Effect of loop length on integration trajectories .",
"( A ) Schematic illustrations of a new EmrE mutant with elongated , 10-bead loops , and a new CB mutant with shortened , 5-bead loops .",
"( B ) Fraction of CG trajectories that reach fully integrated topologies as a function of time for the EmrE and CB mutants with both loop lengths .",
"The integration kinetics for the EmrE mutants clearly show that longer loops increase the timescale of post-translational annealing , due to the reduction of the loop-flipping frequency for the longer soluble loops .",
"The corresponding effect is less clear for the two CB mutants , since the timescale for the loop-flipping frequency is dominated by the large positive charges on these loops , masking the effect of changing the loop length .",
"( C ) Average loop positions in the end-of-translation ( EOT ) ensemble for both the EmrE and CB mutants , presented in terms of the fraction of configurations for which each loop occupies the cytoplasm .",
"For the EmrE mutants , changing the loop length leads to relatively small shifts in the EOT ensemble; however , for the CB mutants , the effect of loop length on the EOT ensemble is much larger , with the new short-loop CB mutant exhibiting a much weaker bias towards the Ncyto/Ccyto topology in the EOT ensemble . DOI: http://dx . doi . org/10 . 7554/eLife . 08697 . 012 The slow post-translational integration of the dual-topology EmrE mutants is due to the fact that a significant fraction of trajectories exhibit configurations in which some TMDs are not fully integrated at the end of translation .",
"As a function of time , Figure 3B shows the fraction of CG trajectories in which each TMD is integrated for both the CB mutant ( top ) and EmrE ( bottom ) .",
"TMDs in the CB mutant integrate sequentially with near 100% efficiency during translation , which is consistent with the standard cotranslational model of topogenesis ( c . f . Figure 1 ) and explains the rapid timescale for fully integrating into a multispanning topology shown in Figure 3A .",
"In contrast , the TMDs of EmrE exhibit only partial integration , even at long times following the completion of translation .",
"Snapshots of a typical misintegrated TMD in EmrE are shown in Figure 3B .",
"Various experiments have indicated that such configurations with misintegrated TMDs arise due to frustration from charges placed in consecutive loops ( Gafvelin and von Heijne , 1994 ) , the strong orientational preference of a neighboring TMD ( Öjemalm et al . , 2012 ) , or the weak stop-transfer efficiency of marginally hydrophobic TMDs ( Moss et al . , 1998 ) .",
"Consistent with these experimental observations , the simulations in Figure 3B find that the weakly hydrophobic TMD1 of EmrE integrates the least efficiently , followed by TMD4 which is flanked by two charged loops ."
],
[
"In this work , we utilize a recently developed CG computational approach ( Zhang and Miller , 2012a ) that enables the direct simulation of Sec-facilitated membrane integration of proteins on biological timescales to investigate the topogenesis of the dual-topology EmrE protein and its mutants .",
"In addition to demonstrating excellent agreement with the experimentally observed topologies of EmrE and its mutants ( Seppälä et al . , 2010 ) , the simulations reveal a novel mechanism for the regulation of topogenesis in multi-spanning membrane proteins , in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach final , fully integrated topologies .",
"The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins .",
"The inclusion of charged residues on the soluble loops of the IMP can lead to significant changes in the distribution of fully integrated topologies by both altering the ensemble of protein configurations at the end of ribosomal translation , as well as by altering the available kinetic pathways that lead to fully integrated topologies .",
"This analysis leads to a number of experimentally testable predictions regarding IMP topogenesis .",
"In particular , the results of Figure 8 predict that the mutation of charged residues near the cytoplasmic or periplasmic openings of the translocon channel will lead to significant shifts in the observed topology of several EmrE mutants .",
"More generally , we note that any effect of channel mutations on the fully integrated IMP topology would indicate that kinetic effects during translation influence topogenesis , as suggested by the proposed mechanism .",
"Additionally , we predict that the introduction of IMP mutations that significantly alter the EOT ensemble with respect to the cytosolic localization of the slowest-flipping soluble loop , either by introducing charge mutations or by changing TMD hydrophobicity , will influence the multispanning IMP topology .",
"Although the current manuscript primarily focuses on the mechanism of topogenesis in the dual-topology EmrE protein , the mechanism and simulation analysis presented here has broader implications for topogenesis in other multispanning IMPs .",
"For EmrE and its mutants , we find that a significant fraction of the IMP configurations are misintegrated upon completion of ribosomal translation and undergo subsequent post-translational annealing to reach fully integrated topologies .",
"In contrast , a CB mutant exhibits an essentially fully integrated ensemble of configurations at the time that ribosomal translation completes .",
"For other IMPs , a combination of these behaviors may well be expected ( Lu et al . , 2000; Lambert and Prange , 2001; Kanki et al . , 2002; Skach , 2009; Öjemalm et al . , 2012; Bowie , 2013; Virkki et al . , 2014 ) , with some fraction of the nascent protein configurations reaching fully integrated topologies at the completion of ribosomal translation and some fraction reaching misintegrated configurations that subsequently undergo post-translational annealing .",
"Indeed , the importance of chaperone proteins such as YidC or Sec62 that post-translationally rescue misintegrated TMDs ( Sommer et al . , 2013; Zhu et al . , 2013; Aviram and Schuldiner , 2014; Jung et al . , 2014 ) may be connected to this necessary process of annealing initially misintegrated IMP configurations towards fully integrated topologies .",
"The emerging understanding of the role of the Sec translocon in regulating IMP topogenesis , as well as advances in the methodologies for probing and modifying interactions between the nascent protein and the translocon complex , hold intriguing possibilities for the prediction and control of protein folding in cellular environments ."
],
[
"Loop-flipping frequencies are calculated by monitoring loop positions with respect to the membrane in 1-ms time intervals .",
"A loop-flipping event is counted if a given loop switches from a cytoplasmic to periplasmic ( or vice versa ) position according to the definitions in the main text and if the y-position of the reference bead in the loop ( defined in the main text ) is at least 4 . 5σ after the flip ( to exclude counting the rapid flipping of loops within the translocon channel ) .",
"The loop-flipping frequencies presented in Figure 6 are obtained by averaging the frequency of loop-flipping events in each trajectory .",
"Sufficiently infrequent loop flips will not occur in every trajectory , but such events are observed in the combined ensemble of trajectories .",
"To examine the effect of neighboring TMDs on the loop-flipping frequency , we separately calculate the loop-flipping frequency of each loop for configurations in which zero , one , or two of the neighboring TMDs is misintegrated ( discussed in the Rate of loop-flipping depends on charge mutations section of the ‘Results’ ) .",
"In Seppälä et al . ( 2010 ) , the dominant topologies of EmrE mutants are determined by measuring the growth of E . coli cells in the presence of ethidium bromide ( EtBr ) .",
"EtBr is toxic to E . coli , but antiparallel EmrE dimers , in which the two monomers forming the dimer have opposite topologies , confer drug resistance .",
"EmrE dimerization can also be suppressed by including an E14D mutation .",
"The topology of an EmrE variant with the E14D mutation can thus be inferred by coexpressing the mutant with another EmrE variant of known topology , as any resulting drug resistance ( and cell growth ) can be attributed to the formation of antiparallel dimers .",
"To enable a direct comparison between the topologies measured from simulations and the experimental results , we convert the experimentally-measured cell activities from Seppälä et al . ( 2010 ) to the fraction of Ncyto/Ccyto topologies by assuming a linear relationship between cell growth and the population of antiparallel EmrE dimers .",
"The fraction of Ncyto/Ccyto topologies is calculated as ( 1 ) f ( Ncyto/Ccyto ) =A ( Nperi ) A ( Ncyto ) +A ( Nperi ) , where A ( Ncyto ) and A ( Nperi ) are the experimentally-measured cell activities for cells coexpressing the EmrE ( Ncyto ) and EmrE ( Nperi ) mutants , respectively .",
"Greater cell growth in the presence of the EmrE ( Nperi ) mutant , which exhibits a single dominant Nperi/Cperi topology , indicates that the mutant of interest exhibits a larger fraction of the opposite Ncyto/Ccyto topology , and vice versa for growth in the presence of the EmrE ( Ncyto ) mutant .",
"Experimental values for the activities of the EmrE and nEmrE mutants are taken from Figure 2 and Figure S1 of Seppälä et al . ( 2010 ) , respectively; these values are used to compute the fraction of Ncyto/Ccyto topologies reported in Figure 2 of the current paper .",
"Values for the activities of the ( Nout ( E14D ) + Nin ) and the Nout constructs from Figure 1 of Seppälä et al . ( 2010 ) are used to approximate the topology of the EmrE ( Nperi ) mutant , while the activities of the ( Nin ( E14D ) + Nout ) and the Nin constructs from the same figure are used to approximate the topology of the EmrE ( Ncyto ) mutant .",
"Error bars are approximated via standard error propagation techniques based on Equation",
"1 . In Figure 7A , the average position of the slowest-flipping loop relative to the membrane in the EOT ensemble is compared with the average position of that same loop in the ensemble of fully integrated configurations at the end of the CG trajectories .",
"Four mutants ( K3 , L85R , nEmrE , and nT28R1 ) , however , have two slow-flipping loops with similar loop-flipping frequencies ( Figure 6 ) , and two of these mutants ( K3 and L85R ) deviate most significantly in terms of the correlation in Figure 7A .",
"To better understand the effect of multiple slow-flipping loops on the correlation between the EOT ensemble and the final topology , the current section provides additional analysis in which a more sophisticated definition of the ‘slowest-flipping loop’ is employed for the four mutants that exhibit a pair of slow-flipping loops .",
"Below , we present this alternative definition , which leads to a slightly better correlation between the EOT ensemble and the ensemble of fully integrated configurations , as plotted in Figure 7—figure supplement",
"1 . The alternative definition of the slowest-flipping loop for mutants with two slow-flipping loops is given by ϕEOT and ϕFI , which report on the average position of the two slow-flipping loops in the EOT ensemble and in the ensemble of fully integrated configurations , respectively .",
"The quantity ϕFI reports on the average position of the two slow-flipping loops in the ensemble of fully integrated configurations at the end of the CG trajectories .",
"For the L85R and nEmrE mutants , the two slow-flipping loops ( L2/L4 and L1/L5 , respectively ) reach positions on the same side of the membrane in either fully integrated topology; for these mutants , ϕFI is defined as the fraction of fully integrated configurations for which both slow-flipping loops are positioned in the cytoplasm .",
"For the K3 and nT28R1 mutants , the two slow-flipping loops ( L1 and L2 ) reach positions on opposite sides of the membrane in either fully integrated topology; for these mutants , ϕFI is defined as the fraction of fully integrated configurations for which L1 is positioned in the cytoplasm and L2 is positioned in the periplasm .",
"For the nEmrE , K3 , and nT28R1 mutants , ϕFI is equivalent to the fraction of CG trajectories that reach the fully integrated Ncyto/Ccyto topology , whereas for the L85R mutant , ϕFI is equivalent to the fraction of CG trajectories that reach the fully integrated Nperi/Cperi topology .",
"The quantity ϕEOT reports on the average position of the two slow-flipping loops in the EOT ensemble .",
"For each mutant , ϕEOT is defined as ( 2 ) ϕEOTL85R=0 . 5[f ( cyto ) L2+f ( cyto ) L4]ϕEOTnEmrE=0 . 5[f ( cyto ) L1+f ( cyto ) L5]ϕEOTK3=0 . 5[1+f ( cyto ) L1−f ( cyto ) L2]ϕEOTnT28R1=0 . 5[1+f ( cyto ) L1−f ( cyto ) L2] , where f ( cyto ) Li is the fraction of configurations in the EOT ensemble for which loop Li is in the cytoplasm .",
"As for the previous definition of ϕFI , this definition accounts for the fact that the two slow-flipping loops of the L85R and nEmrE mutants reach the same side of the membrane in the fully integrated topologies , while the two slow-flipping loops of the K3 and nT28R1 mutants reach opposite sides of the membrane in the fully integrated topologies .",
"The definition in Equation 2 additionally assumes that the post-translational annealing of misintegrated configurations in the EOT ensemble is equally rate-limited by the two slow-flipping loops .",
"Using these alternative definitions for the position of the slowest-flipping loop ( i . e . , ϕFI and ϕEOT ) , Figure 7—figure supplement 1 compares the average position of the slowest-flipping loop in the EOT ensemble to the average position of that same loop in the ensemble of fully integrated configurations .",
"Having more carefully accounted for the effect of both slow-flipping loops in the K3 , L85R , nEmrE , and nT28R1 mutants , this figure reveals a slight improvement in the correlation ( R2 = 0 . 88 vs R2 = 0 . 85 ) in comparison to the results in Figure 7A .",
"Alternative trajectory termination criteria are tested to ensure the robustness of the simulated distribution of multispanning topologies presented in Figure",
"2 . As a first alternative , the original set of CG trajectories are extended by 50 s , and the distribution of topologies is determined from the position of the slowest-flipping loop at the end of the extended trajectories .",
"As a second alternative , the distribution of topologies is calculated from the subset of original CG trajectories that reach fully integrated topologies in which all beads in the TMDs are at least 20σ , rather than 4 . 5σ , from the origin .",
"These robustness checks are presented in Figure 2—figure supplement 1 and exhibit excellent correlation with the results obtained using the original protocol .",
"Additionally , Figure 7B shows results in which the CG trajectories are terminated at a range of fixed times following the end of ribosomal translation .",
"Again , the results using this alternative trajectory termination criterion are in good agreement with the results obtained using the original protocol ( indicated in dashed lines in Figure 7B ) .",
"From the ensemble of CG trajectories , it is possible to examine the pathways by which individual TMDs undergo Sec-facilitated cotranslational integration .",
"In particular , following the definitions of Cymer et al . ( 2014 ) , it is possible to characterize each cotranslational TMD integration event as corresponding to either the ‘channel-sliding’ , ‘interface-sliding’ , or ‘in-out’ pathways .",
"Simulation snapshots illustrating the three pathways are shown in Figure 3—figure supplement",
"1 . Each pathway is defined in terms of the series of intermediate states that are visited by the TMD prior to membrane integration .",
"To characterize these intermediate states , the following geometric regions are defined ( see Figure 1—figure supplement 2 ) .",
"The channel region is defined as that for which −2σ ≤ x ≤ 2σ and −2σ ≤ y ≤ 2σ , the membrane region is defined as that for which −2σ ≤ x ≤ 2σ and y > 2σ , the ribosome region is defined as that for which −11σ ≤ x < −2σ and −8 . 5σ ≤ y ≤ 4 . 5σ , and the cytoplasm region is defined as the region outside of the ribosome for which x < −2σ .",
"Finally , a bead is considered to overlap the LG if it is within a distance of σ to any lateral-gate bead .",
"We now define the four intermediate states .",
"Intermediate state 1 ( IS1 ) is that for which the TMD partially enters the channel; it is defined as the set of configurations for which at least two TMD beads are in the channel region and zero TMD beads are in the membrane region .",
"Intermediate state 2 ( IS2 ) is that for which the TMD fully spans the membrane while in the channel; it is defined as the set of configurations for which all four TMD beads are in the channel region and the two hydrophilic beads that flank the TMD occupy opposite sides of the membrane .",
"Intermediate state 3 ( IS3 ) is that for which the TMD accesses the membrane interior via the LG; it is defined as the set of configurations for which at least one TMD bead occupies the membrane region , the remaining three TMD beads occupy either the channel or membrane regions , and at least one TMD bead overlaps with the LG .",
"Intermediate state 4 ( IS4 ) is that for which the TMD accesses the cytoplasm region without accessing the channel region; it is defined as the set of configurations for which each of the four TMD beads occupies either the ribosome , membrane , or cytoplasm regions and for which at least one of the hydrophilic beads that flank the TMD occupies the cytoplasm region .",
"In this analysis , cotranslational TMD integration events are defined as those for which the TMD reaches a membrane integrated configuration ( for which all four beads of the TMD span the membrane region and the two hydrophilic flanking beads occupy opposite sides of the membrane ) before reaching a misintegrated configuration ( for which both hydrophilic flanking beads occupy the same side of the membrane and for which all TMD beads and both flanking beads lie outside of the channel and ribosome regions ) .",
"Using the definitions of intermediate states , the cotranslational integration pathways are defined as follows .",
"In the ‘channel-sliding’ pathway , the TMD partially enters the channel , then crosses the LG , then fully integrates into the membrane; a trajectory thus exhibits this pathway if a TMD visits IS1 , IS2 , and membrane integration in chronological order and without visiting any other intermediate states .",
"In the ‘interface-sliding’ pathway , the TMD enters the cytoplasm through the gap between the translocon and ribosome , prior to undergoing membrane integration; a trajectory thus exhibits this pathway if a TMD visits IS4 on the way to membrane integration .",
"In the ‘in-out’ pathway , the TMD fully spans the channel prior to membrane integration; a trajectory thus exhibits this pathway if a TMD visits IS3 on the way to membrane integration without visiting IS4 .",
"At right , Figure 3—figure supplement 1 shows the relative fraction of cotranslational TMD integration events that exhibit each of these three pathways .",
"It is clear that the dominant cotranslational integration pathway for all four TMDs in both the EmrE and nEmrE mutants is the ‘channel-sliding’ pathway .",
"This same pathway was also observed in the previous study of single-spanning proteins using the CG model ( Zhang and Miller , 2012a ) and similar configurations were observed in long-timescale atomistic molecular dynamics simulations of the early stages of cotranslational membrane integration ( Zhang and Miller , 2012b ) .",
"We find that only a small number of CG trajectories exhibit the ‘interface-sliding’ pathway .",
"Finally , we note that the dominant cotranslational integration pathway is likely to depend on the IMP sequence , and the ‘channel-sliding’ behavior may be less dominant in other IMPs with less hydrophobic TMDs ."
]
] | [
"The canonical mechanism for multispanning membrane protein topogenesis suggests that protein topology is established during cotranslational membrane integration .",
"However , this mechanism is inconsistent with the behavior of EmrE , a dual-topology protein for which the mutation of positively charged loop residues , even close to the C-terminus , leads to dramatic shifts in its topology .",
"We use coarse-grained simulations to investigate the Sec-facilitated membrane integration of EmrE and its mutants on realistic biological timescales .",
"This work reveals a mechanism for regulating membrane-protein topogenesis , in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies .",
"The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins .",
"The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis ."
] | [
"Proteins are long chains of smaller molecules called amino acids , and are built inside cells by a molecular machine called the ribosome .",
"Many important proteins must be inserted into the membrane that surrounds each cell in order to carry out their role .",
"As these proteins are being built by the ribosome , they thread their way into a membrane-spanning channel ( called the translocon ) from the inner side of the membrane .",
"Short segments of these integral membrane proteins ( called transmembrane domains ) then become embedded in the membrane , while other parts of the protein remain on either side of the membrane .",
"For a membrane protein to work properly , the end of each of its transmembrane domains must be on the correct side of the membrane ( i . e . , the protein must obtain the correct ‘topology’ ) .",
"The conventional model for this process suggests that topology is fixed when the first transmembrane domain of a protein is initially integrated into the membrane , while the ribosome is still building the protein .",
"This model can explain most integral membrane proteins , which only have a single topology .",
"However , it cannot explain the family of membrane proteins that have an almost equal chance of adopting one of two different topologies ( so-called ‘dual-topology proteins’ ) .",
"Van Lehn et al . have now used computer modeling to simulate how a bacterial protein called EmrE ( which is a dual-topology protein ) integrates into the membrane via the translocon .",
"The results reveal that a few transmembrane domains in EmrE do not fully integrate into the membrane while the ribosome is building the protein .",
"Instead , these transmembrane domains slowly integrate after the ribosome has finished its job .",
"These findings contradict the conventional model and suggest that some membrane proteins only become fully integrated after the protein-building process is complete .",
"The next step in this work is to experimentally test predictions from the computer simulations ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression"
] | The tardigrade damage suppressor protein binds to nucleosomes and protects DNA from hydroxyl radicals | elife-47682-v1 | [
[
"Tardigrades , which are also known as water bears or moss piglets , are small invertebrate animals that are found in marine , freshwater , and terrestrial habitats throughout the Earth ( reviewed in Guidetti et al . , 2012; Møbjerg et al . , 2011; Weronika and Łukasz , 2017 ) .",
"They are typically about 0 . 1 to 1 mm in length , and comprise a head segment in addition to four body segments that each contains two legs with claws .",
"Terrestrial tardigrades require a thin film of water to remain active .",
"In the absence of water , they undergo anhydrobiosis into a dormant dehydrated state from which they can be rehydrated to an active form .",
"In the anhydrobiotic state , tardigrades are resistant to extreme conditions of heat , cold , vacuum , pressure , radiation , and chemical treatments .",
"Remarkably , they have been found to survive exposure to the vacuum and radiation of outer space ( Jönsson et al . , 2008 ) .",
"Thus , the unique properties of tardigrades have led to considerable interest in these animals .",
"The analysis of their singular features should lead to significant new biological insights .",
"The molecular analysis of tardigrades has been advanced by the sequencing of the genomes of Ramazzottius varieornatus ( Hashimoto et al . , 2016 ) and Hypsibius exemplaris ( Yoshida et al . , 2017 ) .",
"[Note: the strain of tardigrades that was designated as Hypsibius dujardini in Yoshida et al . ( 2017 ) has since been found to be a new species that is now termed Hypsibius exemplaris ( GĄsiorek et al . , 2018 ) . We will use the new terminology in this paper . ] In R . varieornatus , the study of chromatin-associated factors revealed a tardigrade-specific protein , termed Dsup , for damage suppressor ( Hashimoto et al . , 2016; Hashimoto and Kunieda , 2017 ) .",
"Dsup is a highly charged and largely unstructured nuclear protein that binds to DNA .",
"Intriguingly , the expression of Dsup gene in human cells was observed to decrease DNA fragmentation induced by X-ray irradiation or treatment with hydrogen peroxide .",
"Moreover , Dsup-containing human cells exhibited higher viability after X-ray irradiation than control cells .",
"These findings suggest that Dsup is a unique protein that either directly or indirectly protects DNA .",
"In this work , we sought to examine the molecular function of Dsup .",
"It was previously shown that Dsup interacts with free DNA in an apparently nonspecific manner ( Hashimoto et al . , 2016 ) .",
"Because the natural form of DNA in the nucleus is chromatin , we investigated the binding of Dsup to nucleosomes .",
"These experiments revealed that R . varieornatus Dsup binds preferentially to nucleosomes relative to free DNA .",
"Then , to test the relevance of these findings to other tardigrades , we examined the properties of a Dsup-like protein from H . exemplaris , and found that this protein appears to be an ortholog of R . varieornatus Dsup .",
"Intriguingly , the Dsup proteins contain a region with sequence similarity to core consensus of the nucleosome-binding domain of vertebrate high mobility group N ( HMGN ) proteins , and the HMGN-like sequence in Dsup is important for its binding to nucleosomes .",
"Furthermore , in a purified biochemical system , both Dsup proteins are able to protect chromatin from cleavage by hydroxyl radicals , which are generated in cells by ionizing radiation as well as by treatment with hydrogen peroxide .",
"These studies thus reveal conserved functions by which Dsup maintains the integrity of chromosomal DNA under extreme conditions ."
],
[
"The R . varieornatus Dsup protein is a largely disordered and highly charged nuclear protein ( Figure 1A ) ( Hashimoto et al . , 2016; Hashimoto and Kunieda , 2017 ) .",
"To study its biochemical properties , we synthesized a FLAG- and His6-tagged version of the full-length protein in Escherichia coli and purified it to greater than 95% homogeneity by affinity chromatography ( Figure 1B ) .",
"We will refer to the R . varieornatus Dsup protein as ‘Rv Dsup’ .",
"Although Rv Dsup was observed to associate with free DNA ( Hashimoto et al . , 2016 ) , we tested the binding of Rv Dsup to nucleosomes , which are the natural form of DNA in the eukaryotic nucleus .",
"To this end , we reconstituted mononucleosomes with the 5S rDNA nucleosome positioning sequence from Xenopus borealis ( Rhodes , 1985; Hayes et al . , 1990; Fei et al . , 2018 ) .",
"We compared the binding of Rv Dsup to 147 bp DNA-containing mononucleosomes relative to the corresponding 147 bp free DNA by gel mobility shift analysis ( Figure 2A and Figure 2—figure supplement 1A ) .",
"These experiments revealed that Rv Dsup binds with a higher affinity to nucleosomes than to free DNA .",
"To test whether this effect is specific for the 5S rDNA sequence , we examined the binding of Rv Dsup to mononucleosomes and free DNA that contain the 601 nucleosome positioning sequence ( Lowary and Widom , 1998 ) .",
"As with the 5S rDNA nucleosomes , we observed more efficient binding of Rv Dsup to the 601 mononucleosomes than to the 601 free DNA ( Figure 2B and Figure 2—figure supplement 1B ) .",
"Thus , Rv Dsup binds preferentially to mononucleosomes relative to free DNA in a manner that appears to be independent of the specific DNA sequence .",
"Because the 147 bp DNA-containing mononucleosomes lack linker DNA that extends beyond the nucleosome core , we compared the binding of Rv Dsup to mononucleosomes that contain either 147 bp DNA or 181 bp DNA ( Figure 2C ) .",
"These experiments showed that the presence of linker DNA did not substantially alter the efficiency of Rv Dsup binding to nucleosomes .",
"It thus appears that Rv Dsup binds primarily to the nucleosome core rather than to the linker DNA .",
"In the gel shift experiments with Rv Dsup and mononucleosomes , there is typically a distinct major shifted band ( denoted by black dots in Figure 2 ) along with a minor faster migrating band .",
"This pattern can be seen particularly clearly in the titration of Rv Dsup with 147 bp 5S rDNA mononucleosomes in Figure 2C , and is suggestive of the binding of two molecules of Rv Dsup to the nucleosome .",
"With the 5S rDNA mononucleosomes and high concentrations of Rv Dsup , we also observed a slower migrating band ( Figure 2A ) , which may reflect additional Rv Dsup binding to the nucleosomes and/or higher-order aggregation of the Rv Dsup-nucleosome complexes .",
"In contrast , a distinct shifted band is not seen with Rv Dsup and free DNA .",
"To complement the studies with mononucleosomes , we tested whether Rv Dsup can be incorporated into extended periodic nucleosome arrays .",
"In these experiments , we assembled nucleosomes onto plasmid DNA by using a purified and defined ATP-dependent chromatin assembly system with the ACF motor protein and the dNLP core histone chaperone ( Fyodorov and Kadonaga , 2003; Khuong et al . , 2017 ) .",
"We also included separate reactions with histone H1 ( reviewed in Woodcock et al . , 2006; Happel and Doenecke , 2009; Kalashnikova et al . , 2016 ) as a positive control for a nucleosome-binding factor .",
"The presence of Rv Dsup does not inhibit the efficiency of ACF-mediated chromatin assembly , as measured by the DNA supercoiling assay ( Figure 3A ) .",
"Partial MNase digestion analysis showed that the assembly of chromatin with Rv Dsup results in a small but distinct increase in the nucleosome repeat length ( Figure 3B ) .",
"To analyze the binding of Rv Dsup to the nucleosome arrays , we extensively digested the ACF-assembled chromatin with MNase , and subjected the resulting mononucleosome species to nondenaturing gel electrophoresis ( Figure 3C ) .",
"This experiment revealed that Rv Dsup-bound mononucleosome particles are released from the chromatin upon extensive MNase digestion .",
"We then compared the Rv Dsup-mononucleosome particles that were generated via ACF assembly followed by MNase digestion , as in Figure 3C , with the Rv Dsup-mononucleosome particles formed by the addition of Rv Dsup to mononucleosomes , as in Figure 2A , and found that the differently prepared Rv Dsup-nucleosome particles migrate at approximately the same rate during nondenaturing gel electrophoresis ( Figure 3D ) .",
"It therefore appears that the interaction of Rv Dsup with nucleosomes in periodic arrays , as in Figure 3 , is similar to the binding of Rv Dsup to mononucleosome particles , as in Figure 2 .",
"Histone H1 is a major nucleosome-binding protein in animals , and it has been found to be present at approximately one molecule per nucleosome ( Bates and Thomas , 1981 ) ( reviewed in Woodcock et al . , 2006; Happel and Doenecke , 2009; Kalashnikova et al . , 2016 ) .",
"We were therefore interested in testing whether histone H1 and Rv Dsup can bind simultaneously to nucleosomes .",
"To this end , we carried out gel mobility shift analyses with 181 bp DNA mononucleosomes in the presence of varying concentrations of Rv Dsup and histone H1 ( Figure 4A ) .",
"( We used 181 bp DNA mononucleosomes to allow the binding of histone H1 to the linker DNA . )",
"These experiments revealed that the addition of Rv Dsup to H1-bound mononucleosomes results in the formation of a slower migrating Rv Dsup-H1-nucleosome complex .",
"Likewise , the addition of histone H1 to Rv Dsup-bound mononucleosomes yields Rv Dsup-H1-nucleosome complexes that migrate at the same rate as the species formed upon addition of Rv Dsup to H1-bound nucleosomes .",
"These results indicate that histone H1 and Rv Dsup can bind simultaneously to mononucleosomes .",
"We then examined whether the presence of both histone H1 and Rv Dsup results in a disruption of chromatin structure .",
"To address this question , we carried out ACF-mediated chromatin assembly reactions in the presence or absence of histone H1 and/or Rv Dsup .",
"These experiments revealed that the addition of both H1 and Rv Dsup did not alter the efficiency of chromatin assembly , as measured by the DNA supercoiling assay ( Figure 4B ) .",
"Furthermore , the presence of histone H1 and Rv Dsup did not disrupt the periodicity of the nucleosome arrays , as assessed with the partial MNase digestion assay ( Figure 4C ) .",
"Thus , histone H1 and Rv Dsup can bind simultaneously to nucleosomes , and this binding does not substantially alter or disrupt chromatin structure .",
"These findings are consistent with the observation that Rv Dsup is not deleterious to histone H1-containing human cells ( Hashimoto et al . , 2016 ) .",
"It was also important to assess whether Dsup is present in tardigrades other than R . varieornatus .",
"In this regard , we were interested in H . exemplaris , a tardigrade that was recently subjected to genome resequencing ( Yoshida et al . , 2017 ) .",
"R . varieornatus and H . exemplaris are limnoterrestrial tardigrades in the family Hypsibiidae , and both animals can undergo anhydrobiosis ( see , for example , Horikawa et al . , 2008; Kondo et al . , 2015; Wright , 1989 ) .",
"Dsup was initially not identified in H . exemplaris ( Yoshida et al . , 2017 ) , but further analysis revealed a Dsup-like protein in this organism ( Hashimoto and Kunieda , 2017 ) .",
"The H . exemplaris Dsup-like protein was found to have 26 . 4% amino acid identity with Rv Dsup as well as hydrophobicity and charge profiles that are similar to those of Rv Dsup .",
"To investigate the evolutionary relationship between these proteins , we examined the chromosomal regions that encompass the Dsup-related genes in R . varieornatus and H . exemplaris .",
"This analysis revealed that the Dsup-related genes in both organisms are flanked by the same neighboring genes ( Figure 5A ) .",
"Hence , based on the similarity of the proteins , including the closely related nuclear localization signals near the C-termini ( Hashimoto and Kunieda , 2017 ) , and the similarity of the gene arrangements ( Figure 5A ) , it appeared likely that the H . exemplaris Dsup-like protein and R . varieornatus Dsup are homologs .",
"It remained to be determined , however , whether the two proteins have related biochemical functions .",
"To this end , we synthesized and purified the H . exemplaris Dsup-like protein , which we will refer to as ‘He Dsup’ ( Figure 5B ) .",
"We then tested the binding of He Dsup to mononucleosomes and to free DNA , and found that He Dsup binds preferentially to nucleosomes relative to free DNA ( Figure 5C ) , as does Rv Dsup ( Figure 2 ) .",
"In addition , we directly compared the binding of He Dsup and Rv Dsup to mononucleosomes , and observed that each of the proteins shifts mononucleosomes to approximately the same position on the gel ( Figure 5D ) .",
"The slower migration of the Rv Dsup-nucleosome complexes relative to that of the He Dsup-nucleosome complexes is consistent with the larger size of Rv Dsup ( 445 amino acid residues ) compared to He Dsup ( 328 amino acid residues ) .",
"Therefore , in the analysis of Rv Dsup and He Dsup , the relationship between the proteins ( Hashimoto and Kunieda , 2017 ) , the similarity of the gene arrangements ( Figure 5A ) , and the conserved biochemical function ( Figures 2 , 5C and D ) lead to the conclusion that the two proteins are orthologs .",
"An intriguing property of Dsup is its ability to protect DNA in human cells from degradation that is induced either by X-ray irradiation or by treatment with hydrogen peroxide ( Hashimoto et al . , 2016 ) .",
"Because ionizing radiation and hydrogen peroxide both generate hydroxyl radicals as a major reactive oxygen species in cells ( Halliwell and Gutteridge , 1992; Stadtman , 1993; Riley , 1994 ) , we investigated whether purified Dsup affects hydroxyl radical-mediated DNA cleavage in a purified and defined biochemical system .",
"To this end , we carried out hydroxyl radical-mediated DNA cleavage reactions in the absence or presence of Dsup .",
"In these experiments , we generated hydroxyl radicals by the Udenfriend modification of the Fenton reaction ( Udenfriend et al . , 1954; Tullius et al . , 1987 ) .",
"Each of the hydroxyl radical reactions was carried out under the same conditions .",
"In the absence of Dsup , the 3 . 3 kb plasmid DNA was mostly degraded to DNA fragments ranging from 100 to 1000 nt ( Figure 6A ) .",
"Notably , the extent of cleavage of free plasmid DNA was nearly the same as the amount of cleavage of chromatin .",
"Thus , the presence of approximately one nucleosome per 200 bp DNA only slightly protects the DNA from hydroxyl radicals .",
"These results are consistent with the previous observation that hydroxyl radicals can cleave DNA in nucleosomes ( Hayes et al . , 1990 ) .",
"Upon addition of Rv Dsup , we observed substantial protection of free DNA as well as chromatin from hydroxyl radicals ( Figure 6A ) .",
"The Dsup-mediated protection was stronger with chromatin than with free DNA , possibly because the specific binding of Dsup to nucleosomes creates a highly resistant structure .",
"In the presence of 4 molecules of Rv Dsup per 200 bp DNA ( approximately 4 Dsup molecules per nucleosome ) in chromatin , there was a considerable amount of full-length linear DNA remaining after hydroxyl radical-mediated cleavage .",
"These results indicate that Rv Dsup is able to protect chromatin from the damaging effects of hydroxyl radicals .",
"Because hydroxyl radicals react primarily with hydrogen atoms that are exposed in the minor groove of DNA ( Balasubramanian et al . , 1998 ) , it appears likely that Dsup blocks access to the minor groove .",
"We additionally tested whether He Dsup and human TFIIB ( a nuclear protein that does not bind to nucleosomes ) can protect chromatin from hydroxyl radical-mediated DNA cleavage ( Figure 6B ) .",
"These experiments revealed that He Dsup , like Rv Dsup , is able to protect nucleosomal DNA from hydroxyl radical-mediated cleavage , and further support the conclusion that He Dsup is an ortholog of Rv Dsup .",
"As seen in Figure 6B , we consistently observed that Rv Dsup provides slightly more protection against hydroxyl radicals than He Dsup .",
"In contrast to Dsup , TFIIB did not confer any detectable protection of chromatin from hydroxyl radical-mediated DNA cleavage .",
"Thus , these findings collectively indicate that Dsup binds to nucleosomes and protects DNA from cleavage by hydroxyl radicals ( Figure 6C ) .",
"The homology between Rv Dsup and He Dsup led us to examine whether any of their conserved regions are related to protein sequences in organisms other than tardigrades .",
"This analysis led to the identification of sequence similarity between a segment in the C-terminal region of the Dsup proteins and a conserved sequence in the nucleosome-binding domain of HMGN proteins ( Figure 7A ) .",
"The HMGN proteins are abundant nucleosome-binding proteins that have been found only in vertebrates ( for reviews , see: Kugler et al . , 2012; González-Romero et al . , 2015 ) .",
"They bind to two high affinity sites on nucleosomes in a manner that is independent of the DNA sequence .",
"The nucleosome-binding domain of the HMGN proteins has been identified , and it contains a conserved core sequence , RRSARLSA , which is critical for the binding of HMGN proteins to nucleosomes ( Ueda et al . , 2008 ) .",
"Strikingly , both Dsup proteins contain a sequence that is similar to the RRSARLSA consensus ( Figure 7A ) .",
"To examine whether the HMGN-like sequence in the Dsup proteins is important for their binding to nucleosomes , we generated two mutant versions of Rv Dsup ( Figure 7A ) .",
"The M1 mutant Dsup is a C-terminal deletion that removes the HMGN-like region , and the M2 mutant Dsup contains three R to E substitution mutations in the HMGN-like region .",
"We purified the mutant Dsup proteins ( Figure 7B ) and then tested their ability to bind to mononucleosomes by gel mobility shift analysis ( Figure 7C ) .",
"These experiments revealed that the M1 mutant Dsup is nearly completely defective for binding to nucleosomes .",
"Hence , the C-terminal region of Rv Dsup ( from amino acid residues 360 to 445 ) is essential for nucleosome binding .",
"In addition , the M2 mutant Dsup exhibits reduced binding to nucleosomes .",
"Thus , the HMGN-like sequence in Dsup is important for its binding to nucleosomes .",
"We further investigated the ability of the mutant Dsup proteins to protect chromatin from hydroxyl radicals .",
"These experiments revealed that the nucleosome-binding-deficient M1 mutant Dsup is nearly completely defective for protection of chromatin against hydroxyl radicals .",
"We also observed that the M2 mutant Dsup is partially defective for protection against hydroxyl radicals .",
"These data support the model that the binding of Dsup to nucleosomes is required to protect chromatin from hydroxyl radical-mediated DNA damage ."
],
[
"Here we have found that Dsup , a tardigrade-specific factor , is a nucleosome-binding protein that protects chromosomal DNA from hydroxyl radical-mediated cleavage .",
"These findings provide a molecular explanation for the ability of Dsup to protect DNA in human cells from degradation by ionizing radiation or by treatment with hydrogen peroxide ( Hashimoto et al . , 2016 ) .",
"More generally , these results suggest that Dsup protects the tardigrade genome from hydroxyl radical-mediated damage under diverse conditions .",
"For instance , protection of the tardigrade genome from hydroxyl radicals may contribute to survival after extended periods in the anhydrobiotic state ( Wełnicz et al . , 2011 ) .",
"Notably , Dsup and histone H1 can bind simultaneously to nucleosomes ( Figure 4A ) .",
"Both R . varieornatus and H . exemplaris appear to contain histone H1 , and it is thus likely that Dsup normally acts in conjunction with H1 .",
"In this regard , it is also notable that Dsup functions in histone H1-containing human cells ( Hashimoto et al . , 2016 ) .",
"Rv Dsup and He Dsup are orthologs .",
"In addition to their sequence similarity and related gene arrangements ( Figure 5A ) , both Rv Dsup and He Dsup are nucleosome-binding proteins that protect chromatin from hydroxyl radicals ( Figures 5 and 6 ) .",
"It had been previously suggested that Dsup is not present in H . exemplaris ( Yoshida et al . , 2017 ) .",
"If true , that would have implied that Dsup is not generally important for tardigrades .",
"Instead , the identification of Dsup as a chromatin-protective protein in two species suggests that it is a key factor that contributes to the unique properties of tardigrades .",
"Unexpectedly , the Dsup proteins contain a region that exhibits sequence similarity to the conserved core of the nucleosome-binding domain of HMGN proteins ( Figure 7A ) .",
"Moreover , the HMGN-like sequence in Rv Dsup is functionally important for its ability to bind to nucleosomes ( Figure 7C ) and to protect chromatin from hydroxyl radicals ( Figure 7D ) .",
"Because the HMGN proteins have been found only in vertebrates ( González-Romero et al . , 2015 ) , the origin of the HMGN-like sequences in the tardigrade Dsup proteins is , at present , a mystery .",
"It is possible but not likely ( see , for example , Doolittle , 1994 ) that the Dsup and HMGN proteins provide a very rare example of sequence convergence .",
"In the future , detailed functional and evolutionary analyses might reveal the relation between these proteins .",
"The Dsup proteins are highly charged and largely disordered ( Hashimoto et al . , 2016; Hashimoto and Kunieda , 2017 ) ( Figure 1A ) .",
"With respect to the latter , examination of the amino acid composition of the Dsup proteins reveals that they are enriched in serine , alanine , glycine , and lysine ( SAGK ) residues .",
"Rv Dsup contains more than 60% SAGK residues , and He Dsup has over 50% SAGK residues .",
"SAGK are disorder-promoting amino acids ( Dunker et al . , 2001 ) , and the SAGK residues may form a diffuse mass of protein that protects the chromosomal DNA from hydroxyl radical-mediated cleavage in a variety of conditions ( Figure 6C ) .",
"This model is consistent with the observation that tardigrades can survive high doses of ionizing radiation in the active as well as anhydriobiotic states ( see , for example , Jönsson et al . , 2005; Horikawa et al . , 2006 ) .",
"In conclusion , these studies reveal molecular aspects of Dsup function and suggest how this intriguing protein may contribute to the unique biological properties of tardigrades .",
"A direct mechanism can be envisaged in which Dsup binds specifically to nucleosomes and protects the DNA from damaging agents such as hydroxyl radicals via coverage of the chromatin with its SAGK-rich disordered regions .",
"In the future , the analysis of Dsup will reveal new insights into the physical and molecular bases of its functions and might further lead to its practical applications .",
"For instance , it is possible that the DNA-protective properties of Dsup could be used to extend the longevity of cells .",
"Thus , this unique protein from an extreme organism could be a new and powerful reagent in biological research ."
],
[
"The cDNA sequence for Rv Dsup was synthesized with an N-terminal His6 tag and a C-terminal FLAG tag ( Integrated DNA Technologies; San Diego , CA ) .",
"The resulting DNA fragment was subcloned into pET21b to give pET21b-His6-Dsup-FLAG .",
"For expression , freshly transformed Escherichia coli strain BL21 ( DE3 ) was grown in LB medium ( 2 L volume; 40 µg/mL ampicillin ) at 37°C to A600 nm of approximately 0 . 6 to 0 . 8 .",
"The synthesis of Dsup was induced by the addition of IPTG to 0 . 4 mM final concentration , and the culture was incubated at 18°C for 16 to 18 hr .",
"The bacteria were collected by centrifugation ( Fiberlite F9−4×1000y rotor; 6 , 000 rpm; 10 min; 18°C ) .",
"Until stated otherwise , the next series of operations were carried out at room temperature ( 22°C ) .",
"The pellet was resuspended in 20 mL of Denaturing Binding Buffer [20 mM sodium phosphate , pH 7 . 8 , containing 8 M urea , 0 . 5 M NaCl , 5 mM 2-mercaptoethanol , 0 . 4 mM PMSF] .",
"The cells were lysed with 3 × 15 strokes in a Wheaton Dounce homogenizer ( B pestle ) , with a 10 min interval between each set of 15 strokes .",
"The mixture was incubated for 1 hr and then subjected to centrifugation ( Fiberlite F21S-8×50y rotor; 15 , 000 rpm; 20 min ) .",
"The supernatant was collected and stored , and the pellet was resuspended with 15 mL of Denaturing Binding Buffer .",
"The suspended pellet was dispersed with 15 strokes in a Wheaton Dounce homogenizer ( B pestle ) , incubated for 1 hr , and subjected to centrifugation ( Fiberlite F21S-8×50y rotor; 15 , 000 rpm; 20 min ) .",
"The second supernatant was combined with the first supernatant , and the resulting mixture was incubated with 3 mL of Ni-NTA agarose beads ( Qiagen; Germantown , MD ) for 2 hr on a rotating wheel at 4°C .",
"At room temperature ( 22°C ) , the mixture was transferred into a Bio-Rad Econo-Pac chromatography column , and the resin was washed with 2 × 10 mL of Denaturing Wash Buffer [20 mM sodium phosphate , pH 6 . 0 , containing 8 M urea , 0 . 5 M NaCl , 5 mM 2-mercaptoethanol , 0 . 4 mM PMSF] .",
"The protein was eluted with Denaturing Elution Buffer [20 mM sodium phosphate , pH 4 . 0 , containing 8 M urea , 0 . 5 M NaCl , 5 mM 2-mercaptoethanol , 0 . 4 mM PMSF] in 5 × 1 mL steps , with a 2 min interval between the addition of each mL of elution buffer .",
"Unless stated otherwise , all subsequent operations were performed at 4°C .",
"The eluate fractions were combined and dialyzed overnight in 4 L of PBS , and then incubated on a rotator wheel with 0 . 5 mL of anti-FLAG M2 Affinity Gel ( Sigma-Aldrich; St . Louis , MO ) for 3 to 4 hr .",
"The beads were washed with 3 × 0 . 5 mL of Buffer A [20 mM Tris-HCl , pH 7 . 9 , containing 150 mM NaCl , 2 mM MgCl2 , 0 . 2 mM EDTA , 15% ( v/v ) glycerol , 1 mM DTT] , and protein was eluted with 4 × 0 . 1 mL portions of Buffer B [Buffer A containing 0 . 4 mg/mL FLAG peptide ( Sigma-Aldrich; St . Louis , MO ) and 0 . 5 mg/mL recombinant human insulin] .",
"The protein fractions were frozen in liquid nitrogen and stored at −80°C .",
"The Rv Dsup that was used in Figures 1–4 was purified by this method .",
"The nondenaturing method works very well for the purification of wild-type and mutant Dsup proteins from R . varieornatus and H . exemplaris .",
"Here , we describe the synthesis and purification of He Dsup .",
"The E . coli codon-optimized cDNA sequence for He Dsup was synthesized with a C-terminal FLAG tag ( Integrated DNA Technologies; San Diego , CA ) and inserted as an NcoI-XhoI fragment into pET21b-His6-Nco ( Kassavetis et al . , 1998 ) .",
"The resulting plasmid was transformed into Rosetta pLysS ( MilliporeSigma ) .",
"The cells were grown at 37°C in LB medium ( 0 . 5 L; 80 and 34 µg/mL , ampicillin and chloramphenicol , respectively ) to an A600 nm of 0 . 8 , induced with IPTG to 0 . 5 mM final concentration , grown for an additional 2 hr , and harvested by centrifugation ( Fiberlite F14S-6×250y rotor; 10 , 000 rpm; 15 min; 4°C ) .",
"Unless stated otherwise , the remaining operations were carried out at 4°C .",
"The cells ( 1 . 8 g ) were resuspended in 12 . 6 mL Lysis Buffer [40 mM potassium phosphate , pH 7 . 6 , 0 . 01% ( v/v ) NP-40 ( Pierce ) , 0 . 1 mM EDTA , 580 mM NaCl , 10% ( v/v ) glycerol , 10 mM 2-mercaptoethanol , 1 µg/mL leupeptin , 1 µg/mL pepstatin , 0 . 5 mM PMSF , and 300 µg/mL lysozyme] , incubated for 30 min on ice , sonicated ( Branson Sonifier 450 with a 0 . 25 inch microtip; 20% output; 10 cycles of sonication of 13 s each; kept below 6°C by chilling in an ice-ethanol bath ) , and clarified by centrifugation ( Fiberlite F21S-8×50y rotor; 18 , 000 rpm; 30 min ) .",
"Dsup was purified from the soluble lysate supernatant fraction on a 0 . 5 mL Ni-NTA agarose column that was washed with 8 mL Buffer E [40 mM potassium phosphate , pH 7 . 6 , 20 mM imidazole , 500 mM NaCl , 10% ( v/v ) glycerol , 10 mM 2-mercaptoethanol , 0 . 5 mM PMSF] , pre-eluted with 300 µL Buffer E containing 200 mM imidazole , and eluted with 700 µL of the same buffer ( yielding 5 . 8 mg/mL He Dsup and 3 . 6 mg/mL Rv Dsup ) .",
"The samples were frozen in liquid nitrogen and stored at −80°C .",
"For further purification , a portion of each sample ( approximately 0 . 1 mg Dsup ) was loaded onto a 300 µL anti-FLAG M2 agarose column that was pre-equilibrated in Buffer A2 [20 mM Tris-HCl , pH 7 . 9 , containing 150 mM NaCl , 2 mM MgCl2 , 0 . 2 mM EDTA , 15% ( v/v ) glycerol , 1 mM 2-mercaptoethanol] .",
"The column was left undisturbed for 5 min and washed with 2 mL Buffer A2 .",
"The Dsup was eluted by the addition of 250 µL ( to give the pre-elution fraction ) followed by 300 µL ( elution fraction ) Buffer A2 containing 300 µg/mL 3XFLAG peptide ( ApexBio Technology ) .",
"The samples were frozen in liquid nitrogen and stored at −80°C .",
"The preparations of He Dsup and Rv Dsup in Figures 6 and 7 were prepared in this manner .",
"The method of preparation described in this paragraph is the most efficient means of obtaining high yields of pure Dsup proteins .",
"Alternate denaturing method .",
"We also purified Dsup from the pellet of the bacterial cell lysate ( prepared as described in the preceding paragraph ) as follows .",
"The pellet was resuspended in Buffer PG [40 mM potassium phosphate , pH 7 . 6 , 20 mM imidazole , 6 M guanidine HCl , 10% ( v/v ) glycerol , 10 mM 2-mercaptoethanol , 1 µg/mL leupeptin , 1 µg/mL pepstatin , 0 . 5 mM PMSF] at room temperature ( 22°C ) , sonicated ( Branson Sonifier 450 with a 0 . 25 inch microtip; 20% output; 4 cycles of sonication of 10 s each; kept below 6°C by chilling in an ice bath ) , and clarified by centrifugation ( Fiberlite F21S-8×50y rotor; 18 , 000 rpm; 15 min; 4°C ) .",
"The supernatant was loaded onto a 1 mL Ni-NTA agarose column , which was then washed with 11 mL Buffer E containing 6 M urea .",
"The Dsup was renatured on the column with sequential 1 . 2 mL washes of Buffer E containing 5 . 0 , 4 . 5 , 4 . 0 , 3 . 5 , 3 . 0 , 2 . 5 , 2 . 0 , 1 . 5 , 1 . 0 and 0 M urea .",
"The column was washed with 0 . 8 mL Buffer E containing 200 mM imidazole to give the pre-elution fraction , and protein was eluted with 1 . 3 mL of the same buffer to give the elution fraction ( containing approximately 1 mg/mL Dsup ) .",
"A portion of the elution fraction ( approximately 0 . 1 mg Dsup ) was loaded onto a 300 µL anti-FLAG M2 agarose column that was pre-equilibrated in Buffer A2 [20 mM Tris-HCl , pH 7 . 9 , containing 150 mM NaCl , 2 mM MgCl2 , 0 . 2 mM EDTA , 15% ( v/v ) glycerol , 1 mM 2-mercaptoethanol] .",
"The column was left undisturbed for 5 min and washed with 2 mL buffer A2 .",
"The Dsup was eluted by the addition of 250 µL ( to give the pre-elution fraction ) followed by 300 µL ( elution fraction; 100 µg yield ) Buffer A2 containing 300 µg/mL 3XFLAG peptide ( ApexBio Technology ) .",
"The protein fractions were frozen in liquid nitrogen and stored at −80°C .",
"The Dsup proteins that were used in the gel mobility shift assays in Figures 5 , 6 and 7 were prepared by the method described in this section .",
"It might be noted that Dsup proteins are hydrophilic and highly-charged proteins that bind weakly to SDS and thus migrate slowly on SDS-polyacrylamide gels relative to ‘average’ proteins such as the molecular mass markers .",
"To confirm the authenticity of the Dsup proteins , we subjected wild-type R . varieornatus Dsup and wild-type H . exemplaris Dsup to LC-ESI-TOF-MS ( liquid chromatography-electrospray ionization time-of-flight mass spectrometry ) .",
"For each of the two proteins , the observed mass was within one mass unit of the predicted ( calculated ) mass .",
"Thus , the Dsup proteins appear to be authentic .",
"Mononucleosomes were reconstituted by using the salt dialysis method ( Stein , 1989; Fei et al . , 2015 ) with purified recombinant Drosophila core histones ( Levenstein and Kadonaga , 2002; Khuong et al . , 2017 ) and the following DNA fragments .",
"The 147 bp Xenopus borealis 5S rDNA fragment corresponds to the nucleosome positioning sequence that was mapped in Fei et al . ( 2018 ) .",
"The sequence of the longer 181 bp X . borealis 5S rDNA fragment ( with the central 147 bp positioning sequence indicated by lower case type ) is as follows: CAGGCTGTCAAGGCCGGgcttgttttcctgcctgggggaaaagaccctggcatggggaggagctgggccccccccagaaggcagcacaaggggaggaaaagtcagccttgtgctcgcctacggccataccaccctgaaagtgcccgatatcgtctgatctcggaAGCCAAGCAGGGTCGGG .",
"The 147 bp synthetic 601 nucleosome positioning sequence is described in Lowary and Widom ( 1998 ) .",
"For nucleosome reconstitution , the DNA fragments were amplified by PCR and purified by 1 . 25% agarose gel electrophoresis and gel extraction ( QIAquick Gel Extraction Kit; Qiagen; Germantown , MD ) .",
"After reconstitution , the quality of the nucleosomes was assessed by nondenaturing 6% polyacrylamide gel electrophoresis .",
"If necessary , the histone:DNA ratios in the reconstitution reactions were adjusted to achieve the efficient conversion of the DNA into mononucleosomes .",
"If multiple nucleosomal species ( due to the presence of mixture of differently positioned nucleosomes ) were observed , the samples were heated at 58°C for 10 min to facilitate the movement of the differently positioned nucleosomes to the most stable location .",
"The resulting mononucleosomes were stored at 4°C prior to use in the gel mobility shift experiments .",
"Mononucleosomes ( approximately 0 . 3 µM stock concentration; 60 nM final concentration ) were combined with the indicated amounts of purified Dsup protein in HEG buffer [25 mM Hepes ( K+ ) , pH 7 . 6 , 0 . 1 mM EDTA , 10% ( v/v ) glycerol] containing 100 mM KCl , and 100 ng bovine serum albumin in a total volume of 15 µL .",
"The samples were incubated at 30°C for 30 min , and then loaded onto a nondenaturing 4 . 5% polyacrylamide gel that had been pre-equilibrated at 4°C and pre-run at 3 . 5 V/cm for 30 min .",
"The gels were run at 4°C for 30 min at 3 . 5 V/cm and then for 2 hr at 8 V/cm .",
"The nucleosomes were visualized by staining of the DNA with ethidium bromide .",
"In experiments that involved testing Dsup and histone H1 , the reaction medium additionally included 0 . 05% ( v/v ) NP-40 ( Pierce ) and 2 mM MgCl2 .",
"The histone H1 was purified from Drosophila embryos by the method of Croston et al . ( 1991 ) .",
"To ensure reproducibility of the data , each of the reported experiments was performed a minimum of two independent times with different preparations ( i . e . , biologically distinct samples ) of Dsup .",
"The ATP-dependent assembly of periodic nucleosome arrays with the ACF motor protein and the dNLP core histone chaperone was carried out essentially as described in Fyodorov and Kadonaga ( 2003 ) and Khuong et al . ( 2017 ) .",
"A standard reaction included purified Drosophila core histones ( 0 . 35 µg ) , purified Drosophila ACF ( 10 nM ) , purified Drosophila dNLP ( 1 . 8 µg ) , 2 mM ATP , purified Drosophila topoisomerase I ( ND423 ) ( 1 nM ) , and pGIE-0 plasmid DNA ( 0 . 35 µg ) ( Pazin et al . , 1994 ) in a total volume of 70 µL .",
"Purified Dsup and histone H1 were included where indicated .",
"The final KCl concentration was 100 mM for samples that were analyzed by the DNA supercoiling and partial MNase assays and 50 mM for samples that were subjected to extensive ( limit ) MNase digestion .",
"The assembly reactions were incubated at 27°C for 90 min .",
"The partial MNase , extensive MNase , and DNA supercoiling assays were performed as described by Fyodorov and Kadonaga ( 2003 ) and Khuong et al . ( 2017 ) .",
"To ensure reproducibility of the data , each of the reported experiments was performed a minimum of two independent times with different preparations ( i . e . , biologically distinct samples ) of Dsup .",
"Nucleosomes were reconstituted onto plasmid pGIE-0 ( 3269 bp ) by the salt dialysis method ( Stein , 1989; Fei et al . , 2015 ) with 16 . 4 pmol Drosophila core histone octamers per pmol pGIE-0 ( average of one nucleosome per 200 bp DNA ) .",
"The final dialysis buffer was Buffer TEN [10 mM Tris-HCl , pH 8 . 0 , 1 mM EDTA , 0 . 01% ( v/v ) NP-40 ( Pierce ) ] containing 50 mM NaCl .",
"Proteins were diluted into Buffer TEN containing 50 mM NaCl prior to use .",
"For each series of reactions with a particular factor ( such as Rv Dsup , He Dsup , or human TFIIB ) , the protein and its corresponding storage buffer were diluted in parallel and then used in a manner that ensured that each reaction was carried out under identical buffer conditions .",
"We also note that the highest concentration of glycerol in any of the reactions was 0 . 014% ( v/v ) .",
"At this concentration of glycerol , we did not detect quenching of the hydroxyl radical cleavage reactions by the buffer medium .",
"Hydroxyl radical-mediated cleavage of DNA was carried out essentially as described by Tullius et al . ( 1987 ) .",
"Briefly , nucleosomal pGIE-0 ( 162 fmol ) along with the indicated amount of added protein were incubated at room temperature ( 22°C ) for 10 min in 90 µL Buffer TEN containing 50 mM NaCl .",
"Then , in each tube , three separate droplets of 60 mM ascorbic acid ( 3 . 3 µL ) , 100 µM Fe ( II ) and 200 µM EDTA ( 3 . 3 µL ) , and 0 . 3% ( v/v ) H2O2 ( 3 . 3 µL ) were deposited onto different locations on the tube wall ( suspended above the chromatin sample at the bottom of the tube ) , mixed together , and vortexed into the 90 µL binding reaction .",
"The reaction was stopped 2 min later by the addition of 10 µL of 200 mM thiourea .",
"The DNA was extracted with phenol-CHCl3-isoamyl alcohol , precipitated with ethanol , and resuspended in 10 µL of 50 mM NaOH-1 mM EDTA and 2 µL of Purple Loading Dye ( New England Biolabs ) .",
"Alkaline agarose gel electrophoresis was performed as described by Shin and Day ( 1995 ) , and the DNA was stained with GelRed ( Biotium ) in 50 mM Tris-HCl , pH 6 . 8 .",
"To ensure reproducibility of the data , each of the reported experiments was performed a minimum of two independent times with different preparations ( i . e . , biologically distinct samples ) of Dsup ."
]
] | [
"Tardigrades , also known as water bears , are animals that can survive extreme conditions .",
"The tardigrade Ramazzottius varieornatus contains a unique nuclear protein termed Dsup , for damage suppressor , which can increase the resistance of human cells to DNA damage under conditions , such as ionizing radiation or hydrogen peroxide treatment , that generate hydroxyl radicals .",
"Here we find that R . varieornatus Dsup is a nucleosome-binding protein that protects chromatin from hydroxyl radicals .",
"Moreover , a Dsup ortholog from the tardigrade Hypsibius exemplaris similarly binds to nucleosomes and protects DNA from hydroxyl radicals .",
"Strikingly , a conserved region in Dsup proteins exhibits sequence similarity to the nucleosome-binding domain of vertebrate HMGN proteins and is functionally important for nucleosome binding and hydroxyl radical protection .",
"These findings suggest that Dsup promotes the survival of tardigrades under diverse conditions by a direct mechanism that involves binding to nucleosomes and protecting chromosomal DNA from hydroxyl radicals ."
] | [
"Tardigrades , also known as water bears and moss piglets , are small animals found in many different environments on land and sea .",
"These animals have the remarkable ability to survive extremes including very low temperatures , high levels of radiation and exposure to chemicals that are harmful to other forms of life .",
"Tardigrades have even been found to survive the harsh conditions of outer space .",
"X-rays are a type of radiation naturally produced by lightning strikes and are also found in cosmic rays from outer space .",
"High doses of X-rays can cause genetic mutations that may lead to serious illness or death .",
"This is because when X-rays come into contact with water they split the water molecules to make particles known as hydroxyl radicals , which in turn damage the DNA inside cells .",
"The genomes of animals and plants are made of DNA , which is packaged into a structure called chromatin .",
"Previous studies identified a protein named Dsup in a tardigrade called Ramazzottius varieornatus that can protect human cells from damage by X-rays .",
"However , it was not known whether Dsup binds directly to chromatin or plays a more indirect role in protecting DNA .",
"Chavez , Cruz-Becerra , Fei , Kassavetis et al . used biochemical approaches to study Dsup .",
"Their experiments revealed that Dsup from R . varieornatus binds to chromatin to protect the DNA from damage by hydroxyl radicals , and that the Dsup protein in another tardigrade species also works in a similar way .",
"Further analysis showed that a region of Dsup that is needed to bind to chromatin is very similar to a region that had been previously found only in chromatin-binding proteins from humans and other vertebrates ( animals with backbones ) .",
"This connection between Dsup and vertebrate chromatin-binding proteins remains a mystery .",
"The new findings about tardigrade Dsup may help researchers develop animal cells that live longer under normal or extreme environmental conditions .",
"In this manner , Dsup could be used to expand the range of applications of cells in biotechnology .",
"It could also increase the effectiveness of current methods , such as the production of some pharmaceuticals , that depend upon the use of cultured cells ."
] | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Accelerated redevelopment of vocal skills is preceded by lasting reorganization of the song motor circuitry | elife-43194-v2 | [
[
"Complex motor skills , such as singing or playing an instrument , are not inherently determined but need to be acquired through repetitive practice .",
"Many specialized skills are used only incidentally however , and skill performance degrades during intermittent periods of non-use .",
"This means that skills need to be re-acquired each time the need arises .",
"For example , trained human surgeons need to re-acquire their surgical proficiency after a period of absence ( Crewther et al . , 2016 ) , Capuchin monkeys need to acquire manipulative foraging skills to retrieve difficult-to-access , seasonally available food items ( Eadie , 2015 ) , and songbirds need to re-develop high-quality songs to attract a mate after wintering or long-distance migration ( Slagsvold , 1976 ) .",
"Whereas learning novel motor skills is generally a lengthy process , the short-term availability of suitable mates and food items imposes a considerable time pressure on the re-acquisition of such specialized skills .",
"Birdsong , an established model system for vocal motor learning ( Brainard and Doupe , 2013 ) , is characterized by a high degree of complex and rapid acoustic modulations .",
"The fine motor skills that are necessary to produce such complex , high quality vocalizations are thought to advertise an individual’s quality through performance-related characteristics such as song complexity and/or production rate ( Podos , 1997; Drăgănoiu et al . , 2002; Holveck and Riebel , 2007; Byers et al . , 2010 ) .",
"When juvenile songbirds are one to two months old they start singing noisy , unstructured ‘subsongs’ akin to human babbling ( Doupe and Kuhl , 1999 ) , which gradually develop into variable , but recognizable species-specific ‘plastic songs’ ( Marler and Peters , 1982a; Nottebohm et al . , 1986; Tchernichovski et al . , 2001; Mori et al . , 2018 ) .",
"With extensive vocal rehearsal the songs slowly consolidate into high performance ‘crystallized songs’ , a process that can take up more than five months in some songbird species ( Nottebohm et al . , 1986 ) .",
"For adult songbirds that annually need to re-acquire their songs this process of juvenile song development greatly exceeds the time that is available to attract a mate during the early breeding season .",
"Thus , adult songbirds must considerably speed-up song re-acquisition to quickly produce songs of adequate quality to impress conspecifics .",
"It is currently unclear how songbirds cope with the seasonally imposed time pressure on song development , and what mechanisms may be involved to ensure reproductive success under such time constraints in nature .",
"The seasonal development and degradation in vocal performance are highly correlated with changes in testosterone levels ( Nottebohm et al . , 1987; Smith et al . , 1997; Tramontin et al . , 2000; Voigt and Leitner , 2008 ) , and are often accompanied by gross anatomical and cytoarchitectural restructuring of the brain areas involved in song production ( Nottebohm , 1981; Gahr , 1990; Kirn et al . , 1994; Kafitz et al . , 1999; Thompson and Brenowitz , 2005; Balthazart et al . , 2008; Vellema et al . , 2014 ) .",
"Thus testosterone-regulated adaptations of the songbird brain may play an important role in optimizing song performance .",
"In this study we used adult female canaries ( Serinus canaria ) to investigate how a periodically acquired motor behavior attains a high performance level under developmental time pressure .",
"Although female canaries rarely sing spontaneously , and never produce high quality songs under natural conditions ( Pesch and Guttinger , 1985 ) , the systemic application of testosterone leads to the development of high-performance song with male-typical features ( Leonard , 1939; Shoemaker , 1939; Vallet et al . , 1996 ) .",
"This trait provides us with a well-defined behavioral baseline , and allows us to manipulate in detail the onset and offset of song development through repeated testosterone treatments .",
"Here we demonstrate that during a protracted testosterone treatment , adult female canaries gradually develop stable , species-typical songs through a process of song crystallization similar to what is known for naturally-raised juvenile male canaries ( Mori et al . , 2018 ) .",
"Re-treatment with testosterone several months after birds have stopped singing , leads to a rapid recurrence of song performance with song features that strongly resemble those after the first treatment .",
"We propose that once developed , vocal motor memories are retained for an extended period of time , enabling adult animals to recover song performance quickly at a later time , a process termed ‘savings’ ( Ebbinghaus , 2013 ) .",
"We further demonstrate that neurons in the songbird’s premotor nucleus HVC ( proper name ) , a central nucleus in the brain circuitry that controls song production ( Nottebohm et al . , 1976 ) , irreversibly loses a significant number of dendritic spines during the initial testosterone-induced development of vocal skills .",
"This state of reduced spine density is subsequently maintained over long periods in which testosterone levels are low and vocal skills are not used .",
"The observed lasting synaptic pruning could play an important role in the formation of vocal skill savings , enabling birds to rapidly re-acquire song performance within a restricted time period ."
],
[
"To study the development of vocal skills in adult female canaries we recorded and analyzed the entire song ontogeny in acoustically separated animals ( 5 . 6 ± 0 . 6 million song syllables per bird ) , while manipulating song output by consecutively implanting , removing , and re-implanting birds with testosterone implants .",
"Whereas no songs were observed prior to treatment , the systemic application of testosterone ( T1+ ) stimulated birds to start singing their first songs after three days ( 3 . 22 ± 0 . 46 days ) .",
"The phases of female song development were similar to those previously reported for male canaries ( Figure",
"1 ) ( Nottebohm et al . , 1986; Mori et al . , 2018; Weichel et al . , 1986 ) .",
"The first subsongs were produced in an irregular fashion and consisted of unstructured syllables with few distinctive features .",
"Songs gradually developed into plastic songs in which different phrases of repeated syllables could be clearly distinguished , albeit with variation between syllables of the same type .",
"During the following six months , female canary songs continued to develop , gradually crystallizing into the species-typical stable songs .",
"Although structurally similar to male canary songs , female songs had relatively small syllable repertoires ( 6 . 2 ± 1 . 1 syllable types ) , consistent with previous reports ( Vallet et al . , 1996; Fusani et al . , 2003; Hartog et al . , 2009 ) .",
"To investigate the birds’ abilities to re-acquire song performance after a period without vocal practice we withdrew testosterone ( T1- ) from the animals , completely abolishing singing behavior in approximately three days ( 3 . 14 ± 0 . 63 days ) .",
"After 2½ months of no song production birds were treated for a second time with testosterone ( T2+ ) , inducing song output after three days ( 3 . 14 ± 0 . 74 days ) .",
"No subsongs were observed after the second testosterone treatment , and all birds were able to produce plastic songs from the first day of singing .",
"We first compared the development of temporal song features during the first and second testosterone treatments ( Figure 2 , Figure 2—figure supplement 1 , Figure 2—figure supplement 2 ) .",
"Particularly the speed at which subsequent syllables are repeated , the syllable repetition rate ( SR ) , has been strongly associated with individual performance ( Podos , 1997; Podos , 1996 ) and has been shown to increase the attractiveness of the song to conspecifics ( Vallet et al . , 1998; Vallet and Kreutzer , 1995 ) .",
"We observed that during the first two weeks of testosterone-induced female song development , all syllables were typically produced at a similar rate ( SR: 10 . 3 ± 1 . 3 Hz ) .",
"While practicing the song , different syllables were gradually sung at different rates , becoming more distinct towards song crystallization ( Figure 2A ) .",
"Once crystallized , syllables could be distinguished as fast syllables ( SR: 21 . 7 ± 1 . 0 Hz ) , medium-speed syllables ( SR: 11 . 9 ± 1 . 1 Hz ) , and slow syllables ( SR: 4 . 9 ± 0 . 7 Hz ) .",
"A similar distribution of syllable rates redeveloped during a 2nd testosterone treatment .",
"The observed range of syllable rates was similar to what has previously been reported for wild-living male canaries ( Leitner et al . , 2001 ) .",
"To determine how fast this syllable rate pattern emerged during subsequent testosterone treatments we took crystallized song from the last week of the first testosterone treatment as a reference pattern , and calculated the correlation coefficient ( CC ) between this reference and each day of song development and re-development ( Figure 2B & C ) .",
"While syllable repetition rates gradually developed and stabilized over the course of 160 ± 8 days during the first testosterone treatment , stable rates were obtained more than seven times faster , within 22 ± 2 days , during the second treatment ( Figure 2D; paired t-test: p<0 . 001 ) .",
"In addition the maximum daily increase in similarity ( dmax ) was also significantly higher during song re-development than during initial song development ( Figure 2E; dmax: 0 . 021 ± 0 . 005 for T1+ , and 0 . 078 ± 0 . 012 for T2+; paired t-test: p<0 . 05 ) , indicating a much accelerated development of song performance in birds with previous singing experience .",
"Since syllable rate is a compound feature that is determined by both syllable duration and the interval between subsequent syllables , we analyzed syllable durations and pause durations separately .",
"Syllable durations developed slowly during the initial song acquisition ( T1+ ) , reaching stable values after 153 ± 19 days , while pause durations stabilized in 82 ± 13 days ( Figure 2—figure supplement 1 , Figure 2—figure supplement 2 ) .",
"Both temporal features re-emerged more quickly during song re-acquisition ( T2+ ) than during the first song developmental phase ( duration: 4 . 8 ± 1 . 3 days; paired t-test: p<0 . 001; pause: 10 . 7 ± 2 . 2 days; paired t-test: p<0 . 01; Figure 2—figure supplement 1 ) .",
"To determine the bird’s ability to recover spectral song patterns we analyzed the recurring patterns of frequency modulation ( FM ) , amplitude modulation ( AM ) , bandwidth ( BW ) , mean frequency ( MF ) and Wiener entropy ( E ) during song acquisition and re-acquisition .",
"Similar to temporal song features , spectral features were more quickly established during song re-acquisition ( T2+ ) than during the initial song development ( T1+ ) ( Figure 3 , Figure 3—figure supplement 1 , Figure 3—figure supplement 2 ) .",
"All spectral features developed gradually over more than 110 days before reaching stable values .",
"After a period in which the birds did not produce any song , testosterone-induced song re-acquisition led to a rapid stabilization of spectral patterns within 16 days ( Figure 3 , Figure 3—figure supplement 1; paired t test: p<0 . 01 ) .",
"Thus both temporal and spectral song features were acquired significantly faster by experienced birds that had developed singing skills once before .",
"Whereas song re-acquisition in female canaries resulted in a rapid recurrence of stable temporal and spectral patterns , not all song features re-developed in the same way .",
"Most notably , some song features demonstrated a clear deterioration in the absence of vocal practice , while other song features remained stable despite the absence of vocal practice ( Figure 3G & H ) .",
"Of the studied temporal song parameters , syllable durations did not show any deterioration in the period that birds did not sing ( T1- ) and thus also did not need to be re-acquired ( Figure 2—figure supplement 1A–C; Figure 2—figure supplement 2A ) .",
"Both the syllable rate and the pause duration between syllables did deteriorate however , and a short phase of re-development was required to recover the originally developed patterns ( Figure 2 , Figure 2—figure supplement 1D–F , Figure 2—figure supplement 2B ) .",
"The accelerated re-acquisition of syllable repetition rates thus appears to be driven by the need to re-optimize the timing between syllables rather than syllable lengths .",
"Of the spectral syllable features that were studied , the FM and AM patterns deteriorated when birds stopped singing ( T1- ) , but were re-acquired during a short developmental phase after a 2nd testosterone treatment ( T2+ ) ( Figure 3A–C , Figure 3—figure supplement 1A–C , Figure 3—figure supplement 2A & B ) .",
"BW , MF , and E did not need to be re-acquired , but maintained strong similarities with the initial acoustic patterns despite the absence of song production in the intermittent period ( T1- ) ( Figure 3D–F , Figure 3—figure supplement 1D–I , Figure 3—figure supplement 2C–E ) .",
"Thus song re-acquisition can be characterized by recalling a combination of sound features that immediately can be reproduced and sound features that require a short phase of redevelopment .",
"The songs that reappeared during the second testosterone treatment were strikingly similar to those that developed after the first treatment ( Figure 4 ) .",
"Visual inspection of the song spectrograms revealed no differences in syllable repertoire when comparing crystallized songs during the first and second testosterone treatment ( Figure 4A & B; 6 . 2 ± 1 . 1 syllable types for both treatments ) .",
"In addition both temporal and spectral song features demonstrated a strong similarity in their distribution patterns between subsequent testosterone treatments ( T1xT2 ) , with correlation coefficients above 0 . 82 for all studied song features ( Figure 4C ) .",
"These values were not significantly different from the correlation coefficients obtained by cross-correlating distribution patterns from consecutive days during the first treatment period ( T1xT1 ) for any of the measured song features ( Figure 4C ) , indicating that the song patterns that developed during the first and second testosterone treatment are similar in structure .",
"To determine the similarity of syllables produced during the 1st and 2nd testosterone treatments we calculated for each bird the Euclidean distance across all eight analyzed temporal and spectral features within and between syllable types ( Figure 4—figure supplement 1 ) .",
"The Euclidean distance between two syllables provides a measure of similarity where a syllable is represented as a point with coordinates that correspond to the eight analyzed sound features .",
"The distance between syllables with similar sound features and thus similar coordinates is low , while the distance between syllables with differing sound features is high .",
"The mean Euclidean distances between syllables of the same type across the 1st and 2nd testosterone treatment ( T1xT2 ) were not different from the Euclidean distances between syllables from subsequent days during the 1st testosterone treatment ( T1xT1: d = 0 . 73 ± 0 . 04 , T1xT2: d = 0 . 76 ± 0 . 06; Tukey’s HSD: p=0 . 99 ) .",
"The mean Euclidean distances between syllables of different types across the two treatment periods were significantly larger than those between syllables of the same type ( T1xT2ext: d = 2 . 12 ± 0 . 23; Tukey’s HSD: p<0 . 001 ) .",
"These results indicate that for a given syllable the sound features that redeveloped after the 2nd testosterone treatment fell within the range of variation observed during the 1st testosterone treatment for that same syllable type , but were clearly distinguishable from the sound features of other syllable types .",
"To investigate if the sequential structure of the song was different between the two testosterone treatments we analyzed the probability that specific phrase transitions occurred in the songs ( Figure 4—figure supplement 2 ) .",
"Because the syllable repertoire is relatively small in testosterone treated female canaries , the number of possible phrase transitions is also small .",
"Nevertheless , on average only 8 . 7% of all possible phrase transitions were used in the songs .",
"Thus whereas the song sequences are not fixed as in for example zebra finches , the song sequences are not random either , following a limited subset of possible transitions similar to what has been observed in male canaries ( Leitner et al . , 2001; Markowitz et al . , 2013 ) .",
"The percentage of phrase transitions that made up the songs during the 1st and 2nd testosterone treatments were not different ( T1+: 9 . 2 ± 4 . 5%; T2+: 8 . 3 ± 4 . 2%; paired t-test: p=0 . 59 ) , and the probability distribution of these transitions were visually similar between treatments ( Figure 4—figure supplement 2A & B ) .",
"The linearity , consistency , and entropy of song phrase transitions did not differ significantly between the songs from the 1st and 2nd treatment periods ( Figure 4—figure supplement 2C ) .",
"Together these data strongly suggest that testosterone-induced song re-acquisition in canaries is driven towards the previously acquired song pattern .",
"The finding that the re-acquisition of song performance progresses considerably faster than the initial acquisition and leads to the production of highly similar song patterns suggests that singing skills are retained during intermittent silent periods .",
"Motor learning and memory are generally considered to rely on the maturation and consolidation of the synaptic connections within the neural circuitry that drives the behavior in question ( Changeux and Danchin , 1976; Yu and Zuo , 2011; Hoshiba et al . , 2017 ) .",
"To investigate changes in synaptic connectivity during vocal motor development we quantified the number of neuronal dendritic spines in the forebrain motor nucleus HVC and the robust motor nucleus of the arcopallium ( RA ) before , during , and after the acquisition of vocal motor skills ( Figure 5 ) .",
"Excitatory projection neurons in the HVC of testosterone-treated , singing female canaries displayed a more than 30% reduction in dendritic spine density compared to untreated , non-singing control birds ( C: 0 . 95 µm−1 , T1+: 0 . 64 µm−1; Dunnett’s test: p<0 . 01 ) , indicating a significant synaptic pruning during the first acquisition of singing skills ( Figure 5B ) .",
"Compared to non-singing control birds , spine densities remained significantly lower in birds that stopped singing for 2 . 5 months after testosterone removal ( C: 0 . 95 µm−1 , T1-: 0 . 69 µm−1; Dunnett’s test: p<0 . 01 ) .",
"Thus , the synaptic pruning in HVC associated with testosterone-induced song acquisition is not reversed when birds stop singing , but instead the pruned state is maintained in periods in which the birds do not sing ( Figure 5B ) .",
"In contrast , we did not observe any changes in dendritic spine densities in RA across the different experimental periods ( Figure 5C; ANOVA: p=0 . 857 ) .",
"Strong differences were observed in the density of dendritic spines between different spinous neurites in HVC ( range: 0 . 15 to 2 . 14 spines/µm ) .",
"To investigate which neurite types were associated with the observed spine pruning we calculated the probability distribution of neuronal dendrites in HVC based on their spine density ( Figure 5D–F ) .",
"We observed a marked shift in the distribution of dendrites from more densely-spined dendrites in control birds towards less densely-spined dendrites in the testosterone treatment ( T1+ ) and removal groups ( T1- ) , reflecting a global decrease in spine density .",
"The strongest reduction was detected in the range of dendrites with more than 0 . 8 spines per µm , which halved in number in both testosterone-treated , singing birds ( T1+ ) , and testosterone-removed , non-singing birds ( T1- ) compared to non-singing controls ( C: 0 . 64 ± 0 . 06 , T1+: 0 . 28 ± 0 . 05 , T1-: 0 . 33 ± 0 . 06; ANOVA: p<0 . 01 ) .",
"The total density of both spinous and aspinous neurites in HVC did not significantly differ between the treatment groups ( ANOVA: p>0 . 36 ) , suggesting that changes in dendrite densities were not caused by specific cell death .",
"These data suggest that the observed vocal motor acquisition is accompanied by a lasting synaptic pruning specifically in nucleus HVC , providing a possible anatomical site for motor memory that could facilitate the observed rapid re-acquisition of previously established song patterns ."
],
[
"The ability to learn new skills enables one to adapt to changes in the surrounding environment .",
"Whereas learning new skills requires time and effort , the survival of many species relies on their capacity to utilize specialized skills in a timely fashion , capitalizing on the short-term availability of foods , mates , and other environmental factors ( Prendergast et al . , 2002 ) .",
"Such a capacity is biologically relevant for numerous skills in a multitude of species , ranging from periodic foraging skills in dolphins ( Patterson et al . , 2016 ) , bats ( Clarin et al . , 2013 ) , and monkeys ( Boinski and Fragaszy , 1989 ) to courtship displays in crabs ( Mowles et al . , 2017 ) and songbirds ( Ota et al . , 2015 ) .",
"As the use of these specialized skills is interrupted over longer periods , for example during long-distance migration or hibernation ( Slagsvold , 1976; Prendergast et al . , 2002 ) , such skills need to be re-acquired quickly to maximize survival and reproductive success .",
"In this study we establish the repeated , hormone-driven acquisition of vocal motor performance in female canaries as a model system for studying motor savings .",
"Using this model , we show that while the initial development of specialized motor skills can be a lengthy process , motor performance can quickly be re-acquired at a later time , even after extended periods of non-use ( Figure 2 and 3 ) .",
"The accelerated song re-acquisition observed in this study indicates that acquiring a motor skill for the first time follows a different developmental trajectory than re-acquiring the same skill at a later time .",
"This is both evident from the accelerated consolidation of song performance during song re-development ( Figure 2 and 3 ) , and the observation that birds skip the subsong phase of song development when re-acquiring their songs .",
"Our data further suggest that songbirds have the ability to retain previously developed singing skills through a process of selective pruning of the neural circuitry ( Figure 5 ) , enabling them to rapidly recover songs of adequate quality to attract a mate before breeding opportunities disappear .",
"Several song parameters were maintained across a period in which birds did not sing , while other song features demonstrated clear deterioration ( Figure 3G & H ) .",
"Birds were able to recover those deteriorated song features with renewed vocal practice .",
"At the peripheral level , song performance depends on the accurate control of the respiratory system , the sound producing organ ( the syrinx ) , and the upper vocal tract .",
"Interestingly , those song features that required a phase of re-development to be optimized seem primarily associated with motor constraints in syrinx function ( Podos , 1997; Geberzahn and Aubin , 2014 ) .",
"Online modulation of frequency and amplitude requires active contractile modulation of syringeal muscles ( Goller and Suthers , 1996; Elemans et al . , 2008 ) .",
"Syllable repetition and the inter-syllabic interval also depend on how rapidly syringeal abductor and adductor muscles can open and close the syringeal aperture .",
"The upper vocal tract may act as a tunable acoustic filter of the sounds produced by the syrinx ( Nowicki , 1987; Beckers et al . , 2003; Riede et al . , 2006 ) and play a more important role in establishing the bandwidth , entropy and mean frequency of song syllables .",
"Thus song parameters linked to vocal tract modulation may be preserved after birds stopped singing , while song parameters linked to contraction speed of syringeal muscles appear to deteriorate .",
"Whereas male canaries are known to annually re-organize their songs , even when deafened ( Mori et al . , 2018 ) , no such modifications were observed in our study .",
"It is not clear to what extent annual changes in male canary song structure are due to relearning or are driven by a selection process from a previously learned collection of syllable types during juvenile development ( Nottebohm et al . , 1986; Marler , 1997; Gardner et al . , 2005; Belzner et al . , 2009 ) .",
"Considering that the female canaries in our study were acoustically separated during the testosterone-induced development of song suggests that the emerging song patterns are either for a large part innate ( Mori et al . , 2018; Gardner et al . , 2005 ) , or are limited to a memorized collection of syllable types that they heard earlier in life ( Belzner et al . , 2009; Marler and Peters , 1981; Marler and Peters , 1982b; Prather et al . , 2010; Kiefer et al . , 2014 ) .",
"The initial song acquisition may thus rely on an innate or memorized auditory template , while the accelerated song re-acquisition may be driven by a motor memory trace of what the birds learned to produce during the first song acquisition phase .",
"The syllable repertoires of our female canaries were much lower than what is common for males .",
"Such a small vocabulary would limit the possibilities to re-arrange or replace syllables without compromising song complexity , and thus few modifications to female songs can be expected .",
"During juvenile brain development in humans , mammals , and birds , synaptic connections are initially overproduced , followed by protracted , activity-dependent synapse elimination during puberty ( Huttenlocher , 1990; Petanjek et al . , 2011; Rakic et al . , 1986; Zuo et al . , 2005; Nixdorf-Bergweiler et al . , 1995 ) .",
"By selectively stabilizing functional synapses while pruning away inactive synapses , brain circuits are thought to reduce neuronal plasticity , consolidating newly learned information and reducing unnecessary redundancy ( Changeux and Danchin , 1976 ) .",
"The lasting spine pruning observed in the forebrain song control area HVC ( Figure 5 ) could reflect such a structural modification facilitating the long-term memorization of vocal patterns .",
"Optical imaging studies have demonstrated a rapid accumulation and stabilization of neuronal dendritic spines in HVC during developmental song learning in zebra finches ( Roberts et al . , 2010 ) .",
"In the mouse cortex , morphological modifications to dendritic spines have been observed that lasted well beyond the learning experience that caused the spine modifications ( Hofer et al . , 2006; Yang et al . , 2009; Xu et al . , 2009; Hofer et al . , 2009 ) , strongly suggesting that long-term memory retention of previously acquired skills is facilitated by stably maintained synaptic connections .",
"Despite such learning-related circuit modifications , the large majority of dendritic spines in the mammalian brain are stably maintained in adulthood ( Petanjek et al . , 2011; Rakic et al . , 1986; Zuo et al . , 2005; Yang et al . , 2009; Xu et al . , 2009; Grutzendler et al . , 2002 ) .",
"In contrast we observed a strong spine reduction in fully adult birds ( Figure 5 ) , suggesting that the observed synaptic pruning in the adult canary HVC during song acquisition may reflect a developmentally delayed , activity-dependent consolidation of the neural motor circuitry .",
"A recent study in zebra finches has shown that singing prevention during juvenile development can also avert developmental spine pruning in the robust motor nucleus of the arcopallium ( RA ) ( Hayase et al . , 2018 ) .",
"In addition , auditory input during sensorimotor learning may play an important role in circuit consolidation , and auditory deprivation has been shown to delay the natural juvenile maturation of HVC in zebra finches ( Huang et al . , 2018 ) .",
"It is not known how long such developmental processes can be postponed , but we did not observe delayed synaptic pruning in RA upon song acquisition in adult female canaries ( Figure 5C ) , suggesting that the time-window for large-scale spine pruning in RA may be more limited than in HVC .",
"The developmentally delayed maturation of HVC ( Figure 5B ) is consistent with our observation that the delayed , testosterone-induced development of song performance in adult female canaries follows a juvenile male-like developmental trajectory ( Figure 1 ) .",
"Thus , in the absence of singing behavior , parts of the vocal motor circuit may remain plastic in adult female canaries , only consolidating once neural activity within the circuitry increases to drive testosterone-induced song output .",
"Testosterone-induced song development in adult female canaries has previously been associated with changes in other neural attributes in HVC as well , including an increase in volume , neuron numbers ( but not densities ) , increased vascularization , and changes in cellular structure ( Vellema et al . , 2014; Hartog et al . , 2009; Gahr and Garcia-Segura , 1996; Nottebohm , 1980; Rasika et al . , 1994; Louissaint et al . , 2002 ) .",
"Many of the observed modifications to the song system follow an annually changing pattern in seasonally breeding male songbirds ( Nottebohm et al . , 1986; Smith et al . , 1997; Nottebohm , 1981; Kirn et al . , 1994 ) , indicating that in males these modifications are not maintained outside the breeding season when birds no longer sing high performance song .",
"In addition , withdrawing testosterone from male Gambel’s white-crowned sparrows , another well-studied seasonal singer , resulted in a rapid regression in HVC size , neuron number , and neuron size ( Thompson et al . , 2007 ) , indicating that these neural modifications are reversible and unlikely to reflect long-term memorization of vocal patterns .",
"The lasting synaptic pruning that we observed in HVC is to our knowledge the first report of a song-related modification to the adult songbird brain that persists after the behavior itself is no longer expressed .",
"HVC is at a central position between the motor circuitry that controls song output and a forebrain-basal ganglia circuit , analogous to the mammalian cortico-basal ganglia ( CBG ) circuit , which feeds back on the motor circuitry for song production ( Brainard and Doupe , 2013; Nottebohm et al . , 1976 ) .",
"Firing patterns in HVC neurons that project to RA are closely associated with millisecond precision of syllable timing ( Yu and Margoliash , 1996; Hahnloser et al . , 2002; Amador et al . , 2013 ) , suggesting that HVC is directly involved in maintaining time-related features of song performance .",
"Burst patterns in RA neurons on the other hand are more closely associated with intrasyllabic structure ( Yu and Margoliash , 1996; Chi and Margoliash , 2001 ) .",
"However , slowing down brain processes by local cooling of HVC has also been shown to change the intrasyllabic structure ( Long and Fee , 2008; Alonso et al . , 2015 ) , indicating that both HVC and RA activity play a role in maintaining the fine temporal structure within syllables .",
"The acquisition of song syllables in zebra finches is accompanied by increased timing accuracy of inhibitory neural firing within the HVC microcircuitry , suggesting that precisely-timed inhibition may play an important role in shaping song-related premotor sequences ( Kosche et al . , 2015; Vallentin et al . , 2016 ) .",
"The lasting synaptic reorganization in HVC during song acquisition may be guided by such inhibitory activity and provide a neural scaffold for both intra and intersyllabic timing .",
"RA-projecting HVC neurons are continuously replaced in the adult songbird brain ( Alvarez-Buylla et al . , 1990 ) , providing a base for ongoing variability in syringeal muscle control .",
"A permanently maintained HVC network may guide dynamically changing circuits in HVC and RA during subsequent periods of singing , quickly driving song features towards previously acquired patterns .",
"Neurons from HVC that project to the avian basal ganglia Area X are not replaced in adulthood , providing a permanent subpopulation of HVC neurons ( Gahr , 1990; Alvarez-Buylla et al . , 1988 ) .",
"Although we could not specifically distinguish between neuron types in our study , we observed the strongest reduction of 50% in the number of densely-spined dendrites in HVC after birds developed song .",
"Previous studies have shown that those HVC neurons that project to Area X predominantly consist of neurons with densely-spined dendrites ( Kornfeld et al . , 2017; Nixdorf et al . , 1989; Kubota and Taniguchi , 1998; Benton et al . , 1998; Mooney , 2000; Mooney and Prather , 2005; Fortune and Margoliash , 1995 ) .",
"Thus our observed synaptic pruning may be related to a major reorganization within the HVC to Area X connection , a reorganization that could reflect the long-term memorization of vocal skills .",
"CBG-like circuits have been strongly implicated in the learning and long-term retention of motor skills in humans ( Albouy et al . , 2008; Debas et al . , 2010; Lehéricy et al . , 2006; Poldrack et al . , 2005; Coynel et al . , 2010 ) , mammals ( Barnes et al . , 2005; Pasupathy and Miller , 2005; Sheth et al . , 2011; Yin et al . , 2009 ) , and birds ( Bottjer et al . , 1984; Scharff and Nottebohm , 1991; Brainard and Doupe , 2000; Kao et al . , 2005; Olveczky et al . , 2005; Aronov et al . , 2008; Sober and Brainard , 2009; Andalman and Fee , 2009; Ölveczky et al . , 2011; Charlesworth et al . , 2012 ) .",
"Vocal exploration is driven by activity in the avian CBG circuit ( Kao et al . , 2005 ) , which may guide synaptic modifications in plastic motor circuits ( Mehaffey and Doupe , 2015 ) , even in adulthood .",
"Although selective elimination of X-projecting HVC neurons did not appear to alter the song structure of adult zebra finches ( Scharff et al . , 2000 ) , altered expression of FoxP2 in Area X of adult male zebra finches has been associated with changes in vocal performance ( Murugan et al . , 2013; Thompson et al . , 2013; Teramitsu and White , 2006 ) .",
"In addition neurotoxic lesions in Area X lead to changes in singing tempo and syllable sequencing in adult zebra finches ( Kubikova et al . , 2014 ) , suggesting that the HVC to Area X connection continues to play a role in the long-term maintenance of vocal sequences .",
"The mechanisms behind learning-related , selective stabilization and elimination of dendritic spines remain largely unknown .",
"Sex hormones may play an important role in the stabilization of such synaptic connections during the development of motor skills .",
"Many of the brain areas that are involved in song learning and production express high numbers of androgen receptors ( Arnold et al . , 1976; Balthazart et al . , 1992; Metzdorf et al . , 1999; Nastiuk and Clayton , 1995; Gahr and Metzdorf , 1997 ) , and X-projecting HVC neurons express estrogen receptors ( Gahr , 1990 ) that can be activated after conversion of testosterone into estradiol .",
"Furthermore , changes in neuronal dendritic growth and synaptic morphology in the songbird RA have been observed to accompany testosterone-induced song development in female canaries ( DeVoogd and Nottebohm , 1981; Devoogd et al . , 1985; Canady et al . , 1988 ) .",
"In addition , Frankl-Vilches et al . ( Frankl-Vilches et al . , 2015 ) recently observed that 85% of the seasonally and testosterone upregulated genes in the canary HVC are related to neuron differentiation , axon , dendrite and synapse organization .",
"In the brain of birds and mammals , sex hormones are known to upregulate the expression of brain-derived neurotrophic factor ( BDNF ) ( Dittrich et al . , 1999; Rasika et al . , 1999; Sohrabji et al . , 1995 ) , which in its turn contributes to the stabilization of synapses through the interaction with tyrosine receptor kinase B ( TrkB ) ( Koleske , 2013 ) .",
"Furthermore , BDNF has been shown to be essential during testosterone-induced song development in female canaries ( Hartog et al . , 2009 ) , providing a likely pathway by which sex hormones can consolidate motor skills by stabilizing synapses .",
"Testosterone levels and song consolidation are tightly linked , and it is difficult to determine if brain anatomical changes in our and other studies are caused by testosterone , singing activity or both .",
"Interestingly , local intracerebral testosterone implants close to nucleus RA did not cause anatomical changes in this androgen receptor-rich brain area ( Brenowitz and Lent , 2002 ) , suggesting that modifications in RA are driven by singing-related , activity-dependent input from HVC .",
"Whether or not testosterone , vocal practice , or both play a role in the observed synaptic pruning in HVC , the vocal circuitry demonstrated synaptic changes that persisted through several months in which testosterone levels were low and birds did not sing , potentially enabling the birds to quickly re-acquire their previously developed song patterns .",
"Sex hormones can be regulated by both environmental and social factors ( Goymann et al . , 2007; Oliveira , 2004; Wingfield et al . , 1990 ) , thereby providing a timed cue for the consolidation of behavioral output and the underlying brain circuitry .",
"Such a cue could facilitate the timely formation and recall of long-term memories of relevant sensory and motor information .",
"Our study suggests that lasting , hormone-driven synaptic pruning of related brain circuitries could form a basis for such long-term memories , enabling a quick recovery of previously acquired skills .",
"The ability to respond quickly to environmental and social cues through such primed hormone-sensitive control mechanisms would constitute an important adaptation to ensure competitive proficiency and maximize reproductive success under environmental restrictions .",
"The results obtained from this study may have implications not only across different animal groups , but may generally apply to different motor , sensory , and motivational systems that rely on a rapid recall of previously learned information ."
],
[
"For this study adult domesticated canaries ( Serinus canaria ) were either purchased from a local breeder in Antwerp or taken from the breeding colony of the Max Planck Institute for Ornithology , Seewiesen .",
"All animals were raised and kept under local natural daylight conditions , until the day length reached 11 hr in early March .",
"Birds were subsequently housed individually in sound attenuating chambers for song recordings .",
"Experimental procedures were conducted according to the guidelines of the Federation of European Animal Science Associations ( FELASA ) and approved by the Ethical Committee on animal experiments of the University of Antwerp .",
"To stimulate song production in naive female canaries , birds were subcutaneously implanted ( T1+ ) with 8 mm silastic tubes ( Dow Corning , Midland , MI; ID: 1 . 47 mm ) filled with crystalline testosterone ( Sigma-Aldrich Co . , St . Louis , MO ) .",
"During the hormone treatments a light cycle of 11 hr light , and 13 hr darkness was maintained to exclude photoperiodic effects on song production .",
"This light cycle was chosen because under natural conditions it corresponds to a phase of testosterone up-regulation and physiological sensitivity to hormone fluctuations in canaries ( Nottebohm et al . , 1987 ) .",
"After consolidation of song we removed the hormone implants ( T1- ) to end song production , and changed the daylight conditions to 8 hr light and 16 hr dark , inducing a molting phase of approximately 1 . 5 months .",
"Once the molt was finished , the day length was gradually returned to 11 hr over the following month .",
"Subsequently , the song-experienced birds received a second testosterone treatment ( T2+ ) , and were sacrificed 5 weeks after implantation .",
"Blood samples were collected from the birds’ right wing veins using heparinized haematocrit capillaries ( Brand , Wertheim , Germany ) .",
"Directly after blood collection , samples were centrifuged at 3000 RPM for 10 min to separate cells from plasma , and stored at −80°C for later analysis .",
"Testosterone concentrations in the blood plasma were determined by radioimmunoassay ( RIA ) after extraction and partial purification on diatomaceous earth ( glycol ) columns , following the procedures described previously ( Goymann et al . , 2001; Goymann et al . , 2008 ) .",
"Briefly , plasma samples were extracted with dichloromethane ( DCM ) after overnight equilibration of the plasma with 1500 dpm of 3H-labeled testosterone ( Perkin-Elmer , Rodgau , Germany ) .",
"After separation of the organic phase , the extracts were re-suspended in 2 × 250 µl 2% ethylacetate in isooctane and fractioned using columns of diatomaceous earth:propylene glycol:ethylene glycol ( 6:1 . 5:1 . 5 , w:v:v ) .",
"Collected testosterone fractions were dried , re-suspended in phosphate-buffered saline with 1% gelatin ( PBSG ) , and left to equilibrate overnight at 4°C .",
"Extraction of plasma testosterone with dichloromethane resulted in 86 ± 11% ( mean ± sd ) recovery .",
"Hormone samples were incubated with antisera against testosterone ( Esoterix Endocrinology , Calabasas Hills , CA ) , and after 30 min 3H-labeled steroids ( 13500 dpm ) were added and left to incubate for 20 hr at 4°C .",
"Free steroids were separated from the bound fractions by absorption on dextran-coated charcoal , centrifuged , and decanted into scintillation vials to be counted .",
"Testosterone concentrations in the plasma samples were measured in one assay .",
"The lower detection limit of the RIA was 0 . 33 pg/tube , and all measured plasma levels were above the lower detection limit .",
"The intra-assay variation of a chicken plasma pool as control sample at the beginning and end of the assay was 6 . 7% .",
"Pooled plasma levels of testosterone for all birds during the different consecutive hormone treatments are shown in Table 1 .",
"Mean detected hormone levels were within range of physiological plasma levels in nest-building male canaries ( range: 360–7970 pg/ml; n = 6 ) and previously reported values for other small passerines ( Wingfield and Hahn , 1994; Kempenaers et al . , 2008; Apfelbeck and Goymann , 2011 ) .",
"To monitor the entire song ontogeny during consecutive testosterone treatments randomly selected , naive female canaries ( n = 6 ) were kept individually in sound attenuating chambers during testosterone treatment ( T1+ ) , testosterone removal ( T1- ) and testosterone re-treatment ( T2+ ) , while song output was continuously recorded .",
"This approach resulted in a song database of 5 . 6 ± 0 . 6 million song syllables per bird , recorded over the course of approximately 1 year .",
"The vocal activity of each bird was recorded using Sound Analysis Pro 2 . 0 ( Tchernichovski et al . , 2000 ) .",
"Omnidirectional condenser microphones ( TC-20; Earthworks , Milford , NH ) connected to a multi-channel microphone preamplifier ( UA-1000; Roland , Los Angeles , CA ) were used to acquire and digitize all sounds produced within the sound attenuating boxes with a sampling frequency of 44 . 1 kHz .",
"The incoming signal was filtered online using an amplitude and Wiener entropy threshold to exclude background noises , and saved in 60 s waveform audio files ( 16-bit PCM format ) .",
"Vocalizations were segmented into individual syllables with the fully automated Feature Batch module in Sound Analysis Pro by applying an amplitude threshold to the sound wave .",
"Because the distance from perch to microphone ranged between 10 and 20 cm only , little variation in recorded amplitude levels can be expected .",
"The amplitude threshold was selected once manually to assure reliable segmentation , and was kept constant during the analyses of all sound phrases within birds .",
"To filter out non-song vocalizations , syllables were only included when produced within a song bout of at least 750 ms . A song bout was defined as a sequence of sounds traversing the amplitude threshold with an interval of no more than 100 ms . We used Sound Analysis Pro to measure duration ( d ) , pause duration ( i ) , syllable rate ( SR ) , frequency modulation ( FM ) , amplitude modulation ( AM ) , syllable bandwidth ( BW ) , mean frequency ( MF ) and Wiener entropy ( E ) for each syllable and stored these syllable features in MySQL 5 . 1 tables ( Oracle , Redwood Shores , CA ) .",
"Bandwidth was calculated for each syllable by subtracting the minimum peak frequency from the maximum peak frequency .",
"The syllable rate was defined for each syllable as: SRa = ( da +ia ) −1 , where da is the duration of syllable ‘a’ , and ia is the interval between the end of syllable ‘a’ and the beginning of the consecutive syllable within the same song bout , irrespective of syllable type .",
"For a detailed computational description of how Sound Analysis Pro calculates the different sound features we refer to the accompanying publication ( Tchernichovski et al . , 2000 ) and the online manual ( http://soundanalysispro . com ) .",
"To investigate the dynamic change of song features over the course of the experiment , daily histograms were obtained from the entire dataset by rounding individual values to the nearest integer , and plotting the number of times those integers occurred each day .",
"To determine the SRs that were most commonly used at the onset and crystallization of song development a non-linear exponential curve ( 1 ) was fitted through the peak values of each SR histogram .",
"( 1 ) Fx=a+b1+ec-xd Further developmental correlation plots for all temporal and spectral song features were produced by calculating the Pearson product-moment correlation coefficient ( CC ) between a 7 day average of the histogram pattern at the time of song stabilization and all other recorded days .",
"This calculation resulted in a value between −1 and 1 for each recorded day , and was used as a measure of similarity where one describes an absolute copy of the stable song pattern , and −1 describes the absolute inverse of the stable song pattern .",
"A non-linear exponential curve ( 1 ) was fitted through the CC values and song parameters were considered stable as soon as the daily increase in similarity dropped below 0 . 001 .",
"For each song feature we used the fit curve to determine the duration between testosterone implantation and feature stabilization , and to determine the maximum daily increase in similarity ( dmax ) .",
"Dmax could only be calculated for birds and song features that demonstrated a developmental increase in similarity , and was only used for statistical purposes if three or more birds demonstrated such an increase .",
"Mean CC plots combining all analyzed birds were calculated by averaging the raw ( non-fitted ) CC values that were calculated for each bird and for each day .",
"We aligned song development in the different birds based on the first day where we detected song .",
"Spectrograms of the original sound files were visually inspected on syllable-like sound structures to determine the syllable repertoire of each bird and the first occurrence of song after testosterone treatments .",
"To assess syllable similarity during consecutive testosterone treatments we calculated the Euclidean distance between each of 100 randomly selected syllables for each syllable type from stable songs during both the T1+ and T2+ periods .",
"The Euclidean distance between two syllables ‘a’ and ‘b’ with coordinates c = SR , d , i , FM , AM , BW , MF , E was defined as:da , b=∑c=1n ( b1-a1 ) 2 To give all analyzed temporal and spectral features equal weight in the analysis , each feature was normalized by dividing its value by its global median value from all syllables produced by all birds recorded in this study .",
"First , for each syllable type the mean Euclidean distance between 100 syllables of the same type during T1+ ( T1xT1 ) was calculated to determine how much baseline variation existed between syllables of the same type .",
"Lower values indicate a higher similarity between syllables .",
"Secondly , we calculated the mean Euclidean distance between 100 syllables of the same type from the T1+ and T2+ ( T1xT2 ) periods to determine if syllables produced during the 2nd testosterone treatment were similar to those produced during the 1st testosterone treatment .",
"Finally , we calculated the mean Euclidean distance between 100 syllables of the same type from the T1 +period with each of 100 syllables from all other types of syllables that a bird produced ( T1xT2ext ) to provide a measure of dissimilarity between syllables of different types .",
"All observed syllable types were included in this analysis with the exception of single transitional syllables connecting two song phrases .",
"To assess similarity of song syntax between subsequent testosterone treatments we determined the probability of song phrase transitions during the T1+ and T2+ periods for each bird .",
"On average 794 . 3 phrase transitions per bird were used for the analysis .",
"From the resulting transition probability distributions we calculated the sequence linearity , consistency and entropy as previously described ( Scharff and Nottebohm , 1991; Daou et al . , 2012 ) .",
"In short , sequence linearity is expressed as the number of syllable types divided by the number of transition types and addresses the way phrases are ordered in a song .",
"Sequence consistency is expressed as the sum of common phrase transitions divided by the sum of total transitions and addresses how often the same sequences are produced .",
"Common phrase transitions were defined as transitions that make up at least 5% of all phrase transitions .",
"The entropy ( S ) of the transition probability distribution with transition type Ti is a measure of the spread of that distribution and was calculated as:S=-∑i=1nTi log2 ( Ti ) To estimate spine densities and dendrite densities in motor nucleus HVC and RA , brains were collected from a control group of adult female canaries that did not receive any testosterone treatment ( C: n = 6 ) , a group of birds that was sacrificed five months after testosterone treatment ( T1+: n = 8 ) , and a group of birds that was sacrificed at least 2 . 5 months after testosterone withdrawal ( T1-: n = 6 ) .",
"All experimental birds were raised and kept in group aviaries together with other male and female canaries throughout the experiment to assure similar social and auditory conditions .",
"Birds were transferred to sound attenuating chambers for two weeks after testosterone treatment to assure that birds were singing , and prior to sacrifice .",
"Individuals were randomly allocated to the different treatment groups .",
"All birds were maintained on a light cycle of 11 hr light , and 13 hr darkness and were between 2 . 5 and 3 years old at the time of sacrifice .",
"Brains were processed for Golgi-Cox staining using the FD Rapid GolgiStain kit ( FD NeuroTechnologies , Columbia , MD ) .",
"After overnight fixation in a 4% formaldehyde solution in phosphate-buffered saline ( PBS; 10 mM; pH 7 . 4 ) , brains were stored in PBS with 0 . 05% sodium azide at 4°C until further processing .",
"Brain hemispheres were separated with a razor blade and left hemispheres were immersed for two weeks in an impregnation solution consisting of equal amounts of FD solutions A and B in total darkness at room temperature .",
"Following three days of incubation in FD solution C , brains were cut into 30 µm sagittal sections using a sliding microtome ( Leica Microsystems GmbH , Wetzlar , Germany ) , which were stored in PBS with 0 . 05% sodium azide at 4°C .",
"Sections with a regular interval of 180 µm were mounted on SuperFrost glass slides ( Menzel GmbH , Brauschweig , Germany ) , developed in a solution of two parts distilled water , one part FD solutions D , and one part FD solution E for ten minutes , and rinsed in distilled water before embedding in CC/Mount tissue mounting medium ( Sigma-Aldrich ) .",
"Slides were incubated at 70°C until the mounting medium had hardened , cleared in xylene , and further embedded in Roti-Histokitt II ( Carl Roth , Karlsruhe , Germany ) before coverslipping .",
"For each bird , z-stacks were obtained from three brain sections of nucleus HVC and two sections of nucleus RA with a Nikon Eclipse Ti microscope ( Nikon , Tokyo , Japan ) , equipped with a 60x oil immersion lens ( CFI Plan Apo VC 60x Oil ) .",
"The outline of HVC and RA in each section was delineated in ImageJ ( http://rsb . info . nih . gov/ij/ ) , and 100 µm2 non-overlapping regions of interests ( ROIs ) were randomly placed within the boundaries of the target nucleus prior to the quantifications .",
"Considering the relatively uniform distribution of X-projecting HVC neurons and RA-projecting HVC neurons ( Fortune and Margoliash , 1995 ) , this approach should result in a homogenous sampling across treatment groups .",
"Neuronal dendritic spine densities were estimated for each brain section by checking ROIs in random order until five ROIs were located that contained spinous dendrite segments ( 15 ROIs per animal for HVC and 10 ROIs for RA ) .",
"The individual dendrite segments within the ROIs were traced in ImageJ to measure the length , and the number of visible spines that originated from the traced segment was manually counted .",
"Spine densities were calculated as the number of visible spines per µm of dendrite .",
"All visible spine types were included in the quantifications , including filopodia , long , thin , stubby , mushroom and branched spines ( Risher et al . , 2014 ) .",
"For branched spines , each spine head was counted separately .",
"To further obtain a density measure of spinous dendrites we counted the total number of spinous dendrite segments in all ROIs examined , and divided the number of observed dendrite segments with the number of ROIs , giving an estimation of the number of spinous dendrites per ROI .",
"Neuronal dendrites were further binned into categories containing 0–0 . 2 , 0 . 2–0 . 4 , 0 . 4–0 . 6 , 0 . 6–0 . 8 , 0 . 8–1 . 0 , 1 . 0–1 . 2 , 1 . 2–1 . 4 , 1 . 4–1 . 6 , 1 . 6–1 . 8 spines per µm .",
"The probability distribution of neuronal dendrite segments was then obtained by dividing the observed number of spinous dendrites per ROI in each bin with the total number of spinous dendrites per ROI .",
"To obtain a density measure of aspinous neurites in HVC , the number of visible spine-less neurite segments were counted in ten new non-overlapping ROIs that were randomly placed within the boundaries of HVC .",
"We refer to these projections as neurites , because we were unable to distinguish between aspinous dendrites and axons in our tissues .",
"All quantifications were conducted by an experimenter that was blind to the experimental condition of the animals .",
"Statistical analyses were performed in SAS 9 . 3 ( SAS Institute , Cary , NC ) .",
"No sample sizes were calculated prior to the experiments .",
"A technical replication was defined as a repeated measurement of the same individual , and biological replications constitute the number of sampled individuals ( n ) .",
"Differences in spine and dendrite densities between treatment groups were tested with a one-way analysis of variance ( ANOVA ) with Dunnett’s correction for multiple comparisons against one control value ( untreated birds ) .",
"Changes in hormone levels were determined using a repeated one-way ANOVA with Dunnett’s correction .",
"Differences in syllable similarity and syllable rates of individual syllable types were tested with one-way ANOVAs , and when significant , Tukey's HSD test was used to analyze differences between time points .",
"Song pattern similarities between the 1st and 2nd hormone treatment were established by comparing the average CC from seven consecutive days of stable song during the 1st treatment with the average CC from seven consecutive days of stable song production during the 2nd treatment .",
"Paired-samples t-tests were used to compare CC’s , sequence parameters , and developmental time parameters between subsequent hormone treatments .",
"To determine song feature deterioration we calculated the difference between the average CC from the last 7 days of stable song production during the 1st testosterone treatment and the average CC from the first 2 days of song production during the 2nd treatment .",
"Feature recovery was determined by subtracting the average CC from the first 2 days of song production from the average CC from the last 7 days of stable song production during the 2nd testosterone treatment .",
"One-sample t-tests were used to determine if feature deterioration and recovery significantly exceeded zero .",
"Measurement values in the text are given as means ± SEM , unless stated otherwise .",
"No outliers or data were excluded from analysis ."
]
] | [
"Complex motor skills take considerable time and practice to learn .",
"Without continued practice the level of skill performance quickly degrades , posing a problem for the timely utilization of skilled motor behaviors .",
"Here we quantified the recurring development of vocal motor skills and the accompanying changes in synaptic connectivity in the brain of a songbird , while manipulating skill performance by consecutively administrating and withdrawing testosterone .",
"We demonstrate that a songbird with prior singing experience can significantly accelerate the re-acquisition of vocal performance .",
"We further demonstrate that an increase in vocal performance is accompanied by a pronounced synaptic pruning in the forebrain vocal motor area HVC , a reduction that is not reversed when birds stop singing .",
"These results provide evidence that lasting synaptic changes in the motor circuitry are associated with the savings of motor skills , enabling a rapid recovery of motor performance under environmental time constraints ."
] | [
"Developing a complex skill , for example learning how to play the violin , takes considerable time and effort .",
"If you then abandon the violin for months or years , your ability to play will deteriorate over time .",
"However , when you do pick up the violin again you will be able to recover your proficiency much faster than you did when learning for the first time .",
"It therefore seems that while first learning a complex motor skill , some sort of memory is formed and stored .",
"Learning and memory in general rely on the patterns of connections , called synapses , among neurons in the brain .",
"Early in development , neurons make too many of these connections .",
"This network is then refined over time as unneeded connections are discarded .",
"However , the structural changes to the network that make it easier to re-acquire a skill were not well understood .",
"Canaries are a useful example of this sort of learning .",
"Young males learn complex songs that are critical for their ability to attract a mate , and can quickly re-acquire their songs at the beginning of each mating season .",
"Female canaries do not normally sing but will develop songs if they are implanted with a testosterone-releasing device .",
"When the implant is removed , they stop singing .",
"To investigate how songbirds re-acquire songs , Vellema et al . gave female canaries a testosterone implant , and the birds gradually developed songs .",
"The implant was then removed , and for two and a half months the birds did not sing .",
"Vellema et al . then gave the canaries a second testosterone treatment .",
"The canaries rapidly began to sing songs that were strikingly similar to the ones they sang during the initial learning period .",
"Examining the brains of these canaries revealed that major structural changes in brain connections occurred while the canaries first developed their songs .",
"After the initial period of testosterone exposure , the female canaries had fewer synapses in a brain region that is associated with learning and producing motor tasks .",
"Importantly , this reorganization of the brain circuitry was irreversible .",
"These findings provide fundamental insights into how we learn and maintain new motor skills .",
"Similar rapid re-learning phenomena are observed in other areas of neuroscience , and future research can explore whether the mechanism described here extends to these areas .",
"Possibly , this mechanism could also illuminate how we can regain skills lost after trauma or injury ."
] | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"stem cells and regenerative medicine",
"research communication"
] | Sox9+ messenger cells orchestrate large-scale skeletal regeneration in the mammalian rib | elife-40715-v1 | [
[
"Whereas amphibians regenerate large portions of their skeletons after injury or amputation , natural large-scale skeletal repair in mammals is more limited .",
"A notable exception is the rib .",
"Craniofacial surgeons often extract large segments of rib cartilage and bone for autologous repair of skeletal defects in other parts of the body , and in post-operative visits have noted extensive regeneration at the donor rib site ( Kawanabe and Nagata , 2006; Munro and Guyuron , 1981; Srour et al . , 2015 ) .",
"To better understand the unique regenerative potential of the rib , we have recently developed analogous large-scale rib cartilage and bone regeneration models in the mouse ( Srour et al . , 2015; Tripuraneni et al . , 2015 ) .",
"In most mammalian bones , healing involves the formation of a callus using a mixture of direct ossification and endochondral ossification via a cartilage callus intermediate ( Gerstenfeld et al . , 2003; Hall , 2014; Marsell and Einhorn , 2011 ) .",
"The identity and regulation of the adult skeletal progenitors that build the callus remain incompletely understood .",
"While markers for a number of different skeletal stem cells have been reported ( Balani et al . , 2017; Bianco and Robey , 2015; He et al . , 2017; Matthews et al . , 2014; Park et al . , 2012; Ransom et al . , 2016; Shi et al . , 2017 ) , their relative roles during bone repair are less clear , particularly in cases of large-scale bone regeneration such as in the rib .",
"During skeletal repair , studies have shown that cells from the periosteum , a heterogeneous connective tissue sheath covering the bone , are major contributors to the callus ( Colnot , 2009; Duchamp de Lageneste et al . , 2018; Murao et al . , 2013 ) .",
"Accordingly , we have found that the periosteum is essential for regeneration of the rib bone ( Tripuraneni et al . , 2015 ) .",
"During normal bone homeostasis , periosteal stem cells generate bone-producing osteoblasts but not cartilage-producing chondrocytes ( Roberts et al . , 2015 ) .",
"How injury stimulates periosteal stem cells to generate chondrocytes is unclear .",
"Although some fractures can heal in the absence of a cartilage callus , for example when the fracture is rigidly stabilized ( Thompson et al . , 2002 ) , the formation of a large cartilage callus appears to be required in large-scale bone regeneration .",
"Unfortunately little is known about the specific periosteal progenitor population that drives the formation of the cartilage callus nor the signaling pathways required .",
"Recent studies have used Cre-based lineage tracing experiments to show that cells marked by expression of Gremlin1 ( Worthley et al . , 2015 ) , Axin2 ( Ransom et al . , 2016 ) , Gli1 ( Shi et al . , 2017 ) , Act2a ( Matthews et al . , 2016 ) , Periostin ( Duchamp de Lageneste et al . , 2018 ) and Sox9 ( Balani et al . , 2017; He et al . , 2017 ) can be found in the periosteum and contribute to the fracture callus during repair .",
"Other than participation , the specific role of any of these progenitor population remains unclear .",
"In this study , we therefore focus on the role of one subpopulation within the periosteum and its specific role in driving callus formation and bone regeneration .",
"As Sox9 has a well-known function in promoting chondrogenesis during embryonic development ( Akiyama et al . , 2002; Lefebvre et al . , 1997 ) , we postulated that Sox9-expressing cells in the adult periosteum may possess a potent ability to form or promote cartilage in response to bone injury .",
"In addition , a requirement for Sox9-expressing cells in repair or homeostasis has not yet been established .",
"The signaling pathways required for generating a cartilage callus from the periosteum in response to injury are also not well-characterized .",
"The formation of the cartilage callus in fractures has been thought to involve a recapitulation of endochondral bone development ( Bahney et al . , 2014; Maes et al . , 2010; Marsell and Einhorn , 2011; Yang et al . , 2014; Zhou et al . , 2014 ) .",
"One of the most well-known signaling pathways during endochondral bone development is the Hh pathway , activation of which promotes chondrogenic proliferation and osteocyte maturation ( Long et al . , 2001; Shi et al . , 2017; St-Jacques et al . , 1999 ) .",
"Studies have investigated Hh signaling during fracture repair , but due to conflicting results it remains unclear if Hh signaling has the same role in bone repair as it does during bone development .",
"Genetic ablation of the obligate Hh co-receptor Smo in mice , using two different ubiquitously inducible Cre lines , resulted in reduced bone formation during fracture repair , yet was not reported to disrupt initial cartilage callus formation ( Baht et al . , 2014; Wang et al . , 2010 ) .",
"Forced activation of Hh signaling throughout the mouse during fracture repair , using an inducible constitutively active Smo allele , resulted in increased bone formation ( Baht et al . , 2014 ) , similar to what was seen upon engraftment of cells overexpressing Hh or treatment with an Hh agonist ( Edwards et al . , 2005; Huang et al . , 2014; Zou et al . , 2014 ) .",
"However , on which cell types Hh acts upon , and whether it regulates the decision to build the cartilage callus and/or other aspects of bone repair in mammals , has remained unknown .",
"In this study we examine the role of the Sox9-expressing periosteal subpopulation ( referred to as Sox9+ cells hereafter ) during large-scale rib repair .",
"This subpopulation appears to be a key player in orchestrating the formation of a large repair callus in the rib that consists of an unusual hybrid osteochondral cell type with properties of both chondrocytes and osteoblasts .",
"Loss of Smo in Sox9+ periosteal cells prior to injury results in a near-complete failure of cartilage callus formation and bone regeneration .",
"This Sox9+ subpopulation must be able to respond to Hh signaling in order to initiate this process , indicating that Hh signaling’s role in bone repair is distinct from its role in bone development .",
"Additionally , since Sox9+ periosteal cells contribute to only a minority of callus cells , we suggest that Sox9+ periosteal cells act as ‘messenger’ cells and orchestrate repair by inducing the differentiation of neighboring callus cells through non-autonomous signals .",
"Overall our results indicate that bone regeneration does not fully recapitulate bone development , and that the periosteum consists of subpopulations that may have different roles/responses during repair ."
],
[
"Like appendicular long bones , the bony portion of the rib develops via an endochondral process including growth plates at either end and a central hollow bone marrow cavity .",
"Both human and murine rib bones display remarkable regenerative potential ( Srour et al . , 2015; Tripuraneni et al . , 2015 ) , however the cellular basis for such large-scale repair remains unknown .",
"To better understand the cellular sequence of events during regeneration , we analyzed 3 mm rib bone defects at sequential time points up to 10 weeks post-resection ( wpr ) ( Figure 1A ) .",
"Histology at 5 days post-resection ( dpr ) revealed cells with a mesenchymal-like morphology filling the entire resected region ( Figure 1B ) .",
"We then observed formation of a substantial alcian-blue positive callus spanning most of the defect by 1 wpr ( Figure 1A ) , with many of these cells displaying a cartilage-like morphology at 10 dpr ( Figure 1C ) .",
"Histology revealed increasing bone formation by 10 and 14 dpr ( Figure 1C , D ) , with extensive alizarin-positive mineralization across the defect at 4 wpr and full remodeling to the pre-injury organization by 10 wpr ( Figure 1A ) .",
"Next , we used double fluorescent RNA in situ hybridization ( RNA-ISH ) to characterize the molecular identity of cells during the regeneration process .",
"At 5 dpr , mesenchymal cells within the lesion co-expressed Sox9 , a master regulator of the chondrocyte lineage ( Akiyama et al . , 2002; Lefebvre et al . , 1997 ) , and Runx2 , a master regulator of the osteoblast lineage ( Lian and Stein , 2003 ) ( Figure 1B ) .",
"While co-expression of chondrocyte and osteoblast markers has been observed during early bone development , normally as differentiation proceeds , a cell will express only chondrocyte or only osteoblast markers .",
"Surprisingly , we observed co-expression of genes associated with chondrocyte and osteoblast differentiation within single cells throughout the repair process .",
"For example , we see co-expression of Sox9 with the major osteoblast collagen gene Col1a1 ( Figure 1—figure supplement 1A ) .",
"As the callus matures bone and cartilage markers continue to be co-expressed at high levels .",
"The major chondrocyte collagen gene Col2a1 was observed to be co-expressed with the late osteoblast marker Bglap ( also known as Osteocalcin ) ( Figure 1C and Figure 1—figure supplement 1C ) , and with Col1a1 at both 10 and 14 dpr ( Figure 1D and Figure 1—figure supplement 1B ) .",
"We observed co-expression of chondrocyte and osteoblast markers in cells of both cartilage and osteoblast morphology , including cells on the surface of newly formed trabecular bone ( Figure 1C , D , Figure 1—figure supplement 1C ) .",
"Similar co-expression results were obtained using mice double transgenic for a reporter of hypertrophic chondrocytes ( Col10a1-mCherry ) and osteoblasts ( Col1 ( 2 . 3 ) -GFP ) as well as using double immunofluorescence for COL1 and COL2 protein ( Figure 1E , Figure 1—figure supplement 1D ) .",
"In contrast , we did not observe co-expression of Col2a1 and Col1a1 in the rib growth plate under the same assay conditions ( Figure 1F ) .",
"Single colorimetric RNA-ISH assays of near-adjacent sections at 7 dpr confirmed co-expression of Sox9 , Runx2 , Col2a1 , Col1a1 , Bglap , and the hypertrophic cartilage collagen gene Col10a1 in cells of cartilage morphology within the callus ( Figure 1—figure supplement 2 ) .",
"Together , these findings indicate that , in marked contrast to the growth plate , cells within the rib repair callus co-express cartilage and bone markers while displaying either a chondrocyte or osteoblast morphology .",
"We therefore refer to these cells as ‘hybrid’ osteochondral skeletal cells .",
"We next examined the source of the skeletal cells that mediate repair .",
"These cells are likely derived from the periosteum , since removal of the periosteum along with the bone , results in a failure of cartilage callus formation ( Figure 2—figure supplement 1 ) and subsequent bone repair ( Tripuraneni et al . , 2015 ) .",
"Sox9 is a master regulatory gene of chondrogenesis , and a previous study indicated that Sox9+ cells in the femur periosteum can contribute to callus formation after fracture ( He et al . , 2017 ) .",
"We therefore examined whether Sox9-expressing periosteal cells contribute to the repair callus during rib regeneration .",
"To do so , we administered three consecutive daily doses of tamoxifen to Sox9-CreERT2; ROSA26-loxP-stop-loxP-tdTomato mice , followed by a 4-day chase to allow clearance of residual tamoxifen ( Figure 2A , “Pre” regimen ) .",
"In the absence of injury , we observed that Sox9-expressing cells were predominantly found in the periosteum and but only constituted 6 ± 0 . 3% of the population , with almost no labeled cells seen in the marrow compartment ( Figure 2B ) .",
"After rib resection , we found that 22 ± 1 . 3% of callus cells were tdTomato+ by 10 dpr with many of these cells having a typical chondrocyte morphology ( Figure 2C ) .",
"At 14 dpr ( Figure 2C ) , we observed tdTomato expression in both chondrocyte-like cells , as well as osteoblast-like cells lining new trabecular bone ( as defined by near-adjacent H&E sections ) , with contribution to osteoblast-like cells also evident by 21 dpr ( Figure 2—figure supplement 2A ) .",
"We observed fewer tdTomato+ cells at 14 dpr suggesting that as the callus matures , Sox9+ lineage cells are remodeled out and replaced by non-Sox9+ lineage cells .",
"Pre-existing -expressing cells , thus contribute to only a minority of the cells that form the initial repair callus , including only a subset of the callus cells with hybrid osteochondral characteristics .",
"Most of the cells in the rib callus are therefore derived from a lineage that did not express Sox9 prior to injury .",
"In addition , similar to reports by He et al . ( He et al . , 2017 ) we found that also after femur fracture , our “Pre” tamoxifen regimen revealed contribution of pre-existing Sox9+ cells to the callus ( Figure 2—figure supplement 3A ) .",
"We further found that some of the cells within the femur fracture callus also co-expressed Col1a1 and Col2a1 strongly , suggesting a similar hybrid identity to that observed in the rib callus ( Figure 2—figure supplement 3B ) .",
"Since only a minority of the callus cells are derived from the Sox9-expressing periosteal subpopulation , as opposed to the majority of the callus cells that expresses Sox9 mRNA at 5 dpr ( Figure 1B ) , we instead applied tamoxifen at the time of injury plus 2 days following ( “Post” regimen , Figure 2D ) .",
"This regimen will capture both the subpopulation that expresses Sox9 prior to injury as well as any cells that turn on Sox9 in response to injury .",
"Consistent with detection of endogenous Sox9 mRNA expression in early callus cells ( e . g . Figure 1B ) , we found that this later tamoxifen regimen labeled the majority of callus cells by 10 dpr ( 85 . 5 ± 2 . 8% ) and most of the osteoblast-like cells lining newly forming trabecular bone at 14 ( Figure 2E ) and 28 dpr .",
"Further , typical of osteocytes , some of the tdTomato+ cells at 28 dpr were found embedded in bone ( Figure 2—figure supplement 2B ) .",
"Thus , depending on when tamoxifen is administered ( Pre vs . Post ) , either a minority or majority of the callus cells are labelled .",
"Since we hypothesized that the Hh pathway may be important for large-scale repair , we first determined the expression of Hh ligands and Ptch1 after rib resection .",
"As expected , based on the expression of Ihh in the growth plate , callus cells with cartilage morphology expressed Ihh and Ptch1 .",
"Interestingly we also detected the related ligand , Shh , in these cells ( Figure 3A and C ) .",
"At earlier stages Ihh expression was undetectable until 7 dpr and then only at very low levels in developing chondrocytes .",
"However , Shh expression was readily detectable at 5 dpr and 7dpr similar to reports by others ( Matsumoto et al . , 2016 ) although at lower levels than found in cells with cartilage morphology ( Figure 3A and C ) .",
"Ptch1 , a read-out of the Hh pathway , was also detected in tdTomato+ periosteal cells a 3 dpr , supporting the idea that these cells are responsive to a Hh signal prior to entering the callus ( Figure 3—figure supplement 1A ) .",
"We then tested the requirement for Hh signaling in large-scale bone regeneration by deleting Smo , the required Hh co-receptor , using the Sox9-CreERT2 transgene and employing either the Pre or Post tamoxifen treatment regimens .",
"Using Smo RNA-ISH , we observed that the Post tamoxifen treatment resulted in deletion of Smo broadly ( Figure 3B ) as predicted given that Cre is active in most cells ( Figure 2D ) .",
"Pre tamoxifen treatment resulted in deletion of Smo in the Sox9-positive lineage subpopulation which were marked by including the tdTomato reporter in the cross ( Figure 3B ) .",
"This was expected , since deletion of Smo occurred prior to surgery and thus only a minority of the cells in the repair callus are expected to be null for Smo ( Figure 2C ) .",
"We then examined the callus at 7 dpr while there is a mix of both immature and differentiating cells .",
"In control Pre tamoxifen treatment animals , tdTomato+ cells could be found scattered across the developing callus and were not preferentially found in clusters of differentiating chondrocytes suggesting that Sox9+ cells do not differentiate ahead of other cells in the callus ( Figure 3—figure supplement 1B ) .",
"In addition , in regions of the control callus where cells appeared immature and in Pre tamoxifen treatment calluses , there was no strong correlation between the tdTomato trace and the expression of Shh , or read-outs of Hh signaling such as Ptch1 or Gli2 ( Pak and Segal , 2016 ) suggesting that in contrast to our earlier observations in the periosteum , that at these later stages , tdTomato cells are not strongly responding to a Hh signal ( Figure 3C and Figure 3—figure supplement 1C ) .",
"Furthermore , in cells neighboring Tdtomato+ cells , we did not see any enrichment of Ptch1 or Gli2 expression suggesting that they were not receiving a potent Hh signal that could be released by Tdtomato+ cells .",
"We then investigated the consequence of Smo deletion on cartilage callus formation .",
"Surprisingly , Smo deletion using either the Pre or Post tamoxifen treatment resulted in a similarly near-complete loss of the cartilage callus at both 7 and 10 dpr ( Figure 4A , Figure 4—source data 1 ) , despite the Pre treatment only deleting Smo in a small subset of callus progenitors ( Figures 2C and 3B ) .",
"After Smo deletion , although cells did not form a mature callus , they still co-expressed Sox9 and Runx2 at 5 dpr ( Figure 4—figure supplement 1A ) .",
"At 10 dpr , in contrast to co-expression of Col2a1 and Col1a1 throughout the control callus , the few Col1a1-expressing cells that formed after Smo deletion lacked high levels of Col2a1 and vice versa , demonstrating a lack of hybrid osteochondral cells ( Figure 3B ) .",
"The lack of callus formation and hybrid cells following Smo deletion was not reflected by altered numbers of pHH3+ proliferative or TUNEL+ apoptotic cells at either 7 or 10 dpr , and lineage tracing with the tdTomato reporter revealed similar numbers of cells within the resection site at 10 dpr ( Figure 4C , Figure 4—source data 2 , Figure 4D , Figure 4—source data 3 , Figure 4—figure supplement 1B , Figure 4-figure supplement-source data 1 ) .",
"Thus , Hh signaling is likely not required for the early proliferative expansion in response to injury or for cell survival .",
"Instead , we conclude that Hh signaling is required in Sox9+ cells for promoting the differentiation of non-Sox9 lineage cells into hybrid osteochondral cells of the callus .",
"When the Sox9+ Pre population is null for Smo , non-Sox9 lineage cells are still unable to differentiate ( despite expressing Ptch1 ) resulting in the near complete failure of callus formation .",
"Thus , this minority Sox9+ periosteal lineage population plays a critical initiating role in callus differentiation .",
"We next assessed the consequence of defective callus formation on subsequent regeneration of rib bone in Smo-deleted animals .",
"In contrast to control animals showing robust Col1a1 and Col2a1 co-expression in cells lining new trabecular bone at 14 dpr , deletion of Smo using either the Pre or Post regimens resulted in a near complete absence of co-expressing cells .",
"Instead , only small numbers of Col2a1-only cells were observed , primarily near the cut ends of the bone , while cells with osteoblast morphology expressed predominantly only Col1a1 ( Figure 5A ) .",
"Analysis of H and E-stained histological sections confirmed a marked decrease in bone formation at 14 dpr , despite substantial mesenchyme still observed in the resection site , with the magnitude of the bone defect similar in both regimens ( Figure 5B ) .",
"We found that ‘Late’ removal of Smo ( injection of tamoxifen at 3–5 dpr targeting the whole callus ) only caused a slight delay in repair with a fully bridged callus evident at 14 dpr ( Figure 5—figure supplement 1 ) suggesting that the decrease in bone formation seen in both the Pre and Post regimens is largely related to reduced cartilage callus formation .",
"Whole-mount staining with alizarin red and alcian blue confirmed a failure of bone union at 4 and 6 wpr in both Pre and Post conditions ( Figure 5C ) .",
"These findings demonstrate that the Sox9+ subpopulation requires Hh signaling not only to build the repair callus but that a substantial cartilage callus may be needed to efficiently regenerate bone ."
],
[
"We find that building an extensive callus of a unique type of hybrid osteochondral skeletal cell is essential for successfully bridging large gaps in adult mammalian rib bone .",
"During this process , Hh signaling plays a critical role , distinct from that in the developing growth plate , in promoting the ultimate differentiation of these hybrid osteochondral skeletal cells .",
"Further , we provide genetic evidence that Hh signaling acts upon a rare periosteal subpopulation of Sox9-expressing cells , that behave as messenger cells .",
"These Sox9+ periosteal cells then stimulate , through a yet to be determined signal , their neighboring non-Sox9-lineage cells , which constitute a majority of the callus , to differentiate and build new callus and bone ( summarized in Figure 6 ) .",
"We observe high-level co-expression of genes typically associated with cartilage ( Sox9 , Col2a1 , Col10a1 ) or bone ( Col1a1 , Bglap ) in callus cells during murine rib bone regeneration .",
"This is in marked contrast to the largely exclusive expression of cartilage versus bone genes in the developing growth plates , although hypertrophic chondrocytes do express low levels of many bone-associated genes ( Gerstenfeld and Shapiro , 1996 ) .",
"The trajectory of hybrid cells is also fundamentally different from the subpopulation that is proposed to ‘transdifferentiate’ from chondrocytes to osteoblasts in the growth plate ( Bahney et al . , 2014; Shimomura et al . , 1975; von der Mark and von der Mark , 1977; Yang et al . , 2014; Zhou et al . , 2014 ) .",
"In the growth plate , these transdifferentiating cells express chondrocyte-associated genes first and then , through a process that remains mysterious , turn off chondrocyte-associated genes and turn on osteoblast-associated genes .",
"Here , we confirm a similar process of ‘first cartilage and then bone’ in the rib growth plate .",
"In contrast , in the regenerating rib , we observe that callus cells co-express cartilage- and bone-associated genes at the earliest stages .",
"We propose that they then maintain this hybrid osteochondral identity as they shift from making a cartilaginous and then a bone-like matrix .",
"We also observe a similarly hybrid osteochondral cell in the femur fracture callus , although further investigations will be required to determine how similar these cells are between the rib and femur callus .",
"Moreover , similar hybrid cells have been reported during zebrafish jawbone repair ( Paul et al . , 2016 ) suggesting that skeletal cells with dual chondrocyte/osteoblast properties may be critical for bone regeneration across vertebrate species .",
"Co-expression of bone and cartilage programs in the same cell is certainly not unique to the regenerating callus .",
"For example , Col1a1 and Col2a1 are also highly expressed in fibrocartilage cells , however this tissue is morphologically quite different in terms of its high fiber to cell ratio when compared to the regenerating rib callus ( Benjamin and Ralphs , 2004 ) .",
"Of note , a rare developmental skeletal type has been described , historically referred to as ‘chondroid bone’ or ‘secondary cartilage’ , that does share many features with the hybrid osteochondral cells we observe during regeneration ( Goret-Nicaise , 1984; Shibata and Yokohama-Tamaki , 2008 ) .",
"Therefore , it may be that these cells are not unique to regeneration , but rather are selectively utilized due to their ability to rapidly proliferate in a relatively avascular environment ( a property of cartilage [Dennis et al . , 2015] ) while directly producing mineralized matrix ( a property of bone ) .",
"Whereas some bone does form in our Smo-deleted mice , this is not sufficient to bridge the lesion and healing fails , resulting in a persistent non-union .",
"Cells within this bone do not display hybrid chondrocyte/osteoblast character , consistent with residual bone forming by direct ossification rather than ossification through a callus intermediate .",
"These findings suggest that during large-scale bone regeneration , Hh signaling in Sox9-expressing periosteal cells is selectively required for the formation of a hybrid osteochondral callus .",
"Hh signaling may play an important but more subtle role in osteocyte maturation as fractures still heal in the absence of Smo but with decreased or delayed bone formation ( Baht et al . , 2014; Wang et al . , 2010 ) .",
"Similarly , we see production of bone ( although delayed ) in our rib resection model with a Late KO of Smo ( Figure 5—figure supplement 1 ) .",
"In contrast to our rib model , however , loss of Smo in these femur/tibia fracture assays still resulted in the formation of a cartilage callus .",
"We do not know why these results contrast from ours where cartilage callus formation is dramatically compromised .",
"One possibility is that the efficacy of Smo removal in these fracture experiments was not efficient during cartilage callus stages , alternatively , there could be different requirements for Hh signaling depending on the type of injury ( resection vs . fracture ) .",
"Interestingly , the formation of a cartilage callus may not be as critical during fracture repair , as fractures can repair solely through direct ossification ( Colnot et al . , 2003 ) .",
"While in contrast , during large-scale repair , the process of direct ossification is not sufficient to build large pieces of bone and instead , building a hybrid osteochondral callus may be particularly important for bridging large bone gaps .",
"Hh signaling is known to promote chondrocyte proliferation and osteoblast differentiation in the developing growth plate ( Long et al . , 2004; Long et al . , 2001 ) .",
"Surprisingly , we found that loss of Hh signaling did not affect the early proliferation of callus cells or the differentiation of osteoblasts in the residual directly ossifying bone .",
"Instead , we provide evidence that Hh signaling has a distinct and essential role in promoting the differentiation of Sox9/Runx2 expressing progenitors into the hybrid osteochondral skeletal cells that form the repair callus .",
"These results indicate that the regeneration of the rib bone , including its dependence on Hh signaling , does not simply the recapitulate developmental processes seen at the growth plate .",
"While the heterogeneity of the cells in the periosteum and their lineage relationships remain incompletely understood , we propose that Sox9+ periosteal cells or a subpopulation within them , play an essential instructive role for callus formation during large-scale bone regeneration that has not been previously described in the context of repair .",
"The Sox9+ periosteal subpopulation may be distinct from the previously described subpopulations that are Gli1+ , αSMA+ , Gremlin1+ , or Axin2+ , as RNA-seq analysis of the periosteum from an uninjured femur indicated that Gli1 , αSMA , Axin2 , and Gremlin1 are not highly expressed in the Sox9+ cells ( He et al . , 2017 ) .",
"Future studies tracing the lineage of these other populations and determining their dependence on Hh signaling will be needed to resolve whether Sox9+ cells represent a subset of one of these other more abundant populations , or alternatively a distinct progenitor subpopulation .",
"In addition , efforts to delineate potential differences that may exist in periosteal populations from different bones , may explain why some bones regenerate well , while others do not .",
"One of the most striking findings from our study is that Sox9+ cells are essential for efficient callus formation and rib bone regeneration , despite contributing to only a minority of cells within the callus and regenerated bone .",
"Based on this observation , we propose that the Sox9+ cells act as ‘messenger’ cells by releasing a yet to be determined signal that promotes the differentiation of neighboring cells Sox9-negative lineage cells .",
"One possibility is that Sox9+ progenitors differentiate early in response to the initial Hh signal ( potentially Shh , since strong expression is evident early ) and then propagate another wave of Hh signaling to neighboring Sox9-negative cells , similar to the role of Hh in the morphogenetic furrow during Drosophila eye development ( Domínguez and Hafen , 1997; Kumar , 2011; Ma et al . , 1993 ) .",
"The failure of Sox9+ cells to differentiate and thus express Shh and Ihh , could then lower the total Hh signal in the callus to below a critical threshold need for cartilage differentiation .",
"However , our results suggest that Sox9+ cells are not the first to differentiate and while they likely respond to a Hh signal early in the periosteum , they are likely not strong propagators of a further Hh signal as cells nearby Sox9+ lineage cells do not display a strong upregulation of Hh pathway read-outs as the callus differentiates .",
"While a response to Hh signaling may require more sensitive assays , we found the expression of both Ptch1 and Gli2 to be very low in undifferentiated cells at 7 dpr in both control and Pre Smo KO calluses Figure 3C and Figure 3—figure supplement 1 ) suggesting that they were not receiving a potent secondary Hh signal .",
"In addition , the expression of Shh was not particularly strong in the tdTomato+ cells at this stage ( Figure 3C ) .",
"Thus , we instead favor a hypothesis , that in response to Hh signaling Sox9+ cells emit another to-be-identified relay signal .",
"This signal then orchestrates repair by promoting the differentiation of neighboring cells from a non-Sox9+ lineage into mature matrix-producing hybrid osteochondral skeletal cells that bridge the lesion ( Figure 6 ) .",
"It is possible that Hh signaling may have an additional role in later stages of repair ( osteocyte differentiation ) , but our study supports a critical role early in large-scale rib repair .",
"Future investigations into factors produced by Sox9-lineage cells that promote callus formation may lead to better strategies of boosting bone repair in other parts of the body that do not heal as effectively ."
],
[
"All procedures were approved by the University of Southern California Institutional Animal Care and Use Committee ( Protocol #: 11256 , 20639 ) .",
"We used the following mouse lines: Sox9-CreERT2 ( Sox9tm1 ( cre/ERT2 ) Haak [Soeda et al . , 2010] ) , R26R-tdTomato ( B6;129S6-Gt ( ROSA ) 26Sortm9 ( CAG-tdTomato ) Hze/J; JAX 007905 ) , Smofl/fl ( Smotm2Amc/J; JAX 004526 , Col1a1 ( 2 . 3-GFP ) ( B6 . Cg-Tg; Col1a1*2 . 3-GFP ) 1Rowe/J;JAX 013134 ) , Col10a1-mCherry ( Maye et al . , 2011 ) .",
"To generate Sox9-CreERT2;tdTomato;Smofl/fl mice , Sox9-CreERT2;tdTomato males were crossed to Smofl/fl females and Sox9CreERT2;tdTomato;Smofl/+ offspring males were back-crossed to Smofl/fl females to generate Sox9-CreERT2;tdTomato;Smofl/fl offspring .",
"Both male and female mice between 6–8 weeks old were used for experiments .",
"Control mice were uninduced siblings or tamoxifen-induced Sox9-CreERT2;tdTomato mice .",
"Rib resections were performed as previously described ( Tripuraneni et al . , 2015 ) with the modification that a bone segment of 3 mm was removed and that post-operative pain was managed with buprenorphine SR ( ZooPharm ) at a dose of 0 . 5 uL/gram .",
"Rib repair was assessed after set healing time points: 0 dpr – 10 wpr .",
"To induce Cre recombination , 100 uL of a 20 mg/ml stock of Tamoxifen ( Sigma-Aldrich: T5648-1G , dissolved in corn oil at 60°C for 2 hr ) was used per injection .",
"Tamoxifen injections were administered intraperitoneally using a 25-gauge needle .",
"Two injection schedules were used:",
"1 ) three consecutive injections starting 7 days before resection ( 'Pre' regimen ) ,",
"2 ) four consecutive injections starting 1 day prior to surgery ( 'Post' regiment ) .",
"Uninduced controls were injected with corn oil only .",
"Femur fracture assays were carried out as previously described ( He et al . , 2017 ) .",
"Samples were fixed with 4% PFA overnight at room temperature , decalcified with 20% ETDA at pH 7 . 5 for 10–14 days , and then processed for paraffin embedding .",
"A microtome ( Shandon Finesse Me+: 77500102 ) was used to cut paraffin sections seven microns thick .",
"The sections were mounted on Superfrost Plus slides ( VWR , 48311–703 ) .",
"After deparaffinizing slides in Citrisolv and rehydrating , H and E or Safranin O staining was carried out using standard protocols .",
"Skeletal staining was performed on EtOH-fixed samples ( Rigueur and Lyons , 2014 ) .",
"To visualize native tdTomato fluorescence , samples were fixed in 4% PFA on ice for 30 min and placed in 30% sucrose overnight at room temperature .",
"The samples were embedded in OCT and flash frozen in an EtOH dry ice bath .",
"10 µM thick sections were cut using a Leica CM3050 S cryostat .",
"Tape ( cryofilm type 3C ( 16UF ) C-FUF303 ) was used to preserve the histology of the bone ( Kawamoto , 2003 ) .",
"OCT was removed with a 1xPBS wash before mounting .",
"Detection of pHH3 and Tdtomato proteins was carried out on paraffin sections .",
"Slides were de-waxed and cells were permeabilized with 0 . 1% Triton-X followed by antigen retrieval in 10 mM sodium citrate , 0 . 05% Tween 20 , pH of 6 . 0 in a 95°C water bath for 30 min .",
"Slides were blocked in 20% serum for 1 hr and then incubated with the primary antibodies overnight at 4°C ( anti-pHH3 , Millipore: 06–570 , 1:200; anti-mCherry which also detects tdTomato , Novus Biological: NBP2-25158SS , 1:200; anti-Col1 , Abcam: ab34710 , 1:250; anti-Col2 , Southern Biotech: 1320–01 , 1:200 ) .",
"Secondary antibodies used were: Alexa Fluor 568 goat anti-rabbit ( ThermoFisher: A-11011 , 1:250 ) , Alexa Fluor 568 goat anti-chicken ( Abcam: ab175477 , 1:500 ) , and Alexa Fluor 488 donkey anti-goat ( Abcam:ab150129 , 1:500 ) .",
"To detect apoptosis , the In Situ Cell Death Detection Kit , Fluorescein ( Sigma-Aldrich: 11684795910 ) was used as directed .",
"Fluorescent and colorimetric RNA in situ hybridization ( RNA-ISH ) was performed on 7 µm paraffin sections as previously described ( Paul et al . , 2016 ) .",
"Complementary DIG or FL labeled RNA probes were generated following kit instructions ( Sigma-Aldrich: 11277073910 and 11685619910 ) and were detected with Anti-Digoxigenin-POD ( Sigma-Aldrich: 11207733910 ) and Anti-Fluorescein-POD ( Sigma-Aldrich: 11426346910 ) .",
"For double fluorescent RNA-ISH the TSA Cyanine three and Fluorescein system from Perkin Elmer was used as directed ( NEL753001KT ) .",
"For colorimetric RNA-ISH Anti-Digoxigenin-AP ( Sigma-Aldrich: 11093274910 ) was used to detect the probes .",
"Probes were generated to the following sequences: Slides with fluorescence were mounted with Vectashield with DAPI ( Vector Laboratories: H1200 ) and were imaged with a Nikon AZ100 Macroscope and photographed ( Nikon Digital sight DS-Fi1 ) .",
"Fluorescent images were edited for contrast and color levels in Adobe Photoshop CS5 .",
"For quantification , all images were taken at the same magnification for each data set .",
"Student’s t-test was used to compare groups .",
"A probability value of 0 . 05 or less was marked as significant .",
"Each data point was plotted on the scatter plot and the mean was defined on the graph , unless the data set was normalized .",
"Statistical tests were performed using GraphPad .",
"To determine the amount of cartilage , a mid-sagittal section through both mutant and control sample was stained with Safranin O and quantified in ImageJ .",
"In brief , the image was thresholded for the Safranin O color ( orange ) and the area of the color was measured .",
"To determine bone area , samples were stained with H and E and analyzed with the BioQuant image analysis program .",
"The bone area was compared to the entire resected area .",
"The values were normalized within each time point .",
"Quantification of the number of cells expressing pHH3 was carried out using the analyze particle function in ImageJ with the repair callus defined as the area of interest .",
"The red channel was used to count the number of cells positive for pHH3 while the blue channel was used to count the total number of cells ( nuclei stained with DAPI ) .",
"The ratio of pHH3 positive cells to total cells was used to calculate the percentage of cells in mitosis .",
"This method was also used to quantify the number of tdTomato+ cells within the callus .",
"To quantify apoptosis , Adobe Photoshop CS5 was used to analyze the green channel .",
"The number of green pixels compared to the total number of blue pixels ( DAPI ) in the region of interest was calculated .",
"Analysis of pHH3 and TUNEL positivity was done on de-identified images by several laboratory members ."
]
] | [
"Most bones in mammals display a limited capacity for natural large-scale repair .",
"The ribs are a notable exception , yet the source of their remarkable regenerative ability remains unknown .",
"Here , we identify a Sox9-expressing periosteal subpopulation that orchestrates large-scale regeneration of murine rib bones .",
"Deletion of the obligate Hedgehog co-receptor , Smoothened , in Sox9-expressing cells prior to injury results in a near-complete loss of callus formation and rib bone regeneration .",
"In contrast to its role in development , Hedgehog signaling is dispensable for the proliferative expansion of callus cells in response to injury .",
"Instead , Sox9-positive lineage cells require Hh signaling to stimulate neighboring cells to differentiate via an unknown signal into a skeletal cell type with dual chondrocyte/osteoblast properties .",
"This type of callus cell may be critical for bridging large bone injuries .",
"Thus despite contributing to only a subset of callus cells , Sox9-positive progenitors play a major role in orchestrating large-scale bone regeneration .",
"Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review .",
"The Reviewing Editor's assessment is that all the issues have been addressed ( see decision letter ) ."
] | [
"Fractures to major bones often heal slowly or incompletely , especially in older people , and large bone injuries do not repair naturally .",
"By comparison , rib bones show an unusual capacity to regrow and repair themselves even when a large portion is damaged .",
"Previous research suggests that the connective tissue around the ribs helps to support and co-ordinate bone healing .",
"Yet it is currently not clear why ribs have a greater capacity to repair these large injuries compared to other bones .",
"Kuwahara et al . have now examined bone repair by studying how ribs heal in mice .",
"The experiments show that around 6% of the connective tissue cells are critical for large-scale repair .",
"Kuwahara et al . named these cells messenger cells .",
"These cells detect the presence of a signal molecule called Hedgehog ( Hh ) , however , if these cells lose the ability to respond to the Hh molecules , the ribs do not heal properly .",
"Further examination revealed that these messenger cells co-ordinate repair by encouraging other cells to build a special kind of bone-healing tissue with hybrid properties of both cartilage and bone .",
"Further research could now examine how messenger cells co-ordinate healing and if their properties could be adapted to help repair other bones .",
"Ultimately , understanding how messenger cells work may even provide insights into new ways to repair and regenerate other tissues and organs too ."
] | 2019 |
[
"Introduction",
"Results and discussion",
"Materials and methods"
] | [
"plant biology",
"structural biology and molecular biophysics"
] | The structure of plant photosystem I super-complex at 2.8 Å resolution | elife-07433-v2 | [
[
"Oxygenic photosynthesis , in which the conversion of sunlight into chemical energy by plants , green algae , and cyanobacteria , occurs , underpins the survival of virtually all life forms .",
"By producing oxygen and assimilating carbon dioxide into organic matter , this process determines , to a large extent , the composition of our atmosphere and provides essential food and fuel .",
"Light photons are captured by pigments in very large membrane–bound complexes and the excitation energy is utilized to form NADPH and ATP .",
"Two reaction centers , photosystem II ( PSII ) and photosystem I ( PSI ) , drive this electron transport chain .",
"PSII oxidizes water to produce oxygen and reduce membrane-embedded quinones .",
"The reduced quinones are then utilized by the cytochrome b6f complex to produce a proton gradient across the membrane and to reduce the small copper protein plastocyanin ( PC ) , the electron donor of PSI .",
"After an additional photon is absorbed by any of the 200 antenna pigments of PSI , its energy migrates through this large network of connected pigments and eventually oxidizes P700 , a special chlorophyll pair located at the center of PSI .",
"The electron removed from P700 by this oxidation event migrates along an internal electron transport chain and finally reduces ferredoxin ( Fd ) , the final electron acceptor of PSI .",
"Reduced Fd is utilized by several cellular pathways , mainly the reduction of NADP to NADPH .",
"This NADPH and ATP generated by the thylakoid ATP-synthase complex powers the Calvin cycle to produce carbohydrates .",
"Oxygenic photosynthesis evolved over 3 billion years ago in cyanobacteria ( Blankenship , 1992; Barber , 2004; Nelson , 2013 ) .",
"Later , approximately 1 . 5 billion years ago , the first photosynthetic eukaryotes appeared , eventually evolving into land plants roughly 0 . 5 billion years ago .",
"The basic building blocks of photosynthesis are remarkably conserved .",
"The architectures of both photosystems have been determined by numerous techniques , but X-ray crystallography has provided the most detailed structural information for the four large membrane complexes catalyzing oxygenic photosynthesis ( Nelson and Ben-Shem , 2004; Nelson and Yocum , 2006; Croce and van Amerongen , 2013; Nelson and Junge , 2015 ) .",
"Structures at the highest resolution has been obtained for thermophilic cyanobacteria , but representative structures from eukaryotic chloroplasts , especially plants , are scarce ( Jordan et al . , 2001; Ben-Shem et al . , 2003; Kurisu et al . , 2003; Stroebel et al . , 2003; Ferreira et al . , 2004; Loll et al . , 2005; Amunts et al . , 2007 , 2010; Umena et al . , 2011 ) .",
"Here , we report the structure of plant PSI super-complex at high resolution ."
],
[
"The crystal structure of plant PSI was first reported at 4 . 4 Å resolution ( Ben-Shem et al . , 2003 ) and has been improved up to 3 . 3 Å resolution in the last decade ( PDB 2WSC ) .",
"This PSI preparation was limited to pea plants from the variety Alaska , and good crystals were hard to come by ( Amunts et al . , 2007 , 2010 ) .",
"Therefore , we screened for new robust crystals that are abundant , stable , and much more uniform .",
"The new crystals could be obtained from several pea plants varieties , a large proportion of them diffracted to 3 Å with several yielding higher resolutions .",
"In contrast to the P21 symmetry of the previous crystal , the current crystal belonged to higher symmetry space group P212121 .",
"The organization of the PSI unit within the new crystal was also markedly different .",
"In the P21 crystal the PSI-LHCI complex was organized as parallel layers in which the iron-sulfur clusters FX , FA and FB face the adjacent P700 ( Figure 1A ) .",
"The complexes inside the new crystal lattice were serially arranged in a crissed-crossed manner in which the polarity of each PSI unit contrasts another ( Figure 1B ) .",
"Consequently , the current crystals generated no net voltage ( data not shown ) , whereas a voltage of up to 50 V was recorded ( Toporik et al . , 2012 ) upon illumination of dried P21 crystals placed on electron conductive material . 10 . 7554/eLife . 07433 . 003Figure 1 . Comparison of two PSI-LHCI crystal lattices .",
"( A ) The previous PSI-LHCI crystal in the P21 space group with a layered arrangement of the complex .",
"This arrangement is capable of generating extremely high voltages upon illumination .",
"( B ) The new crystal lattice in the P212121 space group .",
"Iron sulfur clusters are colored in red and the pigments of the internal electron transport chain in magenta ( chlorophylls ) and blue ( quinones ) .",
"PSI-LHCI complexes are arranged in a crissed-crossed manner from left to right . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 003 The extreme size and complexity of the PSI assembly was a major obstacle for accurate and bias-free modeling .",
"The best way to eliminate model bias in X-ray crystallography is to utilize experimentally measured phase information .",
"Using the new , highly stable crystal form of PSI we were able to measure the weak native anomalous signal from the iron , sulfur , and phosphate atoms in the complex .",
"Starting with a minimal model containing only the three natively bound iron-sulfur clusters , the entire structure was eventually re-built with more than 35 , 000 atoms ( Figure 2 and Figure 2—figure supplement 1 , see ‘Material and methods’ section for details ) . 10 . 7554/eLife . 07433 . 004Figure 2 . Overall structure and organization of the plant PSI-LHCI supercomplex .",
"( A ) A view from the stromal side of the membrane of PSI-LHCI with the PsaL subunit pointing up .",
"The PsaF and PsaJ subunits connecting in the middle of LHCI are colored in magenta and green , respectively .",
"The three subunits of the stromal ridge , PsaC , PsaD , and PsaE , can be seen in the middle of the complex , colored cyan , pink , and blue , respectively .",
"The two iron-sulfur clusters of PsaC can be distinguished as yellow and orange clusters in the middle of the complex .",
"( B ) Pigment organization in PSI-LHCI .",
"The central pigments of the internal electron transport chain are colored red , chlorophylls of the core antenna green , chlorophyll a in LHCI in cyan , and chlorophyll b in magenta .",
"Carotenoids , which are distributed throughout the complex , are colored in blue and lipids in key connecting points and conserved positions in the core , in orange . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 00410 . 7554/eLife . 07433 . 005Figure 2—figure supplement 1 . Phasing substructure and electron density maps .",
"( A ) Positions of the native sulfurs , phosphates ( both in yellow ) , and iron atoms ( orange ) identified in the PSI-LHCI complex based on the measured anomalous signal .",
"( B ) Electron density maps .",
"The 2F − Fc map in blue , contoured at 1 . 3 rmsd , and an anomalous difference map in red , contoured at 2 rmsd around the phosphate atom of lipid 7001 bound by PsaA .",
"Residues 577:580 from PsaA are also shown . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 005 The structure of plant PSI includes 12 core subunits bound with four light-harvesting proteins comprising the LHCI antenna complex .",
"The entire complex contains 214 prosthetic groups , including 156 chlorophylls ( nine assigned as chlorophyll b ) , 32 carotenes , and 14 lipids , many of them located at key contact points of the complex .",
"The core photosynthetic reaction centers have remained virtually unchanged over the entire 2 billion years of their evolution ( Jordan et al . , 2001; Ben-Shem et al . , 2003; Amunts et al . , 2010 ) .",
"Instead , the evolution of PSI is marked by the loss and gain of whole subunits from the complex ( Scheller et al . , 2001; Nelson , 2011; Nelson and Junge , 2015 ) .",
"Compared to our previous model ( PDB 2WSC ) , the root-mean-square deviation ( rmsd ) between the plant and cyanobacterial core ( PDB 1JB0 ) decreased from 1 . 1 Å to 0 . 55 Å .",
"The majority of the changes made in the core subunits involved the configuration of extramembrane loops , which now closely resemble the cyanobacterial configuration .",
"The exceptions to this role are found at the anchor points of LHCI to the core ( discussed below ) and at the interfaces between plant-specific subunits , such as the PsaH–PsaL interaction ( Figure 3A ) .",
"The dramatic change from trimer to monomeric organization that occurred in eukaryotes , was triggered by the addition of the PsaH subunit ( Ben-Shem et al . , 2003 ) .",
"A new configuration for PsaH shows that this subunit binds four other core subunits .",
"Starting from the stromal side of the membrane , the N-terminus is tightly tucked between the N-terminus of PsaD and a eukaryotic-specific loop in the PsaL subunit .",
"PsaH then enters the membrane surrounding PsaL to prevent PSI trimerization and associates with PsaI and PsaB via mostly hydrophobic interactions ( Figure 3A ) . 10 . 7554/eLife . 07433 . 006Figure 3 . PsaH connects the state II and the PC binding sites .",
"( A ) PsaL is presented in blue , and the path of the PsaH subunit ( red ) travels through its extended , eukaryotic specific , loop .",
"The new PsaH chlorophyll ( green , marked as H1 ) connects to the PsaL-coordinated chlorophyll and carotenoid ( green and pink ) , as well as an additional chlorophyll trimer ( middle left , marked as PsaA trimer ) , which is proposed to connect PSI to LHCII in state II .",
"( B ) A luminal view of the PSI surface at the PC binding site .",
"The two hydrophobic helices are colored in grey ( PsaA ) and light blue ( PsaB ) .",
"The luminal PsaA loop is highlighted on the background of the entire PsaA subunit .",
"All the ligands ( with the exception of P700 ) are not shown .",
"( C and D )",
"Side by side view of the cyanobacterial ( C ) and plant ( D ) PC binding sites showing the extended PsaF helices that limit access from the membrane plane and the N terminus of PsaH ( red ) , which parallels the configuration of the PsaA luminal loop .",
"The approximate location of PC is indicated in blue . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 00610 . 7554/eLife . 07433 . 007Figure 3—figure supplement 1 . The proposed configuration of the state II super complex .",
"( A ) monomeric LHCII is shown in magenta near the PsaL subunit , juxtaposed to PsaK .",
"The region blown up in B is circled black .",
"( B ) This arrangement brings sites 5 and 12 from Lhcb next to the PsaA-coordinated chlorophyll trimer . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 007 Eukaryotes can modulate the distribution of excitation energy transfer between their two photosystems via a mechanism called state transitions ( Lunde et al . , 2000; Bellafiore et al . , 2005; Rochaix , 2011; Rochaix et al . , 2012 ) .",
"Under state II conditions , PSI associates with a mobile pool of the LHCII antennae , which increases its absorbance cross-section ( Kargul et al . , 2005; de Bianchi et al . , 2010 ) .",
"Genetic studies suggest that PsaH , PsaL , and PsaK play important roles in this process ( Scheller et al . , 2001; Zhang and Scheller , 2004 ) .",
"Electron microscopy studies have identified the binding site of the additional antennae complexes along the PsaL/PsaH-PsaK side ( Kargul et al . , 2005; Kouril et al . , 2005 ) .",
"A new chlorophyll bound by PsaH was identified at the current resolution .",
"This new chlorophyll , together with pigments bound by PsaL , probably participates in energy transfer into the core ( Figure 3A ) , suggesting that PsaH is not simply a ‘landing pad’ for LHCII , but is also important for energy transfer into the core during state II .",
"Additional pigments bound by PsaA in close proximity to subunit PsaK ( the structure of which is now almost completely defined ) provided the first accurate description of this binding site and suggest a mechanism for energy transfer into the core antenna through the PsaK side ( Figure 3—figure supplement 1 ) .",
"On the luminal side of the membrane , the new position of the N-terminus of PsaH suggests that this subunit also directly contributes to PC binding .",
"One of the distinguishing characteristics of the eukaryotic PSI is the stable complex it forms with its electron donor PC , which results in a thousand-fold acceleration of the electron transfer rate ( Bottin and Mathis , 1985 ) .",
"Three elements make up the PC binding site: the first is a positive patch located along the helix-turn-helix N-terminal domain of PsaF ( Hippler et al . , 1997 , 1998; Ben-Shem et al . , 2003 ) .",
"The second element is a hydrophobic patch composed of two parallel helices in PsaA and PsaB ( Figure 2B ) ( Sommer et al . , 2006; Kuhlgert et al . , 2012 ) .",
"The third conserved feature of the PC binding site is a PsaA luminal loop protruding from the generally flat luminal surface of PSI .",
"This loop is found in both plants and cyanobacteria PSI; the only exception being sequences from marine Prochlorococcus and their phages ( Mazor et al . , 2012 ) .",
"As seen in Figure 3C , D , the binding site of the cyanobacterial and plant complexes are similar .",
"However , it is clear that the plant binding site is buried deeper in the complex , this is achieved by the extension of the PsaF N-terminal and by the new position of the N-terminus of PsaH , which forms a loop mirroring the conformation of the conserved luminal PsaA loop , suggesting a direct role for PsaH in PC binding .",
"The PSI core is a highly efficient hub onto which diverse antennae systems connect such as phycobilisomes and IsiA-like assemblies in cyanobacteria and red algae and LHC type antennae in eukaryotes ( Berera et al . , 2009; Engelken et al . , 2010; Nelson and Junge , 2015; Wahadoszamen et al . , 2015 ) .",
"Remarkably , the core pigment organization is conserved across kingdoms despite this diversity in connected antenna ( Amunts et al . , 2007; Busch and Hippler , 2011; Croce and van Amerongen , 2013 ) , which suggests that the connection points between the core and the antennae are conserved .",
"The existence of red-absorbing pigments ( or ‘red traps’ ) is a general property of PSI ( Morosinotto et al . , 2005; Wientjes et al . , 2012 ) .",
"These pigments affect the rate of trapping in PSI and can affect the path of excitation migration in the complex .",
"Most of the eukaryotic red pigments have been shown to reside at LHCI .",
"However , red pigments may be lost from the core complex during the isolation of LHCI .",
"The first high-resolution PSI structure from thermophilic cyanobacteria revealed the organization of the core antenna ( Jordan et al . , 2001 ) .",
"A stacked chlorophyll trimer supported by an extended loop in PsaB was the best candidate for one of the strong red absorbers in this complex ( Jordan et al . , 2001 ) .",
"PsaB sequences from eukaryotes and many cyanobacteria lack this extended loop , resulting in this chlorophyll trimer being lost , as has been shown in the plant and mesophilic PSI structures ( Amunts et al . , 2010; Mazor et al . , 2014 ) .",
"At the current resolution , we observed new core chlorophyll bound between PsaG and Lhca1 and a newly discovered lipid ( Figure 4A ) .",
"This new chlorophyll restores the stacked chlorophyll trimer independent of the shortened PsaB loop and is responsible for one of the connection points between the core complex and the LHCI antenna , with a Mg–Mg distance of 12 . 5 Å between it and chlorophyll 1010 in Lhca1 ( the nomenclature for LHCII is used to describe Lhcas [Standfuss et al . , 2005] ) .",
"On the stromal side of the membrane , an additional chlorophyll trimer first discovered in Synechocystis is also responsible for an antenna attachment point with a Mg–Mg distance of 13 . 7 Å between the core chlorophyll A40 and chlorophyll 1005 in Lhca1 ( Figure 4—figure supplement 1 ) .",
"We suggest that chlorophyll trimers located at the periphery of the core antenna are extremely important for antenna attachment and are probably general attachment points to the core that are utilized not only by eukaryotes , but also by other antenna systems in cyanobacteria . 10 . 7554/eLife . 07433 . 008Figure 4 . Antenna connections in PSI-LHCI .",
"( A ) The configuration of the PsaG-Lhca1 pigment connection shown from the luminal side of the membrane .",
"The new stacked chlorophyll trimer ( numbered B1231 , B1232 and G1003 ) is shown .",
"The N-terminus of PsaG ( dark red ) supports one of the chlorophylls making up this trimer .",
"The entire trimer is connected with chlorophyll 10 ( numbered 1010 ) in Lhca1 ( blue ) .",
"( B ) The second LHCI-PSI connection between Lhca1 and PsaF ( magenta ) on the luminal side of the membrane is bound by a lipid ( orange ) .",
"( C ) The Lhca3 ( red ) -PsaA connection .",
"Two chlorophyll pairs mediate this interaction .",
"At the lumen face , 13 . 7 Å separate chlorophyll 3010 from chlorophyll 1114 .",
"On the stromal side , chlorophyll 3005 and chlorophyll 1108 are 16 . 5 Å apart .",
"( D ) Lhca2 ( blue ) —PsaJ ( green ) connecting chlorophylls . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 00810 . 7554/eLife . 07433 . 009Figure 4—figure supplement 1 . LhcA1 connects to PSI through chlorophyll trimers . The second chlorophyll trimer connecting Lhca1 to the core on the stromal side of the membrane bound by a short PsaB helix ( teal ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 00910 . 7554/eLife . 07433 . 010Figure 4—figure supplement 2 . PsaG occupy roughly an equivalent position compared to previous PSI-LHCI structures ( PsaG from 2WSC in cyan , PsaG from the current structure in red ) .",
"However the polarity of the transmembrane helices is reversed , as a result the N-terminus which was protruding outward from the complex now faces LHCI .",
"Three chlorophylls , a carotene and a lipid are coordinated by this subunit in the current structure . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 010 In contrast to the previous plant structures , which included a small pool of ‘Gap chlorophylls’ , only six pigment pairs connect LHCI to the core antenna in the current structure .",
"Lhca1 is the main connector for excitation transfer , harboring three chlorophylls that are within 14 Å of reaction center pigments ( Figure 4A , B and Figure 4—figure supplement 1 ) .",
"This close proximity ensures efficient and fast energy transfer .",
"Surprisingly , Lhca3 is also one of the main connection points with two such pairs ( Figure 4C ) .",
"The final excitonic connection between PSI to LHCI is located between chlorophyll J1302 bound by PsaJ and chlorophyll 2010 ( A chlorophyll b molecule ) .",
"The Mg–Mg distance of this pair is quite large ( 17 . 6 Å ) however , since the gap between LHCI and PSI allows for some movement , this distance can change to provide an efficient link between the core and LHCI ( Figure 4D ) .",
"To summarize , the extremely fast and efficient energy transfer processes that typify PSI-LHCI occurs through only six pairs of pigment molecules located at three sites .",
"These sites connect to the core antennae at the PsaG and PsaK poles through Lhca1 and Lhca3 , with Lhca2 playing a relatively minor role .",
"Our structure includes the fully modeled LHCI belt with nearly complete structures of all four Lhca proteins .",
"These structures reveal the essential features of the specific interactions between: each Lhca protein and the core; the red chlorophyll assembly present in Lhca4 and Lhca3; and a previously unknown pigment binding site , which is the probable site for the recently discovered non-photochemical quenching ( NPQ ) at the luminal gap region of LHCI ( de Bianchi et al . , 2010; Ballottari et al . , 2014 ) .",
"The LHCI belt is located on the PsaF side of the PSI core .",
"On the stromal face of the membrane , the four conserved N-terminal domains connect each Lhca subunit to its neighbor through interaction with an Lhca-specific loop ( loop 23 ) ( Figure 5A ) that follows immediately after the second transmembrane helix and supports a new chlorophyll site ( numbered",
"16 ) in Lhca4 and Lhca2 ( Figure 6A , B ) .",
"Lhca1 , which interacts with PsaG in the core complex , completely lacks this loop and instead contains a short , positively charged linker in this region ( Figure 5A ) .",
"One of the major changes in the structure of LHCI is the reversal in the polarity of PsaG .",
"While PsaG occupies roughly an equivalent position compared to previous PSI-LHCI structures , the first transmembrane helix contacts Lhca1 while the second one contacts PsaB ( Figure 4—figure supplement 2 ) .",
"Connections between each Lhca and the core are mediated by small regions immediately preceding the first transmembrane helix and are stabilized by salt bridges between negatively charged residues positioned at the membrane entrance point and a conserved arginine located at a helix turn below them ( Figure 5A ) .",
"Additional Lhca–Lhca interactions are provided by a short C-terminal segment on the luminal side that binds helix 2 as it exits the membrane , mainly via hydrophobic interactions ( Figure 5—figure supplement 1 ) . 10 . 7554/eLife . 07433 . 011Figure 5 . Lhcas connect to the PSI core through conserved structural elements .",
"( A ) A view from the stromal side of the membrane of LHCI .",
"Each Lhca connects to the next one through its conserved N-terminal domain ( marked N ) .",
"All connections to the core are mediated by the short region preceding the entrance of the first transmembrane helix into the membrane ( marked by a circle ) .",
"The extended loop ( L23 ) is conserved in all Lhcas except Lhca1 .",
"( B ) Electrostatic interactions determine the specificity of the Lhca4/5 binding site to PsaF .",
"The conserved E84-R209 interaction occurs within the PsaF ( magenta ) domain , which is partially membrane-buried , and Lhca4 ( raspberry ) .",
"( C ) Superposition of Lhca3 ( red ) and LHCII ( cyan ) .",
"Lhca3 interacts with the core via contacts with PsaA and PsaK through short extension ( red circle ) or deletion ( green circle ) in the otherwise highly conserved N terminus .",
"The extension of loop 23 is also seen at the bottom left corner of the image . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 01110 . 7554/eLife . 07433 . 012Figure 5—figure supplement 1 . Luminal side connections between the core PSI and LHCI . Short sequences immediately preceding the insertion point of the second transmembrane helix in the LHC fold ( marked by a red spot ) interact with the C-terminal sequences of the adjacent subunit ( marked with a blue spot ) .",
"Numbers indicate the sequence range shown in bold for the respective Lhca subunit . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 01210 . 7554/eLife . 07433 . 013Figure 6 . The interface within the Lhca heterodimers provides a mechanism for NPQ .",
"( A and B )",
"Structure of the Lhca1/4 ( A ) and Lhca2/3 ( B ) heterodimer as viewed from the membrane plane extrinsic to the PSI-LHCI complex .",
"The conserved connecting carotenoid in position L3 ( pink ) serves a structural role , connecting the two subunits of the dimer by linking chlorophyll 6 ( bottom right ) to chlorophyll 9 ( upper left corner ) .",
"The position of the new chlorophyll site bound by loop 23 of Lhca2 ( numbered 2016 ) is also shown .",
"( C and D )",
"A new chlorophyll site connects the pigments of the heterodimer .",
"Chlorophyll 17 ( numbered 4017 or 3017 ) is shown as viewed from the luminal side of the membrane .",
"The connections between the red pigment cluster on the right and position 9 in the adjacent subunit are marked with grey lines .",
"The new carotenoid site , L5 , is shown in C . This site lines the gap between LHCI and PSI and is therefore easily interchangeable .",
"The distance between L5 and of chlorophylls 17 π systems is 3 . 7 Å .",
"( E ) Lhca3 is colored in red .",
"A short helix segment from the first transmembrane helix is shown as sticks .",
"Chlorophyll 3005 is part of the red-absorbing dimer .",
"LHCII ( PDB: 2BHW ) was superimposed on the structure and shown in faint grey .",
"Chlorophyll 612 is shown as a ring .",
"The coordinating side chain was changed from histidine 68 in LHCII to asparagine 109 in Lhca3 .",
"Another important change is the leucine to glycine modification at position 105 , which allows chlorophyll 3005 to alter its ring orientation by approximately 12° .",
"( F ) The same site in Lhca4 shown in a similar fashion with Lhca4 colored in raspberry .",
"The same basic coordination is observed , though the ring tilt is smaller because of the alanine occupying position 95 . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 01310 . 7554/eLife . 07433 . 014Figure 6—figure supplement 1 . Identification of chlorophyll b molecules . Shown is the chlorophyll b occupying position 10 from Lhca2 .",
"Simulated annealing omit maps were calculated on a model which lacked the formyl-7 group ( shown in the image ) .",
"All b factors of the model were reset to the Wilson B factor , followed by a single round of simulated annealing and refinement using phenix . refine .",
"The difference map ( green ) is contoured at 2 . 5 rmsd and the 2Fo − Fc map ( blue ) is contoured at 1 . 2 rmsd . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 014 Genetic analysis in plants has revealed that each subunit has a specific binding site and , with the exception of the Lhca4-Lhca5 pair , the various Lhcas are not interchangeable ( Lucinski et al . , 2006; Wientjes et al . , 2009 ) .",
"The specific binding sites are identified in the current structure .",
"At the PsaG pole of LHCI , helix C of Lhca1 interacts with the first transmembrane helix of PsaG .",
"Additional protein–protein interactions occurred between a stromal loop of PsaB ( aa 307–320 ) and the N-terminus of Lhca1 ( Figure 5A ) .",
"The next contact point between LHCI and the core occurs between the N-terminus of Lhca4 and the C-terminus of PsaF , the conformation of which is almost identical to the conformation found in cyanobacteria .",
"This interaction consists of hydrophobic patches surrounding charged residues in the membrane-buried regions of both proteins .",
"This binding site should be shared between Lhca4 and Lhca5 and indeed , arginine 209 of PsaF and glutamate 84 of Lhca4 form electrostatic interactions in the middle of the hydrophobic binding site ( Figure 5B ) .",
"This residue is conserved in Lhca5 as well , but absent from Lhca1-3 sequences , explaining the specificity of the interaction .",
"The PsaF-Lhca4 interaction in the current structure does not include any pigments .",
"Energy transfer from Lhca4 to the core probably occurs through the Lhca1-PsaB pigment cluster or the Lhca2-PsaJ clusters .",
"Lhca2 is connected to the core almost exclusively through interactions with the N-terminus of PsaJ on the stromal side of the membrane ( Figure 5—figure supplement 1 ) .",
"The main contact point of Lhca3 is in the N-terminus of PsaA , which contacts a small patch just before helix 1 enters the membrane as in all other Lhcas ( Figure 5C ) .",
"The structure shows that this site is the main determinant of Lhca binding to the core .",
"In contrast to the previous PSI-LHCI model , Lhca3 follows the general fold of LHCII ( rmsd 0 . 8 Å between the two apoproteins ) .",
"Departures from the LHCII fold are seen in key contact points where small loops were extended or deleted from the otherwise conserved N-terminus domain to facilitate protein–protein interactions with the core ( Figure 5C ) .",
"All four Lhcas are remarkably similar to each other and to LHCII .",
"Most of the differences between them can be explained by their interaction partner , such as the loss of loop 23 in Lhca1 due to its binding to PsaG .",
"Two key differences in pigment organization were found between lhcas and other lhcs .",
"The extended loop 23 supports a new chlorophyll-binding site ( numbered 16 ) , common to Lhca4 and Lhca2 ( Figure 6A , B ) and an additional chlorophyll site , coordinated by transmembrane helix two , connects the two partners of each heterodimer .",
"Chlorophyll b pigments are bound by the different Lhca proteins to various extents and serve as antennae pigments .",
"Four binding sites for chlorophyll b were detected in the high resolution structure of LHCII ( Liu et al . , 2004; Standfuss et al . , 2005 ) as well as in the structure of CP29 ( Pan et al . , 2011 ) .",
"We were able to assign nine chlorophyll b sites in LHCI based on electron density maps ( Figure 6—figure supplement 1 ) .",
"All Lhca proteins contain a glutamine to glutamate change ( similarly to CP29 ) in equivalent positions to position 131 of LHCII , this change makes the binding of Chlorophyll b less likely at three sites ( 10 , 12 and 13 ) .",
"In agreement with this change we find that site 12 is occupied by chlorophyll a in all Lhcas and sites 10 and 13 are occupied differentially .",
"The distribution of chlorophyll b sites in LHCI is markedly uneven , with three sites located in Lhca2 ( 2010 , 2011 and 2013 ) , two sites found at Lhca1 ( site 1009 and 1010 ) , three sites at Lhca4 ( 4010 , 4011 and 4013 ) and a single site on Lhca3 ( site 3011 ) .",
"These findings are consistent with mutational data which identified more chlorophyll b sites on Lhca2 then on Lhca3 ( Castelletti et al . , 2003 ) .",
"Site 13 on Lhca1 , 2 and 4 is probably a mixed site , which can accommodate both chlorophyll a and chlorophyll b .",
"The high number of chlorophyll b pigments in Lhca2 and the fact that its closest connection to the core is mediated by chlorophyll b ( site 2010 , shown in Figure 4D ) is consistent with our suggestion that Lhca1 and Lhca3 are the main junctions for excitation energy transfer from LHCI to the PSI core .",
"The dipole orientations of all but two chlorophylls were identified in the current structure .",
"The most significant changes in LHCI compared to LHCII exist in two sites , 2009 and 4009 , where 90° rotations are observed .",
"Such a rotation is expected to impact the energy transfer processes within these LHCI subunits but the significance of this change cannot be ascertained from the rotation alone .",
"Each of the two Lhca1/4 Lhca2/3 heterodimers contains a blue- and red-absorbing subunit .",
"The structures of Lhca3 and Lhca4 clearly show a coordinating asparagine residue unique to this site ( Arnoux et al . , 2009 ) .",
"The configurations of the pigments themselves differed , with the red pigments of Lhca3 and Lhca4 tilted approximately 12° relative to their orientation in LHCII , Lhca1 , and Lhca2 due to a second change from a bulky leucine residue at position 64 of LHCII to glycine and alanine in Lhca3 and Lhca4 , respectively ( Figure 6E , F ) .",
"Additional connections that stabilize LHCI are formed by three luteins at position L3 .",
"These luteins bridge the chlorophylls at site 6 in Lhca1 , Lhca3 , and Lhca4 to PsaG , Lhca2 , and Lhca1 ( Figure 6A , B ) .",
"Photosynthetic eukaryotes respond to high light conditions by decreasing the efficiency of energy transfer from the antennae in a process called NPQ .",
"First discovered in the PSII complex , NPQ was recently reported in PSI .",
"NPQ sites are located on LHC proteins bound by the hydroxylated carotenoid zeaxanthin , which quenches harmful chlorophyll triplets ( Standfuss et al . , 2005 ) .",
"The main excitonic connections in the two Lhca dimers appear to be intimately linked to NPQ .",
"A new chlorophyll site ( numbered",
"17 ) coordinated by a histidine residue ( histidine 150 in Lhca4 and histidine 170 in lhca3 ) is located at the heterodimer interface of both Lhca1/4 and Lhca2/3 .",
"Site 17 links the putative red chlorophyll pair with site 9 of the adjacent complex .",
"In the Lhca1/4 interface , a new carotenoid site ( numbered 4505 or L5 ) forms co-planar π systems ( plane to plane distance 3 . 7 Å ) with the ring of chlorophyll 4017 , positioning it in a configuration that should provide efficient photo-protection from chlorophyll triplets ( Figure 6C , D ) .",
"We propose that this is also the site of NPQ in LHCI , and this fits well with the experimental observation ( Standfuss et al . , 2005 ) that the zeaxanthin responsible for NPQ is located near the red pair in the PSI-LHCI luminal gap region .",
"In response to lumen acidification , zeaxanthin is synthesized from violaxanthin by violaxanthin deepoxidase ( VDE ) as part of the xanthophyll cycle .",
"Both the activity and location of VDE are regulated by pH changes ( Ballottari et al . , 2014 ) .",
"At low pH , VDE is activated and binds the luminal side of the membrane , gaining access to its substrate .",
"On the luminal side , a large gap ( 25 Å ) separates LHCI from the core .",
"This gap stems from the fact that most of the connections between LHCI and PSI are located at the stromal side ( seen in Figure 7C , also compare Figure 5A and Figure 5—figure supplement 1 ) , leaving the luminal side open . 10 . 7554/eLife . 07433 . 015Figure 7 . The surface electrostatic potential of PSI-LHCI .",
"( A ) Luminal view of the electrostatic potential ( red for negative and blue for positive charges ) generated from PSI-LHCI apoproteins .",
"The gap region is shown as an open cavity ( the ligands occupying this cavity were omitted from the calculation ) lined with negative patches .",
"The plastocyanin binding site can be distinguished as a blue patch generated by the positively charged luminal double helix domain of PsaF .",
"( B ) The stromal electrostatic surface of PSI-LHCI showing the putative ferredoxin binding site as a basic patch on the left side of the stromal ridge .",
"( C ) A side view of the stromal gap shows the large opening facing the lumen .",
"Lhca1 and PsaG are omitted to reveal the internal cavity .",
"This surface was drown around all ligands identified in the gap with only chlorophyll 4017 and carotene 4505 omitted .",
"( D ) We propose that the negative patches lining the gap play a role in preventing the accessibility of VDE to gap pigments during regular growth .",
"While at high light conditions , lumen acidification partially neutralizes these negative charges , allowing VDE activity on gap xanthophylls . DOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 015 The complete modeling of LHCI side chains shows that the luminal side of PSI-LHCI contains patches of negative potential distributed on both sides of the PSI-LHCI gap ( Figure 7 ) .",
"We suggest that the negative charge characteristic of the luminal side of PSI-LHCI is an important feature regulating the accessibility of VDE to the PSI-LHCI gap .",
"Under low light conditions , the relatively high luminal pH will result in strong repulsive forces between these negative patches and the acidic domain of VDE , preventing VDE from binding to PSI .",
"Under high light , acidification of lumen , which is known to trigger VDE activity , will result in partial neutralization of these patches and allow VDE access to the PSI-LHCI gap regions ( Arnoux et al . , 2009 ) ( Figure 7D ) .",
"Carotene L4505 is ideally positioned to be exchanged easily via this mechanism ( Figure 7C ) .",
"Ligands , which are easily exchangeable , are particularly labile and may disassociate from the complex during its purification , accounting for a lack of a second carotene bound at the analogous position in Lhca3 .",
"In the current structure , position L5 is occupied with zeaxanthin; however , the pigment assignment cannot be definite , even at the current resolution .",
"In this work we presented the most complete plant PSI-LHCI structure obtained thus far , revealing the locations of and interactions among its protein subunits and more than 200 non-covalently bound photochemical cofactors .",
"Using the new crystal structure , we examined the network of contacts among the protein subunits from a structural perspective , which provide the basis for elucidating the functional organization of the complex ."
],
[
"Pea seeds ( Pisum sativum var . Kalvadon ) were washed with running water for 6–8 hr and seeded in a vermiculite tray .",
"Plants were germinated in the dark at 22°C for 5 days in shaded sunlight or the dark .",
"After germination , the plants were grown for an additional 10 days under cool-white fluorescent light at a photon flux density of 90–130 μE m−2 s−1 in a 14 hr light/10 hr dark cycle at 22°C .",
"Approximately 200 g of leaves were ground for 25 s in a blender with 1000 ml of ice-cold solution containing 0 . 3 M sucrose , 15 mM NaCl , 30 mM Tricine-NaOH ( pH 8 ) , 1 mM PMSF , 15 μM leupeptin , and 1 μM pepstatin A . The slurry was filtered through eight layers of cheesecloth and chloroplasts pelleted by centrifugation at 1000 g for 9 min .",
"The pellet was suspended in 500 ml of hypotonic medium ( 10 mM Tricine-NaOH , pH 8 ) to disrupt the chloroplasts .",
"Thylakoids were collected by centrifugation at 12 , 000 g for 10 min and resuspended in 500 ml of buffer containing 10 mM Tricine-NaOH ( pH 8 ) and 150 mM NaCl .",
"The thylakoid membranes were then pelleted at 8 , 000 g for 10 min and resuspended in a minimal volume of STN2 buffer ( 0 . 4 M sucrose , 20 mM Tricine-NaOH , pH 8 ) .",
"The thylakoid concentration was adjusted to 3 mg of chlorophyll per ml and 0 . 4% n-dodecyl-α-D-maltoside ( DDM ) added .",
"This concentration of detergent selectively extracts the ATP synthase , b6f , and PSII complexes .",
"After 5 min incubation on ice , the detergent- treated thylakoid membranes were collected by ultra-centrifugation at 200 , 000 g for 30 min .",
"The pellet was resuspended in a minimal volume of STN2 buffer , adjusted to 3 mg chlorophyll per ml , and stored at −80°C .",
"Frozen thylakoid membranes ( 20–25 ml ) containing 3 . 0 mg of chl/ml were thawed in cold water and solubilized with 1 . 5% DDM .",
"Insolubilized material was removed by ultracentrifugation at 120 , 000 g for 15 min .",
"The supernatant was applied to a DEAE-cellulose column ( DE-52 , Whatman , Inc . , 1 . 5 × 18 cm ) pre-equilibrated with 15 mM Tricine-Tris ( pH 8 . 0 ) containing 0 . 25% DDM .",
"The column was washed with the same buffer and PSI eluted with a 0–230 mM tetraethylammonium chloride linear gradient ( 75 ml in each chamber ) in 15 mM Tricine-Tris ( pH 8 . 0 ) containing 0 . 25% DDM .",
"Dark green fractions containing PSI were precipitated by 10% PEG6000 ( Hampton Research , Aliso Viejo , CA ) , followed by centrifugation at 5 , 000 g for 6 min .",
"The pellet was dissolved in 15 mM Tricine-Tris ( pH 8 . 0 ) and 0 . 05% DDM .",
"The green solution was applied to a 10–35% sucrose gradient containing the same buffer and centrifuged using the SW-40 rotor ( Beckman Coulter , Fullerton , CA ) at 37 , 000 rpm ( 170 , 000 g ) for 16 hr .",
"The wide green band containing PSI was collected and loaded onto a second DEAE-cellulose column ( 0 . 5 × 4 cm ) pre-equilibrated with 15 mM Tricine-Tris ( pH 8 . 0 ) and 0 . 05% DDM , mainly to concentrate it for a second sucrose gradient .",
"PSI was eluted with 230 mM tetraethylammonium chloride .",
"The collected dark green fraction was applied to a 10–35% sucrose gradient and centrifuged at 57 , 000 rpm ( 330 , 000 g ) for 4 hr using an SW-60 rotor ( Beckman Coulter ) .",
"Purified PSI appeared as a dark band in the middle of the tube , but only the central part of the band was collected .",
"The material was precipitated with 15% PEG1500 and 100 mM tetraethylammonium chloride and centrifuged at 10 , 000 g for 4 min .",
"The pellet was dissolved in a solution containing 2 mM Tricine ( pH 8 . 75 ) and 0 . 02% n-dodecyl-β-D-thiomaltoside ( DTM , Glycon Biochemicals , Luckenwalde , Germany ) and adjusted to a chlorophyll concentration of 2 . 5 mg/ml .",
"Crystallization was performed manually in 24-well plates using the sitting drop variant of the vapor-diffusion technique at 4°C ( Charles Super Company , Natick , MA ) .",
"Aliquots ( 6–8 μl ) of PSI solution were mixed with equal volumes of reservoir solution ( 50 mM di-potassium phosphate , 50 mM Tris [pH 8] , 12–17% PEG400 , 1% glycerol , 2 mM L-glutathione , and 0 . 03% octyl glucose neopentyl glycol ) and equilibrated against 0 . 5 ml of reservoir solution .",
"Dark green rectangular crystals appeared after 3 days at the higher PEG concentrations , but the best diffracting crystals appeared after 1 month at the lower PEG concentrations .",
"For cryogenic protection , the crystals were moved to a solution containing 50 mM di-potassium phosphate , 50 mM Tris ( pH 8 ) , 20% PEG400 , 2% glycerol , and 2 mM L-glutathione .",
"After a brief incubation the crystals were soaked sequentially in the same buffer containing 5% and 10% glycerol and immediately frozen in liquid nitrogen .",
"X-ray diffraction data were collected at the European Synchrotron Radiation Facility ( ID23- 2 , ID23-1 , ID-29 and MASSIF-3 ) , the Swiss Light Source ( PXI , PXII , and PXIII ) , and BESSYII .",
"Images were collected at 0 . 1° oscillation using full beam at exposures of 0 . 1–0 . 05 s .",
"The large size of the crystals helped mitigate the effect of radiation damage , yet only 60°–90° of data were collected from each crystal using constant translation of the crystal in the beam .",
"Images were integrated using XDS ( Kabsch , 2010 ) and scaled with XSCALE or AIMLESS ( Evans and Murshudov , 2013 ) .",
"Measurements were carried out at the peak of the iron fluorescence scan .",
"Individual datasets generally had I/SIGMA values of ∼1 at 3 . 1 Å with CC1/2 values of ∼0 . 5 calculated by XDS .",
"To obtain a more accurate measure of weak reflections , we combined several datasets measured under similar conditions .",
"This combination was possible because of the consistency of the new crystals .",
"The most effective method for combining datasets was to simply choose the sets with the strongest statistics at lower resolutions , and these crystals were also very similar to each other in terms of their unit cell dimensions .",
"The unified datasets contained 40–100 independent measurements of each reflection with I/SIGMA of ∼1 . 5 at 2 . 8 Å and CC1/2 of ∼0 . 4 at the same resolution ( Table 1 ) .",
"The CCanomalous calculated by XDS or AIMLESS was ∼0 . 3 at 6 Å .",
"Some individual datasets had measurable anomalous signals ( CCanomalous > 0 . 3 ) to 4 . 5 Å , but the final quality of phases was similar , as judged by the figure of merit for substructures from PHASER runs ( typically ∼0 . 6 [McCoy et al . , 2007] ) and visual inspection of the maps . 10 . 7554/eLife . 07433 . 016Table 1 . Data collection and refinement statisticsDOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 016Data collectionBeamlineBESSY PX14 . 1SLS PXI−X06SAESRF ID 23-2Wavelength ( Å ) 1 . 731 . 740 . 873Resolution ( Å ) 40−2 . 8 ( 2 . 85–2 . 8 ) 40−2 . 8 ( 2 . 85−2 . 8 ) 50−3 ( 3 . 1−3 ) Space groupP212121P212121P21Unit cell dimensionsa , b , c ( Å ) 188 . 7 , 200 . 8 , 212 . 4188 . 6 , 201 . 3 , 212 . 7120 . 6 , 189 . 2 , 129 . 7α , β , γ90 , 90 , 9090 , 90 , 9090 , 91 . 1 , 90Measured reflections7 , 831 , 30214 , 841 , 310724 , 010Unique reflections198 , 911200 , 218116 , 039Rpim ( % ) 0 . 051 ( 1 . 276 ) 0 . 030 ( 0 . 180 ) 0 . 099 ( 0 . 812 ) <I/σ ( I ) >10 . 5 ( 1 . 2 ) 13 . 4 ( 1 . 4 ) 5 . 8 ( 1 . 3 ) Completeness ( % ) 99 . 9 ( 98 . 4 ) 99 . 9 ( 98 . 4 ) 99 . 9 ( 99 . 2 ) Redundancy39 . 9 ( 37 ) 74 . 1 ( 33 . 4 ) 6 . 2 ( 5 . 4 ) Refinement statisticsResolution ( Å ) 40−2 . 840−2 . 850−3Rwork/Rfree25 . 6/26 . 524/25 . 225 . 8/29 . 3No .",
"of chains161617No .",
"of ligands214214197Average B-factor ( Å2 ) 98 . 711296 . 6R . M .",
"S deviationsBond angles1 . 922 . 4Bond lengths0 . 0040 . 0050 . 011Ramachandran statisticsFavoured region %90 . 290 . 286 . 6Allowed region %7 . 17 . 18 . 7Outlier region %2 . 72 . 74 . 7Data collection , scaling and merging statistics were calculated using XDS , AIMLESS and PHENIX XTRIAGE .",
"Refinement statistics are from PHENIX .",
"The crystals were initially solved by molecular replacement ( MR ) with a partial model containing only the reaction center subunits with chlorophylls modeled as rings using PHASER .",
"This solution was used to place the three iron-sulfur clusters , which were subsequently used as the initial substructure for locating additional sites .",
"These initial runs located approximately 40 sites of the 100 that were eventually modeled .",
"Phases were improved using DM ( Cowtan , 1994 ) , and the resulting maps showed most of the transmembrane helices of the reaction center with 11 transmembrane helices of LHCI ( missing helix 2 of Lhca3 ) .",
"To improve the phases , information from the visible parts of the reaction center was incorporated as a partial MR solution .",
"This model included the 22 transmembrane helices of PsaA and PsaB , the PsaC subunit , and chlorophyll rings , omitting all of the loops from the proteins .",
"From the maps generated in this step , we proceeded to build the model using Coot ( Emsley et al . , 2010 ) and used the modified phases as restraints during refinement in either PHENIX ( Adams et al . , 2010 ) or REFMAC ( Murshudov et al . , 1997 ) .",
"At various points during the refinement process , the phases were recalculated with the newly modeled sites using PHASER and including the improved model ( Read and McCoy , 2011 ) .",
"Final runs identified 99 sites , 89 of them present in the model .",
"The final model refined to an R-free of 25 . 2% with 3% Ramachandran outliers , a considerable decrease from previous values ( Table 1 ) .",
"A complete model for the lhca subunits of LHCI was obtained , as well as extensions and modifications to some of the other PSI subunits ( Table 2 ) .",
"Images were created using Pymol and electrostatic surfaces calculated using APBS ( Baker et al . , 2001 ) . 10 . 7554/eLife . 07433 . 017Table 2 . Amino acid changes between PSI-LHCI structuresDOI: http://dx . doi . org/10 . 7554/eLife . 07433 . 017SubunitNumber of amino acidsModeled amino acidsNumber of changes4Y282WSCPsaA7587417291PsaB7347327321PsaC8180811PsaD*1561401355PsaE*9268659PsaF*15415015418PsaG*98959515PsaH*95846911PsaJ*4241425PsaK*134798510PsaL*16816016121Lhca1*20419316512Lhca22562061763Lhca324221016216Lhca42521971663The number of modeled amino acids in each subunit is shown and compared to the most recent PSI-LHCI structure ( 2WSC ) .",
"Insertions , deletions and extensions are counted as a single change .",
"Since the genome sequence of Pisum Sativum is not completely known ( genes with no DNA data are marked with an * ) we relied on high-throughput mRNA sequence data for verification ( Franssen et al . , 2011 ) ."
]
] | [
"Most life forms on Earth are supported by solar energy harnessed by oxygenic photosynthesis .",
"In eukaryotes , photosynthesis is achieved by large membrane-embedded super-complexes , containing reaction centers and connected antennae .",
"Here , we report the structure of the higher plant PSI-LHCI super-complex determined at 2 . 8 Å resolution .",
"The structure includes 16 subunits and more than 200 prosthetic groups , which are mostly light harvesting pigments .",
"The complete structures of the four LhcA subunits of LHCI include 52 chlorophyll a and 9 chlorophyll b molecules , as well as 10 carotenoids and 4 lipids .",
"The structure of PSI-LHCI includes detailed protein pigments and pigment–pigment interactions , essential for the mechanism of excitation energy transfer and its modulation in one of nature's most efficient photochemical machines ."
] | [
"Most plants , green algae and some bacteria use a process called photosynthesis to convert energy from sunlight into the chemical energy they need to survive and grow .",
"With this energy , these organisms use carbon dioxide and water to create organic matter and release oxygen into the atmosphere .",
"Therefore , photosynthesis plays a major role in providing the basis for life on earth .",
"During photosynthesis , molecules of pigments known as chlorophyll and carotenoid capture the light energy .",
"These pigments are contained within large groups ( or ‘complexes’ ) of proteins that sit in membrane structures within cells .",
"Two of the protein complexes—called photosystem I and LHCI—interact with each other to form a ‘supercomplex’ that transfers energy to a small protein called ferredoxin .",
"To achieve this , the light energy captured by pigment molecules is transferred to other pigment molecules so that the energy is funneled towards the center of photosystem I . Mazor et al . used a technique called X-ray crystallography to create a very detailed three-dimensional model of photosystem I and LHCI from pea plants .",
"The model shows how the twelve proteins of photosystem I are arranged in relation to the four proteins of the LHCI complex .",
"The super-complex contains more than 200 other molecules , which are mostly chlorophylls and carotenoids .",
"Of these , 61 chlorophyll molecules and ten carotenoid molecules are found in LHCI .",
"The model also provides detailed information about how the pigments interact with each other and with the proteins in the supercomplex .",
"Mazor et al . 's detailed model may help us to understand how these interactions allow photosystem I to harvest light energy with almost 100% efficiency , and aid efforts to develop new technologies that harness light ."
] | 2015 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | Cytoplasmic translocation of the retinoblastoma protein disrupts sarcomeric organization | elife-01228-v1 | [
[
"Skeletal muscle degeneration , which is characterized by the progressive depletion of muscle strength , occurs in a variety of chronic diseases including advanced cancer , congestive heart failure , and AIDS ( Tisdale , 2002 ) .",
"The underlying intercellular mechanism is currently thought to be multifactorial .",
"Inflammatory cytokines , particularly TNF-α , have been shown to be key mediators of cancer-related skeletal muscle degeneration ( Tisdale , 2002; Seruga et al . , 2008 ) .",
"Elevated levels of TNF-α precede the onset of cancer-related skeletal muscle degeneration and act through several cancer-related signaling pathways such as the p53 and nuclear factor kappa B ( NF-κB ) pathways ( Guttridge et al . , 2000; Cai et al . , 2004; Schwarzkopf et al . , 2006 ) .",
"Retinoblastoma protein ( Rb ) prevents tumor formation by inducing differentiation , controlling cell-cycle progression , and maintaining genomic stability ( Burkhart and Sage , 2008 ) .",
"To date , numerous studies of Rb function have focused on the transcriptional regulation of E2F .",
"Rb forms a transcriptional repressor complex with two protein groups , E2F transcription factors and LXCXE motif-containing proteins ( Halaban , 2005; Burkhart and Sage , 2008 ) .",
"Rb activity is regulated by sequential phosphorylation on several serine and threonine residues , first by cyclin D/cyclin-dependent kinase 4 ( CDK4 ) and then by cyclin E/CDK2 complexes ( Halaban , 2005 ) .",
"This serial phosphorylation of Rb induces dissociation of the transcriptional repressor complex , allowing expression of E2F-target genes , which are required for many cellular processes .",
"Loss of Rb function in many cancer cells is frequently caused by aberrant CDK-mediated phosphorylation ( Chau and Wang , 2003; Burkhart and Sage , 2008 ) .",
"Consequently , selective CDK inhibition is considered a potentially useful approach for cancer treatment ( Malumbres and Barbacid , 2009 ) .",
"In addition , inactivation of Rb , which is induced by TNF-α treatment , has been shown to lead to various cellular behaviors including proliferation of vascular smooth muscle cells ( Rastogi et al . , 2012 ) and apoptosis of fibroblasts and aortic endothelial cells ( Chau and Wang , 2003; Rastogi et al . , 2012 ) .",
"Recently , a non-nuclear function of Rb has been reported; where Rb at the mitochondria participates in TNF-α-induced apoptosis ( Hilgendorf et al . , 2013 ) .",
"However , it is still unknown whether the Rb pathway is involved in cancer-related skeletal muscle degeneration mediated by TNF-α .",
"A sarcomere is the basic functional unit of striated muscle and consists of two sets of filaments: thick and thin ( Squire , 1997; Gautel , 2011 ) .",
"The thick filaments are composed of myosin proteins and the thin filaments are assembled from polymerized actin monomers , called filamentous actin ( F-actin ) .",
"The contractile activity of skeletal muscle is achieved through the actin and myosin filaments sliding past one another ( Squire , 1997 ) .",
"The motor function of striated muscle therefore , requires the well-ordered assembly of sarcomeres , which is closely tied to the highly organized actin cytoskeleton .",
"The formins are a large family of proteins and are characterized by the presence of the conserved formin homology 2 ( FH2 ) domain ( Kovar , 2006; Campellone and Welch , 2010 ) .",
"The FH2 domain promotes actin nucleation and polymerization , thereby producing long straight actin filaments and regulating cytoskeletal organization .",
"It has been reported that several members of the formin family serve as key regulators of actin dynamics during sarcomeric organization in striated muscle ( Taniguchi et al . , 2009; Kan et al . , 2012; Mi-Mi et al . , 2012 ) .",
"In this study , we present a potential mechanism underlying TNF-α-induced skeletal muscle degeneration .",
"We propose a novel function for Rb; where Rb disrupts sarcomeric organization in human skeletal muscle myotubes ( HSMMs ) following its phosphorylation and translocation into the cytoplasm .",
"Our study implicates the tumor suppressor protein in the regulation of cytoskeletal organization ."
],
[
"To gain insights into the role of Rb in cancer-related skeletal muscle degeneration , we first examined the phosphorylation kinetics and subcellular localization of Rb in TNF-α-treated HSMMs .",
"For this purpose , cells were pretreated with interferon-gamma ( IFN-γ , 100 ng/ml ) for 8 hr prior to initial TNF-α treatment in order to promote cellular sensitivity to the effects of TNF-α ( Tsujimoto et al . , 1986 ) .",
"During differentiation from myoblasts to myotubes ( from day 0 to day 4 ) , Rb shifted from a phosphorylated to an unphosphorylated state ( Figure 1A ) .",
"Moreover , following TNF-α treatment Rb phosphorylation on the CDK4-specific phosphorylation site , serine 780 ( Kitagawa et al . , 1996 ) , was induced ( Figure 1A ) .",
"This was not observed on threonine 821 , a CDK2-specific phosphorylation site ( Halaban , 2005 ) .",
"In untreated HSMMs , Rb was primarily present in the nucleus , but translocated to the cytoplasm after TNF-α treatment ( Figure 1B , C ) .",
"In addition , we found that phosphorylated Rb in TNF-α-treated HSMMs was predominantly located in the cytoplasm ( Figure 1D ) .",
"In accordance with the induction of Rb phosphorylation on a CDK4-specific phosphorylation site , TNF-α treatment led to an increase in the level of nuclear CDK4 ( Figure 1E ) .",
"The vast majority of nuclear Rb was in an unphosphorylated state , while cytoplasmic Rb that accumulated after TNF-α treatment was in a phosphorylated state ( Figure 1E ) .",
"We then tested whether TNF-α-induced cytoplasmic accumulation of Rb is caused by CDK4-mediated Rb phosphorylation .",
"When CDK4 was depleted by short hairpin RNA ( shRNA ) , cytoplasmic accumulation of Rb induced by TNF-α was decreased ( Figure 1F–H ) .",
"These results suggest that phosphorylation of Rb by CDK4 triggers its cytoplasmic translocation in HSMMs . 10 . 7554/eLife . 01228 . 003Figure 1 . TNF-α induces cytoplasmic translocation of Rb .",
"( A–E )",
"HSMMs were treated with TNF-α for 2 days .",
"( A ) CDK4-mediated phosphorylation of Rb is induced by TNF-α treatment .",
"At the indicated time points after differentiation stimuli , total cell lysates were prepared and immunoprecipitated with anti-Rb antibody , followed by immunoblotting .",
"TNF-α was added for the last 2 days of 6-day cultures .",
"The open and solid arrowheads indicate the position of phosphorylated and unphosphorylated Rb , respectively .",
"( B ) Cytoplasmic translocation of Rb is caused by TNF-α treatment .",
"Z-stack confocal images of Rb and 4′ , 6-diamidino-2-phenylindole ( DAPI ) -stained nuclei were obtained ( 50 slices at 0 . 3-μm intervals ) .",
"X-Y section images for Rb and DAPI-stained nuclei and X-Z section images for Rb along yellow lines in the X-Y section images are shown .",
"Scale bar , 20 μm .",
"( C ) Line plots denote the fluorescence intensities of Rb ( red lines ) and DAPI ( blue lines ) along the yellow lines in B . Intensity values were normalized by the maximum value of each plot .",
"a . u . , arbitrary units .",
"( D ) Phosphorylated Rb is localized in the cytoplasm .",
"Confocal images for Rb phosphorylated at S780 ( red ) and DAPI-stained nuclei ( blue ) .",
"Scale bar , 20 μm .",
"( E ) Nuclear CDK4 expression is increased after TNF-α treatment .",
"Cytoplasmic ( Cyto ) and nuclear ( Nuc ) lysates were analyzed by immunoblotting with the antibodies indicated .",
"Tubulin and TFIIB were used as loading controls for cytoplasmic and nuclear lysates , respectively .",
"The open and solid arrowheads indicate the position of phosphorylated and unphosphorylated Rb .",
"( F–H )",
"HSMMs were infected with adenoviruses expressing control non-target shRNA or shRNA against CDK4 at a MOI of 10 pfu/nucleus and then treated with TNF-α for 2 days .",
"( F ) Experimental design and reference time frame .",
"( G ) The cytoplasmic ( Cyto ) and nuclear ( Nuc ) lysates were analyzed by immunoblotting .",
"( H ) TNF-α-induced cytoplasmic translocation of Rb is prevented by CDK4 depletion .",
"The cytoplasmic and nuclear lysates were subjected to immunoprecipitation and probed by immunoblotting .",
"The open and solid arrowheads indicate the position of phosphorylated and unphosphorylated Rb , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 003 In human skeletal muscle myoblasts , Rb was mainly in a phosphorylated state ( Figure 1A , day 0 ) , although the localization of S780-phosphorylated Rb was confined to the nucleus ( Figure 2A ) .",
"Lamina-associated polypeptide ( LAP ) 2α , a binding partner of nucleoplasmic A-type lamins , interacts with S780-phosphorylated Rb in mitotic myoblasts ( Markiewicz et al . , 2005 ) and is known to play an important role in nuclear tethering of Rb ( Markiewicz et al . , 2002 ) .",
"Indeed , when LAP2α was depleted by shRNA in human myoblasts , Rb localized to the cytoplasm as well as the nucleus ( Figure 2B ) .",
"Given the level of LAP2α decreased during muscle differentiation ( Markiewicz et al . , 2005 ) ( Figure 2C ) , these results suggest that low-level LAP2α expression in HSMMs facilitates the cytoplasmic translocation of Rb .",
"In many types of cancer cells , Rb exists predominantly in a phosphorylated state , but is primarily localized in the nucleus .",
"It has been reported that LAP2α is an E2F-target gene and its expression is enhanced in cancer cells ( Parise et al . , 2006; Ward et al . , 2011 ) .",
"Overexpressed LAP2α may therefore serve to tether Rb to the nucleus in cancer cells and the cytoplasmic translocation of Rb may be triggered in cells that express low levels of LAP2α , such as terminally differentiated cells . 10 . 7554/eLife . 01228 . 004Figure 2 . Loss of LAP2α affects the cytoplasmic translocation of Rb .",
"( A ) Phosphorylated Rb is localized in the nucleus in human skeletal muscle myoblasts .",
"Confocal images for Rb phosphorylated at S780 and DAPI-stained nuclei .",
"Scale bar , 20 μm .",
"( B ) Rb is translocated to the cytoplasm by LAP2α depletion .",
"Human skeletal muscle myoblasts were transfected with an mCherry-HA-Rb expression plasmid together with an shRNA expression plasmid against eGFP or LAP2α .",
"Confocal images for mCherry , LAP2α and DAPI-stained nuclei .",
"The arrowheads indicate transfected cells .",
"Scale bar , 20 μm .",
"( C ) LAP2α expression is decreased in HSMMs .",
"Total cell lysates from human skeletal muscle myoblasts and HSMMs were subjected to immunoblotting . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 004 We next tested whether the motor function of skeletal muscle is affected under this condition .",
"In response to electric pulse stimulation ( EPS ) , which evokes a contractile reaction in myotubes ( Fujita et al . , 2007 ) , approximately 55% of HSMMs displayed beating , an index of contractile reaction ( Figure 3A; Video 1 ) , while it was hardly observed after TNF-α treatment ( Figure 3A; Video 2 ) .",
"The beating was not induced in mitotic myoblasts or prematurely differentiated myoblasts implying that the sarcomeric structure , which is essential for the contractile activity of muscle cells ( Squire , 1997 ) , is organized in HSMMs .",
"To evaluate the effect of TNF-α on the periodic assembly of sarcomeres , we examined the distribution of α-actinin , a major component of Z-disks , as Z-disks define the lateral borders of individual sarcomeres ( Gautel , 2011 ) .",
"In untreated HSMMs , α-actinin was observed at evenly spaced intervals , whereas in TNF-α-treated HSMMs the peak-to-peak distance in line plots of α-actinin intensity along the myofibril was larger and inconsistent ( Figure 3B–D ) .",
"Furthermore , when the repeating pattern of α-actinin distribution was analyzed using an autocorrelation image processing technique , peak values were much lower in TNF-α-treated HSMMs ( Figure 3E ) .",
"These results indicate that the periodic assembly of sarcomeres is disrupted by TNF-α treatment . 10 . 7554/eLife . 01228 . 005Figure 3 . TNF-α disrupts sarcomeric organization .",
"( A–E )",
"HSMMs were treated with TNF-α for 2 days .",
"( A ) Contractile activity of HSMMs is impaired by TNF-α treatment .",
"EPS was applied to HSMMs .",
"The percentage of beating cells from a total of 100 HSMMs is shown .",
"Results are presented as mean ± SD from three independent experiments .",
"*p<0 . 002 , determined by the Student’s t-test .",
"( B–E )",
"Sarcomeric organization of HSMMs .",
"Confocal images for α-actinin ( B ) and merged images of F-actin ( green ) and α-actinin ( red ) ( C ) are shown .",
"Scale bar , 10 μm in B and 5 μm in C . ( D ) Line plots of α-actinin fluorescence intensity along individual myofibrils ( denoted by yellow lines in C ) .",
"Intensity values were normalized by the maximum value for each fibril .",
"a . u . , arbitrary units .",
"( E ) Autocorrelation analyses of the α-actinin distribution .",
"Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 00510 . 7554/eLife . 01228 . 006Video 1 . Image of live beating HSMMs in response to EPS . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 00610 . 7554/eLife . 01228 . 007Video 2 . Image of live beating TNF-α-treated HSMMs in response to EPS . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 007 In striated muscle cells , anti-parallel actin filaments spanning the sarcomeres are crosslinked to the Z-disks ( Sparrow and Schock , 2009 ) .",
"The peak-to-peak distance in line plots of α-actinin intensity therefore reflects a counterbalance between tensile forces generated by the actin filaments .",
"Sarcomeric disorganization represents an imbalance in the tensile forces , which may be caused by defective actin filament formation .",
"The close association of accurate sarcomeric organization with actin polymerization was demonstrated by treatment of the cells with the actin polymerization inhibitor cytochalasin D . After 30 min of treatment , the periodic arrangement of α-actinin was not well ordered ( Figure 4A–C ) and was strongly disordered by 60 min ( Figure 4D ) . 10 . 7554/eLife . 01228 . 008Figure 4 . Inhibition of actin polymerization disorganizes sarcomeric assembly .",
"( A–D )",
"HSMMs were treated with 2 μM cytochalasin D ( Cyto D ) for 30 min ( A–C ) or 60 min ( D ) at room temperature .",
"( A ) The periodic arrangement of α-actinin is not well ordered after 30 min of Cyto D-treatment .",
"Merged images of F-actin ( green ) and α-actinin ( red ) .",
"Scale bar , 5 μm .",
"( B ) Line plots of α-actinin fluorescence intensity along individual myofibrils ( denoted by yellow lines in A ) .",
"Intensity values were normalized by the maximum value for each fibril .",
"a . u . , arbitrary units .",
"( C ) Autocorrelation analyses of the α-actinin distribution .",
"Scale bar , 5 μm .",
"( D ) The lateral periodicity in the α-actinin distribution is strongly disordered by 60 min of treatment .",
"Merged images of F-actin ( green ) and α-actinin ( red ) .",
"Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 008 We then evaluated the role of Rb in the negative regulation of sarcomeric organization using Rb knockdown HSMMs ( Figure 5A , B ) .",
"Rb is known to be required for myogenic differentiation but not for the maintenance of the terminally differentiated state in myotubes ( Huh et al . , 2004 ) .",
"In accordance with this , sarcomeric organization was not affected when Rb was depleted by shRNA ( Figure 5C ) .",
"After TNF-α treatment , the periodic assembly of sarcomeres was disrupted in control shRNA-expressing HSMMs , but TNF-α failed to induce the sarcomeric disorganization in sh-Rb-expressing HSMMs ( Figure 5D–F ) .",
"Next , we directly examined whether cytoplasmic Rb is capable of disrupting sarcomeric organization .",
"To this end , HSMMs were infected with adenoviruses expressing a heterologous nuclear exporting signal ( NES ) -fused form of Rb , which was tagged with monomeric red fluorescent protein mCherry and influenza hemaglutinin ( HA ) at its amino-terminus ( mCherry-HA-NES Rb ) .",
"Given a small population of endogenous Rb was localized in the cytoplasm after TNF-α treatment , we expressed NES Rb at a comparable level to TNF-α-induced cytoplasmic Rb ( Figure 6A , asterisk vs the open arrowhead ) .",
"Under this condition , sarcomeric organization was not well ordered as in TNF-α-treated HSMMs ( Figure 6B–D ) .",
"When we expressed higher levels of NES Rb in HSMMs ( Figure 6E , F ) , the periodic arrangement of α-actinin was significantly disordered when compared to mCherry-HA-Rb-expressing cells ( Figure 6G ) .",
"Taken together , these results suggest that Rb is involved in TNF-α-induced sarcomeric disorganization and TNF-α-induced cytoplasmic Rb may have a pivotal role in this process . 10 . 7554/eLife . 01228 . 009Figure 5 . Rb contributes to TNF-α-induced sarcomeric disorganization .",
"( A–F )",
"HSMMs were infected with adenoviruses expressing control non-target shRNA or shRNA against Rb at a MOI of 10 pfu/nucleus and then treated with TNF-α for 2 days .",
"( A and B )",
"Depletion of Rb protein was verified by immunoblotting ( A ) and epifluorescence microscopy ( B ) .",
"The open and solid arrowheads indicate the position of phosphorylated and unphosphorylated Rb , respectively .",
"Scale bar , 20 μm .",
"( C ) Confocal images for F-actin and α-actinin .",
"Scale bar , 10 μm .",
"( D and E )",
"TNF-α-induced sarcomeric disorganization is attenuated by Rb depletion .",
"Confocal images for α-actinin ( D ) and merged images of F-actin ( green ) and α-actinin ( red ) ( E ) .",
"Scale bar , 10 μm in D and 5 μm in E . ( F ) Autocorrelation analyses of the α-actinin distribution .",
"Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 00910 . 7554/eLife . 01228 . 010Figure 6 . Sarcomeric organization is impaired by cytoplasmic Rb .",
"( A–G )",
"HSMMs were infected with adenoviruses expressing mCherry-HA-Rb or mCherry-HA-NES Rb at a MOI of 10 pfu/nucleus ( A–D ) or 50 pfu/nucleus ( E–G ) for 4 days .",
"( A ) The expression of exogenous Rb proteins .",
"Cytoplasmic ( Cyto ) and nuclear ( Nuc ) lysates were prepared from infected HSMMs in parallel with TNF-α-treated HSMMs .",
"The lysates were subjected to immunoprecipitation and probed by immunoblotting .",
"The open and solid arrowheads indicate the position of phosphorylated and unphosphorylated Rb .",
"Asterisk indicates the position of exogenous Rb .",
"( B ) Sarcomeric structure is not well ordered in NES Rb-expressing HSMMs .",
"Merged images of F-actin ( green ) and α-actinin ( red ) .",
"Scale bar , 5 μm .",
"( C ) Line plots of α-actinin fluorescence intensity along individual myofibrils ( denoted by yellow lines in B ) .",
"Intensity values were normalized by the maximum value for each fibril .",
"a . u . , arbitrary units .",
"( D ) Autocorrelation analyses of the α-actinin distribution .",
"Scale bar , 5 μm .",
"( E and F )",
"The expression and distribution of exogenous Rb proteins were analyzed by immunoblotting ( E ) and confocal microscopy ( F ) .",
"The open and solid arrowheads indicate the position of exogenous and endogenous Rb , respectively .",
"Scale bar , 20 μm .",
"( G ) Sarcomeric structure is strongly disordered in NES Rb-expressing HSMMs .",
"Confocal images for F-actin and α-actinin .",
"Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 010 Similarly to TNF-α-treated HSMMs , the cytoplasmic translocation of Rb has been shown to be induced by its CDK-mediated phosphorylation in certain types of cancer cells ( Jiao et al . , 2008 ) , although the function of cytoplasmic Rb in the cancer cells has not been described thus far .",
"In the cancer cells , Rb is phosphorylated on CDK2 phosphorylation sites as well as CDK4 phosphorylation sites , whereas our data showed that Rb phosphorylation on the CDK2-specific phosphorylation site , threonine 821 , was not induced by TNF-α treatment ( Figure 1A , day 6 ) .",
"We therefore reasoned that the selective phosphorylation of Rb by specific CDKs affects the function of cytoplasmic Rb .",
"Given that it has been reported that threonine 821/threonine 826 phosphorylation disrupts Rb binding to LXCXE motif-containing proteins ( Knudsen and Wang , 1996; Dick and Rubin , 2013 ) , we examined whether the function of cytoplasmic Rb is achieved through its interaction with LXCXE motif-containing proteins .",
"We introduced mCherry-HA-NES Rb lacking exon 22 ( Rb Δexon 22 ) , which is known to be an LXCXE-binding deficient mutant ( Henley et al . , 2010 ) , into HSMMs and found that the inhibitory effect of NES Rb Δexon 22 on sarcomeric organization was less effective as compared to NES Rb-expressing HSMMs ( Figure 7A ) . 10 . 7554/eLife . 01228 . 011Figure 7 . The function of cytoplasmic Rb is rendered through its interaction with LXCXE motif-containing proteins .",
"( A ) Sarcomeric structure is not disordered in NES Rb Δexon 22-expressing HSMMs .",
"HSMMs were infected with an adenovirus expressing mCherry-HA-NES Rb Δexon 22 at a MOI of 50 pfu/nucleus for 4 days .",
"Confocal images for F-actin and α-actinin .",
"Scale bar , 10 μm .",
"( B ) mDia1 contains the LXCXE motif .",
"Sequence alignment of the LXCXE motif of mDia1 and other known Rb-binding proteins .",
"( C ) The primary structure of mDia1 and its mutants .",
"Scheme represents location of GBD and DAD of mDia1 .",
"Numbers denote amino acid positions in mDia1 isform2 .",
"The LXCXE motif is present in GBD ( amino acid positions 153 to 157 ) .",
"ΔGBD/ΔDAD , doubly deleted mDia1 lacking both GBD and DAD .",
"( D ) The LXCXE motif is required for the in vitro interaction between Rb and mDia1 .",
"Purified Flag-tagged mDia1 proteins were mixed with full-length recombinant Rb protein and immunoprecipitates were analyzed by immunoblotting .",
"( E ) Rb interacts with mDia1 after TNF-α treatment .",
"HSMMs were treated with TNF-α for 2 days .",
"The cytoplasmic lysates were subjected to immunoprecipitation and probed by immunoblotting .",
"The open arrowheads indicate the position of phosphorylated Rb .",
"( F ) Expression and purification of GST fusion Rb proteins .",
"The purity of bacterially expressed GST-Rb proteins was evaluated by SDS-PAGE , followed by CBB staining .",
"GST-Rb wild-type protein encompasses amino acids 379–928 .",
"GST-Rb Mut CDK2 contains serine/threonine to alanine substitutions at CDK2-specific phosphorylation sites ( S612 and T821 ) .",
"( G ) GST-Rb proteins were preincubated with CDK4/Cyclin D1 and CDK2/Cyclin E proteins in the presence or absence of ATP and then mixed with purified Flag-mDia1 protein .",
"The interaction between mDia1 and Rb proteins was analyzed by immunoprecipitation with anti-Flag antibody-agarose beads . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 01110 . 7554/eLife . 01228 . 012Figure 7—figure supplement 1 . Identification of NES Rb-binding protein . HSMMs were infected with adenoviruses expressing mCherry-HA-NES Rb WT or mCherry-HA-NES Rb Δexon 22 at a MOI of 50 pfu/nucleus for 4 days .",
"Total cell lysates from adenovirus-infected HSMMs were immunoprecipitated with anti-HA antibody-conjugated agarose beads .",
"The bound proteins were analyzed by SDS-PAGE and visualized by CBB staining .",
"Individual protein bands were excised from SDS-PAGE gel and protein samples were subjected to electrospray ionization mass spectrometric analysis .",
"The lists of proteins from the bands are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 012 Next , to explore how cytoplasmic Rb disorganizes sarcomeric assembly in HSMMs , we searched for the binding proteins of cytoplasmic Rb using mCherry-HA-NES Rb wild-type ( WT ) -expressing HSMMs .",
"Total cell lysates from adenovirus-infected HSMMs were immunoprecipitated with anti-HA antibody-conjugated agarose beads and the bound proteins were subjected to electrospray ionization mass spectrometric analysis ( Figure 7—figure supplement 1 ) .",
"Among the proteins identified , we focused on mammalian diaphanous-related formin 1 ( mDia1 ) , a potent actin nucleation factor ( Watanabe et al . , 1997 ) , as the LXCXE motif is contained in the GTPase binding domain ( GBD ) of mDia1 ( Figure 7B , C ) .",
"It is noteworthy that the band including mDia1 was hardly detected in mCherry-HA-NES Rb Δexon 22-expressing HSMMs ( Figure 7—figure supplement 1 , band 1 ) .",
"Data from purified wild-type and LXCXE motif-deleted mutant mDia1 proteins ( WT and ΔLXCXE ) ( Figure 7C ) showed that binding of mDia1 to Rb was abolished by deletion of the LXCXE motif ( Figure 7D ) .",
"Cytoplasmic Rb , which was phosphorylated and accumulated after TNF-α treatment , interacted with mDia1 ( Figure 7E ) .",
"We then examined whether the selective phosphorylation of Rb affects this interaction .",
"Unphosphorylated Rb protein containing the large pocket , an important domain for interactions with a variety of cellular proteins ( Burkhart and Sage , 2008 ) , bound to mDia1 in vitro and the binding level was strongly reduced according to its phosphorylation catalyzed by cyclin D/CDK4 and cyclin E/CDK2 complexes ( Figure 7F , G ) .",
"Although differences in the patterns of Rb phosphorylation by cyclin D/CDK4 and cyclin E/CDK2 have been reported , we failed to detect the selective phosphorylation of Rb by our in vitro phosphorylation system , which may be due to a supraphysiological activity of CDKs in vitro .",
"We then tested a mutant Rb protein bearing serine/threonine-to-alanine substitutions in the CDK2-specific phosphorylation sites , serine 612 and threonine 821 ( Mut CDK2 ) .",
"The mutant Rb exhibited substantial interaction with mDia1 even after phosphorylation ( Figure 7G ) , suggesting that phosphorylation of Rb on CDK4 phosphorylation sites alone does not impair its interaction with mDia1 and TNF-α-induced cytoplasmic Rb has the potential to interact with mDia1 .",
"Muscle wasting/atrophy accompanies cancer-related skeletal muscle degeneration ( Tisdale , 2002; Acharyya et al . , 2004 ) .",
"We therefore carried out immunohistological analysis on normal and atrophied tibialis anterior muscles excised from cancer patients and examined the localization of Rb in these muscles ( Figure 8A ) .",
"In normal muscles , Z-disks were regularly aligned and the sarcomere striation pattern was clearly observed ( Figure 8B , left ) .",
"In contrast , the Z-disks were misaligned and the sarcomeric banding pattern was not well organized in the atrophied muscles ( Figure 8B , right ) .",
"With regard to the localization of Rb , it was mainly located in the nucleus in normal muscle cells , but Rb could be observed in the cytoplasm , as well as the nucleus in atrophied muscle cells ( Figure 8C ) .",
"In both normal and atrophied skeletal muscles , the localization of mDia1 was mainly confined to the Z-disk ( Figure 8B ) , and cytoplasmic Rb observed in atrophied muscles colocalized with mDia1 ( Figure 8C ) . 10 . 7554/eLife . 01228 . 013Figure 8 . Cytoplasmic Rb colocalizes with mDia1 in atrophied skeletal muscle .",
"( A–C′ )",
"Normal and atrophied tibialis anterior muscles were surgically excised from cancer patients .",
"Cross sections ( A ) and longitudinal sections ( B–C′ ) were stained .",
"( A ) Hematoxylin and eosin-stained cryosections .",
"Scale bar , 400 μm .",
"( B–C′ )",
"Localization of Rb and mDia1 in normal and atrophied tibialis anterior muscles .",
"Confocal images of cryosections stained with anti-mDia1 and anti-α-actinin antibodies ( B ) or anti-Rb and anti-mDia1 antibodies ( C , magnified in C′ ) .",
"Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 013 We next tested whether inhibition of mDia1 activity is responsible for the TNF-α-induced disorganization of the sarcomere .",
"For this purpose , we generated a recombinant adenovirus expressing the constitutively active form of mDia1 ( Watanabe et al . , 1999 ) , which lacks the entire GBD and carboxy-terminal diaphanous autoregulatory domain ( DAD ) ( Figure 7C ) .",
"We introduced mDia1 ΔGBD/ΔDAD tagged with green fluorescent protein ( GFP-mDia1 ΔGBD/ΔDAD ) into HSMMs and treated them with TNF-α ( Figure 9A ) .",
"mDia1 ΔGBD/ΔDAD itself did not affect either α-actinin distribution or the contractile reaction of HSMMs ( Figure 9B , C ) .",
"After TNF-α treatment , however , the lateral alignment of α-actinin in GFP-mDia1 ΔGBD/ΔDAD-expressing HSMMs was well ordered as compared to that in control GFP-expressing HSMMs ( Figure 9D–F ) .",
"Accordingly , the percentage of beating cells decreased by TNF-α treatment was restored by the introduction of mDia1 ΔGBD/ΔDAD ( p<0 . 02 , determined by the Student’s t-test ) ( Figure 9C ) . 10 . 7554/eLife . 01228 . 014Figure 9 . TNF-α-induced sarcomeric disorganization is prevented by constitutively active mDia1 . ( A–F ) HSMMs were infected with adenoviruses expressing GFP or GFP-mDia1 ΔGBD/ΔDAD at a MOI of 1 pfu/nucleus and then treated with TNF-α for 2 days .",
"( A ) The expression was analyzed by immunoblotting .",
"( B ) Confocal images for α-actinin .",
"Scale bar , 5 μm .",
"( C ) TNF-α-induced contractile dysfunction is diminished by constitutively active mDia1 .",
"EPS was applied to HSMMs .",
"The percentage of beating cells from a total of 100 HSMMs is shown .",
"Results are presented as mean ± SD from three independent experiments .",
"*p<0 . 002; **p<0 . 02 , determined by the Student’s t-test .",
"( D and E )",
"Constitutively active mDia1 recovers TNF-α-induced sarcomeric disorganization .",
"Confocal images for α-actinin ( D ) and merged images of F-actin ( green ) and α-actinin ( red ) ( E ) .",
"Scale bar , 10 μm in D and 5 μm in E . ( F ) Autocorrelation analyses of the α-actinin distribution .",
"Scale bar , 5 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 014"
],
[
"In this study , we have elucidated a novel pathway of cancer-related skeletal muscle degeneration .",
"TNF-α induces CDK4 activation and the concomitant phosphorylation of Rb .",
"Subsequently , cytoplasmic translocation of Rb is triggered .",
"Cytoplasmic Rb disrupts sarcomeric organization , which may be caused by dysfunction of mDia1 .",
"The precise role of mDia1 in the regulation of sarcomeric organization is poorly understood ( Sparrow and Schock , 2009 ) , but the contribution of mDia1 to sarcomeric organization is supported .",
"When mDia1 was depleted by shRNA , the lateral periodicity in the distribution of α-actinin was markedly perturbed ( Figure 10A , B ) .",
"The importance of actin nucleation factors for the periodic assembly of sarcomeres is proposed by the recent observations that depletion of actin nucleation factors , such as Fhod3 and leiomodin , results in disordered α-actinin distribution in cardiomyocytes ( Chereau et al . , 2008; Taniguchi et al . , 2009; Iskratsch et al . , 2010 ) .",
"The notion that the degree of sarcomeric disorganization is dependent on the inhibition of actin polymerization is further supported by our data that show Z-disk alignment is more severely impaired by longer-term treatment of cytochalasin D ( Figure 4 ) . 10 . 7554/eLife . 01228 . 015Figure 10 . mDia1 is critical for sarcomeric organization .",
"( A and B )",
"HSMMs were infected with adenoviruses expressing control non-target shRNA or shRNA against mDia1 at a MOI of 5 pfu/nucleus for 4 days .",
"( A ) Depletion of mDia1 protein was verified by immunoblotting .",
"( B ) Confocal images for F-actin and α-actinin .",
"Scale bar , 10 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 015 Phosphorylation of Rb and the subsequent activation of E2F transcriptional activity enhance the expression of E2F-target genes , which include cell-cycle regulators ( e . g . , Tk1 and Dhfr ) .",
"Although Rb phosphorylated at serine 780 is unable to bind to E2F1 ( Kitagawa et al . , 1996 ) , quantitative PCR ( qPCR ) analysis did not reveal any statistically significant differences in the expression of Tk1 and Dhfr after TNF-α treatment ( Figure 11 ) .",
"During muscle differentiation , methylation of histone H3 lysine 9 and DNA methylation occur at several E2F-target gene promoters including Tk1 and Dhfr ( Ait-Si-Ali et al . , 2004; Blanchet et al . , 2011 ) .",
"These epigenetic changes may trigger the permanent silencing of E2F-target gene expression and prevent E2F1 from activating their expression after terminal differentiation . 10 . 7554/eLife . 01228 . 016Figure 11 . The expression of cell-cycle regulators and mitochondrial biogenesis factors after TNF-α treatment . HSMMs were treated with TNF-α for 2 days .",
"Quantification of the expression of cell-cycle regulators and mitochondrial biogenesis factors .",
"Results are presented as mean ± SD from three independent experiments .",
"*p<0 . 02 , determined by the Student’s t-test . DOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 016 It has been reported that phosphorylation of Rb is induced in the skeletal muscles of mice under fasting conditions ( Blanchet et al . , 2011 ) , which leads to the activation of E2F1 transcriptional activity and increases the expression levels of mitochondrial biogenesis factors ( Ppargc1a and Tfam ) .",
"In contrast , although TNF-α treatment induced Rb phosphorylation , the expression of Ppargc1a and Tfam decreased after TNF-α treatment ( Remels et al . , 2010 ) ( Figure 11 ) .",
"These findings suggest that other TNF-α-induced signaling pathways , such as the NF-κB pathway , may affect the transcriptional activity of E2F1 ( Araki et al . , 2008 ) and/or regulation of the expression of mitochondrial biogenesis factors ( Remels et al . , 2010 ) .",
"The contractile activity of skeletal muscle is caused by the sliding of thick and thin filaments in the sarcomere , and this sliding action is intimately linked to the proper control of adenosine 5’-triphosphate ( ATP ) production ( Squire , 1997 ) .",
"Because mitochondria are responsible for cellular ATP production , incomplete restoration of the percentage of beating cells by mDia1 ΔGBD/ΔDAD might be ascribed to an impairment of mitochondrial function caused by TNF-α ( Figure 9C ) .",
"In cancer patients , the circulating levels of TNF-α and IFN-γ would be lower than the amounts used in this study .",
"Monocytes/macrophages infiltrate in atrophied muscles and may produce considerable amounts of these inflammatory cytokines .",
"It may therefore be possible that the local concentrations of TNF-α and IFN-γ are elevated in atrophied muscles , which then contributes to phosphorylation of Rb in the long-term .",
"TNF-α-induced skeletal muscle degeneration is achieved through multiple mechanisms .",
"The results presented in this study propose a novel non-nuclear function for Rb , which is independent of the transcriptional regulation of E2F and may be involved in TNF-α-induced skeletal muscle degeneration .",
"In the future , it would be interesting to clarify the role of mDia1 and determine how cytoplasmic Rb disrupts sarcomeric organization ."
],
[
"Early passage human skeletal myoblasts purchased from Lonza ( Basel , Switzerland ) were cultured according to the manufacturer’s instructions using Lonza-supplied growth medium ( SkGM-2 BulletKit ) .",
"Cells grown to approximately 80% confluence were induced to differentiate into multinucleated myotubes by switching to differentiation medium ( DMEM-F12 containing 2% horse serum ) .",
"Under these conditions , numerous myotubes could be detected on the fourth day .",
"For TNF-α treatment , HSMMs were cultured in fresh serum-free media containing TNF-α ( 100 ng/ml ) for 2 days .",
"TNF-α was repeatedly added every 24 hr .",
"Recombinant human TNF-α and IFN-γ were purchased from PeproTech ( Rocky Hill , NJ ) .",
"Cytochalasin D was obtained from Sigma ( St . Louis , MO ) .",
"Immunoprecipitations were performed using anti-Flag antibody- ( M2; Sigma ) and anti-HA antibody- ( 3F10; Roche , Indianapolis , IN ) conjugated agarose beads .",
"The anti-Rb antibody ( G3-245; BD Biosciences , San Jose , CA ) and anti-mDia1 antibody ( AP50 , a gift from S Narumiya ) ( Watanabe et al . , 1997 ) were used for immunoprecipitation .",
"The following antibodies were used for immunoblotting: anti-Rb ( G3-245 and ab6075; Abcam , Cambridge , UK ) , anti-phospho Rb-S780 ( 9307; Cell Signaling Technology , Danvers , MA ) , anti-phospho Rb-T821 ( a gift from K Tamai , CycLex ) , anti-CDK4 ( C-22; Santa Cruz Biotechnology , Santa Cruz , CA ) , anti-mDia1 ( AP50 and 51 [we used two kinds of mDia1 antibodies: one is AP50 which is mentioned above; the other is 51 , which is a clone name of BD antibody]; BD Transduction Laboratories , Franklin Lakes , NJ ) , anti-LAP2α ( ab5162; Abcam ) , anti-GFP ( 598; MBL , Nagoya , Japan ) , anti-α-Tubulin ( DM1A; Sigma ) , anti-TFIIB ( C-18; Santa Cruz Biotechnology ) , anti-Flag-Peroxidase ( M2; Sigma ) and anti-HA-Peroxidase ( 3F10; Roche ) .",
"The recombinant adenoviruses expressing mCherry-HA-Rb , mCherry-HA-NES Rb , mCherry-HA-NES Rb Δexon 22 , GFP and GFP-mDia1 ΔGBD/ΔDAD were generated using the ViraPower adenoviral expression system ( Invitrogen , Carlsbad , CA ) .",
"The recombinant adenovirus-mCherry-HA-NES Rb contained the NES of MAPKK ( NLVDLQKKLEELELDEQQ ) ( Fukuda et al . , 1996 ) between mCherry and Rb .",
"The recombinant adenoviruses expressing shRNAs were generated using the BLOCK-iT adenoviral RNAi expression system ( Invitrogen ) .",
"For shRNA-mediated gene silencing , the respective target sequences were as follows: CDK4 , 5′-CCTAGATTTCCTTCATGCCAA-3′ ( sh-CDK4 ) ; mDia1 , 5′-GCCCAGAATCTCTCAATCTTT-3′ ( sh-mDia1 ) ; Rb , 5′-CAGAGATCGTGTATTGAGATT-3′ ( sh-Rb ) ; the non-target control , 5′-CAACAAGATGAAGAGCACCAA-3′ ( sh-Control ) .",
"The recombinant adenoviruses were purified with AsEasy virus purification kits ( Agilent technologies , Palo Alto , CA ) and adenovirus infections were performed with ViraDuctin adenovirus transduction reagent ( Cell Biolabs , San Diego , CA ) .",
"Mammalian expression vectors that encode shRNAs against human LAP2α or enhanced GFP ( eGFP ) were constructed by cloning suitable oligonucleotide sequences ( human LAP2α , 5′-CAGAAGAGAATTGATCAGT-3′; eGFP , 5′-ACAACAGCCACAACGTCTA-3′ ) into the pSilencer 2 . 1-U6 Hygro vector ( Ambion , Austin , TX ) .",
"pXJ Flag-mDia1 was obtained from C Koh ( Xie et al . , 2008 ) .",
"Flag-mDia1 plasmids were transfected into 293T cells and total cell lysates were extracted .",
"The mDia1 proteins tagged with a Flag epitope at their amino-termini were captured on anti-Flag antibody-agarose beads and eluted by competition with free 1× Flag peptide ( Sigma ) in 50 mM Tris ( pH 7 . 4 ) and 50 mM NaCl .",
"The purity of the mDia1 proteins , wild-type and ΔLXCXE , was evaluated by SDS-polyacrylamide gel electrophoresis ( SDS-PAGE ) , followed by Coomassie Brilliant Blue staining ( CBB staining ) .",
"Full-length recombinant Rb protein was purchased from QED bioscience ( San Diego , CA ) .",
"The recombinant glutathione S-transferase ( GST ) fusion Rb proteins were produced in BL21 E . coli .",
"GST-Rb proteins were incubated with CDK4/Cyclin D1 ( Merck Millipore , Billerica , MA ) and CDK2/Cyclin E ( Merck Millipore ) proteins in the presence or absence of 100 μM ATP in the kinase buffer ( 20 mM Tris [pH 7 . 5] , 10 mM MgCl2 and 1 mM dithiothreitol [DTT] ) for 30 min at 30°C .",
"The cells were fixed with 4% paraformaldehyde ( PFA ) in phosphate-buffered saline ( PBS ) for 30 min , permeabilized with 0 . 2% ( vol/vol ) Triton X-100/PBS for 5 min , and then blocked with 1% bovine serum albumin ( BSA ) /PBS for 30 min at room temperature .",
"Subsequently , the cells were incubated with anti-Rb ( 1:200 , 9309; Cell Signaling Technology ) , anti-phospho Rb-S780 ( 1:300 , 13H9L5; Novex , Carlsbad , CA ) and anti-LAP2α ( 1:300 , ab5162; Abcam ) antibodies for 1 hr , and further incubated with Alexa Fluor 546-conjugated goat anti-mouse IgG , Alexa Fluor 546-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated goat anti-rabbit IgG antibodies ( Molecular Probes , Carlsbad , CA ) for 30 min .",
"DAPI was used for nuclear staining .",
"For double-staining for α-actinin and F-actin , cytoskeletal stabilizing buffer was used in lieu of PBS , as described previously ( Hirata et al . , 2008 ) .",
"The cells were incubated with anti-α-actinin antibody ( 1:100 , EA53; Sigma ) , followed by incubation with Alexa Fluor 488- or 546-conjugated goat anti-mouse IgG antibody and Alexa Fluor 546- or 633-phalloidin ( Molecular Probes ) .",
"Confocal images were taken using a PerkinElmer Spinning Disk microscope or a Nikon A1Rsi microscope , equipped with oil-immersion objectives ( 60× and 100× ) .",
"Epifluorescence images were taken using a Nikon A1Rsi microscope , equipped with an oil-immersion objective ( 60× ) and an electron multiplying charge-coupled device camera ( DU897; Andor technology , Belfast , UK ) .",
"Images were acquired with the Volocity software and the Nikon NIS-Elements imaging software .",
"To evaluate periodicity in the distribution of α-actinin , 256 × 256 pixel , corresponding to 12 × 12 μm , sample areas of fluorescence images of α-actinin were subjected to computing autocorrelation analyses as described previously ( Peterson et al . , 2004 ) using the ImageJ program ( NIH , version 10 . 2 ) .",
"Each autocorrelation image was then normalized by the value of the central peak in the image .",
"Features of periodicity of its distribution are represented as local peaks other than the central maxima .",
"Sample images of α-actinin and normalized autocorrelation images are shown .",
"Frozen blocks of human skeletal muscle were obtained from Asterand ( Detroit , MI ) , who acquired appropriate informed consent from patients under the Institutional Review Board ( IRB ) approval .",
"Subject characteristics are described in Table 1 . 10 . 7554/eLife . 01228 . 017Table 1 . Subject characteristicsDOI: http://dx . doi . org/10 . 7554/eLife . 01228 . 017Biosample diagnosisNormalAtrophyGender and age , yearsFemale , 15Female , 14Cancer diagnosisOsteosarcomaSynovial sarcomaCancer locationShin boneSoft tissues of shinHeight , cm166159Weight , kg5050BMI , kg/m218 . 1419 . 78BMI , body mass index The sections were fixed in acetone for 10 min , permeabilized with 0 . 2% ( vol/vol ) Triton X-100/PBS for 30 min , and then blocked with CAS-Block ( Invitrogen ) for 10 min at room temperature .",
"Subsequently , the sections were incubated with anti-α-actinin , anti-Rb ( 1:100 , ab24; Abcam ) and anti-mDia1 ( 1:400 , ab11173; Abcam ) antibodies overnight at 4°C .",
"They were further incubated with appropriate fluorescence-labeled secondary antibodies for 1 hr at room temperature .",
"HSMMs grown on 4-well plates ( Nunc , Naperville , IL ) were pretreated with IFN-γ and then placed in a C-Dish electrode chamber ( IonOptix , Milton , MA ) after changing to fresh serum-free media .",
"EPS was applied to HSMMs using a C-Pace pulse generator ( IonOptix ) at 40 V/60 mm , 1 Hz , 10 ms for 2 days in parallel with TNF-α treatment .",
"The media were changed and TNF-α was repeatedly added every 24 hr .",
"Images of live beating cells were taken using Nikon Eclipse Ti-U microscope , equipped with a 20× objective lens .",
"Images were sequentially acquired with Nikon NIS-Elements imaging software at a frame rate of 15 fps .",
"Total RNA extraction , cDNA preparation , and real-time qPCR analyses were performed as described previously ( Kawauchi et al . , 2012 ) .",
"The primer sets used were: Tk1 , 5′-CATTAACCTGCCCACTGT-3′ forward and 5′-GATCACCAGGCACTTGTA-3′ reverse; Dhfr , 5′-TCATGGTTGGTTCGCTAA-3′ forward and 5′-TGAAGAGGTTGTGGTCATT-3′ reverse; Tfam , 5′-TGTAGAAGCCACGGTGTT-3′ forward and 5′-ACAACCATCAACTCTGAATACAAT-3′ reverse; Ppargc1a , 5′-TGAAGAGGCAAGAGACAGAATGA-3′ forward and 5′-CACACGCACACTCCATCAC-3′ reverse; B2m , 5′-GCATTCCTGAAGCTGACA-3′ forward and 5′-CGTGAGTAAACCTGAATCTTT-3′ reverse .",
"After normalization against B2m , data show mRNA expression levels relative to control expression levels for each experiment .",
"Mass spectrometry analysis was performed by Proteomics International ( Perth , Australia ) .",
"Protein samples were enzymatically digested to produce fragmented peptides and the resulting peptides were analyzed by electrospray ionization mass spectrometry using the Ultimate 3000 nano HPLC system ( Dionex , Sunnyvale , CA ) in combination with a 4000 Q TRAP mass spectrometer ( Applied Biosystems , Foster City , CA ) .",
"The spectra were analyzed by Mascot sequence matching software ( Matrix Science , Boston , MA ) ."
]
] | [
"Skeletal muscle degeneration is a complication arising from a variety of chronic diseases including advanced cancer .",
"Pro-inflammatory cytokine TNF-α plays a pivotal role in mediating cancer-related skeletal muscle degeneration .",
"Here , we show a novel function for retinoblastoma protein ( Rb ) , where Rb causes sarcomeric disorganization .",
"In human skeletal muscle myotubes ( HSMMs ) , up-regulation of cyclin-dependent kinase 4 ( CDK4 ) and concomitant phosphorylation of Rb was induced by TNF-α treatment , resulting in the translocation of phosphorylated Rb to the cytoplasm .",
"Moreover , induced expression of the nuclear exporting signal ( NES ) -fused form of Rb caused disruption of sarcomeric organization .",
"We identified mammalian diaphanous-related formin 1 ( mDia1 ) , a potent actin nucleation factor , as a binding partner of cytoplasmic Rb and found that mDia1 helps maintain the structural integrity of the sarcomere .",
"These results reveal a novel non-nuclear function for Rb and suggest a potential mechanism of TNF-α-induced disruption of sarcomeric organization ."
] | [
"Skeletal muscles , such as the biceps and calves , are one of three main muscle groups in the body , and a range of chronic diseases—including cancer , heart disease and AIDS—can cause wasting and a loss of strength in these muscles .",
"Many different cellular processes are known to be involved in the degeneration of skeletal muscle during illness .",
"For example , in people suffering from cancer , the immune response produces large numbers of molecules called inflammatory cytokines to combat the cancer cells , and these molecules are thought to have a role in the breakdown of skeletal muscle .",
"A cytokine called tumour necrosis factor alpha , or TNF-α for short , is thought to cause muscle damage , but the details of this process are not fully understood .",
"One possibility is that TNF-α interacts with a protein called Rb—short for retinoblastoma protein—that suppresses the proliferation of cells that leads to cancer .",
"However , if this protein is modified by a chemical process called phosphorylation , the Rb molecules will not be able to suppress the genes that lead to excessive cell growth .",
"The hyperphosphorylation of Rb has been observed in many cancer cells , and it has been shown that high levels of TNF-α in cells results in Rb not working properly , but it has not been clear if faulty Rb also leads to the breakdown of skeletal muscle .",
"Now Araki et al . provide evidence that the phosphorylation of Rb by TNF-α leads to skeletal muscle degeneration .",
"Araki et al . found that in muscle cells that contain high concentrations of TNF-α , the Rb molecules move from the nuclei of the cells , where they interact with genes , to the cytoplasm , where they disrupt the formation of structural fibres .",
"This means that Rb inhibits the ability of muscle cells to slide over one during contractions and relaxation , as happens in normal muscle tissue .",
"If confirmed by further experiments , these results could lead to the development of new approaches for the treatment of skeletal muscle degeneration ."
] | 2013 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | Neuronal reactivation during post-learning sleep consolidates long-term memory in Drosophila | elife-42786-v2 | [
[
"Animals guide their behavior in part based on the memories of past learning experiences .",
"Temporally , these memories can be either short- or long-lasting; short-term memory ( STM ) shapes future animal behavior within seconds , minutes to hours after a learning experience , whereas long-term memory ( LTM ) holds the learned information for hours , days or even a lifetime .",
"STM is thought to rely on protein synthesis-independent covalent modifications and changes in weight of existing synaptic connections , whereas LTM is believed to reflect protein synthesis-dependent structural changes in specific synapses ( Kandel , 2001 ) .",
"Depending on the salience or duration of the learning experience , some STMs can be transformed into LTMs through the process of memory consolidation ( Dudai et al . , 2015 ) .",
"Numerous studies in vertebrates suggest that sleep has a beneficial effect on memory in various learning tasks ( Marshall and Born , 2007; Smith , 2001 ) .",
"Studies in humans and rodents have shown that an insufficient amount of sleep impairs cognitive functions such as memory formation and retention , while an ample amount of sleep after a learning experience supports memory storage ( Goel et al . , 2009; Diekelmann and Born , 2010 ) .",
"In addition , several studies have found that animals , including humans , exhibit an enhanced amount and quality of sleep after experiencing a novel or enriched environment ( Gais et al . , 2002; Smith and Wong , 1991; Walker and Stickgold , 2006 ) .",
"One hypothesis is that sleep plays an active role in consolidation of newly acquired memories into long-term storage , that are critical for the animal future actions .",
"To date , however , a mechanistic understanding of sleep function in this selective memory consolidation remains elusive .",
"Over the last two decades , studies of sleep and LTM have expanded from the almost exclusive studies in mammals to other animals , including insects .",
"Sleep is now well-documented in Drosophila .",
"Sleep in Drosophila exhibits almost all the hallmarks of sleep in mammals , including homeostatic regulation , diminished behavioral responsiveness , and the existence of different sleep stages as characterized by distinct electrophysiological signatures ( Dissel et al . , 2015a; Yap et al . , 2017; van Alphen et al . , 2013 ) .",
"Recently , a link between sleep and LTM has been established: sleep deprivation before and after learning impairs mnemonic performance ( Ganguly-Fitzgerald et al . , 2006; Seugnet et al . , 2008; Seugnet et al . , 2009 ) , whereas sleep induction enhances memory formation and retention in wild-type ( Donlea et al . , 2011 ) and restores memory in memory mutant flies ( Dissel et al . , 2015b ) .",
"Moreover , flies that were exposed to a socially enriched environment , including courtship learning , exhibit an increased amount of sleep ( Ganguly-Fitzgerald et al . , 2006 ) .",
"Courtship conditioning has been extensively exploited to study both memory and sleep in Drosophila .",
"Courtship conditioning is an ethologically relevant form of complex learning whereby male flies learn to associate the outcome of their own behavior with multisensory cues presented by females during courtship ( Ejima et al . , 2005 ) .",
"Naive Drosophila males eagerly court both virgin and mated females , which are generally receptive and unreceptive , respectively .",
"However , after being repeatedly rejected by mated females , males become less likely to court other mated females ( Ejima et al . , 2005; Keleman et al . , 2012 ) .",
"Like with other types of learning , the resulting memory can last from minutes to days depending on the training protocol .",
"We previously established that activity of the dopaminergic aSP13 neurons ( DAN-aSP13s ) is necessary and sufficient for STM acquisition via the dopamine receptor DopR1 in the γ neurons of the mushroom body known as Kenyon Cells ( γKCs ) ( Keleman et al . , 2012 ) , a neuropil in the Drosophila central brain critical for memory formation ( Heisenberg et al . , 1985 ) .",
"Moreover , we recently demonstrated that the activity of the same DAN-aSP13s is also essential for the consolidation of courtship STM to LTM .",
"This requirement is observed in a discrete time window after learning , and it is mediated via the dopamine receptor DopR1 in the γKCs ( Krüttner et al . , 2015 ) .",
"In this study , we examine the mechanisms of DAN-aSP13 post-learning activation .",
"We show here that DAN-aSP13s are activated during sleep after courtship experience that induces LTM .",
"We present evidence that the specific class of sleep-promoting neurons in the fan-shaped body ( FB ) , a neuropil previously implicated in sleep regulation ( Donlea et al . , 2011 ) , activates DANs-aSP13 in the discrete time window after learning to consolidate courtship LTM ."
],
[
"To investigate the role of sleep in post-learning activation of DAN-aSP13s , and hence LTM consolidation , we first asked whether DAN-aSP13s are active in freely behaving males after a prolonged experience with mated females , which induces LTM .",
"To monitor activity of DAN-aSP13s for several hours in unrestrained males we employed a luminescence-based transcriptional reporter of neuronal activity ( Guo et al . , 2017 ) .",
"Using specific DAN-aSP13s GAL4 driver ( Aso et al . , 2014a ) ( Figure 1—figure supplement 1A ) , we expressed luciferase exclusively in DAN-aSP13s under the control of multimerized binding sites for the neuronal activity regulated gene Lola and FLP recombinase target sites ( FRT ) for its activity dependent cell specific expression ( MB315B-GAL4>UAS-FLP , Lola ( FRT ) stop ( FRT ) LUC ) ( Chen et al . , 2016 ) .",
"We measured luminescence as a proxy for neuronal activity in males that had undergone prolonged training with mated females and in naive males without prior courtship experience .",
"As a control we used males after prolonged training with virgin females which does not induce memory when tested 24 hr later with mated females ( our unpublished results ) .",
"For training , single males were paired with a single recently-mated or virgin female for 6 hr .",
"Afterwards , trained , naïve and control males were individually transferred to a 96-well luminescence reading plate .",
"Luminescence measurements were taken every 15 min over 16 hr beginning at a common starting time for all males , at the hour seven from the onset of training .",
"Notably , flies that had undergone training with mated females displayed a gradual increase of the luminescence signal , which was significantly different from naïve males between 7–10 hr from the onset of training ( Figure 1A ) .",
"In contrast , control males that had undergone training with virgin females also displayed an increase in luminescence however , it was identical to that in naïve males ( Figure 1—figure supplement 1B ) .",
"That specific time window of DAN-aSP13 activation after onset of training with mated females was preserved in males that were trained in a later circadian time during the day ( Figure 1—figure supplement 1C ) .",
"Together , these data show that DAN-aSP13s are activated in a specific time window after a learning experience that leads to LTM .",
"It was previously shown that flies that had been subjected to social enrichment , including courtship experience , display an increased amount of day time sleep ( Ganguly-Fitzgerald et al . , 2006 ) .",
"To determine whether Drosophila males sleep in the time period when DAN-aSP13s are activated , we measured the amount of sleep in males after prolonged courtship training and in naïve males .",
"Sleep was analyzed with Drosophila Activity Monitors , and periods of inactivity lasting for at least 5 min were classified as sleep ( Shaw et al . , 2000; Hendricks et al . , 2000 ) .",
"Both trained and naive wild-type males showed a significant amount of day time sleep , particularly during hours 7–10 when DAN-aSP13s are active .",
"Importantly , males that had undergone training for LTM slept significantly more in this time window in comparison to naïve males ( Figure 1B ) .",
"Both groups displayed the same amount of sleep throughout the night .",
"Notably , enhancement of post-training sleep occurs between 7–10 hr from the start of training regardless of the circadian time of training .",
"Males that were trained for LTM in the afternoon instead of the usual morning session , displayed an increased amount of sleep between 7–10 hr after onset of training ( Figure 1—figure supplement 1D ) .",
"They also had a normal LTM when tested 24 hr later ( Figure 1—figure supplement 1E , Figure 1—figure supplement 2A ) .",
"To evaluate their memory , we used automated video analysis to derive a courtship index ( CI ) for each male; CI is defined as the percentage of time over a 10 min test period during which the male courts the female .",
"Memory is represented as a suppression index ( SI ) , which is the relative reduction in the median courtship indices of trained versus naïve males: SI = 100*[1-CItrain+/CItrain-] ( Keleman et al . , 2012 ) .",
"In contrast , males that were trained for 1 hr to induce STM did not exhibit an increased amount of sleep ( Figure 1—figure supplement 1F ) .",
"Together , these data suggest that only training that can induce LTM leads to sleep enhancement between 7–10 hr after the start of training .",
"To investigate whether the enhancement of post-training sleep is caused by learning or a prolonged intense activity such as courtship towards mated female , we monitored sleep pattern and courtship memory of mutants for the dopamine receptor DopR1 that do not form courtship memory due to an impairment in memory acquisition ( Keleman et al . , 2012 ) .",
"DopR1 mutant males , although underwent the same courtship experience and courted mated females as vigorously or more than the wild-type males during 6 hr training ( Figure 1—figure supplement 1G ) , neither displayed an increased amount of post-training sleep ( Figure 1C ) nor formed LTM ( Figure 1—figure supplement 1H , Figure 1—figure supplement 2B ) .",
"Together , these data show that learning is essential for the sleep enhancement in the specific time window after training .",
"To determine whether post-learning sleep is necessary for LTM consolidation , we deprived flies of sleep during specific time intervals after training and tested their memory 24 hr later .",
"Single wild-type males were trained with mated females for 6 hr and immediately after were exposed to intermittent gentle mechanical perturbation spanning 2 hr intervals .",
"Naïve males were sleep deprived during the same time periods .",
"Control males that were trained with mated females and allowed to sleep , and males that were sleep deprived after training between 9–11 and 10–12 hr had normal SIs of approximately 30–40% .",
"However , males that were deprived of sleep between 7–9 and 8–10 hr had SIs indistinguishable from 0 ( Figure 2A , Figure 2—figure supplement 2A ) .",
"Deprivation of sleep during the night did not impair LTM significantly , except for a small but statistically-significant effect between 14–16 hr ( Figure 2—figure supplement 1A , Figure 2—figure supplement 2E ) .",
"These results show that sleep deprivation between 7–9 hr after the start of training blocks LTM , and thus post-learning sleep is necessary for LTM consolidation .",
"Previously , we had shown that silencing of DAN-aSP13s in a discrete time window after training impairs LTM ( Krüttner et al . , 2015 ) .",
"To test whether the temporal effect of sleep deprivation mimics the effect of DAN-aSP13s silencing , we expressed specifically in DAN-aSP13s ( Figure 2—figure supplement 1B ) a temperature-sensitive inhibitory form of dynamin shibire ( shits ) ( Kitamoto , 2002 ) ( VT005526-LexA>LexAop-shits ) , which blocks synaptic transmission at 32°C but not at 22°C .",
"We silenced DAN-aSP13s at 2 hr intervals after a 6 hr training session .",
"Inhibition of DAN-aSP13s between 7–9 or 8–10 hr , but not between 9–12 hr , after the onset of training abolished LTM .",
"Control males , in which DAN-aSP13s remained functional throughout the assay , had a normal SI between 30–40% ( Figure 2B , Figure 2—figure supplement 2B ) .",
"These results show that sleep and DAN-aSP13 activity are essential for memory consolidation in the same time window after training .",
"To investigate whether sleep can consolidate STM into LTM , we tested whether artificial enhancement of sleep would lead to memory consolidation .",
"We trained males for 1 hr , which does not generate LTM ( Krüttner et al . , 2015 ) , and then induced sleep at various time intervals afterwards .",
"Since activation of the FB neurons induces sleep ( Donlea et al . , 2011 ) , we expressed the thermosensitive cation channel TrpA1 ( Viswanath et al . , 2003; Rosenzweig et al . , 2005 ) ( open at 30°C and closed at 20°C ) in FB neurons ( 104y-GAL4 > UAS-TrpA1 ) and monitored sleep for 24 hr .",
"Consistent with previous reports , males in which FB neurons were activated slept significantly more than males that were kept at 20°C and the empty-GAL4 control males ( pBDP-GAL4 > UAS-TrpA1 ) ( Figure 2—figure supplement 1C ) .",
"To induce sleep precisely with 2 hr temporal resolution after a 1 hr training session , we used the optogenetic activator CsChrimson ( Klapoetke et al . , 2014 ) .",
"We found that activating FB neurons ( 104y-GAL4 > UAS CsChrimson ) in the period between 5–7 hr after the onset of training led to a SI of ~ 30% , which is similar to that obtained after LTM training ( Figure 2C , Figure 2—figure supplement 2C ) .",
"In contrast , males activated at other time intervals or not activated at all failed to consolidate LTM and had SIs that were indistinguishable from 0 .",
"This temporal window was identical to that observed for DAN-aSP13s activation to consolidate LTM ( VT005526-LexA > LexAop-CsChrimson ) ( Figure 2D , Figure 2—figure supplement 2D ) .",
"When we activated FB neurons ( 104y-GAL4 > UAS-CsChrimson ) while silencing DAN-aSP13s ( VT005526-LexA > LexAop-Shits ) between 5–7 hr after the onset of training we did not observe LTM ( Figure 2C , Figure 2—figure supplement 2C ) .",
"Taken together , these data show that enhancement of sleep after a learning experience that induces STM can consolidate it to LTM , in a manner that requires activation of DAN-aSP13s .",
"This LTM consolidation is a specific effect of DAN-aSP13s activation after training since activation of DAN-aSP13s ( VT005526-LexA > LexAop-CsChrimson ) in naïve males between 5–7 hr when they normally display a significant amount of sleep does not induce courtship suppression towards mated females and thus ‘courtship LTM’ ( Figure 2—figure supplement 1D ) .",
"To test whether FB activation excites DAN-aSP13s , we optogenetically activated FB neurons ( 104y-GAL4 > UAS-Chrimson88 ) and monitored calcium levels in DANs located in the protocerebral anterior medial ( PAM ) cluster in explant brains ( R58E02-LexA > LexAop-GCamP6s ) ( Pfeiffer et al . , 2008; Chen et al . , 2013 ) .",
"The PAM cluster of DANs consists of multiple neuronal classes , including a class of DAN-aSP13s , which is thought to respond to rewarding stimuli and promote their avoidance ( Aso et al . , 2014b ) ( Figure 3—figure supplement 1A ) .",
"Since the expression pattern of 104y-GAL4 is not exclusive to the FB ( Donlea et al . , 2011 ) , to activate FB neurons selectively , we used a digital mirror device ( DMD ) to target the illumination activating Chrimson to specific layers in the FB ( Strother et al . , 2018 ) .",
"104y-FB neurons form three layers of projections: dorsal ( dFB ) , medial ( mFB ) and ventral ( vFB ) ( Donlea et al . , 2011 ) .",
"To test the targeted illumination , we expressed in 104y neurons both Chrimson88 and GCamP6s ( 104y-GAL4 > UAS-Chrimson88 > UAS-GCamP6s ) and selectively activated one of two layers of the FB ( dFB or vFB ) while monitoring calcium levels in the presence of tetradotoxin ( TTX ) .",
"TTX inhibits propagation of action potentials ( Boccaccio et al . , 1999 ) , and thus calcium signals should only be observed in the projections located in the targeted layer .",
"Upon activation of either dFB or vFB layers , a robust calcium response was observed only in the activated layer , implying that this local activation was highly restricted ( Figure 3A ) .",
"We next activated all FB layers using targeted illumination and monitored calcium responses in DAN-aSP13s ( 104y-GAL4 > UAS-Chrimson88 , R58E02-LexA > LexAop-GCamP6s ) .",
"Local activation of FB neurons elicited an excitatory response in DAN-aSP13s ( Figure 3B ) .",
"Given that the FB has a multilayered organization , we next aimed to identify the specific layer of the 104y-FB expression pattern that provides excitatory input to DAN-aSP13s .",
"We individually activated all three 104y-FB layers and monitored calcium levels in DAN-aSP13s .",
"Surprisingly , activation of the dFB layer , which has been recently implicated in homeostatic sleep regulation ( Donlea et al . , 2011 ) , resulted in a mixture of small inhibitory and excitatory responses ( Figure 3C ) , whereas activation of the vFB layer induced excitatory responses in DAN-aSP13s ( Figure 3D ) .",
"Activation of the mFB had no effect on DAN-aSP13 activity ( Figure 3—figure supplement 1B ) .",
"These data show that neurons that project to the vFB and potentially the dFB layers provide an excitatory input onto DAN-aSP13s and may thus have a role in LTM consolidation .",
"To identify the specific neurons in the 104y-GAL4 line that project to the different FB layers , we performed multicolor flip-out experiments ( Nern et al . , 2015 ) .",
"We identified two distinct neuronal populations that project to either the dFB or vFB layers ( Figure 4—figure supplement 1A ) .",
"Next , we searched the Janelia ( Jenett et al . , 2012 ) and Vienna Tiles ( VT ) ( Tirian and Dickson , 2017 ) collections for specific GAL4 driver lines with expression in these two cell types .",
"We identified one sparse GAL4 line with expression in the dFB neurons ( R23E10 ) and one in the vFB neurons ( VT036875 ) .",
"Since the expression pattern of the VT036875-GAL4 line also includes a class of DAN-β’1 neuron , we combined this line with PAM-GAL80 to restrict its expression to vFB neurons only ( VT036875-GAL4 , R58E02-GAL80 ) .",
"In addition , we generated a split line ( SS057264-GAL4 ) ( Pfeiffer et al . , 2010; Luan et al . , 2006 ) with the expression restricted to the vFB neurons only ( Figure 4—figure supplement 1B , C , D , E ) .",
"As previously reported , thermogenetic activation of dFB over a 24 hr time period resulted in an increased amount of sleep ( R23E10-GAL4 > UAS-TrpA1 ) ( Figure 4A ) .",
"Sleep increase was displayed mainly during the day time since males sleep almost continuously during night .",
"We found that activation of vFB ( VT036875-GAL4 > UAS-TrpA1; VT036875-GAL4 > UAS-TrpA1 , R58E02-GAL80; SS057264-GAL4 > UAS-TrpA1 ) also robustly induced day time sleep ( Figure 4B , C , D ) in comparison to the genetic control ( pBDP-GAL4 > UAS-TrpA1 ) ( Figure 4—figure supplement 1G ) .",
"Loss of night time sleep observed upon prolonged vFB activation with VT036875-GAL4 ( Figure 4B ) was likely caused by co-activation of wake promoting DAN-β’1 neurons ( Sitaraman et al . , 2015a ) ( Figure 4—figure supplement 1C ) .",
"Accordingly , males upon activation with the same line while excluding DAN-β’1 neurons displayed a higher level of both day and night sleep ( Figure 4C , Figure 4—figure supplement 1D ) .",
"Similarly , acute optogenetic activation of the dFB and vFB neurons with CsChrimson for 1 hr also significantly enhanced the amount of sleep ( R23E10-GAL4 > UAS -CsChrimson; VT036875-GAL4 > UAS-CsChrimson; VT036875-GAL4 > UAS-CsChrimson , R58E02-GAL80; SS057264-GAL4 > UAS-CsChrimson; 104y-GAL4 > UAS-CsChrimson ) in comparison to the genetic control ( pBDP-GAL4 > UAS-CsChrimson ) but to a lesser degree when DAN-β’1 neurons were co-activated ( Figure 4—figure supplement 1F ) .",
"To test whether these neurons provide an excitatory input on DAN-aSP13s , we activated either dFB ( R23E10-GAL4 > UAS-Chrimson88 ) or vFB ( VT036875-GAL4 > UAS-Chrimson88; VT036875-GAL4 > UAS-Chrimson88 , R58E02-GAL80; SS057264-GAL4 > UAS-Chrimson88 ) neurons and monitored calcium levels in DAN-aSP13s .",
"Since specific DANs innervate MB lobes in well-defined discrete areas , we used a broad PAM-DAN GAL4 driver ( R58E02-LexA > LexAop-GCamP6s ) .",
"To monitor activity specifically in DAN-aSP13s upon activation of FB neurons we focused on the region at the tip of the MBγ lobe ( Figure 3—figure supplement 1A ) .",
"Activation of dFB neurons did not elicit calcium changes in DAN-aSP13s ( Figure 4A ) however , activation of vFB neurons elicited a robust increase of calcium levels in DAN-aSP13s ( Figure 4B , C , D ) .",
"These data show that neurons in the vFB provide excitatory input onto DAN-aSP13s .",
"Consistent with the functional connectivity results , optogenetic activation of dFB neurons ( R23E10-GAL4 > UAS-CsChrimson ) between 5–7 hr after a 1 hr training period did not lead to LTM consolidation .",
"However , activation of either a combination of dFB and vFB neurons ( 104y-GAL4 > UAS-CsChrimson ) or just the vFB neurons ( VT036875-GAL4 > UAS-CsChrimson; VT036875-GAL4 > UAS-CsChrimson , R58E02-GAL80; SS057264-GAL4 > UAS-CsChrimson ) in that same time window fully consolidated STM to LTM ( Figure 5A , Figure 5—figure supplement 1A ) .",
"In addition , silencing of dFB neurons ( R23E10-GAL4 > UAS-Shits ) in the period between 7–10 hr after the onset of a 6 hr training period did not affect LTM persistence , while silencing of a combination of dFB and vFB or just the vFB neurons ( VT036875-GAL4 > UAS-Shits; SS057264-GAL4 > UAS-Shits and 104y-GAL4 > UAS-Shits ) in the same time window strongly impaired LTM consolidation ( Figure 5B , Figure 5—figure supplement 1B ) .",
"These results show that a class of sleep-promoting neurons in the vFB layer of the 104y-FB expression pattern is necessary and sufficient to consolidate LTM during post-learning sleep .",
"dFB neurons , although they promote sleep , appear to have no role in courtship LTM consolidation ."
],
[
"The activity of DAN-aSP13s , which is essential for courtship memory acquisition ( Keleman et al . , 2012 ) , is also necessary during a discrete post-learning time window for LTM consolidation ( Krüttner et al . , 2015 ) .",
"Because neuronal reactivation occurs during sleep in rodents ( Wilson and McNaughton , 1994; Smith , 2001 ) , we hypothesized that post-learning activation of DAN-aSP13s involves a sleep-dependent mechanism .",
"Using behavioral analysis and neuronal activity monitoring and perturbation approaches , we have shown here that DAN-aSP13s display an increased activity in freely behaving animals during sleep after a prolonged learning experience .",
"We demonstrated that this sleep is necessary for LTM consolidation , and it can be mediated by a specific class of sleep promoting neurons in the ventral layer of the FB ( vFB ) .",
"These vFB neurons consolidate courtship LTM in a discrete time window and provide an excitatory input to DAN-aSP13s .",
"Thus , the data we present here provide a causal link between sleep promoting neurons in the vFB , post-learning activation of dopaminergic neurons , and LTM consolidation .",
"Based on our data , we propose the following model for sleep-dependent consolidation of courtship LTM in Drosophila ( Figure 6 ) .",
"During a prolonged learning experience , γKCs and DAN-aSP13s are repeatedly activated by olfactory and behavioral cues presented by an unreceptive female , respectively .",
"Whereas prolonged wakefulness leads to an increase in homeostatic sleep drive in ellipsoid body neurons that in turn is conveyed to dFB ( Liu et al . , 2016 ) , we hypothesize that an extended learning experience generates a learning-dependent sleep drive that is transmitted to vFB .",
"In turn , the vFB neurons enhance sleep after learning and provide an excitatory input back on DAN-aSP13s .",
"We believe that one potential site of a learning-dependent sleep drive are the MB neurons since they have been implicated in both memory formation ( Heisenberg et al . , 1985 ) and sleep regulation ( Sitaraman et al . , 2015b ) .",
"Dopamine released upon DAN-aSP13 reactivation stimulates molecular processes in γKCs ( Krüttner et al . , 2015 ) that are different from those engaged during STM acquisition and involve protein synthesis that is essential for LTM formation and persistence .",
"Dopaminergic pathways are thought to convey information about whether an experience is rewarding or punishing and thus , worth remembering ( Gais et al . , 2002; Schultz , 2010 ) .",
"Post-learning neuronal activity of the dopaminergic hippocampal inputs from the Ventral Tagmental Area ( VTA ) has been implicated in the consolidation of fear memory in rodents .",
"Interestingly , this activity is critical during a discrete time window after learning ( Rossato et al . , 2009 ) .",
"Post-learning activity of the VTA dopamine neurons has been also implicated in the reactivation during sleep of the hippocampal cells involved earlier in encoding of the spatial experience ( Gomperts et al . , 2015 ) .",
"Here we show that post-learning activation of DAN-aSP13s mediates the consolidation of courtship LTM in Drosophila .",
"We propose that reactivation during sleep of the dopamine neurons that were previously active during memory acquisition ensures that spurious experiences are not admitted into LTM storage and thus only experiences that are either sufficiently salient or persistent become long-lasting memories .",
"Specifically , reactivation of DAN-aSP13s during post-learning sleep enhances reactivation of the γKCs and cognate MBON-M6 , which together with DAN-aSP13s form a recurrent circuit necessary for courtship memory acquisition ( Zhao et al . , 2018 ) .",
"We consider two hypotheses to account for this selectivity .",
"During sleep , vFB neurons might selectively reactivate only the relevant DANs , or alternatively , they might activate all DANs but only the relevant subset is able to consolidate LTM .",
"The selective-reactivation model would require some marker to distinguish which DANs were activated , whereas the selective-consolidation model would require a marker in the synapses of the γKCs that were earlier active during memory acquisition , for example translational regulator Orb2 , which regulates translation upon neuronal activity during LTM consolidation ( Krüttner et al . , 2015 ) .",
"Activity of dopaminergic neurons regulates sleep-wake states in animals , including flies ( Dzirasa et al . , 2006 ) .",
"Artificial activation of DAN-aSP13s has been shown to increase wakefulness ( Sitaraman et al . , 2015a ) .",
"In contrast , the data we present here imply that activation of vFB neurons , although they activate DAN-aSP13s , do not promote wakefulness .",
"These results suggest that activation of DAN-aSP13s by vFB neurons during sleep is qualitatively different from a direct optogenetic or thermogenetic activation used in previous studies .",
"One potential explanation is that post-learning sleep involves activation of the vFB circuit , which provides both excitatory stimulus to DAN-aSP13s and inhibitory input to motor neurons .",
"Another possibility is that post-learning sleep activates a subset od DAN-aSP13s that do not affect wakefulness .",
"We have identified here a class of sleep-promoting neurons in the ventral layer of FB that are distinct from the well-studied sleep-promoting neurons in the dorsal layer of FB , which regulate sleep homeostasis ( Donlea et al . , 2011; Donlea et al . , 2014; Pimentel et al . , 2016 ) .",
"Given that vFB neurons enhance sleep and activate DAN-aSP13s for LTM consolidation , whereas dFB neurons are neither necessary nor sufficient for LTM consolidation , we hypothesize that dFB and vFB neurons promote distinct components of sleep that have different functions .",
"Homeostatic sleep is thought to facilitate memory encoding by downscaling synaptic weights and clearing metabolites from the brain accumulated during wakefulness ( Bushey et al . , 2011; Xie et al . , 2013; Tononi and Cirelli , 2014 ) .",
"In contrast , the function of learning-dependent sleep might be to facilitate memory consolidation by strengthening synaptic connections that were engaged earlier during memory acquisition ( Frank , 2012 ) .",
"Thus , the co-operation of homeostatic- and experience-dependent sleep would facilitate optimal conditions for learning new information and , if appropriate , incorporating it into long-term storage .",
"Recent studies have implied that sleep in flies , as in humans and rodents , exhibits sleep stages characterized by distinct electrophysiological signatures ( Yap et al . , 2017; van Alphen et al . , 2013 ) .",
"Interestingly , the signature of sleep that is induced by activation of the dFB neurons seems to have a simpler oscillatory pattern , recorded by local field potentials , than sleep that is induced by activation of the FB neurons comprising both dFB and vFB neurons ( Yap et al . , 2017 ) .",
"Thus , these data support our hypothesis that dFB and vFB neurons promote sleep with different properties and likely different functions .",
"It is thought that sleep evolved in animals that are capable of complex learning which requires selective attention ( Kirszenblat and van Swinderen , 2015 ) .",
"Courtship learning is a multisensory form of learning that requires selective attention of a male to associate multiple learning cues presented by the mated female with the outcome of his own behavior ( Kirszenblat and van Swinderen , 2015 ) .",
"Accordingly , studies in bees have shown that sleep affects a complex form of learning such as spatial memory but has no role in the simple learning paradigm of proboscis extension ( Vorster and Born , 2015 ) .",
"Hence , it would be interesting to investigate whether post-learning sleep is involved in the consolidation of other types of memory in Drosophila , such as the well-studied Pavlovian olfactory associative learning whereby animals associate an individual learning cue with a behavioral contingency .",
"In this work , we establish a functional link between a novel class of sleep-promoting neurons in the FB , post-learning reactivation of dopaminergic neurons and consolidation of courtship LTM .",
"Moreover , our data suggest that sleep promoting vFB neurons mediate a learning-dependent regulation of sleep that is distinct from the homeostatic control which is facilitated by dFB neurons ( Liu et al . , 2016 ) .",
"Thus , we uncover a causal link between sleep-mediated neuronal reactivation and LTM consolidation in Drosophila .",
"In addition , we establish courtship LTM in Drosophila as a tractable model to investigate the mechanisms that link learning-dependent sleep , neuronal reactivation and LTM consolidation ."
],
[
"Flies for behavioral experiments were reared in vials with standard cornmeal food at 25°C , or as indicated , at 60% humidity in a 12 hr light:12 hr dark cycle .",
"Detailed information regarding specific strains and genotypes is provided in the Key resources table section .",
"Courtship conditioning was performed as described ( Siwicki and Ladewski , 2003 ) .",
"Briefly , solitary males aged for 5–6 days after eclosion were placed for training ( during day time as indicated ) in food chambers for 1 hr ( STM ) or 6 hr ( LTM ) either with ( trained ) or without ( naïve ) a single mated female .",
"After training each male was recovered , allowed to rest for 30 min or 24 hr and tested with a fresh mated female .",
"Tests were performed in 10 mm diameter chambers and videotaped for 10 min ( Prosilica GT cameras , Allied Vison Technologies ) .",
"Automated video analysis was used to derive a courtship index ( CI ) for each male , defined as the percentage of time over a 10 min test period during which the male courts the female .",
"Memory was calculated as a suppression index ( SI ) that is a relative reduction in the mean courtship indices of trained ( CI+ ) versus naïve ( CI- ) populations: SI = 100*[1-CItrain+/CItrain-] .",
"To monitor sleep over a 24 hr time period , male flies were individually inserted into 65 mm glass tubes containing standard fly food , loaded into TriKinetics Drosophila activity monitors ( DAM ) , and housed under 12 hr light:12 hr dark schedule ( Donelson et al . , 2012 ) .",
"Periods of inactivity lasting at least 5 min were classified as sleep .",
"Total 24 hr sleep quantity ( day time and night time sleep ) was extracted from DAM system as described ( Donelson et al . , 2012 ) .",
"To monitor sleep upon acute induction with csChrimson , individual males were reared at 25°C on retinal supplemented ( 0 . 1 mM ) cornmeal medium in darkness for 4–5 days after eclosion .",
"For sleep induction single males were placed in 10 mm diameter behavioral chambers in a temperature and illumination-controlled box and videotaped ( Prosilica GT cameras , Allied Vison Technologies ) .",
"The amount of sleep was scored manually with periods of inactivity lasting at least 5 min being considered as sleep .",
"For sleep deprivation , flies were subjected to intermittent mechanical perturbation method while housed in TriKinetics DAM monitors .",
"Flies received mechanical perturbations on a horizontal shaker with a total cycle of 15 s/min delivered in eight pulses of 1–3 s each occurring intermittently at random times .",
"A MATLAB script ( Kamyshev et al . , 1999 ) ( permutation test ) implemented in Keleman et al . ( 2012 ) was used for statistical comparison of SIs between two groups .",
"Briefly , the entire set of courtship indices for both naïve and trained flies were pooled and then randomly assorted into simulated naïve and trained groups of the same size as the original data .",
"A SI was calculated for each of 100 , 000 randomly permutated data sets , and P values were estimated for the null hypothesis that learning equals 0 ( H0: SI = 0 ) or for the null hypothesis that experimental and control males learn equally well ( H0: SI = SIc ) .",
"Luminescence assay for detecting neuronal activity in freely behaving adult flies was modified from Guo et al . ( 2017 ) .",
"Flies were starved on filter soaked in water overnight prior to training .",
"They were transferred to fresh chambers with filter paper containing 40 mM Luciferin ( GOLDBIO ) in a 2% sucrose solution .",
"For luminescence measurements after training , flies were placed into 96-well plates ( Greiner Bio-one ) with 40 mM Luciferin in a 2% sucrose solution .",
"CLARIOstar microplate reader ( BMG Labtech ) was used for luminescence detection .",
"Luminescence was measured every 15 min over 16 hr .",
"Relative Luminescence ( R . Luminescence ) was calculated by dividing the luminescence of the experienced and naïve groups by the luminescence of the genetic controls ( males with luciferase reporter only , fed with luciferin ) at every measurement time point .",
"Single housed male flies were reared at 25°C on retinal supplemented ( 0 . 1 mM ) cornmeal medium in darkness for 4–5 days after eclosion .",
"For sleep induction or memory consolidation specific classes of neurons were optogenetically activated in behavior chambers housed in a temperature and illumination-controlled box .",
"During activation , the behavior chamber was illuminated with 617 nm LEDs ( Red-Orange LUXEON Rebel LED—122 lm; Luxeon Star LEDs , Brantford , Ontario , Canada ) with a 3 mm thick diffuser between the LED and flies .",
"The LED was driven by a customized linear current controller .",
"20 HZ red light was used to activate neurons with intensity varying from 20 uW/mm2 to 70 uW/mm2 .",
"The LED board was cooled by a customized liquid cooling system to maintain the temperature in the chamber .",
"Single housed male flies were reared at 25°C on retinal supplemented ( 0 . 1 mM ) cornmeal medium in darkness for 4–5 days after eclosion .",
"Brains of the immobilized flies on ice were dissected out in saline ( 103 mM NaCl , 3 mM KCl , 2 mM CaCl2 , 4 mM MgCl2 , 26 mM NaHCO3 , 1 mM NaH2PO4 , 8 mM trehalose , 10 mM glucose , 5 mM TES , bubbled with 95% O2/5% CO2 ) and mounted anterior up on the cover slip in a Sylgard-lined dish in a 20°C saline bath .",
"Brains were imaged with a resonant scanning two-photon microscope with near-infrared excitation ( 920 nm , Spectra-Physics , INSIGHT DS DUAL ) and a 25x objective ( Nikon MRD77225 25XW ) .",
"The microscope was controlled by ScanImage 2015 . v3 ( Vidrio Technologies ) .",
"Images of brain volume were acquired with ~ 157 μm x 157 μm field of view at 512 × 512 pixels resolution .",
"Each frame and each volume was sampled by 42 frames with 3 μm per step , at approximately 1 Hz volume rate .",
"Times series of volume images were acquired and the excitation power for calcium imaging ~ 12 mW .",
"For the optogenetic activation , the light-gated ion channel Chrimson88 was activated with a 660 nm LED ( M660L3 Thorlabs ) coupled to a digital micromirror device ( DMD ) ( Texas Instruments DLPC300 Light Crafter ) and combined with the imaging light path using a FF757-DiO1 dichroic ( Semrock ) .",
"On the emission side , the primary dichroic was Di02-R635 ( Semrock ) , the detection arm dichroic was 565DCXR ( Chroma ) , and the emission filters were FF03-525/50 and FF01-625/90 ( Semrock ) .",
"Photostimulation light was delivered in a pulse train that consisted of six 8 s or 15 s pulses ( 100% duty cycle during each pulse ) with a 30 s inter-pulse interval .",
"The light intensity was ~ 0 . 6 mW/mm2 , as measured using Thorlabs S170C power sensor .",
"All image data were analyzed off-line .",
"Region of interest ( ROI ) of DAN-aSP13 at mushroom body γ5 compartment was manually defined in Fiji or CircuitCatcher ( a customized python program by Daniel Bushey ) and the average GCaMP signal intensity within the DAN-aSP13 ROI was taken as the calcium activity of DAN-aSP13 .",
"Time series calcium activity of DAN-aSP13 ( f ( t ) ) was extracted from the image data and then analyzed with customized Matlab ( MathWorks ) programs .",
"The normalized Calcium activity of DAN-aSP13 dF/F is defined as:dF/F= f ( t ) −F0F0 where the f ( t ) is the calcium signal intensity and the F0 is the mean F of the first 10 s of the image session before optogenetic activation .",
"dF/F traces of six stimulation were aligned to the LED onset and averaged to represent the DAN-aSP13 activity upon neuron activation .",
"To determine the connectivity from the activated neuron to DAN-aSP13 , the mean dF/F during 10 s pre-stimulation and the mean dF/F during stimulation were taken as baseline activity and stimulated activity in a fly .",
"Groups of baseline activities and stimulated activities of different flies were tested with student’s test or Wilcoxon rank sum test to determine if optogenetic activation had evoked significant calcium activity changes in DAN-aSP13 against the hypothesis that the baseline activity and stimulated activity were the same level .",
"p value smaller than 0 . 05 was taken as the criteria of connectivity ( either inhibitory or excitatory ) .",
"Correlation of determination ( r2 ) was used to measure the correlation between the time series calcium trace ( f ( t ) ) of each voxel and a predicted model .",
"Here we used the voltage driving the stimulation LED ( V ( t ) ) as the model .",
"The calcium trace of each voxel was firstly normalized to z-score , calculated with the following functionZ ( t ) = f ( t ) −μσ Where μ is the mean and σ is the standard deviation estimated of the f ( t ) .",
"Then by a linear fit of the model V ( t ) , we have the predicted calcium response Zp ( t ) .",
"Correlation coefficient ( r ) between Z ( t ) and Zp ( t ) was calculated by:r= cov ( Z ( t ) , Zp ( t ) ) σZ∗σZpwhere r= covZt , ZptσZ*σZp is the covariance of Z ( t ) and Zp ( t ) , σZ and σZp are the standard deviation of Z ( t ) and Zp ( t ) .",
"The correlation of determination ( R2 ) was simply the square of the r .",
"R2 is by definition in the range of 0 to 1 .",
"The higher the value is , the higher is the correlation ( both positive and negative ) between a voxel’s calcium trace and stimuli .",
"The r2 indexes of a whole brain volume are projected to a 16-bit image stack , which demonstrates the calcium response pattern evoked by stimuli .",
"Multicolor Flip-out ( MCFO ) was performed according to the protocol described in Nern et al . ( 2015 ) .",
"Immunostaining was performed according to a protocol described in Zhao et al . ( 2018 ) ."
]
] | [
"Animals consolidate some , but not all , learning experiences into long-term memory .",
"Across the animal kingdom , sleep has been found to have a beneficial effect on the consolidation of recently formed memories into long-term storage .",
"However , the underlying mechanisms of sleep dependent memory consolidation are poorly understood .",
"Here , we show that consolidation of courtship long-term memory in Drosophila is mediated by reactivation during sleep of dopaminergic neurons that were earlier involved in memory acquisition .",
"We identify specific fan-shaped body neurons that induce sleep after the learning experience and activate dopaminergic neurons for memory consolidation .",
"Thus , we provide a direct link between sleep , neuronal reactivation of dopaminergic neurons , and memory consolidation ."
] | [
"Why do some memories fade after only a few seconds , whereas others last a lifetime ?",
"Studies suggest that part of the explanation has to do with sleep .",
"Experiments in rodents show that neural circuits that are active during learning become active again when an animal sleeps .",
"This process of reactivation , which may be akin to dreaming , helps strengthen specific memories and move them into long-term storage .",
"But the complexity of the mammalian brain has made it difficult to pin down the underlying mechanisms .",
"One possible solution is to study the mechanisms in a simpler brain with fewer neurons , such as that of the fruit fly Drosophila .",
"Dag , Lei et al . have now used molecular genetic tools to explore how sleep supports a specific type of learning in male fruit flies , called courtship learning .",
"Female fruit flies that have recently mated will reject the courtship efforts of other males .",
"A male fly that experiences repeated rejections therefore learns to avoid mated females in future .",
"This type of memory can last for at least a day – a long time in the life of a fly .",
"Dag , Lei et al . show that males that experience repeated rejections subsequently spend more time asleep than control males .",
"Preventing this sleep hinders the males from learning from their experience .",
"But how does this process work ?",
"During sleep , specific dopamine neurons that were active during the learning episode become active once again .",
"Blocking this reactivation prevents the flies from learning from their rejections .",
"By contrast , artificially activating the dopamine neurons enables flies with only limited experience of rejection to learn to avoid mated females .",
"Dag , Lei et al . show that neurons called vFB cells control this process .",
"The vFB neurons both induce sleep and reactivate the memory-inducing dopamine neurons .",
"These findings in fruit flies thus reveal a direct causal link between sleep , reactivation of memory traces , and persistence of memories .",
"They also show that fruit flies are a valid model for exploring the neural and molecular mechanisms connecting sleep and long-term memory ."
] | 2019 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience"
] | A longitudinal study of Caenorhabditis elegans larvae reveals a novel locomotion switch, regulated by Gαs signaling | elife-00782-v1 | [
[
"The life cycle of the nematode Caenorhabditis elegans is comprised of the embryonic stage , four larval stages termed L1–L4 , and adulthood .",
"Each larval stage lasts 8–12 hr at 20°C , and under standard conditions , and ends with a molt , when epidermal cells synthesize a new cuticle and the old one is shed .",
"Although C . elegans larvae are mostly continuously motile , each molt is accompanied by a 2–3 hr period of behavioral quiescence ( Singh and Sulston , 1978 ) , referred to as ‘lethargus’ .",
"Thus , the larval stages can be divided into motile intermolt sub-stages ( L1int–L4int ) and their corresponding lethargus sub-stages ( L1leth-L4leth ) .",
"However , behavior during distinct developmental sub-stages ( Singh and Sulston , 1978 ) has not previously been examined in detail .",
"Specifically , both the modulation of body posture and locomotion on developmental timescales remain largely unexplored .",
"C . elegans move directionally by propagating dorsoventral undulations along their body .",
"Upon receiving input from interneurons , motoneurons provide output to an array of longitudinal body-wall muscles in order to propagate body bends ( White et al . , 1986; Stetina et al . , 2006; Karbowski et al . , 2006; Stephens et al . , 2008 ) .",
"Head/neck motoneurons and muscles independently control head movements , such as exploratory head swings ( White et al . , 1986; Stetina et al . , 2006; Pirri et al . , 2009; Maguire et al . , 2011 ) .",
"The gsa-1 gene encodes a Gαs subunit , which activates the adenylyl cyclase ACY-1 , and the GSA-1 ( Gαs ) pathway has been previously shown to affect locomotion .",
"Activation ACY-1 by GSA-1 leads to the production of cyclic adenosine monophosphate ( cAMP ) .",
"The binding of cAMP to the protein kinase A ( PKA ) regulatory subunit KIN-2 dissociates it from the inactive holoenzyme , releasing the PKA catalytic subunit , KIN-1 .",
"Increased PKA activity enhances signaling at the neuromuscular junction , as well as increases cAMP response element ( CRE ) mediated transcription in C . elegans neurons ( Kimura et al . , 2002 ) .",
"Thus , upregulating this signaling pathway has been reported to result in hyperkinetic phenotypes , typically described as a nonspecific increase in the rate of locomotion ( Schade et al . , 2005; Reynolds et al . , 2005; Raizen et al . , 2008; Perez-Mansilla and Nurrish , 2009 ) , as well as in reduced quiescence during lethargus ( Raizen et al . , 2008; Belfer and Raizen , 2013 ) .",
"Here we present the first detailed analysis of locomotion patterns during developmentally relevant timescales , that is , periods in which significant developmental changes occur .",
"We have analyzed the initiation , propagation and eventual demise of individual dorsoventral body bends over a 14-hr period , from the mid-L4int stage to the mid-young-adult stage .",
"We found that some locomotion patterns undergo a gradual modulation , while others display abrupt switching .",
"In particular , two behavioral states , active wakefulness and quiet wakefulness , were observed during the mid- to late- L4int stage .",
"Active wakefulness was dominated by forward locomotion ( propagation of body-bends from the anterior to the posterior ) , but included intervals of backward locomotion and ‘dwelling’ ( non-directional dynamics of body bends ) .",
"In contrast , quiet wakefulness was dominated by dwelling behavior , although it included intervals of directed locomotion .",
"In individual animals , active wakefulness was observed to persist for epochs of 1–100 min .",
"Moreover , the switching between active and quiet wakefulness was abrupt , suggesting that they are distinct behavioral states .",
"The process underlying these states exhibited the signature of ergodicity breaking , characteristic of a scale-free switch , as opposed to having a simple rate constant that governs the dynamics of switching .",
"We further show that the transitions between behavioral states were regulated by the GSA-1 ( Gαs ) pathway: increased Gαs signaling stabilized the active wakefulness state both within and outside of lethargus , while decreased Gαs signaling destabilized this state , but only outside of lethargus ."
],
[
"To assay the modulation of behavior during development , we developed PyCelegans: a high-speed , modular image-processing tool for analyzing posture and locomotion of C . elegans on high performance computing resources .",
"The function we used for processing a single frame was limited to tracking a single animal .",
"However , the modular design of PyCelegans can accommodate multi-animal tracking once an appropriate substitute for this function is implemented , without further changes .",
"The rate of data capture for recording throughout a larval developmental stage at a sufficiently high temporal resolution can exceed 3 , 000 , 000 images per day .",
"For a dataset of this magnitude , the required post-processing is the rate-limiting step of the experiment .",
"Using PyCelegans , the rate-limiting component of the analysis scaled linearly with the number of available processing-cores .",
"By using 256 cores we achieved a speed-up of two orders of magnitude relative to previous implementations .",
"For proof of principle , analyses have been run on up to 1024 processors .",
"The number of processors that could be utilized , for example , from publically available clusters , is in the tens of thousands for a single dataset .",
"The objective of the image-processing portion of PyCelegans is to identify the head , tail , and body of the animal and to compute secondary properties based on this identification ( e . g . , the body midline , perimeter , and orientation ) .",
"Tertiary properties can then be computed from the resulting raw data .",
"Our analysis of posture and locomotion was based on dividing the midline of the body of each animal into 20 segments and measuring the local angles between them ( Figure 1A–B ) .",
"This method extended previous analyses based on body curvature as a function of time and body coordinate ( Fang-Yen et al . , 2010; Vidal-Gadea et al . , 2011 ) : we tagged individual body-bends , followed them from initiation to eventual demise , and recorded their origin , velocity , amplitude , and life-time .",
"The identification of the midlines and individual body-bends enabled measurements of local properties as a function of the body-coordinate ( e . g . , quiescence of individual body-segments , Figure 1C ) , as well as global behavioral patterns such as mean curvature and growth rate ( Figure 1D ) or modes of locomotion ( Figure 2 ) .",
"For instance , a 2% growth of body-length per hour was observed outside of lethargus , but during lethargus a 5% shortening of the body-length was observed ( Figure 2D ) .",
"This shortening may be due to continuous growth in body-volume at a period where adding surface area to the ( old ) cuticle was restricted .",
"PyCelegans enabled us to conduct such high-resolution measurements over extended periods for the first time . 10 . 7554/eLife . 00782 . 003Figure 1 . Measurements of angles associated with body-segments: the midline of the body of the worm was fitted to a spline and divided into 20 equal segments . The local angle at each of the 18 inner segments , θi , was defined as the angle between its two flanking segments , si−1 and si+1 .",
"( A ) Body-bends propagating from the head ( left arrow ) to the tail ( right arrow ) during forward locomotion in a wild-type mid L4 larva .",
"For the purpose of demonstration , the local angles of only eight segments along the body were plotted .",
"The color map corresponds to angles from the most anterior ( brown ) , through the middle ( orange and yellow ) , to the most posterior ( grey ) body-regions .",
"Each bend appears as a peak or a trough in the local angle .",
"A successive bend is initiated at the head ∼4 s before its predecessor reaches the tail .",
"( B ) Angles at the same eight body segments during early L4 lethargus .",
"When the animal is quiescent the local angles remain constant or relax slowly .",
"( C ) The fraction of time , calculated from a 10 min running window , that individual body segments ( as denoted on the worm schematic ) are quiescent .",
"Body-segments that are further removed from the head of the animal exhibit more quiescence than more anterior segments , such that the overall quiescent behavior associated with L4 lethargus reflects the quiescence of the head and the neck .",
"( D ) The length of the body ( top ) and the mean angle along its midline between the mid L4 and mid young adult stages .",
"Shaded area denotes the L4 lethargus period .",
"The reduction in body-length during lethargus may be a signature of growth in the volume of the body at a time when expanding the surface area of the body is constrained .",
"Panels ( C and D ) depict N = 37 animals , mean ± SEM .",
"Standard errors are illustrated as shadowed areas surrounding the plotted averages . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 00310 . 7554/eLife . 00782 . 004Figure 2 . Modulation of locomotion between the mid-L4 and mid-young-adult stages in wild-type animals .",
"( A ) The locomotion behavior of each animal at each time-point was determined to belong to one of four characteristic classes: forward ( Fwd ) , dwelling ( Dwl ) , quiescence ( Qsc ) or backward ( Bck ) .",
"The average fractions of time out of a 10-min running window in which animals exhibited each of the four characteristic behaviors were plotted .",
"( B ) The frequency of generation of body-bends ( top ) and the velocity of bends that propagated from the anterior to the posterior of the body , that is , during forward locomotion .",
"During lethargus , rearward propagating bends were rare , and a meaningful average could not be obtained .",
"Panels ( A and B ) depict N = 37 animals , mean ± SEM .",
"Standard errors are illustrated as shadowed areas surrounding the plotted averages .",
"( C and D )",
"The modulation of locomotion in single animals .",
"The bars above each plot indicate epochs of active wakefulness ( dark brown ) or quiet wakefulness ( light grey ) .",
"In all panels the shaded rectangular area indicates the period of L4 lethargus . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 004 Although the term lethargus , describing reduced activity associated with molting in a variety of nematode species , was already in use about a century ago ( Veglia , 1915; Sommerville , 1960; Singh and Sulston , 1978 ) , the detailed dynamics of this behavior have only recently been studied ( Raizen et al . , 2008; Iwanir et al . , 2013; Belfer and Raizen , 2013 ) .",
"In C . elegans , head/neck and body motor circuits govern the generation and propagation of body-bends ( White et al . , 1976 , 1986; McIntire et al . , 1993; Stetina et al . , 2006; Chen et al . , 2007; Wen et al . , 2012 ) , and the body motoneuron network exhibits an iterative pattern of six interconnected modules along the anterior-posterior axis ( Haspel and O’Donovan , 2011 ) .",
"It was thus possible that different body regions would exhibit distinct temporal dynamics of quiescence .",
"To identify such patterns , we divided the midline of the animal to 20 equi-length segments , and quantified the fraction of time spent in quiescence by the individual angles between pairs of segments , during the period from mid L4int to the mid young adult stage ( Figure 1C ) .",
"While an abrupt increase in the fraction of quiescence at the onset of lethargus occurred in all of the individual body-regions , most of them exhibited elevated quiescence during the preceding 2-hr period .",
"Interestingly , the most anterior region was the least quiescent throughout the measurement .",
"Subsequent regions exhibited successively increasing quiescence , with the mid-body being the most quiescent .",
"As a result of this ordering , the temporal dynamics of quiescence of the entire animal mirrored that of the head/neck region .",
"We thus concluded that the activity of the head/neck motoneuron circuits determined the outcome of previously reported measurements of whole-animal quiescence ( Raizen et al . , 2008; Singh et al . , 2011; Bringmann , 2011; Iwanir et al . , 2013; Belfer and Raizen , 2013 ) .",
"Although behavioral quiescence provides the most prominent example of modulation of posture and locomotion during development ( Raizen et al . , 2008; Singh et al . , 2011; Iwanir et al . 2013; Ghosh and Emmons 2008; Schwarz et al . , 2012 ) , additional instances of modulation could occur outside of lethargus .",
"To identify such phenomena , we focused on four categories of locomotion behavior: forward and backward locomotion were defined by the appropriate directional propagation of body-bends along the anterior-posterior axis , dwelling was defined as non-directional propagation of body-bends , and quiescence was defined as sub-threshold changes in body angles ( see ‘Materials and methods’ ) .",
"Aligning behavioral data by the onset of lethargus for each 10 hr measurement and averaging between animals , we observed a decrease in the percentage of time spent in forward locomotion during the 4 hr preceding L4leth and a corresponding increase in the percentage of time spent in dwelling ( Figure 2A ) .",
"During the first 1–2 hr following L4leth termination , wild-type behavior was characterized by a high , decreasing fraction of dwelling , and a low , increasing fraction of forward locomotion .",
"Backward locomotion was modulated more weakly during the period of the measurement .",
"In agreement with our previous findings using complementary methods ( Iwanir et al . , 2013 ) we found that the overall curvature , quantified as the mean absolute angle , was modulated during lethargus , and that the overall growth rate of the body length outside of lethargus was 2% per hour ( Figure 1D ) .",
"In addition , the propagation velocity of anterior-to-posterior body-bends and the frequency of generation of bends were gradually downregulated during late L4int , and upregulated during the early young adult stage ( Figure 2B ) .",
"Although the frequency of bend generation was naturally limited by how quickly bends propagated away from their point of origin , there was no a priori restriction on how low this frequency could be .",
"The similarity between the dynamics of bend generation frequency and of bend velocity suggested that the former could serve as an adequate proxy for the latter throughout the measurement period .",
"The gradual modulation of the average behavioral dynamics shown in Figure 2A could result from a progressive modulation in individual animals or from abrupt but asynchronous events .",
"To distinguish between these possibilities , we examined the behavior of the individual animals in our dataset .",
"We found that the forward velocities of individual animals decreased gradually ( data not shown ) .",
"In contrast , the fraction of time spent in forward locomotion and dwelling alternated between high and low values , giving rise to the definition of two behavioral states: active wakefulness , characterized by a high proportion of forward locomotion , and quiet wakefulness , characterized by a high proportion of dwelling ( Figure 2C–D ) .",
"Transitions between active and quiet wakefulness were in the form of rapid behavioral switches , suggesting that they represented two distinct behavioral states .",
"In order to further characterize the observed behavioral dynamics we measured the durations of the epochs of active wakefulness prior to the onset of L4leth .",
"The simplest model , a two-state Markov chain with constant rates of transitions into and out of the active wakefulness state , would yield Poisson statistics , that is , an exponential distribution of epoch durations .",
"The histogram of epochs longer than 3 min was thus fit to exponential ( N",
"( t ) ∼ e−t/τ ) and power-law ( N",
"( t ) ∼ t− ( 1+α ) ) distributions , and the Akaike information criteria ( AIC ) were calculated in order to compare the two models ( Burnham and Anderson , 2002; Burnham and Anderson , 2004 ) .",
"Interestingly , the power law fit ( Figure 3A , AIC = −3 . 1 ) was strongly favored over the exponential fit ( AIC = 13 . 1 ) : the Akaike weight was 0 . 9997 , indicating that the probability that the power-law model better described the data was 99 . 97% .",
"The exponent obtained from the power law fit was − ( 1+α ) = −1 . 83 ± 0 . 31 ( 95% confidence intervals , R = 0 . 95 ) .",
"These results indicated that a simple , Poissonian , model would not adequately describe the observed transitions between active and quiet wakefulness .",
"Rather , the long tail of the power-law distribution suggested that the active wakefulness state was stabilized during L4int . 10 . 7554/eLife . 00782 . 005Figure 3 . The dynamics of the active wakefulness state during the three hours prior to L4 lethargus in wild-type animals .",
"( A ) A histogram of the durations of epochs of active wakefulness plotted on a log-log scale .",
"Epoch durations longer than 3 min exhibited a power-law distribution with an exponent – ( 1+α ) = −1 . 83±0 . 31 .",
"( B ) Two displacements along the y-axis of a sample trace of the fraction of forward locomotion , Δy1 ( between filled circles ) and Δy2 ( between empty circles ) .",
"Both displacements correspond to an identical time interval , Δt .",
"The time-averaged mean square displacement ( TMSD ) is calculated in two steps:",
"( i ) using a sliding window to calculate the mean squared displacements along traces of each of the individual animals ( Golding and Cox , 2006 ) ;",
"( ii ) averaging the results obtained from the previous step for all animals .",
"( C ) The TMSD plotted on a log-log scale as a function of the time-interval , Δt .",
"The TMSD was calculated for the subset of N = 20 animals where data 3 hr prior to the onset of L4leth was available ( N = 20 ) .",
"The TMSD exhibited power-law growth with the exponent ( 1−α ) = 0 . 32 ± 0 . 03 , consistent with a value of α ≈ 0 . 7 .",
"Inset: for the purpose of illustration , the TMSD for a two-state Markov chain with a comparable mean duration of epochs is shown to reach its saturation value at Δt ≈ 400 s ( vertical dashed line ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 005 An additional quantity that can be used to detect the signature of an underlying non-Poissonian process is the time-averaged mean squared displacement ( TMSD ) : the mean squared difference between the values of the process at two time-points that are Δt apart , as illustrated in Figure 3B .",
"In simple cases such as a two-state Markov chain , when the TMSD is plotted as a function of Δt it saturates at a constant value at long measurement times ( Figure 3C , inset ) .",
"In contrast , in processes with no underlying timescale , that is , where the duration of the intervals follow a power-law distribution , the TMSD increases as the time interval increases , exhibiting its own , related , power-law long-term behavior ( S ( Δt ) ∼ Δt ( 1−α ) ) with an exponent 1−α , where α is determined by the durations of the epochs ( Stefani et al . , 2009; Burov et al . , 2010 ) .",
"We thus measured the TMSD for the fraction of time spent in active wakefulness during the 3 hr prior to L4leth .",
"As shown in Figure 3C , the resulting long-term behavior was characterized by the exponent 1−α = 0 . 32 ± 0 . 03 ( 95% confidence intervals , R = 0 . 96 ) , consistent with a value of α ≈ 0 . 7 .",
"These findings support the hypothesis of a mechanism for stabilizing the active wakefulness state during L4int , resulting in the observed distribution of epoch durations .",
"Both the loss of function mutation of the kin-2 gene—encoding a negative regulatory subunit of the cAMP-dependent protein kinase ( PKA ) KIN-1—and the gain of function mutation of the adenylyl cyclase gene acy-1 were inferred to result in increased activity of the holoenzyme ( Schade et al . , 2005; Reynolds et al . , 2005; Charlie et al . , 2006 ) .",
"Correspondingly , both mutations result in hyperkinetic behavior outside of lethargus and reduced quiescence during lethargus ( Schade et al . , 2005; Reynolds et al . , 2005; Charlie et al . , 2006; Perez-Mansilla and Nurrish , 2009; Belfer and Raizen , 2013 ) .",
"We thus assayed the dynamics of locomotion of these mutants from mid L4int to the mid young adult stage .",
"In particular , we asked whether both their velocity and the overall structure of the active and quiet wakefulness states were different from wild-type and from each other .",
"We found that in both cases active wakefulness persisted from mid L4int until the onset of L4leth , and resumed at the end of L4leth ( Figure 4A–B , D–E ) .",
"Correspondingly , as compared to wild-type , these mutants spent a significantly reduced amount of time in quiet wakefulness before and after lethargus ( Figures 4 and 7 ) , and exhibited an approximately two-fold increase in peak velocity during L4int and milder downregulation of velocity in anticipation of L4leth ( Figure 4C , F ) .",
"In addition , consistent with previous reports ( Belfer and Raizen , 2013 ) , during bouts of non-quiescent behavior in lethargus ( ‘motion bouts’ ) we observed a large increase in the prevalence of forward locomotion in kin-2 and acy-1 ( gf ) mutants .",
"Taken together , these results suggest that increased PKA activity stabilizes active wakefulness . 10 . 7554/eLife . 00782 . 006Figure 4 . Increased Gαs signaling stabilizes active wakefulness outside of L4leth . The effects of increased Gαs signaling on locomotion were assayed using animals mutant in two genes: a loss of function mutation of kin-2 , encoding a negative regulatory subunit of PKA , and a gain of function mutation of acy-1 , encoding an adenylyl cyclase .",
"( A and D )",
"Locomotion dynamics of a single animal between the mid L4 and the mid young adult stages .",
"In contrast to wild-type behavior , forward locomotion was also prominently observed during L4leth ( see also Figures 7 and 8 ) .",
"( B and E )",
"The average fractions of time out of a 10-min running window in which animals exhibited each of the four characteristic types of locomotion .",
"( C and F )",
"The frequency of generation of body-bends of mutant ( dark grey ) and wild-type ( light grey , data from Figure 2B ) animals .",
"Panels ( B–F ) depict Nkin-2 = 16 animals , Nacy-1 ( gf ) = 17 animals , mean ± SEM .",
"Standard errors are illustrated as shadowed areas surrounding the plotted averages . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 006 Animals carrying a null mutation of kin-1 or acy-1 die as embryos or first stage larvae respectively .",
"However , animals carrying a partial loss of function allele of the acy-1 gene appear superficially wild-type .",
"We assayed these mutants to determine whether the partial loss of function of the cyclase would confer the opposite phenotype from that of the gain of function , and found this to be the case outside of lethargus but not during lethargus .",
"During the last 4 hr of L4int quiet wakefulness was significantly exaggerated and the active wakefulness state was fragmented—animals displayed a larger number of switches out of brief epochs of active wakefulness ( Figure 5 ) .",
"Similarly , after the termination of L4leth , quiet wakefulness was exaggerated and active wakefulness was reduced .",
"However , during L4leth the dynamics of quiescence and locomotion of the mutants were similar to wild-type ( Figures 7 and 8 ) .",
"These results are consistent with the idea that PKA activity stabilizes the active wakefulness state .",
"They further suggest that during lethargus , when active wakefulness is strongly suppressed , the native role of PKA signaling in modulating locomotion and quiescence may be minor . 10 . 7554/eLife . 00782 . 007Figure 5 . Decreased Gαs signaling destabilizes active wakefulness outside of L4leth . The effects of decreased Gαs signaling on locomotion were assayed using a loss of function mutation of the adenylyl cyclase gene , acy-1 .",
"( A ) Locomotion dynamics of a single animal between the mid L4 and the mid young adult stages ( see also Figures 7and 8 ) .",
"( B ) The average fractions of time out of a 10-min running window in which animals exhibited each of the four characteristic types of locomotion .",
"( C ) The frequency of generation of body-bends of mutant ( dark grey ) and wild-type ( light grey , data from Figure 2B ) animals .",
"Panels ( B and C ) depict Nacy-1 ( lf ) =15 animals , mean ± SEM .",
"Standard errors are illustrated as shadowed areas surrounding the plotted averages . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 007 Tomosyn , a target of PKA ( Baba et al . , 2005 ) , was shown to negatively regulate both unc-13-dependent synaptic transmitter release and unc-31-dependent neuropeptide release in C . elegans ( Gracheva et al . , 2006; Gracheva et al . , 2007a , 2007b ) .",
"Since hyper-activation of PKA could bypass the requirement for UNC-31 in the docking of dense core vesicles ( DCVs ) , the GSA-1 ( Gαs ) and the UNC-31/CAPS pathways were suggested to converge to control DCV release ( Schade et al . , 2005; Reynolds et al . , 2005; Charlie et al . , 2006; Zhou et al . , 2007; Perez-Mansilla and Nurrish , 2009 ) .",
"We could not analyze the behavior of unc-13 mutants due to their severe locomotion defects , but in order to assess whether the modulation of locomotion during L4int depended on the release of DCVs , we assayed unc-31 mutants ( Figure 6 ) .",
"The resulting phenotype was similar to that of acy-1 ( lf ) mutants .",
"As compared to wild-type animals , outside of lethargus unc-31 mutants exhibited increased occupation of the quiet wakefulness state , a fragmented active wakefulness state , and overall lower velocities .",
"In contrast , during L4leth , the dynamics of quiescence and locomotion of unc-31 mutants were similar to wild-type .",
"Unlike acy-1 ( lf ) mutants , the mutation in the unc-31 gene resulted in elevated levels of backward locomotion outside of lethargus and a decrease in directed locomotion during motion bouts in lethargus ( Figures 7 and 8 ) .",
"Taken together with the acy-1 ( lf ) phenotype , these results support a model in which PKA activity stabilizes active wakefulness during L4int ( at least in part ) by regulating DCV exocytosis , but only plays a minor role in regulating locomotion during lethargus . 10 . 7554/eLife . 00782 . 008Figure 6 . Decreased neuropeptide release ( in animals mutant for the gene encoding UNC-31/CAPS ) , or decreased CREB activity ( in animals mutant for the gene encoding the C . elegans CREB ortholog , CRH-1 ) , destabilize active wakefulness outside of L4leth .",
"Both UNC-31/CAPS and CRH-1 act downstream of PKA .",
"( A and D )",
"Locomotion dynamics of a single animal between the mid L4 and the mid young adult stages ( see also Figures 7 and 8 ) .",
"( B and E )",
"The average fractions of time out of a 10-min running window in which animals exhibited each of the four characteristic types of locomotion .",
"( C and F )",
"The frequency of generation of body-bends of mutant ( dark grey ) and wild-type ( light grey , data from Figure 2B ) animals .",
"Panels ( B–F ) depict Nunc-31 = 19 , Ncrh-1 = 16 animals , mean ± SEM .",
"Standard errors are illustrated as shadowed areas surrounding the plotted averages . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 00810 . 7554/eLife . 00782 . 009Figure 7 . Comparisons of mutant and wild-type behavior before , during and after lethargus . Panels ( A–D ) aggregate the data collected during the 2–4 hr prior to the onset of L4leth , the 0–2 hr prior to the onset of L4leth , L4leth , and the 0–2 hr after the termination of L4leth .",
"Each column summarizes the data for a given type of locomotion ( Forward , Dwelling , Backward , and Quiescence ) for all genotypes .",
"Asterisks denote behavioral patters that were significantly different from wild-type ( p<0 . 05 ) .",
"Each bar depicts mean ± SEM , and the sizes of the datasets are the same as in Figures 2 , 4–6 . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 00910 . 7554/eLife . 00782 . 010Figure 8 . Forward and backward locomotion during motion bouts , that is , bouts of non-quiescent behavior during lethargus .",
"( A ) The percentage of the duration of individual motion bouts that was spent in directed locomotion ( forward—left , backward—right ) on each of the genetic backgrounds .",
"( B ) Venn diagrams depicting the fraction of motion bouts ( out of the total number of motion bouts that were longer than 10 s ) where any forward or backward locomotion was detected .",
"Increased Gαs signaling resulted in a larger fraction of motion bouts that included directed motion , and a larger percentage of the total duration of individual bouts that was spent in directed locomotion .",
"Partially decreased Gαs signaling resulted in wild-type-like motion bouts .",
"Strongly decreased neuropeptide release resulted in a mild reduction in forward locomotion and a mild increase in backward locomotion during motion bouts .",
"Loss of CREB function increased the fraction of motion bouts that included directed locomotion , as compared with wild-type .",
"Asterisks denote behavioral patters that were significantly different from wild-type ( p<0 . 05 ) .",
"( C–D )",
"The effects of Gαs signaling on the duration of bouts and the fraction of time spent in quiescence during two time intervals: t = 10–40 min ( C ) , and t = 105–250 min ( D ) , where t = 0 corresponds to the onset of L4leth . DOI: http://dx . doi . org/10 . 7554/eLife . 00782 . 010 Another major target of PKA is the cAMP response element-binding protein ( CREB ) , a transcription factor that , after phosphorylation by PKA , induces gene expression through promoters containing the cAMP-response element ( CRE ) enhancer ( Mayr and Montminy , 2001 ) .",
"Although the C . elegans ortholog of CREB , CRH-1 , was primarily implicated in long-term habituation and memory ( Kimura et al . , 2002; Kauffman et al . , 2010; Nishida et al . , 2011; Timbers and Rankin , 2011 ) it was possible that it could also have a role in regulating larval locomotion .",
"To address this question , we assayed crh-1 mutants ( Figure 6 ) .",
"Outside of lethargus , crh-1 mutants exhibited more poorly-defined global locomotion states , although a clear active wakefulness state was observed in 4 out of 16 animals 5–6 hr prior to L4leth .",
"Gradual transitions between directed locomotion and dwelling were commonly observed during this period , in contrast to the abrupt wild-type switching .",
"In addition , the mutants exhibited lower overall velocities and increased backward locomotion .",
"During L4leth , the dynamics of quiescence of crh-1 mutants were similar to wild-type ( Figures 6 and 7 ) .",
"However , the mutants did show significantly increased levels of directed ( forward and backward ) locomotion during motion bouts as compared to wild-type ( Figures 6 and 8B ) .",
"The levels of directed motion during lethargus of crh-1 mutants were similar to those of kin-2 and acy-1 ( gf ) mutants , possibly indicating a disruption to lethargus behavior .",
"We concluded that two of the downstream targets of PKA , namely neuropeptide release and CREB , acted coherently to stabilize active wakefulness during L4int , while playing a minor role in regulating quiescence and locomotion during lethargus ."
],
[
"Changes to neural circuits induced by experience or development can occur on timescales of hours to months .",
"Neuromodulators such as biogenic amines or neuropeptides often act on such long timescales , modifying the output of neural circuits by altering the activity of neurons and affecting synaptic connections ( Bargmann , 2012; Marder , 2012 ) .",
"Yet there are few techniques for tracking long-term physiological and behavioral dynamics and mostly offer limited resolution ( Clark et al . , 2010; Fonio et al . , 2012; Hart , 2006 ) .",
"Counter-intuitively , despite the simplicity of invertebrate models , detailed longitudinal studies designed to follow their behavioral trends across development are rare as well .",
"In order to study these processes , we have established a novel high-throughput assay , and obtained the first analysis of C . elegans locomotion on developmental timescales .",
"Since data analysis is the bottleneck of prolonged , detailed studies of behavior , we developed PyCelegans , a suite of tools that leverages high performance parallel computing to speed up the computational analysis in our workflow .",
"Accordingly , bottlenecks were shifted back to experimental elements , permitting increased throughput .",
"PyCelegans leverages only open source , freely available tools and libraries .",
"These utilities ( Python , NumPy , SciPy , mpi4py ) are top tier open source scientific computing tools , among the most widely used by researchers developing their own analyses .",
"As such , it is virtually guaranteed that they will be available at any dedicated research computing facility .",
"Moreover , the suite is easily extendable—additional analyses and features are simple to incorporate within the existing framework .",
"Quiet wakefulness was previously reported in diverse species such as various mammals , birds and reptiles , and can comprise a large percentage of the waking state ( Ruckebusch , 1972; Flanigan , 1973; Campbell and Tobler , 1984 ) .",
"However , to the best of our knowledge , quiet wakefulness was not previously reported in nematodes .",
"During its larval development the nematode C . elegans is required to integrate newly differentiated neurons into its neural circuits ( Sulston , 1976; Sulston and Horvitz , 1977; White et al . , 1986 ) and to reshape the connections of existing cells ( White et al . , 1978; Thomas et al . , 1990 ) .",
"At the same time , the animal grows , develops new organs , and undergoes four molts .",
"Maintaining the functionality of essential motor programs such as feeding , locomotion and defecation concurrently with these anatomical and physiological changes imposes constraints on the developing animal; modulation of behavioral patterns may contribute towards satisfying these constraints .",
"In particular , 2–4 hr prior to lethargus , the seam cells exhibit increased synthetic activity and begin to deposit the new cuticle ( Singh and Sulston , 1978; Monsalve et al . , 2011 ) .",
"The timing of quiet wakefulness that we observed coincided with the timing of this activity .",
"Thus , it is possible that this state may assist with maintaining an appropriate energy balance .",
"Our analysis revealed a novel behavioral switch that toggled between the active and quiet wakefulness states prior to L4leth .",
"The distribution of durations of the epochs of active wakefulness supported a model in which this state was actively stabilized and ergodicity was weakly broken ( Stefani et al . , 2009; Burov et al . , 2010 ) .",
"We note that the global locomotion states that we report are distinct from the cGMP-dependent short intervals of continuous roaming or dwelling behavior , previously described in adults ( Fujiwara et al . , 2002 ) .",
"We have shown that upregulating or downregulating Gαs signaling stabilized or destabilized active wakefulness respectively .",
"Downregulated neuropeptide release in unc-31 mutants , a gene encoding the calcium-dependent activator protein for secretion ( CAPS ) required for the priming and docking of DCVs ( Speese et al . , 2007; Lin et al . , 2010 ) , affected behavior similarly to downregulated Gαs signaling .",
"These findings extend the previously reported effects of PKA signaling on locomotion and quiescence ( Schade et al . , 2005; Reynolds et al . , 2005; Charlie et al . , 2006; Raizen et al . , 2008; Perez-Mansilla and Nurrish , 2009; Belfer and Raizen , 2013 ) .",
"Moreover , PKA was shown to catalyze the phosphorylation of tomosyn , a highly-conserved syntaxin-binding protein .",
"Phosphorylation of tomosyn by PKA reduced its interaction with syntaxin and enhanced the formation of the SNARE complex ( Baba et al . , 2005 ) .",
"In C . elegans , tomosyn ( TOM-1 ) was shown to negatively regulate synaptic transmitter release and UNC-31/CAPS-dependent neuropeptide release ( Gracheva et al . , 2007a ) , and KIN-1 alleviated the suppression of both processes by phosphorylating TOM-1 ( J Richmond , personal communications , February 2013 ) .",
"Interestingly , a mutation in the tom-1 gene suppressed behavioral deficits and DCV accumulation in unc-31 mutants ( Gracheva et al . , 2007a ) .",
"Similarly , enhanced PKA activity was shown to be sufficient for bypassing the requirement for UNC-31 for the release of DCVs ( Zhou et al . , 2007; Perez-Mansilla and Nurrish , 2009 ) .",
"Taken together , these findings support a model in which KIN-1 and TOM-1 regulate locomotion .",
"Upregulating Gαs signaling resulted in enhanced active wakefulness outside of lethargus and enhanced directed locomotion during L4leth , mirroring previous findings in Drosophila melanogaster and in C . elegans ( Joiner et al . , 2006; Belfer and Raizen , 2013 ) .",
"These results were also consistent with a previous observation that acute activation of the photoactivated adenylyl cyclase PACα in cholinergic neurons evoked an enhanced activity response ( Weissenberger et al . , 2011 ) .",
"Downregulating Gαs signaling resulted in a clear locomotion phenotype outside of but not during L4leth .",
"However , loss of CREB function resulted in an enhancement of directed locomotion , as well as changes in the dynamics of bouts of motion and quiescence ( Figure 8C , D ) , during lethargus .",
"In addition , crh-1 mutants exhibited strong locomotion defects outside of lethargus .",
"Studies of the effects of cAMP signaling on sleep in D . melanogaster and in mice have demonstrated that CREB , when activated by PKA , promotes the duration of wakefulness ( Hendricks et al . , 2001; Graves et al . , 2003; Shaw and Franken , 2003 ) .",
"The mild loss of function phenotypes of acy-1 ( lf ) and unc-31 during lethargus may be explained by a floor effect , while the implications of the crh-1 phenotype remain to be understood .",
"Interestingly , a recent study reported that adenylyl cyclase activity increases the intensity of nighttime sleep in Drosophila ( van Alphen et al . , 2013 ) .",
"Taken together , our analyses indicated that both PKA-dependent pathways acted in concert to regulate active wakefulness during L4int and the early young adult stages .",
"Finally , the approach to quantifying locomotion presented here provides several distinct advantages .",
"Visible phenotypes were invaluable for uncovering molecular pathways that regulated behavior , but were largely based on crude classifications such as hypo- or hyper-kinesis or a focus on arbitrarily defined features .",
"It was recently shown that the space of shapes adopted by the nematode C . elegans is low dimensional , and that these dimensions ( ‘eigenworms’ ) can provide a quantitative description of behavior ( Stephens et al . , 2008 ) .",
"This exquisitely sensitive analysis revealed the underlying simplicity of complex locomotion dynamics as well as subtle behavioral phenotypes that had been previously undetectable ( Stephens et al . , 2010 , 2011; Brown et al . , 2013 ) .",
"However , this analysis does not provide an intuitive interpretation of the detected phenotypes that may directly relate to the underlying neuronal activity .",
"Tracking of individual body-bends opens the door to a heuristic description of C . elegans locomotion , composed of intuitive building blocks .",
"As in the case of the eigenworms , one underlying assumption of this analysis is that C . elegans does not maintain a ‘mental map’ of its environment , nor does it keep track of its own acceleration in the laboratory frame of reference .",
"Rather , it responds to external and internal cues solely by altering its own body-posture .",
"Specifically , the primary task of the motor neurons during directional motion is to generate body-bends and propagate them along the body in the appropriate direction ( Gray et al . , 2005; Stetina et al . , 2006; Stephens et al . , 2008; Haspel and O’Donovan , 2011; Boyle et al . , 2011; Wen et al . , 2012 ) .",
"It follows that a natural , heuristic model for describing directional locomotion can be constructed using the rates of generation , propagation , and decay of body-bends .",
"Tracking each body-bend from initiation to eventual demise thus provides direct experimental measurements of the basic building blocks of nematode locomotion ."
],
[
"C . elegans strains were maintained and grown according to standard protocols ( Brenner , 1974 ) .",
"The wild-type strain used was C . elegans variety Bristol , strain N2 .",
"The following mutant strains were obtained from the Caenorhabditis Genetics Center: KG518 acy-1 ( ce2 ) III ( gf ) , KG532 kin- 2 ( ce179 ) X , CB169 unc-31 ( e169 ) IV , KP1182 acy- 1 ( nu329 ) III ( lf ) and YT17 crh-1 ( tz2 ) III .",
"Animals were synchronized by restricting the duration of egg-laying ( placing gravid adults on plates for 6 hr , removing the parents , and selecting L4s 3 days later ) , grown at 20°C on standard NGM plates , and fed OP50 bacteria until their mid L4int larva stage .",
"They were then transferred to individual 0 . 8 × 3 . 7 mm ‘artificial dirt’ chambers ( Lockery et al . , 2008 ) .",
"The chambers , containing an overnight OP50 Escherichia coli culture that was concentrated 10-fold and suspended in NGM liquid ( Singh et al . , 2011; Iwanir et al . , 2013 ) , were sealed with a coverslip held in place with VALAP ( a mix of equal parts of vaseline , lanolin and paraffin wax ) at the corners and submerged , facedown , in NGM buffer inside a petri dish to prevent drying .",
"Each observation chamber contained a single animal .",
"Behavior was recorded for 10 hr at a rate of 10 frames per second using a 5 megapixels CCD camera ( Prosilica GC2450 , 2448 × 2050 pixels , Allied Vision Technologies , Stadtroda , Germany ) .",
"We recorded at a magnification of 4 . 2X , resulting in a resolution of 1 . 54 × 1 . 54 μm2/pixel; the body length of a typical L4leth larva was approximately 500 pixels ( 750 μm ) .",
"The contrast at the edges of the animals was maximized , typically by setting background levels to 230–250 on a greyscale of 0–255 , and by focusing slightly away from the middle plane of the pharynx .",
"We developed a suite of tools , called PyCelegans , for image analysis on high performance parallel computing resources .",
"Similar to previously described methods ( Husson et al . , 2013 ) , PyCelegans identifies the body of the animal in each frame and calculates the coordinates of 100 points along its midline , ordered from head to tail .",
"The rate of segmentation failure , where the animals could not be properly identified , was typically 5% of all frames .",
"Frames in which the animal was not identified were not included in the dataset , but their timing was accounted for when time-stamping subsequent frames .",
"Each midline was divided into 20 equal segments and the local angle at each of the inner 18 segments was calculated as shown in Figure 1A .",
"The raw angle dynamics data was smoothed with a Gaussian filter with a width of 5 frames ( 0 . 5 s ) .",
"Body-bends were defined as positions of local spatial maxima or minima of the angles ( Figure 1A ) .",
"The position of each bend was tracked from the time of its initiation until it decayed ( typically at the head or the tail ) or was interrupted by 10 consecutive missing frames .",
"Forward locomotion was defined by the propagation of bends in the anterior-posterior direction that persisted for at least three consecutive midline segments .",
"Backward locomotion was defined analogously .",
"Quiescence of an individual segment was defined as an interval in which the rate of change of the corresponding angle did not exceed a threshold of 0 . 01 radians/sec .",
"Neither missing frames in which the inferred change in angle was below a threshold of 0 . 3 radians , nor motion occurring in isolated single frames , were considered to interrupt a bout of quiescence .",
"At each time point , the whole-animal behavior was classified as forward , backward or dwelling by applying a majority rule to the dynamics of the individual body-bends .",
"Dwelling occurred when the number of bends propagating in both directions was equal ( typically zero ) .",
"Whole-animal quiescence was defined as the state where the most anterior angle ( between the head and neck segments ) and at least 16 of the remaining 17 angles were quiescent .",
"The fraction of time spent in each behavior was calculated with a running-average window of 10 min for Figures 1 and 2 and 4–6 min for Figure 3 .",
"The onset/termination of lethargus was defined as the first/last point of increasing/decreasing whole-animal quiescence fraction that was followed by 20 consecutive minutes of the quiescence fraction remaining above/below a threshold of 5% .",
"Source code , documentation , and a sample dataset are available to download from GitHub ( https://github . com/labello/pycelegans ) .",
"Mutant behavior was compared to wild-type in Figures 7 and 8 using a one-way analysis of variance ( ANOVA ) with Bonferroni adjustments to compensate for multiple comparisons .",
"Exponential and power-law models for the data presented in Figure 3 were compared by calculating Akaike information criteria ( AIC ) ( Burnham and Anderson , 2002; Burnham and Anderson , 2004 ) ."
]
] | [
"Despite their simplicity , longitudinal studies of invertebrate models are rare .",
"We thus sought to characterize behavioral trends of Caenorhabditis elegans , from the mid fourth larval stage through the mid young adult stage .",
"We found that , outside of lethargus , animals exhibited abrupt switching between two distinct behavioral states: active wakefulness and quiet wakefulness .",
"The durations of epochs of active wakefulness exhibited non-Poisson statistics .",
"Increased Gαs signaling stabilized the active wakefulness state before , during and after lethargus .",
"In contrast , decreased Gαs signaling , decreased neuropeptide release , or decreased CREB activity destabilized active wakefulness outside of , but not during , lethargus .",
"Taken together , our findings support a model in which protein kinase A ( PKA ) stabilizes active wakefulness , at least in part through two of its downstream targets: neuropeptide release and CREB .",
"However , during lethargus , when active wakefulness is strongly suppressed , the native role of PKA signaling in modulating locomotion and quiescence may be minor ."
] | [
"The roundworm C . elegans is a key model organism in neuroscience .",
"It has a simple nervous system , made up of just 302 neurons , and was the first multicellular organism to have its genome fully sequenced .",
"The lifecycle of C . elegans begins with an embryonic stage , followed by four larval stages and then adulthood , and worms can progress through this cycle in only three days .",
"However , relatively little is known about how the behaviour of the worms varies across these distinct developmental phases .",
"The body wall of C . elegans contains pairs of muscles that extend along its length , and when waves of muscle contraction travel along its body , the worm undergoes a sinusoidal pattern of movement .",
"A signalling cascade involving a molecule called protein kinase A is thought to help control these movements , and upregulation of this cascade has been shown to increase locomotion .",
"Now , Nagy et al . have analysed the movement of C . elegans during these different stages of development .",
"This involved developing an image processing tool that can analyze the position and posture of a worm’s body in each of three million ( or more ) images per day .",
"Using this tool , which is called PyCelegans , Nagy et al . identified two behavioral macro-states in one of the larval forms of C . elegans: these states , which can persist for hours , are referred to as active wakefulness and quiet wakefulness .",
"During periods of active wakefulness , the worms spent most ( but not all ) of their time moving forwards; during quiet wakefulness , they remained largely still .",
"The worms switched abruptly between these two states , and the transition seemed to be regulated by PKA signaling .",
"By using PyCelegans to compare locomotion in worms with mutations in genes encoding various components of this pathway , Nagy et al . showed that mutants with increased PKA activity spent more time in a state of active wakefulness , while the opposite was true for worms with mutations that reduced PKA activity .",
"In addition to providing new insights into the control of locomotion in C . elegans , this study has provided a new open-source PyCelegans suite of tools , which are available to be extended and adapted by other researchers for new uses ."
] | 2013 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"cell biology"
] | SWELL1 regulates skeletal muscle cell size, intracellular signaling, adiposity and glucose metabolism | elife-58941-v3 | [
[
"Maintenance of skeletal muscle mass and function is associated with improved metabolic health and is thought to protect against obesity and obesity-related diseases such as diabetes , nonalcoholic fatty liver disease , heart disease and osteoarthritis , and correlates with overall health in the aging population .",
"Skeletal muscle atrophy , the loss of skeletal muscle mass , is associated with cancer ( cachexia ) , heart failure , chronic corticosteroid use , paralysis or denervation ( disuse atrophy ) , chronic positive pressure ventilation ( diaphragm atrophy , inability to extubate ) , prolonged space flight ( Fitts et al . , 2001 ) or bed rest ( unloading ) and aging ( Schiaffino et al . , 2013 ) .",
"Each of these clinical scenarios also contribute to poor metabolic health and increase frailty .",
"Thus , a deeper understanding of the molecular mechanisms that regulate skeletal muscle maintenance , growth and function has important implications for human health .",
"Skeletal muscle growth is regulated by a multitude of stimuli , including growth factor signaling , cytokines , mechanical load , integrin signaling and hormones ( Schiaffino et al . , 2013 ) .",
"Activation of AKT-mTOR signaling downstream of insulin and IGF-1 is well established as a critical regulator of skeletal muscle differentiation and growth ( Schiaffino et al . , 2013 ) .",
"However , mechanical loading of muscle , as occurs with regular activity , exercise , and resistance training also induces mTOR-mediated skeletal muscle growth ( Ben-Sahra and Manning , 2017; Hornberger et al . , 2006; Yoon , 2017 ) , possibly via a growth-factor independent mechanosensory mechanism .",
"β1-integrin and focal adhesion kinase ( FAK ) signaling has been proposed as this mechanosensory system - sensing mechanical load on skeletal muscle and regulating hypertrophic signaling ( Carson and Wei , 2000; Schlaepfer et al . , 1999; Flück et al . , 1999; Klossner et al . , 2009 ) .",
"However , mechanosensitive ion channels , or ion channel complexes , may also regulate intracellular signaling , including PIEZO1 , TRPV4 and the recently identified LRRC8 channel complex that functionally encodes the volume-regulated anion current ( VRAC ) ( Syeda et al . , 2016; Voss et al . , 2014; Qiu et al . , 2014 ) .",
"Lrrc8a ( leucine-rich repeat containing protein 8A , also known as SWELL1 ) encodes a ~ 95 kDa membrane protein with four transmembrane domains and an intracellular , C-terminal leucine-rich repeat domain ( LRRD ) ( Osei-Owusu et al . , 2018 ) .",
"It was first described , in 2003 , as the site of a translocation mutation in a young woman with an immunodeficiency characterized by agammaglobulinemia and absent B-cells ( Sawada et al . , 2003; Kubota et al . , 2004 ) .",
"This phenotype led to studies linking LRRC8A to lymphocyte differentiation defects arising from impaired LRRC8A dependent GRB2-mediated PI3K-AKT signaling , in part based on data from a global Lrrc8a knock-out ( KO ) mouse ( Kumar et al . , 2014 ) .",
"Although LRRC8A was speculated in 2012 to form a hetero-hexameric ion channel complex with other LRRC8 family members ( Abascal and Zardoya , 2012 ) , it was not until 2014 that LRRC8A was experimentally identified as an essential component of the volume-regulated anion channel ( VRAC ) ( Voss et al . , 2014; Qiu et al . , 2014 ) , forming hetero-hexamers with LRRC8B-E ( Syeda et al . , 2016; Voss et al . , 2014 ) .",
"So , for about a decade , LRRC8A was considered a membrane protein that regulates PI3K-AKT-mediated lymphocyte function ( Sawada et al . , 2003; Kubota et al . , 2004 ) , putatively via non-ion channel , protein-protein interaction mediated signaling , and only later discovered to also form the long-studied VRAC ion channel signaling complex .",
"Indeed , since its first description >30 years ago ( Cahalan and Lewis , 1988; Hazama and Okada , 1988; Pedersen et al . , 2015 ) , VRAC has been associated with a multitude of complex physiological and pathophysiological functions , including cell proliferation , cell migration , angiogenesis , cell death and apoptosis ( Pedersen et al . , 2016; Eggermont et al . , 2001 ) ; however , the molecular mechanisms underlying these diverse functions had remained elusive without knowledge of the molecular identity of this ion channel complex .",
"We recently identified LRRC8A as a swell or stretch-activated volume sensor in adipocytes that regulates glucose uptake , lipid content , and adipocyte growth via a LRRC8A-GRB2-PI3K-AKT signaling pathway – providing a putative feed-forward amplifier to enhance adipocyte growth and insulin signaling during caloric excess ( Zhang et al . , 2017; Gunasekar et al . , 2019 ) .",
"Intriguingly , others have shown that mechanical stimuli applied by pulling on β1-integrins on cardiac muscle cells with magnetic beads activates a VRAC-like current , suggesting a putative connection between β1-integrin/focal adhesion kinase signaling and LRRC8A ( Browe and Baumgarten , 2003; Browe and Baumgarten , 2006 ) .",
"Taken together , these findings suggest that LRRC8A may connect β1-integrin-mediated mechano-transduction with insulin/IGF1-PI3K-AKT-mTOR signaling , which , in skeletal muscle is anticipated to regulate skeletal muscle differentiation , function and potentially also adiposity and systemic glucose metabolism ( Brüning et al . , 1998; Moller et al . , 1996; Kim et al . , 2000 ) .",
"In this study , we tested this hypothesis by examining LRRC8A dependent intracellular signaling and myotube differentiation in both C2C12 and primary skeletal muscle cells in vitro , and by performing skeletal muscle targeted LRRC8A loss-of-function experiments in vivo .",
"We find that myotube differentiation and insulin and stretch-mediated PI3K-AKT , ERK1/2 , mTOR signaling is strongly regulated by LRRC8A protein expression , and provide evidence that GRB2 signaling mediates these LRRC8A dependent effects .",
"Finally , using skeletal muscle Lrrc8a knock-out mice , we reveal the requirement of skeletal muscle LRRC8A for maintaining normal skeletal muscle cell size , muscle endurance , force generation , adiposity and glucose tolerance , under basal conditions and in the setting of overnutrition ."
],
[
"LRRC8A is the essential component of a hexameric ion channel signaling complex that encodes ICl , SWELL , or the volume-regulated anion current ( VRAC ) ( Voss et al . , 2014; Qiu et al . , 2014 ) .",
"While the LRRC8A complex has been shown to regulate cellular volume in response to application of non-physiological hypotonic extracellular solutions , the physiological function ( s ) of this ubiquitously expressed ion channel signaling complex remain unknown .",
"To determine the function of the LRRC8A channel complex in skeletal muscle , we genetically deleted Lrrc8a from C2C12 mouse myoblasts using CRISPR/cas9-mediated gene editing as described previously ( Zhang et al . , 2017; Kang et al . , 2018 ) , and from primary skeletal muscle cells isolated from Lrrc8a fl/fl mice transduced with adenoviral Cre-mCherry ( KO ) or mCherry alone ( WT control ) ( Zhang et al . , 2017 ) .",
"LRRC8A protein western blots confirmed robust Lrrc8a ablation in both Lrrc8a KO C2C212 myotubes and Lrrc8a KO primary skeletal myotubes ( Figure 1A ) .",
"Next , whole-cell patch clamp revealed that the hypotonically activated ( 210 mOsm ) outwardly rectifying current present in WT C2C12 myoblasts is abolished in Lrrc8a KO C2C12 myoblasts ( Figure 1B ) , confirming LRRC8A as also required for ICl , SWELL or VRAC in skeletal muscle myoblasts .",
"Remarkably , Lrrc8a ablation is associated with impaired myotube formation in both C2C12 myoblasts and in primary skeletal satellite cells ( Figure 1C ) , with an 58% and 45% reduction in myotube area in C2C12 and skeletal muscle myotubes , respectively , compared to WT .",
"As an alternative metric , myoblast fusion is also markedly reduced by 80% in Lrrc8a KO C2C12 compared to WT , as assessed by myotube fusion index ( number of nuclei inside myotubes/total number of nuclei; Figure 1C ) .",
"In order to further characterize the observed LRRC8A-dependent impairment in myotube formation in C2C12 and primary muscle cells , we performed genome-wide RNA sequencing ( RNA-seq ) of Lrrc8a KO C2C12 relative to control WT C2C12 myotubes .",
"These transcriptomic data revealed clear differences in the global transcriptional profile between WT and Lrrc8a KO C2C12 myotubes ( Figure 1D and Supplementary file 1 ) , with marked suppression of numerous skeletal muscle differentiation genes including Mef2a ( 0 . 2-fold ) , Myl2 ( 0 . 008-fold ) , Myl3 ( 0 . 01-fold ) , Myl4 ( 0 . 008-fold ) , Actc1 ( 0 . 005-fold ) , Tnnc2 ( 0 . 005-fold ) , Igf2 ( 0 . 01-fold ) ( Figure 1E ) .",
"Curiously , this suppression of myogenic differentiation is associated with marked induction of Ppargc1α ( PGC1α; 14-fold ) and Pparγ ( 3 . 7-fold ) .",
"PGC1α and Pparγ are positive regulators of skeletal muscle differentiation ( Haralampieva et al . , 2017; Ruas et al . , 2012; Singh et al . , 2007 ) , suggesting that the LRRC8A-dependent defect in skeletal muscle differentiation lies downstream of PGC1α and PPARγ .",
"To further define putative pathway dysregulation underlying disruptions in myogenesis observed in Lrrc8a KO C2C12 myotubes , we next performed pathway analysis on the transcriptome data .",
"We found that numerous signaling pathways essential for myogenic differentiation are disrupted , including insulin ( 2 × 10−3 ) , MAP kinase ( 5 × 10−4 ) , PI3K-AKT ( 1 × 10−4 ) , AMPK ( 6 × 10−5 ) , integrin ( 3 × 10−6 ) , mTOR ( 2 × 10−6 ) , integrin linked kinase ( 4 × 10−7 ) and IL-8 ( 1 × 10−7 ) signaling pathways ( Figure 1F and Supplementary file 2 ) .",
"Guided by the results of the pathway analysis , and the fact that skeletal myogenesis and maturation is regulated by insulin-PI3K-AKT-mTOR-MAPK ( Conejo et al . , 2001; Schiaffino et al . , 2013 ) , we directly examined a number of insulin-stimulated pathways in WT and Lrrc8a KO C2C12 myotubes , including insulin-stimulated AKT2-AS160 , FOXO1 and AMPK signaling .",
"Indeed , insulin-stimulated pAKT2 , pAS160 , pFOXO1 and pAMPK are abrogated in Lrrc8a KO myotubes compared to WT C2C12 myotubes ( Figure 2A and C ) .",
"Importantly , insulin-AKT-AS160 signaling is also diminished in Lrrc8a KO primary skeletal muscle myotubes compared to WT primary myotubes ( Figure 2B and D ) , consistent with the observed differentiation block ( Figure 1C ) .",
"This confirms that LRRC8A-dependent insulin-AKT and downstream signaling is not a feature specific to immortalized C2C12 myotubes but is also conserved in primary skeletal myotubes .",
"It is also notable that reduction in total AKT2 protein is associated with Lrrc8a ablation in both C2C12 and primary skeletal muscle cells , and this is consistent with threefold reduction in AKT2 mRNA expression observed in RNA sequencing data ( Figure 2E ) .",
"Moreover , transcription of a number of critical insulin signaling and glucose homeostatic genes are suppressed by Lrrc8a ablation , including GLUT4 ( Slc2a4 , 51-fold ) , FOXO3 ( 2-fold ) , FOXO4 ( 2 . 8-fold ) and FOXO6 ( 18-fold ) ( Figure 2E ) .",
"Indeed , FOXO signaling is thought to integrate insulin signaling with glucose metabolism ( Lee and Dong , 2017; Gross et al . , 2008 ) in a number of insulin sensitive tissues .",
"Collectively , these data indicate that impaired LRRC8A-dependent insulin-AKT-AS160-FOXO signaling is associated with the observed defect in myogenic differentiation upon Lrrc8a ablation in cultured skeletal myotubes , and also predict putative impairments in skeletal muscle glucose metabolism and oxidative metabolism .",
"We next asked if these LRRC8A-dependent effects on downstream skeletal myotube insulin signaling are due to impaired myotube differentiation , and associated impairments in insulin signaling , or if LRRC8A also regulates these signaling pathways in differentiated skeletal myotubes .",
"To test this , we first fully differentiated C2C12 myotubes , and then knocked down ( KD ) LRRC8A using Ad-shLRRC8A-mCherry post-differentiation and compared myotube size and insulin-stimulated signaling to Ad-shSCR-mCherry treated C2C12 myotubes ( Figure 2—figure supplement 1 ) .",
"Similar to Lrrc8a ablation prior to differentiation , shRNA-mediated LRRC8A KD of fully differentiated C2C12 myotubes mildly reduced myotube size ( Figure 2—figure supplement 1A ) , and significantly reduced insulin-stimulated pAKT2 , pAKT1 , pAS160 , pAMPK and pS6 ribosomal proteins ( Figure 2—figure supplement 1B and C ) , although in some cases , these differences were less marked than in C2C12 Lrrc8a KO myoblasts that were subsequently differentiated ( Figure 2 ) .",
"Also , the reductions in total AKT2 observed at both the mRNA ( Figure 2E , and Supplementary file",
"1 ) and protein levels ( Figure 2A ) in differentiated Lrrc8a KO C2C12 myoblasts are also observed upon LRRC8A KD in differentiated C2C12 myotubes ( Figure 2—figure supplement 1B and C ) , suggesting that total AKT2 expression is regulated by LRRC8A in fully differentiated myotubes .",
"As a complementary approach , we confirmed these findings in fully differentiated , primary skeletal myotubes isolated from Lrrc8afl/fl mice upon adenoviral transduction of Ad-CMV-Cre-mCherry ( KO ) compared to Ad-CMV-mCherry ( WT ) , and noted similar reductions in myotube size ( Figure 2—figure supplement 1D ) , and in basal levels of pAS160 , pAKT2 , AKT2 and pAKT1 signaling ( Figure 2—figure supplement 1E and F ) .",
"Taken together , these data indicate that impaired insulin signaling observed in LRRC8A-depleted myotubes is in part due to impaired myotube differentiation with secondary effects on insulin signaling , and in part due to a direct contribution of LRRC8A to intracellular signaling in fully differentiated myotubes .",
"To further validate LRRC8A-mediated effects on muscle differentiation and signaling , we re-expressed LRRC8A in Lrrc8a KO C2C12 myoblasts ( LRRC8A O/E ) and then examined myotube differentiation and basal activity of multiple intracellular signaling pathways by western blot , including pAKT1 , pAKT2 , pAS160 , p-p70S6K , pS6K and pERK1/2 as compared to WT and Lrrc8a KO C2C12 myotubes .",
"LRRC8A O/E to 2 . 12-fold WT levels fully rescues myotube development in Lrrc8a KO myotubes ( Figure 3A ) , as quantified by restoration of Lrrc8a KO myotube area to levels above WT ( Figure 3B ) .",
"This rescue of Lrrc8a KO myotube development upon LRRC8A O/E ( Figure 3A&B ) is associated with either restored ( pAS160 , AKT2 , pAKT1 , AKT1 , p70S6K ) or supra-normal ( pAKT2 , p-p70S6K , pS6K , pERK1/2 ) signaling ( Figure 3C&D ) compared to WT C2C12 myotubes .",
"These data demonstrate that LRRC8A protein expression level strongly regulates skeletal muscle insulin signaling and myogenic differentiation .",
"In a cellular context , there are numerous reports that VRAC and the LRRC8A complex that functionally encodes it is mechano-responsive ( Browe and Baumgarten , 2003; Browe and Baumgarten , 2006; Osei-Owusu et al . , 2018; Barakat et al . , 1999; Nakao et al . , 1999; Nilius and Droogmans , 2001; Romanenko et al . , 2002; Strange et al . , 2019 ) .",
"It is well established that mechanical stretch is an important regulator of skeletal muscle proliferation , differentiation and skeletal muscle hypertrophy and may be mediated by PI3K-AKT-MAPK signaling ( Schiaffino et al . , 2013; Ma et al . , 2017; Fu et al . , 2018 ) and integrin signaling pathways ( Carson and Wei , 2000; Schlaepfer et al . , 1999; Flück et al . , 1999; Klossner et al . , 2009 ) .",
"To determine if LRRC8A is also required for stretch-mediated AKT and MAP kinase signaling in skeletal myotubes , we subjected WT and Lrrc8a KO C2C12 myotubes to 0% or 5% equiaxial stretch using the FlexCell stretch system .",
"Mechanical stretch ( 5% ) is sufficient to stimulate PI3K-AKT2/AKT1-pAS160-MAPK ( ERK1/2 ) signaling in WT C2C12 in a LRRC8A-dependent manner ( Figure 4A and B ) .",
"These data position LRRC8A as a co-regulator of both insulin and stretch-mediated PI3K-AKT- pAS160-MAPK signaling .",
"It has been reported earlier in both lymphocyte and adipocytes that the LRRC8A complex interacts with Growth factor Receptor-Bound 2 ( GRB2 ) and regulates PI3K-AKT signaling ( Kumar et al . , 2014; Zhang et al . , 2017; Gunasekar et al . , 2019 ) , whereby GRB2 binds with IRS1/2 and negatively regulates insulin signaling ( Shan et al . , 2013 ) .",
"Indeed , GRB2 knock-down augments insulin-PI3K-MAPK signaling and induces myogenesis and myogenic differentiation genes ( Shan et al . , 2013; Liu et al . , 2009; Mitra and Thanabalu , 2017 ) .",
"To determine if LRRC8A and GRB2 interact in C2C12 myotubes , we overexpressed C-terminal 3XFlag tagged LRRC8A in C2C12 cells followed by immunoprecipitation ( IP ) with Flag antibody .",
"We observed significant GRB2 enrichment upon Flag IP from lysates of LRRC8A-3xFlag expressing C2C12 myotubes , consistent with a GRB2-LRRC8A interaction ( Figure 5A ) .",
"Based on the notion that LRRC8A titrates GRB2-mediated suppression of AKT/MAPK signaling , and that Lrrc8a ablation results in unrestrained GRB2-mediated AKT/MAPK inhibition ( Gunasekar et al . , 2019 ) , we next tested if GRB2 knock-down ( KD ) may rescue myogenic differentiation in Lrrc8a KO C2C12 myotubes .",
"shRNA-mediated GRB2 KD in Lrrc8a KO C2C12 myoblasts ( LRRC8A KO/shGRB2; Figure 5B ) stimulates myotube formation ( Figure 5C ) and increases myotube area ( Figure 5D ) , to levels equivalent to WT/shSCR ( Figure 5C and D ) .",
"Similarly , GRB2 KD in Lrrc8a KO C2C12 myotubes induces myogenic differentiation markers IGF1 , MyoHCl , MyoHClla and MyoHCIIb relative to both LRRC8A KO/shSCR and WT/shSCR ( Figure 5E and F ) .",
"These data are consistent with GRB2 suppression rescuing myotube differentiation in Lrrc8a KO C2C12 , and supports a model in which LRRC8A regulates myogenic differentiation by titrating GRB2-mediated signaling .",
"To examine the physiological consequences of Lrrc8a ablation in vivo , we generated skeletal muscle-specific Lrrc8a KO mice using Cre-LoxP technology by crossing Myf5-Cre mice with Lrrc8afl/fl mice ( Skm KO; Figure 6A ) , and confirmed robust skeletal muscle LRRC8A depletion in Skm KO gastrocnemius muscle , 12 . 3-fold lower than WT controls ( Figure 6B ) .",
"Remarkably , in contrast to the severe impairments in skeletal myogenesis observed in both Lrrc8a KO C2C12 and primary skeletal myotubes in vitro ( Figures 1 , 3 and 5 ) , Skm KO mice develop skeletal muscle mass comparable to WT littermates , based on Echo/MRI body composition ( Figure 6C ) and gross muscle weights ( Figure 6D ) , and are born at normal mendelian ratios ( Supplementary file 3 ) .",
"However , histological examination reveals a 27% reduction in skeletal myocyte cross-sectional area in Skm KO as compared to WT mice ( Figure 6E ) , suggesting a requirement for LRRC8A in skeletal muscle cell size regulation in vivo .",
"This is potentially due to reductions in myotube fusion , as observed in C2C12 and primary skeletal muscle cells in vitro ( Figure 1 ) , but occurring to a lesser degree in vivo .",
"These data indicate that the profound impairments in myogenesis observed in vitro may reflect a very early requirement for LRRC8A signaling in skeletal muscle development ( prior to LRRC8A protein elimination by Myf5-Cre-mediated LRRC8A recombination ) , or other fundamental differences in myogenic differentiation processes in vitro versus in vivo .",
"Since insulin signaling is an important regulator of skeletal muscle oxidative capacity and endurance ( Affourtit , 2016 ) , we next examined exercise tolerance on treadmill testing in Lrrc8afl/fl ( WT ) compared to Skm KO .",
"Skm KO mice exhibit a 14% reduced exercise capacity , compared to age and gender matched WT controls ( Figure 7A; Figure 7—Video 1 ) .",
"Hang-times on inversion testing are also reduced 29% in Skm KO compared to controls , further supporting reduced skeletal muscle endurance upon skeletal muscle LRRC8A depletion in vivo ( Figure 7B ) .",
"To determine if these reductions in muscle function in vivo are due to muscle-specific functional impairments , we next performed ex vivo experiments in which we isolated the soleus muscle from mice and performed twitch/train testing .",
"We observed that peak developed tetanic tension is 15% reduced in Skm KO soleus muscle compared to WT controls ( Figure 7C ) , suggesting a skeletal muscle autonomous mechanism , with no difference in time to fatigability ( TTF , Figure 7D ) or time to 50% decay ( Figure 7E ) .",
"To determine whether these LRRC8A dependent differences in muscle endurance and force were due to impaired oxidative capacity , we next measured oxygen consumption rate ( OCR ) and extracellular acidification rate ( ECAR ) in WT and Lrrc8a KO primary skeletal muscle cells , under basal and insulin-stimulated conditions ( Figure 7F ) .",
"Oxygen consumption of Lrrc8a KO primary myotubes are 26% lower than WT and , in contrast to WT cells , are unresponsive to insulin-stimulation ( Figure 7F ) , consistent with abrogation of insulin-AKT/ERK1/2 signaling upon skeletal muscle LRRC8A depletion .",
"These relative changes persist in the presence of Complex V and III inhibitors , Oligomycin and Antimycin A ( Figure 7F and G ) , suggesting that insulin-stimulated glycolytic pathways are primarily dysregulated upon LRRC8A depletion .",
"In contrast , FCCP , which maximally uncouples mitochondria , abolishes differences in oxygen consumption between WT and Lrrc8a KO primary muscle cells , suggesting no differences in functional mitochondrial content in Lrrc8a KO muscle .",
"Consistent with this finding , we observe no differences in muscle fiber-type based on Myosin Heavy Chain ( MHC ) Type 1 , MHC Type IIa , MHC Type IIx and MHC Type IIb in both superficial and deep tibialis anterior muscle in Skm KO as compared to WT mice ( Figure 7—figure supplement 1 ) , indicating the reductions in muscle force and endurance in Skm KO mice is not due to muscle fiber-type switching .",
"Indeed , it is increasingly appreciated that changes in muscle metabolic potential and morphology may occur independent from adaptive fiber-type switching as measured by a change in MHC expression ( Egan and Zierath , 2013 ) .",
"To examine an alternative mechanism , such as glycolysis , we measured extracellular acidification rate ( ECAR ) in WT and Lrrc8a KO primary myotubes .",
"Insulin-stimulated ECAR increases are abolished in Lrrc8a KO compared to WT cells , and these differences persist independent of electron transport chain modulators ( Figure 7H ) .",
"These data suggest that LRRC8A regulation of skeletal muscle cellular oxygen consumption occurs at the level of glucose metabolism - potentially via LRRC8A-dependent insulin-PI3K-AKT-AS160-GLUT4 signaling , glucose uptake and utilization .",
"These findings in primary skeletal muscle cells are supported by marked transcriptional suppression of numerous glycolytic genes: Aldoa , Eno3 , Pfkm , and Pgam2; and glucose and glycogen metabolism genes: Phka1 , Phka2 , Ppp1r3c and Gys1 , upon Lrrc8a ablation in C2C12 myotubes ( Figure 7—figure supplement 2 ) .",
"Guided by evidence of impaired insulin-PI3K-AKT-AS160-GLUT4 signaling observed in Lrrc8a KO C2C12 and primary myotubes , we next examined systemic glucose homeostasis and insulin sensitivity in WT and Skm KO mice by measuring glucose and insulin tolerance .",
"On a regular chow diet , there are no differences in either glucose tolerance or insulin tolerance ( Figure 8A ) between WT and Skm KO mice .",
"However , over the course of 16–24 weeks on chow diet Skm KO mice develop 29% increased adiposity based on body composition measurements ( Figure 8B ) compared to WT , with no significant difference in lean mass ( Figure 8C ) or in total body mass ( Figure 8C ) .",
"When Skm KO mice are raised on a high-fat-diet ( HFD ) for 16 weeks , there is no difference in adiposity observed ( Figure 8—figure supplement",
"1 ) compared to WT mice , but glucose tolerance is impaired ( Figure 8D ) and there is mild insulin resistance in HFD Skm KO mice as compared to WT ( Figure 8E ) .",
"Since Myf5 is also expressed in brown fat ( Seale et al . , 2008 ) , it is possible that these metabolic phenotypes arise from LRRC8A-mediated effects in brown fat and consequent changes in systemic metabolism .",
"To rule out this possibility , we repeated a subset of the above experiments in a skeletal muscle-targeted KO mouse generated by crossing the Myl1-Cre and Lrrc8afl/fl mice ( Myl1Cre/Lrrc8afl/fl ) , since Myl1-Cre is restricted to mature skeletal muscle ( Figure 8—figure supplement 2B ) , and excludes brown fat ( Bothe et al . , 2000 ) .",
"Similar to Myf5Cre/Lrrc8afl/fl mice , Myl1Cre/Lrrc8afl/fl mice fed a regular chow diet , have normal glucose tolerance ( Figure 8—figure supplement 2C ) , but exhibit 24% reduced exercise capacity on treadmill testing , as compared to WT ( Figure 8—figure supplement 2D ) .",
"Also , Myl1Cre/Lrrc8afl/fl mice develop increased visceral adiposity over time on regular chow , based on 24% increased epididymal adipose mass normalized to body mass ( Figure 8—figure supplement 2E ) , with no differences in inguinal adipose tissue , muscle mass ( Figure 8—figure supplement 2F ) , or total body mass ( Figure 8—figure supplement 2G ) .",
"These data suggest that impaired skeletal muscle glucose uptake in Skm KO ( Myl1Cre/Lrrc8afl/fl and Myf5Cre/Lrrc8afl/fl ) mice are compensated for by increased adipose glucose uptake and de novo lipogenesis , which contribute to preserved glucose tolerance , at the expense of increased adiposity in skeletal muscle-targeted Lrrc8a KO mice raised on a regular chow diet .",
"However , overnutrition-induced obesity , and the associated impairments in adipose and hepatic glucose disposal may uncover glucose intolerance and insulin resistance in skeletal muscle-targeted Lrrc8a KO mice ."
],
[
"Our data reveal that the LRRC8A channel complex regulates insulin/stretch-mediated AKT-AS160-GLUT4 , MAP kinase and mTOR signaling in differentiated myoblast cultures , with consequent effects on myogenic differentiation , insulin-stimulated glucose metabolism and oxygen consumption .",
"In vivo , skeletal muscle-targeted Lrrc8a KO mice have smaller skeletal muscle cells , impaired muscle endurance , and force generation , and are predisposed to adiposity , glucose intolerance and insulin resistance .",
"Insulin/stretch-mediated PI3K-AKT , mTOR signaling are well known to be important regulators of myogenic differentiation ( Rotwein and Wilson , 2009; Héron-Milhavet et al . , 2008 ) , metabolism and muscle function ( Schiaffino et al . , 2013 ) suggesting impaired LRRC8A-AKT-mTOR signaling may underlie the defect in myogenic differentiation .",
"Indeed , consistent with our previous findings and proposed model in adipocytes , in which LRRC8A mediates the interaction of GRB2 with IRS1 to regulate insulin-AKT signaling ( Zhang et al . , 2017; Gunasekar et al . , 2019 ) , LRRC8A also associates with GRB2 in skeletal myotubes , and GRB2 knock-down rescues impaired myogenic differentiation in Lrrc8a KO muscle cells .",
"Thus , our working model for LRRC8A-mediated regulation of insuln-PI3K-AKT and downstream signaling in adipocytes ( Gunasekar et al . , 2019 ) appears to be conserved in skeletal myotubes .",
"The in vitro phenotype that we observe in CRISPR/cas9-mediated Lrrc8a KO C2C12 myotubes and in Lrrc8a KO primary myotubes is consistent with the observation of Chen et al . , 2019 that used siRNA-mediated LRRC8A knock-down to demonstrate that the LRRC8A channel complex is required for myogenic differentiation .",
"However , the ability of both GRB2 KD and LRRC8A O/E to rescue myogenic differentiation and augment insulin-AKT , MAP kinase and mTOR signaling in Lrrc8a KO myotubes implicates non-canonical , non-conductive signaling mechanisms .",
"Based on our work and also previous studies ( Voss et al . , 2014; Qiu et al . , 2014 ) , LRRC8A O/E does not increase ICl , SWELL/VRAC to supranormal levels at the plasma membrane .",
"However , pAKT , pERK1/2 and mTOR levels are augmented by twofold to threefold above endogenous levels , upon twofold LRRC8A O/E in C2C12 myotubes .",
"These data suggest that alternative/non-canonical signaling mechanisms underlie LRRC8A signaling , as opposed to canonical/conductive signaling mechanisms .",
"Another finding that warrants further study is the requirement for LRRC8A in stretch-induced AKT and MAP kinase signaling in C2C12 myotubes upon static stretching .",
"Mechanical stretch is known to regulate myoblast proliferation and differentiation and myofiber hypertrophy via PI3K-AKT-MAPK signaling ( Schiaffino et al . , 2013; Ma et al . , 2017; Fu et al . , 2018 ) .",
"Also , there are numerous reports that VRAC , and presumably LRRC8A , is mechano-responsive ( Browe and Baumgarten , 2003; Browe and Baumgarten , 2006; Osei-Owusu et al . , 2018; Barakat et al . , 1999; Nakao et al . , 1999; Nilius and Droogmans , 2001; Romanenko et al . , 2002; Strange et al . , 2019 ) .",
"Therefore , it may not be surprising that LRRC8A complexes co-regulate both insulin and stretch-mediated PI3K-AKT , ERK1/2 signaling in skeletal myotubes - potentially integrating mechanical and hormonal stimuli to tune downstream signaling .",
"Indeed , the concept of mechano-tuning insulin signaling has been proposed and demonstrated in other cell systems ( Chen and Chalfie , 2014; Kim et al . , 2018 ) which implicate integrin signaling ( Kim et al . , 2018 ) as the mechano-sensory mechanism .",
"Curiously , it has been reported that VRAC can be activated in cardiac muscle cells by applying mechanical tension to β1-integrins ( Browe and Baumgarten , 2003; Browe and Baumgarten , 2006 ) , supporting the notion that , in striated muscle , integrin-LRRC8A may indeed participate in mechano-tuning insulin-AKT and downstream signaling .",
"Further studies to more fully delineate the putative molecular mechanisms are necessary .",
"It is also notable that the profound myogenic differentiation block observed upon Lrrc8a ablation in both C2C12 myotubes and primary myotubes in vitro is significantly milder in vivo , where only a 30% reduction in skeletal myocyte cross-sectional area is observed , with no change in total muscle mass , or lean content , in Skm KO mice .",
"This discordance in phenotype may reflect fundamental differences in the biology of skeletal muscle differentiation in vitro versus the in vivo milieu .",
"Alternatively , it may be that , although early , the time interval between Myf5-Cre expression early in myogenesis , and ultimate reductions in LRRC8A protein ( potentially ~3 days ) may extend beyond the critical period during which LRRC8A is required for myogenic differentiation .",
"Directly testing this hypothesis would require examining mice expressing Cre-recombinase at the precursor stage , in skeletal muscle satellite cells , such as Pax3 or Pax7 promoters ( Relaix et al . , 2006; Buckingham et al . , 2006 ) - these experiments are currently underway .",
"Although overall muscle development is grossly intact in both LRRC8A skeletal muscle KO ( Myl1Cre/Lrrc8afl/fl and Myf5Cre/Lrrc8afl/fl ) mice , there is a consistent reduction in exercise capacity , muscle endurance and force generation , and a propensity for increased adiposity over time compared to age and gender matched controls .",
"The impaired exercise capacity observed in skeletal muscle Lrrc8a KO mice are consistent with some level of insulin resistance , as in db/db mice ( Ostler et al . , 2014 ) and in humans ( Reusch et al . , 2013 ) , and may be due to impaired skeletal muscle glycolysis and oxygen consumption in LRRC8A-depleted skeletal muscle .",
"Furthermore , the increased gonadal adiposity , with preserved glucose and insulin tolerance , observed in LRRC8A skeletal muscle KO ( Myl1Cre/Lrrc8afl/fl and Myf5Cre/Lrrc8afl/fl ) mice phenocopy both skeletal muscle-specific insulin receptor KO mice ( MIRKO ) ( Brüning et al . , 1998 ) and transgenic mice expressing a skeletal muscle dominant-negative insulin receptor mutant ( Moller et al . , 1996 ) , wherein skeletal muscle-specific insulin resistance is compensated for by re-distribution of glucose from skeletal muscle to adipose tissue , to promote adiposity ( Kim et al . , 2000 ) .",
"In the case of LRRC8A skeletal muscle KO mice , overnutrition and HFD feeding unmasks this underlying mild insulin resistance and glucose intolerance , as adipose-tissue insulin resistance also begins to set in .",
"Recent findings from skeletal muscle specific AKT1/AKT2 double KO mice indicate that these effects may not attributable to solely to muscle AKT signaling ( Jaiswal et al . , 2019 ) , but potentially involve other insulin-sensitive signaling pathways .",
"In summary , we show that LRRC8A regulates myogenic differentiation and insulin-PI3K-AKT-AS160 , ERK1/2 , and mTOR signaling in myotubes via GRB2-mediated signaling .",
"In vivo , LRRC8A is required for maintaining normal exercise capacity , muscle endurance , adiposity under basal conditions , and systemic glycemia in the setting of overnutrition .",
"These findings contribute further to our understanding of LRRC8A channel complexes in the regulation of systemic metabolism ."
],
[
"The Institutional Animal Care and Use Committee of the Washington University in St . Louis and the University of Iowa approved all experimental procedures involving animals .",
"All the mice were housed in temperature , humidity , and light-controlled room and allowed free access to water and food .",
"Male and female Lrrc8a fl/fl ( WT ) , Myl1Cre/Lrrc8a fl/fl , Myf5Cre/Lrrc8a fl/fl ( skeletal muscle targeted Lrrc8a KO ) , were generated and used in these studies .",
"Myl1Cre ( JAX# 24713 ) and Myf5Cre ( JAX# 007893 ) mice were purchased from Jackson labs .",
"For high-fat diet ( HFD ) studies , we used Research Diets Inc ( Cat # D12492 ) ( 60 kcal% fat ) regimen starting at 14 weeks of age .",
"Lrrc8afl/fl mice were generated as previously described ( Zhang et al . , 2017 ) .",
"Briefly , Lrrc8a intronic sequences were obtained from Ensembl Transcript ID ENSMUST00000139454 .",
"All CRISPR/Cas9 sites were identified using ZiFit Targeter Version 4 . 2 ( http://zifit . partners . org/ZiFiT/ ) .",
"Pairs of oligonucleotides corresponding to the chosen CRISPR-Cas9 target sites were designed , synthesized , annealed , and cloned into the pX330-U6-Chimeric_BB-CBh-hSpCas9 construct ( Addgene plasmid # 42230 ) , following the protocol detailed in Cong et al . , 2013 .",
"CRISPR-Cas9 reagents and ssODNs were injected into the pronuclei of F1 mixed C57/129 mouse strain embryos at an injection solution concentration of 5 ng/μl and 75–100 ng/μl , respectively .",
"Correctly targeted mice were screened by PCR across the predicted loxP insertion sites on either side of Exon",
"3 . These mice were then backcrossed >8 generations into a C57BL/6 background .",
"Rabbit polyclonal anti-LRRC8A antibody was generated against the epitope QRTKSRIEQGIVDRSE ( Pacific Antibodies ) .",
"All other primary antibodies were purchased from Cells Signaling: anti-β-actin ( #8457 s ) , p-AKT1 ( #9018 s ) , Akt1 ( #2938 s ) , pAKT2 ( #8599 s ) , Akt2 ( #3063 s ) , p-AS160 ( #4288 s ) , AS160 ( #2670 s ) , AMPKα ( #5831 s ) , pAMPKα ( #2535 s ) , FoxO1 ( #2880 s ) and pFoxO1 ( #9464 s ) , p70 S6 Kinase ( #9202 s ) , p-p70 S6 Kinase ( #9205 s ) , pS6 Ribosomal ( #5364 s ) , GAPDH ( #5174 s ) , pErk1/2 ( #9101 s ) , Total Erk1/2 ( #9102 s ) .",
"Purified mouse anti-Grb2 was purchased from BD ( 610111 s ) .",
"Purified anti-flag mouse antibody was purchased from sigma .",
"Rabbit IgG Santa Cruz ( sc-2027 ) .",
"All primary antibodies were used at 1:1000 dilution , except for anti-flag at 1:2000 dilution .",
"All secondary antibody ( anti-rabbit-HRP and anti-mouse-HRP ) were used at 1:10 , 000 dilution .",
"Adenovirus type five with Ad5-CMV-mCherry ( 1 × 1010 PFU/ml ) , Ad5-CMV-Cre-mCherry ( 3 × 1010 PFU/ml ) were obtained from the University of Iowa viral vector core facility .",
"Adenovirus type five with Ad5-CMVmCherry-U6-hLRRC8A-shRNA , 2 . 2 × 1010 PFU/ml , ( AD3535 ) , ( Vector Biolabs ) .",
"Scrambled non-targeting control ( shSCR: Ad5-U6-scramble-mCherry , 1 × 1010 PFU/ml ) , ( Vector biolabs #3086 ) .",
"Ad5-CAG-LoxP-stop-LoxP-3XFlag- LRRC8A ( 1 × 1010 PFU/ml ) were obtained from Vector Biolabs .",
"Ad5-U6-shGRB2-GFP ( 1 × 109 PFU/ml ) and Ad5-U6-shSCR-GFP ( 1 × 1010 PFU/ml ) were obtained from Vector Biolabs .",
"All recordings were performed in the whole-cell configuration at room temperature , as previously described ( Zhang et al . , 2017; Kang et al . , 2018 ) .",
"Briefly , currents were measured with either an Axopatch 200B amplifier or a MultiClamp 700B amplifier ( Molecular Devices ) paired to a Digidata 1550 digitizer , using pClamp 10 . 4 software .",
"The intracellular solution contained ( in mM ) : 120 L-aspartic acid , 20 CsCl , 1 MgCl2 , 5 EGTA , 10 HEPES , 5 MgATP , 120 CsOH , 0 . 1 GTP , pH 7 . 2 with CsOH .",
"The extracellular solution for hypotonic stimulation contained ( in mM ) : 90 NaCl , 2 CsCl , 1 MgCl2 , 1 CaCl2 , 10 HEPES , five glucose , five mannitol , pH 7 . 4 with NaOH ( 210 mOsm/kg ) .",
"The isotonic extracellular solution contained the same composition as above except for mannitol concentration of 105 ( 300 mOsm/kg ) .",
"The osmolarity was checked by a vapor pressure osmometer 5500 ( Wescor ) .",
"Currents were filtered at 10 kHz and sampled at 100 μs interval .",
"The patch pipettes were pulled from borosilicate glass capillary tubes ( WPI ) using a P-87 micropipette puller ( Sutter Instruments ) .",
"The pipette resistance was ~4–6 MΩ when the patch pipette was filled with intracellular solution .",
"The holding potential was 0 mV .",
"Voltage ramps from −100 to +100 mV ( at 0 . 4 mV/ms ) were applied every 4 s .",
"Satellite cell isolation and differentiation were performed as described previously with minor modifications ( Hindi et al . , 2017 ) .",
"Briefly , gastrocnemius and quadriceps muscles were excised from Lrrc8aflfl mice ( 8–10 weeks old ) and washed twice with 1XPBS supplemented with 1% penicillin-streptomycin and fungizone ( 300 µl/100 ml ) .",
"Muscle tissue was incubated in DMEM-F12 media supplemented with collagenase II ( 2 mg/ml ) , 1% penicillin-streptomycin and fungizone ( 300 ul/100 ml ) and incubated at shaker for 90 min at 37°C .",
"Tissue was washed with 1X PBS and incubated again with DMEM-F12 media supplemented with collagenase II ( 1 mg/ml ) , dispase ( 0 . 5 mg/ml ) , 1% penicillin-streptomycin and fungizone ( 300 µl/100 ml ) in a shaker for 30 min at 37°C .",
"Subsequently , the tissue was minced and passed through a cell strainer ( 70 µm ) , and after centrifugation; satellite cells were plated on BD Matrigel‐coated dishes .",
"Cells were stimulated to differentiate into myoblasts in DMEM‐F12 , 20% fetal bovine serum ( FBS ) , 40 ng/ml basic fibroblast growth factor ( bfgf , R and D Systems , 233‐FB/CF ) , 1X non‐essential amino acids , 0 . 14 mM β‐mercaptoethanol , 1X penicillin/streptomycin , and Fungizone .",
"Myoblasts were maintained with 10 ng/ml bfgf and then differentiated in DMEM‐F12 , 2% FBS , 1X insulin–transferrin–selenium , when 80% confluency was reached .",
"WT C2C12 and Lrrc8a KO C2C12 cell line were cultured at 37°C , 5% CO2 Dulbecco’s modified Eagle’s medium ( DMEM; GIBCO ) supplemented with 10% fetal bovine serum ( FBS; Atlanta Bio selected ) and antibiotics 1% penicillin-streptomycin ( Gibco , USA ) .",
"Cells were grown to 80% confluency and then transferred to differentiation media DMEM supplemented with antibiotics and 2% horse serum ( HS; GIBCO ) to induce differentiation .",
"The differentiation media was changed every two days .",
"Cells were allowed to differentiate into myotubes for up to 6 days .",
"Subsequently , myotube images were taken for quantification of myotube surface area and fusion index .",
"After differentiation ( Day 7 ) , cells were imaged with Olympus IX73 microscope ( 10X objective , Olympus , Japan ) .",
"For each experimental condition , 5–6 bright field images were captured randomly from six-well plate .",
"Myotube surface area was quantified manually with ImageJ software .",
"The morphometric quantification was carried out by an independent observer who was blinded to the experimental conditions .",
"For fusion index , differentiated myotube growing on coverslip were washed with 1X PBS and fixed with 2% PFA .",
"After washing with 1XPBS three times , cells were permeabilized with 0 . 1% TritonX100 for 5 min at room temperature and subsequently blocking was done with 5% goat serum for 30 min .",
"Cells were stained with DAPI ( 1 µM ) for 15 min and after washing with 1X PBS , coverslip were mounted on slides with ProLong Diamond anti-fading agent .",
"Cells were imaged with Olympus IX73 microscope ( 10X objective , Olympus , Japan ) with bright field and DAPI filter .",
"Fusion index ( number of nuclei incorporated within the myotube/total number of nuclei present in that view field ) were analyzed by ImageJ .",
"RNA quality was assessed by Agilent BioAnalyzer 2100 by the University of Iowa Institute of Human Genetics , Genomics Division .",
"RNA integrity numbers greater than eight were accepted for RNAseq library preparation .",
"RNA libraries of 150 bp PolyA-enriched RNA were generated , and sequencing was performed on a HiSeq 4000 genome sequencing platform ( Illumina ) .",
"Sequencing results were uploaded and analyzed with BaseSpace ( Illumina ) .",
"Sequences were trimmed to 125 bp using FASTQ Toolkit ( Version 2 . 2 . 0 ) and aligned to Mus musculus mmp10 genome using RNA-Seq Alignment ( Version 1 . 1 . 0 ) .",
"Transcripts were assembled and differential gene expression was determined using Cufflinks Assembly and DE ( Version 2 . 1 . 0 ) .",
"Ingenuity Pathway Analysis ( QIAGEN ) was used to analyze significantly regulated genes which were filtered using cutoffs of >1 . 5 fragments per kilobase per million reads , >1 . 5 fold changes in gene expression , and a false discovery rate of <0 . 05 .",
"Heatmaps were generated to visualize significantly regulated genes .",
"For insulin stimulation , differentiated C2C12 myotubes were incubated in serum free media for 6 hr and stimulated with 0 and 10 nM insulin for 15 min; while differentiated primary myotubes were incubated in serum free media for 4 hr and stimulated with 0 and 10 nM insulin for 2 hr .",
"To examine intracellular signaling upon LRRC8A overexpression ( LRRC8A O/E ) , we overexpressed LRRC8A-3xFlag by transducing C2C12 myotubes with Ad5-CAG-LoxP-stop-LoxP-LRRC8A-3xFlag ( MOI 50–60 ) and Ad5-CMV-Cre-mCherry ( MOI 50–60 ) and polybrene ( 4 µg/ml ) in DMEM ( 2% FBS and 1% penicillin-streptomycin ) for 36 hr .",
"Ad5-CMV-Cre-mCherry alone with polybrene ( 4 µg/ml ) ( MOI 50–60 ) was transduced in WT C2C12 or Lrrc8a KO C2C12 as controls .",
"Viral transduction efficiency ( 60–70% ) was confirmed by mCherry fluorescence .",
"Cells were allowed to differentiate further in differentiation media up to 6 days .",
"Myotube images were taken before collecting lysates for further signaling studies .",
"GRB2 knock-down was achieved by transducing myotubes with Ad5-U6- shSCR-GFP ( Control , MOI 50–60 ) or Ad5-U6- sh LRRC8A-GFP ( GRB2 KD , MOI 50–60 ) in DMEM ( 2% FBS and 1% penicillin-streptomycin ) supplemented with polybrene ( 4 µg/ml ) for 24 hr .",
"Cells were allowed to differentiate further in differentiation media up to 6 days .",
"Differentiated myotube images were taken for myotube surface area quantification before collecting the cells for RNA isolation .",
"WT C2C12 cells were cultured in differentiation media for 6 days to form myotubes .",
"Subsequently myotubes were transduced either with Ad5-U6-scramble-mCherry ( sh-SCR ) or Ad5-mCherry-U6-shLRRC8A ( sh LRRC8A ) shRNA ( KD ) ( MOI 50–60 ) for 2 days and grown for 1 additional day prior to analysis .",
"Myotubes were then incubated in serum free media for 6 hr and stimulated with 0 and 10 nM insulin for 15 min .",
"Primary skeletal muscle cells isolated from Lrrc8a fl/fl mice were grown in differentiation media for 3 . 5 days .",
"To generate Lrrc8a KO primary myotubes , cells were transduced with either Ad5-CMV-CMV-mCherry ( WT ) or Ad5-CMV-Cre-mCherry ( KO ) ( MOI 50–60 ) for 2 days and then grown for 1 additional day prior to analysis .",
"Myotubes were then harvested to measure intracellular signaling under basal culture conditions .",
"C2C12 myotubes were plated in each well of a six-well BioFlex culture plate .",
"Cells were allowed to differentiate up to 6 days in differentiation media , and then placed into a Flexcell Jr .",
"Tension System ( FX-6000T ) and incubated at 37°C with 5% CO2 .",
"C2C12 myotubes on flexible membrane were subjected to either no tension or to static stretch of 5% for 15 min .",
"Cells were lysed and protein isolated for subsequent western blots .",
"Cells were washed with ice cold 1X PBS and lysed in ice-cold lysis buffer ( 150 mM NaCl , 20 mM HEPES , 1% NP-40 , 5 mM EDTA , pH 7 . 5 ) with added proteinase/phosphatase inhibitor ( Roche ) .",
"The cell lysate was further sonicated ( 20% pulse frequency for 20 s ) and centrifuged at 14 , 000 rpm for 20 min at 4°C .",
"The supernatant was collected and estimated for protein concentration using DC protein assay kit ( Bio-Rad ) .",
"For immunoblotting , an appropriate volume of 4 x Laemmli ( Bio-rad ) sample loading buffer was added to the sample ( 10–20 μg of protein ) , then heated at 90°C for 5 min before loading onto 4–20% gel ( Bio-Rad ) .",
"Proteins were separated using running buffer ( Bio-Rad ) for 2 hr at 110 V . Proteins were transferred to PVDF membrane ( Bio-Rad ) and membrane blocked in 5% ( w/v ) BSA or 5% ( w/v ) milk in TBST buffer ( 0 . 2 M Tris , 1 . 37 M NaCl , 0 . 2% Tween-20 , pH 7 . 4 ) at room temperature for 1 hr .",
"Blots were incubated with primary antibodies at 4°C overnight , followed by secondary antibody ( Bio-Rad , Goat-anti-mouse #170–5047 , Goat-anti-rabbit #170–6515 , all used at 1:10 , 000 ) at room temperature for one hour .",
"Membranes were washed three times and imaged by chemiluminescence ( Pierce ) by using a Chemidoc imaging system ( BioRad ) .",
"The images were further analyzed for band intensities using ImageJ software .",
"β-Actin or GAPDH levels were quantified for equal protein loading .",
"C2C12 myotubes were plated on 10 cm dishes in complete media and grown to 80% confluency .",
"For LRRC8A-3xFlag overexpression , Ad5-CAG-LoxP-stop-LoxP-3XFlag- LRRC8A ( MOI 50–60 ) and Ad5-CMV-Cre-mCherry ( MOI 50–60 ) along with polybrene ( 4 ug/ml ) were added to cells in DMEM media ( 2% FBS and 1% penicillin-streptomycin ) allowed to grow for 36 hr .",
"Cells were then switched to differentiation media for up to 6 days .",
"After that myotubes were harvested in ice-cold lysis buffer ( 150 mM NaCL , 20 mM HEPES , 1% NP-40 , 5 mM EDTA , pH 7 . 5 ) with added protease/phosphatase inhibitor ( Roche ) and kept on ice with gentle agitation for 15 min to allow complete lysis .",
"Lysated were incubated with anti-Flag antibody ( Sigma #F3165 ) or control rabbit IgG ( Santa Cruz sc-2027 ) rotating end over end overnight at 4°C .",
"Protein G sepharose beads ( GE ) were added for 4 hr and then samples were centrifuged at 10 , 000 g for 3 min and washed three times with RIPA buffer and re-suspended in laemmli buffer ( Bio-Rad ) , boiled for 5 min , separated by SDS-PAGE gel followed by the western blot protocol .",
"Differentiated cells were solubilized in TRIzol and the total RNA was isolated using PureLink RNA kit ( Life Technologies ) and column DNase digestion kit ( Life Technologies ) .",
"The cDNA synthesis , qRT-PCR reaction and quantification were carried out as described previously ( Zhang et al . , 2017 ) .",
"All experiments were performed in triplicate and GAPDH were used as internal standard to normalize the data .",
"All primers used for qRT-PCR are listed in Supplementary file",
"4 . Mice were euthanized and gastrocnemius muscle excised and washed with 1X PBS .",
"Muscles tissue were minced with surgical blade and kept in 8 vol of ice cold homogenization buffer ( 20 mM Tris , 137 mM NaCl , 2 . 7 mM KCl , 1 mM MgCl2 , 1% Triton X-100 , 10% ( w/v ) glycerol , 1 mM EDTA , 1 mM dithiothreitol , pH 7 . 8 ) supplemented with protease/phosphatase inhibitor ( Roche ) .",
"Tissues were homogenized on ice with a Dounce homogenizer ( 40–50 passes ) and incubated for overnight at 4°C with continuous rotation .",
"Tissue lysate was further sonicated in 20 s cycle intervals for 2–3 times and centrifuged at 14 , 000 rpm for 20 min at 4°C .",
"The supernatant was collected for protein concentration estimation using DC protein assay kit ( Bio-Rad ) .",
"Due to the high content of contractile protein in this preparation , coomassie gel staining was performed to demonstrate equal protein loading , and for quantification normalization of Western blots .",
"Mice were anesthetized with isoflurane followed by cervical dislocation .",
"Tibialis anterior ( TA ) muscle was carefully excised and gently immersed into the tissue-tek O . C . T medium placed on wooden cork .",
"Orientation of the tissue maintained while embedding in the medium .",
"Subsequently , wooden cork with tissue gently immersed into the liquid N2 pre-chilled isopentane bath for 10–14 s and store at −80°C .",
"Tissue sectioning ( 10 µm ) were done with Leica cryostat and all sections collected on positively charged microscope slide for H and E staining as described earlier ( Bonetto et al . , 2015 ) .",
"Briefly , TA sectioned slides were stained for 2 min in hematoxylin , 1 min in eosin and then dehydrated with ethanol and xylenes .",
"Subsequently , slides were mounted with coverslip and image were taken with EVOS cell imaging microscope ( 10X objective ) .",
"For quantification of fiber cross-sectional area , images were processed using ImageJ software to enhance contrast and smooth/sharpen cell boundaries and clearly demarcate muscle fiber cross sectional area .",
"All measurements were performed with an independent observer who was blinded to the identity of the slides .",
"Skeletal muscle fiber-type was quantified in WT and Skm KO tibialis anterior ( TA ) muscle as previously described ( Biltz et al . , 2020 ) .",
"Briefly , sections were immunostained against myosin heavy chain isoforms ( type I , type IIa , and type IIb BA‐F8 , SC‐71 and BF‐F3 , respectively , Developmental Studies Hybridoma Bank , Iowa City , IA ) and against laminin ( ab11575; Abcam , Cambridge , UK ) to quantify fiber types and areas .",
"Fiber types were identified on four non‐overlapping 20 × images in controlled locations: two from the superficial region and two from the deep .",
"Unstained fibers were assumed to be of type IIx .",
"Fiber areas were detected with a custom ImageJ macro using the automated Huang threshold and particle analyzer .",
"Regions of interest with a circularity of less than 0 . 5 were excluded as being out of the axial plane of that fiber .",
"Mouse treadmill exercise protocols were adapted from Dougherty et al . , 2016 .",
"Briefly , mice were first acclimated with the motorized treadmill ( Columbus Instruments Exer3/6 Treadmill Columbus , OH ) for 3 days by running 10–15 min ( with 3 min interval ) for three consecutive days at 7 m/min , with the electric shocking grid ( frequency 1 Hz ) installed in each lane .",
"During the treadmill testing , mice ran with a gradual increase in speed ( 5 . 5 m/minute to 22 m/minute ) and inclination ( 0o-15o ) at time intervals of 3 min each .",
"The total running distance for each mouse was recorded at the end of the experiment .",
"The predefined criteria for removing the mouse from the treadmill and recording the distance travelled was: continuous shock for 5 s or receiving 5–6 shocks within a time interval of 15 s .",
"These mice were promptly removed from the treadmill and total duration and distance were recorded for further analysis .",
"Mouse inversion test was performed using a wire-grid screen apparatus elevated to 50 cm .",
"Mice were stabilized on the screen inclined at 60o , with the mouse head facing towards the base of the screen .",
"The screen was slowly pivoted to 0o ( horizontal ) , such that the mouse was fully inverted and hanging upside down from the screen .",
"Soft bedding was placed underneath the screen to protect mouse from any injury , were they to fall .",
"The inversion test for each mouse was repeated two times with an interval of 45 min ( resting period ) .",
"The hang time for each mouse was repeated three times with an interval of 5 min .",
"The maximum hanging time limit for each mouse was set for 3 min .",
"Soleus muscle was carefully dissected and transferred to a specialized muscle stimulation system ( 1500A , Aurora Scientific , Aurora , ON , Canada ) where physiology tests were run in a blinded fashion .",
"Muscle was immersed in a Ringer solution ( in mM ) ( NaCl 137 , KCI 5 , CaCl2 2 , NaH2PO4 1 , NaHCO3 24 , MgSO4 1 , glucose 11 and curare 0 . 015 ) maintained at 37°C .",
"The distal tendon was secured with silk suture to the arm of a dual mode ergometer ( 300C-LR , Aurora Scientific , Aurora , ON , Canada ) and the proximal tendon secured to a stationary post .",
"Muscles were stimulated with an electrical stimulator ( 701C , Aurora Scientific , Aurora , ON , Canada ) using parallel platinum plate electrodes extending along the muscle .",
"Muscle slack length was set by increasing muscle length until passive force was detectable above the noise of the transducer and fiber length was measured through a micrometer reticule in the eyepiece of a dissecting microscope .",
"Optimal muscle length was then determined by incrementally increasing the length of the muscle by 10% of slack fiber length until the isometric tetanic force plateaued .",
"At this optimum length , force was recorded during a twitch contraction and isometric tetanic contraction ( 300 ms train of 0 . 3 ms pulses at 225 Hz ) .",
"The muscle was then fatigued with a bout of repeated tetanic contractions every 10 s until force dropped below 50% of peak .",
"At this point , the muscle was cut from the sutures and weighed .",
"This weight , along with peak fiber length and muscle density ( 1 . 056 g/cm3 ) , was used to calculate the physiological cross-sectional area ( PCSA ) and convert to specific force ( tension ) .",
"The experimental data were analyzed and quantified using Matlab ( Mathworks ) , and presented as peak tetanic tension ( Tetanic Tension ) – peak of the force recording during the tetanic contraction , normalized to PCSA; Time to fatigue ( TTF ) – time for the tetanic tension to fall below 50% of the peak value during the fatigue test; Half relaxation time ( HRT ) – half the time between force peak and return to baseline during the twitch contraction .",
"Cellular respiration was quantified in primary myotubes using the XF24 extracellular flux ( XF ) bioanalyzer ( Agilent Technologies/Seahorse Bioscience , North Billerica , MA , USA ) .",
"Primary skeletal muscle cells isolated from Lrrc8a fl/fl mice were plated on BD Matrigel‐coated plate at a density of 20 × 103 per well .",
"After 24 hr , cells were incubated in Ad5-CMV-mCherry or Ad5-CMV-Cre-mCherry ( MOI 90–100 ) in DMEM-F12 media ( 2% FBS and 1% penicillin-streptomycin ) for 24 hr .",
"Cells were then switched to differentiation media for another 3 days .",
"For insulin-stimulation , cells were incubated in serum free media for 4 hr and stimulated with 0 and 10 nM insulin for 2 hr .",
"Subsequently , medium was changed to XF‐DMEM , and kept in a non‐CO2 incubator for 60 min .",
"The basal oxygen consumption rate ( OCR ) was measured in XF‐DMEM .",
"Subsequently , oxygen consumption was measured after addition of each of the following compounds: oligomycin ( 1 μg/ml ) ( ATP-Linked OCR ) , carbonyl cyanide 4‐ ( trifluoromethoxy ) phenylhydrazone ( FCCP; 1 μM ) ( Maximal Capacity OCR ) and antimycin A ( 10 μM; Spare Capacity OCR ) ( Wende et al . , 2015 ) .",
"For the glycolysis stress test , prior to experimentation , cells were switched to glucose‐free XF‐DMEM and kept in a non‐CO2 incubator for 60 min .",
"Extracellular acidification rate ( ECAR ) was determined in XF‐DMEM followed by these additional conditions: glucose ( 10 mM ) , oligomycin ( 1 μM ) , and 2‐DG ( 100 mM ) .",
"Data for Seahorse experiments ( normalized to protein ) reflect the results of one Seahorse run/condition with six replicates .",
"Mouse body composition ( fat and lean mass ) was measured by nuclear magnetic resonance ( NMR; Echo-MRI 3-in-1 analyzer , EchoMRI , LLC ) .",
"For glucose tolerance test ( GTT ) , mice were fasted for 6 hr and intraperitoneal injection of glucose ( 1 g/kg body weight for lean mice and 0 . 75 g/kg of body weight for HFD mice ) administered .",
"Glucose level was monitored from tail-tip blood using a glucometer ( Bayer Healthcare LLC ) at the indicated times .",
"For insulin tolerance test ( ITT ) , mice were fasted for 4 hr and after an intra-peritoneal injection of insulin ( HumulinR , 1 U/kg for lean mice and 1 . 25 U/kg for HFD mice ) glucose level was measured by glucometer at the indicated times .",
"Data are represented as mean ± s . e . m . Two-tail paired or unpaired Student’s t-tests were used for comparison between two groups .",
"For three or more groups , data were analyzed by one-way analysis of variance and Tukey’s post hoc test .",
"For GTTs and ITTs , two-way analysis of variance ( Anova ) was used .",
"A p-value<0 . 05 was considered statistically significant .",
"* , ** and *** represents a p-value less than 0 . 05 , 0 . 01 and 0 . 001 respectively ."
]
] | [
"Maintenance of skeletal muscle is beneficial in obesity and Type 2 diabetes .",
"Mechanical stimulation can regulate skeletal muscle differentiation , growth and metabolism; however , the molecular mechanosensor remains unknown .",
"Here , we show that SWELL1 ( Lrrc8a ) functionally encodes a swell-activated anion channel that regulates PI3K-AKT , ERK1/2 , mTOR signaling , muscle differentiation , myoblast fusion , cellular oxygen consumption , and glycolysis in skeletal muscle cells .",
"LRRC8A over-expression in Lrrc8a KO myotubes boosts PI3K-AKT-mTOR signaling to supra-normal levels and fully rescues myotube formation .",
"Skeletal muscle-targeted Lrrc8a KO mice have smaller myofibers , generate less force ex vivo , and exhibit reduced exercise endurance , associated with increased adiposity under basal conditions , and glucose intolerance and insulin resistance when raised on a high-fat diet , compared to wild-type ( WT ) mice .",
"These results reveal that the LRRC8 complex regulates insulin-PI3K-AKT-mTOR signaling in skeletal muscle to influence skeletal muscle differentiation in vitro and skeletal myofiber size , muscle function , adiposity and systemic metabolism in vivo ."
] | [
"Skeletal muscles – the force-generating tissue attached to bones – must maintain their mass and health for the body to work properly .",
"It is therefore necessary to understand how an organism can regulate the way skeletal muscles form , grow and heal .",
"A multitude of factors can control how muscles form , including mechanical signals .",
"The molecules that can sense these mechanical stimuli , however , remain unknown .",
"Mechanoresponsive ion channels provide possible candidates for these molecular sensors .",
"These proteins are studded through the cell membranes , where they can respond to mechanical changes by opening and allowing the flow of ions in and out of a cell , or by changing interactions with other proteins .",
"The SWELL1 protein is a component of an ion channel known as VRAC , which potentially responds to mechanical stimuli .",
"This channel is associated with many biological processes such as cells multiplying , migrating , growing and dying , but it is still unclear how .",
"Here , Kumar et al . first tested whether SWELL1 controls how skeletal muscle precursors mature into their differentiated and functional form .",
"These experiments showed that SWELL1 regulates this differentiation process under the influence of the hormone insulin , as well as mechanical signals such as cell stretching .",
"In addition , this work revealed that SWELL1 relies on an adaptor molecule called GRB2 to relay these signals in the cell .",
"Next , Kumar et al . genetically engineered mice lacking SWELL1 only in skeletal muscle .",
"These animals had smaller muscle cells , as well as muscles that were weaker and less enduring .",
"When raised on a high-calorie diet , the mutant mice also got more obese and developed resistance to insulin , which is an important step driving obesity-induced diabetes .",
"Together , these findings show that SWELL1 helps to regulate the formation and function of muscle cells , and highlights how an ion channel participates in these processes .",
"Healthy muscles are key for overall wellbeing , as they also protect against obesity and obesity-related conditions such as type 2 diabetes or nonalcoholic fatty liver disease .",
"This suggests that targeting SWELL1 could prove advantageous in a clinical setting ."
] | 2020 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"chromosomes and gene expression",
"biochemistry and chemical biology"
] | The insulin receptor cellular IRES confers resistance to eIF4A inhibition | elife-00542-v1 | [
[
"During times of stress the cell changes its gene expression profile to better manage the cause of the stress .",
"Coordinate changes in both transcription and translation occur ( Sengupta et al . , 2010; Spriggs et al . , 2010 ) .",
"A central pathway that responds to stress stimuli by controlling both protein and RNA synthesis is the insulin and insulin-like receptor-signaling ( IIS ) pathway .",
"The fundamental molecular architecture of the IIS pathway is conserved from flies to man ( Figure 1 ) ( Oldham , 2011 ) .",
"When IIS signaling is high , the protein kinase AKT is activated ( Ruggero and Sonenberg , 2005 ) .",
"AKT directly phosphorylates the Foxo family of transcription factors and consequently prevents activated transcription of Foxo target genes ( Brunet et al . , 1999 ) .",
"AKT also stimulates the activation of the mechanistic target of rapamycin ( mTOR ) protein ( Zoncu et al . , 2011 ) . 10 . 7554/eLife . 00542 . 003Figure 1 . Simplified insulin/insulin-like growth factor signaling diagram .",
"( A ) When Insulin receptor or Insulin-like growth factor receptor is active signaling through AKT inhibits Foxo transcription factors and activates mTOR .",
"mTOR in turn inhibits 4E-BP and activates S6K .",
"S6K in turn inhibits Pdcd4 and activates eIF4B .",
"When insulin signaling is low inhibition of Foxo is relieved and Foxo activates the transcription of Insulin receptor and 4E-BP .",
"The broken line indicates the proposed activation of Pdcd4 by Foxo .",
"( B ) Alignment of human ( Hs top ) and Drosophila ( Dm bottom ) Pdcd4 proteins .",
"Conserved Akt and S6K phosphorylation sites are indicated by asterisk .",
"Conserved MA3 domains are indicated by shaded boxes .",
"Arrowheads indicate conserved acidic residues important for eIF4A binding in humans .",
"( C ) eIF4A interacts with Pdcd4 in Drosophila cells .",
"Cytoplasmic extracts from a saturated culture of S2 cells were subjected to immunoprecipitation with antisera directed against eIF4A or preimmune serum .",
"Pdcd4 was detected with antisera against Pdcd4 .",
"( D ) Mutant Pdcd4 binds less efficiently to eIF4A than wildtype .",
"Cytoplasmic extracts from cultures of S2 cells expression wild-type Myc-Pdcd4 or mutant Myc-Pdcd4 ( AA ) were subjected to immunoprecipitation with antisera directed against eIF4A .",
"Myc-Pdcd4 was detected with mouse monoclonal antibody to the Myc tag .",
"Immunoprecipitated eIF4A was detected with rabbit antisera .",
"( E ) Immobilized Drosophila Pdcd4 interacts with Drosophila eIF4A .",
"On the top is a cartoon of approach .",
"On the bottom is an immnoblot of proteins eluted from the affinity columns .",
"Position of the recombinant eIF4A is indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 00542 . 003 Activated mTOR stimulates general translation , in part , by influencing the activity of the translation initiation complex eIF4F .",
"The eIF4F complex consists of eIF4E , the 7-methyl-Guanosine-cap ( m7G ) binding protein , eIF4A , an RNA helicase , and eIF4G , a large scaffolding protein .",
"In addition , the RNA binding protein eIF4B can associate with eIF4F to stimulate the helicase activity of eIF4A ( Ma and Blenis , 2009; Sonenberg and Hinnebusch , 2009; Zoncu et al . , 2011 ) .",
"mTOR stimulates general translation in part by inactivating translational inhibitors .",
"mTOR phosphorylates and inactivates the translation repressor eIF4E binding protein ( 4E-BP ) ( Gingras et al . , 1999 ) allowing efficient formation of the eIF4F complex .",
"In addition , mTOR activates ribosomal protein S6 kinase ( S6K ) ( Sarbassov et al . , 2005 ) .",
"S6K stimulates the helicase eIF4A by activating eIF4B and inhibiting programmed cell death protein 4 ( Pdcd4 ) , a known eIF4A inhibitor ( Figure 1 ) ( Yang et al . , 2003; Raught et al . , 2004; Dorrello et al . , 2006 ) .",
"Thus under conditions of high signaling through AKT and mTOR , cap-dependent translation is stimulated .",
"In times of stress , low levels of signaling through the IIS pathway lead to activated Foxo and 4E-BP in addition to inactive S6K .",
"Foxo moves to the nucleus and controls the transcription of its target genes ( Salih and Brunet , 2008 ) .",
"4E-BP prevents formation of the translation initiation complex eIF4F , thereby inhibiting m7G-dependent translation , and S6K no longer stimulates eIF4A .",
"This in turn leads to lower levels of global protein synthesis .",
"Thus the IIS pathway controls gene expression with two different branches: transcription of Foxo target genes and m7G-cap-dependent translation through 4E-BP and S6K .",
"The IIS pathway in Drosophila contains a mechanism that functionally couples activated transcription to translation .",
"A portion of the system includes a signaling and gene expression feedback loop for direct genetic targets of Drosophila Foxo .",
"The insulin-like receptor ( INR ) and 4E-BP genes are conserved transcriptional targets of Foxo ( Figure 1 ) ( Puig et al . , 2003; Puig and Tjian , 2005; Marr et al . , 2007; Hu et al . , 2008 ) .",
"Paradoxically , the insulin receptor protein , as well as mRNA , is being synthesized and accumulating under the same conditions when 4E-BP activity and expression is induced and S6K is inhibited ( Marr et al . , 2007 ) .",
"Foxo activates the transcription of the Drosophila insulin receptor gene from three promoters .",
"Each promoter produces a transcript with a distinct 5′ untranslated region ( UTR ) but identical coding region ( Casas-Tinto et al . , 2007; Marr et al . , 2007 ) .",
"Transcripts derived from promoter 1 are by far the most abundant and ubiquitous form of INR transcript ( Casas-Tinto et al . , 2007; Marr et al . , 2007 ) .",
"The Drosophila INR 5′ UTRs contain an internal ribosome entry site ( IRES ) that allows the message to escape 4E-BP inhibition of cap-dependent translation .",
"This mechanism provides a functional coupling of transcription and translation in times of stress that allows amplification of insulin receptor expression ( Marr et al . , 2007 ) .",
"Because IRES containing transcripts can outcompete cap-dependent transcripts under these conditions their translation is actually stimulated ( Svitkin et al . , 2005; Marr et al . , 2007 ) .",
"This leads to an effective switch of the cellular translation machinery to targets of the IIS pathway .",
"Thus , Foxo targets impose a translational program by activation of genes that repress general translation while simultaneously activating targets that are immune to this translational control .",
"This provides these targets with a competitive advantage allowing them to utilize the translation machinery that is freed by the general inhibition .",
"Here we identify Drosophila Pdcd4 as an additional Foxo target further enhancing the coupling of transcription and translation regulation in the IIS pathway .",
"Since the IIS pathway targets translation initiation through control of both eIF4E and eIF4A , we wondered if the most abundant and ubiquitous Drosophila INR 5′ UTR would also provide resistance to inhibition of eIF4A activity .",
"To answer this question we used both an in vitro translation system and a cell based assay to investigate the eIF4A requirements for efficient translation of reporters containing the INR 5′ UTR from Drosophila .",
"Because mammalian systems show the same type of regulation , we also investigated the role of eIF4A inhibition in the murine insulin receptor and insulin-like growth factor receptors .",
"( Giraud et al . , 2001; Meng et al . , 2008; Spriggs et al . , 2009b ) .",
"We find , in both the Drosophila and mouse systems , that the 5′ UTRs of the mRNAs for these receptors provide resistance to both eIF4E and eIF4A inhibition .",
"Taken together , these results indicate that these cellular messages have some of the lowest requirement for eIF4F activity for translation identified to date ."
],
[
"A connection between Foxo activation and translation inhibition was identified when it was discovered that 4E-BP expression is controlled by Foxo in Drosophila and mouse cells ( Junger et al . , 2003; Puig et al . , 2003; Hu et al . , 2008 ) .",
"Since the IIS pathway is also known to control eIF4A activity through Pdcd4 ( Figure 1A ) , we tested if this gene is under direct Foxo control in Drosophila .",
"Blast analysis of human Pdcd4 with the Drosophila genome identifies a single homologous protein encoded by CG10990 .",
"Alignment of human PDCD4 and CG10990 indicate that important regions of the protein are conserved ( Figure 1B ) ( Cash and Andrews , 2012 ) .",
"The two MA3 domains , including the acidic residues shown to be important for the interaction with eIF4A are conserved ( Chang et al . , 2009; Waters et al . , 2011 ) .",
"In addition , the Akt and S6K phosphorylation sites are conserved ( Palamarchuk et al . , 2005; Dorrello et al . , 2006 ) .",
"To determine if the interaction with eIF4A is conserved , we immunoprecipitated Drosophila eIF4A from cytoplasmic extracts derived from a saturated culture of Drosophila S2 cells .",
"Associated Pdcd4 was detected by immunoblot .",
"Pdcd4 is co-precipitated with antisera against eIF4A but not with preimmune serum indicating that Pdcd4 and eIF4A interact in Drosophila cells ( Figure 1C ) .",
"We next created Myc-tagged expression constructs for Drosophila Pdcd4 , one wildtype construct and a construct containing mutations in conserved residues in the first MA3 domain ( E282A , D286A ) .",
"The analogous mutations in human PDCD4 destabilize the interaction with eIF4A ( Chang et al . , 2009; Waters et al . , 2011 ) .",
"The constructs were transfected into growing Drosophila S2 cells .",
"Subsequent immunoprecipitation of eIF4A from these cells reveals a decreased association of the mutant Pdcd4 with eIF4A ( Figure 1D ) .",
"This indicates the mutations induce the same destabilization of the eIF4A–Pdcd4 interaction in Drosophila cells .",
"Interestingly under these conditions we detect multiple forms of Pdcd4 by western blot , most likely phosphorylated forms of Pdcd4 , and only the fastest migrating species associates with eIF4A .",
"To determine if Drosophila Pdcd4 can interact directly with eIF4A we used an affinity chromatography assay using recombinant proteins purified from Escherichia coli .",
"A GST fusion to Drosophila Pdcd4 was immobilized on glutathione agarose and recombinant Drosophila eIF4A was passed over the column ( Figure 1D ) .",
"As a control GST was also immobilized on glutathione agarose .",
"The eIF4A bound to the immobilized GST-Pdcd4 but not to GST alone indicating that Drosophila Pdcd4 can interact directly with Drosophila eIF4A .",
"Taken together these data are consistent with the notion that CG10990 is the Drosophila homologue of human PDCD4 .",
"There are hints in the literature , based on microarray experiments , indicating this gene is induced in response to nutrient stress and might be controlled by Foxo ( Gershman et al . , 2007 ) .",
"In an effort to determine if Foxo binds to the Pdcd4 gene in nutrient stressed animals we reanalyzed the only publically available Foxo ChIP ( Chromatin immunoprecipitation ) dataset ( Teleman et al . , 2008 ) .",
"These experiments were performed on starved larva .",
"We find Foxo binds the Pdcd4 gene in both the promoter and intronic regions with enrichment values as high as 16-fold over background ( Figure 2A ) .",
"To corroborate this finding , we performed ChIP on genomic DNA from a cell line with an inducible Foxo cDNA gene that has been modified so the Foxo protein produced is constitutively active because it is immune to the negative regulation by insulin signaling ( FoxoCA ) ( Puig et al . , 2003; Gershman et al . , 2007 ) .",
"This allows us to induce Foxo under conditions of high nutrient signaling and remove possible crosstalk from upstream signaling pathways .",
"We tested the enrichment of genomic sequences by qPCR using primers to the Pdcd4 promoter region ( Figure 2A ) compared to a region in the first intron of CG15414 , a gene just downstream of 4E-BP .",
"We find FoxoCA binds to the promoter region of Pdcd4 at levels comparable to a well-defined direct target , 4E-BP ( Junger et al . , 2003; Puig et al . , 2003; Marr et al . , 2007 ) ( Figure 2B ) .",
"To determine the effect on mRNA production under these conditions we performed quantitative RT-qPCR on induced cells .",
"We find that the steady-state level of Pdcd4 mRNA is increased about threefold in cells expressing active Foxo ( Figure 2C ) .",
"To determine if the effect is due to mRNA stability changes or new transcription we assayed intron-containing pre-mRNAs by RT-qPCR .",
"Since most splicing is co-transcriptional in Drosophila this is a good assay for new RNA synthesis ( Khodor et al . , 2011 ) .",
"We find that Pdcd4 pre-mRNA is increased , indicating an increase in transcription of the gene .",
"The increased mRNA also leads to increased protein synthesis as determined by immuno-blot with antibodies directed against Drosophila Pdcd4 ( Figure 2D ) .",
"This is likely an underestimate of the effect since these experiments are all done under high serum and insulin conditions that should result in the rapid turnover of Pdcd4 protein ( Dorrello et al . , 2006 ) . 10 . 7554/eLife . 00542 . 004Figure 2 . Foxo activates Pdcd4 in Drosophila cells .",
"( A ) Reanalysis of ChIP-chip data from Teleman et al . ( 2008 ) .",
"Genomic Browser view of Foxo binding to the Pdcd4 locus in starved larva .",
"The data are plotted as the enrichment ( log2 ) over mock precipitated samples .",
"Primers used for ChIP and qPCR are indicated .",
"( B ) ChIP of Foxo at 4E-BP promoter and Pdcd4 locus in Drosophila S2 cells expressing constitutively active Foxo ( FoxoCA ) .",
"The data are plotted as fold enrichment over a background region 1 kb downstream of 4E-BP .",
"Uninduced samples are plotted in white , induced samples in black ( error bars indicate SD ) .",
"( C ) RT-qPCR of Pdcd4 mRNA and pre-mRNA in Drosophila S2 cells expressing FoxoCA .",
"Data are plotted as fold-induction ( error bars indicate SD ) .",
"( D ) Immunoblot of total protein from Drosophila S2 cells expressing FoxoCA .",
"Positions of Pdcd4 and tubulin are indicated .",
"( E ) 4E-BP , GstD1 , and Pdcd4 RNA levels in untreated and paraquat-treated animals .",
"The levels of RNA were normalized to RP49 and are plotted as fold-induction relative to untreated animals ( error bars indicate SEM ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00542 . 004 We tested if Foxo controls Pdcd4 in adult flies subjected to stress .",
"Adult wildtype or Foxo null flies ( Slack et al . , 2011 ) were treated with paraquat to induce oxidative stress and assayed for expression of 4E-BP , Pdcd4 , and GstD1 by RT-qPCR .",
"Consistent with previous results ( Wang et al . , 2005 ) , 4E-BP is induced by paraquat in a Foxo dependent manner .",
"Like 4E-BP , we find that Pdcd4 is induced in response to paraquat in a Foxo dependent manner ( Figure 2E ) .",
"To determine if the effect was due to loss of general oxidative stress response or if it is Foxo specific we examined the induction of GstD1 , a gene controlled by Nrf2 in response to oxidative stress ( Misra et al . , 2011 ) .",
"We find that GstD1 is still responsive indicating that the effects at Pdcd4 and 4E-BP are Foxo-specific and not due to a loss of responsiveness in the mutant flies ( Figure 2E ) .",
"These results are consistent with the idea that in addition to controlling the cap-binding complex through 4E-BP , active Foxo can influence eIF4A through activation of the Pdcd4 gene in response to stress .",
"Given that Foxo is activating transcription of the INR gene while Pdcd4 is also active , we hypothesized that since the INR mRNA is translated efficiently under these conditions ( Marr et al . , 2007 ) it must be at least partially immune to diminished eIF4A activity .",
"To determine the effect of increased Pdcd4 on the translation of insulin receptor UTR containing RNAs we modified a dicistronic mRNA assay which we previously used to investigate the effects of 4E-BP on insulin receptor translation in Drosophila ( Marr et al . , 2007 ) .",
"In this assay a construct is used that produces a RNA in which the open reading frames of renilla luciferase and firefly luciferase are present on the same transcript ( Figure 3A ) .",
"Renilla luciferase levels are an indication of total message produced in the cell and firefly luciferase levels are an indication of IRES dependent translation .",
"As previously reported , insertion of the INR 5′ UTR between the ORFs promotes translation of the second ORF ( Marr et al . , 2007 ) .",
"The levels of IRES activity of the INR UTR are comparable to the well-characterized IRES from Hepatitis C Virus ( Figure 3B ) ( Tsukiyama-Kohara et al . , 1992 ) .",
"The INR UTR and the HCV IRES both produce more firefly signal than the empty vector ( Figure 3D ) . 10 . 7554/eLife . 00542 . 005Figure 3 . Drosophila insulin receptor 5′UTR provides resistance to Pdcd4 . ( A ) Diagram of dicistronic reporters .",
"Translation of the Firefly ORF requires internal ribosome entry .",
"Firefly to renilla activity ratio provides an indication of IRES activity .",
"( B ) The Drosophila Insulin receptor UTR provides IRES activity comparable to the activity of HCV .",
"( C ) Renilla activity of the reporters .",
"( D ) Firefly activity of the reporters .",
"( E ) Dicistronic reporter activities in the presence of 4E-BP or Pdcd4 expression .",
"4E-BP and Pdcd4 stimulate the IRES activity of the insulin receptor UTRs .",
"Mutation of the critical acidic residues of Pdcd4 prevents the stimulation .",
"( F ) Renilla activity of the reporters in the presence of expressed proteins .",
"( G ) Firefly activity of the reporters in the presence of the expressed proteins ( error bars indicate SEM ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00542 . 005 To determine the effect of Pdcd4 on the INR UTR we expressed Pdcd4 in Drosophila S2 cells and measured the activity of the dicistronic reporter .",
"In order to more accurately determine the effects of the protein in question we modified the assay so the production of the dicistronic mRNA is inducible .",
"This allows accumulation of the experimental protein in the cell before the dicistronic mRNA is produced giving a more precise measure of the effects on the activity of the dicistronic message .",
"The levels of expression of the second ORF relative to the first ORF are an indication of the cap-independent translation potential of the insert .",
"To validate this assay we reproduced our previous results with 4E-BP ( Marr et al . , 2007 ) .",
"As reported previously , expression of 4E-BP stimulates the translation of the second open reading frame in a reporter containing the INR 5′UTR ( Figure 3E , G ) .",
"Expression of Pdcd4 also stimulates translation of the second ORF dependent on the INR 5′UTR similar to the effects seen with the HCV IRES ( Figure 3E , G ) .",
"These effects are specific .",
"Mutation of the key acidic residues ( Figure 1B ) in Pdcd4 shown to disrupt eIF4A binding in the human system ( Waters et al . , 2011 ) prevent the IRES stimulation .",
"To address the role of eIF4A in insulin receptor translation , we used a highly specific small molecule inhibitor of eIF4A , hippuristanol ( Bordeleau et al . , 2006; Lindqvist et al . , 2008 ) .",
"Hippuristanol is a potent translation inhibitor that works in eukaryotes from yeast to human ( Lindqvist et al . , 2008 ) .",
"Hippuristanol inhibits the ATPase activity and RNA binding of eIF4A ( Bordeleau et al . , 2006; Lindqvist et al . , 2008 ) .",
"The small molecule binds to the protein in conserved regions V and VI in eIF4A homologues ( Lindqvist et al . , 2008 ) .",
"Importantly , the effects on translation can be rescued by addition of either wild-type or mutant forms of eIF4A that are immune to hippuristanol indicating that the effects are highly specific for eIF4A ( Bordeleau et al . , 2006; Lindqvist et al . , 2008 ) .",
"This small molecule had been used previously in the Drosophila system to investigate eIF4A requirements ( Iwasaki et al . , 2009 ) .",
"We performed in vitro competitive translation experiments with capped and polyadenylated firefly luciferase reporters ( Figure 4A ) and a Drosophila embryo extract translation system that has not been treated with micrococcal nuclease ( Gebauer et al . , 1999; Marr et al . , 2007 ) in the presence of hippuristanol .",
"The RNA reporters contain the 5′ UTR from the Drosophila insulin receptor .",
"In addition we include two control RNAs .",
"One control RNA contains a non-specific UTR derived from plasmid sequences .",
"The other RNA contains the IRES from the Hepatitis C virus ( HCV ) ( Tsukiyama-Kohara et al . , 1992 ) .",
"This IRES does not require eIF4A activity and controls for non-specific effects on the extract ( Pestova et al . , 1998 ) .",
"Under the experimental conditions , translation of the first control RNA is strongly inhibited by hippuristanol while the reporter containing the HCV IRES is completely resistant to eIF4A inhibition ( Figure 4A ) .",
"This small molecule inhibitor exposed a greatly diminished role for eIF4A in the Drosophila INR UTR mediated translation ( Figure 4A ) .",
"At the low and moderate concentrations of hippuristanol , the Drosophila INR UTR reporter retains almost complete activity comparable to the HCV UTR .",
"Even at the highest concentration of hippuristanol tested , the reporter containing the INR UTR retains >50% of the original translation activity .",
"To determine the effect of Pdcd4 in this system we added recombinant Pdcd4 to the translation extract .",
"Consistent with the data using hippuristanol , we find Pdcd4 can inhibit the control RNA but not the Drosophila INR UTR or the HCV IRES ( Figure 4B ) .",
"These finding suggests that translation of the most abundant Drosophila INR transcript can tolerate inhibition of eIF4A . 10 . 7554/eLife . 00542 . 006Figure 4 . Drosophila insulin receptor 5′UTR provides resistance to eIF4a inhibition .",
"( A ) Top: Diagram of RNAs used in the in vitro translation assays .",
"Bottom: Titration of hippuristanol in in vitro translation assays .",
"The shade of the bars indicates the final concentration of hippuristanol in the assay .",
"The legend appears above the graph .",
"Data are plotted as the fraction activity of the carrier treated extracts ( error bars indicate SEM ) .",
"( B ) Top: Diagram of RNAs used in the in vitro translation assays .",
"Bottom: Activity of these RNAs in in vitro translation assays in the absence ( white bars ) and presence ( black bars ) of Drosophila Pdcd4 ( error bars indicate SEM ) .",
"( C ) Dicistronic RNA translation in vitro .",
"Top: Diagram of RNAs used in the in vitro translation assays .",
"Bottom: Firefly to renilla ratio in the absence ( white bars ) and presence ( black bars ) of hippuristanol .",
"( D ) Top: Renilla activity in the dicistronic assay .",
"Bottom: Firefly activity in the dicistronic assay .",
"Shading as in C . Percentage above the bars indicates activity after hippuristanol addition relative to carrier treated samples . DOI: http://dx . doi . org/10 . 7554/eLife . 00542 . 006 We used a dicistronic RNA in the in vitro translation assay to directly test the IRES activity under hippuristanol treatment ( Figure 4C top ) .",
"Dicistronic RNAs were synthesized in vitro using T7 RNA polymerase .",
"The RNA was capped and polyadenylated and used to program the same Drosophila embryo translation system described above .",
"The extracts were treated with either hippuristanol or carrier .",
"Consistent with the monocistronic assay , both the INR and the HCV IRES containing transcripts show increased relative translation of the second ORF upon eIF4A inhibition ( Figure 4C ) .",
"The increase is due both to a resistance of the second ORF to the inhibition and a decrease in the cap-dependent translation of the first ORF ( Figure 4D ) .",
"Under these conditions there is a small amount of cryptic translation of the second ORF in the control transcript .",
"However both the renilla and firefly activites respond the same to the hippuristanol treatment .",
"Because the molecular architecture of the IIS signaling pathway is conserved in mammals , we wondered if this level of regulation would be conserved in mammals .",
"To address this , we cloned the 5′ UTR from the longest mRNAs for the mouse insulin receptor ( mINR ) and the mouse insulin-like growth factor receptor-I ( IGFR ) and created firefly luciferase reporters under the control of these UTRs ( Figure 5A ) .",
"Previously , it was reported that INR and IGFR UTRs confer cap-independent translation activity in human and rat ( Giraud et al . , 2001; Spriggs et al . , 2009b ) .",
"To extend this finding to the mouse system and ensure that our competitive rabbit reticulocyte system was capable of supporting cap-independent translation , we assayed translation of the mINR and IGFR reporters in the presence of excess m7G cap along with our control RNA and the HCV IRES reporters described above ( Figure 5A ) .",
"While the control RNA is inhibited by excess cap , translation from the 5′ UTR of mINR and IGFR is not only resistant to excess m7G cap but the activity actually increases in the presence of excess m7G cap .",
"This observation is common with UTRs that contain an IRES ( Svitkin et al . , 2005 ) .",
"The increase in activity for both the mINR and IGFR UTRs exceeded the IRES activity of the HCV UTR .",
"This indicates that mINR UTR and IGFR UTR are capable of conferring cap-independent translation initiation . 10 . 7554/eLife . 00542 . 007Figure 5 . Mammalian insulin receptor and insulin-like growth factor receptor 5′UTR provide resistance to eIF4a inhibition .",
"( A ) Diagram of RNAs used in the in vitro translation assays .",
"( B ) In vitro Translation in the absence ( white bars ) and presence ( black bars ) of excess m7G analogue .",
"Data are plotted as the fraction activity of the mock treated extracts ( error bars indicate SEM ) .",
"( C ) Titration of hippuristanol in in vitro translation assays .",
"Data are plotted as the fraction activity of the mock treated extracts ( error bars indicate SEM ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 00542 . 007 To directly test the ability of these UTRs to allow internal ribosome entry we performed a dicistronic assay in mammalian cells .",
"The UTRs were subcloned into a plasmid construct between the renilla and firefly open reading frames controlled by the RSV LTR .",
"Both the mINR and the IGFR UTRs supported substantial firefly activity compared to the original vector .",
"This is apparent both in the firefly to renilla ratio and in the raw firefly activity units ( Figure 5B ) .",
"This combined with the in vitro translation assays strongly suggest that the mINR and IGFR UTRs can support cap-independent translation .",
"To test for conservation of resistance to eIF4A inhibition , hippuristanol was titrated into the rabbit reticulocyte translation system .",
"As expected , the activity of the control RNA is reduced to <10% of the mock treated extract , and the HCV IRES is completely resistant to hippuristanol ( Figure 5C ) .",
"In fact , the HCV IRES is stimulated fourfold by addition of hippuristanol under these conditions .",
"Both mINR and IGFR UTRs confer resistance to hippuristanol ( Figure 5C ) .",
"Even at the highest concentrations of the small molecule the mINR and mIGFR UTRs remain roughly 50% active indicating a decreased requirement for eIF4A relative to the control RNA ."
],
[
"Stress responses controlled through the IIS pathway result in changes in both mRNA synthesis and protein synthesis .",
"These processes are coordinated to ensure proper expression of the downstream targets .",
"Under the same low IIS signaling conditions that activate 4E-BP , Pdcd4 is stabilized resulting in the inhibition of the DEAD box helicase eIF4A .",
"Thus , under these conditions the eIF4F complex is repressed by two mechanisms .",
"At the same time , the Foxo family of transcription factors is active and increasing the synthesis of certain mRNAs , one of which is the INR transcript itself .",
"Previously we identified a connection between 4E-BP mediated inhibition of the cap-binding complex and INR mRNA translation in Drosophila ( Marr et al . , 2007 ) .",
"The INR message is immune to the 4E-BP translational repression and thus is preferentially translated under low signaling conditions coupling the increase in mRNA expression to an increase in protein synthesis .",
"In the work presented here we extend this observation of gene expression coordination of INR mRNA to the eIF4A branch of the IIS signaling pathway .",
"First , we show that active Foxo is capable of directly stimulating the transcription of Pdcd4 , analogous to the activation of 4E-BP seen previously under these same conditions ( Junger et al . , 2003; Puig et al . , 2003 ) .",
"This provides a mechanism for the pathways controlling Foxo to enhance the inhibition of eIF4A when stressed or when nutrients are low .",
"Second we show that the 5′ UTR of the most abundant Drosophila INR transcript provides resistance to eIF4A inhibition comparable to the resistance seen with the HCV IRES that does not require eIF4A .",
"A similar finding has been seen for the Drosophila reaper 5′ UTR ( Hernandez et al . , 2004; Iwasaki et al . , 2009 ) .",
"The data presented above also support the conservation of the cap-independent mechanism of translation initiation of the insulin receptor and insulin-like growth factor receptor mRNAs in mammals .",
"The mouse transcripts show resistance to hippuristanol under conditions that almost completely inhibit a control RNA indicating that the resistance to eIF4A inhibition is also conserved .",
"There are no easily recognizable conserved sequence elements between the Drosophila and mouse UTRs , but the mode of regulation is conserved suggesting an important role for this type of translational control .",
"This defines a functional characteristic of the insulin receptor and IGF receptor transcripts that is conserved across hundreds of millions of years of evolution ( from flies to mammals ) .",
"Taken together with previous work , these data indicate that the coupling of transcription to translation of insulin receptor mRNA mediated by Foxo targets can culminate in an activated translational response .",
"Our findings highlight a unique characteristic of the insulin receptor and IGF receptor UTRs that differentiates them from other cellular transcripts .",
"In addition to being immune to 4E-BP , these IRESes are resistant to eIF4A inhibition .",
"While viral IRESes are fairly common , cellular IRESes are rare and relatively unexplored .",
"Where it has been explored , most cellular IRESes have a strong requirement for eIF4A ( Thoma et al . , 2004; Spriggs et al . , 2009a ) .",
"The INR and IGFR UTRs seem to require neither eIF4E nor eIF4A .",
"In fact , these UTRs have the lowest identified requirement for eIF4F activity of any cellular transcript thus far .",
"In addition , they are immune to two of the most important types of translational control , namely 4E-BP control and eIF4A inhibition .",
"Both of these features make sense given the cellular environment when these mRNAs are to be translated .",
"The conserved resistance to eIF4E and eIF4A inhibition of the insulin receptor transcripts should make them capable of out-competing other cellular transcripts with greater need for eIF4A or eIF4E .",
"Using these exceptional characteristics , the insulin receptor mRNA could out-compete more abundant transcripts under times of stress or when nutrients are limiting and 4E-BP and Pdcd4 are active .",
"We focused on the insulin receptor UTRs as a mechanism for continued translation under conditions of general inhibition of protein synthesis as this is one of the initial components of the pathway identified as a direct Foxo target .",
"In more recent work other components of the pathway have been identified as transcriptionally controlled by Foxo .",
"If these targets are to be translated when Foxo is active they should also require mechanisms to escape 4E-BP and Pdcd4 inhibition .",
"It remains to be seen if they will use the same mechanism as the INR mRNA or another mechanism ."
],
[
"Wildtype Canton S flies are from the Bloomingtion Stock Center .",
"foxOΔ94 has been described ( Slack et al . , 2011 ) .",
"Chromatin immunoprecipitation using Foxo antibodies followed by tiling array analysis was performed previously on starved larva ( Teleman et al . , 2008 ) .",
"The raw .",
"CEL files for Foxo precipitated and mock precipitated arrays were downloaded from the Teleman lab web page ( http://www . dkfz . de/en/signal-transduction-cancer/pages/Data . html ) .",
"Triplicate samples were combined and the Foxo precipitated samples were compared to mock precipitated samples using using the Affymetrix Tiling analysis ( TAS ) software .",
"Combined mock arrays were used to set the background signal intensities for the ChIP arrays .",
"The Integrated Genome Browser ( Nicol et al . , 2009 ) was used to visualize the resultant profile .",
"Oligonucleotides were synthesized corresponding to annotated transcripts with the longest 5′ for both Insulin receptor ( corresponding to EST G430111A11 ) and IGF-1 receptor ( corresponding to ESTs CJ180736 and CJ173921 ) ( Supplementary file 1 ) .",
"The oligos were used to clone the UTRs from cDNA derived from NIH 3T3 cells by PCR .",
"Drosophila eIF4A and Pdcd4 were cloned into pET28 in frame with the 6x HIS tag from cDNA using PCR and standard cloning methods .",
"The plasmid was transformed into BL21* ( DE3 ) cells ( Life Technologies , Grand Island , NY ) containing pLacIRARE2 ( Novagen , EMD Millipore , Billerica , MA ) .",
"After induction with 1 mM IPTG overnight at 25°C , eIF4A or PDCD-4 was purified using HisPur Ni-NTA Resin ( Thermo Fisher Scientific , Rockford , IL ) according to manufacturer’s directions .",
"PDCD-4 was eluted from the resin using 500 mM imidazole in PBS .",
"eIF4A was eluted using 50 mM EDTA in 1X PBS .",
"Purified 6His-Pdcd4 and 6His eIF4A was used to make rabbit polyclonal antisera ( Cocalico Biologicals , Inc . , Reamstown , PA ) .",
"Full length Drosophila Pdcd4 was cloned into pGEX2TKN in frame with GST .",
"GST-PDCD-4 or GST alone was expressed in BL21* ( DE3 ) cells ( Invitrogen , Grand Island , NY ) containing pLacIRARE2 ( Novagen , EMD Millipore , Billerica , MA ) .",
"Expression was induced with 1 mM IPTG overnight at 25°C .",
"Cells were resuspended and lysed in 1X PBS with lysozyme .",
"GST or GST-PDCD4 was immobilized on glutathione sepharose 4B ( GE Healthcare Biosciences , Pittsburgh , PA ) .",
"Equal amounts of recombinant eIF4A were applied to 50 µl of resin containing either GST or GST-Pdcd4 .",
"The resin was incubated for 1 hr at 4°C .",
"The resin was poured into a small spin column ( Pierce , Thermo Fisher Scientific , Rockford , IL ) and washed with 100 column volumes wash buffer ( 20 mM Tris pH 7 . 5 , 100 mM NaCl , 1 mM DTT , 0 . 1 mM EDTA , 0 . 1 mg/ml BSA ) .",
"Bound proteins were eluted with wash buffer containing 10 mM reduced glutathione for 1 hr at 4°C .",
"Samples were separated by SDS-PAGE , transferred to nitrocellulose and recombinant eIF4A was detected with a monoclonal antibody directed against the 6x HIS tag on eIF4A ( A00186 GenScript , Piscataway , NJ ) and a fluorescent secondary antibody against mouse IgG using a Li-Cor Odyssey Infrared imaging system .",
"15 ml of a saturated culture of Drosophila S2 cells were harvested by centrifugation .",
"The cells were washed once with 1X PBS .",
"The cells were resuspended in two packed cell volumes hypotonic buffer ( 10 mM HEPES pH 7 . 4 , 10 mM KCl , 5 mM MgCl2 , 1 mM DTT , 1X protease inhibitors [Sigma-Aldrich Corp . , St . Louis , MO] ) .",
"Triton X-100 was added to 0 . 5% and the cells were left on ice 30 min .",
"Nuclei were pelleted 10 min at 6000×g .",
"Supernatant was transferred to a new tube .",
"10% of the sample was saved for input analysis .",
"Polyclonal rabbit antisera to Drosophila eIF4A was added to the remainder of the sample and the sample was incubated at 4°C overnight with constant mixing .",
"Samples were centrifuged 2 min at 23 , 000×g .",
"The supernatant was combined with 50 µl protein-A Sepharose ( GE healthcare ) and incubated at 4°C for 2 hr .",
"The mixture was poured into a small spin column ( Pierce , #89869 ) connected to a needle and washed with 5 ml 1XPBS 0 . 1% Triton X-100 followed by 1 ml 1X PBS .",
"Proteins were elute by addition of 2X SDS-PAGE buffer ( 62 . 5 mM Tris-HCl , pH 6 . 8 , 25% glycerol , 2% SDS , 0 . 01% Bromophenol Blue , 5% β-mercaptoethanol ) incubation at room temperature for 10 min and collected by centrifugation .",
"Samples were separated by SDS-PAGE .",
"The gel was transferred to nitrocellulose and probed with rabbit α-Drosophila Pdcd4 antisera directed against full length 6His-Pdcd4 ( 1:1000 ) .",
"To avoid detection of the IgG used for precipitation the immunoblot was developed with biotinylated protein-A ( 1:5000 ) ( Lal et al . , 2005 ) followed by fluorescent labeled streptavidin ( 1:5000 ) using a Li-Cor Odyssey Infrared imaging system .",
"For experiments with the mutant Pdcd4 , 10 ml of Drosophila S2 cells were plated at 1 × 106 cells/ml in Schneider’s media supplemented with 10% FBS in a 10-cm dish .",
"Constructs expressing wild-type myc-Pdcd4 or mutant myc-Pdcd4-282 A286A were transfected using effectene ( Qiagen Inc . , Valencia , CA ) .",
"4 days later the cells were harvested and immunoprecipitation of eIF4A was performed as described above except transfected Pdcd4 was detected with a monoclonal antibody directed against the myc tag ( 9E10 ) and a fluorescent secondary antibody against mouse IgG using a Li-Cor Odyssey Infrared imaging system .",
"Protein concentrations were determined using BCA assay ( Pierce ) and equal amounts of protein were loaded onto a 10% SDS-PAGE gel .",
"The gel was transferred to nitrocellulose and probed with rabbit α-Drosophila Pdcd4 antisera directed against full length 6His-Pdcd4 ( 1:500 ) and mouse α-tubulin antibody ( 1:1000 ) .",
"Transcription templates for monocistronic RNAs were created using PCR containing a template-specific forward primer with a T7 promoter incorporated and a vector specific reverse primer .",
"Dicistronic RNA templates were made by digesting the cellular reporter vectors downstream of the firefly luciferase coding sequence and utilizing a T7 promoter incorporated in the vector .",
"Templates were purified using column clean up protocol and eluted in 50 µl 10 mM Tris pH 8 . 0 ( Epoch Life Science , Missouri City , TX ) .",
"Templates were transcribed using T7 polymerase and subsequently purified using LiCl precipitation .",
"Transcripts were capped using vaccinia virus capping enzyme ( New England Biolabs , Ipswich , MA ) as recommended and purified using RNeasy column protocol ( Qiagen ) .",
"Transcripts were tailed using poly ( A ) polymerase ( New England Biolabs , Ipswich , MA ) and purified using RNeasy columns ( Qiagen ) .",
"Embryo translation extracts were prepared as described from 0- to 4-hr embryos ( Marr et al . , 2007 ) .",
"Extracts were left untreated ( no Micrococcal nuclease treatment ) to allow translation under competitive conditions .",
"Translation assays were performed in 6 µl of Drosophila embryo extract , 0 . 1 mM spermidine , 60 µm Amino Acids , 16 . 8 mM creatine phosphate , 800 ng of creatine kinase , 24 mM HEPES ( pH 7 . 4 ) , 0 . 4 mM Mg acetate , 30 mM K acetate , 1 µg of calf liver tRNA , and 100 ng of template RNA in a 10-µl reaction .",
"Hippuristanol was added to a final concentration of 2µM or otherwise indicated .",
"For assays containing PDCD-4 , protein was added to a final concentration of 320 nM and was preincubated for 15 min with extract before the addition of RNA templates .",
"Translation reactions were incubated at 27°C for 1 hr and luciferase activity was measured using 100 µl of luciferase substrate ( Promega Corp . , Madison , WI ) .",
"Firefly luciferase was measured by adding 100 µl of 75 mM HEPES pH 8 . 0 , 5 mM MgSO4 , 20 mM DTT , 100 µM EDTA , 530 µM ATP , 0 . 5 mM coenzyme A , and 0 . 5 mM D-luciferin and renilla was measured by adding 100 µl 25 mM Na4PPi , 10 mM NaOAc , 15 mM EDTA , 0 . 5 M Na2SO4 , 1 . 0 M NaCl , and 0 . 1 mM Coelenterazine , pH 5 . 0 .",
"All experiments were performed at least twice in triplicate .",
"Translation assays were performed in 6 µl of untreated rabbit reticulocyte extract ( no Micrococcal nuclease treatment to allow translation under competitive conditions ) .",
"( Green Hectares , McFarland , WI ) , 0 . 1 mM spermidine , 60 µm Amino Acids , 16 . 8 mM creatine phosphate , 800 ng of creatine kinase , 24 mM HEPES ( pH 7 . 4 ) , 0 . 4 mM Mg acetate , 30 mM K acetate , 1 µg of calf liver tRNA , and 100 ng of template RNA in a 10-µl reaction .",
"Translation reactions were incubated at 37°C for 30 min and luciferase activity was measured using 100 µl of luciferase substrate ( Promega ) .",
"For assays containing excess m7G cap , cap structure analogue ( New England Biolabs , #S1407S ) was added to a final concentration of 1 mM .",
"All experiments were performed at least twice in triplicate .",
"Drosophila S2 cells with a stable transfection of constitutively active Foxo under the control of the metallothionein A promoter were maintained in Schneider’s Insect Media supplemented with 10% fetal bovine serum ( Puig et al . , 2003 ) .",
"These cells were plated at 1 . 25 × 106 cell/ml , and expression was induced by addition of 500 µm CuSO4 for 16 hr .",
"During induction the media was supplemented with 1 µg/m bovine insulin .",
"For protein samples , cells were lysed in RIPA buffer ( PBS containing 10 mM EDTA , 1% Triton X-100 , 1% SDS , 1% deoxycholate , 1× complete protease inhibitor [Roche , Indianapolis , IN] , 10% glycerol ) .",
"Total RNA was extracted from mock-treated and induced cells using TRI Reagent according to manufacturer’s protocol ( Molecular Research Center , Inc . , Cincinnati , OH ) .",
"5 µg of total RNA were digested by DNaseI ( New England Biolabs ) .",
"First strand cDNA sythesis was done using a mix of oligo-dT and random hexamers with MMLV reverse transcriptase .",
"The final concentrations of the cDNA reaction were 50 mM Tris-HCl ( pH 8 . 3 ) , 50 mM KCl , 3 mM MgCl2 , 10 mM DTT , 400 µM dNTPs , 1–2 µg RNA , 500 ng primers , 200 units MMLV RT . cDNAs were diluted 1:10 in TE pH 8 .",
"qPCR was run using 5 µl cDNA , GoTaq qPCR Master Mix ( Promega ) , and primers at a final concentration of 100 nM in a 20-µl reaction .",
"qPCR was done using specific primers against Drosophila Pdcd4 , RP49 , GstD1 , and 4E-BP ( Supplementary file 1 ) .",
"Pdcd4 fold-expression was calculated as a fraction of RP49 and normalized to mock-treated expression levels .",
"All experiments were performed at least twice in triplicate .",
"7-day-old adult male flies were starved for 5 hr and transferred to vials containing 5% sucrose or 5% sucrose+5 mM paraquat .",
"After 24 hr flies were harvested and total RNA was extracted from mock-treated and paraquat-treated flies using TRI Reagent according to manufacturer’s protocol ( Molecular Research Center , Inc . ) .",
"Previously described dicistronic reporter constructs ( Marr et al . , 2007 ) were subcloned into a plasmid containing the metallothionein A promoter for metal inducible expression ( Marr et al . , 2006 ) .",
"The IRES sequence from HCV ( Kieft et al . , 1999 ) was subcloned into the inducible expression vector .",
"For expression in S2 cells , Pdcd4 was cloned under a minimal actin promoter .",
"For the Pdcd4 double mutant construct , amino acids 282 , and 286 ( Figure 1B ) were mutated to alanine by site directed mutagenesis .",
"S2 cells were maintained in Schneider’s media with 10% FBS and Penicillin/Streptomycin .",
"For transfection , cells were plated at 1 . 25×106 cells/ml in Schneider’s supplemented with 10% FBS and an additional 1 µg/ml bovine insulin .",
"DNA was transfected at a 4:1 ratio expression plasmid to reporter plasmid using effectene transfection reagent ( Qiagen ) following instructions for S2 cells .",
"Cells were induced with 500 µM CuSO4 36 hr after transfection .",
"Cells were lysed in passive lysis buffer ( Promega ) and assayed 36 hr after induction using a dual luciferase assay .",
"Firefly expression was measured in 75 mM HEPES pH 8 . 0 , 5 mM MgSO4 , 20 mM DTT , 100 µM EDTA , 530 µM ATP , 0 . 5 mM coenzyme A , and 0 . 5 mM D-luciferin .",
"Renilla expression was measured by addition of an equal volume of 25 mM Na4PPi , 10 mM NaOAc , 15 mM EDTA , 0 . 5 M Na2SO4 , 1 . 0 M NaCl , and 0 . 1 mM Coelenterazine .",
"All experiments were performed at least twice in triplicate .",
"Drosophila S2 cells expressing constitutively active Foxo were formaldehyde crosslinked .",
"Nuclei were isolated , lysed , and chromatin was sonicated to 500–1000 bp in length .",
"Chromatin was incubated with polyclonal sera against full length Foxo .",
"The chromatin/antibody mix was then incubated with protein A beads to isolate Foxo-bound chromatin from the sample .",
"Purified DNA was assayed by qPCR to determine enrichment for genomic sites bound by Foxo .",
"Enrichment is based on signal increase compared to a region of the genome in the first intron of CG15414 ( Supplementary file 1 ) .",
"The mouse insulin receptor and insulin-like growth factor one receptor 5′ UTRs were subcloned into the plasmid pGLRSVRF .",
"This plasmid contains the RSV LTR followed by the renilla luciferase open reading frame , the firefly luciferase open reading frame and the SV40 early polyadenylation signal .",
"The UTRs were cloned between the renilla and firefly open reading frames .",
"For transfection NIH3T3 cells were trypsinized and counted .",
"350 µl of cells at a concentration of 1 × 105 cells per milliliter were plated in each well of a 24-well plate .",
"Cells were transfected using Effectene ( Qiagen ) according to manufacturer’s instructions .",
"Cells were lysed in passive lysis buffer ( Promega ) and assayed 36 hr after induction using a dual luciferase assay ( Promega ) ."
]
] | [
"Under conditions of stress , such as limited growth factor signaling , translation is inhibited by the action of 4E-BP and PDCD4 .",
"These proteins , through inhibition of eIF4E and eIF4A , respectively , impair cap-dependent translation .",
"Under stress conditions FOXO transcription factors activate 4E-BP expression amplifying the repression .",
"Here we show that Drosophila FOXO binds the PDCD4 promoter and stimulates the transcription of PDCD4 in response to stress .",
"We have shown previously that the 5′ UTR of the Drosophila insulin-like receptor ( dINR ) supports cap-independent translation that is resistant to 4E-BP .",
"Using hippuristanol , an eIF4A inhibitor , we find that translation of dINR UTR containing transcripts are also resistant to eIF4A inhibition .",
"In addition , the murine insulin receptor and insulin-like growth factor receptor 5′ UTRs support cap-independent translation and have a similar resistance to hippuristanol .",
"This resistance to inhibition of eIF4E and eIF4A indicates a conserved strategy to allow translation of growth factor receptors under stress conditions ."
] | [
"Protein synthesis in eukaryotes occurs in two stages: transcription of DNA into messenger RNA ( mRNA ) in the nucleus , and then translation of that mRNA into a protein by ribosomes in the cytoplasm .",
"These processes are regulated by a complex network of signaling pathways that enables cells to tailor protein synthesis to match current conditions .",
"This involves regulating the expression of the genes that code for these proteins .",
"When cells experience stressful events , such as a shortage of oxygen or nutrients , they reduce the synthesis of most proteins .",
"This response is regulated , in part , by a signaling pathway known as the insulin and insulin-like receptor pathway .",
"In particular , stressful events inhibit a protein complex called eIF4F , which normally initiates the translation of mRNA molecules by binding to a structure on one end of the mRNA called the 5′ cap .",
"Despite this general inhibition , the production of certain other proteins—including the insulin receptor itself—is actually increased in response to stress .",
"Olson et al . have carried out a series of experiments to explore how inhibition of the eIF4F protein complex influences the translation of the mRNA for the insulin receptor .",
"The eIF4F complex is made up of three proteins , including one that binds to the 5′ cap and a helicase that unwinds the RNA .",
"Previous work in the fruit fly Drosophila showed that translation of this mRNA can continue even if formation of the eIF4F complex is inhibited by targeting the cap binding protein .",
"Olsen et al . now show that translation of this mRNA is also independent of the helicase .",
"Instead , translation is maintained under these conditions because the insulin receptor mRNA contains a sequence called an internal ribosome entry site , which allows ribosomes to bind to the mRNA without the influence of the 5′ cap .",
"Olson et al . reveal the details of this regulatory pathway in Drosophila and show that similar mechanisms are at work in mammalian cells , suggesting this pathway represents a crucial regulatory process that has been conserved during evolution .",
"A key question for future research is whether other genes within the insulin and insulin-receptor like signaling pathway use this same trick to evade translational inhibitors ."
] | 2013 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"neuroscience",
"cancer biology"
] | Autophagy linked FYVE (Alfy/WDFY3) is required for establishing neuronal connectivity in the mammalian brain | elife-14810-v1 | [
[
"The Autophagy linked FYVE domain protein ( Alfy ) [gene name , WD40 repeat and FYVE domain protein 3 ( Wdfy3 ) ] is a member of the Beige and Chediak-Higashi ( BEACH ) domain containing proteins , a family of proteins implicated in vesicle trafficking and membrane dynamics ( Isakson et al . , 2013; Simonsen et al . , 2004 ) .",
"As its name implies , Alfy has been implicated in the degradative pathway macroautophagy , by acting as a molecular scaffold between select cargo and core members of the mammalian autophagic machinery such as Atg5 , p62 and Atg8 homologs ( Clausen et al . , 2010; Filimonenko et al . , 2010; Lystad et al . , 2014; Simonsen et al . , 2004 ) .",
"In addition , its functional FYVE zinc finger domain at the COOH-terminus permits partial co-localization to phosphatidylinositol-3-monophosphate ( PtdIns3P ) , especially at autophagosome membranes ( Simonsen et al . , 2004 ) .",
"Wdfy3 is evolutionarily conserved and the most extensively studied homolog is Blue Cheese ( bchs ) in D . melanogaster ( Finley et al . , 2003 ) .",
"In the developing and adult fly central nervous system ( CNS ) , Bchs is abundantly expressed , with preferential accumulation in axon terminals and at the growth cone ( Finley et al . , 2003; Khodosh et al . , 2006 ) .",
"Adult bchs null flies have a shortened life span and show signs of adult onset neurodegeneration , including the accumulation of ubiquitinated aggregates ( Filimonenko et al . , 2010; Finley et al . , 2003; Khodosh et al . , 2006 ) .",
"Loss-of-function ( LoF ) mutations in bchs disrupt the axonal transport of endolysosomal vesicles ( Lim and Kraut , 2009 ) , however no defects in axon guidance have been reported in bchs null larva ( Khodosh et al . , 2006 ) .",
"Recently it has been reported that in vertebrates , genetically diminished levels of Alfy disrupts neurogenesis leading to altered forebrain morphology ( Orosco et al . , 2014 ) .",
"Furthermore , genetic screening has revealed a possible role for the human homolog WDFY3 as a genetic risk factor for intellectual and developmental disabilities ( IDD ) , microcephaly and neuropsychiatric disorders ( Bonnet et al . , 2010; Iossifov et al . , 2012; Kadir et al . , 2016 ) .",
"These findings raise the possibility that Alfy could have an important function in mammalian CNS development .",
"Here , we present two new mouse models that eliminate Alfy expression and identify an essential role for Alfy during murine development .",
"Constitutive elimination of Alfy leads to perinatal lethality , in conjunction with developmental brain wiring defects throughout the CNS , involving forebrain commissures , internal capsule , optic chiasm , spinal cord and longitudinal tracts such as the medial forebrain bundle .",
"In the ventral midbrain , dopaminergic cell populations retain an immature morphology and their axons aberrantly project into the hypothalamic region , forming an ectopic commissure near the optic chiasm .",
"Consistent with a failure of axon guidance mechanisms , localization of glial guidepost cells for callosal axons were disrupted , and sensitivity of Alfy knockout axons to the trophic effect of Netrin-1 was significantly diminished .",
"Moreover , Alfy is enriched in membrane fractions , suggesting that it may play a key role in membrane trafficking events to establish neural connectivity in the mammalian brain ."
],
[
"To characterize the role of Alfy in mouse , we initially determined when and where Alfy/Wdfy3 is expressed .",
"Multiplex , semi-quantitative RT-PCR revealed that Wdfy3 mRNA could be detected as early as embryonic day ( E ) 11 in CNS tissue , and remains detectable throughout gestation ( Figure 1A ) .",
"Similar analysis in adult tissue revealed that the Wdfy3 transcript is ubiquitously expressed , and that the highest concentration of Alfy was observed in the brain ( Figure 1—figure supplement 1 ) , confirming previous results ( Simonsen et al . , 2004 ) .",
"Wdfy3 transcript is detected throughout the both the perinatal and adult brain , as determined by in situ hybridization ( ISH ) ( Figure 1B and not shown ) .",
"Immunoblotting revealed that expression of the protein was present uniformly throughout the brain ( Figure 1C ) .",
"Using both primary neuronal and purified astroglial cultures , endogenous Alfy expression was detected in both cell types ( Figure 1—figure supplement 2 ) , supporting recent transcriptome analysis of the mouse cortex ( Zhang et al . , 2014 ) .",
"Therefore , we conclude that Alfy is a CNS-enriched protein that is present in various neuronal and non-neuronal cell types in the developing and adult brain . 10 . 7554/eLife . 14810 . 003Figure 1 . Alfy is highly expressed throughout the developing and adult mouse CNS .",
"( A ) ( Top ) RT-PCR demonstrates Alfy/Wdfy3 can be detected as early as embryonic day 11 ( E11 ) and remains abundant throughout gestation ( E19 ) .",
"( A ) Multiplex PCR for the 5’ region of the gene encoding Alfy , Wdfy3 with Class III β-tubulin ( βIII , top ) and Glial fibrillary acid protein ( Gfap , bottom ) .",
"( Btm )",
"Quantification of transcript levels relative to βIII ( black ) and Gfap ( gray ) , n = 4 , bars represent mean ± SEM .",
"( B ) In situ hybridization of sections from P0 mice reveals strong expression of Wdfy3 throughout the brain .",
"Low magnification images stitched together to reveal a complete sagittal brain section representative of Wdfy3 mRNA distribution in a wildtype mouse .",
"An abundance of mRNA is observed throughout the newborn CNS ( n = 4 ) , whereas no mRNA staining above background is observed in Alfy KO mice ( data not shown ) .",
"The ISH reflects how Alfy is most highly expressed in brain ( Figure 1—figure supplement 1 ) and this expression is both neuronal and glial ( Figure 1—figure supplement 2 ) .",
"Abbreviations: cerebellum ( cb ) , cerebral cortex ( ctx ) , hippocampus ( hp ) , hypothalamus ( ht ) , midbrain ( mb ) , olfactory bulb ( ob ) , striatum ( st ) , superior colliculus ( sc ) and thalamus ( th ) .",
"( C ) Immunblotting reveals that Alfy expression is maintained throughout the adult brain .",
"Lysates generated from different regions of the adult brain , including the brain stem ( bs ) were probed with an Alfy antibody raised against its N-terminus .",
"γ-tubulin is shown as a loading control ( n = 4 ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 00310 . 7554/eLife . 14810 . 004Figure 1—figure supplement 1 . Alfy/Wdfy3 expression is highest in the brain .",
"( A ) Semi-quantitative RT-PCR for Wdfy3 mRNA in different organs from adult mice .",
"Relative levels of Wdfy3 mRNA ( corrected for β-Actin ) across the different organs is quantified below ( n = 5 ) .",
"Bars represent mean ± SEM .",
"Wdfy3 RNA is most highly expressed in brain , although the transcript can be detected in all tissues examined .",
"( B ) Immunoblotting for Alfy from tissue samples collected from adult mice .",
"Relative protein levels were normalized using the cytoskeletal protein vinculin as shown below ( n = 6 ) .",
"Bars represent mean ± SEM .",
"In confirmation with previous results , Alfy protein is most abundant in brain . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 00410 . 7554/eLife . 14810 . 005Figure 1—figure supplement 2 . Alfy is expressed in neurons and astroglia .",
"( A ) Primary cortical cultures were grown for seven days with or without mitotic inhibitors .",
"Mitotic inhibitors were added to cultures after plating to prevent glial proliferation and generate cultures that are enriched for neurons .",
"After seven days , lysates were prepared from the cultures and western blots containing 5 µg of total protein were probed with antibodies against Alfy , the glial marker GFAP , and the neuronal specific tubulin , BIII .",
"As expected , mitotically inhibited cultures had a minimal GFAP expression but contained abundant BIII Tubulin .",
"Alfy was detected in mitotically inhibited cultures , implying neurons endogenously express Alfy .",
"The experiment was replicated twice , using two litters of mice ( Control n = 3; KO n = 3 ) .",
"( B ) Alfy was detected in lysates prepared from primary astroglia cultures that were passaged and allowed to differentiate for fourteen days .",
"Western blots containing 3 µg of total protein were probed with antibodies against Alfy and GFAP .",
"Results from two independent cultures are shown . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 005 To investigate the consequence of the genetic deletion of Wdfy3 in mice , we generated and characterized two different Alfy deficient mouse lines: One using gene trap ( GT ) -mediated disruption and a second using a conditional strategy ( Figure 2 , Figure 2—figure supplement 1 ) .",
"Whereas several GT lines disrupting the Wdfy3 locus were found , one that contained a GT insertion within the first intron was predicted to completely abolish production of the full length transcript .",
"To confirm , mice heterozygous for this mutation ( Alfy GT Het ) were interbred .",
"Homozygous mice ( Alfy GT ) were born at close to the expected Mendelian ratios ( WT: 19% , n = 20; Alfy GT Het: 52% , n = 54; Alfy GT: 28% , n= 29 ) .",
"Primer pairs spanning the transcript indicated that the full length Wdfy3 transcript is not produced in Alfy GT mice ( Figure 2—figure supplement 1B ) , and antibodies directed against the NH3- or COOH-terminus of Alfy confirmed that full length Alfy protein was not detectable in brain lysates ( not shown and Figure 2B ) .",
"This demonstrates that the GT insertion successfully mutagenized the Wdfy3 locus , leading to the loss of Alfy expression . 10 . 7554/eLife . 14810 . 006Figure 2 . Two lines of knockout ( KO ) mice reveal Alfy is essential for postnatal survival .",
"( A , B )",
"Alfy GT mice .",
"( A ) The GT cassette introduces a splice acceptor site ( SA ) and causes the premature termination of transcription through the introduction of a poly-adenylation ( pA ) tail .",
"The translation start site for Wdfy3 is located in exon IV .",
"RT-PCR indicates that the gene trap insertion leads to a loss of Wdfy3 transcript ( Figure 2—figure supplement 1A , B ) .",
"( B ) Western blot of brain lysates probed with an antibody against the COOH-terminus of Alfy and βIII ( n = 5 ) .",
"Abbreviations: long terminal repeats ( LTR ) , neomycin ( NEO ) , Phosphoglycerate kinase-1 promoter and Bruton tyrosine kinase splice donor site ( PGK-BTK-SD ) .",
"( C , D )",
"Alfy KO mice .",
"( C ) A conditional ( flox ) Wdfy3 allele is created by insertion of two loxP sites ( red triangles ) to flank exon 5 , leading to its excision upon exposure to Cre and the creation of the smaller knockout ( KO ) ‘Δ’ allele .",
"The two forward and one reverse primers ( arrows ) used for genotyping are noted ( also see Figure 2—figure supplement 1C ) .",
"( D ) Immunoblotting detects Alfy in brain lysates from heterozygous ( Alfy Het , n = 6 ) but not in Alfy KO mice ( n = 7 ) .",
"( E–G )",
"Characterization of newborn Alfy GT mice .",
"( E ) Newborn Alfy GT and littermatecontrol ( Ctrl ) mice .",
"Arrows highlight that control pups have stomachs full of milk whereas their GT littermates do not .",
"( F ) Alfy GT pups are consistently smaller than control .",
"After genotyping , the weights of the animals were percentile ranked and binned into four groups .",
"Approximately 50% of Alfy GT mice ( 8/15 ) had birth weights in the lowest 25th percentile , whereas heterozygous and wildtype littermates made up 92% ( 12/13 ) of mice with birth weights in the 75th percentile or greater .",
"Data were collected from 10 litters of mice , n = 55 .",
"Similar differences were observed between the Alfy KO pups and their heterozygous littermates ( data not shown ) .",
"( G ) Alfy GT pups lack a righting reflex .",
"The amount of time it took each pup to perform the task was averaged over three trials and recorded as the latency to right .",
"Alfy GT failed to rotate from back to their bellies within 60 sec .",
"A single factor ANOVA revealed genotype had a significant effect on pup behavior ( n = 6 WT , 9 Alfy GT Het , 7 Alfy GT; F ( 2 , 17 ) = 20 . 90 , p < 0 . 001 ) .",
"Fisher’s PLSD test indicated a significant difference between Alfy GT and WT ( p < 0 . 001 ) or heterozygous ( p < 0 . 001 ) littermates .",
"Bars represent mean ± SEM .",
"Similar differences were observed between Alfy KO pups and their heterozygous littermates ( Figure 2—figure supplement 1D ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 00610 . 7554/eLife . 14810 . 007Figure 2—figure supplement 1 . Creation and characterization of Alfy GT and Alfy KO mice .",
"( A , B )",
"Creation of Alfy GT mice .",
"( A ) PCR based genotyping of wildtype ( WT ) , Alfy GT heterozygotes ( Alfy GT Het ) and Alfy GT mice .",
"( B ) Semi-quantitative RT-PCR of Alfy GT mice .",
"Both the 5’ and 3’ portions of the Wdfy3 mRNA transcript are undetectable in Alfy GT mice by RT-PCR .",
"β-actin is shown as control .",
"n = 2 WT , 3 Alfy GT Het , 3 Alfy GT ) ( C ) PCR genotyping of Alfy KO mice .",
"PCR based genotyping of the homozygous and heterozygous conditional Wdfy3 allele ( Wdfy3loxP/+ and Wdfy3loxP/loxP , respectively ) and WT , Alfy KO heterozygous ( Alfy KO Het ) and Alfy KO mice .",
"( D ) Mice are born with an inherent ability to right themselves from a supine to prone position .",
"To determine if the loss of Alfy affected this behavior , marked and warmed pups were placed on their backs on a flat surface and given 60 s to rotate to a prone position .",
"The amount of time it took each pup to perform the task was averaged over three trials and recorded as the latency to right .",
"Alfy KO mice were not able to rotate from their backs to their bellies within 60 s , similar to Alfy GT mice ( Figure 2 ) .",
"A single factor ANOVA revealed genotype had a significant effect on pup behavior ( n = 4/genotype; F ( 1 , 6 ) = 43 . 14 , p < 0 . 001 ) .",
"Bars represent mean ± SEM . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 007 The second Alfy-deficient mouse line was produced using a conditional strategy ( Figure 2C ) .",
"Mice carrying a conditional Wdfy3 allele were crossed to HprtCre/+ females to generate a constitutive null '∆' allele ( Figure 2C , Figure 2—figure supplement 2C ) ( Tang et al . , 2002 ) .",
"Similar to Alfy heterozygous for the GT insertion , mice heterozygous for the '∆' allele ( Alfy Het ) were healthy and fertile .",
"Breeding of Wdfy3+/∆::HprtCre/+females with Wdfy3loxP/loxP::Hprt+/Y males generated mice heterozygous ( Alfy Het ) and homozygous ( Alfy KO ) for the null allele close to the expected Mendelian ratios ( Alfy Het: 56% , n = 40; Alfy KO: 44% , n = 32 ) .",
"Alfy protein also was not detected in Alfy KO mice ( Figure 2D ) .",
"Although neonatal Alfy mutant pups appeared grossly normal , were reactive , and showed no sign of cyanosis , they all died several hours after birth .",
"Unlike their littermates , Alfy mutant pups failed to thrive and consistently showed an absence of milk in their stomachs ( Figure 2E ) .",
"Despite being properly cleaned , live Alfy mutant pups were frequently the smallest pups within the litter ( Figure 2F ) and were selectively buried under nesting material , indicating that the dams were rejecting them due to diminished or uncoordinated movement ( Turgeon and Meloche , 2009 ) .",
"To test this hypothesis , we performed a righting reflex test ( Figure 2G , Figure 2—figure supplement 1D , Video 1 ) .",
"Littermates with at least one copy of Alfy were able to right themselves within the allotted test time .",
"In contrast , mice lacking Alfy consistently failed at this test , and demonstrated uncoordinated behavior . 10 . 7554/eLife . 14810 . 008Video 1 . Mice lacking Alfy have an abnormal righting reflex . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 008 Histological examination of the newborn ( P0 ) brain by Nissl staining revealed that brains from Alfy GT and Alfy KO both demonstrated striking abnormalities in the forebrain , midbrain and hindbrain , including visibly smaller brains and gross enlargement of the lateral ventricles ( Figure 3A , B and Figure 3—figure supplement 1A ) .",
"Concurrently , there was an apparent loss and disorganization of interhemispheric axonal tracts throughout the brain .",
"Brains of Alfy GT Het or Alfy Het mice were indistinguishable from wildtype brains ( Figure 3A , B ) . 10 . 7554/eLife . 14810 . 009Figure 3 . Commissures fail to cross the midline appropriately in Alfy GT and KO brains .",
"( A , B )",
"Nissl stained forebrain from newborn ( P0 ) control or Alfy mutant ( Alfy GT or Alfy KO ) mice .",
"Coronal sections are shown .",
"Alfy mutants have many abnormal features , including enlarged ventricles ( * ) , and apparent white matter abnormalities , including absence of a corpus callosum ( cc ) at the midline ( black arrow ) , undetectable anterior commissure ( ac , white arrow ) , and dysmorphic internal capsule ( rectangle ) .",
"More caudal sections can be found in Figure 3—figure supplement 1A .",
"n = 6 WT , 3 Alfy GT Het , 7 Alfy GT; n = 3 Alfy Het , 3 Alfy KO .",
"( C , D )",
"Immunostaining highlights axonal abnormalities in Alfy mutant mice .",
"Coronal sections are shown .",
"The three major forebrain commissures fail to cross the midline in Alfy mutant brains .",
"( C ) The cc , hippocampal commissure ( hc ) and ( D ) ac are absent in Alfy mutants .",
"‘*’ denotes midline and lack of axonal connectivity .",
"Boxed regions are shown enlarged below .",
"n = 5/genotype .",
"The habenular and posterior commissures can be found in Figure 3—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 00910 . 7554/eLife . 14810 . 010Figure 3—figure supplement 1 . Alfy mutant mice have aberrant and disorganized axon projections .",
"( A ) H&E staining of neonatal brains reveal altered brain morphology in Alfy GT mice .",
"Coronal sections are shown .",
"In the absence of Alfy , the cerebral cortex ( ctx ) , thalamus ( th ) and hippocampus ( hp ) are dysmorphic .",
"Additional abnormalities in the Alfy null mice include a reduced size of the thalamus .",
"Alfy heterozygotes are indistinguishable from WT littermates ( data not shown ) .",
"Wildtype mice are shown as control ( Ctrl ) .",
"n = 3/genotype .",
"Scale bar = 250 μm .",
"( B ) NF staining reveals aberrant and disorganized axonal projections of Alfy mutant brains .",
"Coronal sections are shown .",
"Alfy GT mice show reduced staining in the ctx and abnormal NF staining in the internal capsule ( ic ) and tracts within the hp .",
"The fasciculus retroflexus ( fr , square box ) , mammalothalamic tract ( mt , circle ) and cerebral peduncle ( arrow ) are evident in the Ctrl but present as aberrant , disorganized axonal tracts in Alfy GT ( dashed box ) .",
"The external capsule ( arrowhead ) is present in Ctrl and Alfy null brains .",
"n = 5/genotype .",
"Additional abbreviations: hypothalamus ( ht ) , periaqueductal gray ( pag ) , striatum ( st ) , and superior colliculus ( sc ) .",
"DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 01010 . 7554/eLife . 14810 . 011Figure 3—figure supplement 2 . Aberrant and disorganized projections of the habenular and posterior commisural axons .",
"( A ) Low-power magnification of Ctrl and Alfy GT coronal sections are presented in the top panels .",
"The arrow points to the habenular commissure ( hac ) , which is presented at higher magnification below .",
"( B ) The posterior commissure ( pc ) .",
"Both appeared morphologically abnormal in Alfy GT mice , although both crossed the midline .",
"n = 3/genotype .",
"Scale bar as indicated . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 011 To examine the axonal defects further , P0 Alfy mutant brains were stained for neurofilament ( NF ) ( Figure 3C , D and Figure 3—figure supplement 2 ) .",
"When compared to control , NF labeling of Alfy null brains was greatly reduced , suggesting fewer axonal tracts .",
"A notable abnormality was the failure of the three major forebrain commissures to cross the midline .",
"Axons of the corpus callosum , hippocampal commissure ( Figure 3C ) , and anterior commissure ( Figure 3D ) failed to decussate , leading to a separation of the two hemispheres .",
"Two smaller , caudal commissures , the habenular and posterior commissures ( Figure 3—figure supplement 2 ) , were morphologically abnormal and reduced in size however a portion of their tracts crossed the midline .",
"We next examined the retinal axon decussation at the optic chiasm by anterograde-labeling with DiI ( Figure 4A–C ) .",
"Ipsilateral and contralateral projections were quantified by calculating the ipsilateral index ( Kuwajima et al . , 2012 ) .",
"At P0 , the ipsilateral projection in Alfy GT mice ( 1 . 40 ± 0 . 06 ) was 40% larger than that in wildtype ( 1 . 0 ± 0 . 08 ) or heterozygous mice ( 1 . 1 ± 0 . 06 ) ( Figure 4C ) .",
"This significant difference suggests that in Alfy mutants , contralateral axons less readily cross the optic chiasm midline .",
"These data reinforce the findings that Alfy is required to properly establish commissures throughout the developing mouse brain . 10 . 7554/eLife . 14810 . 012Figure 4 . Midline crossing defects extend to the optic chiasm in Alfy GT and KO brains , possibly due to disrupted localization of guidepost glial cells .",
"( A–C )",
"Retinal decussation defects are observed in Alfy mutant mice .",
"( A ) Whole mounts of P0 optic chiasm are unilaterally labeled with DiI at the optic disc .",
"The ipsilateral projection denoted in the white dashed box , which is shown enlarged below .",
"WT and GT heterozygous littermates were indistinguishable .",
"A heterozygous mouse is shown as a control ( Ctrl ) .",
"( B ) Schematic representation of how the ipsilateral index is calculated .",
"Pixel intensities of contralateral and ipsilateral optic tracts are measured within 500 x 500 µm2 .",
"( C ) The ipsilateral index of Alfy mutant mice is greater than both WT and heterozygous ( GT Het ) littermate controls , indicating the abnormally enlarged ipsilateral projection .",
"Bars represent mean ± SEM , and statistical analysis was determined using one-way ANOVA followed by the Tukey’s post hoc test .",
"Respective n-values per genotype are noted in the bars .",
"*p < 0 . 05 .",
"( D , E )",
"Immunostaining for GFAP in P0 forebrain reveals mislocalization of guidepost glial cells .",
"Coronal sections are shown .",
"( D ) The glial wedge ( gw ) , the indusium griseum ( ig ) and midline zipper ( mz ) glia populations of glial cells were in the expected locations in WT littermate brains ( Ctrl ) .",
"In Alfy GT brains , the ig was not detectable ( expected location denoted by '*' ) , and the gw and mz cells were disorganized .",
"n = 3/genotype .",
"( E ) Glial populations within the fimbria ( fi ) and dentate gyrus ( dg ) of the hippocampus were apparent in both genotypes .",
"Boxed areas are shown at higher magnification below .",
"n = 3/genotype .",
"The examination of other potential intrinsic alterations that may contribute to forebrain axonal connectivity can be found in Figure 4—figure supplements 1 and 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 01210 . 7554/eLife . 14810 . 013Figure 4—figure supplement 1 . The loss of Alfy leads to modest changes in proliferation but not cell death in the neocortex .",
"( A ) Loss of Alfy leads to a moderate but significant decrease in cortical plate thickness .",
"Measurements of P0 cortex reveal that the cortical plate/subplate ( CP/SP ) of Alfy GT brains ( n = 3 ) are modestly but significantly thinner than control ( Ctrl , n = 3 ) brains , ( t ( 4 ) = 5 . 30 , p < 0 . 01 ) .",
"The marginal zone ( MZ ) and intermediate zone ( IZ ) do not reach significance , ( t ( 4 ) = 1 . 67 , p = 0 . 17 and t ( 4 ) = 2 . 38 , p = 0 . 076 , respectively ) .",
"Bars denote mean ± St . Dev; * , p < 0 . 01 .",
"( B , C )",
"Loss of Alfy leads to a subtle defect in cell proliferation at E15 . 5 but not at E13 . 5 .",
"A single injection of BrdU was administered to time-pregnant dams at E13 . 5 or E15 . 5 and embryos were collected 1 hr post-injection .",
"( B ) A clear band of BrdU-positive cells ( red ) was detected in the subventricular zone and deep cortical layer in all three littermate genotypes ( WT: n = 2 , heterozygous: n = 3 , and Alfy GT n = 3; two litters ) .",
"Hoechst 33342 nuclear DNA stain is in blue .",
"( C ) Colorimetric BrdU was used to quantify the number of proliferative cells in the developing cortical plate .",
"At E13 . 5 , Quantification revealed no significant difference in the number of BrdU positive ( BrdU+ ) cells between Alfy KO and heterozygous littermate control ( Ctrl ) t ( 8 ) = 1 . 59 , p = 0 . 15 ( Ctrl: n = 5 , Alfy KO: n = 5; two litters ) .",
"At E15 . 5 , there was a modest , but significant reduction in the absence of Alfy , t ( 8 ) = 3 . 12 , p < 0 . 01 ( Ctrl: n = 4 , Alfy KO: n = 6; two litters ) .",
"Bars denote mean ± SEM .",
"ns , not significant; * , p<0 . 01 .",
"( D ) Proliferation , indicated by Ki67-positivity indicates the normal proliferation in newborn Alfy GT mice .",
"Boxed regions highlight the sub-ventricular zone ( VZ ) and are shown at higher magnification in images below .",
"Stereology was performed to quantify the number of Ki67 positive cells in the sub-VZ ( Gundersen Coeffecient of Error = 0 . 15 , Ctrl ( wildtype littermate , n = 3 ) = 187 , 221 cells , S . D . = 19 , 634 compared to Alfy GT ( n = 3 ) = 179 , 074 cells , S . D . = 28 , 917 , t ( 4 ) = 0 . 40 , p = 0 . 71 . ) ( E ) Immunohistochemistry ( brown ) against cleaved caspase-3 reveals no significant difference in cell death at embryonic age E15 . 5 in the absence of Alfy .",
"The enlarged images of the inset for the images are provided immediately below .",
"No cell loss was detected in cortex at this age .",
"As a positive control , the naturally occurring cell death observed in the trigeminal ganglia at this age is included ( leftmost panels ) .",
"No difference was observed across genotype ( n = 2 WT , 2 Alfy GT Het; 3 Alfy GT ) .",
"Tissue sections are counterstained with Nissl , which is shown in purple . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 01310 . 7554/eLife . 14810 . 014Figure 4—figure supplement 2 . Focal cortical dysplasias are observed in Alfy GT brains .",
"( A ) No gross difference in Doublecortin ( DCX , green ) staining can be observed in E15 . 5 Alfy GT brains ( n = 3 ) compared with littermate controls ( Ctrl , n = 3 ) .",
"( B ) Specification of deep layer , Tbr1-positive cortical projection neurons are observed in Alfy GT mice when compared to littermate controls ( Ctrl ) .",
"Three mice per genotype were stained Tbr1 ( Top , red ) and counterstained with Hoeschst 33342 ( bottom , blue ) , and all mice demonstrated this pattern .",
"( C–E )",
"Focal cortical dysplasia ( FCD ) in Alfy null brains .",
"H&E staining reveals the presence of FCD in coronal ( C ) and sagittal ( D ) sections of Alfy GT brains .",
"Boxed areas are shown at higher magnification below .",
"FCDs are labeled with arrows .",
"( E ) FCDs can be detected by Nissl staining as early as E15 in Alfy GT mice ( 6/6 ) but not in control embryos ( 0/6 ) .",
"Arrowheads point to multiple FCDs located within the same hemisphere of an Alfy GT embryo . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 014 Two cell populations that help axons navigate the developing forebrain during the genesis of the corpus callosum are the glial wedge ( gw ) and indusium griseum ( ig ) glia ( Shu and Richards , 2001; Shu et al . , 2003 ) .",
"In light of the presence of Alfy in astroglial cells , as well as the midline crossing defects , we hypothesized that migration abnormalities could contribute to the connectivity defects in Alfy GT and KO mice .",
"Thus , we next examined the callosal guidepost cells , a key population implicated in providing intermediate guidance cues to developing cortical axons .",
"Immunostaining against GFAP on P0 mouse brains revealed abnormalities in Alfy GT brains ( Figure 4D , E ) .",
"Unlike control brain , the ig cells were undetectable and the gw appeared disorganized and loosely packed .",
"The midline zipper glia ( mz ) , cells proposed to promote fusion of the two telencephalic hemispheres ( Silver , 1993 ) , were present but distributed abnormally ( Figure 4D ) , whereas cells in the fimbria and dentate gyrus of the hippocampus appear largely normal ( Figure 4E ) .",
"Taken together , the disorganization of the ig , gw and mz is consistent with the agenesis of the corpus callosum observed in the absence of Alfy .",
"These findings suggest that cells that act as important guideposts for decussating callosal axons ( Shu and Richards , 2001 ) fail to migrate properly in the absence of Alfy , contributing to the connectivity defects observed .",
"In addition to the localization of the midline glial cells , we also determined if intrinsic alterations in the proliferation or differentiation of cortical projection neurons were also contributing to the disruptions in forebrain axonal connectivity .",
"Nissl staining revealed that cortical layering appeared normal , however there was a significant but modest thinning of the cortical plate/subplate at P0 ( Figure 4—figure supplement 1A ) .",
"Although this may be due to the diminished NF staining observed in Figure 3 , we next determined if cell proliferation was affected .",
"Pulse-labeling with bromo-6-deoxy-uridine ( BrdU ) at E15 . 5 revealed cell division in the developing cortex at the expected location in Alfy GT mice ( Figure 4—figure supplement 1B ) .",
"To determine if the amount of proliferation was impacted by the loss of Alfy , stereology was performed on BrdU labeled embryos at ages E13 . 5 and E15 . 5 ( Figure 4—figure supplement 1C ) .",
"No significant difference across genotypes was detected at E13 . 5 , but a modest yet significant reduction in the number of BrdU-positive cells was detected in Alfy KO embryos at E15 . 5 .",
"This difference appeared limited to the cortex , since staining against Ki67 within the subventricular zone at P0 revealed no significant difference between Alfy null mice and controls ( Figure 4—figure supplement 1D ) .",
"Further , despite this modest decrease in embryonic proliferation , there were no detectable differences across genotypes in the amount of cell death in E15 . 5 brains , as indicated by immunohistochemistry against cleaved caspase-3 ( Figure 4—figure supplement 1E ) .",
"We next sought to determine if the loss of Alfy disrupted proliferation in the embryonic cerebral cortex .",
"Although qualitative immunofluorescence against expression markers for immature migrating neurons ( DCX ) and for early born layer VI neurons ( Tbr1 ) also were not significantly altered in the absence of Alfy , histological staining revealed focal cortical malformations known as focal cortical dysplasias ( FCD ) present throughout the cerebral cortex of Alfy KO mice , as described previously in a Wdfy3 hypomorph ( Figure 4—figure supplement 2C–E ) ( Orosco et al . , 2014 ) .",
"These structures were observed as early as E15 . 5 .",
"Thus , the loss of Alfy leads to subtle defects in proliferation , but no changes in cell death or specification in the developing cortical projection neurons .",
"Although columnar organization of discrete regions of the cerebral cortex can be disrupted by the absence of Alfy during embryonic development , it is unlikely that these defects can account for the midline crossing defects observed in Alfy null mice .",
"In addition to the absence of interhemispheric commissures , another striking feature revealed by NF staining in Alfy GT and KO brains was the evident disorganization and apparent loss of other CNS axon tracts .",
"One particularly prominent defect was found in the organization of the internal capsule , which carries the corticospinal tract , and corticothalamic and thalamocortical projections; the internal capsule of Alfy GT brains was disorganized and followed an unusual trajectory through the striatum ( Figure 3 and Figure 3—figure supplement 1 ) .",
"To gain a temporal perspective on the development of the internal capsule , we examined its development during E15 . 5 ( Figure 5A ) and E17 . 5 ( Figure 5B ) .",
"NF staining revealed the expected fasciculated axonal projections of the developing internal capsule en route to and from the cerebral cortex in controls .",
"In contrast , the thalamocortical projections in Alfy GT mice formed ventrally displaced bundles , with fewer projections observed traveling through the ganglionic eminence .",
"Similarly , Alfy GT embryonic brain sections labeled with the axonal marker NCAML1 also highlighted abnormal bundle-like structures in the ventral diencephalic-telencephalic border ( Figure 5—figure supplement 1 ) 10 . 7554/eLife . 14810 . 015Figure 5 . Axonal tracts develop abnormally in the Alfy mutant brain and spinal cord .",
"( A , B )",
"Coronal sections of diencephalon at ( A ) E15 . 5 ( n = 3/genotype ) and ( B ) E17 . 5 ( n = 3/genotype ) stained for neurofilament ( NF , red ) and counter-stained with the nuclear stain Hoechst 33342 ( blue ) .",
"The developing internal capsule is highlighted with a yellow box ( top ) and higher magnification , confocal images of NF staining are shown with ( middle ) and without ( bottom ) Hoechst 33342 .",
"In control brains ( Ctrl ) , axons that comprise the internal capsule form a fan-shaped projection of bundled axons within the ganglionic eminence .",
"The abnormal , ventrally displaced , knot-like bundles of axons found in Alfy GT are marked with white arrows .",
"Neural cell adhesion molecular L1 ( NCAML1 ) staining reveals similar defects ( Figure 5—figure supplement 1 ) .",
"( C , D )",
"TH staining reveals abnormal projections in DAergic cell populations .",
"( C ) Coronal sections .",
"A9 and A10 DAergic populations are present in Alfy GT brains appear immature ( top ) .",
"The hypothalamic A13 DAergic cell population and medium forebrain bundle ( mfb ) are present in the diencephalon in Alfy GT brains; however aberrant projections into the hypothalamus and abnormal midline-crossing events are observed ( arrows , bottom ) .",
"n = 3/genotype .",
"( D ) Sagittal sections counterstained with Nissl .",
"In Alfy GT brains , the mfb has ectopic ventral projections into the hypothalamus ( arrows , bottom ) .",
"Boxed regions are shown at higher magnification below .",
"n = 3/genotype .",
"( E ) NF staining of E13 . 5 cervical spinal cord .",
"Coronal sections are shown .",
"Higher magnification images of the dorsal spinal cord are shown below .",
"The abnormal NF patterning observed in Alfy GT embryos is highlighted with white arrows .",
"n = 3/genotype . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 01510 . 7554/eLife . 14810 . 016Figure 5—figure supplement 1 . NCAML1 staining confirms axonal defects of the internal capsule in the developing Alfy GT brain . Immunofluorescence against NCAML1 ( red ) in E15 . 5 brains counterstained with Hoechst 33342 ( blue ) ( top ) .",
"Boxed areas are shown at higher magnification with and without the counterstain ( bottom ) .",
"Arrow indicates abnormal bundle-like structures formed by the projections in the Alfy GT brain .",
"n = 4 littermate controls , 4 Alfy GT . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 016 We next examined the ventral midbrain ( vMB ) dopaminergic ( DAergic ) neurons and their projections to the striatum ( Figure 5C , D ) .",
"Tyrosine hydroxylase ( TH ) staining revealed that the projections and morphology of vMB DAergic neurons were clearly abnormal in the P0 Alfy KO brains .",
"Notably , the A9 and A10 cell populations , which represent the ventral tegmental area ( A9 ) and substantia nigra ( A10 ) , revealed that the morphology of the regions had an immature appearance relative to control littermates at the same age ( Figure 5C ) .",
"Moreover , rather than follow their typical trajectory through the medium forebrain bundle to striatal targets in the forebrain , in Alfy KO brain , ectopic TH fibers were observed projecting ventrolaterally into the hypothalamus , near the location of the supraoptic decussation ( Figure 5C , D ) .",
"Axonal abnormalities were not limited to the developing brain but were also detected in the developing spinal cord of mice lacking Alfy .",
"At E13 . 5 , disorganized projections into gray matter and the loss of axon bundles were evident in the cervical , thoracic and lumbar levels of the spinal cord in the absence of Alfy , but not in control littermates ( Figure 5E ) .",
"In summary , these data indicate that Alfy is essential for establishing axonal tracts throughout the CNS , from forebrain commissures to the spinal cord .",
"Using two independent mouse models , we find that the loss of Alfy leads to erroneous pathfinding throughout the CNS .",
"Several of these findings are suggestive that the anatomical phenotype observed in Alfy mutant mice may in part be due to defects in axon guidance .",
"For example , an abnormal dopaminergic commissure at the level of the diencephalon traveling through the hypothalamus from the MFB has been previously reported in the Slit1/Slit2 double KO ( Bagri et al . , 2002 ) , the Deleted in colorectal cancer ( Dcc ) KO ( Xu et al . , 2010 ) , and the NK2 Homeobox 1 ( Nkx2 . 1 ) KO ( Kawano et al . , 2003 ) mice .",
"Moreover , diminished guidance cue responsiveness could also account for the failure of the midline astroglia to migrate properly .",
"We therefore hypothesized that Alfy may be involved in the ability of neural cells to respond to guidance cues .",
"Prior to determining if Alfy is involved in axon guidance , we first determined where Alfy is expressed .",
"To do so , we reintroduced full length Alfy into Alfy null primary dissociated cultures to visualize its localization ( Figure 6A , Figure 6—figure supplement 1 ) .",
"Full length Alfy constructs with an N-terminus tag were transfected into DIV7 dissociated primary cortical cultures .",
"Alfy localized throughout the neuron , including within the axon .",
"Neighboring astroglia also showed a diffuse localization of Alfy ( Figure 6—figure supplement 1B ) .",
"Next , we performed subcellular fractionation of adult cortical lysates ( Figure 6B , C , Figure 6—figure supplement",
"2 ) ( Hallett et al . , 2008 ) .",
"Fractionation revealed that Alfy enriched in the light membrane fraction ( P3 ) along with synaptosomal membrane protein synaptophysin and a classic marker for autophagic vesicles , the membrane bound form of microtubule associated protein light chain 3 ( LC3-II ) ( Kabeya et al . , 2000 ) ( Figure 6B , C ) .",
"Additionally , Alfy was enriched in the crude synaptic vesicle fraction ( LP1 ) .",
"Taken together , these results suggest that Alfy may be involved in regulating the trafficking , sorting or signaling events that are required for brain wiring . 10 . 7554/eLife . 14810 . 017Figure 6 . Alfy localizes to axons and enriches to membrane fractions .",
"( A ) Immunofluorescence images showing MAP2-positive neurons expressing Alfy .",
"Merged color image demonstrates co-localization of mCherry-Alfy within a MAP2-positive neuron .",
"Alfy is found within the soma and co-localizes with MAP2 positive projections .",
"Transfections were replicated three times across three independent cultures .",
"Colocalization to βIII Tubulin projections is shown in Figure 6—figure supplement 1 .",
"( B , C )",
"Fractionation of adult cortical lysates reveals that Alfy enriches into membrane fractions .",
"( B ) Equal amounts of protein per fraction were analyzed by immunoblotting .",
"Alfy was present in membrane fractions that also enriched with LC3-II and synaptophysin ( P3 , LP1; boxes .",
"( C ) The fold enrichment measured relative to the total homogenate fraction ( H ) .",
"Bars represent mean enrichment ( n = 3 ) ± SEM .",
"A schematic depiction of the fractionation can be found in Figure 6—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 01710 . 7554/eLife . 14810 . 018Figure 6—figure supplement 1 . Alfy is expressed in the soma and axons of neurons .",
"( A , B )",
"Alfy control mixed primary cortical cultures were transfected with plasmids containing full-length or truncated tagged-Alfy cDNA .",
"( A ) Representative confocal images are shown of Alfy-tagged constructs detected with antibodies directed against the fluorophore tag and co-stained MAP2 or BIII tubulin .",
"Full-length and C-terminal fragments of Alfy are detected throughout MAP2- and BIII tubulin-positive projections ( arrows ) .",
"( B ) Representative confocal images are shown of Alfy-tagged constructs detected with antibodies directed against the fluorophore tag ( left ) and co-stained with GFAP .",
"Alfy are expressed throughout astroglia , although large vesicles and the nucleus are devoid of staining .",
"Studies were replicated across three independent cultures .",
"( C , D )",
"To determine the sub-cellular distribution of Alfy , Alfy KO or Ctrl ( A , B ) mixed primary cortical cultures were transfected with plasmids containing full-length tagged-Alfy .",
"To confirm transfection , COS-7 cells were transfected in parallel and immunoblotted .",
"Vinculin is shown as a loading control .",
"( C ) Full-length Alfy-tagged proteins migrate at the expected size and are detected with anti-mCherry and anti-Alfy antibodies .",
"Over-exposed images are necessary to show endogenous Alfy , since it is at a much lower abundance in non-neuronal cells ( circle ) .",
"( D ) Full length and truncated constructs of Alfy .",
"Studies were replicated across three cultures . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 01810 . 7554/eLife . 14810 . 019Figure 6—figure supplement 2 . Alfy enriches in membrane fractions from brain . A schematic depiction of the modified synaptosome preparation used for Figure 6A , based on a previously published protocol ( Hallett et al . , 2008 ) .",
"Abbreviations: H , homogenate; P1 , nuclei and large debris; S1 , soluble cytoplasmic; P2 , crude synaptosomal membranes; S2 , soluble cytoplasmic; P3 , light membrane; S3 , soluble cytoplasmic; LP1 , enriched synaptosomal membranes; LS1 , synaptic vesicles and synaptic proteins . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 019 To test this hypothesis , we next explored whether Alfy is required for the outgrowth of axons .",
"Dissociated cultures from P0 cortices were plated and examined for morphological differences at day in vitro 7 ( DIV7 ) ( Figure 7A , B; Figure 7—figure supplement 1A–C ) .",
"Alfy KO neurons displayed healthy growth in vitro and Scholl analysis confirmed that they were morphologically similar to control ( Figure 7B ) .",
"Staining also revealed that the neuronal cytoskeletal markers Tau-1 , βIII Tubulin and microtubule associated protein 2 ( MAP2 ) were expressed comparably across genotypes ( data not shown ) .",
"Taken together , these data indicate Alfy is not required for processes controlling non-directed neuronal outgrowth in a cell autonomous manner . 10 . 7554/eLife . 14810 . 020Figure 7 . Alfy is required for the ability of axons to respond to Netrin-1 . ( A , B ) Primary cortical neurons are morphologically similar across genotype .",
"( A ) Individual , GFP-transfected neurons were imaged by confocal microscopy and traced in ImageJ .",
"Representative maximum projection images of two heterozygous ( Het , n=5 brains ) and two Alfy KO neurons ( n = 5 brains ) are shown .",
"Neuronal cytoskeletal markers are expressed comparably across genotype ( Figure 7—figure supplement 1A , B ) .",
"( B ) Scholl analysis of neurons at DIV7 as represented in A . One-way ANOVA revealed no effect of genotype on the number of crossings ( n = 50 neurons/genotype; F ( 1 , 97 ) = 0 . 66 , p = 0 . 41 ) .",
"Analysis of branching ( Figure 7—figure supplement 1C ) similarly showed no effect of genotype .",
"( C , D )",
"Alfy KO cortical explants have attenuated responsiveness to Netrin-1 .",
"( C ) Representative images of Alfy Het or Alfy KO cortical explants in the presence of control ( Ctrl ) or Netrin-1 as described ( Figure 7—figure supplement",
"2 ) and stained with βIII Tubulin .",
"( D ) Quantification of the average length of outgrowth after 72 hr in culture on data collected from 54 explants ( Alfy Het treated with Ctrl: n = 15; Alfy Het treated with Netrin-1: n = 12; Alfy KO treated with Ctrl = 12 , Alfy KO treated with Netrin-1: n = 15 ) , from two litters of mice .",
"A two-way ANOVA revealed a significant effect of genotype ( F ( 1 , 50 ) = 7 . 69 , p < 0 . 01 ) and the presence of Netrin-1 ( F ( 1 , 50 ) = 7 . 71 , p < 0 . 01 ) on outgrowth , as well as a significant interaction between genotype and Netrin-1 exposure ( F ( 1 , 50 ) = 12 . 29 , p < 0 . 001 ) .",
"Fisher PLSD post hoc analysis revealed that exposure to Netrin-1 resulted in a significant increase in outgrowth in Alfy Hets ( p < 0 . 001 ) , but not in Alfy KO ( p = 0 . 56 ) .",
"Under control conditions , there is no difference in the amount of outgrowth between the genotypes ( p = 0 . 56 ) .",
"Netrin-1 attractive guidance was also assessed by determining the Guidance Ratio , which is achieved by measuring the amount of outgrowth from the side of the explant closest to the cue-expressing cell block ( proximal ) over the amount of growth on the side of the explant furthest from the cell block ( distal ) .",
"Under the conditions of our assay , directional growth in response to Netrin-1 was not observed in the control explants ( t ( 25 ) = 1 . 98 , p = 0 . 059 ) or KO explants ( t ( 22 ) = 1 . 77 , p = 0 . 091 ) , suggesting that under the conditions of our assay HEK293T cells secrete Netrin-1 above the threshold for selectively promoting attractive guidance .",
"Similar results were achieved after 48 hr in culture .",
"Bars represent mean ± SEM .",
"* , p < 0 . 001; ns , not significant . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 02010 . 7554/eLife . 14810 . 021Figure 7—figure supplement 1 . Primary cortical neurons are similar across genotype .",
"( A , B )",
"The loss of Alfy does not affect the growth and differentiation of dissociated neurons in culture .",
"Day in vitro 7 ( DIV7 ) neurons from Ctrl and Alfy KO mice express cytoskeletal markers ( A ) Tau-1 ( red ) or ( B ) MAP2 ( red ) and βIII Tubulin ( βIII , green ) positive processes .",
"Merged images are shown in the top panel , whereas individual channels for each fluorophore are shown in grayscale images below .",
"Representative images shown from three independent cultures generated from Alfy Het or Alfy KO brains .",
"( C ) The loss of Alfy had no effect on neurite outgrowth in dissociated cortical neurons .",
"A one-way ANOVA reveals no significant difference across genotype ( F ( 1 , 97 ) = 0 . 19 , p = 0 . 66 ) .",
"Data represented as mean ± St . Dev .",
"Cortical cultures were prepared from five independent cultures with 10 neurons/culture/genotype imaged , resulting in 50 Alfy Het and 50 Alfy KO neurons analyzed . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 02110 . 7554/eLife . 14810 . 022Figure 7—figure supplement 2 . Responsiveness to guidance cues is attenuated in Alfy KO axons .",
"( A , B )",
"Cortical explant culture to examine axon outgrowth in response to Netrin-1 as described in Figure 7C , D .",
"( A ) Diagram of an E14 . 5 embryo with developing cerebral cortex highlighted in red .",
"Cortical explants were co-cultured with agarose-embedded HEK293T cells that were mock transfected ( Control ) or transfected with Netrin-1-Myc ( Netrin-1 ) .",
"Transiently transfected HEK293T cells robustly secrete Netrin-1-MYC into the culture media as revealed by immunoblotting for the MYC tag .",
"Probing for the cytoskeletal protein Vinculin demonstrates that the culture media fraction is predominately cell-free .",
"( B ) Low-power images , stitched together to show the entire amount of outgrowth after 72 hrs in culture , from cortical explants stained with βIII Tubulin ( red ) in the presence of control or Netrin-1 expressing HEK293T cells ( location of HEK293T cells denoted by white arrow ) .",
"( C ) The loss of Alfy does not affect guidance cue receptor levels in the telencephalon .",
"Lysates prepared from E14 . 5 telencephalon were probed on a Western blot with antibodies directed against Alfy , Robo1 , DCC .",
"The amount of DCC and Robo1 in Alfy KO telencephalon was equivalent to the abundance of the receptors detected in Alfy control samples ( DCC: t ( 5 ) = 0 . 45 , p = 0 . 67; Robo1: t ( 4 ) = 0 . 13 , p = 0 . 90 ) .",
"-DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 022 We next examined if Alfy is essential for neurons to respond appropriately to trophic agents , such as the bi-directional guidance cue Netrin-1 ( reviewed in [Leyva-Diaz and Lopez-Bendito , 2013] ) .",
"Explants from control and Alfy KO mice were co-cultured with mock transfected or Netrin-1-Myc transfected HEK293T cells embedded in agarose ( Figure 7—figure supplement 2A ) .",
"After 48 hr , the distance of growth outward from cortical explants was measured ( Figure 7C , D , Figure 7—figure supplement 2B ) .",
"In the presence of Netrin-1 , control explants demonstrate significantly increased growth whereas Alfy KO explants did not respond , despite maintaining their ability to extend their processes under non-stimulated conditions ( Figure 7D ) .",
"Under the conditions used , directional responsiveness could not be ascertained ( Figure 7D ) , likely due to the abundance of Netrin-1 expression due to the use of HEK293T cells ( Shirasaki et al . , 1996 ) .",
"These data indicate that Alfy is required for cortical neurons to respond to the trophic effects of Netrin-1 .",
"The loss of Alfy did not affect the total levels of the guidance cue receptor DCC ( Figure 7—figure supplement 2C ) .",
"Therefore , our data suggest that Alfy function could involve the regulation of intracellular membrane-based signaling events in response to changes in the extracellular milieu of the developing brain .",
"We and others previously found using stable cell lines that Alfy was an adaptor protein for the degradation of aggregated proteins by selective macroautophagy ( Clausen et al . , 2010; Filimonenko et al . , 2010; Lystad et al . , 2014 ) .",
"A key step in response to a guidance cue signaling is the recycling and degradation of large signaling protein complexes .",
"Moreover , upon responding to cues , the resultant neuronal remodeling is also accompanied by the local sequestration and turnover of various cellular components , including cytoskeletal proteins .",
"In light of the degradative capacity of macroautophagy , it is possible that this degradative pathway may be involved , and Alfy acts to sequester these cargoes .",
"As an adaptor protein , the absence of Alfy should not impact macroautophagic degradation overall .",
"Consistent with previous findings , non-selective macroautophagy remains intact in tissues and cells derived from Alfy null mice ( Filimonenko et al . , 2010 ) ( Figure 8A–D ) .",
"Mice lacking Alfy clearly mounted an autophagic response to starvation as indicated by the increased levels of LC3-II in lysates collected from the perinatal liver ( Figure 8A ) , which is consistent with the lack of feeding observed in the pups ( Figure 2E ) .",
"The presence of LC3 conversion is consistent with the formation of APs , which we find forms in response to starvation in the presence or absence of Alfy ( Figure 8—figure supplement 1A ) .",
"Although neurons can properly mount an autophagic response ( Figure 8D ) , no abnormal accumulation of LC3-II was observed in the brain ( Figure 8B ) , suggesting that autophagosome maturation also is unaffected .",
"Consistent with this finding , macroautophagic degradation of long lived proteins was also independent of Alfy expression ( Figure 8C ) . 10 . 7554/eLife . 14810 . 023Figure 8 . Alfy is an adaptor protein for selective macroautophagy ( A–D ) Non-selective macroautophagy proceeds normally in the absence of Alfy .",
"( A ) Liver lysates from perinatal Alfy GT pups reveal greater LC3 conversion to LC3-II , reflecting the starvation response due to their inability to feed properly ( Figure 2E ) .",
"Lysates from normally fed WT and heterozygous littermates mice predominantly contain LC3-I .",
"The bottom graphs quantify LC3 conversion as the ratio of LC3-II/LC3-I .",
"Bars represent mean ± St . Dev .",
"ANOVA reveals a significant effect of genotype on LC3 conversion ( n = 7 WT , 6 Alfy GT Het , 6 Alfy GT; F ( 2 , 16 ) = 11 . 07; p < 0 . 001 ) .",
"Fisher PLSD post hoc analyses indicate that LC3 conversion of Alfy GT mice differed significantly from mice with at least one copy of Alfy .",
"( B ) In the brain , there is no abnormal accumulation of LC3-II in the Alfy GT mice , suggesting that autophagosome maturation proceeds normally .",
"A single-factor ANOVA reveals no significant difference between the three genotypes ( n = 5/genotype; F ( 2 , 12 ) = 5 . 52; p = 0 . 90 ) .",
"( C ) Macroautophagic protein degradation in response to starvation is normal in the absence of Alfy .",
"Long lived protein degradation assay .",
"( LC3 puncta formation is shown in Figure 8—figure supplement 1A ) .",
"A two-factor ANOVA revealed a significant main effect for treatment ( n = 4/genotype; F ( 2 , 27 ) = 24 . 93 , p < 0 . 001 ) , but no significant effect of genotype on percent ( % ) Proteolysis ( n = 4/genotype; F ( 2 , 27 ) = 1 . 82 , p = 0 . 18 ) .",
"Fisher PLSD post hoc analysis revealed % Proteolysis was significantly increased in all genotypes during starvation compared with complete media ( p < 0 . 001 ) and the addition of 5 mM 3MA significantly attenuated the starvation-induced proteolysis ( p < 0 . 001 ) .",
"( D ) In the absence of Alfy , LC3 conversion is normal in primary neurons in response to trophic factor withdrawal ( n = 4/genotype ) .",
"To accumulate LC3-II , cells were starved in the presence of 20 µM leupeptin .",
"( E ) The loss of Alfy impedes the selective macroautophagic turnover of its cargo , ALIS .",
"Although ubiquitinated structures are readily found when all forms of macroautophagy is impeded ( Figure 8—figure supplement 1B ) , the turnover of only specific cargoes is affected by the loss of Alfy .",
"Primary Alfy GT versus Alfy heterozygous ( HET ) MEFs after 72 hr of Veh or 5 μM AraC .",
"Ubiquitin-positive ALIS bodies form in response to mitotic inhibition , as revealed by FK2 immunofluorescence ( green ) .",
"In the absence of Alfy , these structures accumulate more rapidly ( white arrows ) .",
"n = 3 .",
"Similar Alfy dependence can be observed with the aggregation prone expanded polyQ proteins ( Figure 8—figure supplement 1C , D ) .",
"For all graphs , bars represent mean ± SEM .",
"ns , not significant; * , p < 0 . 05 . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 02310 . 7554/eLife . 14810 . 024Figure 8—figure supplement 1 . Alfy is an adaptor for selective macroautophagy .",
"( A ) In the presence of complete media , LC3 immunoreactivity ( green ) produces a diffuse staining pattern throughout the cytoplasm .",
"When MEF cultures are starved by incubation in HBSS , LC3 reactivity becomes localized to distinct puncta regardless of genotype , suggesting the formation of LC3-positive autophagosomes ( n = 4 ) .",
"( B ) The inhibition of macroautophagy leads to ALIS body accumulation in the absence of stress .",
"Immortalized Atg5 KO MEFs ( Kuma et al . , 2004 ) demonstrate the accumulation of ubiquitin positive aggregates ( white arrows ) under basal conditions as revealed by FK2 antibody ( green ) ( n = 3 ) .",
"( C , D )",
"The selective macroautophagy cargo , expanded polyglutamine inclusion , accumulates more rapidly in the absence of Alfy .",
"Transient transfection of 17aahtt103Q-eGFP in Ctrl and Alfy GT MEFs .",
"Aggregation over time is quantified in ( D ) .",
"The percentage of transfected cells with aggregates is higher in the Alfy GT MEFs compared with Ctrl .",
"Repeated measures ANOVA revealed a significant difference of genotype in the% transfected cells with aggregate genotype ( n = 3/genotype; F ( 1 , 8 ) = 154 . 643 , p = 0 . 002 ) , and a significant interaction between time and genotype ( n = 3/genotype; F ( 2 , 8 ) = 55 . 332 , p < 0 . 0001 ) .",
"Post hoc analyses ( Fisher PLSD ) revealed that in Ctrl MEFs , ‘% transfected cells with agg’ do not change between 24 and 48 hr ( p = 0 . 4144 ) , but decrease after 72 hr ( p = 0 . 005 ) .",
"In contrast , in Alfy GT MEFs , ‘% transfected cells with agg’ continuously increase over time ( comparison between 24 and 48 hr , p = 0 . 0047; comparison between 48 and 72 hr , p = 0 . 0105 ) .",
"Scale bar = 25 μm .",
"Data represent mean ± St . Dev . DOI: http://dx . doi . org/10 . 7554/eLife . 14810 . 024 Since we confirmed that nonselective macroautophagy was unaffected , we next determine if the loss of Alfy impeded the turnover of its substrates .",
"One naturally occurring Alfy substrate are known as aggresome-like induced structures ( ALIS ) ( Clausen et al . , 2010 ) .",
"Cytosolic ubiquitinated ALIS were formed in MEFs upon mitotic inhibition ( Figure 8E ) .",
"In contrast to control MEFs , these structures accumulated more rapidly in the absence of Alfy , suggesting that in its absence , the turnover of ALIS are impeded .",
"Moreover , unlike in MEFs that lack all forms of macroautophagy ( Figure 8—figure supplement 1B ) ( Kuma et al . , 2004 ) , Alfy GT MEFs do not demonstrate basal protein accumulation , suggesting that Alfy is not required for the turnover of all ubiquitinated substrates .",
"Nonetheless , over-expression of a canonical aggregation-prone polyQ protein , a 17 amino acid fragment of the mutant huntingtin protein with 103 glutamines and an eGFP tag ( 17aahtt ( 103Q ) eGFP ) ( Kazantsev et al . , 1999 ) , accumulated more rapidly by the absence of Alfy ( Figure 8—figure supplement 1C , D ) .",
"Thus , in the absence of Alfy , the elimination of ubiquitinated proteins by selective macroautophagy , but not basal macroautophagy , is impaired .",
"Taken together with the rest of our findings , we hypothesize that Alfy-mediated sequestration of cargoes may help selectively eliminate cargoes at or around the membrane , thereby affecting the ability of axons to properly respond to guidance cues ."
],
[
"In this study , our genetic models revealed that Alfy influences how growing axons interact with their environment to form stereotyped axonal connectivity in the central nervous system ( CNS ) .",
"The homozygous loss of Wdfy3 disrupts the formation of many axonal connections , causing agenesis of all three forebrain commissural tracts and leading to profound changes in brain wiring throughout the CNS , including the optic chiasm , internal capsule , cerebral peduncle , medial forebrain bundle and spinal cord tracts .",
"At the cellular level , the loss of Alfy led to defects in the development of midline glial population , impacted embryonic cortical neuron proliferation and impeded the ability of axons to respond to Netrin-1 .",
"Furthermore , Alfy KO mice present a phenotype unlike mice lacking core autophagy genes such as Atg5 , suggesting the perinatal lethality is not due to a loss of general autophagic degradation , as Alfy mutant tissue and cells display the classic response to starvation .",
"We hypothesize that Alfy functions as a selective macroautophagy adaptor protein and that Alfy-dependent regulation of intracellular vesicle trafficking and signaling pathways upstream of macroautophagy can have a significant influence over brain development .",
"The widespread disruption of axonal wiring identified in the absence of Alfy supports a functional role for this protein in the sequestration and trafficking of substrates into the autophagic pathway .",
"In the adult brain , Alfy facilitates the sorting of ubiquitinated cargo into APs , and one possibility is that during development Alfy has an analogous function .",
"For instance , APs are present in the growth cone and axon tips ( Hollenbeck , 1993; Maday and Holzbaur , 2014; Maday et al . , 2012 ) .",
"We propose that Alfy could regulate the ability of growth cones to respond to cues by sequestering and sorting proteins into degradative vesicles .",
"Such regulation could be critical for responding to cues in the environment through changes in cell shape and motility .",
"It has been suggested previously that the ubiquitination of guidance receptor complexes could regulate the responsiveness of axons to guidance cues ( O'Donnell et al . , 2009 ) .",
"The trafficking and responsiveness of the axonal guidance receptors Robo1 and UNC5 has been shown to be regulated by the E3 ubiquitin ligases rpm-1 in C . elegans and USP33 in murine commissural axons , respectively ( Li et al . , 2008; Yuasa-Kawada et al . , 2009 ) .",
"Furthermore , deletion of the rpm-1 homologue , Phr1 , in mice , causes axonal wiring defects reminiscent of Alfy mutants ( Bloom et al . , 2007 ) .",
"One intriguing possibility is that Alfy might sequester cargo ubiquitinated by E3 ligases such as Phr1 or USP33 .",
"Alternatively , the association of Alfy with the GABAA receptor-associated protein ( GABARAP ) ( Lystad et al . , 2014 ) , implies Alfy could be involved in the selective sorting of plasma membrane receptors .",
"Based on this model , we predict that in the absence of Alfy , the efficient degradation of Alfy-associated cargo would be slowed , or the cargo would be trafficked into alternative pathways , disrupting the temporal regulation of signaling events within the growth cone .",
"Similarly , the disruption of the midline glial structures may also reflect the disruption of signaling events that promote the differentiation and migration of these cell populations .",
"Examination of substrates labeled with a Lys63 polyUb chain , which are also associated with APs ( Shaid et al . , 2013 ) , may identify other Alfy-interacting substrates .",
"Wdfy3 and its homologue bchs have been implicated in trafficking events associated with lysosomes , yet genetic disruptions yield distinct phenotypes in their respective model systems .",
"Both homologues have been shown to co-localize with markers for degradative vesicles , including APs in mammals and endolysosomes in Drosophila ( Lim and Kraut , 2009; Lystad et al . , 2014; Simonsen et al . , 2004 ) yet , despite the conservation of domain structure , bchs loss of function ( LoF ) mutants have no reported developmental neuroanatomical defects ( Khodosh et al . , 2006 ) .",
"Overexpression of bchs , however can impact synaptic and axonal morphology at the neuromuscular junction and the retina , revealing that bchs dosage plays a role in CNS development and function ( Khodosh et al . , 2006; Kraut et al . , 2001 ) .",
"The difference in phenotype between bchs LoF flies and Alfy mutant mice might be explained by the emergence of novel biological functions due to the evolutionary expansion of the BEACH family of genes from six in invertebrates to nine in vertebrates ( Cullinane et al . , 2013 ) .",
"Evolutionary divergence also has been described for the BEACH protein , Neurobeachin ( Nbea ) , which has evolved distinct biochemical properties from its Drosophila homologue rugose ( Volders et al . , 2012 ) .",
"We propose Alfy , which shares 48% protein sequence identity with bchs , may have similarly diverged , acquiring new functions that are essential for vertebrate development .",
"Whereas proteasome-mediated degradation is thought to be the primary catabolic pathway regulating neurodevelopmental signaling ( Yi and Ehlers , 2007 ) , recent studies indicate that macroautophagy may also play a critical role in morphogenesis .",
"For instance , the loss of Ambra1 , a regulator of the autophagy protein Beclin1 , causes neural tube closure defects in mice ( Fimia et al . , 2007 ) .",
"In humans , recessive mutations in TECPR2 and EPG5 , genes implicated in AP maturation ( Stadel et al . , 2015; Tian et al . , 2010 ) , are responsible , respectively , for spastic paraplegia 49 with brain malformations , including hypogenesis of the corpus callosum [OMIM 615031] and Vici syndrome with brain malformations , including agenesis of the corpus callosum [OMIM 242840] ( Cullup et al . , 2013; Oz-Levi et al . , 2012 ) .",
"Why loss of these autophagy-related proteins causes corpus callosum defects remains unclear .",
"Although little is known about the importance of core autophagy genes such as Atg5 , Atg7 and Atg14 in neurodevelopment , no obvious defects in axonal connectivity have been reported ( Hara et al . , 2006; Komatsu et al . , 2006; Liang et al . , 2010 ) .",
"One exception is that the conditional elimination of Atg7 in POMC neurons leads to a defect in postnatal axonal outgrowth ( Coupe et al . , 2012 ) , and recently , a partial LoF mutation in a core autophagy gene , Atg5 , have been reported to lead to cerebellar hypoplasia and developmental delay ( Kim et al . , 2016 ) , but connectivity of the callosum appears unaffected .",
"Given that degradation by autophagy is slower than proteasome-mediated turnover , these data suggest that the sorting and sequestration of cargo , rather than elimination of cargo , is critical for the timing of cellular processes regulated by macroautophagy .",
"Alternatively , as a scaffold for ubiquitinated structures for degradation by macroautophagy , the loss of Alfy might have indirect effects on proteasome activity .",
"For example , Alfy is recruited to aggregated structures that form upon proteasome-inhibition ( Clausen et al . , 2010; Simonsen et al . , 2004 ) ; when Alfy is absent , the persistence of structures that might otherwise be eliminated may impact the efficiency of proteasome-mediated degradation .",
"We have previously reported that the deletion of Alfy does not measurably affect the enzymatic activity of the proteasome ( Filimonenko et al . , 2010 ) and does not lead to the accumulation of ubiquitinated structures in Alfy null MEFs ( Figure 8 ) .",
"Nonetheless , it is still possible that localized , discrete disruptions of proteasome activity or modifications in activity enough to disrupt timing might be present , leading to the profound phenotype we observe here .",
"Genetic screening has revealed a possible role for the human Wdfy3 homolog ( WDFY3 ) as a genetic risk factor for intellectual and developmental disabilities .",
"Most recently , a missense mutation in WDFY3 has been linked to autosomal dominant primary microcephaly ( Kadir et al . , 2016 ) .",
"Furthermore , in autism spectrum disorder ( ASD ) , rare de novo nonsense mutations within WDFY3 have been identified across two independent studies ( De Rubeis et al . , 2014; Iossifov et al . , 2012 ) .",
"WDFY3 resides on chromosome 4q21 . 23 and this region of chromosome 4 was suggested to harbor a genetic predisposition to ASD ( Chen et al . , 2006 ) and to schizophrenia ( Faraone et al . , 2006; Paunio et al . , 2004 ) .",
"In addition , the dosage of WDFY3 is affected in a subset of individuals with a rare chromosome disorder known as 4q21 deletion syndrome [OMIM 613509] , characterized by intellectual disabilities ( ID ) , language impairment and congenital birth defects ( Bonnet et al . , 2010; Cooper et al . , 2011 ) .",
"Taken together , these data imply WDFY3 is a rare genetic risk factor for childhood neurological disorders .",
"Future studies should aim to determine whether WDFY3 dosage is critical for human brain development and how mutations could potentially disrupt the biological function of Alfy .",
"A network analysis study on genomic data found that CNVs in genes associated with autophagy were enriched in patients with ASD ( Poultney et al . , 2013 ) .",
"While it remains to be determined whether computational modeling can be used to accurately predict the cellular pathways disrupted in ASD , in light of our findings , we would propose that further study is warranted .",
"In conclusion , mice lacking Alfy survive embryogenesis , but the loss of Alfy function produces widespread CNS defects in axonal tracts , including loss of the major forebrain commissures .",
"The results of our study provide new insight into the genetic control of brain wiring and open the door to many new questions surrounding the physiological role of Alfy and selective autophagy in CNS development ."
],
[
"The following antibodies were used for western blot , immunocytochemistry , immunohistochemistry , in situ hybridization and immunoprecipitation experiments: Anti-Alfy ( rabbit polyclonal anti-COOH Alfy; generously provided by Dr . Anne Simonsen and used for western blots and immunoprecipitation ) , anti-Alfy-N1 ( rabbit polyclonal anti-NH3 Alfy; generously provided by Dr . Masaaki Komatsu and used for western blots ) , anti-BrdU clone BU-33 ( B2531 , Sigma-Aldrich [St . Louis , MO] ) , anti-Caspase 3 active/cleaved form ( AB3623 , EMD Millipore [Germany] ) , anti-Class βIII-Tubulin ( PRB-435P , Covance [Princeton , NJ] ) , anti-Human DCC ( 554222 , BD Biosciences [San Jose , CA] ) , anti-Digoxigenin-AP , Fab fragments ( Roche [Switzerland] ) , anti-Doublecortin ( ab18723 , Abcam [Cambridge , MA] ) , anti-GFAP , clone GA5 ( MAB3402 , EMD Millipore ) , anti-LC3 ( for immunofluorescence , rabbit polyclonal; generously provided by Ron Kopito ) , anti-LC3B ( for western blots , ab48394 , Abcam ) , anti-Ki67 clone B56 ( 550609 , BD Biosciences ) , anti-MAP-2 ( ab11267 , Abcam ) , anti-NCAML1 , clone 324 ( MAB5272 , EMD Millipore ) , anti-Neurofilament clone2H3 ( developed by TM Jessell and J Dodd and was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa ) , anti-Robo1 ( H-200 , Santa Cruz Biotechnology [Santa Cruz , CA] ) , anti-synaptophysin ( 101 002 , Synaptic Systems [Germany] ) , anti-Tau-1 , clone PC1C6 ( MAB3420 , EMD Millipore ) , anti-Tbr1 ( AB2261 , EMD Millipore ) , and anti-Ubiquitin , clone FK2 ( Enzo Life Sciences [Farmingdale , NY] ) .",
"The following reagents were generously provided: Atg5 knockout and wildtype MEFs ( Noboru Mizushima , [Itakura and Mizushima , 2010] ) and MYC-tagged Netrin-1 ( pGNET1-myc ) ( Marc Tessier-Lavigne [Serafini et al . , 1994] ) .",
"All animals and procedures complied with the Guide for Care and Use of Laboratory Animals and were approved by the IACUC committee at Columbia University .",
"Mice were maintained in a 12h/12h light/dark cycle in a temperature and humidity controlled environment , with ad libitum access to food and water .",
"Alfy GT mice ( 129/SvEv x C57BL/6; Wdfy3Gt ( OSTGST_5258_D3 ) Lex ) were generated at the Texas Institute for Genomic Medicine ( TIGM [College Station , TX] ) .",
"A complete description of the gene trap vector can be found in ( Zambrowicz et al . , 2003 ) .",
"The Alfy GT mice used in this study were derived from mating heterozygous mice .",
"Wdfy3 floxed mice were generated on a 129/SvEv x C57BL/6 background under contract with the University of Connecticut .",
"The floxed Wdfy3 allele has loxP sites flanking exon V that is predicted to produce a 66 amino acid peptide following Cre-mediated recombination , instead of the 3508 amino acid full-length Alfy protein .",
"The Wdfy3 deletion allele ( ∆ ) is created when male mice carrying Wdfy3 floxed alleles are crossed with Bl6/J female carrier of HprtCre/+ , a Cre-deleter stain with 100% Cre-mediated recombination in oocytes ( Tang et al . , 2002 ) .",
"All experiments included control and experimental littermates and represent data across multiple litters .",
"Genomic DNA was extracted and the alleles were identified using the following primers: Common Alfy forward: 5’-CTTGTTACACTTGTCCCACAGC-3’ , Alfy WT reverse 5’– TTAGACTTCTAAGCCCACGAGTACC-3’ , and Alfy GT reverse: 5’-ATAAACCCTCTTGCAGTTGCATC-3’ .",
"Genotyping for the Alfy WT , loxP and ∆ alleles was performed using following the primers in a multiplex PCR reaction: LoxP-F 5’-gaaagcaagctcgtttacgg-3’ , Frt-R 5’-aggttaccagccacaaccag -3’ , and Frt-F 5’-acttgggaagagggaagctc-3’ .",
"RNA from whole embryo was isolated using Trizol reagent ( Life Technologies [Carlsbad , CA] ) according to the manufacture’s recommendation .",
"This RNA was used in a reverse transcription reaction containing random 9mers ( NEB Biosciences ) and reverse transcriptase ( Superscript , Qiagen [Germany] ) according to manufacturer’s instructions .",
"Negative controls ( minus reverse transcriptase ) were run for each sample .",
"The resulting cDNA was used to determine the abundance of Wdfy3 mRNA relative to GFAP , βIII Tubulin or Actin .",
"The following primers were used: βIII Tubulin: Tuj1-1F: 5’-ctacgacatctgcttccgca -3’ , Tuj1-1R: 5’-gaagggaggtggtgactcca-3’; GFAP: GFAP-F: 5’-gagctcaatgaccgctttgc -3’ , GFAP-R: 5’-tccttggctcgaagctggt -3’ .",
"Primary dissociated cortical cultures were prepared from postnatal day 0 mouse pups ( P0 ) .",
"The cortical lobes were dissected in ice cold DMEM/F12 ( Life Technologies ) containing 10% heat- inactivated FBS and antibiotics .",
"Cortical tissue were trypsinized for 30 min at 37°C , then triturated in fresh 10% FBS DMEM/F12 through a fire-polished glass pipette and filtered through a 40 micron nylon cell strainer ( BD Biosciences ) .",
"For immunocytochemistry , cells were plated at a density of 1 . 0 x 105 cells/well onto coverslips coated with 20 μg/mL polyD-lysine ( Sigma Aldrich ) and 5 µg/mL mouse laminin ( Life Technologies ) .",
"The medium was changed 2 hr after plating to NB media ( Neurobasal media [Life Technologies] ) containing 0 . 5 mM L-glutamine , 1X B27 supplement ( Life Technologies ) , 2% heat inactivated FBS ( Life Technologies ) and 1X antibiotic ( Life Technologies ) .",
"Embryos were collected from deeply anesthetized pregnant dams at E14 . 5 .",
"Uterine horns were placed into ice cold Hank’s buffered saline solution ( HBSS ) .",
"Heart , liver and head were removed for genomic DNA extraction , the remaining tissue was trypsinized in equal volume of HBSS and 0 . 25% trypsin for 15 min . at 37°C .",
"Samples were triturated with a fire-polished glass pipette , then diluted into MEFs complete media: DMEM ( Life Technologies ) containing 10% FBS ( Life Technologies ) , 1X L-glutamine ( Life Technologies ) , 0 . 1 mM beta-mercaptoethanol ( Life Technologies ) , 1X Pen/strep ( Life Technologies ) .",
"Cells were filtered through a 100 micron sieve and plated .",
"In addition to genotype confirmation , RNA was extracted from the cultures to measure transcript levels and MEF cultures were confirmed to be mycoplasma free ( Takara ) .",
"To starve the MEF cultures , the complete media was aspirated off the cultures , followed by three PBS washes and cultures were placed in HBSS supplemented with 10 mM HEPES for four hours .",
"The subcellular fractionation of the brain tissue protocol was performed as described ( Hallett et al . , 2008 ) .",
"Briefly , adult control mice were euthanized and cerebral cortex was rapidly dissected and frozen on dry ice .",
"An aliquot of each fraction was saved for analysis .",
"Brain tissue was homogenized in TEVP buffer ( 10 mM Tris , 5 mM NaF , 1 mM Na3VO4 , 1 mM EDTA , 1 mM EGTA , pH . 7 . 4 ) containing 320 mM sucrose .",
"Homogenates ( H ) were fractionated sequentially as shown in Figure 6—supplement figure 2 at 800 xg , resulting in the first supernatant ( S1 ) and pellet ( P1 ) fractions , and 9200 xg to generate the second supernatant ( S2 ) and crude synaptosomal membranes ( P2 ) .",
"P2 was resuspended in TEVP buffer containing 35 . 6 mM sucrose for 30 min on ice to release vesicles and organelles .",
"P2 was then spun at 25 , 000 xg to segregate the supernatant ( LS1 ) from the membrane fraction ( LP1 ) .",
"Lastly , S2 was centrifuged for 2 hr at 165 , 000 xg to generate the supernatant fraction ( S3 ) and light membrane fraction ( P3 ) .",
"Protein quantification was performed on all fractions and equal amounts of protein were loaded onto SDS-PAGE .",
"Alfy MEFs as well as Atg5 knockout and wildtype MEFs were maintained in MEF complete media .",
"Starvation was achieved using a starvation medium of HBSS + 10 mM HEPES for 4 hr .",
"To slow lysosome-mediated degradation , cells were treated with 20 μM leupeptin .",
"Mitotic inhibition was achieved by treating cells with 10 μM arabinofuranosylcytidine ( AraC ) for 72 hr .",
"Transfections of cells were achieved with Lipofectamine 2000 ( Life Technologies ) in serum-free media .",
"Cells were transfected with constructs encoded by 17aahtt103Q tagged with eGFP as previously described ( Filimonenko et al . , 2010 ) .",
"Cells were fixed 24 , 48 and 72 hr later and imaged by epifluorescence microscopy .",
"Long lived protein degradation assay was performed as previously described ( Filimonenko et al . , 2010 ) .",
"Whole cell lysates and tissue lysates from freshly dissected brain and liver were generated using a modified RIPA buffer ( 50 mM Tris-HCl , pH 7 . 4 , 150 mM NaCl , 10 mM EDTA , 0 . 1% Triton-X 100 ) containing protease and protein phosphatase inhibitors ( Halt Inhibitor Cocktail , Roche ) .",
"Ten strokes in a dounce homogenizer were used to extract protein from tissues and samples were cleared by centrifugation at 15 , 000 xg for 60 min at 4°C unless otherwise indicated .",
"Protein concentration was determined using the DC Protein Assay ( Bio-rad Laboratories [Hercules , CA] ) and equal amounts of protein were prepared and loaded onto 4–12% Bis-Tris or 3–8% Tris-Acetate NuPAGE gels and blotted to PVDF membranes as described by the manufacture’s recommendations ( Life Technologies ) .",
"PVDF membranes were blocked in 5% BSA in TBS containing 1% Tween-20 ( TBST ) .",
"Antibodies were diluted in 1% BSA in TBST and incubated overnight at 4°C .",
"The appropriate HRP-conjugated secondary antibodies ( ThermoFisher Scientific ) were diluted in 1% BSA TBST and a chemiluminescent reaction ( West Dura SuperSignal , ThermoFisher Scientific ) was detected by the Versadoc Imaging System ( Bio-rad ) .",
"Fresh frozen tissue was sectioned at 15 μm onto Fisherband Superfrost plus slides and stored at −80°C until use .",
"On day one , slides were warmed at 37°C for 30 min , fixed in 4% paraformaldehyde for 10 min and washed in DECP-treated phosphate buffered saline ( PBS ) .",
"Slides were acetylated with 0 . 3 M acetic anhydride in 0 . 1 M triethanolamine for 10 min followed by three PBS washes .",
"Slides were prehybridized in formamide diluted 2X Prehyb buffer ( 5 M NaCl , 1 M Tris , pH 7 . 4 , 6% Ficoll , 6% polyvinylpyrrolidone , 6% Bovine serum albumin , 50 mg total Yeast RNA , 5 mg Yeast tRNA , 250 mM EDTA and 50 mg SS DNA ) for 1 hr at room temperature .",
"Wdfy3 riboprobes were prepared from a pCR II plasmid ( Life Technologies ) containing a PCR fragment of the 5’ UTR region of mouse Wdfy3 from 320–540 nt ( ref NM_172882 . 3 ) .",
"In vitro transcription was performed as described by the manufacturer ( Promega Corporation [Madison , WI] ) using the T7 polymerase for the antisense probe on template linearized with KpnI and Sp6 polymerase for the sense probe on template linearized with NotI .",
"Riboprobes were cleaned using the RNeasy MinElute Cleanup Kit ( Qiagen ) according the manufacturer’s protocol .",
"Riboprobes were heated to 80°C for 5 min , quenched on ice and added to 2X Hyb buffer ( 5 M NaCl , 1 M Tris , 6% Ficoll , 6% polyvinylpyrrolidone , 6% Bovine serum albumin , 50 mg total Yeast RNA , 5 mg Yeast tRNA , 250 mM EDTA , 50 mg SS DNA and 20% dextran sulphate ) diluted 1:1 with formamide .",
"Hyb solution was added directly to slides and they were incubated overnight at 68°C in a humidified chamber .",
"On day two , slides were washed in 5X SSC at 68°C for 10 min and coverslips were removed .",
"Three more washes 0 . 2X SSC were carried out at 68°C , followed by a 0 . 2X SSC wash at room temperature .",
"Slides were incubated in B1 buffer ( 0 . 1M Tris , pH 7 . 5 , 0 . 15M NaCl ) for 5 min then blocked in B1 containing 10% heat-inactivated sheep serum for 1 hr in a humidified chamber .",
"Anti-DIG antibody diluted 1:5000 in B1 buffer containing 1% heat-inactivated sheep serum and slides were incubated overnight at 4°C in a humidified chamber .",
"On day two , slides were washed in B1 buffer three times and equilibrated in B3 buffer ( 0 . 1 M Tris , pH 9 . 5 , 0 . 1 M NaCl , 50 mM MgCl2 ) for 5 min at room temperature .",
"The staining reaction was carried out using NBT/BCIP ( Promega ) diluted in B3 buffer in the dark at room temperature overnight in a humidified chamber and the staining reaction was stopped by washing the slides three times in TE ( 10 mM Tris , pH 8 . 0; 1 mM EDTA ) .",
"On DIV3 , cultures were transfected with 1 µg pEGFP-N3 ( Clontech ) using Lipofectamine 2000 ( Life Technologies ) in Optimem for 4 hr .",
"The next day , the media was changed to NB media + mitotic inhibitors: 10 µM AraC ( Sigma-Aldrich ) , 10 µM Uridine ( Sigma-Aldrich ) , and 10 µM 5-Fluoro-2’-deoxyuridine ( FDU , Sigma-Aldrich ) to prevent the excessive growth of glial cells .",
"On DIV7 , cells were washed with TBS , fixed for 15 min with 4% paraformaldehyde , permeabilized in 0 . 1% Triton-X 100 in TBS , blocked in 5% BSA in TBS and stained with primary antibodies diluted in 1% BSA in TBS overnight at 4°C .",
"Alexa Fluor conjugated secondary antibodies ( Life Technologies ) were diluted in 1% BSA TBS , incubated with coverslips for 1 hr at room temperature , counterstained with Hoechst 33 , 342 ( Life Technologies ) and mounted with prolong gold antifade ( Life Technologies ) onto glass slides .",
"A Leica SP5 confocal microscope was used to image cortical cultures .",
"GFP-transfected cells with the characteristic neuronal morphology , including a small triangular shaped soma were analyzed to compare morphology between Alfy null and Alfy control cultures .",
"Z-stacks were taken every micron using a 20X objective and 3X digital zoom .",
"For each culture , approximately10 individual neurons were imaged totaling 50 control and 49 knockout cortical neurons .",
"Confocal z-stacks were traced and analyzed using the simple neurite tracer segmentation plug-in for the FIJI version of ImageJ ( Longair et al . , 2011 ) .",
"Time-pregnant dams were sacrificed in accordance with humane protocols established by the Columbia University IACUC .",
"All dissections were carried out in ice-cold PBS .",
"The embryonic cerebral cortex was dissected out and finely cut into pieces , then 28 gauge needles were used to transfer and position explant pieces inside a drop ( 5 µL ) of Matrigel ( BD Biosciences ) on a collagen-coated glass bottom petri dish ( MatTek ) .",
"Once explants were placed , 350 µm diameter punches of solidified agarose cell blocks containing either untransfected or Netrin1-MYC transfected HEK293T cells were placed alongside cortical explant pieces .",
"Explants were grown for 48 hr , and then fixed with 4% PFA for 15 min .",
"Explants were imaged and measured using phase contrast microscopy using a 10X objective on a Nikon TiE microscope and NIS Elements software ( Nikon ) .",
"Across two experiments , outgrowth in microns towards the HEK293T cellblock was recorded for the three longest processes in a single plane .",
"The measurements were averaged for each explant and statistical analysis was performed using two-way ANOVA followed by the Fisher LSD post hoc test .",
"Following analysis of outgrowth , explants were further processed for immunocytochemistry as described above .",
"To prepare HEK293T agarose cell blocks , HEK293T cells were seeded at 600 , 000 cells per six well , then 16 hr later transiently transfected with pGNET1-myc using Lipofectamine 2000 .",
"The following day , untransfected HEK293T or HEK293T cells transfected with Netrin1-MYC were trypsinized , pelleted and resuspended in 900 µL of warm ( ~50°C ) 1% agarose dissolved in DMEM .",
"Punches of agarose cell blocks containing HEK293T cells were prepared with a 0 . 35 mm diameter Harris Uni-Core tissue punch .",
"To visualize retinal ganglion cell ( RGC ) axon projections through the optic chiasm , unilateral whole eye anterograde labeling with DiI ( Molecular Probes ) was performed on fixed tissue as described previously ( Kuwajima et al . , 2012 ) .",
"Samples were incubated in PBS + 0 . 02% sodium azide for 14 days at 37°C .",
"Ipsilateral versus contralateral projections were quantified by measuring the pixel intensity of ipsilateral and contralateral optic tracts in a 500 x 500 μm area lateral to the optic chiasm midline with the MetaMorph image analysis software .",
"An ipsilateral index was calculated by dividing the intensity of the ipsilateral projection by the sum of the contralateral and ipsilateral pixel intensities as described previously ( Erskine et al . , 2011; Kuwajima et al . , 2012 ) .",
"The ipsilateral index in Alfy null mice was normalized to the littermate WT ipsilateral index .",
"The number of Ki67 positive cells labeled with DAB within the subventricular zone of the ganglionic eminence was determined by stereology using StereoInvestigator software ( MicrobrightField ) by an experimenter blind to genotype .",
"For each animal , measurements were made at 60X bilaterally in five matched , adjacent sections spaced 200 μm apart .",
"There were three animals per genotype .",
"Timed pregnant dams were weighed and received an intraperitoneal injection with 50 mg/kg of BrdU at E15 . 5 .",
"One hour following the injection , the dams were deeply anesthetized and the uterine horns were dissected out .",
"Embryo heads were drop fixed in 4% PFA for 48 hr and then cryoprotected in 30% sucrose , embedded in OCT and sectioned at 25 µm .",
"Sections were processed using an antibody against BrdU as described in the immunohistochemistry section .",
"Statistical analyses were performed using Statview 5 . 0 ( SAS Institute ) .",
"Normally distributed data were subject to student t-test , or for multiple comparisons , analysis of variance ( ANOVA ) followed by the appropriate post hoc comparisons as indicated in the figure legends .",
"Complete F-statistics and calculated p-values are also available in the figure legends .",
"Power analyses for quantitative studies were performed to achieve a power of 0 . 8 with a confidence of 0 . 95 using G*Power 3 . 1 ( Faul et al . , 2007 , 2009 ) .",
"Effect sizes were based upon pilot studies &/or previously published or unpublished materials .",
"n-values are indicative of biological replicates and no data were excluded from analyses ."
]
] | [
"The regulation of protein degradation is essential for maintaining the appropriate environment to coordinate complex cell signaling events and to promote cellular remodeling .",
"The Autophagy linked FYVE protein ( Alfy ) , previously identified as a molecular scaffold between the ubiquitinated cargo and the autophagic machinery , is highly expressed in the developing central nervous system , indicating that this pathway may have yet unexplored roles in neurodevelopment .",
"To examine this possibility , we used mouse genetics to eliminate Alfy expression .",
"We report that this evolutionarily conserved protein is required for the formation of axonal tracts throughout the brain and spinal cord , including the formation of the major forebrain commissures .",
"Consistent with a phenotype reflecting a failure in axon guidance , the loss of Alfy in mice disrupts localization of glial guidepost cells , and attenuates axon outgrowth in response to Netrin-1 .",
"These findings further support the growing indication that macroautophagy plays a key role in the developing CNS ."
] | [
"Unlike many other cells in the body , neurons typically survive throughout the life of a mammal .",
"This long life suggests that they may be more vulnerable to damage from cellular debris .",
"Previous research has found that a protein called Alfy , which is abundant in the brain , is involved in cleaning up debris , such as those involved in neurodegenerative diseases , by a pathway known as autophagy .",
"Alfy guides the formation of spherical compartments called autophagosomes , which deliver the debris to another compartment known as the lysosome to permit degradation .",
"In developing embryos , neurons need to migrate to the right location within the central nervous system and extend projections called axons to communicate with other cells .",
"However , it was not clear whether this process requires cell materials to be selectively sent to lysosomes , and whether this involves the Alfy protein .",
"Dragich et al . addressed this question by studying mouse embryos that lack Alfy .",
"The brains of these mice developed abnormally and were missing the corpus callosum ( the dense band of fibers that normally connects the two halves of the brain ) .",
"Without Alfy , the growing axons could not navigate their way to the right places to connect with other neurons .",
"Furthermore , some neurons migrated to the wrong places in the developing brain , which resulted in the abnormal formation of cell-clusters .",
"The findings of Dragich et al . suggest that autophagy also plays an important role in normal brain development .",
"Future studies are now needed to work out exactly how Alfy controls neuron migration and the growth of axons .",
"The human gene WDFY3 is nearly identical to the gene that encodes the Alfy protein , and has been implicated in neurodevelopmental disorders such as autism and microencephaly .",
"Studying Alfy therefore may help us to understand human conditions that affect the developing or aging brain ."
] | 2016 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"short report",
"cancer biology"
] | Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells | elife-30224-v2 | [
[
"Amongst the most significant therapeutic breakthroughs in cancer has been the discovery of drug-sensitising genomic alterations .",
"Drugs such as the tyrosine kinase inhibitors ( TKIs ) developed against the BCR-ABL fusion product in chronic myeloid leukaemia ( CML ) and the receptor products of HER2 mutations in breast cancer have transformed the prognosis of these cancers ( Druker et al . , 2006 ) .",
"Malignant mesothelioma ( MM ) currently has no biomarker-driven therapies in routine clinical use .",
"The mainstay of medical therapy for all patients remains systemic cytotoxic chemotherapy that offers only limited survival benefit in unselected populations; as such the disease remains invariably fatal ( Vogelzang et al . , 2003 ) .",
"A plethora of genomic studies in MM has identified recurrent mutations in several genes considered to be tumour drivers .",
"CDKN2A , NF2 , BAP1 and TP53 are the most frequently mutated ( Guo et al . , 2015; Bueno et al . , 2016 ) and there has been increased focus on these genes and their associated signaling pathways as potential therapeutic targets ( LaFave et al . , 2015 ) .",
"We aimed to determine if the mutational status of these tumour driver genes could predict response to a range of existing anti-cancer compounds with a view to identifying genomic biomarkers for responsive subsets of MM .",
"We have previously reported on the ability of such unbiased high-throughput chemical screens in cancer cell lines to identify drug-sensitising mutations in other cancer types ( Garnett et al . , 2012 ) .",
"To this end , we conducted a high-throughput chemical screen of molecularly characterised MM cell lines seeking associations between MM driver gene mutations and compound response .",
"This strategy led to the discovery of a subset of MM cell lines defined by loss-of-function ( LOF ) mutations in BRCA associated protein-1 ( BAP1 ) that demonstrated heightened sensitivity to the death receptor agonist recombinant tumour necrosis factor ( TNF ) -related apoptosis-inducing ligand ( rTRAIL ) .",
"We validated this finding using in vitro , in vivo and ex vivo models supporting the use of BAP1 as a genomic biomarker to identify TRAIL-sensitive MM tumours and a novel stratified approach to treat MM . rTRAIL and other death receptor agonists selectively induce apoptosis in cancer cells and have long held promise as anti-cancer agents owing to their broad clinical utility and minimal off-target effects ( Wiley et al . , 1995; Pitti et al . , 1996; Ashkenazi et al . , 1999 ) .",
"Despite this , successful preclinical studies have not translated to clinical efficacy in trials of unselected populations ( Herbst et al . , 2010; Wainberg et al . , 2013; Soria et al . , 2010; Lemke et al . , 2014a ) ; there have been no trials to date in MM .",
"However , within these trials some patients showed signs of therapeutic benefit and differential sensitivity within cell lines is well known .",
"Retrospective biomarker identification has led to the stratified use of other anti-cancer therapies that initially failed in unselected trials such as activating EGFR mutations and EGFR TKIs ( Lynch et al . , 2004 ) .",
"We propose that BAP1 could potentially act as such a biomarker for the death receptor agonists .",
"BAP1 is a nuclear deubiquitinase and forms multi-protein complexes that regulate the transcription of genes involved in key cellular functions including cell cycle regulation and DNA repair ( Ismail et al . , 2014; Machida et al . , 2009 ) .",
"We investigated which BAP1 protein-binding partners , and thus which regulatory complexes , mediate TRAIL sensitivity identifying the BAP1-ASXL1 complex , the Polycomb repressive deubiquitinase ( PR-DUB ) , as key .",
"We further found that loss of BAP1 function modulates mRNA and protein expression of components of the extrinsic apoptotic pathway ."
],
[
"A 6 day viability screen using 94 drugs including small molecule inhibitors and cytotoxic chemotherapeutics ( Supplementary file",
"1 ) was performed on 15 MM cell lines ( Supplementary file",
"2 ) that had been characterised using whole-exome sequencing , copy number analysis and gene expression arrays .",
"We generated 1425 single agent activity data profiles across the 15 cell lines ( Figure 1A and Supplementary file 3 ) .",
"To detect novel markers of drug sensitivity , we sought statistical associations between drug response and the mutational status of the cell lines based on five genes identified as candidate drivers of tumourigenesis in MM ( Guo et al . , 2015 ) ( Figure 1—figure supplement 1 ) .",
"There were 24 significant associations ( false discovery rate ( FDR ) < 0 . 2 ) between single agent response and the presence of a genomic alteration .",
"The most statistically significant sensitising association seen was between BAP1 LOF mutations ( mt BAP1 ) and treatment with recombinant TRAIL ( rTRAIL; FDR = 0 . 18 , effect size −0 . 48 ) ( Figure 1B , C and Supplementary file 4 ) .",
"No significant effect on cell viability was observed in BAP1 wild-type ( wt BAP1 ) lines when treated with rTRAIL .",
"We subsequently confirmed this association in a larger panel of MM cell lines ( Figure 1D and Supplementary file 5 ) .",
"Strikingly , 6 of the 8 cell lines ( 75% ) harbouring a BAP1 LOF mutation were sensitive or partially sensitive to a dose range of rTRAIL while 7 of the 9 cell lines ( 78% ) harbouring wild-type BAP1 were resistant .",
"BAP1 LOF mutations correlated with a loss of BAP1 protein expression in the majority of cell lines ( Figure 1E ) .",
"No sensitising association with BAP1 was observed for pemetrexed or cisplatin , which are current first line agents for the treatment of MM ( Figure 1—figure supplement 2A and B ) .",
"A marginal trend towards increased sensitivity in BAP1 mutant MM lines in response to treatment with the agonistic FAS receptor antibody CH11 and a TNF-α/IAP inhibitor combination was observed .",
"However , this was not as pronounced as that observed with rTRAIL or the multivalent death receptor five superagonist MEDI3039 ( Figure 1—figure supplement 2C , D and E ) .",
"Thus , while the significant sensitising association observed in the screen appears most specific to death receptor agonists , the trend observed with other TNF superfamily agonists indicates the BAP1-rTRAIL association to be mediated by an underlying mechanism common to this family such as the cytoplasmic extrinsic apoptotic machinery .",
"To determine if knockdown of BAP1 in wild-type MM cells led to TRAIL sensitivity , we silenced BAP1 expression in four wt BAP1 MM cell lines using a lentiviral shRNA construct .",
"Knockdown of BAP1 resulted in increased cell death following rTRAIL treatment compared with empty vector ( EV ) control shRNA and the parental cell line in all four MM cell lines ( Figure 2A and Figure 2—figure supplement 1B and C ) .",
"Loss of BAP1 expression has also been identified in several other tumour types including uveal melanoma ( 47% ) ( Harbour et al . , 2010 ) , clear cell renal carcinoma ( CCRC ) ( 14% ) ( Peña-Llopis et al . , 2012 ) and cholangiocarcinoma ( 7% ) ( Fujimoto et al . , 2015 ) .",
"Notably , knockdown of BAP1 in two CCRC lines resulted in increased sensitivity to rTRAIL in addition to the MDAMB-231 breast cancer line ( Figure 2B and Figure 2—figure supplements 2 and 3 ) .",
"We also analysed a panel of 1001 cancer cell lines submitted for whole exome and copy number analysis as part of the COSMIC cell lines project ( Forbes et al . , 2015 ) and identified nine additional non-mesothelioma cell lines harbouring truncating mutations in BAP1 ( http://cancer . sanger . ac . uk/cancergenome/projects/cell_lines/ ) .",
"These include CCRC , bladder and breast cancer lines .",
"Treatment of cancer cell lines harbouring nonsense mutations in BAP1 with rTRAIL resulted in markedly reduced cell viability compared with cancer cell lines harbouring missense mutations ( Figure 2—figure supplement 4 ) .",
"BAP1 is a nuclear deubiquitinase that forms multi-protein complexes with transcription factors to regulate gene transcription ( Jensen et al . , 1998; Ventii et al . , 2008 ) .",
"To elucidate the mechanism by which BAP1 modulates sensitivity to TRAIL we generated expression vectors containing wild-type or mutant forms of BAP1 , each with an inactive functional site or protein-binding domain .",
"These included C91A ( mutation in the deubiquitination catalytic site ) ( Jensen et al . , 1998; Ventii et al . , 2008 ) , ΔHBM ( deletion of the HCF-1-binding site ) ( Misaghi et al . , 2009 ) , T493A ( mutation in the FOXK2-binding site ) ( Ji et al . , 2014 ) , ΔASXL ( deletion of the ASXL1/2 protein-binding site ) ( Daou et al . , 2015 ) and ΔCTD ( deletion of the C-terminal domain containing the nuclear localisation signal ) ( Ventii et al . , 2008 ) .",
"H226 MM cells , which harbour a homozygous deletion of BAP1 and demonstrate complete loss of BAP1 expression , were transduced with a GFP ( vector control ) , a wild-type BAP1 expression vector or one of these five mutant BAP1 expression vectors .",
"rTRAIL sensitivity of the parental BAP1-null H226 MM line was significantly diminished following expression of wild-type BAP1 and each of the mutant constructs except those with an inactive deubiquitinating or ASXL protein-binding site ( Figure 2C ) , implicating the function of these sites in BAP1-induced TRAIL resistance .",
"These effects were replicated using MEDI3039 ( Figure 2D ) .",
"Transduction of two further BAP1-mutant rTRAIL-sensitive cell lines , H28 and H2804 , with wild-type BAP1 also induced resistance to rTRAIL while sensitivity was maintained with transduction of the deubiquitinase mutant ( Figure 2—figure supplement 5 ) .",
"The BAP1 deubiquitinase and ASXL-binding sites are key to the function of the PR-DUB , an epigenetic transcriptional regulatory complex composed of BAP1 and ASXL1 .",
"Deubiquitination of the main substrate of the PR-DUB , H2AK119Ub , alters chromatin architecture to modulate gene transcription ( Scheuermann et al . , 2010 ) .",
"This led us to hypothesise that PR-DUB , rather than exclusively BAP1 , function might underlie rTRAIL sensitivity .",
"Consistent with this shRNA silencing of ASXL1 , but not ASXL2 , induced sensitivity to MEDI3039 and rTRAIL in the BAP1/ASXL1/ASXL2-wild-type MM line MPP-89 ( Figure 2E and Figure 2—figure supplement 6 ) .",
"Furthermore , H2AK119Ub expression was unaltered in the rTRAIL-sensitive H226 cells transduced with mutant constructs that disrupt PR-DUB activity , while the rTRAIL-resistant H226 cells transduced with a wild-type BAP1 construct exhibited lower H2AK119Ub levels ( Figure 2—figure supplement 7 ) .",
"Thus , as the PR-DUB complex is implicated in transcriptional regulation , differential modulation of specific transcriptional programmes by BAP1 may determine rTRAIL sensitivity .",
"We therefore compared differential gene expression data from BAP1-null H226 cells transduced with the C91A BAP1 mutant or with wild-type BAP1 and carried out a signalling pathway impact analysis ( SPIA ) ( ( Figure 2—figure supplements 8 and 9 [SPIA_H226 C91A mutant vs WT] ) ( http://www . genome . jp/dbget-bin/www_bget ? path:map04210 ) .",
"Among those pathways significantly altered when comparing wild-type versus C91A BAP1 ( FDR < 0 . 2 ) was that of apoptosis .",
"In particular , there was altered mRNA expression of components of the extrinsic death pathway ( Figure 2F and Supplementary file 6 ) .",
"This manifested as an imbalance in levels of pro- and anti-apoptotic mRNA expression with , for example , significantly decreased levels of the anti-apoptotic cIAP1/2 ( p=2 . 32E-10 ) and increased levels of the pro-apoptotic death receptor 5 ( p=7 . 79E-10 ) in the rTRAIL sensitive C91A BAP1-transduced cells relative to the rTRAIL resistant BAP1-wild-type transduced cells .",
"Immunoblot analysis confirmed reduced protein expression of cIAP1/2 and c-FLIP in both C91A and ΔASXL BAP1-transduced cells relative to BAP1-wild-type transduced cells ( Figure 2G ) .",
"Flow cytometry analysis confirmed reduced DR4 and DR5 expression in C91A BAP1 transduced relative to BAP1-wild-type-transduced cells .",
"Knockdown of BAP1 in the BAP1 wild-type H2818 line resulted in a significant increase in DR4 expression only ( Figure 2H ) .",
"To support the clinical relevance of our finding we extended our assays to two further models derived from primary tumour tissue .",
"25 human early passage MM lines from the UK Mesobank ( Rintoul et al . , 2016 ) were assessed for BAP1 expression by immunohistochemistry , a technique known to correlate strongly with BAP1 LOF mutations in the absence of strong nuclear staining ( Nasu et al . , 2015 ) .",
"When treated with rTRAIL , those without strong nuclear staining were significantly more sensitive than those with strong nuclear staining ( p=0 . 0067 ) .",
"Of the 12 lines that did not express nuclear BAP1 9 were sensitive , 2 partially sensitive and only one resistant to rTRAIL ( Table 1 , Figure 3A and Figure 3—figure supplement 1 ) .",
"Remarkably , rTRAIL treatment of tumour explants derived from three patients with MM also revealed increased levels of apoptosis ( as measured by poly ( ADP-ribose ) polymerase ( PARP ) cleavage ) in explants with low BAP1 expression compared with those with high BAP1 expression ( Figure 3B and C , Figure 3—figure supplement 2 ) .",
"To test the in vivo efficacy of TRAIL in inducing apoptosis in BAP1-mutant MM cells , we transduced the H226 BAP1-wild-type and the H226 C91A BAP1-mutant cell lines with luciferase and injected equal numbers of wild-type and mutant cells into the opposite flanks of mice ( Figure 3—figure supplement 3A ) .",
"On day 14 after injection the mice were divided into two groups and injected intraperitoneally with rTRAIL or vehicle for 6 days per week until day 40 .",
"At sacrifice rTRAIL-treated BAP1-mutant tumours weighed significantly less than rTRAIL-treated BAP1-wild-type tumours ( p=0 . 020 ) and vehicle-treated BAP1-mutant tumours ( p=0 . 019 ) ( Figure 3D and Figure 3—figure supplement 3B ) .",
"BAP1-wild-type tumours showed no response to rTRAIL compared with vehicle .",
"The growth rate of rTRAIL-treated BAP1-mutant tumours was also significantly suppressed compared with rTRAIL-treated BAP1-wild-type and vehicle-treated tumours ( p<0 . 05 ) as assessed by longitudinal bioluminescence intensity ( Figure 3E and F ) ."
],
[
"Malignant mesothelioma remains a devastating disease with limited systemic treatment options ( Vogelzang et al . , 2003 ) .",
"Biomarker-driven therapies have significantly improved the prognosis for subsets of patients within other cancer types however this strategy has yet to impact MM .",
"Our data support the use of loss of function of BAP1 as a genomic stratification tool to identify rTRAIL-sensitive MM tumours , an approach that may extend to other cancer subtypes .",
"We propose the underlying mechanism involves the transcriptional regulation of expression of components of the apoptotic pathway by the PR-DUB .",
"Our finding has potentially significant and immediately actionable clinical implications for both MM treatment and for the death receptor agonist field .",
"BAP1 has emerged as a key driver of tumorigenesis in MM ( Bueno et al . , 2016 ) .",
"As such , there has been increased focus on this nuclear deubiquitinase and its associated pathways ( LaFave et al . , 2015 ) .",
"While next-generation sequencing reveals MM BAP1 mutation rates in the order of 20–30% ( Guo et al . , 2015; Bueno et al . , 2016; Bott et al . , 2011 ) , immunohistochemical analysis has identified loss of BAP1 function in up to 67% of MM tumours ( Nasu et al . , 2015 ) opening our biomarker-driven approach to a significant proportion of MM patients .",
"BAP1 immunohistochemistry accurately identifies loss of BAP1 function as a consequence of genetic and non-genetic mechanisms ( Nasu et al . , 2015 ) and is already in clinical use as a diagnostic tool; hence the clinical tools for our proposed approach are validated and ready .",
"Our data indicate the BAP1-TRAIL association extends beyond MM to other tumours with loss of BAP1 function .",
"Chromosomal deletions and somatic inactivating mutations have been identified at high frequency in uveal melanoma ( Harbour et al . , 2010 ) , clear cell renal carcinoma ( Peña-Llopis et al . , 2012 ) and cholangiocarcinoma ( Fujimoto et al . , 2015 ) , increasing the potential clinical impact of our discovery .",
"Although loss of BAP1 function is seen at far lower rates in breast carcinoma ( 1% ) ( Stephens et al . , 2012 ) and non-small cell lung carcinoma ( 1% ) ( Owen et al . , 2017 ) , the high incidence of these cancers translates to a large cohort of patients .",
"Focus on death receptor agonists as anti-cancer agents has generated two decades of preclinical studies and the development of numerous clinically tested compounds , all of which have demonstrated limited therapeutic efficacy at phase I/II trials ( Herbst et al . , 2010; von Pawel et al . , 2014; Paz-Ares et al . , 2013; Forero-Torres et al . , 2013 ) .",
"Strategies to overcome this have included the development of increasingly potent death receptor agonists and combination therapies to address resistance factors within the apoptosis pathway ( Holland , 2013; Lemke et al . , 2014b ) .",
"As differential sensitivity has been observed in trials , it has been accepted that identification of a biomarker predicting the therapeutic outcome is of paramount importance ( Ashkenazi , 2015; von Karstedt et al . , 2017 ) .",
"There have been previous attempts to identify predictive biomarkers largely focused on molecular expression panels ( Passante et al . , 2013 ) .",
"Ours is the first unbiased approach to address how the genetic make-up of tumours predicts response to rTRAIL treatment .",
"The identification of BAP1 as a potential genomic biomarker has the potential to reignite the death receptor agonist field of research into which significant investment has already been made .",
"The value of retrospective analysis of clinical trials based on the genomic landscape has clearly been demonstrated in the past ( Lynch et al . , 2004 ) and we wait with interest whether this will be performed on archived tumour tissue , in the context of BAP1 status , from previous trials .",
"Notably there have been no trials of any death receptor agonists in MM or indeed any cancer with a high proportion of BAP1 mutations .",
"We suspect a significantly higher proportion of responders would have been identified in such trials .",
"Our findings also have implications for death receptor agonists as a therapy for BAP1-wild-type tumours as delineation of the underlying mechanism would offer a novel avenue by which to sensitise these tumours .",
"Our mechanistic data implicate transcriptional regulation by the PR-DUB as key to the capacity of BAP1 to modulate death receptor agonist sensitivity .",
"BAP1 is a master genetic regulator and is known to influence the transcription of thousands of genes as supported by our and others’ gene expression data ( Dey et al . , 2012 ) .",
"While we highlight the extrinsic apoptotic pathway and proteins as being significantly altered by BAP1 status , identifying a single factor to explain BAP1-induced TRAIL resistance is extremely challenging .",
"Of more direct clinical significance is our finding that loss of function of either component of the PR-DUB , BAP1 or ASXL1 , results in an increase in death receptor agonist sensitivity .",
"ASXL1 mutations have an important role in the pathogenesis of myeloid neoplasms primarily consisting of nonsense , missense and frameshift mutations resulting in a truncated ASXL1 protein that retains the BAP1-binding domain ( Boultwood et al . , 2010 ) .",
"It has yet to be clarified if this truncated protein possesses dominant-negative or gain-of-function properties in the context of PR-DUB activity ( Balasubramani et al . , 2015 ) .",
"In the case of the former , ASXL1 could potentially predict death receptor agonist sensitivity in myeloid neoplasms .",
"Further research is needed in these malignancies to determine this .",
"Confirmation of the clinical value of BAP1 as a targeting biomarker for death receptor agonists in early phase clinical trials of mesothelioma is the first priority .",
"The clinical tools for this approach are already validated and established facilitating the translation of our discovery into a desperately needed new therapy for this fatal thoracic cancer ."
],
[
"All cell lines were sourced from the Wellcome Trust Sanger Institute except the H226 line that was a kind gift from Dr Peter Szlosarek , Barts Cancer Institute .",
"All cell lines were authenticated by genotyping using Short Tandem Repeat ( STR ) and Sequenom profiling of a panel of 92 single nucleotide polymorphisms for each cell line to ensure non-synonymous cell lines were not used .",
"As a cell line classified as mesothelioma , H513 ( on the list of commonly misidentified cell lines ) was included in the drug screen of 15 mesothelioma cell lines conducted .",
"Use of this cell line however was not carried forward to further experiments in the paper .",
"The 25 early passage MM cultures were purchased from MesobanK ( Rintoul et al . , 2016 ) .",
"All cell lines and cultures were tested for mycoplasma contamination and confirmed to be negative .",
"Cell lines were cultured in RPMI-1640 or Dulbecco's modified Eagle's medium and nutrient mix 12 medium ( DMEM:F12 ) supplemented with 10% fetal bovine serum ( FBS ) , penicillin/streptavidin and sodium pyruvate .",
"Early passage human mesothelioma cultures were cultured in RPMI-1640 medium supplemented with 5% FBS , 25 mM HEPES , penicillin/streptavidin and sodium pyruvate .",
"293 T cells were cultured in Dulbecco's modified Eagle's medium ( DMEM ) supplemented with 10% fetal bovine serum ( FBS ) and 2 mM L-glutamine .",
"All cells were maintained in a humidified environment at 37°C and 5% CO2 .",
"Cells were lysed in radioimmunoprecipitation assay ( RIPA ) buffer ( Sigma-Aldrich , St . Louis , MO ) with protease inhibitors ( Complete-mini; Roche , Switzerland ) on ice to extract protein .",
"20 μg of protein samples were separated by SDS–PAGE and transferred onto nitrocellulose membranes .",
"Membranes were incubated with specific primary antibodies , washed , incubated with secondary antibodies and visualised using an ImageQuant LAS 4000 imaging system ( GE Healthcare , Little Chalfont , NY ) .",
"Antibodies used include BAP1 ( Santa Cruz Biotechnology , Santa Cruz , CA ) Cat# sc-28383 , RRID:AB_626723 ) , caspase 8 ( Cell Signaling Technology , Danvers , MA ) Cat# 9746 , RRID:AB_2275120 ) , c-FLIP ( Enzo Life Sciences , Farmingdale , NY ) Cat# ALX-804-961-0100 RRID:AB_2713915 ) , cIAP1 ( Cell Signaling Technology Cat# 7065S , RRID:AB_10890862 ) , cIAP2 ( Cell Signaling Technology Cat# 3130S , RRID:AB_10693298 ) , FADD ( Cell Signaling Technology Cat# 2782 , RRID:AB_2100484 ) , XIAP ( Cell Signaling Technology Cat# 2045 , RRID:AB_2214866 ) , survivin ( Cell Signaling Technology Cat# 2803 , RRID:AB_490807 ) , α-tubulin ( Cell Signaling #2125 ) , H2AK119Ub ( Cell Signaling Technology Cat# 8240P , RRID:AB_10891618 ) , H2A ( Cell Signaling Technology Cat# 12349 , RRID:AB_2687875 ) , anti-mouse HRP ( Cell Signaling Technology Cat# 7076 , RRID:AB_330924 ) and anti-rabbit HRP ( Cell Signaling Technology Cat# 7074 , RRID:AB_2099233 ) .",
"To detect the ubiquitination status of the histones , the cells were lysed with TBS buffer containing 1% SDS , protease and phosphatase inhibitors .",
"The cell extract was denatured by heating up at 95°C for 10 min and centrifuged at 13000 rpm for 10 min .",
"The supernatant was collected and immunoblotted as described above .",
"Cells were seeded in 96-well plates in 100 μl media per well at a density of 40 , 000 cells/ml 1 day prior to treatment with soluble recombinant TRAIL ( rTRAIL; Peprotech , UK ) or MEDI3039 ( Medimmune , UK ) .",
"XTT ( Applichem , UK; A8088 ) or MTT ( M-2128 , Sigma-Aldrich ) reagent was added on day 3 .",
"The absorbance was measured with a spectrophotometer at a wavelength of 490 nm or 560 nm for XTT or MTT respectively .",
"Relative cell viability was calculated as a fraction of viable cells relative to untreated cells .",
"Full-length BAP1 cDNA was amplified by PCR from pCMV6-AC BAP1 plasmid ( Origene ( Rockville , MD; SC117256 ) and cloned into the lentiviral plasmid pCCL-CMV-flT vector previously described ( Yuan et al . , 2015 ) in place of flT via BamHI and SalI sites , creating the BAP1 vector designated pCCL-CMV-BAP1 .",
"Vectors expressing mutant BAP1 constructs were generated by site-directed mutagenesis ( New England Biolabs ) of the pCCL-CMV-BAP1 vector .",
"The primers used are listed below .",
"All mutations were confirmed by sequencing .",
"BAP1-F CGTGGATCCGCCACCATGAATAAGGGCTGGCTGGA BAP1-R GTCGGTCGACTCACTGGCGCTTGGCCTTGTA C91A-F ATACCCAACTCTGCTGCAACTCATGCCTTGCTG C91A-R CAGCTGGTGGGCAAAGAACATGTTATTCACAATATCATC HBM-F CGCTGCTGCCAAGTCCCCCATGCAGGAGGA HBM-R GCAGCGTCTAGAAAGGCCGGCAGCCGCT CTD-F CGTGGATCCGCCACCATGAATAAGGGCTGGCTGGA CTD-R GTCGTTCGAATCAGTCAGGCTTCCGCTGCTTGTGG T493A-F GCAGACACGGCCTCTGAGATCGGCAGTGCT T493A-R ACTCTCATTGCTGGGGGTGGGTGA ASXL-F AACTACGATGAGTTCATCTGCACCT ASXL-R CTGGTCATCAATCTTGAACTTCTTCCTC The ZS-green luciferase plasmid , pHIV-Luc-ZsGreen ( a gift from Bryan Welm , Addgene plasmid #39196 ) was used for generating ZS-Green luciferase-expressing lentivirus to transduce the H226 cells used in animal experiments .",
"Short hairpin RNAs ( shRNAs ) were expressed as part of a mir30-based GIPZ lentiviral vector ( Dharmacon , Lafayette , CO ) .",
"The clones used in this study include BAP1 ( V2LHS_41473 ) , ASXL1 ( V2LHS_78829 ) , ASXL2 ( V3LHS_313940 ) and the empty GIPZ control vector .",
"Lentiviral vectors were produced by co-transfection of 293 T cells with construct plasmids together with the packaging plasmids pCMV-dR8 . 74 and pMD2 . G ( kind gifts from Dr Adrian Thrasher , UCL , Addgene plasmid #22036 and #12259 ) in the presence of a DNA transfection reagent jetPEI ( Source Bioscience UK Ltd ) .",
"Lentiviruses were concentrated by ultracentrifugation at 17 , 000 rpm ( SW28 rotor , Optima LE80K Ultracentrifuge , Beckman Coulter , Brea , CA ) for 2 hr at 4°C .",
"To determine the titres of prepared lentiviruses 293 T cells were transduced with serial dilutions of viruses in the presence of 8 μg/ml Polybrene ( Sigma-Aldrich ) and BAP1 expression was assessed by flow cytometry .",
"shRNA- and luciferase-expressing vectors were assessed by analysis of GFP expression .",
"Cell lines were transduced in the presence of 8 μg/ml Polybrene at a range of MOIs and transduction efficacy was assessed by flow cytometry for BAP1 expression .",
"We pre-processed and normalised raw CEL files from Affymetrix Human Genome U219 array plate hybridisations with the Multi-Array Average ( RMA ) method ( Irizarry et al . , 2003 ) .",
"We discarded transcripts with low sample variance and consolidated duplicated genes by averaging their expression values across duplicates .",
"The resulting data were subsequently normalised ( μ = 0 , σ = 1 ) sample-wise and gene-median centred .",
"Gene expression was averaged across three biological replicates of H226 transduced cells with either a C91A mutant or a wild-type BAP1 construct .",
"SPIA pathway analysis as described in Tarca et al ( Tarca et al . , 2009 ) was performed on those genes with an adjusted p<0 . 05 and a fold change of >1 .",
"All flow cytometry analysis was performed on a LSR Fortessa analyser ( Becton Dickinson , Franklin Lakes , NJ ) .",
"For analysis of BAP1 expression cells were stained with primary antibody to BAP1 ( Santa Cruz Biotechnology Cat# sc-28383 , RRID:AB_626723; 1:50 ) and then with an AlexaFluor 488-conjugated anti-mouse antibody ( Thermo Fisher Scientific Cat# A-21202 , RRID:AB_141607; 1:200 ) .",
"For analysis of apoptosis and cell death all floating and adherent cells were harvested and stained with an Annexin V AlexaFluor 647-conjugated antibody ( Thermo Fisher Scientific Cat# A23204 , RRID:AB_2341149 ) and 4’ , 6-diamidino-2-phenylindole ( DAPI; Sigma-Aldrich , 200 μg/ml ) .",
"For analysis of DR4 and DR5 expression on cell surface cells were stained with PE-conjugated antibody ( DR4 - BioLegend , San Diego , CA ) Cat# 307205 , RRID:AB_314669 , DR5 - BioLegend Cat# 307405 , RRID:AB_314677 , Isotype control - Biolegend #400112; 1:100 ) .",
"FlowJo software was used to analyse all data .",
"H226 cells were seeded at 2 . 5 × 103 cells per well into 96-well Greiner micro-clear imaging plates in DMEM 10% FBS .",
"After 48 hr , cells were fixed in 4% PFA for 10 min at room temperature and permeabilised in 0 . 3% NP-40 in PBS for 10 min .",
"Cells were blocked in 1% BSA in 0 . 1% PBS tween for 1 hr at room temperature .",
"Ubiquityl-histone H2A ( Lys119 ) primary antibody ( Cell Signaling , #8240 ) was incubated overnight at 4°C , before incubating for 1 hr at room temperature with Alexafluor 488-conjugated anti-rabbit secondary antibody .",
"Nuclei were stained with Hoechst 33342 ( Thermo Fisher Scientific Cat# 62249 ) .",
"Images were acquired ( n = 3 ) with a BioTek Cytation3 Multimode reader .",
"Using a 10x objective 4 fields of view were acquired per well ( n = 3 ) and the level of nuclear ubiquityl-histone H2A intensity was determined within the primary nuclear mask and normalised to total cell number .",
"BAP1 immunohistochemistry of human early passage cell lines was conducted on sections of cell pellets mounted on slides .",
"Automated staining on a Leica Bond III staining platform was used .",
"Slides were incubated with BAP1 primary antibody ( Santa Cruz Biotechnology Cat# sc-28383 , RRID:AB_626723; 1:150 ) for 15 min at room temperature .",
"Epitope retrieval was completed using HIER using Leica Bond ER2 ( high pH ) for 30 min and a Leica Bond Polymer Refine with DAB chromogen detections system used .",
"Appropriate ethical approval was obtained from the NHS Health Research Authority National Research Ethics Service to carry out this work ( reference 14/LO/1527 ) .",
"Informed consent to conduct research on samples collected and to publish results was obtained from patients .",
"The diagnosis of mesothelioma was confirmed histologically for all patients prior to consent and surgery .",
"Patients underwent pleurectomy , following which primary pleural tissue was sectioned into fragments measuring approximately 2 mm3 .",
"These tissue explants were cultured in 50% neurobasal and 50% DMEM:F12 , supplemented with B27 ( 2% ) , EGF ( 20 ng/ml ) and FGF ( 10 ng/ml ) .",
"After 24 hr the explants were treated with rTRAIL ( vehicle , 50 ng/ml , 100 ng/ml or 200 ng/ml ) for a further 24 hr , following which explants were either fixed for PARP immunohistochemistry .",
"The explants were fixed in 10% neutral-buffered formalin ( NBF ) for 24 hr and then transferred into 70% ethanol followed by paraffin embedding .",
"Subsequently , 5 μm sections were used for immunohistochemistry , as previously described ( Busacca et al . , 2016 ) .",
"All animal studies were approved by the University College London Biological Services Ethical Review Committee and licensed under the UK Home Office regulations and the Evidence for the Operation of Animals ( Scientific Procedures ) Act 1986 ( Home Office , London , UK ) .",
"Mice were purchased from Charles River , kept in individually ventilated cages under specific pathogen-free conditions and had access to sterile irradiated food and autoclaved water ad libitum .",
"12 8 week old NOD . CB17-Prkdcscid/NcrCrl ( NOD SCID ) mice ( Charles River , UK; RRID:IMSR_CRL:394 ) were injected with 1 × 106 H226 cells transduced with a plasmid containing wild-type BAP1 and luciferase on the right flank and with a plasmid containing a catalytically inactive BAP1-mutant ( C91A ) and luciferase on the left flank in a 1:1 mixture of Matrigel ( Corning , Corning , NY ) and medium .",
"Tumour size was assessed by bioluminescence in vivo imaging system ( IVIS , PerkinElmer , Waltham , MA ) 15 min following intraperitoneal injection of 0 . 2 ml ( 2 mg ) luciferin .",
"Tumours were allowed to establish for 2 weeks prior to baseline assessment of size at day 13 .",
"Mice were then divided into two groups each of which received either 600 μg TRAIL or vehicle 6 days a week from day 14 until day",
"40 . Bioluminescence was measured on days 0 , 13 , 19 , 26 and",
"41 . Mice were sacrificed on day 42 and tumours harvested for measurement .",
"TRAIL used in the mouse experiment was made in Henning Walczak’s laboratory as per the established protocol ( Ganten et al . , 2006 ) .",
"Statistical analysis was performed using GraphPad Prism ( GraphPad Software , CA , USA ) .",
"t-test was used to analyse differences between two groups whilst the analysis of variance ( ANOVA ) test with a Tukey post-hoc analysis was used to analyse differences between three groups .",
"For multiple groups measured over multiple time points repeated measures ANOVA was used .",
"All in vitro tests were performed in triplicate and all data are represented as mean values ± standard error of mean unless otherwise stated ."
]
] | [
"Malignant mesothelioma ( MM ) is poorly responsive to systemic cytotoxic chemotherapy and invariably fatal .",
"Here we describe a screen of 94 drugs in 15 exome-sequenced MM lines and the discovery of a subset defined by loss of function of the nuclear deubiquitinase BRCA associated protein-1 ( BAP1 ) that demonstrate heightened sensitivity to TRAIL ( tumour necrosis factor-related apoptosis-inducing ligand ) .",
"This association is observed across human early passage MM cultures , mouse xenografts and human tumour explants .",
"We demonstrate that BAP1 deubiquitinase activity and its association with ASXL1 to form the Polycomb repressive deubiquitinase complex ( PR-DUB ) impacts TRAIL sensitivity implicating transcriptional modulation as an underlying mechanism .",
"Death receptor agonists are well-tolerated anti-cancer agents demonstrating limited therapeutic benefit in trials without a targeting biomarker .",
"We identify BAP1 loss-of-function mutations , which are frequent in MM , as a potential genomic stratification tool for TRAIL sensitivity with immediate and actionable therapeutic implications ."
] | [
"Two patients with the same disease who receive the same treatment may respond in different ways .",
"This variation often arises from differences in each patient’s genetic code .",
"Genes encode proteins , and proteins are the targets of most medical drugs and thus determine the patient’s response to treatment .",
"A major advance in the 21st century is that doctors recognise that patients can respond differently to the same treatment and now try to predict which patients will respond best to which drug – an approach known as personalised medicine .",
"Cancer treatment has been at the forefront of personalised medicine because mutations in different genes underlie each different cancer .",
"By analysing which mutations are present in a cancer , doctors can thus predict which drug ( or combination of drugs ) will be most effective .",
"This approach has been used successfully in several cancers , including breast and lung cancer , leading to fewer patients being exposed to ineffective treatments and their associated side effects and costs .",
"Mesothelioma is a cancer of the lining of the lung that is associated with exposure to the mineral asbestos .",
"Current treatment options for mesothelioma are unfortunately limited and not very effective .",
"No personalised treatments are currently in use and new treatment approaches are desperately needed .",
"Kolluri , Alifrangis , Kumar , Ishii et al . set out to determine if any of the mutations commonly seen in mesothelioma affected how the cancer would respond to 94 anticancer drugs that are either in use or in development .",
"In the laboratory , mesothelioma cells that have mutations in the gene that codes for a protein known as BRCA associated protein-1 ( or BAP1 for short ) were killed much more effectively by a drug known as TNF-related apoptosis-inducing ligand ( TRAIL ) .",
"The same link was seen in experiments with tumours of mesothelioma cells that had been transplanted into mice , and for fragments of mesothelioma tumours taken from patients .",
"When Kolluri et al . studied why these tumours might be killed more effectively with TRAIL , they found that mutations in the gene for BAP1 result in a change in the levels of proteins that transmit the signal from the receptors targeted by the TRAIL drug .",
"These findings may one day result in a new approach to treating patients with mesothelioma .",
"But first , the next step would be to conduct a clinical trial of TRAIL in patients with mesothelioma and assess if those with tumours that have mutations in the gene for BAP1 do indeed respond better .",
"If this proves to be the case , this would result in a new personalised treatment option for patients that suffer from this disease ."
] | 2018 |
[
"Introduction",
"Results",
"Discussion",
"Materials and methods"
] | [
"developmental biology",
"neuroscience"
] | Imp/IGF2BP levels modulate individual neural stem cell growth and division through myc mRNA stability | elife-51529-v2 | [
[
"The many cells of the brain are produced through the highly regulated repeated divisions of a small number of neural stem cells ( NSCs ) .",
"NSCs grow and divide rapidly in order to supply the cells of the developing brain , but must be restrained to prevent tumour formation .",
"Individual NSCs produce characteristic lineages of progeny cells ( Kriegstein and Alvarez-Buylla , 2009; Merkle et al . , 2007 ) , which vary in number suggesting differences in division and growth rates during development .",
"However , the mechanisms differentially regulating the growth and division of individual NSCs are currently unknown .",
"Many of the processes and factors regulating neurogenesis are conserved between mammals and insects , making Drosophila an excellent model system to study NSC regulation ( Homem and Knoblich , 2012 ) .",
"During Drosophila neurogenesis , NSCs , also known as neuroblasts ( NBs ) , divide asymmetrically , budding-off a small progeny cell , the ganglion mother cell ( GMC ) , which divides into neurons that progress through differentiation .",
"During larval neurogenesis , the NB divides on average once every 80 min ( Homem et al . , 2013 ) and regrows between divisions to replace its lost volume , maintaining the proliferative potential of the cell ( Homem and Knoblich , 2012 ) .",
"However , average measurements of growth and division mask considerable heterogeneity between the behaviour of individual NBs in the brain over developmental time .",
"Individual NBs produce unique lineages of neurons ( Pereanu and Hartenstein , 2006 ) , with characteristically different clone sizes ( Yu et al . , 2013 ) .",
"Individual NBs also have differing division frequencies ( Hailstone et al . , 2019 ) and terminate division at different times ( NB decommissioning ) ( Yang et al . , 2017a ) .",
"This individual control ensures that the appropriate number of each neuron type is produced in the correct location during the construction of the brain .",
"Systemic signals such as insulin and ecdysone signalling drive NB growth and division , with a particularly strong influence at the transitions between developmental stages ( Chell and Brand , 2010; Géminard et al . , 2009; Homem et al . , 2014; Ren et al . , 2017; Rulifson et al . , 2002; Sousa-Nunes et al . , 2011; Syed et al . , 2017 ) .",
"However , the reproducible heterogeneity between individual NBs implies the existence of an unknown local or cell-intrinsic signal , acting in addition to the systemic signals to determine the proliferation of each NB .",
"The temporal regulation of NB proliferation and progeny fate has been well studied in the embryo and larva , and many key factors have been identified ( Doe , 2017; Li et al . , 2013; Miyares and Lee , 2019; Rossi et al . , 2017 ) .",
"The developmental progression of larval NBs is characterised by the levels of two conserved RNA-binding proteins ( RBPs ) , IGF2 mRNA-binding protein ( Imp/IGF2BP2 ) and Syncrip ( Syp/hnRNPQ ) ( Liu et al . , 2015 ) .",
"Imp and Syp negatively regulate each other and are expressed in opposing temporal gradients through larval brain development ( Liu et al . , 2015 ) : Imp level in the NB declines through larval development while Syp level correspondingly increases .",
"Imp and Syp play numerous key roles in larval neurogenesis .",
"The levels of Imp and Syp are known to determine the different types of neuron produced by the NBs over time , through post-transcriptional regulation of the transcription factor ( TF ) chinmo ( Liu et al . , 2015; Ren et al . , 2017 ) .",
"The loss of Syp results in an enlarged central brain , in part due to an increase in NB proliferation rate ( Hailstone et al . , 2019 ) .",
"In pupal NBs , declining Imp expression allows NB shrinkage and Syp promotes NB termination ( Yang et al . , 2017a ) .",
"Temporal regulation of the Imp/Syp gradients depends on the upstream temporal patterning system ( Narbonne-Reveau et al . , 2016; Ren et al . , 2017; Syed et al . , 2017 ) .",
"The timing and rates of change of these RBP levels differ substantially between classes of NB , and to a lesser degree between NBs of the same class ( Liu et al . , 2015; Syed et al . , 2017; Yang et al . , 2017a ) .",
"However , it is unknown if the intrinsic levels of Imp and Syp in each NB play a role in controlling the growth and division rates of individual NBs during their main proliferative window in the larva .",
"Imp and Syp are RBPs and can modify the protein complement of a cell via post-transcriptional modulation of mRNA localisation , stability and translation rates ( Boylan et al . , 2008; Geng and Macdonald , 2006; Hobor et al . , 2018; McDermott et al . , 2012; McDermott et al . , 2014; Medioni et al . , 2014; Munro et al . , 2006 ) .",
"Cell growth and proliferation are classically thought to be regulated at the level of transcription by pro-proliferative TFs .",
"Various signalling pathways converge to promote cell growth and proliferation through transcriptional upregulation of the conserved TF and proto-oncogene , Myc ( Dang , 2012; Delanoue et al . , 2010; Levens , 2010; Teleman et al . , 2008 ) .",
"Myc interacts with a binding partner , Max , to exert widespread transcriptional effects , binding upwards of 2000 genes in Drosophila ( Orian et al . , 2003 ) .",
"In Drosophila , Myc is best known for its role in promoting cell growth through increased ribosome biogenesis ( Grewal et al . , 2005 ) , and also accelerates progression through the G1 phase of the cell cycle in the developing wing , though this does not affect overall cell cycle length ( Johnston et al . , 1999 ) .",
"It is unclear whether the transcriptional activation of pro-proliferative TFs , such as Myc and its downstream targets , is overlaid by post-transcriptional regulatory mechanisms executed by RBPs , such as Imp and Syp , which could increase the precision and flexibility of the system .",
"Here , we examine the role of the Imp/Syp temporal gradient in regulating NB size and division during larval neurogenesis .",
"We show that the upregulation of Imp increases NB division and size , while Syp influences these processes indirectly via its negative regulation of Imp .",
"We use a genome-wide approach to determine the mRNA targets bound by Imp in the brain and identify myc mRNA among the top 15 targets of Imp .",
"Single molecule fluorescent in situ hybridisation ( smFISH ) shows that myc mRNA is stabilised by Imp , leading to increased Myc protein levels , NB growth and proliferation .",
"We compare NB types with different Imp levels and find that low Imp levels result in unstable myc mRNA , which restrains NB growth and division .",
"Finally , at an earlier time point , when Imp expression is heterogeneous between individual NBs , we find that higher Imp correlates with increased myc mRNA half-life .",
"We propose a model in which Imp post-transcriptionally regulates myc mRNA stability to fine-tune individual NB size and division rate in their appropriate developmental context ."
],
[
"To investigate the roles of the opposing Imp and Syp gradients in the NB , we used RNAi knockdown to manipulate the level of these RBPs ( Figure 1—figure supplement 1 ) .",
"We studied the type I NBs , the most numerous NB type in the brain , which are also very convenient to analyse , as they have a simple division hierarchy with each asymmetric division producing a GMC that divides only once more to produce two neurons or glia ( Bello et al . , 2008; Boone and Doe , 2008; Bowman et al . , 2008 ) .",
"In the wandering L3 stage ( wL3 ) brains all type I NBs express high levels of Syp and low of Imp ( Figure 1—figure supplement 1A ) .",
"We depleted Syp or Imp from the NBs with Syp knockdown and Imp knockdown RNAi constructs using the GAL4-UAS system , driven by insc-GAL4 ( Betschinger et al . , 2006 ) .",
"In NBs Imp and Syp negatively regulate each other and therefore the Syp knockdown results in Imp upregulation ( Figure 1—figure supplement 1B ) ( Liu et al . , 2015 ) .",
"We distinguished between direct effects of Syp depletion and indirect effects due to upregulated Imp expression by analysing Imp Syp double knockdown mutants ( Figure 1—figure supplement 1C ) ( Yang et al . , 2017a ) .",
"We also examined Imp overexpression brains , but the UAS overexpression construct only produces a very limited upregulation of Imp in the type I NB at the wL3 stage ( Figure 1—figure supplement 1D ) , as previously observed ( Liu et al . , 2015; Yang et al . , 2017a ) .",
"Therefore we primarily use the Syp knockdown to upregulate Imp .",
"We first examined the roles that Imp and Syp play in influencing type I NB size .",
"Our results show that higher Imp promotes larger size of type I NBs at wL3 , and Syp acts indirectly through its negative regulation of Imp .",
"Imp-depleted NBs are almost half the size of wild type NBs and NBs that overexpress Imp are 1 . 4-fold larger in midpoint area ( Figure 1A , A’ , Materials and methods ) .",
"Syp-depleted NBs are 1 . 5-fold larger than wild type .",
"We tested whether this effect is direct or indirect by studying the size of NBs in the Imp Syp double knockdown .",
"Our results show that Imp depletion suppresses the increase in NB size observed in Syp knockdown mutants , which indicates that Syp only plays an indirect role in type I NB size , through its repression of Imp .",
"NB size is affected by both cell growth and division rate so we then tested whether NB division rate is also sensitive to Imp levels .",
"We incubated ex vivo explanted brains in 5-ethynyl-2’-deoxyuridine ( EdU ) -containing media for four hours to label the progeny cells produced during this time ( see Materials and methods ) .",
"The number of labelled progeny was decreased by more than half in the Imp RNAi brains compared to wild type ( Figure 1B , B’ ) , which suggests that the decreased NB size in the Imp knockdown is not due to an increased division rate .",
"The number of progeny was increased 1 . 4-fold in the Imp overexpressing brains and increased 1 . 6-fold in the Syp RNAi brains , in which Imp is strongly upregulated , compared to wild type .",
"This phenotype is consistent with the increased proliferation rate previously observed in Syp knockdown brains with ex vivo culture and live imaging ( Hailstone et al . , 2019 ) .",
"However , the increased proliferation was lost in the Imp Syp double knockdown brains .",
"These results , together with our previous findings that Imp overexpression prevents NB shrinkage in the pupa and extends NB lifespan ( Yang et al . , 2017a ) , suggest that low levels of Imp in the late larval NBs restrains NB growth and division , ensuring the brain growth is limited appropriately during its development .",
"Imp is an RBP , so is likely to exert its function in the NB through regulation of the RNA metabolism of its key target mRNA transcripts .",
"In an effort to identify strong candidate targets , we identified the transcripts bound by Imp in the brain .",
"To achieve this aim we performed Imp RNA immunoprecipitation and sequencing ( RIPseq ) in larval brain lysates ( see Materials and methods ) .",
"We identified 318 mRNA targets that were significantly enriched in the Imp pulldown compared to input brain RNAseq ( using the thresholds DESeq2 . padj < 0 . 01 and DESeq2 . log2FoldChange > 2 ) ( Figure 2—figure supplement 1A , B , Supplementary file 1 ) .",
"The list of targets includes known Imp targets such as chickadee ( target rank: 37 ) ( Medioni et al . , 2014 ) , as well as mRNAs that have previously been shown to be regulated by Imp .",
"Imp binds syp mRNA ( target rank: 103 ) , which indicates a post-transcriptional mechanism for the previously observed negative regulation of Syp by Imp ( Liu et al . , 2015 ) .",
"Another Imp target is chinmo ( target rank: 55 ) , which is known to be post-transcriptionally regulated by Imp to determine the progeny fate of NBs in the mushroom body ( MB ) , the centre for memory and learning .",
"Chinmo is also regulated by Imp in type II NBs ( Liu et al . , 2015; Ren et al . , 2017; Syed et al . , 2017 ) and during NB self renewal ( Dillard et al . , 2018; Narbonne-Reveau et al . , 2016 ) .",
"Imp binds a number of long non-coding RNAs , including CR43283/cherub ( target rank: 5 ) .",
"cherub is also a binding target of Syp and facilitates Syp asymmetric segregation during type II NB division ( Landskron et al . , 2018 ) .",
"The large number of Imp targets identified by RIPseq indicates that Imp has a broad range of roles in the developing brain .",
"Imp has been shown to regulate mRNA localisation , stability , and translation ( Degrauwe et al . , 2016 ) .",
"Our results suggest that examining the Imp targets will provide further insight into the role of Imp in neurogenesis and the critical importance of post-transcriptional regulation .",
"To identify the key candidate mRNA targets responsible for the Imp NB size and division phenotypes , we examined the gene ontology ( GO ) annotations of the top 40 Imp targets ( Figure 2A ) .",
"We searched for genes annotated to play a role in cell growth , cell size , cell cycle and neural development , as well as regulatory genes with RNA-binding or DNA-binding function ( Figure 2B , Supplementary file 1 ) .",
"We identified myc ( target rank: 13 ) as the top candidate that could explain the Imp phenotype , based on these GO categories .",
"As discussed in the introduction , myc is a master transcription factor regulator of growth and division in diverse model systems .",
"In Drosophila it is primarily known as a driver of cell growth ( Grewal et al . , 2005 ) , and is a determinant of self renewal in the type II NB ( Betschinger et al . , 2006 ) .",
"We also identified a second member of the Myc transcriptional network , mnt , as an mRNA target bound by Imp ( target rank: 36 ) .",
"Mnt competes with Myc for binding to Max , and promotes opposed transcriptional effects ( Loo et al . , 2005; Orian et al . , 2003 ) .",
"We first focussed on myc , and later investigated mnt .",
"myc is the 13th most enriched target of Imp and is a very promising candidate as a direct mediator of the Imp phenotype in NBs .",
"To further examine the interaction between Imp and myc mRNA , we reanalysed a previously published dataset of Imp iCLIP ( individual nucleotide resolution cross-linking and immunoprecipitation ) performed in S2 cells ( Hansen et al . , 2015 ) .",
"The iCLIP data shows that Imp directly binds the myc transcript ( Figure 2—figure supplement 1C ) , which supports our identification of myc mRNA as an Imp target in the brain .",
"The iCLIP experiment identifies Imp binding sites primarily in the myc untranslated regions ( UTRs ) and binding signal is enriched in the extended 3’ UTR of the longer mRNA isoform .",
"In our brain Imp RIPseq dataset , we also see reads throughout the extended 3’ UTR , suggesting that Imp binds to the long myc mRNA isoform ( Figure 2—figure supplement 1D ) .",
"Notably , the full myc 3’ UTR extension is expressed in the brain ( Figure 2—figure supplement 1E ) but it is truncated early in the S2 cells ( Figure 2—figure supplement 1F ) , so the fully extended transcript in the brain may contain additional Imp binding sites .",
"The results in S2 cells support our identification of myc mRNA as a target of Imp in the brain , highlighting the hypothesis that Imp is a key regulator of myc in the NB .",
"To test the hypothesis that Myc protein levels are regulated by Imp , we used antibody staining in wild type and knockdown type I NB lineages .",
"We found that Imp is required to maintain correct Myc levels in the NB .",
"We observed Myc protein expression in type I NBs , but not in the surrounding GMCs or neurons ( Figure 3A ) .",
"Myc protein level was increased more than 2-fold in the Syp RNAi NBs compared to wild type ( Figure 3B , quantitated in 3C ) , while this effect was lost in the double Imp Syp depleted NBs .",
"Directly overexpressing Imp resulted in a small increase in Myc protein level ( 1 . 2-fold increase on wild type level ) ( Figure 3C ) .",
"The effect of Imp overexpression on Myc protein level is smaller than that in Syp knockdown NBs as the overexpression construct produces a smaller upregulation of Imp ( Figure 1—figure supplement 1 ) .",
"Imp knockdown produced a small decrease in Myc protein level ( Figure 3C ) , as expected because Imp levels are already very low in wild type type I NBs .",
"These data indicate that Imp upregulation increases Myc protein level in the NB , while Syp’s effect on Myc is indirect , as it requires Imp .",
"We next examined the effect of Imp and Syp on Mnt , the antagonist of Myc , also identified as an Imp target .",
"Using antibody staining , we found that Mnt protein is expressed in the type I NB , as well as in the progeny cells of the lineage ( Figure 3—figure supplement 1A ) .",
"However , knockdowns of Imp and Syp have no effect on the levels of Mnt protein .",
"Therefore , we conclude that Mnt is not likely to be a key target responsible for the NB growth and division phenotype of Imp .",
"We then asked whether the upregulation of Myc by Imp could be responsible for the phenotype of increased type I NB growth and division .",
"We overexpressed the Myc open reading frame ( ORF ) in type I NBs ( Figure 3—figure supplement 1B , Materials and methods ) and found a significant 1 . 3-fold increase in NB size ( Figure 3D ) .",
"Myc knockdown produced a small and not significant decrease in NB size .",
"We used a Myc Syp double knockdown to confirm that upregulated Myc is responsible for the increased size of Syp knockdown NBs ( in which Imp is upregulated ) .",
"We found that the increased NB size in the Syp knockdown is lost in the Myc Syp double knockdown brains ( Myc_Syp RNAi NBs are 0 . 7x the size of wild type ) , supporting the hypothesis that Imp regulates NB size through upregulation of Myc .",
"We tested the effect of Myc overexpression on type I NB division rate , and observed an increased division rate in the Myc OE compared to wild type ( Myc OE: 4 . 04 EdU-labelled progeny per NB , Figure 3E ) .",
"The observed increase in division rate is a surprising result as previous work in the wing disc showed that Myc overexpression increased cell size without affecting division rate ( Johnston et al . , 1999 ) , highlighting that Myc could regulate cell size and division rate in distinct ways in different tissue contexts .",
"In the NB , we find that increased Myc protein levels can explain the increased size and division rate that occur in response to overexpressing Imp .",
"However , Imp levels are very low in wL3 wild type type I NBs ( Figure 1—figure supplement 1 ) , which may limit Myc protein expression and restrain NB growth and division .",
"In order to further characterise the regulation of myc mRNA by Imp , we visualised myc mRNA transcripts using smFISH in type I NBs ( Yang et al . , 2017b ) .",
"The two annotated RNA isoforms of myc are identical except that the longer isoform includes a 3’ UTR extension of 5 . 7 kb ( Figure 4A ) ( FlyBase , Thurmond et al . , 2019 ) .",
"This additional UTR sequence potentially includes substantial regulatory sequence , including multiple binding sites for Imp according to iCLIP in S2 cells ( Hansen et al . , 2015 ) ( Figure 2—figure supplement 1C ) , which could allow differential regulation of the two isoforms .",
"smFISH probes against the myc intron and common exon show myc transcription and mature myc transcripts in the type I NB ( Figure 4A , B , Figure 4—figure supplement 1A , Supplementary file 2 ) .",
"Co-staining with the common exon probe and a long-UTR-specific probe , showed that all cytoplasmic transcripts in the type I NB are positive for both probes ( Figure 4A , C ) .",
"This result shows that the extended UTR isoform of myc ( myclong ) is the predominant isoform expressed in the NB .",
"Therefore , we used probes specifically against the myclong isoform for the following quantitative experiments .",
"Imp binds to myc mRNA and could upregulate Myc protein either through increasing myc mRNA levels or increasing Myc translation .",
"To distinguish between these possibilities , we stained brains with myclong-specific smFISH probes and quantitated the RNA expression in individual NBs within the mixed-cell tissue ( Figure 4—figure supplement 1B , C , Materials and methods , Mueller et al . , 2013 ) .",
"We measured the effects of Imp knockdown , Imp upregulation using the Syp knockdown , and suppression in the Imp Syp double knockdown .",
"Due to the minimal upregulation of Imp with the Imp overexpression construct ( Figure 1—figure supplement 1 ) and correspondingly small upregulation of Myc protein ( Figure 3C ) , we did not quantitate the myc mRNA expression in the Imp overexpression brains ( Figure 4—figure supplement 1B ) .",
"The number of myclong transcripts per NB is significantly reduced in the Imp knockdown , and is significantly increased in the Syp knockdown ( Figure 4D , E ) .",
"The transcript number is similar to wild type levels in the Imp Syp double knockdown , showing that Imp , rather than Syp , is the primary regulator of the number of myclong transcripts observed in the NB .",
"We interpret our results as showing that the increase in myc transcript number observed when Imp is upregulated causes the observed increase in Myc protein level .",
"In contrast , Imp is unlikely to upregulate Myc protein levels primarily through an increase in myc translation efficiency , although the data does not exclude the possibility that this mechanism makes a minor contribution to Myc protein upregulation .",
"The number of mature transcripts is affected by both transcription rate and mRNA stability .",
"In order to distinguish between a role for Imp in regulating myc transcription rate or myc transcript stability , we used smFISH measurements to estimate the transcription rate and mRNA half-life of myclong in each NB ( Bahar Halpern and Itzkovitz , 2016 ) .",
"We used the average intensity of a single transcript to calculate the number of nascent transcripts at the transcription foci , which indicates the relative transcription rate ( Mueller et al . , 2013 , Materials and methods ) .",
"We found that while the number of nascent transcripts is not significantly changed in the Imp knockdown or the Syp knockdown , it is significantly reduced in the Imp Syp double knockdown ( Figure 4F ) .",
"We used this measurement to estimate the transcription rate and showed that myclong transcription is unchanged in the single knockdowns , but is significantly reduced in the Imp Syp double knockdown ( Figure 4G , Materials and methods , [Bahar Halpern and Itzkovitz , 2016] ) .",
"This change in myc transcription in Imp Syp double knockdown NBs is unexpected , and may be an indirect effect through other transcription factors that Imp and Syp regulate , or a feedback loop of Myc autoregulation .",
"To determine the post-transcriptional role of Imp in regulating myc transcript level we calculated the myc mRNA half-life , allowing direct comparison between genotypes despite differing transcription rates ( Materials and methods , [Bahar Halpern and Itzkovitz , 2016] ) .",
"We found that the half-life of myclong is not significantly changed in the Imp knockdown , but is significantly increased in the Syp knockdown , in which Imp is upregulated ( wild type = 18 . 6 mins , Syp RNAi = 43 . 2 mins ) ( Figure 4H ) .",
"This increase in myclong mRNA half-life is suppressed in Imp Syp double knockdown NBs , in which there is no significant difference compared to wild type .",
"It is not surprising that the Imp knockdown has no effect on myc mRNA half-life when compared to wild type NBs , because Imp levels are very low in wild type type I NBs at the wL3 stage .",
"We find that Imp’s main direct role is to promote myclong mRNA stability and this results in upregulation of Myc protein , which promotes NB growth and division .",
"To characterise the regulation of Myc in other cells in the type I NB lineage , we used smFISH to observe myc transcription and cytoplasmic transcripts in the whole lineage ( Figure 4B , C , Figure 4—figure supplement 1 ) .",
"We found that while myc is transcribed and transcripts are present in all cells in the lineage , Myc protein is limited to the NB only ( Figure 3A ) , suggesting that myc transcripts are translationally repressed in the progeny GMCs and neurons .",
"The repression of Myc protein expression in the progeny cells was unaffected by manipulation of Imp and Syp levels , driven by insc-GAL4 ( Figure 3B ) , suggesting that these two RBPs are not responsible for translational regulation of myc .",
"While in the type II NB lineage , Brat is thought to translationally repress myc in progeny cells ( Betschinger et al . , 2006 ) , it is not known to act in the type I lineage .",
"We conclude that Myc is regulated in the NB lineages by mRNA stability through Imp and by translation , perhaps through a different RBP .",
"The gradient of Imp level decline with developmental age is different between different NB types ( Liu et al . , 2015; Syed et al . , 2017; Yang et al . , 2017a ) .",
"Therefore , we used smFISH to explore whether myc mRNA is also differentially stable in distinct NB types .",
"Imp level declines more slowly in MB NBs compared to the rest of the type I NBs in the central brain and higher Imp expression remains in the MB NBs at wL3 ( Liu et al . , 2015; Yang et al . , 2017a ) .",
"In each NB , we used smFISH to measure myclong transcription , myclong mRNA half-life and myclong transcript number as well as NB size and Imp protein level ( Figure 5A ) .",
"We identified MB NBs by their elevated Imp expression ( Figure 5A , B ) .",
"We found that MB NBs are 1 . 5-fold larger than type I NBs ( Figure 5C ) .",
"The myc mRNA half-life is 2 . 5-fold higher in the MB NBs ( type I NBs = 18 . 79 mins , MB NBs , 51 . 34 mins ) ( Figure 5D , Materials and methods ) , while myc transcription rate is slightly reduced in the MB NBs compared to the type I NBs ( Figure 5E ) .",
"Plotting these variables together shows clear differences between the type I NBs and MB NBs .",
"While type I NBs show low Imp , unstable myc mRNA and small NB size , the MB NBs have higher Imp , more stable myc mRNA and larger NB size ( Figure 5F ) .",
"These results support our earlier finding that higher Imp promotes myc mRNA stability and NB growth and indicates that Imp is a key regulator of differences between different classes of NBs .",
"We also measured Myc protein levels and NB division rates in MB NBs and type I NBs , although these could not be multiplexed into the same images as the smFISH measurements .",
"We found that Myc protein level is 1 . 4-fold higher in MB NBs compared to type I NBs ( Figure 5G ) .",
"Finally , we measured NB division rate by incubation with EdU , which showed that MB NBs have a faster division rate than type I NBs ( Figure 5H ) .",
"Collectively , these results suggest that the higher level of Imp maintained into the late L3 stage in the MB NBs increases myc mRNA stability , causing increased Myc protein levels and increased NB growth and division relative to type I NBs at the same stage .",
"Imp levels decline in NBs as larval development progresses ( Liu et al . , 2015 ) so we next asked what role Imp plays in myc regulation in earlier larval neurogenesis .",
"We studied brains at 72 hr after larval hatching ( ALH ) when the Imp protein level in the NB is higher than at the later wL3 stage and there is substantial heterogeneity in Imp expression level between the individual NBs ( Figure 6A ) .",
"We first compared the average populations of 72 hr ALH NBs to wL3 NBs .",
"Imp protein levels were measured from endogenous GFP-tagged Imp and found to be significantly increased in the 72 hr ALH NBs compared to wL3 , as expected ( Figure 6B ) .",
"We then measured NB size and found that NBs are significantly larger at 72 hr ALH ( Figure 6C ) .",
"smFISH quantitation of myclong transcription and half-life at 72 hr ALH showed that myclong half-life is increased at 72 hr ALH ( Figure 6D ) , but there was no significant difference in myclong transcription rate ( Figure 6E ) .",
"To validate the role of Imp in early larval neurogenesis , we measured NB size in Imp-depleted early NBs .",
"NBs were much smaller in the Imp knockdown than in Imp::GFP ( wild type ) brains at 72 hr ALH ( Figure 6F ) .",
"This data supports the model that the decline in Imp levels during larval development reduces myc mRNA stability , restraining NB growth and division at the end of the larval stage .",
"Pooled averages hide the substantial variation in between individual NBs at 72 hr ALH so we asked whether the Imp level in each NB determines myclong half-life .",
"We used a correlation matrix to examine the relationships between the variables measured in each individual NB at 72 hr ALH ( Figure 6G , Figure 6—figure supplement 1 ) and found that Imp level correlates with myclong half-life ( r = 0 . 344 , p<0 . 01 ) in individual NBs .",
"We also found a significant correlation between myclong transcript number and NB size ( r = 0 . 281 , p<0 . 05 ) , which supports the hypothesis that Myc is a significant regulator of NB size at this stage .",
"However , we found no significant correlation between Imp levels and myclong transcript numbers or NB size .",
"The myc transcript number is controlled on multiple levels through both transcriptional and post-transcriptional mechanisms , and transcriptional activation of myc is a downstream consequence of many signalling pathways in the brain .",
"Imp regulates myc mRNA stability to modify the final number of transcripts in each cell and as Imp levels decline through development myc mRNA stability also decreases .",
"These results support the hypothesis that intrinsic Imp levels provide a mechanism to fine-tune the amount of Myc protein produced in each NB , allowing NB growth and division to be determined in each NB independently throughout its lifespan ."
],
[
"Myc is known to promote stem cell character and must be switched off in progeny cells to allow correct differentiation ( Betschinger et al . , 2006; Gallant , 2013 ) .",
"We found that Myc overexpression increases both type I NB size and division rate , which is a very interesting result since Myc is best known to drive cell growth through activation of ribosome biogenesis ( Grewal et al . , 2005 ) .",
"Myc also promotes a shortened G1 phase in the wing disc , but this does not increase division rate as the G2 phase is proportionately lengthened ( Johnston et al . , 1999 ) .",
"In the NB , the increased division rate we observe with Myc overexpression could be the result of a direct effect of Myc driving cell cycle progression , which would be mechanistically different from the cells of the wing disc .",
"Alternatively , division rate may be increased indirectly as a result of the larger cell size .",
"Further experiments will be required to uncover the precise mechanism of Myc action in the NB .",
"Our discovery of Imp-dependent modulation of Myc levels adds another dimension of regulation allowing cell-intrinsic modulation of NB growth and division tailored to individual NBs .",
"It has been shown that Brat , an RBP , translationally represses Myc in type II NB progeny cells ( intermediate neural progenitors ) to prevent formation of ectopic NBs ( Bello et al . , 2008; Betschinger et al . , 2006; Boone and Doe , 2008; Bowman et al . , 2008 ) .",
"Together these findings emphasise the importance of the complex network of RBPs that play crucial post-transcriptional roles to control growth and division in individual NBs and their progeny in brain development .",
"Our work also suggests a new potential mechanism by which NB growth and division is restrained toward the end of the stem cell lifespan , in preparation for the terminal division in the pupa .",
"The intrinsic regulation of myc mRNA stability by Imp could explain why NBs are insensitive to the general growth signalling pathways at their late stages ( Homem et al . , 2014 ) .",
"Homem et al . , show that activation or inhibition of signalling through insulin-like peptides or their effector FOXO , has no effect on NB shrinkage or termination .",
"Our results demonstrate that in the late larval NBs , there is insufficient Imp to stabilise myc mRNA , so that upregulation of myc transcription would still lead to low levels of Myc protein .",
"MB NBs are the longest lived NBs in the larval brain and their growth and division only finally slows at about 72 hr after pupal formation ( Siegrist et al . , 2010 ) , 24 hr after the termination of the other type I NBs ( Yang et al . , 2017a ) .",
"It was previously shown that NB decommissioning is initiated through a metabolic response to ecdysone signalling , via Mediator ( Homem et al . , 2014 ) .",
"Elevated Imp level inhibits Mediator in the MB NBs to extend their lifespan by preventing NB shrinkage ( Yang et al . , 2017a ) .",
"However , Yang et al . ( 2017a ) , found that inhibition of the Mediator complex only partially explained the lack of cell shrinkage in the long-lived MB NBs , suggesting that other targets of Imp also play a role in MB NBs .",
"Imp stabilisation of myc mRNA might additionally promote NB growth to contribute to extending the MB NB proliferative lifespan .",
"In contrast , Imp levels decline faster in the other type I NBs , which would restrain their growth and division in preparation for their earlier decommissioning .",
"We also examined the role of Imp earlier in larval development , at 72 hr ALH when Imp levels are higher and heterogeneous between individual NBs .",
"Type I NBs at 72 hr ALH have higher myc mRNA stability and increased cell size compared to type I NBs at wL3 .",
"Our measurements of multiple variables in single cells allowed us to examine the function of Imp expression heterogeneity between individual NBs .",
"We found that Imp levels correlate with myc mRNA stability in individual NBs at 72 hr ALH , providing a cell intrinsic mechanism to modulate NB growth and division .",
"However , Imp levels do not correlate with NB size , unlike at the later wL3 stage .",
"In the early larva , Imp and Myc levels are rapidly changing so a snapshot measurement of NB size may not be a suitable proxy for cell growth at each time point .",
"Resolving this issue will require more sophisticated methods for long-term imaging of live whole brains that allow direct measurement of the growth and division rates of each NB at the same time as the Imp and Myc levels .",
"We have identified a mechanism of cell-intrinsic regulation of individual NB division and growth , which we suggest plays a key role in ensuring the correct number of progeny is produced in each lineage to build the correct sub-regions and circuits in the brain .",
"This intrinsic regulatory mechanism must be integrated with extrinsic growth signals in the brain to determine the growth and division of each stem cell throughout development .",
"Systemic insulin and ecdysone signalling are known to promote the timing of developmental switches in NBs , at the exit from quiescence after larval hatching and the decommissioning of the NB in the pupa .",
"In the final stages of larval development , brain growth is also driven locally to protect it from nutrient restriction , in a process called brain sparing , by which Jelly-Belly expressed by the glial niche bypasses the insulin signalling pathway ( Cheng et al . , 2011 ) .",
"It is plausible that this local extrinsic regulation might also be specific to individual NBs , for example through controlled expression level of Jelly-Belly in each glial niche .",
"Future experiments will determine the interplay between the intrinsic regulation of myc stability by Imp that we have shown here , and other extrinsic systemic and local regulators of NB growth and division .",
"c-myc , the mammalian homologue of Drosophila myc , is best known for its role in cancers , and so its regulation has been studied extensively ( reviewed in Conacci-Sorrell et al . , 2014; Farrell and Sears , 2014 ) .",
"It is therefore interesting to consider to what extent the mechanism we have uncovered is conserved between c-myc and Drosophila myc .",
"The mammalian homologue of Imp , IGF2BP1 , binds to c-myc mRNA and regulates its stability .",
"However , IGF2BP binds to c-myc mRNA in the coding sequence , whereas Imp binds to myc UTRs in Drosophila .",
"IGF2BP1 is known to stabilise c-myc transcripts by blocking translation-coupled decay ( Bernstein et al . , 1992; Doyle et al . , 1998; Lemm and Ross , 2002; Weidensdorfer et al . , 2009 ) , but in Drosophila , Imp’s exact mechanism of stabilisation is not yet known .",
"Nevertheless , the similarity of the two cases suggests that Imp regulation of myc stability might play a conserved role , coordinating stem cell growth and division with developmental progression .",
"The activity of stem cells in every context must be precisely restrained to prevent uncontrolled proliferation , and produce the correct numbers of each cell type to build the organ .",
"We have discovered an important new regulatory mechanism , that Imp acts through myc mRNA stability to modulate cell growth and division appropriately in each stem cell and each stage of development .",
"During development , lengthening of the G1 phase to extend the cell cycle length of NSCs is correlated with a switch from expansion to differentiation in the mouse ventricular zone ( Takahashi et al . , 1995 ) .",
"It has been proposed that Myc is a critical link between cell cycle length and pluripotency ( Singh and Dalton , 2009 ) .",
"In parallel , Imp expression levels have been shown to occur in declining temporal gradients in diverse stem cells including the Drosophila testis ( Toledano et al . , 2012 ) and , in vertebrates , mouse foetal NSCs ( Nishino et al . , 2013 ) .",
"These diverse studies support our proposal of a new general principal that Imp temporal gradients limit stem cell proliferative potential towards the end of their developmental lifespan , by reducing myc mRNA stability and leading to low Myc protein level .",
"Future experiments in a wide range of other organs and systems will now be required to test our model , and to examine the extent of Imp expression heterogeneity in other stem cell systems ."
],
[
"Drosophila melanogaster fly stocks were kept at 18°C , but transferred to 25°C for crosses and experimental use .",
"OregonR was the wild type strain .",
"Flies were raised on standard cornmeal-agar medium .",
"smFISH probes were designed using the Stellaris Probe Designer version 4 . 2 .",
"The sequences against which the probes were designed are shown in Supplementary file 2 .",
"Stellaris DNA probes were gently resuspended in 95 μl fresh TE buffer and 5 ul RNAse inhibitor ( RNAsin Plus RNase Inhibitor , Promega ) , and frozen at −80°C in 10 μl aliquots .",
"Dissected brains from male larvae were rinsed once with 0 . 3% PBSTX and then fixed in 4% PFA ( in 0 . 3% PSTX ) for 25 min ( for wL3 ) or 15 min ( for 72 hr ALH ) at RT .",
"Samples were rinsed briefly and then washed 3 × 15 min in 0 . 3% PBSTX at RT .",
"Samples were washed for 5 min in Wash Buffer ( 10% deionised formamide ( stored at −80°C ) and 2x SSC in DEPC water ) and then incubated with 250 nM Stellaris DNA probes in Hybridisation Buffer ( 10% deionised formamide , 2x SSC and 5% dextran sulphate in DEPC water ) overnight at 37°C on a rocker .",
"Samples were rinsed briefly 3x in Wash Buffer , and then washed 3 × 15 min in Wash Buffer at 37°C .",
"For nuclear staining DAPI ( 4’ , 6-diamidino-2-phenylindole ) was included at 1:500 in the second wash .",
"Brains were mounted in VECTASHIELD anti-fade mounting medium ( Vector Labs ) .",
"Slides were either imaged immediately or stored at −20°C .",
"DAPI was used to stain nuclei , and was added at 1:500 in one of the final wash steps before mounting .",
"Phalloidin was used to label F-actin and was added in one of the final wash steps and incubated for 1 hr at 37°C .",
"Fluorescein 488 phalloidin was used at 5 μl per 100 μl , 647 Phalloidin was used at 2 . 5 μl per 100 μl .",
"Brains were dissected in Schneider’s medium and then transferred to Brain Culture Medium ( 80% Schneider’s medium , 20% fetal bovine serum ( Gibco ThermoFisher ) , 0 . 1 mg/ml insulin ( Sigma ) ) with 25 μM EdU for 4 hr .",
"Brains were then washed with Schneider’s medium and fixed for 25 min in 4% PFA in 0 . 3% PBSTX at RT .",
"The samples were rinsed and then washed 3 × 15 min in 0 . 3% PBSTX at RT before blocking for 1 hr at RT in Blocking Buffer .",
"Samples were incubated with anti-Dpn antibody in Blocking Buffer overnight at 4°C .",
"The following day , samples were washed in Blocking Buffer and then incubated with Alexa Fluor secondary antibody ( Thermofisher ) at 1:200 in Blocking Buffer and samples were incubated for 1 hr at RT in the dark .",
"Samples were washed 3 × 15 min in 0 . 3% PBSTX at RT and then fixed in 1% PFA in 0 . 3% PBSTX at RT for 15 min .",
"Samples were washed and then incubated in Blocking Buffer for 1 hr .",
"The Click-iT reaction was carried out with the Click-iT EdU Alexa Fluor 488 Imaging Kit ( Invitrogen ) following manufacturer’s instructions for 30 min at RT .",
"Samples were washed in 0 . 3% PBST with 5 mM EDTA , once including DAPI , and then mounted in VECTASHIELD anti-fade mounting medium ( Vector Labs ) .",
"Samples were imaged on the same day .",
"An inverted Olympus FV3000 Laser Scanning Microscope was used for fixed imaging of larval brains .",
"Images were acquired using 60x/1 . 30 NA Si UApoN objective .",
"For smFISH quantitation images , pixel size was 74 nm in x and y , and 200 nm in z .",
"The presented RNA sequencing data has been deposited with Gene Expression Omnibus ( GEO ) , with accession number GSE140704 .",
"Further details of the analysis and code are available in Source code 1 ."
]
] | [
"The numerous neurons and glia that form the brain originate from tightly controlled growth and division of neural stem cells , regulated systemically by important known stem cell-extrinsic signals .",
"However , the cell-intrinsic mechanisms that control the distinctive proliferation rates of individual neural stem cells are unknown .",
"Here , we show that the size and division rates of Drosophila neural stem cells ( neuroblasts ) are controlled by the highly conserved RNA binding protein Imp ( IGF2BP ) , via one of its top binding targets in the brain , myc mRNA .",
"We show that Imp stabilises myc mRNA leading to increased Myc protein levels , larger neuroblasts , and faster division rates .",
"Declining Imp levels throughout development limit myc mRNA stability to restrain neuroblast growth and division , and heterogeneous Imp expression correlates with myc mRNA stability between individual neuroblasts in the brain .",
"We propose that Imp-dependent regulation of myc mRNA stability fine-tunes individual neural stem cell proliferation rates ."
] | [
"The brain is a highly complex organ made up of huge numbers of different cell types that connect up to form a precise network .",
"All these different cell types are generated from the repeated division of a relatively small pool of cells called neural stem cells .",
"The division of these cells needs to be carefully regulated so that the correct number and type of nerve cells are produced at the right time and place .",
"But it remains unclear how the division rate of individual neural stem cells is controlled during development .",
"Controlling these divisions requires the activity of countless genes to be tightly regulated over space and time .",
"When a gene is active , it is copied via a process called transcription into a single-stranded molecule known as messenger RNA ( or mRNA for short ) .",
"This molecule provides the instructions needed to build the protein encoded within the gene .",
"Proteins are the functional building blocks of all cells .",
"The conventional way of controlling protein levels is to vary the number of mRNA molecules made by transcription .",
"Now , Samuels et al . reveal a second mechanism of determining protein levels in the brain , through regulating the stability of mRNA after it is transcribed .",
"Samuels et al . discovered that a key regulatory protein called Imp controls the growth and division of individual neural stem cells in the brains of developing fruit flies .",
"The experiments showed that Imp binds to mRNA molecules that contain the code for a protein called Myc , which is known to drive cell growth and division in many different cell types .",
"Both human Imp and Myc have been implicated in cancer .",
"Using a technique that images single molecules of mRNA , Samuels et al . showed that the Imp protein in stem cells stabilises the mRNA molecule coding for Myc .",
"This means that when more Imp is present , more Myc protein gets produced .",
"Thus , the level of Imp in each individual neural stem cell fine-tunes the rate at which the cell grows and divides: the higher the level of Imp , the larger the stem cell and the faster it divides .",
"These findings underscore how important post-transcriptional processes are for regulating gene activity in the developing brain .",
"The methods used in this study to study mRNA molecules in single cells also provide new insights that could not be derived from the average measurements of many cells .",
"Similar methods could also be applied to other developmental systems in the future ."
] | 2020 |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.